Handbook of Diagnostic Endocrinology [3 ed.] 9780128182772

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Handbook of Diagnostic Endocrinology [3 ed.]
 9780128182772

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Table of contents :
Cover
Handbook of Diagnostic Endocrinology
Copyright
Dedications
Contents
List of Contributors
Preface and Acknowledgments
Acknowledgments
1 Maximizing the value of laboratory tests
Interferences associated with mass spectrometry
Exogenous interference
Hemolysis, lipemia, and icterus
Matrix effects
Blood collection tubes
Tube wall
Rubber stopper
Anticoagulants
Surfactants
Clot activators
Separator gel
Label interferences
Drugs and herbal medicine
Timing of sample collection
Storage/freeze-thaw
Analytical errors
Carryover
Endogenous interference
Excess antigen interference (hook effect)
Antibody specificity (cross-reactivity)
Antibody interference
Heterophile antibodies
Human antianimal antibodies
Human antimouse antibodies
Autoantibodies
Rheumatoid factors
Endogenous hormone-binding proteins
Other plasma proteins
Fibrinogen
Complement proteins
Lysozymes
Paraproteins
Albumin
Detection and testing for interference in suspected samples
Biotin
Prevalence of elevated biotin
Impact of biotin interference on diagnostic assays
Detection of biotin interference
Anti-ruthenium and anti-streptavidin antibody interference
Serial monitoring and reference change value: Determining whether changes are significant
Physiological variation (intra-individual variation)
Analytic variation
Calculating significant variation
Example 1—Is the statin working?
Example 2—Is the DPP-4 inhibitor working?
Example 3—Sestamibi scan or surgical consult?
Automated calculations
Postanalytical errors
Reference ranges, normal values, and desired results
Sensitivity, specificity, and predictive value
Receiver operating characteristic plot3
Likelihood ratio
Summary of postanalytical errors
Conclusion
References
2 Laboratory investigation of disorders of the pituitary gland
Anterior pituitary
Hypothalamic regulators of anterior pituitary hormones
Anterior pituitary hormone physiology and biochemistry
Features of hypopituitarism
Investigation of hypopituitarism
Secretory pituitary tumors
Prolactinomas
Acromegaly
Other secretory tumors
Posterior pituitary
Diabetes insipidus
SIADH
Conclusion
References
Suggested reading
3 Thyroid disease and laboratory assessment
The thyroid gland
Physiology of the thyroid–pituitary–hypothalamic axis
Metabolism
Signs and symptoms
Screening for thyroid dysfunction
Variables affecting testing of thyroid function
The serum TSH/FT4 relationship
Effects of chronological age on thyroid test reference ranges
Pregnancy
Pathological variables
Medications
Nonthyroidal illness
Specimen variables
Disease-specific variation
Caloric deprivation
Hepatic disorders
Renal failure
Acquired immunodeficiency syndrome and acquired immunodeficiency syndrome—related complex
Psychiatric disorders
Helpful clues to distinguish thyroid disease from nonthyroidal causes of abnormal hormone levels
Thyroid disorders
Hyperthyroidism
Iatrogenic hyperthyroidism
Transient hyperthyroidism
High thyroid-stimulating hormone with hyperthyroidism
Hypothyroidism
Primary hypothyroidism
Central hypothyroidism
Hypothyroidism after radioactive iodine therapy
Treated hypothyroidism
Neonatal hypothyroidism
Effects of binding protein aberrations on thyroid function tests
Screening for subclinical thyroid disease
Laboratory tests used in the assessment of thyroid function
Thyroid-stimulating hormone (thyrotropin)
Specificity
Sensitivity
Thyroid-stimulating hormone reference intervals
Thyroid-stimulating hormone upper reference limits
Thyroid-stimulating hormone lower reference limits
Clinical use of serum thyroid-stimulating hormone measurements
Screening for thyroid dysfunction in ambulatory patients
Elderly patients
L-T4 replacement therapy
L-T4 suppression therapy
Serum thyroid-stimulating hormone measurement in hospitalized patients with nonthyroidal illness
Central hypothyroidism
Inappropriate thyroid-stimulating hormone secretion syndromes
Thyroid-stimulating hormone–secreting pituitary tumors
Thyroid hormone resistance
Thyrotropin-releasing hormone test
FT4
Influence of methods of analysis on test results
Interferences/considerations for interpretation of results
Normal range
T4
Normal range
Resin uptake ratio
Normal range
Free thyroid index
Normal range
Interpretation
T3
Normal range
Free T3
Normal range
Reverse T3
Tg
Analytical methods
Normal range
Thyroid autoantibodies
Analytical methods
Utilization of laboratory tests in the diagnosis and monitoring of thyroid disease: recommendations for testing
Interpretive hints when thyroid-stimulating hormone and FT4 results seem discrepant
Further reading
4 Disorders of the adrenal gland*
Adrenocortical insufficiency
Detecting cortisol deficiency
Clinical symptoms and signs of adrenocortical insufficiency
Causes of adrenocortical insufficiency
Laboratory investigation of adrenocortical insufficiency
Patients presenting with Addisonian crisis
Patients with suspected glucocorticoid deficiency who do not present acutely ill
Tests of adrenal function
Adrenocorticotropic hormone stimulation tests
The 1-h adrenocorticotropic hormone stimulation test
8-h, 2-day, and 3- to 5-day adrenocorticotropic hormone stimulation tests
Corticotropin-releasing hormone stimulation test
Insulin-induced hypoglycemia test
Glucagon stimulation test
Mineralocorticoid assessment
Evaluation for specific causes of primary adrenocortical insufficiency
Autoimmune Addison disease
Congenital adrenal hyperplasia
Adrenoleukodystrophy and adrenomyeloneuropathy
Zellweger spectrum disorder
Congenital adrenal hypoplasia
Steroidogenic factor-1
Wolman disease
Mitochondrial causes of Addison disease
Smith–Lemli–Opitz syndrome
Adrenocortical excess
Clinical symptoms of glucocorticoid excess (Cushing syndrome)
The differential diagnosis of glucocorticoid excess
Cortisol resistance and ectopic adrenocorticotropic hormone Cushing syndrome
The biochemical diagnosis of glucocorticoid excess
Plasma and salivary cortisol
Urinary free cortisol measurements
Overnight dexamethasone suppression test
Low-dose dexamethasone-corticotropin-releasing hormone test
DDAVP stimulation test
Adrenocorticotropic hormone measurements
Bilateral inferior petrosal venous sinus sampling and corticotropin-releasing hormone stimulation testing
Evaluation for adrenal tumors
Mineralocorticoid excess
Adrenal tumors
Glucocorticoid-remediable aldosteronism
Apparent mineralocorticoid excess
Biochemical evaluation of primary aldosteronism
Pheochromocytoma and paraganglioma
Clinical overview
Isolated and familial pheochromocytoma/paraganglioma
Neurofibromatosis Type 1
Von Hippel–Lindau disease
Laboratory diagnosis of the pheochromocytoma/paraganglioma
Urine catecholamines and metanephrines
Plasma catecholamines and metanephrines
Clonidine suppression test
Chromogranin A
Neuroblastoma, ganglioneuroma, and ganglioneuroblastoma
List of abbreviations
References
5 Endocrine disorders of the reproductive system
Hypothalamic-pituitary-gonadal axis
Female reproductive disorders
Prepubertal disorders
Hirsutism
Irregular menses
Polycystic ovarian syndrome
Infertility
Ovulatory dysfunction
Ovarian reserve
Structural factors
Treatments
Male reproductive disorders
Prepubertal disorders
Hypogonadotropic hypogonadism
Hypothalamic causes
Hypergonadotropic hypogonadism
Klinefelter syndrome
Defects in androgen action
Erectile dysfunction
Gynecomastia
Infertility
Semen analysis
Endocrine parameters
Treatment
References
6 Gastroenteropancreatic neuroendocrine tumors
Biochemistry and physiology of the more common gastrointestinal hormones
Gastrin
Somatostatin
Secretin
Cholecystokinin
Pancreatic polypeptide
Pancreatic neuroendocrine tumors
Glucagonoma
Somatostatinoma
VIPoma
Gastrointestinal neuroendocrine tumors
Carcinoid tumor
Nonfunctioning neuroendocrine tumor (pancreatic neuroendocrine tumors)
Conclusion
References
7 Evaluation of hypoglycemia
Introduction
Clinical symptoms of hypoglycemia
Fasting hypoglycemia versus reactive hypoglycemia
Diagnosis of hypoglycemia
Biochemical definition of hypoglycemia
Causes of hypoglycemia
Diagnostic workup for hypoglycemia
Evaluation of acute hypoglycemia
Prolonged fasts in the evaluation of hypoglycemia
Testing strategy for evaluation of hypoglycemia
Hypoglycemia syndromes with hyperinsulinism
Hyperinsulinism with elevated C-peptide
Neonatal hyperinsulinism
Hyperinsulinism in children and adults
Hyperinsulinism with suppressed C-peptide
Hyperinsulinism: biochemical findings compatible with hyperinsulinism in the absence of measured hyperinsulinism
Nonhyperinsulinemic hypoglycemia
Drugs as causes of hypoglycemia
Endocrinopathies as causes of hypoglycemia
Carbohydrate and amino acid inborn errors of metabolism causing hypoglycemia
Disorders of carbohydrate metabolism
Galactosemia and hereditary fructose intolerance
The glycogen storage diseases
Defects in gluconeogenic enzymes
Aminoacidopathies with associated hypoglycemia
Liver and renal disorders as causes of hypoglycemia
Limited substrate/increased utilization as causes of hypoglycemia
Disorders of fatty acid oxidation
Summary of the evaluation of hypoglycemia
References
8 Evaluation of hyperglycemia
Hyperglycemia: acute versus chronic
Clinical symptoms of diabetes
Classification of diabetes
Type 1 diabetes
Type 2 diabetes
Metabolic syndrome
Metabolic syndrome consequences that result from hyperinsulinism
Metabolic syndrome consequences that result from inadequate insulinization
Other specific types of diabetes
Genetic defects of β cell function
Maturity-onset diabetes of youth
Neonatal diabetes
Mitochondrial diabetes
Insulinopathies and hyperproinsulinopathies
Other specific types of diabetes: considerations
Genetic defects in insulin action
Diseases of the exocrine pancreas
Endocrinopathies causing diabetes
Drug-induced diabetes
Infection and diabetes
Uncommon forms of immune-mediated diabetes
Genetic syndromes and diabetes
Gestational diabetes mellitus
Diagnosis of diabetes
Nonpregnant adults and children
Choice of the blood specimen for measuring glucose
Choice of tube type for phlebotomy
The oral glucose tolerance test
Measuring insulin or C-peptide
Testing for gestational diabetes mellitus
Screening strategies for diabetes mellitus
Adults
Children
Biochemical monitoring in diabetes
Self-monitoring of blood glucose and point-of-care testing
Assessment of diabetic control
Fructosamine
Evaluation of lipid status
Renal evaluation
Ketone testing
Other testing
Conclusion
References
9 Lipoproteins
Background
Classification of dyslipidemias
Hypercholesterolemia
Hypertriglyceridemia
Combined hyperlipidemia
Hypolipidemia
Hypoalphalipoproteinemia
Hypotriglyceridemia
Laboratory assessment of dyslipidemia
Conclusion
References
10 Disorders of calcium metabolism
Introduction
Calcium distribution in the body
Circulating calcium
Calcium measurements
Phosphate measurements
Clinical indications to measure calcium, phosphate, and related analytes
Calcium and phosphate physiology
Parathyroid glands and parathyroid hormone
Calcium and the renal tubules
Phosphate and the renal tubules
Parathyroid hormone and bone
Calcitonin and bone
Procalcitonin—a marker of bacterial infection
Vitamin D physiology
25-OHD and 1,25-OH2D metabolism
Calcium and phosphate absorption from the gut
Integrating parathyroid hormone and vitamin D actions
Parathyroid hormone assays
Clinical manifestations of disordered calcium or phosphate metabolism
Hypocalcemia
Decreased parathyroid hormone action
Parathyroid hormone deficiency—acquired hypoparathyroidism
Parathyroid hormone deficiency—genetic causes of hypoparathyroidism
Parathyroid hormone resistance
Parathyroid hormone receptor defects: parathyroid hormone receptor-1 mutations
Postparathyroid hormone receptor-1 signaling
Guanine nucleotide-binding protein (G protein), alpha guanine nucleotide-binding protein, α stimulating defects
Defects distal to guanine nucleotide-binding protein, α stimulating
Vitamin D disorders—reduced vitamin D activity
Vitamin D-deficient rickets/osteomalacia
Vitamin D–dependent rickets/osteomalacia
Hepatic rickets
Accelerated vitamin D metabolism
Hyperphosphaturia causing hypophosphatemia
Renal tubular disorders: primary and secondary conditions
Renal phosphate wasting tumor-induced osteomalacia
Hereditary forms of hypophosphatemic rickets/osteomalacia
X-linked hypophosphatemic rickets
Autosomal-dominant hypophosphatemic rickets
Autosomal-recessive hypophosphatemic rickets
Hereditary hypophosphatemic rickets with hypercalciuria
Other causes of hypocalcemia
Calcium deposition in necrotic tissue
Healing phase of bone disease
Dietary calcium deficiency
Hyperphosphatemia
Miscellaneous causes of hypocalcemia
Laboratory approach to hypocalcemia
Hypercalcemia
Hyperparathyroidism
Intraoperative parathyroid hormone measurements
Nonparathyroid hormone-dependent causes of hypercalcemia
Laboratory approach to hypercalcemia
Laboratory monitoring of bone turnover
Other bone diseases with possible laboratory implications
Conclusions
Acknowledgment
References
11 Laboratory evaluation of endocrine hypertension
Introduction
Definition of hypertension
Definition of hypertension in children
Causes of hypertension
Laboratory evaluation of hypertension in adults
Laboratory evaluation of hypertension in children
Endocrine hypertension and mechanisms
Physiology
Laboratory notes
Mechanisms of sodium retention (mechanism 1)
Hyperaldosteronism (mechanism 1a1)
Fludrocortisone suppression test
Oral salt loading or IV saline infusion
Captopril challenge test
Laboratory notes
Renin-dependent hyperaldosteronism (also known as hyperreninemic hyperaldosteronism) (mechanism 1a1)
Renin-secreting tumors (mechanism 1a1)
Renovascular hypertension (mechanism 1a1)
Renin-independent hyperaldosteronism (mechanism 1a1)
Renin-independent hyperaldosteronism, sporadic causes (mechanism 1a1)
Aldosterone-producing adenoma
Bilateral adrenal hyperplasia
Renin-independent hyperaldosteronism, inherited causes (mechanism 1a1)
Glucocorticoid-remediable aldosteronism (familial hyperaldosteronism type I) (mechanism 1a1)
Laboratory notes
Familial aldosterone-producing adrenal adenomas or hyperplasia (familial hyperaldosteronism type II) (mechanism 1a1)
Laboratory notes
KCNJ5 mutation (familial hyperaldosteronism type III) (mechanism 1a1)
Laboratory notes
Excess desoxycorticosterone (mechanism 1a1)
Desoxycorticosterone-secreting tumors (mechanism 1a1)
11β-Hydroxylase deficiency (mechanism 1a1)
CYP17 deficiency (mechanism 1a1)
Laboratory notes
Excess cortisol (mechanism 1a1)
Cushing syndrome (mechanism 1a1)
Laboratory notes
Cortisol resistance (mechanism 1a1)
Laboratory notes
Excess sex steroids (mechanism 1a1)
Laboratory notes
End-organ disorders causing ENaC activation: Apparent mineralocorticoid excess, mineralocorticoid receptor gain-of-function...
Apparent mineralocorticoid excess (mechanism 1a2)
Laboratory notes
Mineralocorticoid receptor gain-of-function mutations (mechanism 1a2)
Laboratory notes
ENaC gain-of-function mutations (mechanism 1a2)
Laboratory notes
Insulin resistance (mechanism 1b): obesity, type 2 diabetes, the metabolic syndrome, and acromegaly: multiple mechanisms of...
Laboratory notes
Pheochromocytoma: direct effects on vascular smooth muscle and myocardium (mechanism 2a)
Laboratory testing
Hyperthyroidism (mechanism 2a) and hypothyroidism
Laboratory testing
Hypercalcemia: Direct effects on vascular smooth muscle (mechanism 2b)
Laboratory testing
SIADH: vasopressin excess with direct effects on vascular smooth muscle (mechanism 2c)
Laboratory testing
Hypertension in pregnancy—abnormal angiogenesis (mechanism 2c)
Laboratory testing
Approach to the patient
References
12 Malignancy-associated endocrine disorders
Introduction
Multiple endocrine neoplasia syndromes [4–6]
MEN1 [4–10]
Epidemiology
Risk and inheritance factors
Characterization
Diagnostic and screening laboratory tests
MEN2 [5,6,7,10–17]
Classification
Epidemiology
Risk and inheritance factors
Characterization
Diagnostic and screening laboratory tests
MEN4 [5–8]
Gastrointestinal neuroendocrine tumors
Carcinoid tumors
Gastrinoma (Zollinger–Ellison syndrome) [5,6,8,18–22]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features and laboratory tests
Glucagonoma [8,19,21–23]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features and laboratory tests
Insulinoma [6,8,19,21,24–26]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features and laboratory tests
Somatostatinoma [19,21–23,27]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features and laboratory tests
VIPoma (vasoactive intestinal polypeptide) [6,8,19,21–23,28,29]
Epidemiology
Pathology
Clinical features and laboratory tests
Adrenal tumors
Adrenocortical carcinoma [30–36]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features
Laboratory tests
Tumor imaging
Neuroblastoma [1,37–42]
Epidemiology
Risk and inheritance factors
Pathology
Clinical features
Laboratory tests
Tumor imaging
Pheochromocytoma [17,26,43–46]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features and laboratory tests
Tumor imaging
Parathyroid and thyroid tumors
Parathyroid [47–53]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features
Laboratory tests
Tumor imaging and surgery
Thyroid [1,11–13,15,54–60]
Epidemiology
Risk and hereditary factors
Pathology
Clinical features
Laboratory tests
Other tests
Tumor imaging
References
13 Laboratory assessment of acquired immunodeficiency syndrome endocrinopathies
Lipid, glucose, and fat metabolism
Thyroid disorders
Infections and neoplasms
Thyroid function test profile
Adrenal disorders
Infection and neoplasm
Glucocorticoids
Mineralocorticoids
Gonadal function
Females
Pituitary function
Hyponatremia
Bone and mineral metabolism
Tenofovir-induced hypophosphataemia
Other
Autoantibodies
Endocrine and metabolic emergencies
Conclusions
References
14 Laboratory evaluation of short stature in children
Introduction
Definition of short stature
Short stature with normal growth velocity
Familial short stature
Constitutional delay in growth and adolescence
Primordial short stature
Short stature with low growth velocity
Chronic disease
Turner syndrome
Psychosocial short stature
Endocrinopathies
Hypothyroidism
Growth hormone deficiency
Growth hormone testing
Growth hormone treatment
Insulin-like growth factor-I, insulin-like growth factor-II, and insulin-like growth factor binding protein-3
Insulin-like growth factor-I as a therapeutic agent
Insulin-like growth factor-I mutations
Type I insulin-like growth factor receptor mutations
Evaluation of individuals diagnosed with growth hormone deficiency
Other endocrine and genetic causes of short stature
Conclusion
Acknowledgment
References
15 Pregnancy and the fetus
Introduction
Feto-placental unit
Diagnosis and dating of pregnancy
Conception and implantation
Placenta
Placental hormones: pituitary
Human chorionic gonadotropin
Placental growth hormone
Human placental lactogen
Placental adrenocorticotropic hormone
Placental hormones: hypothalamus
Gonadotropin-releasing hormone
Corticotropin-releasing hormone
Thyrotropin-releasing hormone
Other placental hormones
Pregnancy-associated plasma protein A
Insulin-like growth factors I and II
Angiogenic factors
Inhibin and activin
Placental steroid hormones
Progesterone
Estrogens
Fetal endocrine function
Hypothalamus and pituitary
Fetal thyroid
Fetal gonads
Fetal adrenal glands
Maternal endocrine function
Hypothalamus and pituitary
Parathyroid glands
Thyroid
Normal thyroid function in pregnancy
Hyperemesis gravidarum
Maternal thyroid disease
Screening for thyroid disease during pregnancy
Laboratory assessment of thyroid function in pregnancy
Maternal adrenal function
Abnormal pregnancies
Ectopic pregnancy
Preeclampsia
Laboratory testing in preeclampsia
Trophoblastic disease
HELLP
Preterm delivery and premature rupture of membranes
Preterm delivery
Premature rupture of membranes
Fetal lung maturity
Surfactant/albumin ratio
Lamellar body count
Lecithin to sphingomyelin ratio
Phosphatidylglycerol
Prenatal screening for fetal defects
Multiple of the median
Neural tube defects
Trisomy 21
Trisomy 18
Trisomy 13
Sex chromosome aneuploidies
Biochemical prenatal screening
First trimester serum screen
Quad screen
Combined first and second trimester screening
Cell-free DNA screening
References
16 Disorders of sexual development
Introduction
Genitalia: structure and development
A clinical and laboratory approach to disorders of sexual development
Initial evaluation of persons with a disorder of sexual development
Sex chromosome disorders of sexual development
Sex chromosome disorders of sexual development—X-chromosome anomalies
Turner syndrome
Laboratory findings –
Trisomy (triple) X
Laboratory findings –
46, XX sex-reversed male
Laboratory findings –
Sex chromosome disorders of sexual development—Y-chromosome anomalies
Klinefelter syndrome
Laboratory findings –
47, XYY
Laboratory findings –
46, XYp-
Laboratory findings –
46, XY sex-reversed female
Laboratory findings –
Sex chromosome disorders of sexual development—X- and Y-chromosome anomalies
Mixed gonad dysgenesis
Laboratory findings –
Sex chromosome disorders of sexual development—ovotesticular disorders of sexual development
Laboratory findings
Sex chromosome disorders of sexual development—summary
46, XY disorders of sexual development
46, XY disorders of sexual development—failure of normal testicular development
Complete gonadal dysgenesis
Laboratory findings –
Partial gonadal dysgenesis
Laboratory findings –
Gonadal regression
Laboratory findings
Testosterone—a Leydig cell marker
Use of testosterone measurements as a testicular marker –
INSL3—a Leydig cell marker
Use of INSL3 as a testicular marker –
Inhibin B—a Sertoli cell marker
Use of inhibin B as a testicular marker –
Anti-Mullerian hormone—a Sertoli cell marker
Use of anti-Mullerian hormone as a testicular marker –
46, XY disorders of sexual development—gonadotropin deficiency
Laboratory findings
46, XY disorders of sexual development—inborn errors in testosterone synthesis
CYP17 deficiency
Laboratory findings –
17-ketosteroid reductase deficiency
Laboratory findings –
Note for completeness –
3 beta-hydroxysteroid dehydrogenase-delta4,5isomerase deficiency
Laboratory findings –
Cytochrome P450 oxidoreductase (POR) deficiency
Laboratory findings –
46, XY disorders of sexual development— dihydrotestosterone synthesis abnormalities
Laboratory findings
46, XY disorders of sexual development—disorders of androgen action
Complete androgen insensitivity
Incomplete (partial) androgen insensitivity
Laboratory findings –
46, XY disorders of sexual development—deficient anti-Mullerian hormone action
Laboratory findings
46, XY disorders of sexual development—isolated and/or complex Genitourinary (GU) malformations
Laboratory findings
46, XY-chromosome disorders of sexual development—summary
46, XX disorders of sexual development
46, XX disorders of sexual development—disorders of ovarian development
46, XX disorders of sexual development—fetal androgen excess
Virilizing forms of congenital adrenal hyperplasia
21-hydroxylase deficiency
Laboratory findings –
11-beta hydroxylase deficiency
Laboratory findings –
CYP17 deficiency
Laboratory findings –
Nonadrenal fetal androgen excess
Laboratory findings –
Maternal androgen exposure
Laboratory findings –
46, XX disorders of sexual development—isolated or complex malformations affecting the female
46, XX chromosome disorders of sexual development—summary
Approaching the laboratory diagnosis of disorders of sexual development
Sex chromosome disorders of sexual development
46, XY disorders of sexual development
46, XX disorders of sexual development
Considerations
References
17 Transgender endocrinology
Introduction
Transgender adolescents
Diagnosing gender dysphoria in minors
Monitoring for puberty onset
Beginning pubertal suppression
Initiating gender-affirming hormones
Fertility concerns
Feminizing therapies
Masculinizing
Overarching concerns
References
18 The endocrinology of aging
Background
Hypothalamic-pituitary-gonadal/adrenal androgen axes
Menopause and its biochemical features
Gonadotropins, inhibins, and anti-Müllerian hormone
Estradiol and progesterone
Sex hormone binding globulin
Pituitary human chorionic gonadotropin in females
Testosterone deficiency in older males
Declines in dehydroepiandrosterone its sulfate ester
Hypothalamic-pituitary-somatotropic axis
Growth hormone
Insulin-like growth factor 1
Hypothalamic-pituitary-adrenal axis
Dexamethasone suppression
Twenty-four-hour urinary cortisol and salivary cortisol
Salivary cortisol
Conclusions on screening for Cushing’s syndrome
Adrenocorticotropic hormone stimulation
Hypothalamic-pituitary-thyroidal axis
Mild abnormalities: normal aging or disease?
Paradoxes and presumed benefits
Overt thyroid dysfunction
The effect of comorbidities
Clinical management
Conclusion
Appendix 1: Tabular summary of physiological changes
References
Index

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Handbook of Diagnostic Endocrinology

Handbook of Diagnostic Endocrinology

Third Edition Edited by William E. Winter Departments of Pathology, Immunology & Laboratory Medicine, Pediatrics, and Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, United States

Brett Holmquist Endocrine Sciences, Laboratory Corporation of America, Calabasas, CA, United States

Lori J. Sokoll Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, United States

Roger L. Bertholf Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, United States

Published in cooperation with AACC

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. About AACC Dedicated to achieving better health through laboratory medicine, AACC brings together more than 50,000 clinical laboratory professionals, physicians, research scientists, and business leaders from around the world focused on clinical chemistry, molecular diagnostics, mass spectrometry, translational medicine, lab management, and other areas of progressing laboratory science. Since 1948, AACC has worked to advance the common interests of the field, providing programs that advance scientific collaboration, knowledge, expertise, and innovation. For more information, visit www.aacc.org. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-818277-2 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals Publisher: Stacy Masucci Acquisitions Editor: Ana Claudia Garcia Editorial Project Manager: Pat Gonzalez Production Project Manager: Maria Bernard Cover Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

Dedications William E. Winter, MD—This book is dedicated to the untiring support provided to me by my spouse Nancy S. Hardt, MD, my children, William P. Winter, MPH, RN and Katherine H. Winter, MPH, MD, my son-in-law, Abid H. Ahmed, MD, and my grandson, Kian Winter Ahmed. I also wish to recognize my fellowship mentors Arlan L. Rosenbloom, MD and Noel K. Maclaren, MD for launching my academic career and Roger L. Bertholf, PhD, Ishwarlal Jialal, MD, PhD, and Robert Dufour, MD for their support during my professional career. Roger L. Bertholf, PhD—To my family, Marsha, Aaron, and Abby, for their love and support, and to the many professional colleagues who have enriched my career with their knowledge and generosity, I dedicate my contributions to this handbook. Lori J. Sokoll, PhD—My contributions to this handbook honor my mentors at Hahnemann University, Ray Vanderlinde, PhD and Fred Kayne, PhD, and at Johns Hopkins University, Daniel W. Chan, PhD, Jim Nichols, PhD, and Martin Kroll, MD, for their wisdom, guidance, and encouragement. Brett Holmquist, PhD—For my wife Carrie and my children Avery and Grant for their support, and to the countless mentors I have had the privilege to work with since donning my first lab coat.

Contents List of contributors Preface and Acknowledgments

1.

Maximizing the value of laboratory tests

xv xvii 1

Raffick A.R. Bowen, Roger L. Bertholf and Brett Holmquist Interferences associated with mass spectrometry Exogenous interference Analytical errors Endogenous interference Serial monitoring and reference change value: Determining whether changes are significant Postanalytical errors Conclusion References

Section 1 Organ systems 2.

Laboratory investigation of disorders of the pituitary gland

2 3 9 9 22 25 35 36

47 49

Verena Gounden, Charlotte C. Ellberg and Ishwarlal Jialal

3.

Anterior pituitary Posterior pituitary Conclusion References Suggested reading

49 60 63 64 66

Thyroid disease and laboratory assessment

69

Sridevi Devaraj and Emily Garnett The thyroid gland Physiology of the thyroid pituitary hypothalamic axis Metabolism

69 69 72

vii

viii

4.

Contents

73 73 74 77 79 79

Signs and symptoms Screening for thyroid dysfunction Variables affecting testing of thyroid function Pathological variables Specimen variables Disease-specific variation Helpful clues to distinguish thyroid disease from nonthyroidal causes of abnormal hormone levels Thyroid disorders Hypothyroidism Effects of binding protein aberrations on thyroid function tests Screening for subclinical thyroid disease Laboratory tests used in the assessment of thyroid function Influence of methods of analysis on test results Interferences/considerations for interpretation of results T4 Resin uptake ratio Free thyroid index T3 Free T3 Reverse T3 Tg Thyroid autoantibodies Utilization of laboratory tests in the diagnosis and monitoring of thyroid disease: recommendations for testing Interpretive hints when thyroid-stimulating hormone and FT4 results seem discrepant Further reading

99 100

Disorders of the adrenal gland

103

80 81 83 85 85 86 93 94 94 95 95 96 97 97 97 98 99

Roger L. Bertholf Adrenocortical insufficiency Laboratory investigation of adrenocortical insufficiency Patients presenting with Addisonian crisis Patients with suspected glucocorticoid deficiency who do not present acutely ill Tests of adrenal function Evaluation for specific causes of primary adrenocortical insufficiency Adrenocortical excess The biochemical diagnosis of glucocorticoid excess Mineralocorticoid excess Pheochromocytoma and paraganglioma List of abbreviations References

103 111 111 113 114 118 129 132 138 140 147 148

Contents

5.

Endocrine disorders of the reproductive system

ix 157

Angela M. Ferguson and Mark A. Cervinski

6.

Hypothalamic-pituitary-gonadal axis Female reproductive disorders Male reproductive disorders References

157 157 168 177

Gastroenteropancreatic neuroendocrine tumors

181

Neeraj Ramakrishnan, Seong Hyun Ahn and Ishwarlal Jialal Biochemistry and physiology of the more common gastrointestinal hormones Pancreatic neuroendocrine tumors Gastrointestinal neuroendocrine tumors Conclusion References

181 183 190 196 198

Section 2 Analytes

201

7.

203

Evaluation of hypoglycemia William E. Winter and Neil S. Harris

8.

Introduction Clinical symptoms of hypoglycemia Fasting hypoglycemia versus reactive hypoglycemia Diagnosis of hypoglycemia Biochemical definition of hypoglycemia Causes of hypoglycemia Diagnostic workup for hypoglycemia Testing strategy for evaluation of hypoglycemia Summary of the evaluation of hypoglycemia References

203 203 204 204 205 205 206 210 228 229

Evaluation of hyperglycemia

237

William E. Winter, David L. Pittman, Sridevi Devaraj, Danni Li and Neil S. Harris Hyperglycemia: acute versus chronic Clinical symptoms of diabetes Classification of diabetes Diagnosis of diabetes Screening strategies for diabetes mellitus Biochemical monitoring in diabetes

237 238 240 262 267 268

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Conclusion References

274 276

Lipoproteins

287

Anna Wolska and Alan T. Remaley Background Classification of dyslipidemias Laboratory assessment of dyslipidemia Conclusion References

10. Disorders of calcium metabolism

287 290 300 305 305 309

William E. Winter and Neil S. Harris Introduction Calcium distribution in the body Circulating calcium Calcium measurements Phosphate measurements Clinical indications to measure calcium, phosphate, and related analytes Calcium and phosphate physiology Parathyroid glands and parathyroid hormone Calcium and the renal tubules Phosphate and the renal tubules Parathyroid hormone and bone Calcitonin and bone Procalcitonin—a marker of bacterial infection Vitamin D physiology 25-OHD and 1,25-OH2D metabolism Calcium and phosphate absorption from the gut Integrating parathyroid hormone and vitamin D actions Parathyroid hormone assays Clinical manifestations of disordered calcium or phosphate metabolism Hypocalcemia Decreased parathyroid hormone action Vitamin D disorders—reduced vitamin D activity Hyperphosphaturia causing hypophosphatemia Hypercalcemia Laboratory monitoring of bone turnover Other bone diseases with possible laboratory implications Conclusions Acknowledgment References

309 309 310 310 312 312 314 314 318 321 323 324 325 326 329 330 332 332 334 335 335 349 351 358 368 369 370 370 370

Contents

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Section 3 Specific topics

389

11. Laboratory evaluation of endocrine hypertension

391

William E. Winter and Neil S. Harris Introduction Physiology Mechanisms of sodium retention (mechanism 1) End-organ disorders causing ENaC activation: Apparent mineralocorticoid excess, mineralocorticoid receptor gain-offunction mutations, and ENaC gain-of-function mutations Approach to the patient References

12. Malignancy-associated endocrine disorders

391 401 409

426 434 438 449

Lori J. Sokoll and Daniel W. Chan Introduction Multiple endocrine neoplasia syndromes MEN1 MEN2 MEN4 Gastrointestinal neuroendocrine tumors Adrenal tumors Parathyroid and thyroid tumors References

13. Laboratory assessment of acquired immunodeficiency syndrome endocrinopathies

449 450 450 454 456 457 460 465 472

477

Verena Gounden and Manisha Chandalia Lipid, glucose, and fat metabolism Thyroid disorders Adrenal disorders Gonadal function Pituitary function Bone and mineral metabolism Other Conclusions References

14. Laboratory evaluation of short stature in children

477 480 483 485 487 488 489 490 491 497

William E. Winter Introduction Definition of short stature

497 497

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Short stature with normal growth velocity Short stature with low growth velocity Insulin-like growth factor-I, insulin-like growth factor-II, and insulin-like growth factor binding protein-3 Evaluation of individuals diagnosed with growth hormone deficiency Conclusion Acknowledgment References

15. Pregnancy and the fetus

500 505 521 528 532 533 533

543

K. Aaron Geno, Mark A. Cervinski and Robert D. Nerenz Introduction Feto-placental unit Diagnosis and dating of pregnancy Conception and implantation Placenta Maternal endocrine function Abnormal pregnancies Preterm delivery and premature rupture of membranes References

16. Disorders of sexual development

543 543 544 544 545 556 560 564 575

581

William E. Winter, Paul Hiers and Dina N. Greene Introduction Genitalia: structure and development A clinical and laboratory approach to disorders of sexual development Initial evaluation of persons with a disorder of sexual development Sex chromosome disorders of sexual development Sex chromosome disorders of sexual development—Ychromosome anomalies Sex chromosome disorders of sexual development—Xand Y-chromosome anomalies Sex chromosome disorders of sexual development—ovotesticular disorders of sexual development 46, XY disorders of sexual development 46, XX disorders of sexual development Approaching the laboratory diagnosis of disorders of sexual development Considerations References

581 583 585 587 587 591 593 593 596 615 624 630 630

Contents

17. Transgender endocrinology

xiii 639

Dina N. Greene, Tamar Reisman and Zil Goldstein Introduction Transgender adolescents Feminizing therapies Masculinizing Overarching concerns References

18. The endocrinology of aging

639 641 646 653 657 658 663

Daniel T. Holmes and Gregory Kline Background Hypothalamic-pituitary-gonadal/adrenal androgen axes Hypothalamic-pituitary-somatotropic axis Hypothalamic-pituitary-adrenal axis Hypothalamic-pituitary-thyroidal axis Conclusion Appendix 1: Tabular summary of physiological changes References Index

663 664 669 670 673 676 677 677 687

List of Contributors K. Aaron Geno Department of Pathology and Laboratory Medicine, DartmouthHitchcock Health System, Lebanon, NH, United States; The Geisel School of Medicine at Dartmouth, Hanover, NH, United States Seong Hyun Ahn California Northstate University, College of Medicine, CA, United States Roger L. Bertholf Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, United States; Department of Pathology, Stanford University, Palo Alto, CA, United States Raffick A.R. Bowen Department of Pathology, Stanford University, Palo Alto, CA, United States Mark A. Cervinski The Geisel School of Medicine at Dartmouth, Hanover, NH, United States; Department of Pathology and Laboratory Medicine, DartmouthHitchcock Health Systems, Lebanon, NH, United States Daniel W. Chan Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, United States Manisha Chandalia Bay Area Metabolic Health, Diabetes Lipids & Endocrinology Clinics, Baytown, TX, United States Sridevi Devaraj Texas Children’s Hospital, Houston, TX, United States; Division of Clinical Chemistry, Texas Children’s Hospital, Houston, TX, United States Charlotte C. Ellberg California Northstate University College of Medicine, Elk Grove, CA, United States Angela M. Ferguson Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, University of Missouri-Kansas City School of Medicine, Kansas City, MO, United States Emily Garnett Division of Clinical Chemistry, Texas Children’s Hospital, Houston, TX, United States Zil Goldstein Callen-Lorde Community Health Center, New York, NY, United States Verena Gounden Department of Chemical Pathology, Inkosi Albert Luthuli Central Hospital, National Health Laboratory Service, University of Kwa-Zulu Natal, Durban, South Africa Dina N. Greene Washington Kaiser Permanente, Renton, WA, United States; University of Washington Medical Center, Seattle, WA, United States Neil S. Harris Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States

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List of Contributors

Paul Hiers Norton Children’s Hospital, University of Louisville, Louisville, KY, United States Daniel T. Holmes Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Brett Holmquist Endocrine Sciences, Laboratory Corporation of America, Calabasas, CA, United States Ishwarlal Jialal Sacramento VA Medical Center, CA, United States; Retired Distinguished Professor, UCDAVIS, CA, United States; California Northstate University College of Medicine, CA, United States Gregory Kline Division of Endocrinology, University of Calgary, Calgary, AB, Canada Danni Li Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, United States Robert D. Nerenz Department of Pathology and Laboratory Medicine, DartmouthHitchcock Health System, Lebanon, NH, United States; The Geisel School of Medicine at Dartmouth, Hanover, NH, United States David L. Pittman Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States Neeraj Ramakrishnan California Northstate University, College of Medicine, CA, United States Tamar Reisman Icahn School of Medicine at Mount Sinai, New York, NY, United States Alan T. Remaley Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States Lori J. Sokoll Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, United States William E. Winter Department of Pathology, Immunology & Laboratory Medicine, Pediatrics, and Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States Anna Wolska Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States

Preface and Acknowledgments This third edition of the Handbook of Diagnostic Endocrinology is intended for laboratory directors, technologists, clinicians, trainees, and industry scientists who seek a comprehensive one-volume treatise that focuses on the diagnosis of both common and uncommon endocrine disorders. Genetics and personalized medicine reveal the variety and complexity of disorders examined in this handbook. New chapters focus on disorders of sexual development, transgender medicine, and the aging endocrine system. A discussion of the metabolic syndrome has been included in the chapter on hyperglycemia. The editors are from diverse backgrounds, representing academics and industry, and coming from both east and west coasts, south and south-central United States. The authors of individual chapters are an international gathering of experts. Tables, graphs, and illustrations help demonstrate the organization and breadth of disorders that affect hormones, their receptors, and postreceptor signaling. Many chapters include detailed studies of the molecular biology of endocrine systems; examples include disorders of sexual development, endocrine-associated hypertension, hyper- and hypoglycemia, and calcium homeostasis.

Acknowledgments The editors wish to thank the staff at Elsevier, particularly Pat Gonzalez for the assembly of this handbook and Tari S. Broderick for her initial development of the concept. We also thank the authors whose tireless efforts made this volume possible. Lastly, the editors wish to thank the previous handbook editors, Daniel W. Chan, PhD (first edition) and Ishwarlal Jialal, MD, PhD (first and second editions), for their contributions.

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Chapter 1

Maximizing the value of laboratory tests Raffick A.R. Bowen1, Roger L. Bertholf2 and Brett Holmquist3 1

Department of Pathology, Stanford University, Palo Alto, CA, United States, 2Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, United States, 3 Endocrine Sciences, Laboratory Corporation of America, Calabasas, CA, United States

The primary function of a clinical laboratory is to provide accurate and clinically relevant data for the diagnosis of medical conditions in patients. Laboratory data can also be used to provide patients with a management plan that will help them achieve a desirable outcome. Errors in the laboratory test results affect patient care when they mislead clinicians into false diagnoses or indicate a test is normal when it is not. With regard to laboratory tests, there are three potential sources of error: errors may occur before testing begins (preanalytical errors), during the test procedure (analytical errors), and following completion of the test (postanalytical errors). Although errors can occur in any laboratory test result, immunoassays seem to be particularly vulnerable to analytical errors, due to the nature of antibodyantigen interactions and the potential for interference from crossreactive species. By their very nature, antibodies have high but not absolute specificity for the antigen they are intended to detect, and therefore detection of known or unknown antigens with which the antibody reacts limits the specificity of immunoassays. In addition, reagent antibodies can also be antigenic, causing false-positive or false-negative signals due to the reaction of endogenous antibodies with the reagent. Although the frequency of medically important immunoassay interference is probably less than a few percent [1] when it occurs, it can often lead to unnecessary treatment or undetected disease. Many of the analytes measured by immunoassays, such as tumor markers, cannot easily or cost-effectively be corroborated by other laboratory tests. Immunoassay interference, therefore, can lead to misinterpretation of patient’s status, misdiagnosis, unnecessary and costly radiological procedures, and/or unnecessary treatment.

Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00001-7 © 2021 Elsevier Inc. All rights reserved.

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Immunoassay interferences can originate from either exogenous or endogenous sources. Exogenous interferences are caused by the addition of external factors or conditions either in vivo or in vitro that are not normally present in a native, properly collected, and stored sample [2]. For example, hemolysis, lipemia, blood collection tube additives, and administration of radioactive or fluorescent compounds, drugs, herbal medicines, nutritional supplements, and sample storage are all exogenous interferences that can adversely affect immunoassays [25]. Interference from exogenous factors is an often-overlooked problem in immunoassays. Endogenous interferences are caused by factors that may exist in the patient’s blood under either physiological or pathophysiological conditions [2]. Endogenous factors often are difficult to detect and eliminate because they can vary considerably between patients, and from time to time in any given patient. Examples of endogenous interferences include human antianimal, heterophilic antibodies, autoantibodies [68], rheumatoid factors, and binding proteins [2,9,10]. Knowledge of the exogenous and endogenous interferences and the mechanisms through which they interfere with immunoassays is essential to minimize the potential for analytical errors. Although much of this chapter will be devoted to sources of error in immunoassay, because it is the most common analytical method used for measurement of hormones, many of the mechanisms by which endogenous and exogenous compounds interfere with immunoassays are analogous to interferences in chemical tests. The laboratory evaluation of endocrine disorders is not limited to immunoassays, and in this handbook, you will find many references to laboratory tests that do not involve antibodies. However, more considerable attention is given to immunoassays because they are commonly used in this domain and are uniquely susceptible to some of the interferences discussed in this book.

Interferences associated with mass spectrometry Beginning in the early 2000s, the use of high-performance liquid chromatography (LC) with tandem mass spectrometric detection has transformed and vastly improved the analysis of small molecules, including steroid hormones. Beginning in 2010, the Centers for Disease Control (CDC) has worked to standardize steroid hormone analysis with their Hormone Standardization (HoST) project and Vitamin D Standardization Project (VDSP) [11]. In 2013 the endocrine society began requiring mass spectrometry (MS) for sex steroid assays for publication in the Journal of Clinical Endocrinology and Metabolism [12,13]. The use of MS or more often LC-MS/MS has historically been laboratory-developed tests; however, in 2017 the first LC-MS/MS test for 25-hydroxyvitamin D was cleared by Food and Drug Administration (FDA) [14]. The LC-MS/MS platform, owing to its core technology and granularity of data collection, is widely considered more specific, more

Maximizing the value of laboratory tests Chapter | 1

3

selective, and less prone to interference than most immunoassays for small molecule (steroid) analysis. There are, however, some particular areas that laboratorians must address during validation of LC-MS/MS assays [15]. When choosing a laboratory and diagnostic test, it is important to work with a laboratory that is experienced, accredited, using modern tools, and validating to current expectations based on Clinical Laboratory Standards Institute (CLSI) guidelines including C50-A and C62 [16,17]. In addition to steroid analysis, MS is now being used for peptide and protein analysis within the endocrine space for selected analytes, including insulin-like growth factor 1, thyroglobulin, and plasma renin activity (angiotensin I). Protein analysis by MS is more challenging than the analysis of small molecules but offers extraordinary potential for biomarker analysis.

Exogenous interference Hemolysis, lipemia, and icterus Hemolysis, icterus, and lipemia can interfere with many laboratory tests. Hemolysis can affect the absorbance of light in spectrophotometric measurements since hemoglobin is a chromophore that absorbs broadly in the ultraviolet (UV)-visible range of the electromagnetic spectrum. Hemolysis occurs when erythrocytes are disrupted; the normal concentration of hemoglobin in the plasma is very low. When erythrocytes are disrupted, their cytoplasmic contents add soluble factors to the plasma fraction that remain after centrifugation. These soluble factors may bind to the analyte and block antibodybinding sites or cross-react with the reagent antibodies. Hemolyzed specimens may be unacceptable for immunoassays of labile analytes such as insulin, glucagon, calcitonin, parathyroid hormone (PTH), adrenocorticotropic hormone (ACTH), and gastrin because proteolytic enzymes released from erythrocytes can degrade peptide hormones [18]. Hemolysis may also interfere with signal generation steps included in various immunoassays [18]. Hemolysis may interfere with spectrophotometric measurements due to absorption by hemoglobin; this is a hemolysis interference that is not specific to immunoassays. Finally, for some analytes, the intracellular concentration is much greater than the extracellular concentration; therefore disruption of the erythrocytes increases the analyte concentration in the plasma above its physiological level. In general, grossly hemolyzed specimens should not be used for either chemical assays or immunoassays. Lipemia can result from high concentrations of triglycerides, cholesterol, or both, and may produce erroneous results in some assays by interfering with antigen binding, even when antibodies are linked to a solid support. Lipemia has also been shown to cause interference with immunoassays that have turbidimetric end-points because of the light scatter caused by lipid micelles [2]. Interferences by nonesterified fatty acids have been well

4

Maximizing the value of laboratory tests

documented for free thyroxine assays. Nonesterified fatty acids compete with thyroxine and its derivatives used as labels for endogenous protein binding sites and, depending on the assay format, may cause either falsely high or falsely low free thyroxine values [1921]. Nonesterified fatty acids may also inhibit steroid binding to proteins. Hypertriglyceridemia has been shown to cause falsely elevated results in some endocrinology assays, using second antibody and polyethylene glycol separation techniques [22]. It is desirable to collect specimens from individuals after an overnight fast to reduce the immunoassay interference from lipids. Alternatively, ultracentrifugation to remove any excess lipids or enzymatic cleavage by lipase may be used to treat samples before analysis [18]. It should also be kept in mind that blood contains three distinct fractions: a cellular fraction that includes erythrocytes, leukocytes, and platelets; an aqueous fraction that includes primarily water and various small molecules, including peptides; and an extracellular nonaqueous fraction that consists of lipid micelles and globular proteins. The cellular fraction comprises approximately 45% of the total blood volume. When a specimen is centrifuged, the noncellular fraction is usually about 95% aqueous, with the remaining 5% comprised of lipid micelles (such as lipoproteins) and globular proteins, including immunoglobulins. Therefore any analyte that is exclusively in the aqueous fraction of blood (electrolytes, organic acids, catecholamines, unbound steroid hormones) is only occupying around 95% of the total volume of plasma. The excluded nonaqueous volume introduces an error when concentrations are based on the total volume of the specimen. For most assays, this small error is inconsequential. However, in hyperlipidemic or hyperproteinemic states, the nonaqueous volume can be 10%15%, which can cause a significant error in the calculation of analyte concentrations. For electrolytes, direct potentiometry solves this problem because measurements made in undiluted specimens are volume-independent—that is, the volume occupied by nonaqueous components in plasma is not relevant. However, there are no direct potentiometric methods for measuring hormones, and it is another reason why hyperlipidemic specimens should not be used for laboratory tests unless the lipids are removed by ultracentrifugation. Excess bilirubin affects many different types of assays, either due to spectrophotometric interference or chemical oxidation of the analyte by bilirubin. As with the case for hemolysis and lipemia, the user should follow the recommendations of the manufacturer to determine the suitability of icteric samples for analysis.

Matrix effects Clinical specimens are incredibly complex and contain a variable mixture of proteins, carbohydrates, lipids, small molecules, and salts. A matrix effect is an interference arising from any of the above substances present at high

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enough concentration to affect the measurement of an analyte. In addition, the viscosity, pH, and ionic strength of the specimen may also contribute to matrix effects. Matrix interference in immunoassays has been thoroughly investigated and reviewed [2]. Antigen and antibody reactions are often quite sensitive to variations in protein and lipid concentrations, pH, and ionic strength. Differences in the matrix may alter the efficiency of separation of bound and unbound fractions and the extent of nonspecific binding of the tracer. Matrix effects are highly dependent on the assay format and the antibody selected to capture analyte; therefore various immunoassays are affected in different ways. Matrix effects can be important sources of discrepancies among patient samples, calibrators, standards, quality controls, and external proficiency materials that are not based on human serum [2326]. Additionally, matrix effects can make certain types of body fluids unsuitable for analysis [27]. Thus matrix interferences in clinical assays can be minimized by careful assay design. Examples of methods to reduce or eliminate matrix interferences include the use of high-affinity antibodies of certain subtypes or antibody fragments, dilution of the specimen, addition of an immunoglobulin to the assay buffer to saturate any interfering antibodies, and optimizing temperature and incubation times. When it is necessary to dilute a sample with a high concentration beyond the linear range of the assay, it is important to use a diluent recommended by the manufacturer with the proper matrix to avoid any artifacts.

Blood collection tubes Blood collection tubes are not inert containers for blood but have several constituents, including anticoagulants, surfactants, and lubricants for rubber stoppers, clot activators, and separator gels that can potentially interfere with the assays.

Tube wall Serum separator tubes and Vacuette tubes are made from polyethylene terephthalate. Siliconized plastic tubes have been shown to cause a 30%60% decrease in corticotropin (ACTH) measurements when measured by radioimmunoassay, possibly due to interference with the formation of either the biotin-avidin complex or the antibodyantigenantibody sandwich [28]. Besides tube walls, it is important to note that other synthetic materials that come in contact with blood, such as indwelling catheter tubing, have also been reported to affect immunoassay results [29]. Certain drugs, such as tacrolimus, can tightly bind to the catheter tubing wall, resulting in spuriously elevated drug concentrations when blood is collected through catheters, even after flushing the line with saline [29].

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Maximizing the value of laboratory tests

Rubber stopper To maintain the vacuum in blood collection tubes, it is common to use stoppers made from isobutylene-isopropene rubber or chlorinated isobutyleneisoprene rubber along with a stopper lubricant [30,31]. Rubber stoppers used in some commercial blood collection tubes have been found to contain a plasticizer, tris(2-butoxyethyl) phosphate, which can displace quinidine, propanolol, lidocaine, tricyclic antidepressants, fluphenazine, and chlorpromazine from α1-acid glycoprotein [18,32,33]. Displacement of these drugs from their binding protein results in the redistribution of the drug in blood, causing an increase in drug uptake by red blood cells and a decrease in plasma or serum concentration [32,33]. Most manufacturers have reformulated their rubber stoppers with low extractable rubber to minimize this interference. Anticoagulants Historically, most assays were performed in serum, but plasma may be preferable because it eliminates the time required for clotting, thereby reducing the processing time [34]. If plasma is used, care must be taken to select an anticoagulant that does not interfere with the assay. Ethylenediaminetetraacetic acid (EDTA) binds divalent metal ions and prevents coagulation by binding to calcium. EDTA also binds some metal ions that are a constituent of labels: for example, europium, or enzyme cofactors essential for their activity (e.g., alkaline phosphatase requires zinc ions) [34]. Elevated EDTA concentrations in a sample-reagent mixture due to insufficient sample volume can result in the more efficient chelation of magnesium and zinc and can affect the activity of the alkaline phosphatase enzyme label used in chemiluminescence assays [34]. Filling of EDTA sample tubes to ,50% affects, for example, intact PTH [35] and ACTH measurements by the Immulite assays [34]. Many proteins bind divalent cations—typically calcium or magnesium— and the antibodies against these proteins may recognize an epitope that is altered when the cation is absent [36]. Heparin prevents coagulation primarily by forming a complex with antithrombin III. The heparin-antithrombin III complex enhances the inhibitory effects of thrombin and activated Factor X to prevent clotting or activation of thrombin, which in turn prevents the formation of fibrin from fibrinogen. However, heparin may interfere with some antibodyantigen reactions. Heparin decreases the rate of reaction of some antibodies, particularly at the precipitation step in second-antibody systems; however, the use of solidphase systems has minimized this problem. Heparin can precipitate cryofibrinogen; therefore this anticoagulant should not be used for cryoprotein measurements. The influence of exogenously administered heparin on serum levels of thyroid hormones and other analytes has also been investigated. Heparin has also been shown to cause in vivo stimulation of lipoprotein lipase with subsequent release of nonesterified fatty acids. Nonesterified fatty

Maximizing the value of laboratory tests Chapter | 1

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acids inhibited the binding of radiolabeled thyroxine to thyroid-binding globulin with an apparent increase in the thyroxine result. Samples collected into tubes containing sodium fluoride may be unsuitable for some enzymatic immunoassays due to inhibition of the enzyme activity by fluoride.

Surfactants Surfactants are commonly added to immunoassay reagents [37] to decrease or eliminate nonspecific adsorption of the analyte, improve reagent stability, or modify the solid-phase surface to render it less hydrophobic, thus minimizing the loss of noncovalently bound antibody [30,31]. At high concentration, surfactants may inhibit passive adsorption of antibody from the solid phase, among other nonspecific effects. Previous reports have shown that silicone-coated collection tubes can interfere with ion-specific electrode determinations of ionized magnesium [24,3840] and lithium [41], causing falsely increased concentrations. In addition, the water-soluble silicone polymer coating the interior of serum separator tubes can interfere with the formation of an avidin-biotin complex in immunoradiometric assays for thyrotropin, prolactin, and human chorionic gonadotropin (hCG) [22]. Bowen et al. [42] identified a common organosilane surfactant (Silwet L720) in Becton Dickinson SST blood collection tubes that caused a falsely elevated triiodothyronine and other analytes. When present in excess amounts in blood collection tubes, this surfactant causes interferences by desorbing the capture antibody from the solid phase used in the Immulite 2000/2500 triiodothyronine immunoassay and had a similar effect on immunoassays from other manufacturers [42]. Other studies have shown that silicone forms a complex with C-reactive protein, enhancing the antigenantibody reaction in the Vitros Creactive protein assay and falsely elevating results [43]. Clot activators Blood collected in evacuated tubes without anticoagulants should form a dense clot as rapidly and completely as possible to enable clear separation of the clot from the serum layer by centrifugation [4245]. To achieve this end, blood collection tubes include a clot activator with a carrier such as polyvinylpyrrolidone [31,44]. Examples of clot activators include diatomaceous earth, particles of inorganic silicates, and biochemicals such as ellagic acid, thrombin, and thromboplastin. Occasionally, clot activator particles may not pellet completely with the clot and instead remain in the serum layer, causing interferences with some assays. Separator gel Separator gels are widely used in blood collection tubes to form a barrier between serum or plasma and the cellular fraction upon centrifugation. The separator gel is a thixotropic liquid that is solid at rest, but when compressed by centrifugation, it becomes liquid [45]. It has been shown that the

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Maximizing the value of laboratory tests

separator gel can adsorb some hydrophobic leading to falsely low results [4649]. Fragments of separator gel or droplets of oil may be seen within the separated serum or plasma following the centrifugation of some gelcontaining blood collection tubes. The gel or oil droplets can clog the sample probe, coat tubes, and cuvettes, and physically interfere with solid-phase immunoassay systems. To minimize interferences, it is important to follow the tube manufacturer’s recommendations and not use the tubes at temperatures, centrifugation speeds, or orientations that are not within limits specified by the manufacturer.

Label interferences All immunoassay requires a measurable indicator to quantify the antigenantibody complex. Some samples may contain compounds that increase or decrease the magnitude of the indicator response without affecting antigenantibody binding. Diagnostic or therapeutic administration of radioisotopes has the potential to interfere with radioimmunoassays if the same isotope is used as the indicator [50]. When the indicator is a fluorophore, interference can occur due to endogenous fluorescence, fluorescent drugs, or fluorescein administered for retinal angiography [23]. Fluorescence quenchers may also be present in some clinical specimens. For enzyme-labeled immunoassays, the presence of enzyme inhibitors or activators in the sample may alter the enzyme activity, and thus the results from the immunoassay [27,51,52].

Drugs and herbal medicine A major source of exogenous interference in immunoassays is the presence of drugs, nutritional supplements, and herbal medicine in the blood. These interferences have been widely reviewed [5358]. Many of the interferences involve cross-reactivity of exogenous compounds with the capture antibody. Cross-reacting substances may be a precursor of the compound to be measured, or it can be the metabolite of the analyte.

Timing of sample collection Although not strictly an interference, it is important to realize that some analytes have a marked diurnal pattern of physiological release and/or can be affected by variable physiologic factors or therapies. As a result, these analytes must be measured at specific times to ensure that results can be interpreted appropriately. Cortisol concentration, for example, varies significantly from a peak near midnight to a nadir at 78 a.m., and reference ranges are applied accordingly [59].

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Storage/freeze-thaw Inappropriate sample type, specimen processing, and storage can change the properties of the sample over time and affect immunoassay results. For example, ACTH is only stable for 18 h at 4 C, in contrast to 18 other hormones that are stable for .120 h [60]. Most hormones are relatively stable in serum or urine if they are rapidly frozen and stored at 270 C; however, repeated freezing and thawing of analytes can lead to denaturation, aggregation, and loss of antigenicity of some proteins [60]. Specimens collected in EDTA often are more stable than serum or heparinized plasma specimens because EDTA chelates calcium and magnesium ions, which are cofactors for some protease enzymes. The addition of protease inhibitors (e.g., aprotinin) to blood samples may also improve analyte stability [59,61].

Analytical errors Carryover Automated chemistry and immunoassay systems use automated samplehandling devices. If a sample to be assayed is preceded by a sample with a very high concentration of an analyte, there is a potential for the analyte in the first sample to contaminate the second sample due to inadequate rinsing of the probe between specimens. Therefore it is important to routinely test any new analyzer for potential carryover. It is the responsibility of the laboratory to assess the potential for carryover in each assay it performs and incorporate the appropriate action to take in the standard operating procedure.

Endogenous interference Excess antigen interference (hook effect) Excess antigen interference occurs when there is an unusually high concentration of antigen, which paradoxically may lead to a falsely low result. This phenomenon is commonly referred to as a “hook” or prozone effect [62,63]. This type of interference is most likely to occur when the analyte can have a very wide range of concentrations, as do hCG, many tumor markers, and serological tests [64,65]. As shown in Fig. 1.1, 2-site immunometric assays are especially prone to the hook effect. These assays depend upon the formation of a complex between the antigen and the primary (or “capture”) and secondary antibodies to generate a signal. These assays are usually designed so that there are always excess antibodies relative to the antigen. When there is an excess antigen, however, a complex does not form because all of the antigen-binding sites are occupied by a single antigen, thus preventing a single antigen molecule from bridging the primary and secondary

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Maximizing the value of laboratory tests

FIGURE 1.1 Diagram of immunoassay interference on competitive and noncompetitive assays by antibody interference.

antibodies [66]. After a final wash step, the secondary antibodies that generate the signal are removed when there is an excess antigen, thus resulting in a spuriously low value. Antigen excess interference can be prevented by performing the assay on a diluted sample or by the addition of a vast excess of antibody to ensure that there is always a sufficient concentration of antibodies, even for those samples with unusually high concentrations of the analyte. Manufacturers of assays for analytes that are commonly affected by antigen excess usually provide a sufficient amount of antibodies to prevent this problem. In addition, sequential two-step immunometric assays are less prone to this problem because a wash step is included before the addition of the second antibody; this wash removes any excess antigen, preventing an erroneously low result. Finally, an important consideration in implementing any new assay that may be susceptible to the hook effect is to determine whether the linear range described by the manufacturer encompasses the likely concentration range that will be measured in the laboratory.

Antibody specificity (cross-reactivity) Specificity is an important characteristic of any assay because it describes its ability to react with only the analyte of interest in a sample. A major source of bias in an immunoassay is the lack of specificity of the antibodies used, resulting in cross-reactivity. Cross-reactivity is more problematic with polyclonal antibodies compared to monoclonal antibodies. Polyclonal

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antibodies typically have high avidity, but monoclonal antibodies have a higher affinity and therefore are more selective toward a specific antigen. Selection of monoclonal antibodies may reduce cross-reactivity, but closely related molecules may still be recognized by the antibody, especially analytes, such as steroids and drugs. Cross-reactivity is more common with competitive immunoassays compared to noncompetitive immunoassays since the use of an antibody against a second epitope on the analyte reduces the likelihood of a cross-reacting antigen having both epitopes tested for by the noncompetitive immunoassays. However, noncompetitive immunoassays do not always exhibit good specificity because nonspecific binding may arise due to many endogenous substances present in the patient sample, such as rheumatoid factor, complement proteins, or bacterial proteins [67].

Antibody interference Circulating antibodies in patient samples may interfere with immunoassays [68]. Usually, endogenous antibodies interfere by reacting with the reagent antibodies in immunoassays, but all the other components of immunoassays, such as the antigen, enzyme-substrate, and signal molecule, can also be a target of endogenous antibodies, adversely affecting immunoassay results. Previous studies have shown that interfering endogenous antibodies are present in up to 30%40% of patient samples [68], but estimates vary widely [1,69], and their practical effect is far less. Interfering antibodies include heterophilic antibodies that have multiple specificities for antigens, as well as proteins, such as rheumatoid factor; human antianimal antibodies (HAAAs), especially human antimouse antibodies (HAMAs); or autoantibodies. The presence of interfering antibodies can affect both noncompetitive and competitive immunoassays and produce either falsely elevated or falsely decreased results, depending on the specific antibody or protein with which the heterophile reacts. The nature of the antibody interference depends on the type of assay used and the site where the antibody binds to the analyte [2,70]. Heterophile antibodies Heterophile antibodies can be immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin A, and IgG isotypes that are poorly defined and react with a wide spectrum of antigens [6,71,72]. If exposure to a specific animal immunogen is known, the correct term for that heterophile antibody should refer to the specific animal that produced the antigen such as “human antimouse antibody,” or “human antirabbit antibody.” Heterophile antibodies are usually low-titer, weak-avidity antibodies found in the serum of patients with no history of treatment or diagnostic procedures involving antianimal immunoglobulins [73,74]. Heterophile antibodies have been reported to be present in 30%40% of patient samples [68,75] and are a well-recognized cause of

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FIGURE 1.2 Diagram of immunoassay interference from excess antigen.

interference in immunoassays [76,77]. When heterophile antibodies are present, it is often very difficult to predict the direction and magnitude of the immunoassay interference. Interferences may occur in competitive or noncompetitive assays, but the latter is more common [36,78]. In competitive immunoassays, the presence of heterophile antibodies may decrease the number of available binding sites on the primary antibody by steric hindrance of the specific antigen, (Fig. 1.2) and in some competitive immunoassay formats, reduce the binding of a second antibody, causing a positive interference. In noncompetitive immunoassays, heterophile antibodies can interfere by bridging the capture and detection antibodies [68,75], causing false-positive results (Fig. 1.2). In contrast, heterophile antibodies can also cause false-negative results by binding directly to the capture antibody, thus blocking the reactive site from binding the analyte of interest. The same antibody may react differently for different antibody combinations, thus causing falsely elevated results in one assay but a lower result in another assay. Immunoassay manufacturers typically add blocking agents (nonimmune globulins of various species) to their assay formulations to saturate and minimize or eliminate the effects of heterophile antibodies; however, not all heterophile interference can be prevented by blocking agents [79]. Heterophile antibodies may show reactivity to idiotypes that are not present in the blocking agent [80]. Prior extraction of the analyte from the sample by protein A, protein G, cation-exchange, or gel filtration chromatography is also effective in removing heterophile antibodies [6,63,72,8184]. Reaction conditions can also be modified to minimize heterophile antibody interference [80] Heat and acid treatment of samples are not very useful since these antibody-denaturing conditions will destroy most analytes.

Human antianimal antibodies HAAAs are high-affinity, specific polyclonal antibodies that react with immunoglobulins from a specific animal. For antianimal antibodies elicited

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by animal immunoglobulins, the HAAAs can have antiidiotype or antiisotype specificity. Antiidiotype antibodies bind to the hypervariable region of the immunoglobulin molecule, and antiisotype antibodies are directed against the constant regions of the immunoglobulin molecule. Anti-antiidiotype antibodies can also be produced; these antibodies recognize the binding region of the antiidiotype antibody; thus the antigen-binding region of an antiantiidiotype antibody resembles the antigen that elicited the original antiidiotype HAMA. The prevalence of HAAAs in serum samples has been shown to vary widely among studies with ranges anywhere from 0.1% to 80%, depending on the assay used to detect them and the patient population [68,8587]. HAAAs may be produced from iatrogenic and noniatrogenic causes, including the use of mouse monoclonal antibodies for therapeutic and imaging purposes, blood transfusions, vaccinations, exposure to microbial antigens, animal husbandry or keeping animals as pets, transferring of a dietary antigen across the gut wall, and autoimmune diseases that give rise to autoantibodies. Antianimal antibodies are known to interfere unpredictably with immunoassays, the effect on laboratory results depending on the nature and concentration of the interfering antibody, and the immunoassay format. HAAAs most commonly interfere with 2-site immunometric assays by linking the capture and detection antibodies, giving a false-positive result [8,81,8891].

Human antimouse antibodies HAMAs are heterophile antibodies that react with mouse immunoglobulins [92]. Many cases of immunoassay interference due to HAMAs have been reported and reviewed. In one study evaluating the incidence of HAMAs, eight commonly used immunoassays were tested in 500 serum samples [93]. It was found that the incidence of HAMAs ranged from 0.2% to 3.7%. The incidence of HAMAs has increased since the introduction of in vivo techniques using mouse monoclonal antibodies as carriers for radiological or chemotherapeutic agents to tumor sites and for other therapeutic and imaging purposes. Two-site immunometric methods are more susceptible to interference from antibodies to animal IgG in human serum and may cross-react with reagent antibodies to animal IgG in human serum, as well as with reagent antibodies, especially those from the same species. Reaction with both capture and detection antibodies forms an antigenantibody complex that behaves immunochemically like the analyte-antibody complex, resulting in false-positive results. Falsenegative results can also occur in 2-site immunometric assays due to HAMAs reacting with one of the reagent antibodies and blocking the formation of the antigenantibody complexes with the analyte of interest. Methods that use only one mouse monoclonal antibody in immunometric assays are less prone to interference from HAMAs.

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Autoantibodies Autoantibodies are antibodies that exist in a patient’s blood that bind preferentially to the analyte of interest. Autoantibody prevalence is between 4% and 27% in the normal population. Endogenous circulating autoantibodies have been described for a variety of analytes, including thyroid hormones, thyroxine, and triiodothyronine, as well as thyroglobulin [9496], creatine kinase, amylase, insulin [97,98], prolactin [99], and testosterone [100]. The nature of autoantibody interference on immunoassay results depends on the assay technique used [101] as well as the autoantibody titer, affinity, and specificity [85]. In competitive immunoassays, autoantibody interference can lead to an apparent increase or decrease in measured concentration, depending on whether the autoantibody-analyte complex partitions into the free or bound fraction. In noncompetitive immunoassays, autoantibodies can bind to the solid support or block attachment of detection antibody and cause falsely low results by decreasing the amount of label bound to the solid-phase antibody. Besides interfering with immunoassays, autoantibodies against an analyte can delay its in vivo clearance, resulting in a true serum elevation. This is commonly encountered with prolactin, which can lead to a diagnosis of a pituitary adenoma, but the prolactin bound to the autoantibody is not biologically active and does not lead to disease [102]. Autoantibodies to reagent components, such as alkaline phosphatase, may also yield incorrect results for reagents using that enzyme [103]. Thus regardless of whether competitive or noncompetitive immunoassays are used, interference from autoantibodies can occur in both formats. Rheumatoid factors Rheumatoid factors are IgM autoantibodies directed against the Fc portion of IgG [86,104]. These factors bind to a hydrophobic domain on the antibody that is close to the binding site of staphylococcal protein A [105,106]. It has been estimated that B5% of healthy individuals have positive rheumatoid factors [9]. Positive rheumatoid factors have been found in varying percentages in other connective tissue disorders, such as systemic lupus erythematosus, systemic sclerosis, polymyositis, and Sjo¨gren syndrome. Rheumatoid factors can interfere with immunoassays by binding to the Fc portion of immunoglobulins and causing steric hindrance to the binding site, preventing or decreasing binding to the antigen or by cross-linking the reagent antibody, thereby mimicking the antigen. Monoclonal antibody-based immunometric assays are especially sensitive to the presence of heterophile antibodies [68]. The nonspecific binding by rheumatoid factor can be overcome in the same manner as for heterophile antibodies by blocking reagents such as nonimmune homologous immunoglobulin [5,85].

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Endogenous hormone-binding proteins Endogenous hormone-binding proteins are present in varying concentrations in all serum and plasma samples and may significantly affect immunoassay performance. Immunoassays may also be affected by changes in specific binding proteins due to congenital variations; for example, in familial dysalbuminemic hyperthyroxinemia, up to 50% of the albumin molecules bind thyroxine with an affinity up to 50-fold higher than normal. Measurement of total hormone concentration requires the displacement of the hormone from its binding site. Unless the protein binding of both labeled and unlabeled ligands is inhibited or removed, equilibrium will form among the binding proteins, the labeled and unlabeled ligands, and the analytical antibody, producing erroneous results. Therefore total hormone assays need to remove the endogenous binding protein to prevent the binding of the added signal-ligand complex to the endogenous binding sites. Various releasing agents have been used to disassociate the hormones from the binding protein, such as pH changes, 8-anilino-1-naphthalenesulfonic acid, salicylate, and thimerosal. Other plasma proteins Other plasma proteins that can affect antibody binding and interfere in immunoassay include fibrin, complement, lysozyme, paraprotein, and albumin. Fibrinogen Immunoassays are susceptible to interference by fibrin. Residual fibrin may be present because of improper specimen handling procedures. It can be present in the primary tube samples either as a visible clot, which may physically occlude the sample probe on the automated chemistry or immunoassay instrument, or more insidiously, as an invisible microfiber or as strands. Fibrin interference is usually not reproducible and can disappear with time as the fibrin settles out of the sample. Although newer immunoassay analyzers often have clot detectors, they are not infallible, and it is still common laboratory practice to repeat the analysis for those samples with an analyte level below the detection limit to minimize this type of problem. Excess fibrin can also have specific deleterious effects on immunoassays. Some immunoassays are directly affected by fibrin interference [107109], especially with high surfacearea solid supports, presumably caused by nonspecific binding of a conjugate molecule to fibrin or other coagulation intermediates deposited on the solid surface. Complement proteins Many immunoassays use antibodies bound to a solid phase. When a serum sample is added to the immobilized reagent antibodies, the various complement factors in the samples are activated and bind to the reagent antibodies [110]. This binding may partly or totally block the antigen-binding sites, and

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it has been shown that complement activation may interfere with antigen binding to the capture antibody and cause falsely negative results. Complement binding may also lead to the destruction of the antigenantibody complexes. Mouse immunoglobulin IgG2a and IgG2b subtypes are particularly sensitive to complement binding and activation. The effect of complement can be eliminated by diluting or heating the samples or by using EDTA-containing buffers, which chelate calcium and inactivate complement [111]. In addition, since complement can interfere with immunoassay by binding to the Fc fragment of immunoglobulins, it is advantageous to use F (ab) fragments that are resistant to complement binding [2]. Lysozymes Lysozyme is a basic protein with an isoelectric and isoionic pH of 11.1. It is widely distributed and possesses bacteriolytic properties. As a highly basic protein, lysozymes can bind anionic proteins with low isoelectric points like immunoglobulins (pI values between 5.0 and 9.0) [112]. Lysozymes also nonspecifically bind to the immunoglobulin and can form a bridge between solid-phase IgG and detection antibody [36]. The inclusion of copper ions or ovalbumin in some enzyme-labeled immunoassays has been shown to minimize lysozyme interference [112]. Paraproteins Elevated concentrations of paraproteins due to multiple myeloma and similar diseases in serum may result in nonspecific binding of either analytes or reagents in immunoassays [113] or interference in nephelometric assays [114116]. Paraproteins also greatly increase the viscosity of the serum, which can produce short samples and cause falsely low immunoassay values. Albumin Albumin may interfere with immunoassay results by binding, as well as releasing a large quantity of ligands. Methods measuring the unbound analog of free thyroxine may actually measure the albumin-bound hormone fraction and be influenced by the level of albumin in the serum. Interpretation of free thyroxine values should be evaluated with caution in patients with abnormal concentrations of serum albumin, for example, those with nonthyroid severe illness. In conditions associated with low or absent albumin (hereditary analbuminemia) or abnormal albumin binding of analog tracer (familial dysalbuminemic hyperthyroxinemia), false low or false high levels of free thyroxine, respectively, may be obtained. In some solid-phase assays, albumin may even inhibit the binding of labeled thyroxine to the immobilized thyroxine antibody, producing falsely high results. If a patient is on heparin therapy, the concentration of the nonesterified fatty acids is increased due to release of lipoprotein lipase from the vascular epithelium, and nonesterified fatty acids will bind strongly to albumin, displacing

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residual labeled thyroxine, the concentration of which increases in the specific bound fraction, resulting in apparently lower values for free thyroxine.

Detection and testing for interference in suspected samples There is no universal way to detect all types of potential interference in chemical and immunoassays; therefore processes are needed to make both medical laboratory workers and physicians aware of the potential for interference. Once the laboratory is alerted to a potential problem, several laboratory procedures can be used for investigating these specimens. Additional studies to investigate potential interferences include: (1) retesting the same specimen, (2) testing a new specimen from the same patient, (3) testing another body fluid (e.g., urine hCG) to corroborate the result, (4) dilution studies to assess linearity, (5) spiked recovery, (6) mixing experiments with a normal serum or plasma sample, (7) heterophile antibody-blocking reagents; (8) sample pretreatment, and (9) testing with alternate technology. Not all of these above procedures are needed or applicable to all situations, and frequently, repeat testing or testing on a new specimen will resolve a problem. If an interference is still suspected, dilution studies may confirm whether the diluted concentrations produce a linear signal response. Diluting the sample may lower the concentration of the interfering substance below the limit at which it interferes with the assay. Dilution studies should only be attempted with the diluent recommended by the manufacturer, and ideally, another specimen without a suspected interference should be diluted as a control. Another approach is to pretreat the sample with various commercial additives to neutralize any problems from antibody interference. Because many immunoassay interferences are specific to certain assays, it is also helpful to perform the assay using another method. Methods are available to detect heterophile antibodies, but often this is not useful because heterophile antibodies are relatively common and usually do not cause problems. In addition, assays to detect endogenous antibodies are usually very specific to a certain class of antibodies, and no single test is sufficient to exclude the presence of an interfering antibody. A very high titer of endogenous interfering antibodies may be a clue to the source of an immunoassay interference, but this can also be tested empirically by adding blocking agents to neutralize these antibodies. Rarely, interferences can result from an inherent and/or systematic limitation of a specific assay or blood collection tube. In these instances, it is important to notify not only the physicians ordering the test but also the manufacturer of the assay and any relevant regulatory agency, such as the FDA, of the interference.

Biotin Biotin is a B vitamin found in small amounts in a variety of foods. Biotin also is sold in nail, hair, and skin supplements, and is prescribed in high

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doses for patients with neurological diseases such as progressive multiple sclerosis [117121]. Biotin-streptavidin coupling is frequently used in immunoassays to capture biotinylated antibodyantigen complexes [122124]. High concentrations of biotin can interfere with biotinstreptavidin-based immunoassays, producing erroneous test results [125,126]. Although the concentrations of biotin necessary to interfere with an immunoassay are substantially higher than normal blood biotin levels, or those associated with ingesting 30 μg/day of biotin, the risk of biotin interference with immunoassays is increasing, as many contemporary supplements incorporate higher-dose biotin of up to 20 mg [125127].

Prevalence of elevated biotin Estimates of the prevalence of biotin supplement use in the United States range from 8% to 29% [128]. However, as biotin is sold under various names, patients may not be aware of their biotin supplement use and may not report it to their health care provider [127]. Efforts to quantify the prevalence of elevated biotin levels in the blood have produced inconsistent results. One study found B0.74% of the routine samples assessed contained concentrations of .20 ng/mL [129], while another study found that B 7.4% of the samples contained concentrations of $ 10 ng/mL [127]. Cardiac troponin assays have also been found to have a relatively low biotin interference threshold, which could result in a missed diagnosis of acute myocardial infarction [130]. Peak serum or plasma levels of biotin are achieved 12 h after ingestion and afterward decline quickly [124,131,132]. Median serum peak concentrations in healthy volunteers were 41, 91, and 184 ng/mL 1 h after biotin consumption of 5, 10, and 20 mg, respectively; peak biotin concentrations following a 20 mg biotin dose did not exceed 355 ng/mL. The time required to achieve serum biotin concentrations of 10 and 30 ng/mL was 1.5147 and 0105 h, respectively, for individuals who received a daily dose of biotin from 1 to 300 mg. The maximum plasma biotin levels were shown to be ,1200 ng/mL following a 300 mg biotin dose. Impact of biotin interference on diagnostic assays The risk of erroneously low (noncompetitive “sandwich” immunoassays) or high (competitive immunoassays) results varies according to the assay design, mechanism of interference, and the analyte and biotin concentrations in the sample [124,125,131,133]. In noncompetitive immunoassays, the signal- and biotinylated antibodies bind the analyte, which subsequently captures the analyte-antibody sandwich complex on a streptavidin-coated solid phase. In the absence of interference, the signal rises as the analyte concentration increases. Excess biotin can saturate the streptavidin binding sites and prevent the capture of the analyte-antibody sandwich complex on the

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streptavidin-coated solid phase, causing erroneously low results (Fig. 1.3). Labeled (signal) and endogenous analytes compete for biotinylated antibody linking sites in competitive immunoassays, and the biotinylated antibody is subsequently linked to the streptavidin-coated solid phase. In the absence of interference, the signal decreases as the analyte concentration increases.

FIGURE 1.3 Mechanism of biotin interference in (A) noncompetitive “sandwich” and (B) competitive immunoassays.

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When biotin concentrations are high, excess biotin binds to the solid phase and prevents the antigenantibody complexes from binding to the solid phase. Unbound antibodies are removed during washing, deceptively reducing the signal and generating inaccurately high assay results (Fig. 1.3). Biotin interference can affect assays for thyroid and reproductive hormones, immunosuppressants, and cardiac markers [134144]. Some immunoassays exhibit a higher threshold for biotin interference than is publicized, which could be due to differences in how biotin interference thresholds are calculated [130,145]. The lack of consistency between thyroid function test results and clinical symptoms is frequently attributed to biotin interference. Between 2016 and 2018, the Manufacturer and User Facility Device Experience (MAUDE) database maintained by the U.S. FDA reported that 78 of 92 incidents of potential biotin interference with immunoassays determined that biotin interference was “most likely” or “not ruled out” [146]. However, it is difficult to verify biotin interference, and not all laboratories report incidents of potential biotin interference. Therefore incidents of biotin interference are probably underreported in the MAUDE database [147]. The concentration of biotin at which interference can occur varies from one assay to another [130]. For example, biotin interferes with cardiac troponin assays at levels between 2.5 and 10,000 ng/mL [148]. Most assays manufactured by Roche Diagnostics (Indianapolis, IN) are not significantly affected by biotin concentrations between 15.6 and 31.3 ng/mL. However, Roche thyroid and cardiac troponin T assays exhibit greater sensitivity to biotin interference [130,132]. A high concentration of biotin and a biotinsensitive assay are both required for a clinically relevant interference to occur. Although the Roche Diagnostics Elecsys Troponin T Gen 5 assay is sensitive to biotin interference [133], the risk of false-negative results is significantly lower than the misclassification risk due to the overall clinical performance of the assay [149].

Detection of biotin interference All diagnostic algorithms should place test results within the context of the patient’s clinical condition. If test results are not consistent with the patient’s symptoms, it is possible that an analytical interference has occurred, and this possibility should be investigated. If biotin interference is suspected, biotin supplements should be discontinued, and the test should be repeated after biotin levels have normalized [150153]. Note that anomalies in immunoassay test results may be due to interferences other than biotin, and the steps that can be taken to identify the source of interference include: 1. Perform a serial dilution study. Dilution decreases the biotin concentration and limits the likelihood that it will interfere with the assay. Serially dilutions should display the recovery of analyte consistent with each

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dilution. Nonlinear results suggest the presence of an interferent. Both the analyte and the interferent are diluted; the level of interference should be reduced at greater dilutions, and a reliable measure of the concentration occurs when the dilutions become linear with the signal. However, dilutions also affect the equilibrium between bound and free hormone fractions and can generate unreliable results. 2. After exogenous biotin has cleared, the tests should be repeated, preferably using alternative assays/methods, such as direct assays for biotin concentrations by LC-MS or using immunoassays that are not based on biotin-streptavidin linkage. 3. Various compounds can be added to the sample to adsorb biotin. For example, streptavidin agarose beads have been incorporated into the sample (10% by volume) before incubation for 60 min with intermittent mixing. After centrifugation, the supernatant was extracted, and the sample was retested. If was a discrepancy between the results before and after the depletion protocol, biotin interference was suspected. Studies have found that depletion protocols can be very effective [154]. However, depletion protocols are considered lab-developed tests and should be thoroughly validated before use. Additional approaches can be used to detect biotin interference, and no single approach is fully reliable because other interferents, such as heterophile antibodies, may also be present. In some laboratories, immunoassays that are not based on biotin-streptavidin linkage may be unavailable. Troponin assays are an example of time-critical laboratory results, and there may not be alternative methods available to help detect biotin interference. The use of dietary biotin supplements is widespread. Laboratory staff and health care professionals should be fully informed of the risks of biotin interference and should implement appropriate procedures to minimize or eliminate this source of laboratory error. For example, the risk of an inaccurate diagnosis arising can be reduced by ensuring laboratories are operated in strict accordance with guidelines, patients are methodically assessed, and efficient and open communication channels are maintained between patients, clinicians, and laboratory staff. There is also a requirement for the implementation of continuous improvement opportunities and education that enhances laboratory testing protocols [155]. Best practice documents should provide outlines for when patients should cease taking biotin-containing supplements prior to a blood test.

Anti-ruthenium and anti-streptavidin antibody interference Electrochemiluminescent immunoassays that use ruthenium (Ru) chelate as labels can be a target of interfering antibodies (anti-Ru antibodies). The prevalence of anti-Ru antibodies interference has been estimated at ,0.24% [156,157]. The anti-Ru antibodies have been shown to cause falsely elevated

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free triiodothyronine (FT3) concentrations on the Elecsys platform [156,157]. Anti-Ru interference on free thyroxine (FT4), thyroid stimulating hormone, and other hormones have also been described [158160]. Analysis of specimens with immunoassay methods that do not contain Ru label may be useful to detect anti-Ru antibodies [156,157]. For immunoassays that use streptavidin (SA) and biotin molecules, interference from antibodies against SA can cause erroneous hormone results [161]. The prevalence of anti-SA antibody interference is unknown. Lam et al. [162] reported discrepant thyroid hormone results that were determined to be due to IgM anti-SA antibodies. Anti-SA antibodies have been reported to cause spurious hyperthyroidism or misdiagnosis of Graves’ disease by interfering with anti-thyroid stimulating hormone receptor measurements [163]. Using immunoassays without SA-biotin, dilution test, and polyethylene glycol precipitation procedures and incubation of the patient’s specimen with SA-linked agarose may be useful for the identification of anti-SA antibodies [163,164]. With anti-Ru and anti-SA antibody interference, sending specimens to the immunoassay manufacturer for further testing is beneficial to help identify these antibodies that can produce erroneous test results and subsequent patient misdiagnosis and mismanagement.

Serial monitoring and reference change value: Determining whether changes are significant Laboratory tests for endocrine biomarkers frequently involve monitoring patients over time. It is essential to know whether significant changes in concentration of the analyte have occurred. Failure to recognize when changes are not significant can result in inappropriate changes in patient management. Conversely, failure to recognize significant changes in analyte concentrations can result in delayed diagnosis and treatment. The term reference change value (RCV) refers to the threshold for significance of changes in laboratory results [165]. Whether a change in biomarker concentration is significant can be calculated based on two factors: the physiological or intra-individual variation of the biomarker and the analytic variation of the test method. These two factors combined represent the total variability for each analyte or biomarker.

Physiological variation (intra-individual variation) Intra-individual variation (CVi) has been calculated for many analytes; however, many common endocrine biomarkers are not well characterized with regard to their CVi values. Online resources that curate such lists are available; examples include westgard.com and biologicalvariation.eu. CVi values

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can vary from 0.5% for analytes such as plasma sodium, or as high as 37% for insulin [166].

Analytic variation Analytic variation (CVa) varies considerably between analytes, methodologies, instruments, and laboratories. The CVa for any test performed in a clinical laboratory is known and typically can be found in the manufacturer’s product literature or the method standard operating procedure. Variability may change across the dynamic range of the assay, so the precision at high concentrations may not be the same as at low concentration. For some biomarkers, CVa values are near 1%. However, for biomarkers that have very low concentrations, the CVa can be as high as 20%. When the CVa of an assay exceeds 20%, the method is generally considered only semiquantitative.

Calculating significant variation The calculations presented here assume that the same methodology and instrument are being used to monitor a patient. Comparing serial results from different laboratories, methodologies, or instruments is difficult and should be avoided. The total variation is a product of the intra-individual and analytic variation, but unfortunately, these two parameters cannot be added together directly. CV values (or standard deviations, SD) must be converted to variances to be additive [167]. Fortunately, the conversion of CV to variance is only a matter of summing the squares of the two CV values, then taking the square root: First, calculate the total variation: For two results to be significantly different (at P , .05), they have to differ by at least 2.8 SD. Multiply the CV% 3 2.8. This is the percentage difference between the two results required to be statistically different. The difference between the two results is calculated as (Result 1 2 Result 2)/Result 1 3 100%. Some examples of when this approach is helpful are outlined below. Each example is based on a 95% confidence value threshold (P , .05).

Example 1—Is the statin working? A patient taking atorvastatin has a steady-state total cholesterol of 275 mg/ dL. After changing to fluvastatin, the total cholesterol lowered to 235 mg/dL. The ordering physician is satisfied as a result is now below the 239 mg/dL threshold recommendation for “intermediate risk.” Is this change significant? For total cholesterol, the intra-individual variation is 5.9%, and the analytic variation is 2.4%.

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The least significant change will be 6.4%. Taking 6.4% and multiplying by 2.8 yields 17.9%. The minimum change needed for significance is 17.9%. For this patient, the results are 275 and 235 mg/dL. The percent difference is calculated to be: (275235)/275 3 100% 5 14.5%. Because the change was only 14.5% and a minimum 17.9% change is needed for significance, this reduction in total cholesterol can be attributed to a combination of physiological and analytic variability.

Example 2—Is the DPP-4 inhibitor working? A physician has a diabetic patient with a hemoglobin A1c of 9.1% after being on a sulfonylurea. While watching sports on television, a physician saw an advertisement for sitagliptin, which claimed that adding this medicine could reduce A1c by 0.7 percentage points. If the median intra-individual CVi is 1.3% and the analytic CVa is 3.8%, would the reduction in A1c from the advertisement be considered significant for an individual patient? The least significant change will be 4.0%. Taking 4.0 and multiplying by 2.8 yields 11.2%. The minimum change needed for significance is 11.2%. For this patient with a result of 9.1% A1c, a 0.7 percentage point reduction would be 8.4%. The percent difference is calculated to be: (9.18.4)/9.1 3 100% 5 7.7% Therefore a change in A1c of 0.7 percentage units would be only 7.7%, whereas a result would need to be 11.2% different to be significant for an individual patient. Example 3—Sestamibi scan or surgical consult? A patient has a PTH concentration of 95 pg/mL, which is above the laboratory reference interval of 1065 pg/mL. Six months later the PTH is measured again, this time at 156 ng/mL. Is this change significant? For PTH, the intra-individual variation is 25% and the analytic variation is 12%. The least significant change will be 27.7%. Taking 27.7 and multiplying by 2.8 yields 78%. The minimum change needed for significance is 78%. For this patient the results are 95 and 156 mg/dL. The percent difference is calculated to be: (95156)/95 3 100% 5 2 65%. Because the change was only 65% and a minimum 78% change is needed for significance, this increase in PTH could be due to physiological and analytic variability.

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Automated calculations The RCV tool is not particularly computationally challenging. Performing the operation manually to determine 95% confidence is simple and determining the percent likelihood of a significant change is slightly more challenging but within the capability of any modern computing device. This calculation can be implemented in laboratory information systems (LIS), physician electronic health record systems, Excel spreadsheets, and even smartphone apps [168170].

Postanalytical errors Postanalytical errors occur after a laboratory result has been generated by an analytical instrument. Incorrect transcription or data entry of laboratory results is a major source of postanalytical errors. Studies of point-of-care laboratory tests, where results are often manually entered into the patient record, have revealed error rates of approximately 5 in 1000 recorded results [171]. Although transcription errors remain a problem in point-of-care settings where the tests are visually interpreted (e.g., fecal occult blood, pregnancy tests, urine dipsticks), most core clinical laboratories use automated instrument platforms that are digitally interfaced to the LIS, which, in turn, is interfaced with the electronic medical record (EMR), so data are passed from the analytical instrument to the patient record without human intervention. Digital interfaces greatly reduce the frequency of errors in the postanalytical data processing. Many point-of-care instruments are capable of digital communication with the LIS or EMR, as well. These technological advances have vastly improved the integrity of the postanalytical processing of laboratory results compared to manual entry. Preanalytical and analytical errors can affect the accuracy of a laboratory result; however, most of these errors are identifiable, and their frequency can be minimized by vigilance and engineering. On the other hand, many postanalytical errors are very difficult to detect and prevent because they involve how a laboratory result is interpreted and used to influence the treatment of a patient. For most laboratory tests, the involvement of laboratory personnel (e.g., technologists, directors, pathologists) ends when the test result is reported. Surveys have shown that consultation with laboratory personnel or a pathologist is not common even when the requesting physician is uncertain about which test to order or the meaning of a test result [172]. To make matters worse, medical students in the United States receive an average of just 9 h of instruction in the proper selection and interpretation of laboratory tests [172]. Errors in the interpretation of laboratory test results fall into three broad categories: 1. Misunderstanding of the reference range, and the various ways “expected,” or “desired” results are presented with a laboratory result;

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2. Failure to correctly interpret the statistical probability that a laboratory result correctly classifies the disease status of a patient, which can lead to incorrect assessment of risk and, often, unnecessary follow-up testing and procedures; 3. Misinterpretation of the meaning of an abnormal laboratory result, and the specific diagnostic possibilities it might reveal. The third category is far beyond the scope of this introductory chapter, as it requires detailed knowledge of endocrine function and testing, which will be addressed in subsequent chapters. However, the first two categories are more general and merit introductory review because they apply to virtually all laboratory tests.

Reference ranges, normal values, and desired results Accreditation agencies and the Centers for Medicare and Medicaid Services require all laboratory test results to be presented with a reference range or other interpretive guideline to provide advice about whether the patient’s result is normal or abnormal. However, the terminology in reference ranges or interpretive guidelines can be confusing, particularly to laypersons. For example, a “positive” laboratory test is typically an abnormal result, whereas in common (nonmedical) usage “positive” usually means something good. The same is true for “negative,” which is good news in a medical context, but in common usage denotes something undesirable. The use of “normal” is imprecise because it implies “healthy,” and laboratory results that fall within “normal limits” do not necessarily indicate health, just as results that exceed those limits can occur in healthy patients. Even the term “reference range” is slightly problematic, since it raises the question: “in reference to what?” The answer to that question can differ from one laboratory test to another. Fundamentally, reference ranges reflect interpretative advice that is based on one or more of the following: 1. Population-based reference intervals that are generated by testing healthy individuals in a population that mirrors the characteristics of the patients to which the reference interval is being applied; or 2. Desired results based on research and epidemiological data that indicate what level of a particular biomarker is associated with good health or absence of disease. Population-based reference intervals typically reflect the central 95% of results obtained by testing a suitable reference population. The selection of an appropriate reference population may be the most critical challenge in generating a reference interval because it should account for changes in analyte concentration that may occur due to age, sex, ethnicity, geography, diet, or other factors. Fortunately, differences based on these factors are minimal

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or nonexistent for most common laboratory tests. However, some analytes show marked differences based on age, sex, and sometimes ethnicity. Influences due to geography, diet, and other factors are relatively rare but do exist. For example, vitamin D3 is produced in the skin by the action of UV light on 7-dehydrocholesterol, so people who live at latitudes that receive more direct sunlight typically have higher vitamin D levels [173]. People who live at high altitudes usually compensate for the lower oxygen tension by hematopoiesis and have higher average hemoglobin [174,175]. Vegetarian diets have been shown to lower average total, low density lipoprotein, and high density lipoprotein cholesterol levels [175]. A common method used to generate a reference range is to calculate the mean and SD of results in the reference population and then set the reference range as the mean 6 2 SD. Note that defining the central 95% as the mean 6 2 SD is valid only if the data conform to a Gaussian distribution. While some analytes may fit a bell-shaped distribution curve, the data are usually unevenly distributed (i.e., skewed toward higher or lower values). For this reason, the CLSI recommends, in their 2010 document EP28-A3c “Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline-Third Edition,” that reference intervals be determined by a nonparametric method that involves discarding the highest and lowest 2.5% of values, with the remaining limits defining the central 95% reference interval. The drawback of this approach is that it requires at least 120 reference values. It may be difficult for most laboratories to recruit enough volunteers to donate specimens for a reference range study. The mean and SD of Gaussian distribution can be estimated with approximately 30 data points, but if the distribution of results is not truly Gaussian, then the mean 6 2 SD approach will generate a misleading reference interval. There is another subtle issue in the way that “central 95%” reference intervals are generated: the range does not include all healthy subjects. On the contrary, 5% of the reference population had test results that exceeded the newly established reference interval. Therefore their test results would be considered abnormal. From a statistical perspective, the odds that a perfectly healthy patient will have an abnormal result in a single laboratory test is about 1 in 20. If two tests are performed on a healthy patient, the odds go up to about 1 in 10 that at least one of the results will be abnormal. For a basic metabolic profile, which includes eight tests, the statistical probability that at least one result in a healthy patient will be outside its reference range is 0.33 (33%). In a comprehensive metabolic profile, which includes 14 tests, it is more likely than not (probability 5 0.51) that a healthy patient will have at least one test result that is outside the limits of its reference range. The relationship between the number of tests and the probability of an abnormal result is illustrated in Fig. 1.4. Increasingly, the conventional “central 95%” approach to reference intervals is being replaced by a “desired value.” Prominent tests in this category

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FIGURE 1.4 Since reference intervals include only the central 95% of healthy subjects, 5% of healthy subjects will have a laboratory test result outside of the reference range. The probability of this occurring increases with the number of tests performed.

include blood glucose, hemoglobin A1c, cholesterol (total, high density lipoprotein, and low density lipoprotein), estimated glomerular filtration rate (eGFR), troponin, and vitamin D. There are clinical data for these tests that provide a diagnostic decision threshold for disease risk. The American Diabetes Association, for example, has established upper limits for blood glucose and hemoglobin A1c in nondiabetic individuals. Recommended total, high density lipoprotein, and low density lipoprotein cholesterol values have changed over past three decades based on guidelines published by the National Cholesterol Education Program, as the relationship between these parameters and cardiovascular health has become more precisely defined. Chronic renal failure is now assigned a stage of 15 based on the eGFR, where a “desirable” result is .90 mL/min/1.73 m2. Cardiac troponin levels are interpreted relative to the 99th percentile of results in healthy individuals. Desirable levels of Vitamin D have been recommended by several organizations, including the United States Preventive Services Task Force (USPSTF), but remain controversial because epidemiological studies relating high Vitamin D to health have produced contradictory results [176]. In the realm of diagnostic endocrinology, reference intervals are generally useful for screening purposes, but provocative tests usually negate reference ranges, and results must be interpreted in the context of the study. Often, the chemical challenge is expected to produce a measurable change in a biomarker concentration, and it is the magnitude of the change that informs the decision about whether the test results are normal or abnormal. In addition, interventions such as surgical removal or radioactive ablation of endocrine glands render conventional reference ranges useless since they are generated using a reference population with intact endocrine glands and function. For example, in parathyroidectomy surgery, the target PTH is not a value within the normal reference interval, but rather it is a value no more than one-half of the presurgery PTH concentration.

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The correct interpretation of the reference interval requires consideration of how it was generated, whether it represents desirable values or population-based reference data, and whether it applies to the patient being tested. This judgment can only be made in the context of the clinical picture. Uninformed or misguided judgment represents a significant potential source of postanalytical error.

Sensitivity, specificity, and predictive value In general, laboratory tests are designed to detect disease. In some cases, a single test may have the capability of detecting many different diseases. This is particularly true for much of endocrine testing, where specific hormone may be elevated, deficient, or normal, depending on particular disorder in the system that either produces or responds to it. By contrast, a test such as blood glucose has essentially one purpose, which is to diagnose or treat diabetes and its consequences. To discuss the clinical interpretation of laboratory test results without undue complexity, we will use the terms normal, negative, and within the reference range, all to indicate a laboratory test result that is within either the reference interval or a desired threshold. If a test result does not fall within either the reference interval or the desired threshold, we will refer to the result as positive, abnormal, or outside the reference range. “Disease” will refer to any disorder the laboratory test is intended to detect, whereas “healthy” will refer to the absence of any disease associated with abnormal results of the laboratory test in question. The two most recognized measures of the clinical performance of a laboratory test are its sensitivity and specificity. These are based on the likelihood of true-positive (TP), true-negative (TN), false-positive (FP), and false-negative (FN) results. These important parameters are summarized in Table 1.1. The sensitivity of a laboratory test is sometimes referred to as its “positivity in the presence of disease,” and it represents the probability (typically converted to a percentage) that a subject with the disease will have a positive test result.1 Table 1.1 shows that subjects with the disease can produce either TP or FN results. Therefore the sensitivity of laboratory tests is defined as: Sensitivity 5

TP TP 1 FN

1. What is described is the clinical sensitivity of a laboratory test, and it should not be confused with the analytical sensitivity of the measurement. Analytical sensitivity has a different definition and is determined by a very different method. The analytical sensitivity reflects the smallest concentration of analyte that can be accurately measured by the assay. An improvement in analytical sensitivity may also improve the clinical sensitivity by detecting smaller amounts of the biomarker, thereby allowing detection at an earlier stage or milder form of disease, but the two are still distinct quantities.

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TABLE 1.1 Definitions of TP, FP, TN, and FN. Was the test positive?

Did the subject have a disease the test is intended to detect?

True positive (TP)

Yes

Yes

False positive (FP)

Yes

No

True negative (TN)

No

No

False negative (FN)

No

Yes

The numerator is the number of subjects with the disease who produced a positive test, and the denominator is the total number of subjects who had the disease. The sensitivity of a new laboratory test is typically determined by performing the test on a cohort of subjects already confirmed to have the disease the test is designed to detect. Specificity, in a sense, is the mirror image of sensitivity and is sometimes described as “negativity in the absence of disease.” The specificity of a laboratory test is the probability (once again, usually converted to a percentage) that a healthy subject will produce a negative test result2. Table 1.1 confirms that healthy subjects will produce either TN or FP results, so the specificity is defined as: Specificity 5

TN TN 1 FP

The numerator is the number of healthy subjects that produced a negative test result, and the denominator is the total number of healthy subjects. Validation of the clinical performance of a laboratory test is usually performed on a cohort of healthy subjects to determine the specificity. Note that 1-specificity (or, if specificity is expressed as a percentage, 100-specificity) is a useful parameter known as the false-positive rate. The FP rate is the probability that a healthy subject will produce a positive test result. The sensitivity and specificity of a laboratory test are useful measures of the degree to which the assay can distinguish between subjects with and without the disease, which in turn usually reflects the extent to which the biomarker is associated with the disease process. When the marker is only epiphenomenally related to the disease (e.g., hypoproteinemia in renal failure), then the sensitivity 2. In symbolic logic, specificity would be considered the inverse of sensitivity, as the inverse of “if P then Q” is “if not P then not Q.”

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of the test might be high since it is nearly always present with the disease, but specificity may be low because the abnormal result can have many other causes. Some tests have very high sensitivity and specificity for the disease they are intended to detect because the biomarker they measure is directly related to the pathophysiology of the disease. An example is cardiac troponin, which is released by cardiac myocytes during ischemia or infarction. Therefore the cardiac troponins (both I and T) are exquisitely sensitive and specific markers for acute coronary syndrome and myocardial infarction. By contrast, myoglobin is highly sensitive for detecting myocardial damage but lacks specificity because it is produced in all muscle tissue. Sensitivity and specificity provide estimates of the probability that subjects with or without disease will produce positive or negative test results, respectively, and they are a valid basis for comparing the clinical performance of one assay to another. However, they are of limited value in a clinical setting because both measures assume you know whether the patient has the disease. In other words, both sensitivity and specificity tell you how good the test is at confirming what you already know about the patient’s status! That is backwards from how laboratory tests are used in clinical practice, for if a clinician already knows whether the patient has the disease, then nothing is to be gained by running a test. In clinical practice, a test is ordered to reveal, or at least suggest, whether the patient has the disease. If the test is positive, what is the probability that the patient has the disease, and if the test is negative, what is the probability the patient does not have the disease? It should be obvious that neither the sensitivity nor specificity of the test answers these questions; the answers come from the positive (PPV) and negative predictive values (NPV), respectively. Consider first a positive test result in a patient for whom a diagnosis has not been established. You would like to know the probability that the patient has the disease, based on the positive test result. Therefore you need to know what percentage of all positive results are true positives: Positive predictive value 5

TP TP 1 FP

Whereas sensitivity and specificity are inherent characteristics of a particular assay, the PPV of a test result depends on the pretest probability that the patient has the disease. This is because the number of false-positive results depends on how many disease-free individuals are tested, unless the test has 100% specificity. Very few laboratory tests have specificity approaching 100% (by definition, a specificity of 100% means the FP rate is zero, so the PPV would always be 100%). As a result, when the test is applied to a population in which only a very few actually have the disease the test is intended to detect, the TP rate will be low, while the number of false positives will be high due to the much larger number of healthy subjects. This will be the case even when the FP rate is fairly low.

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FIGURE 1.5 Positive predictive value of a hypothetical test with 100% sensitivity and specificities of 90%99.9%, as a function of disease prevalence from 100% to 0.001% (one case per 10,000).

As an example, suppose 100 subjects are tested for a disease that normally afflicts only 1 in 100 the subjects (pretest probability 5 1%), and the test has a sensitivity of 100% and a specificity of 95%. There will be 1 TP and 5 FPs so that the PPV will be 1/6, or about 17%. Fig. 1.5 shows the relationship between disease frequency and PPV for a hypothetical test that has a sensitivity of 100% and a specificity of 99.9%, 99.0%, 95.0%, or 90.0%. Similar calculations can be made for the NPV, which is the percentage of negative results that identify healthy individuals: Negative predictive value 5

TN TN 1 FN

In contrast to PPV, NPV increases as the pretest probability of disease decreases, as long as the sensitivity of the test is less than 100% (by definition, when sensitivity is 100% there are no false-negative results, so the NPV would always be 100%). If a test that is 100% specific but only 90% sensitive is applied to 100 patients, of whom half have the disease, the NPV will be 50/55, or about 91%. If only 10 patients in the group have the disease, the NPV increases to 99%. There are several important points in the preceding analysis: 1. The PPV of a laboratory test increases as the pretest probability of disease increases, so applying the test only in patients at higher risk for disease is an effective strategy to maximize the value of the test. 2. The NPV for most laboratory tests is reasonably high unless the test is used in a patient that is very likely to have the disease. 3. Both PPV and NPV are influenced by the sensitivity and specificity of the test, as well as the pretest probability of disease.

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The third point deserves comment: The sensitivity and specificity of laboratory tests are usually inversely related: the sensitivity can be maximized only at the expense of specificity, and vice versa. To illustrate this, consider a laboratory test in which the threshold separating positive and negative results is set so low that it virtually ensures all patients will test positive. The sensitivity of this test will be 100% because there will be no false-negative results, but the specificity will zero because there will be no true-negative results, either. Conversely, if the positive/negative threshold is set so high that the test is negative in all patients, the sensitivity will be zero, and the specificity will be 100%. Therefore these parameters depend on where the positive/negative threshold is set, and that, in turn, depends on whether maximum sensitivity or specificity is the primary goal. For screening tests designed to detect clinically silent diseases that have a significantly better prognosis when detected early (e.g., most cancers), high sensitivity is the chief goal. However, as the preceding analysis shows, using a test in which sensitivity has been optimized at the expense of specificity has drawbacks, particularly when the pretest risk of disease is small. This scenario essentially guarantees many false-positive results, and the clinical consequences of those FPs must be considered: will it result in a risky procedure or an expensive follow-up test, or will it cause unnecessary patient anxiety from their fear of having a serious disease? The use of tumor markers to screen for cancer is an excellent example of this dilemma. Tests with limited sensitivity but high specificity should be reserved for confirmatory testing when the disease is strongly suspected. In practical terms, this means that the pretest probability is very high, and therefore the PPV is maximized.

Receiver operating characteristic plot3 The interplay between the sensitivity and specificity of laboratory tests lends itself to a graphical representation, in the form of a receiver operating characteristic (ROC) curve. In this graphical approach, the sensitivity is plotted versus 1-specificity (the FP rate) at positive/negative thresholds from lowest to highest. An example ROC curve is shown in Fig. 1.6. A ROC curve visually displays the best compromise between sensitivity and specificity as the positive/negative threshold corresponding to the point closest to the upper-left corner of the plot, which represents 100% sensitivity and 100% specificity. If the entire plot area is normalized to unity, a perfect test (100% sensitivity and 100% specificity) would have an area under the 3. The term “receiver operating characteristic” was first applied to the specifications of radar units, in which the sensitivity and selectivity can be adjusted, but display an inverse relationship similar to the sensitivity and specificity of laboratory tests.

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FIGURE 1.6 In a receiver operating characteristic (ROC) curve, the sensitivity is plotted against 1-specificity at positive/negative thresholds over the entire range of the assay. The upper-left corner of the graph corresponds to 100% sensitivity and specificity, and the dotted line represents identity (sensitivity 5 1-specificity), where the test is of no diagnostic value. The entire area of the plot is normalized to unity, so an area under the curve (AUC) closer to 1 reflects greater diagnostic value. In this example, the test represented by the red curve is slightly more diagnostically useful than the test represented by the black curve, based on the relative areas under the curve.

curve (AUC) of 1.0. To be useful, a test must have an AUC that exceeds 0.5, since the dotted line in Fig. 1.4 corresponds to a test that has no diagnostic value at all (i.e., TP 5 FP and TN 5 FN at every point on the line). An AUC of less than 0.5 suggests a test that is inversely correlated with disease. ROC plots are useful when comparing two diagnostic tests; the larger the AUC, the greater the ability of the test to discriminate between diseased and healthy patients.

Likelihood ratio A final parameter that reflects the value of a laboratory test for detecting a specific disease is the likelihood ratio (LR). Like the predictive value, the LR can be applied to either positive or negative results. The positive LR is the ratio of the probability that someone with the disease will have a positive test and the probability that someone without the disease will have a positive

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test. To use the previously introduced terminology, the positive LR is the ratio of TP to FP and is calculated by: Likelihood ratio ð 1 Þ 5

Sensitivity 1 2 specificity

Tests with a high positive LR are more valuable because they significantly increase the patient’s risk for the disease over pretest estimates. A positive test with an LR of 1 does not change the patient’s risk because it is equally likely to be a false-positive result. However, a positive result with a test that has an LR of 2 increases the patient’s risk of disease by 15%, and an LR of 10 increases it by nearly half (45%).

Summary of postanalytical errors Interpretation is the final step in the sequence of events that occur between the time an order for a laboratory test is placed, and when a treatment plan is initiated based on the result of that test. Clinical laboratories usually provide only a reference interval to aid in the interpretation of results. However, some knowledge of laboratory medicine is required to correctly interpret reference intervals. Every laboratory test has limitations based on its performance characteristics and the attributes of the setting in which it is being applied. An understanding of the factors that influence the interpretation of a laboratory test result will minimize the risk of misinterpretation and misinformed treatment decisions.

Conclusion Laboratory errors can be preanalytical, analytical, or postanalytical. This discussion expands on these three types of errors that can lead to misdiagnoses or failure to treat an undetected condition. Immunoassays are emphasized because many of the analytes relevant to diagnosing endocrine disorders fall into this category of analytical method, but most of the concepts apply to nonimmunochemical methods, as well. Despite the many different types of potential interferences of immunoassays, most immunoassay results are, fortunately, free from such effects. Nevertheless, when they occur, they can often lead to misdiagnosis and other negative consequences for the patient. Therefore it is imperative that the laboratory be vigilant of such problems. Despite this awareness and the ongoing improvement of the robustness of immunoassays, interference problems will often go undetected by the laboratory. It is therefore important that the laboratory actively communicates with physicians who order and interpret test results, provide follow-up, and troubleshoot any results that appear to be discordant with other clinical information on a patient. Although immunoassays have been in routine use in clinical laboratories for several decades, they are continually undergoing

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improvements, which lead to greater sensitivity, improved convenience due to automation, rapid turn-around time, and the development of immunoassays for many new analytes. These improvements, however, will likely be accompanied by new types of interference. As the sensitivity of immunoassay increases and lower concentration of analytes is measured, smaller amounts of endogenous interferents will likely affect these newer assays [50]. A big challenge for laboratorians, therefore, is not only to deal with the current known causes of interference of immunoassays but be able to identify and resolve future problems that will inevitably develop. Finally, the results of laboratory tests should be interpreted within the context of the clinical performance characteristics of the test and the condition of the patient. Proper interpretation of laboratory test results and the clinical decisions based on those results are a largely unknown source of postanalytical error.

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[162] L. Lam, W. Bagg, G. Smith, W.W. Chiu, M.J. Middleditch, J.C. Lim, et al., Apparent Hyperthyroidism caused by biotin-like interference from IgM anti-streptavidin antibodies, Thyroid. 28 (8) (2018) 10631067. [163] L. Peltier, C. Massart, M.P. Moineau, A. Delhostal, N. Roudaut, Anti-streptavidin interferences in Roche thyroid immunoassays: a case report, Clin. Chem. Lab. Med. 54 (1) (2016) e11e14. [164] N.J. Rulander, D. Cardamone, M. Senior, P.J. Snyder, S.R. Master, Interference from anti-streptavidin antibody, Arch. Pathol. Lab. Med. 137 (8) (2013) 11411146. [165] C.G. Fraser, Reference change values, Clin. Chem. Lab. Med. 50 (5) (2011) 807812. [166] IFCC, Biological variations database. Available from: https://biologicalvariation.eu. [167] A. Deacon, Calculations in Laboratory Medicine, ACB Venture Publications;, London, 2009. [168] M.J. O’Kane, B. Lopez, Explaining laboratory test results to patients: what the clinician needs to know, BMJ 351 (2015) h5552. [169] F. Lund, P.H. Petersen, C.G. Fraser, G. Soletormos, Calculation of limits for significant unidirectional changes in two or more serial results of a biomarker based on a computer simulation model, Ann. Clin. Biochem. 52 (Pt 2) (2015) 237244. [170] J.M. Hilderink, R. Rennenberg, F.H.M. Vanmolkot, O. Bekers, R.P. Koopmans, S.J.R. Meex, Labtracker 1 , a medical smartphone app for the interpretation of consecutive laboratory results: an external validation study, BMJ Open. 7 (9) (2017) e015854. [171] J.A. Mays, P.C. Mathias, Measuring the rate of manual transcription error in outpatient point-of-care testing, J. Am. Med. Inform. Assoc. 26 (3) (2019) 269272. [172] M. Laposata, Putting the patient first—using the expertise of laboratory professionals to produce rapid and accurate diagnoses, Lab. Med. 45 (1) (2014) 45. [173] P.F. Leary, I. Zamfirova, J. Au, W.H. McCracken, Effect of latitude on vitamin D levels, J. Am. Osteopath. Assoc. 117 (7) (2017) 433439. [174] J.S. Windsor, G.W. Rodway, Heights and haematology: the story of haemoglobin at altitude, Postgrad. Med. J. 83 (977) (2007) 148151. [175] F. Wang, J. Zheng, B. Yang, J. Jiang, Y. Fu, D. Li, Effects of vegetarian diets on blood lipids: a systematic review and meta-analysis of randomized controlled trials, J. Am. Heart Assoc. 4 (10) (2015) e002408. [176] A.B. Hassan, R.F. Hozayen, R.A. Alotaibi, Y.I. Tayem, Therapeutic and maintenance regimens of vitamin D3 supplementation in healthy adults: a systematic review, Cell Mol. Biol. (Noisy-le-Grand) 64 (14) (2018) 814.

Chapter 2

Laboratory investigation of disorders of the pituitary gland Verena Gounden1, Charlotte C. Ellberg2 and Ishwarlal Jialal3,4,5 1

Department of Chemical Pathology, Inkosi Albert Luthuli Central Hospital, National Health Laboratory Service, University of Kwa-Zulu Natal, Durban, South Africa, 2California Northstate University College of Medicine, Elk Grove, CA, United States, 3Retired Distinguished Professor, UCDavis, CA, United States, 4Sacramento VA Medical Center, CA, United States, 5California Northstate University College of Medicine, CA, United States

Anterior pituitary The anterior pituitary secretes the peptide hormones growth hormone (GH), prolactin (PRL), and adrenocorticotropin (ACTH) and the glycoprotein hormones thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). The glycoproteins TSH, LH, and FSH are each dimers composed of a common α-subunit and a unique β-subunit. The hypophysiotropic regulation of the anterior pituitary is shown in Table 2.1.

TABLE 2.1 Hypothalamic regulation of the anterior pituitary. Hypothalamus

Effect

Pituitary cell type (hormone)

Target gland product or effect

CRH

1

Corticotroph (ACTH)

Cortisol

a

GnRH

1

Gonadotroph (LH and FSH)

Testosterone, estradiol

GHRH

1

Somatotroph (GH)

IGF-1

TRH

1

Thyrotroph (TSH) and lactotroph (PRL)

T4 and T3

SRIF (somatostatin)

2

Somatotroph (GH) and thyrotroph (TSH)

IGF-1

Dopamine

2

Lactotroph (PRL)

Lactation

a

Previously referred to as luteinising hormone releasing hormone (LHRH).

Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00002-9 © 2021 Elsevier Inc. All rights reserved.

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Hypophysiotropic hormones from the hypothalamus reach the anterior pituitary via a portal venous system and either stimulate or inhibit the release of pituitary hormones. The hypothalamus serves as a relay station that receives chemical messages from higher brain centers and feedback information from target glands upon which the pituitary hormones act. The hypothalamus then integrates these messages before exerting its effect on the pituitary. These axes are described as closed-loop or negative feedback systems because hormones of target glands modulate hypothalamic and pituitary hormone release.

Hypothalamic regulators of anterior pituitary hormones Thyrotropin-releasing hormone (TRH) is a tripeptide that primarily stimulates the release of TSH. TRH also stimulates PRL secretion; elevated TRH due to primary hypothyroidism is a well-known cause of hyperprolactinemia. Gonadotropin-releasing hormone (GnRH) is a decapeptide that stimulates the release of both gonadotropins LH and FSH. GnRH is secreted episodically, resulting in pulsatile secretion of gonadotropins. Variability in the frequency of GnRH pulses determines the relative amounts of LH and FSH released. GnRH pulsatility maintains basal gonadotropin secretion, generates the phasic release of gonadotropins for ovulation, and determines the onset of puberty. Somatostatin [somatotropin-release inhibiting factor (SRIF)] is a 14amino acid peptide that inhibits GH and TSH secretion. In addition to its inhibitory role in the pituitary, SRIF plays an inhibitory role in the release of numerous other hormones throughout the body and is also known to have antitumor effects [1]. GH-releasing hormone (GHRH) is a 44-amino acid peptide that specifically triggers release of GH. Ghrelin also stimulates GH release. Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide that stimulates the secretion of ACTH. ACTH is derived from a precursor molecule, pro-opiomelanocortin (POMC), made in the pituitary gland. Unlike the relatively transient response seen with other hypophysiotropic hormones, ACTH and cortisol levels remain elevated for several hours following CRH release. Dopamine is an important physiological inhibitor of PRL secretion. In the absence of dopamine, lactotrophs spontaneously secrete PRL [2].

Anterior pituitary hormone physiology and biochemistry Histological staining techniques classify the cells of the pituitary as acidophils, basophils, or chromophobes. Somatotrophs and lactotrophs are acidophils that secrete GH and PRL, respectively, whereas mammosomatotrophs secrete both GH and PRL. Thyrotrophs, corticotrophs, and gonadotrophs are basophils that secrete TSH, ACTH, and the gonadotropins LH and FSH, respectively. The remaining chromophobe cells do not stain immunocytochemically. Newer

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immune-cytochemical staining techniques and electron microscopy now allow the cells of the anterior pituitary to be histologically classified according to their specific secretory products. The main function of GH (a 191-amino acid polypeptide) is to stimulate linear growth during childhood. This effect is mediated by the somatomedins or insulin-like growth factors (IGFs), a group of small peptides produced by the liver and local tissues in response to GH. GH also directly stimulates lipolysis and has an antagonistic effect on insulin action. Its secretion is stimulated by sleep, exercise, stress, hypoglycemia, dopamine, certain amino acids, GHRH, β-blockers, and glucagon and inhibited by somatostatin and IGF-1. GH is secreted in an episodic and pulsatile manner, with the highest amplitude pulses occurring with sleep overnight. IGF-1 levels generally reflect the integrated secretory activity of GH given their much longer half-life [2]. PRL is a 198-amino acid polypeptide under tonic inhibition by dopamine. The primary effect of PRL is the initiation and maintenance of lactation in the postpartum period. Prolactin also plays a role in suppressing fertility [3]. PRL is secreted in a pulsatile, episodic manner, with highest levels of secretion during sleep [3]. Its secretion is increased by nipple stimulation, stress, exercise, sleep, TRH, and dopamine antagonists [4]. Estrogen also enhances PRL secretion. Women taking oral contraceptives can have mild PRL elevations, and pregnant women have PRL levels up to 10 times the reference range [5]. TSH, a glycoprotein hormone is trophic to the thyroid gland and stimulates the synthesis and secretion of the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). TSH secretion is finely regulated by the levels of thyroid hormones in the blood and by TRH. ACTH is a 39-amino acid peptide, of which the first 24 amino acids are necessary for biological activity. Although ACTH mainly stimulates the synthesis and secretion of glucocorticoids, it also has an effect on the adrenal androgens. ACTH is secreted in a pulsatile manner with a diurnal rhythm (highest secretion in the early morning hours). Many types of stressors, including hypoglycemia, stimulate ACTH release, and ACTH secretion is inhibited by cortisol. Due to the melanocyte-stimulating action of POMC precursor molecule, excessive ACTH secretion can result in increased pigmentation. This has important implications when ACTH levels are excessively elevated for example in Addison’s disease. The gonadotropins (LH and FSH), like TSH, are glycoprotein hormones for which the β-subunit confers the biological and immunodiagnostic specificity. In men, LH acts on Leydig cells to increase the synthesis and secretion of testosterone, whereas FSH (in concert with testosterone) acts on Sertoli cells to stimulate spermatogenesis. In women, LH stimulates estradiol and progesterone production by the ovary. A surge of LH in the midmenstrual cycle is responsible for ovulation, and continued LH secretion

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subsequently maintains the corpus luteum and progesterone production. FSH regulates development of the ovarian follicle, and the secretion of estradiol from the follicle is dependent on variable ratios of LH and FSH. Both FSH and LH are under dual control of the hypothalamus and gonads. This sentence should not be alone, please move up to join the paragraph above Sertoli cells within the testes and granulosa cells of the ovary produce inhibin-B that suppresses FSH secretion.

Features of hypopituitarism Hypopituitarism is defined as a deficiency of one or many pituitary hormones. Isolated hormone deficiency (monohypopituitarism) is usually due to a hypothalamic disorder, whereas multiple hormone deficiencies (panhypopituitarism) can result from disorders of the pituitary gland or hypothalamus. Frequently, a stepwise loss of pituitary function occurs as a result of pituitary insult; GH or gonadotropin deficiency may precede the onset of thyrotroph and corticotroph injury. Clinical manifestations of hypopituitarism are not evident until at least 75% of the pituitary gland is destroyed. Causes of hypopituitarism are shown in Table 2.2. The most common cause is pituitary neoplasm. In the postpartum period, spontaneous hemorrhage of a hypervascular and enlarged pituitary gland leads to infarction of the pituitary and acute hypopituitarism. This is referred to as Sheehan syndrome and classically presents with secondary amenorrhea and failure of lactation. Sheehan syndrome is an important cause of hypopituitarism, especially in developing countries. Anorexia nervosa, a psychological disorder characterized by self-imposed starvation and a preoccupation with body size, can resemble hypopituitarism with weight loss, amenorrhea, and decreased gonadotropins; however, cortisol and GH levels are usually increased.

TABLE 2.2 Causes of hypopituitarism. Tumors

Pituitary adenomas, craniopharyngiomas, metastatic carcinomas, meningiomas, gliomas, chordomas, pituitary hamartoma

Vascular disease

Sheehan syndrome, vascular malformations, sickle cell disease, pituitary apoplexy

Infiltrative disease

Sarcoidosis, tuberculosis, syphilis, hemochromatosis, histiocytosis-X, lymphocytic hypophysitis, histoplasmosis, amyloidosis

Trauma

Head injury, surgical

Iatrogenic

Irradiation, hormonal therapy

Miscellaneous

Congenital absence of the pituitary, isolated hormone deficiencies

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In adults, the manifestations of GH deficiency are usually subtle and nonspecific and include increased fat mass, decreased muscle mass, low energy, osteopenia, hypercholesterolemia, altered cardiac function, and poor quality of life [6,7]. Hypoglycemia from GH deficiency in adults is rare, whereas in children, lack of GH commonly causes hypoglycemia. In children, height below 2 SD of the gender-specific population mean is the most obvious manifestation of GH deficiency. The major consequence of PRL deficiency is failure of lactation. This is usually seen in Sheehan syndrome (postpartum pituitary necrosis with infarction), in which PRL deficiency is invariably accompanied by other hormone deficiencies. In men or nonlactating women, PRL insufficiency does not appear to be associated with any identifiable clinical manifestation. Secondary hypothyroidism resulting from TSH deficiency produces a picture similar to that of primary hypothyroidism with weakness, lethargy, hypothermia, bradycardia, constipation, and delayed relaxation of deep tendon reflexes, although without a goiter. In children, TSH deficiency causes growth retardation [8]. ACTH deficiency leads to adrenocortical insufficiency with decreased production of cortisol and adrenal androgens. The major clinical features of ACTH deficiency are fatigue, anorexia, nausea, and vomiting. Presenting features can be indolent and chronic or acute and life threatening, especially in the setting of acute stressors. Hypoglycemia and life-threatening hypotension may occur. Unlike patients with primary hypoadrenalism, patients with ACTH deficiency do not have hyperpigmentation. Furthermore, the renin-angiotensinaldosterone axis is intact with ACTH deficiency such that hyperkalemia is usually not present. Hyponatremia, however, may be present due to impaired free water excretion [9]. Deficiency of gonadotropins in children results in delayed or partial puberty. If GH secretion is intact, patients have a eunuchoid habitus [tall with arm span . height and lower segment (pubis to ground) . upper segment (pubis to crown)] because fusion of the epiphyses is dependent on sex steroid production. Isolated GnRH deficiency can be associated with midline defects, nerve deafness, color blindness, and anosmia (Kallmann syndrome). When GnRH deficiency occurs after puberty, clinical features in males include loss of libido, impotence, loss of secondary sex characteristics (facial and body hair), and infertility (oligo- or azoospermia). Women may present with dyspareunia from vaginal mucosal atrophy or oligomenorrhea, infertility, or breast atrophy [10].

Investigation of hypopituitarism Routine investigation of patients suspected of having hypopituitarism includes assessment of the visual fields and imaging studies of the pituitary fossa with magnetic resonance imaging (MRI). Initial biochemical screening

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to assess pituitary function includes measurement of TSH and free T4, PRL, IGF-1, LH, FSH, testosterone in males, and morning cortisol. Laboratory investigations should also be guided by clinical features suggestive of excess or deficiency of a particular hormone. The pulsatility and variability of GH and ACTH release limit the utility of random measurements. Ideally, a pooled sample should be obtained (three samples attained at 20-min intervals over 1 h) for an accurate assessment of LH, FSH, PRL, and testosterone. If this is not possible, an early morning 8 9 a.m. sample should be utilized for LH, FSH, PRL, TSH, FT4, and testosterone. The most useful test to diagnose TSH deficiency is serum free T4 because TSH levels can be low, normal, or high since immunoassays measure immune-reactive and not biologically active hormone. The diagnosis of secondary hypothyroidism can be difficult in the face of major illness because the sick euthyroid syndrome can have similar biochemical features. In sick euthyroid syndrome TSH levels are often elevated above the upper reference interval (,10 mU/mL) but may be suppressed. This pattern is often dependent on stage of illness (acute vs resolving). The best assessment of gonadotropin deficiency in premenopausal women is the menstrual history because menses usually cease with inadequate LH and FSH production. A useful index of ovulation is measurement of serum progesterone in the luteal phase because a value $ 5 ng/mL is supportive of ovulation. Men with gonadotropin deficiency have decreased levels of testosterone (total and bioavailable) with low or normal concentrations of FSH and LH. A semen analysis may also be useful especially if fertility is desired. Although TRH and GnRH have previously been used to test for TSH and gonadotropin deficiency, they are no longer commercially available and are used only at highly specialized research institutions. A basal morning cortisol ,3 μg/dL suggests hypoadrenalism [11]. Morning cortisol concentrations above 15 μg/dL make hypoadrenalism unlikely. The most useful laboratory test to confirm a diagnosis of adrenal insufficiency is the ACTH stimulation test. Cosyntropin (ACTH 1 24) is injected intravenously at a dose of 250 μg, and cortisol concentrations are obtained at baseline and 30 60 min following the injection. In normal subjects, the peak cortisol is .20 μg/dL, and the increment over basal is .7 μg/dL. Low-dose ACTH testing (1 μg) has been suggested as a more sensitive test of impaired pituitary reserve [12]. Whilst the use of the high-dose ACTH test is still very common, there is increased use of the low-dose test particularly in the pediatric population [13] A subnormal response to ACTH is consistent with adrenocortical insufficiency but does not distinguish between primary and secondary causes because adrenocortical atrophy may be present with the latter. A plasma ACTH concentration and/or the clinical presentation of the patient will help delineate whether it is primary or secondary. However, a normal response to cosyntropin does not rule out impaired ACTH reserve (partial secondary insufficiency) due to hypothalamic or pituitary disease. In order to rule out

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secondary hypoadrenalism, other stimulation tests are required, such as the metyrapone test or the insulin-induced hypoglycemia challenge. The simplest metyrapone test entails giving metyrapone (30 mg/kg) at night and measuring plasma cortisol and 11-deoxycortisol the next morning. Metyrapone inhibits 11β-hydroxylase, which is the last step in cortisol synthesis by the adrenal glands. Cortisol production is therefore inhibited, resulting in elevated ACTH and 11-deoxycortisol production. Following metyrapone, the morning cortisol should be below 5 μg/dL, and the 11-deoxycortisol levels should exceed 7 μg/ dL [9] in normal subjects. A normal response to metyrapone denotes adequate function of the hypothalamic-pituitary-adrenal axis. Insulin-induced hypoglycemia can also be a useful test to rule out central adrenal insufficiency. Insulin is given intravenously (0.1 0.15 unit/kg) until glucose falls to ,40 mg/dL. Samples for glucose and cortisol are collected every 15 min until hypoglycemia is achieved and for 90 min afterward. A peak cortisol ,20 μg/dL is suggestive of adrenocortical deficiency. Conveniently, this test can simultaneously be used to test for GH deficiency. It should not be performed in elderly patients or in patients with seizure disorders or clinical atherosclerotic cardiovascular disease due to the risk of inducing hypoglycemic seizure or a cardiovascular event. Because GH is secreted in a pulsatile fashion, random GH measurements are useless in the diagnosis of GH deficiency. GHRH, arginine, insulin-induced hypoglycemia, clonidine, levodopa, glucagon, propranolol, sleep, fasting, and exercise have all been used as stimulatory tests for GH. Traditionally, insulininduced hypoglycemia has been the gold standard; however, safety concerns have led investigators to compare the insulin tolerance test (ITT) with other stimuli. For the ITT, samples are obtained at 30-min intervals following adequate hypoglycemia (#40 mg/dL); GH levels should exceed 5 10 ng/mL in normal patients. Combined GHRH and arginine testing has been shown to have equivalent sensitivity and specificity to ITT in both isolated GH deficiency and panhypopituitarism (with a GH cut-off of 5.1 μg/L for the ITT and 4.1 μg/L for the GHRH-arginine test) [14]. Importantly, the ITT has the benefit of testing the entire hypothalamic-pituitary axis, whereas GHRH testing is likely to be normal in patients with hypothalamic causes of GH deficiency. Levels of IGF-1 and its binding protein, IGFBP-3, are dependent on GH secretion and are low with GH deficiency but may overlap into the normal reference range. Low concentrations of serum IGF-1 are confirmatory evidence of GH deficiency provided the patient is not malnourished, is hypothyroid, has hepatic insufficiency or poorly controlled type 1 diabetes mellitus, is taking high-dose oral estrogen, is chronically ill, or is elderly because all of these conditions are known to decrease IGF-1 levels [15]. In patients with multiple preexisting pituitary hormone deficiencies or well-established hypothalamic or pituitary dysfunction, low IGF-1 levels alone may suffice in making the diagnosis of GH deficiency, and dynamic testing is not likely to provide further benefit [7,15]. It is important to interpret IGF-1 concentrations with reference ranges for age and sex.

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When provocative testing is used, a peak GH value of ,5 μg/L is confirmatory of deficiency [7,14,16]. Some experts advocate for at least two provocative testing modalities in order to confirm the diagnosis [7]. The simplest test is to have the child exercise for 10 min and obtain a GH value at baseline and 10 and 20 min later [17]. L-Arginine can be given (0.5 g/kg intravenously over 30 min; maximum dose, 30 g) with GH value obtained at 15-min intervals for 1 h [16]. During the L-dopa test, GH values are obtained at 30-min intervals for 90 min after oral ingestion of L-dopa (125 mg for up to 15 kg of body weight, 250 mg for 15 30 kg, and 500 mg for .30 kg) [16]. The GH response to oral clonidine can also be used (0.1 0.15 mg/m2). However, clonidine can cause postural hypotension and must be used with caution. Glucagon can be given intramuscularly (0.03 mg/kg; maximum, 1 mg) with GH measurement every 30 min for 3 h; however, nausea and vomiting are common side effects. Lastly, GHRH can be given (1 μg/kg) with serial GH measurements obtained over 120 min [17]; however, a normal response to GHRH will not rule out a hypothalamic cause of GH deficiency. During the pubertal growth spurt, rising levels of estrogen (from ovarian production in girls and from conversion of testosterone to estradiol in boys) cause increases in GH pulse amplitude secretion. For this reason, provocative testing for GH deficiency in prepubertal children may be falsely negative in the absence of endogenous estrogen. “Priming” patients for GH testing implies the administration of estrogen or testosterone prior to testing in order to decrease the likelihood of a falsely abnormal (deficient) GH response. Priming should be considered before GH testing procedures in prepubescent children, although a consensus on this practice is lacking [18]. Although one can use estrogen priming in boys and androgen priming in girls, it is most reasonable to use androgens in boys and estrogens in girls. Androgens are most commonly administered as 50 mg of testosterone enanthate intramuscularly 2 3 weeks before the GH test. Ethinyl estradiol, Premarin, and diethylstilbestrol (DES) have been used as estrogen preparations in girls. Patients ,22.7 kg (,50 pounds) receive 20 mg of ethinyl estradiol per dose 18, 12, and 1 h prior to GH testing. Patients .22.7 kg ( . 50 pounds) receive 50 mg per dose on the same schedule. Alternatively, 100 mg/day ethinyl estradiol for 3 days prior to testing can be used. In patients ,13.6 kg (,30 pounds), 2.5 mg of Premarin is used twice daily for 3 days. In patients $ 13.6 ($30 pounds), 5 mg of Premarin are used twice daily for 3 days. For DES, 5 mg/day for 3 days is used prior to GH testing. Alternative protocols for priming that do not involve sex steroids include the use of propranolol in a dose of 0.5 0.75 mg/kg (maximum 40 mg) or, for children ,20 kg, 20 mg 2 h pretest and for children .20 kg, 40 mg 2 h pretest.

Secretory pituitary tumors Pituitary tumors are the most common cause of hypopituitarism, but they may also be secretory tumors that cause unique clinical syndromes of

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hormone excess. Pituitary adenomas constitute about 10% 15% of all intracranial neoplasms. Pituitary tumors may manifest themselves as local spaceoccupying effects (with bitemporal visual field defects), hypopituitarism, hypersecretion of hormones, or as an incidental radiological finding. Investigations (other than assessment of pituitary function) in patients with pituitary tumors include charting of visual fields and imaging studies of the pituitary fossa with computed tomography or MRI. Tumors with a diameter of ,10 mm are referred to as microadenomas, whereas macroadenomas are .10 mm in maximal diameter. Less than 1% of all pituitary tumors are associated with multiple endocrine neoplasia type 1 (MEN1). The presence of other features of MEN1 or a suggestive family history should raise suspicion for MEN1 in patients with primary pituitary tumors. The initial tests should be directed at assaying the hormone whose excess is suspected. Additionally, the possibility of deficiencies of other pituitary hormones should also be considered and relevant testing performed as detailed previously [19,20]. Patterns of excessive secretion of hormones are not uniform and may cycle between normal and excessive secretion due to the pulsatile and episodic secretion.

Prolactinomas The causes of hyperprolactinemia are numerous (Table 2.3) and include interruption of dopaminergic inhibition of lactotrophs (pituitary stalk compression or stalk injury and dopamine antagonists) and autonomous production by lactotrophs (prolactinomas or cosecreting tumors). Primary hypothyroidism can

TABLE 2.3 Causes of hyperprolactinemia. Physiological

Sleep, stress, meals, exercise, pregnancy, lactation, nipple stimulation

Drugs

Antipsychotics, butyrophenones, tricyclic antidepressants, SSRIs, monoamine oxidase inhibitors, opiates, cocaine, metoclopramide, methyldopa, reserpine, verapamil, estrogen, domperidone

Hypothalamic-pituitary stalk lesions

Craniopharyngioma, infiltrative disorders of the hypothalamus, pituitary stalk compression, stalk section after surgery, head injury, or prior irradiation

Pituitary tumors

Prolactinomas, mixed GH- or ACTH- and PRL-secreting adenomas

Miscellaneous

Primary hypothyroidism, chronic renal failure, polycystic ovarian disease, liver disease, spinal cord lesions, macroprolactinemia, seizures, idiopathic

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cause hyperprolactinemia due to elevated TRH. Prolactinomas are the most common secretory tumors of the pituitary and occur eight times more frequently in women than in men. Forty percent of all pituitary tumors are PRL secreting [1]. Cosecretion of GH and prolactin is relatively common, occurring in 25% of secretory pituitary adenomas [21]. Tumor size varies greatly from microadenomas to large invasive macroadenomas with extrasellar extension. Generally, PRL concentrations parallel prolactinoma size [22]. Large nonfunctional pituitary tumors may cause mild elevations in PRL from stalk compression, leading to interruption of dopaminergic inhibition of PRL secretion. However, a mild elevation in PRL in the setting of a pituitary macroadenoma should prompt a dilutional assay for PRL because of the potential for a hook effect artifact when PRL concentrations are excessively high [23]. Hyperprolactinemia causes inhibition of GnRH, LH, FSH, and gonadal steroidogenesis. Hence, female patients may present with amenorrhea and infertility in addition to galactorrhea and the local effects of tumor expansion. Males with prolactinomas may present with impotence, loss of libido, infertility, or gynecomastia from low testosterone concentrations in addition to galactorrhea and the effects of tumor expansion. Rarely, serum PRL may be elevated because of macroprolactinemia in the absence of a pituitary tumor. In this condition, the increase in serum PRL is due to large complexes of PRL and immunoglobulins. This “big PRL” has reduced biological activity, and the clinical consequences, if any, are unclear. The vast majority of patients with macroprolactinemia are asymptomatic; however, some may have a few nonspecific symptoms that may also occur in hyperprolactinemia, thus confounding the clinical differentiation of those with true monomeric increase from those with macroprolactin [24 26]. In suspected macroprolactinemia, laboratory procedures for confirming macroprolactin are recommended [25,27]. This may be done by one of the following methods: polyethylene glycol (PEG) precipitation, gel filtration chromatography, ultrafiltration, and immunoadsorption [28,29]. Basal levels of prolactin are useful with values of .100 μg/L generally associated with the presence of a prolactinoma usually a microadenoma, and .200 μg/L due to a macroadenoma. Prolactin levels generally parallel the tumor size. In patients with microadenomas, other pituitary functions are usually normal; however, patients with macroadenomas should be assessed for hypopituitarism as outlined in previous sections. The most reliable tests for the diagnosis of a prolactinoma are several random measurements of PRL concentrations (preferably with pooled samples). Because the causes of hyperprolactinemia are numerous (pregnancy, drugs, hypothyroidism, chronic renal failure, etc.), a careful history, clinical examination, and routine laboratory tests are essential in evaluation of elevated PRL concentrations. PRL concentrations between the upper limits of normal and 100 ng/mL may be indicative of stalk compression, drugs, prolactinoma, or other causes, whereas PRL concentrations .100 ng/mL are usually caused by a prolactinoma. Concentrations .200 ng/mL are virtually diagnostic of prolactinoma [22]. Provocative tests

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such as TRH, L-dopa, and insulin-induced hypoglycemia have not proven useful in the diagnosis of prolactinomas and are not indicated [22].

Acromegaly Increased secretion of GH prior to epiphyseal closure results in gigantism, whereas in adults, excess GH secretion results in acromegaly; both are usually due to a GH-producing pituitary adenoma. Gigantism is exceptionally rare. Other causes of acromegaly include ectopic GH or GHRH secretion in patients with carcinoid tumors, lymphomas, islet cell tumors, lung carcinomas, or hypothalamic hamartomas. Pituitary adenomas are the most common cause of acromegaly, and .70% are macroadenomas. Approximately 25% of GH-producing pituitary tumors cosecrete GH and PRL. Acral enlargement (skin, subcutaneous tissue, and skeletal overgrowth) is recognized by increasing ring, glove, or shoe size, prominent facial features, enlargement of the mandible, and macroglossia. Other features may include sleep apnea, hyperhidrosis, skin tags, hypertriglyceridemia, headaches, hypertension, impaired glucose tolerance, cardiomyopathy, osteoarthritis, carpal tunnel syndrome, and a propensity for colon polyps. Development of acromegaly can be subtle; hence, the diagnosis is often delayed by many years [30]. Single measurements of GH are not reliable because of the pulsatile nature of GH secretion and because other conditions such as anxiety, acute illness, anorexia nervosa, exercise, diabetes mellitus, cirrhosis, and chronic renal failure can alter GH levels. Serum IGF-1 is elevated and is useful as an initial screening test. In normally nourished persons with normal renal and hepatic function, serum IGF-1 levels reflect the integrated effect of GH at the tissue level and correlate with 24-h GH concentrations. IGF-1 reference ranges must be adjusted for age and sex. Confirmation of the diagnosis of acromegaly should be made by measuring the GH response to a 75-g oral glucose load. In normal subjects, GH levels are suppressed to ,1 μg/L (or ,0.3 μg/L by ultrasensitive assays) at 60 120 min [31], whereas values do not suppress adequately in acromegaly. Ideally, GH concentrations should be based on commonly accepted reference calibrations for recombinant human GH and expressed in mass units, which would permit an accurate diagnosis of acromegaly, even in the context of subtle clinical features. Rarely, there is a paradoxical increase in GH levels during the oral glucose tolerance test (OGTT). The GH level following OGTT can be impaired by aging, female gender, and obesity [32]. GH concentrations cannot suppress in patients with liver disease, renal insufficiency, uncontrolled diabetes, malnutrition, or anorexia, or in those who are pregnant or are receiving estrogens. Once treatment for acromegaly is underway, criteria that constitute an adequate response to therapy include a fasting GH below 5 ng/ mL, adequate suppression of GH following oral glucose (GH ,1 μg/L), and an IGF-1 level within the reference range [30,31,33].

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Other secretory tumors The third most common secretory disorder of the pituitary is Cushing’s disease due to excess ACTH secretion from a basophil adenoma; the clinical features and diagnosis of Cushing’s disease are discussed in the chapter on adrenal disorders (Chapter 3: Thyroid Disease and Laboratory Assessment). Briefly, tests useful in confirming a diagnosis of Cushing’s syndrome include an overnight dexamethasone suppression test, loss of the diurnal variation, and increase in salivary cortisol and urinary free cortisol levels with ACTH levels and petrosal sinus sampling to delineate and confirm Cushing’s disease which is the commonest cause of Cushing’s syndrome. Pituitary tumors secreting glycoprotein hormones (TSH, LH, and FSH) are very uncommon. Also uncommon are α-subunit-producing adenomas. Clinically, TSHsecreting tumors present with symptoms of hyperthyroidism with goiter [34], increased T3 and T4 levels, inappropriately elevated TSH levels that display a blunted response to TRH (unlike thyroid hormone insensitivity), and increased α-subunit concentrations and abnormal TSH response to T3 suppression [35]. The majority of gonadotropin-secreting tumors secrete FSH and α-subunit, but tumors secreting both LH and FSH have been described [9]. They are usually large chromophobe adenomas that present clinically with visual impairment and compressive effects. Most patients with these large tumors have clinical features of hypogonadism, including amenorrhea, impotence, and infertility. In men, testosterone concentrations are usually decreased but may rarely be increased due to increased secretion of bioactive LH.

Posterior pituitary Both antidiuretic hormone (ADH) and oxytocin are synthesized in the cell bodies of neurons in the supraoptic and paraventricular nuclei of the hypothalamus. After synthesis, ADH and its respective carrier protein, neurophysin, are packaged into neurosecretory granules that migrate along the axons to secretory terminals. Large stores of ADH exist in these secretory terminals. Although most secretory terminals are in the posterior pituitary, others are at higher levels in the pituitary stalk and median eminence. The major action of ADH is to allow the urine to become concentrated in the collecting tubules by facilitating passive movement of water along the osmotic gradient from the collecting duct lumen to the renal medullary interstitium. ADH release is mediated by osmotic and nonosmotic stimuli. The major determinant of ADH release is plasma osmolality via osmoreceptors in the hypothalamus. A large decrease in plasma volume also evokes ADH release. Other stimuli of ADH release include stress, nausea, nicotine, and hypoglycemia. Numerous drugs, including selective serotonin reuptake inhibitors (SSRIs), chlorpropamide, vincristine, omeprazole, and angiotensin-converting enzyme

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inhibitors (ACEIs) can cause the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Oxytocin stimulates uterine contraction during parturition and causes milk release from alveoli in lactating women. There are no major clinical syndromes due to the absence or overproduction of oxytocin. The two major disorders of ADH secretion are diabetes insipidus (DI) and SIADH.

Diabetes insipidus DI can result from either failure of ADH secretion (central DI) or failure of the kidneys to respond to ADH (nephrogenic DI). Permanent central DI will usually not develop until 80% of the ADH secretory pathways have been destroyed. Causes of central DI are shown in Table 2.4. Some patients with the idiopathic variety have antibodies to ADH-secreting hypothalamic neurons. Rarely, DI is part of Wolfram syndrome, which also includes diabetes mellitus, optic atrophy, and deafness. The presence of hypopituitarism and/or hyperprolactinemia should prompt a search for a causative lesion with imaging studies and hormone assays. During pregnancy, DI can result from increased placental degradation of ADH. This is termed gestational DI. The essential feature of DI is the elaboration of a large volume of inappropriately dilute urine relative to serum osmolality. The polyuria is of varying severity (3 15 L/day) and leads to hypertonic dehydration (hypernatremia with serum osmolality .295 mOsm/kg). The hyperosmolality in turn stimulates thirst, resulting in polydipsia. DI can be differentiated from psychogenic polydipsia because serum osmolality in the latter is low (#280 mOsm/kg). If the patient with DI has access to water and intact thirst regulation, hypernatremia should not develop. However, if access to water is restricted or the patient has a damaged thirst center, life-threatening hypernatremia will develop.

TABLE 2.4 Causes of central DI. Idiopathic (some idiopathic cases may be autoimmune-mediated.) Familial

Dominant, recessive

Trauma

Head injury, surgery

Tumors

Pituitary tumors, craniopharyngioma, meningioma, germinoma, metastases, granulomas, sarcoidosis, histiocytosis, tuberculosis, inflammatory lymphocytic hypophysitis

Infections

Meningitis, encephalitis

Vascular

Sheehan syndrome, aneurysms, infarction, hemorrhage, pregnancy

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A random urine osmolality that exceeds 750 mOsm/kg excludes DI. The most commonly used provocative test to diagnose this disorder is the water deprivation test. The water deprivation test should not be performed in the presence of hypothyroidism or adrenal insufficiency or if an osmotic diuresis is present, such as uncontrolled diabetes mellitus. Patients are deprived of all fluids until hourly urine osmolalities are constant (,10% difference between consecutive samples) for 3 successive hours. At this point, plasma osmolality and serum ADH are measured, followed by ADH administration. Desmopressin, 1 2 mg subcutaneously or 10 20 μg intranasally, is given, and urine osmolality and urine volume are measured at 60 and 120 min. In normal persons and patients with psychogenic polydipsia, urine osmolality is higher than plasma osmolality at the end of water deprivation and does not increase by more than 10% following ADH administration. Patients with partial DI may have urine osmolality greater than plasma osmolality (300 mOsm) after dehydration, but urine osmolality increase between 10% and 49% following ADH. In severe central DI, urine osmolality is less than plasma osmolality at the end of dehydration and increases by over 50% after ADH. The test should be terminated if body weight falls by more than 3% or serum sodium exceeds 145 mmol/L. Frost et al. described an increased sensitivity of 100% if the cut-off value for urine osmolality was set at 680 mOsmol/kg. Some patients with chronic psychogenic polydipsia may show some impairment in concentrating ability and may not be able to concentrate urine to .750 mOsm/kg [35]. If reliable ADH assays are available, an ADH level should be obtained following dehydration because it may clearly differentiate central from nephrogenic DI when osmolality measurements are inconclusive [36]. In central DI, plasma ADH is subnormal relative to plasma osmolality following dehydration, whereas in nephrogenic DI plasma, ADH is in the upper end of the reference range or elevated. There are several difficulties with ADH measurement particularly related to its short half-life and preanalytical instability. The measurement of the precursor copeptin that is cosecreted with ADH has been described as a possible diagnostic test to assist in the assessment of DI, though this test is not readily accessible and limited data have been available regarding diagnostic sensitivity [35]. However, in a recent study examining over 100 patients with hypotonic polyuria, the measurement of plasma copeptin following hypertonic-saline stimulation showed greater diagnostic accuracy than the water deprivation test [36].

SIADH SIADH is a disorder in which there is increased ADH secretion despite low plasma osmolality. Water retention results in hyponatremia and serum hypoosmolality. Suppression of aldosterone secretion and increased secretion of atrial natriuretic peptide in response to high intravascular volume lead to increases in urinary sodium excretion. The main causes of SIADH are shown

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TABLE 2.5 Major causes of SIADH. Neoplasia

Carcinoma of bronchus, duodenum, pancreas, nasopharynx, ureter, or prostate; leukemia; thymoma; mesothelioma

Pulmonary disorders

Pneumonia, tuberculosis, lung abscess, positive pressure ventilation, chronic obstructive airway disease

Central nervous system disorders

Encephalitis, meningitis, head injury, Guillain Barre´ syndrome, acute intermittent porphyria, brain abscess, brain tumor, subdural hematoma, subarachnoid hemorrhage, vasculitis

Drugs

Chlorpropamide, vincristine, cyclophosphamide, phenothiazines, tricyclic antidepressants, SSRIs, ACEIs, carbamazepine, amiodarone

Other

Acquired immunodeficiency syndrome, strenuous exercise, idiopathic

in Table 2.5. Most patients with SIADH do not have specific clinical features unless the hyponatremia is severe (Na1 ,125 mmol/L); however, mild hyponatremia has been associated with falls, inattention, and gait impairment in elderly populations [37]. Symptoms of moderately severe hyponatremia include anorexia, nausea, vomiting, and headaches. With profound hyponatremia (Na1 ,110) or with rapidly developing hyponatremia, confusion, convulsions, hemiparesis, and coma become evident. The biochemical features that favor the diagnosis of SIADH include hyponatremia, low serum osmolality (,275 mOsm/kg), urine Na1 .40 mmol/L, and inappropriately elevated urine osmolality ( . 100 mOsm/kg) in the setting of low plasma osmolality [38]. Supplemental findings include a serum uric acid of ,4 mg/dL and blood urea nitrogen ,10 mg/dL. Adrenal insufficiency, hypothyroidism, and diuretic use should be excluded before making the diagnosis of SIADH.

Conclusion A comprehensive array of tests are used to assess pituitary function. In some instances, basal hormone levels are adequate for diagnosis. In other instances, evaluation of target organ function (T4 levels, menstrual history, IGF-1 measurement) is more useful than direct measurement of pituitary hormones. Stimulation or suppression tests often provide the best confirmatory evidence of deficiency or excess of pituitary hormones, respectively. The biochemical evaluation of pituitary function detailed in this chapter is summarized in Table 2.6.

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TABLE 2.6 Summary of pituitary hormone assessment. Hormone

Deficiency

Excess issues with measurement

GH

GH response to GHRH, arginine, ITT, exercise, L-dopa I

IGF-1 levels

PRL

PRL

PRL

TSH

T4, TSH

T4, TSH, α-subunit TSH response to TRH

Gonadotropins

Testosterone, estradiol, LH, FSH, menstrual history

LH, FSH, testosterone, α-subunit

ACTH

Cortisol, cosyntropin test, metyrapone test, insulin tolerance test

Urine-free cortisol, overnight or low-dose dexamethasone suppression tests (Chapter 3: Thyroid Disease and Laboratory Assessment)

ADH

Water deprivation test (S and U osmol), ADH levels at maximum dehydration

S and U osmol, S and U Na1

GH response to oral glucose (glucose tolerance test)

S, Serum; U, urine.

References [1] N.M. Gardner-Roehnelt, Update on the management of neuroendocrine tumors: focus on somatostatin antitumor effects, Clin. J. Oncol. Nurs. 16 (1) (2012) 56 64. [2] V. Gounden, Y.D. Rampursat, I. Jialal, Secretory tumors of the pituitary gland: a clinical biochemistry perspective, Clin. Chem. Lab. Med. 57 (2) (2018) 150 164. Available from: https://doi.org/10.1515/cclm-2018-0552. [3] A. Ignacak, M. Kasztelnik, T. Sliwa, R.A. Korbut, K. Rajda, T.J. Guzik, Prolactin not only lactotrophin. A “new” view of the “old” hormone, J. Physiol. Pharmacol. 63 (5) (2012) 435 443. [4] P. Fitzgerald, T.G. Dinan, Prolactin and dopamine: what is the connection? A review article, J. Psychopharmacol. 22 (2_suppl.) (2008) 12 19. [5] F.F. Casanueva, M.E. Molitch, J.A. Schlechte, R. Abs, V. Bonert, M.D. Bronstein, et al., Guidelines of the Pituitary Society for the diagnosis and management of prolactinomas, Clin. Endocrinol. 65 (2006) 265 273. [6] J. Ayuk, M.C. Sheppard, Growth hormone and its disorders, Postgrad. Med. J. 82 (2006) 24 30. [7] M.E. Molitch, D.R. Clemmons, S. Malozowski, G.R. Merriam, S.M. Shalet, M.L. Vance, Evaluation and treatment of adult growth hormone deficiency: an Endocrine Society Clinical Practice Guideline, J. Clin. Endocrinol. Metab. 91 (2006) 1621 1634.

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[8] S. Melmed, J. Jameson, Hypopituitarism, in: J. Jameson, A.S. Fauci, D.L. Kasper, S.L. Hauser, D.L. Longo, J. Loscalzo (Eds.), Harrison’s Principles of Internal Medicine, McGraw-Hill, New York. [9] D.C. Aron, J.W. Findling, J.B. Tyrrell, Hypothalamus and pituitary gland, in: D.G. Gardner, D. Shoback (Eds.), Basic and Clinical Endocrinology, eighth ed., McGraw-Hill, New York, 2007, pp. 101 154. [10] P. Ascoli, F. Cavagnini, Hypopituitarism, Pituitary 9 (4) (2006) 335 342. [11] T. Struja, L. Briner, A. Meier, A. Kutz, E. Mundwiler, A. Huber, et al., Diagnostic accuracy of basal cortisol level to predict adrenal insufficiency in cosyntropin testing: results from an observational cohort study with 804 patients, Endocr. Pract. 23 (8) (2017) 949 961. Available from: https://doi.org/10.4158/ep171861 Or PMID 28614010. [12] L.M. Thaler, L.S. Blevins, The low dose (1 μg) adrenocorticotropin stimulation test in the evaluation of patients with suspected central adrenal insufficiency, J. Clin. Endocrinol. Metab. 83 (1998) 2726 2729. [13] A.S. Cross, E.H. Kemp, A. White, L. Walker, S. Meredith, P. Sachdev, International survey on high and low dose synacthen test assessment of accuracy in preparing low dose synacthen, Clin. Endocrinol. 88 (2018) 744 751. [14] W. Oelkers, Invited commentary: the role of high and low dose corticotrophin tests in the diagnosis of secondary adrenal insufficiency, Eur. J. Endocrinol. 139 (1998) 567 570. [15] M.L. Hartman, B.J. Crowe, B.M.K. Biller, K.Y. Ho, D.R. Clemmons, J.J. Chipman, Which patients do not require a GH stimulation test for the diagnosis of adult GH deficiency? J. Clin. Endocrinol. Metab. 87 (2002) 477 485. [16] D.M. Styne, Pediatric Endocrinology, Lippincott Williams & Wilkins, Philadelphia, PA, 2004, pp. 70 71. [17] E.O. Reiter, R.G. Rosenfeld, Normal and aberrant growth, in: P.R. Larsen, H.M. Kronenberg, S. Melmed, K.S. Polonsky (Eds.), Williams Textbook of Endocrinology, tenth ed., Elsevier, Philadelphia, PA, 2003, pp. 1003 1014. [18] AACE Growth Hormone Task Force, American Association of Clinical Endocrinologists medical guidelines for clinical practice for growth hormone use, Endocr. Pract. 9 (2003) 65 76. [19] B.R. Javorsky, D.C. Aron, J.W. Findling, J.B. Tyrell, Hypothalamus and the pituitary gland, in: D.G. Gardner, D. Shoback (Eds.), Greenspan’s Basic and Clinical Endocrinology, tenth ed., McGraw-Hill Publishers, 2018, pp. 69 118. [20] C.J. McCabe, J.S. Khaira, K. Boelaert, A.P. Heaney, L.A. Tannahill, S. Hussain, et al., Expression of pituitary tumor transforming gene (PTTG) and fibroblast growth factor-2 (FGF-2) in human pituitary adenomas: relationships to clinical tumor behaviour, Clin. Endocrinol. 58 (2003) 141 150. [21] L. Katznelson, E.R. Laws Jr, S. Melmed, M.E. Molitch, M.H. Murad, A. Utz, et al., Acromegaly: an Endocrine Society Clinical Practice guideline, J. Clin. Endocrinol. Metab. 99 (2014) 3933 3951. [22] J.A. Schlechte, Prolactinoma, N. Engl. J. Med. 349 (2003) 2035 2041. [23] T.W. Frieze, D.P. Mong, M.K. Koops, “Hook effect” in prolactinomas: case report and review of the literature, Endocr. Pract. 8 (2002) 296 303. [24] A. Glezer, M. Bronstein, Endotext [Internet], in: L.J. De Groot, G. Chrousos, K. Dungan, K.R. Feingold, A. Grossman, J.M. Hershman, et al. (Eds.), Hyperprolactinemia, MDText. com, Inc., South Dartmouth, MA, 2000. ,https://www.ncbi.nlm.nih.gov/books/ NBK278984/..

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[25] G. Lippi, M. Plebani, Macroprolactin: searching for a needle in a haystack? Clin. Chem. Lab. Med. 54 (2016) 519 522. [26] L. Kavanagh, J. McKenna, M. Fahie-Wilson, J. Gibney, T.P. Smith, Specificity and clinical utility of methods for the detection of macroprolactin, Clin. Chem. 52 (2006) 1366 1372. [27] V. Gounden, I. Jialal, Hypopituitarism (panhypopituitarism), StatPearls [Internet], StatPearls Publishing, Treasure Island, FL, 2018. ,https://www.ncbi.nlm.nih.gov/books/ NBK470414/.. [28] C.R. McCudden, J.L. Sharpless, D.G. Grenache, Comparison of multiple methods for identification of hyperprolactinemia in the presence of macroprolactin, Clin. Chim. Acta 411 (2010) 155 160. [29] J. Barth, C. Lippiatt, S. Gibbons, et al., Observational studies on macroprolactin in a routine clinical laboratory, Clin. Chem. Lab. Med. 56 (8) (2018) 1259 1262. Available from: https://doi.org/10.1515/cclm-2018-0074. [30] AACE Acromegaly Guidelines Task Force, American Association of Clinical Endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of acromegaly, Endocr. Pract. 10 (2004) 213 225. [31] S. Melmed, Acromegaly, N. Engl. J. Med. 355 (2006) 2558 2573. [32] A.P. Heaney, Pituitary tumor pathogenesis, Br. Med. Bull. 75 (2005) 81 97. [33] C.L. Ronchi, M. Arosio, E. Rizzo, A.G. Lania, P. Beck-Peccoz, A. Spada, Adequacy of current postglucose GH nadir limit (,1 microg/L) to define long-lasting remission of acromegalic disease, Clin. Endocrinol. 66 (2007) 538 542. [34] P. Beck-Peccoz, L. Persani, D. Mannavola, I. Campi, TSH-secreting adenomas, Best. Pract. Res. Clin. Endocrinol. Metab. 23 (5) (2009) 597 606. [35] M.D. Frost, et al., The water deprivation test and a potential role for the arginine vasopressin precursor copeptin to differentiate diabetes insipidus from primary polydipsia, Endocr. Connect. 4 (2) (2015) 86 91. [36] W. Fenske, J. Refardt, I. Chifu, S. Ingeborg, et al., A co-peptin based approach in the diagnosis of diabetes insipidus, N. Engl. J. Med. 379 (2018) 428 439. [37] B. Renneboog, W. Musch, X. Vandemergel, M.U. Manto, G. Decaux, Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits, Am. J. Med. 119 (71) (2006) e1 e8. [38] D.H. Ellison, T. Berl, The syndrome of inappropriate antidiuresis, N. Engl. J. Med. 356 (20) (2007) 2064 2072.

Suggested reading D.C. Aron, J.B. Tyrrell, C.B. Wilson, Pituitary tumors: current concepts in diagnosis and management, West. J. Med. 162 (1995) 340 352. T.G. Barrett, S.E. Bundey, Wolfram (DIDMOAD) syndrome, J. Med. Genet. 34 (1997) 838 841. C.W. Burke, The pituitary megatest: outdated? Clin. Endocrinol. 36 (1992) 133 134. R.N. Clayton, Short Synacthen test versus insulin stress test for assessment of the hypothalmopituitary-adrenal axis, Clin. Endocrinol. 44 (1996) 147 149. G. Decaux, Is asymptomatic hyponatremia really asymptomatic? Am. J. Med. 119 (2006) S79 S82. R. Dexter, Hypopituitarism, in: K. Becker (Ed.), Principles and Practice of Endocrinology and Metabolism, second ed., JB Lippincott, Philadelphia, PA, 1995, pp. 169 180.

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M.N. Fahie-Wilson, R. John, A.R. Ellis, Macroprolactin; high molecular mass forms of circulating prolactin, Ann. Clin. Biochem. 42 (2005) 175 192. L.A. Frohman, Diseases of anterior pituitary, in: P. Felig, J.D. Baxter, L.A. Frohman (Eds.), Endocrinology and Metabolism, third ed., McGraw-Hill, New York, 1995, pp. 289 383. I. Jialal, R. Norman, C. Naidoo, Pituitary function in Sheehan’s syndrome, Obstet. Gynecol. 63 (1984) 15 19. I. Jialal, M.A.K. Omar, B.C. Nathoo, S. Mahabeer, Disorders of the hypothalamus and pituitary gland, SA J. Cont. Med. Educ. 5 (1987) 25 33. J.O. Jorgensen, E. Vestergaard, L. Gormse, N. Jessen, H. Norrelund, J.S. Christiansen, et al., Metabolic consequences of GH deficiency, J. Endocrinol. Invest. 28 (Suppl. 5) (2005) 47 51. E.F. Kozyra, R.S. Wax, L.D. Burry, Can 1 μg of cosyntropin be used to evaluate adrenal insufficiency in critically ill patients? Ann. Pharmacother. 39 (2005) 691 698. A. Krishna, L. Phillips, Management of acromegaly: a review, Am. J. Med. Sci. 308 (1994) 370 375. S.W.J. Lamberts, W.W. de Herder, A.J. van der Lely, Pituitary insufficiency, Lancet 352 (1998) 127 134. A. Levy, S.L. Lightman, Diagnosis and management of pituitary tumors, BMJ 308 (1994) 1087 1091. A.N. Makaryus, S.I. McFarlane, Diabetes insipidus: diagnosis and treatment of a complex disease, Cleve Clin. J. Med. 73 (2006) 65 71. H.J. Milionis, G.L. Liamis, M.S. Elisaf, The hyponatremic patient: a systematic approach to laboratory diagnosis, Can. Med. Assoc. J. 166 (2002) 1056 1062. W. Oelkers, Adrenal insufficiency, N. Engl. J. Med. 335 (1996) 1206 1212. A. Rai, A. Whaley-Connell, S. McFarlane, J.R. Sowers, Hyponatremia, arginine vasopressin dysregulation, and vasopressin receptor antagonism, Am. J. Nephrol. 26 (2006) 579 589. S. Rasmuson, T. Olsson, E. Hagg, A low dose ACTH test to assess the function of the hypothalamic-pituitary-adrenal axis, Clin. Endocrinol. 44 (1996) 151 156. G.L. Robertson, Posterior pituitary, in: P. Felig, J.D. Baxter, L.A. Frohman (Eds.), Endocrinology and Metabolism, third ed., McGraw-Hill, New York, 1995, pp. 385 432. M. Samuels, E.C. Ridgway, Glycoprotein-secreting pituitary adenomas, Baillieres Clin. Endocrinol. Metab. 9 (1995) 337 358. O. Serri, C.L. Chik, E. Ur, S. Ezzat, Diagnosis and management of hyperprolactinemia, Can. Med. Assoc. J. 169 (2003) 575 581. M.L. Vance, Hypopituitarism, N. Engl. J. Med. 330 (1994) 1651 1661. J.G. Verbalis, Disorders of body water and homeostasis, Best. Pract. Res. Clin. Endocrinol. Metab. 17 (2003) 471 503. A.A. Toogood, P.M. Stewart, Hypopituitarism: clinical features, diagnosis, and management, Endocrinol. Metab. Clin. North. Am. 37 (1) (2008) 235 261. Available from: https://doi. org/10.1016/j.ecl.2007.10.004. M.E. Molitch, Disorders of prolactin secretion, Endocrinol. Metab. Clin. North. Am. 30 (3) (2001) 585 610. D.S. Ross, Serum thyroid-stimulating hormone measurement for assessment of thyroid function and disease, Endocrinol. Metab. Clin. North. Am. 30 (2) (2001) 245 264. D. Ozdemir, S. Dagdelen, T. Erbas, Endocrine involvement in systemic amyloidosis, Endocr. Pract. 16 (6) (2010) 1056 1063.

Chapter 3

Thyroid disease and laboratory assessment Sridevi Devaraj and Emily Garnett Division of Clinical Chemistry, Texas Children’s Hospital, Houston, TX, United States

The thyroid gland The thyroid gland is one of the largest endocrine glands in the body and is located immediately below the larynx and anterior to the upper part of the trachea. It consists of two lateral lobes connected by a narrow band of tissue, called the isthmus. The isthmus usually overlies the region from the second to the fourth cartilages. The lobes of the thyroid contain many hollow, spherical structures referred to as follicles, the functional units of the thyroid gland. Each follicle is filled with a thick substance called the colloid. The major constituent of the colloid is a large glycoprotein called thyroglobulin (Tg). Unlike other endocrine glands, which secrete their hormones once they are produced, the thyroid gland stores considerable amounts of thyroid hormones in the colloid until they are required for the body. The thyroid gland secretes two major hormones, which are required for the normal operation of a variety of physiologic processes affecting virtually every organ system in the body. Regulation of thyroid hormone secretion is tightly controlled by the hypothalamicpituitarythyroid axis.

Physiology of the thyroidpituitaryhypothalamic axis The thyroid gland synthesizes the two hormones, thyroxine (T4) and 3,5,30 triiodothyronine (T3). The thyroid cells extract iodine from the circulation. In the thyroid cells, the iodine is oxidized in the presence of H2O2 and thyroid peroxidase (TPO) and immediately bound to Tg. This iodinated protein moves to the apex of the thyroid cell where tyrosine is iodinated into monoand diiodotyrosine. At the apex of the thyroid cell, the mono-iodotyrosine and diiodotyrosine molecules are coupled to form T3 and T4, respectively. The synthesis of T4 and T3 takes place on the surface of Tg. Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00003-0 © 2021 Elsevier Inc. All rights reserved.

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These hormones are then stored in the lumen of the thyroid follicle. During secretion, the intrafollicular contents are incorporated by endocytosis, then hydrolyzed, and T4, T3, and Tg molecules are secreted into circulation. All these processes (the uptake of iodine, oxidation, hormone synthesis, and secretion of the thyroid hormones) are stimulated by thyroid-stimulating hormone (TSH), which is secreted from the anterior pituitary gland (Fig. 3.1) in response to thyrotropin-releasing hormone (TRH) release from the hypothalamus. Virtually every aspect of thyroid hormone synthesis and secretion is stimulated by TSH. In addition to regulating thyroid hormone secretion, it maintains structural integrity of the gland. When TSH is absent, the thyroid gland atrophies, and sustained TSH stimulation results in increased follicular cell size (hypertrophy) and number (hyperplasia). When serum thyroid hormone concentrations decline, TRH is released and circulates to the anterior pituitary to stimulate secretion of TSH, and release of TSH by the pituitary stimulates the thyroid gland to produce thyroid hormones and secrete them into circulation. Conversely, circulating T3 and T4 inhibit TSH secretion, thereby decreasing subsequent thyroid hormone synthesis and secretion. Peripheral tissues, particularly liver and kidney, play an important role in storage, metabolism, and production of active thyroid hormone (T3). In these organs, an enzyme, deiodinase, converts T4 to T3. Eighty percent of T3 in circulation is derived through this peripheral mechanism, whereas the rest of the

1000 1000 10 4.0

TSH reference range

TSH mIU/L 0.4 0.1

>x100 x2

0.01 Undetectable Hypothyroid

Free T4

FT4 reference range

Hyperthyroid 23 pmol/L

0.7

1.8 ng/dL

FIGURE 3.1 The relationship between serum TSH and FT4 concentrations in individuals with stable thyroid status and normal hypothalamicpituitary function.

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T3 is secreted directly from the thyroid gland. The liver also synthesizes three proteins that bind thyroid hormones in circulation: thyroid-binding globulin (TBG), transthyretin (TTR; previously called thyroid-binding prealbumin), and albumin. In addition to intrathyroidal stores, the extrathyroidal tissues also store thyroid hormone with a volume of distribution of about 10 L. T4 is the principal hormone secreted by the thyroid gland. All the T4 in the circulation is derived from thyroidal secretion. In contrast, only about 20% of circulating T3 is of thyroidal origin. Most of the T3 in blood is produced enzymatically in nonthyroidal tissues by 50 -monodeiodination of T4. In fact, T4 appears to function as a prohormone for the production of the more biologically active form of thyroid hormone, T3. In the circulation, most (B99.98%) of the T4 is bound to specific plasma proteins, TBG (60%75%), TTR/prealbumin (TBPA) (15%30%), and albumin (B10%). Approximately 99.7% of T3 in the circulation is bound to plasma proteins, specifically TBG. This represents a 10-fold weaker protein binding than seen for T4. Thus only 0.02% of T4 and 0.3% of T3 are free. Protein-bound thyroid hormones do not enter cells and are thus considered to be biologically inert and function as storage reservoirs for circulating thyroid hormone. In contrast, the minute-free hormone fractions readily enter cells and exert their biological effects. In the pituitary, the negative feedback of thyroid hormone on TSH secretion is mediated primarily by T3 that is produced at the site from the free T4 (FT4) entering the thyrotroph cells. A dynamic equilibrium exists in circulation between the free and bound forms. The tissue activity of the thyroid hormone is provided chiefly by the free hormones, although some bound thyroid hormone is also available to tissues for utilization. In most of the tissues, the circulating free T3 (FT3) is responsible for the biological effect of the thyroid hormone. Some tissues, however, utilize circulating T4 as well. The circulating FT4 is converted in the pituitary and probably brain tissues by local deiodinase type II into intracellular T3. This T3 produced within the cells augments the biological effect of the FT3 derived from circulation. The cellular effects of the thyroid hormones are produced through T3 receptors. Two types of thyroid hormone receptors (TRs) have been identified, each with two isoforms: Thyroid receptor (TR) α-I, TR α-II, TR β-I, and TR β-II. Although α-I and the β isoforms are utilized by T3 to produce the thyroid hormone effect, the α-II isoform may antagonize the T3 function. This T3 receptor is unique in the superfamily in that it remains attached to chromatin in the absence of T3 and inhibits the transcription of genes stimulated by T3. Although in most of the tissues, the ligand-bound receptor bound to thyroid hormone response elements (TRE) increases the transcription of T3-responsive genes, in the pituitary gland, T3-bound receptor bound to TRE will suppress the production of TSH. Roughly 90 μg of T4, 30 μg of T3, and 4750 mmol of reverse T3 (rT3) are produced daily. Both the thyroid gland and extrathyroidal tissues are

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involved in T3 formation. The secretion of the daily amounts of hormones by the thyroid is fairly constant and is stimulated by the hypothalamicpituitary axis but inhibited by circulating FT4 and FT3. TSH is secreted episodically. The hypothalamic hormone TRH stimulates the synthesis and secretion of TSH by the pituitary gland. Somatostatin and dopamine dampen the secretion of TSH. Environmental influences can also affect the hypothalamicpituitary axis. For example, the exposure to cold immediately after birth results in a sudden rise in TSH secretion. The hypothalamicpituitary axis attempts to maintain a constant supply of thyroid hormones to peripheral tissues. The circulating free thyroid hormones T4 and T3 exercise an inhibitory influence on the secretion of TSH and TRH. Thus a regulated hypothalamic-pituitary-thyroid-peripheral tissue axis maintains a fairly constant circulating free thyroid hormone blood level. When the free hormone levels decline, the TSH increases, acting to normalize the circulating FT4 and FT3 concentrations. On the other hand, when the FT4 and FT3 concentrations increase, the TSH secretion is inhibited.

Metabolism Thyroid hormones are metabolized by a number of processes including deiodination, sulfation, conjugation, and side-chain cleavage. The major route of metabolism is through deiodination. T4 is metabolized chiefly by deiodinases that remove iodine atoms sequentially. Ultimately, thyronine with no iodine atoms is produced. The removal of iodine from the outer ring as the first step produces T3, the active hormone. The enzyme deiodinase I is present in the liver and kidney. It has an active site for selenium and at pH 6.57.5 removes the iodine from the outer ring, producing T3. A similar enzyme present in the brain (deiodinase II) also produces T3 by removing iodine from the outer ring. It does not require selenium for its activity and is active at pH 6.57.5. It produces T3 in the brain and pituitary gland. The inner ring deiodination by deiodinase I at pH 8.08.5 and by deiodinase III (skin, brain, and placenta) produces rT3, which is inactive. The subsequent deiodination of the T3 and rT3 produces inactive molecules. The production of T3 by deiodinase I can be decreased in diseases of the liver and kidney, systemic diseases, as well as by certain drugs. Many bodily functions are influenced by thyroid hormones. Although thyroid hormones do not target a specific site, they affect nearly every tissue in the body, causing a variety of physiologic responses. One of the most significant effects of the thyroid hormones is on the basal metabolic rate, through oxygen consumption and heat production. Through the action of thyroid hormones, cellular oxidation is induced in most tissues, resulting in increased oxygen consumption and heat production. The thyroid hormones also stimulate carbohydrate metabolism. Use of glucose by cells is increased as is the rate of glucose absorption by the gastrointestinal tract. Calcium,

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water balance, and liver function are also influenced by thyroid hormones. In hypothyroidism, hypercalcemia occurs and glucuronic acid conjugation by the liver is impaired. Also, water is retained in extracellular compartments. Furthermore, because thyroid hormones stimulate carbohydrate metabolism, they are necessary for lipogenesis in liver and fat cells. Mobilization of fatty acids from fat tissue occurs in presence of thyroid hormones, which increases plasma free fatty acids (FFAs). Thyroid hormones also influence cholesterol levels.

Signs and symptoms A number of signs and symptoms are well-established manifestations of thyroid dysfunction. Risk factors identifiable in personal history include previous thyroid dysfunction, goiter, surgery or radiotherapy affecting the thyroid gland, diabetes, vitiligo, pernicious anemia, leukotrichia, medications such as lithium, or iodine-containing compounds. Risk factors identifiable in family history include thyroid disease, pernicious anemia, diabetes, and primary adrenal insufficiency. Abnormal results in commonly obtained laboratory tests are an indication of thyroid dysfunction. In hypothyroidism, there may be accompanying hypercholesterolemia, hyponatremia, anemia, creatine kinase and lactate dehydrogenase elevations, and hyperprolactinemia, and for hyperthyroidism, accompanying features may include hypercalcemia, alkaline phosphatase elevation, and hepatocellular enzyme elevation. The above clinical/laboratory findings warrant thyroid function testing, especially if they are sustained for 2 weeks or more, occur in combination, have not been present previously during documented euthyroidism, or occur in individuals with increased risk of thyroid disease.

Screening for thyroid dysfunction Thyroid dysfunction meets many criteria for a condition that completely justifies population screening: The prevalence of various forms of thyroid dysfunction are substantial; overt hypothyroidism and hyperthyroidism have well-established clinical consequences; the serum TSH assay is an accurate, widely available, safe, and relatively inexpensive test, diagnostic for all common forms of hypo- and hyperthyroidism; and effective therapies for both hypo- and hyperthyroidism are available. Screening of all newborn children for hypothyroidism is well accepted. In addition, serum TSH measurement in adults every 5 years has been shown in decision analysis to be cost-effective, especially in women and older persons. The American Thyroid Association guidelines recommend that all adults have serum TSH measured beginning at age 35 years and every 5 years thereafter. More frequent screening may be appropriate in individuals

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at high risk of developing thyroid dysfunction. With the advent of sensitive TSH measurements, serum TSH measurement is the single most reliable test to diagnose all common forms of hypo- and hyperthyroidism, especially in the ambulatory setting.

Variables affecting testing of thyroid function For practical purposes, variables such as age, gender, race, season, phase of menstrual cycle, cigarette smoking, exercise, fasting, or phlebotomy- induced stasis have minor effects on the reference intervals for thyroid tests in ambulatory adults. Physiological variables include the serum TSH/FT4 relationship, age, pregnancy, and biologic variation.

The serum TSH/FT4 relationship With the advent of ultrasensitive TSH assays, the strategy for screening for thyroid dysfunction is a TSH-first algorithm with testing for FT4 when the TSH is above or below range. It is important to understand the normal relationship between serum levels of FT4 and TSH when interpreting thyroid tests. An intact hypothalamicpituitary axis is a prerequisite for TSH measurements to be used to determine primary thyroid dysfunction. When hypothalamicpituitary function is normal, a log/linear inverse relationship between serum TSH and FT4 concentrations is produced by negative feedback inhibition of pituitary TSH secretion by thyroid hormones. Thus it follows that high TSH and low FT4 are characteristic of primary hypothyroidism, and low TSH and high FT4 are characteristic of primary hyperthyroidism. In fact, now that the sensitivity and specificity of TSH assays have improved, it is recognized that the indirect approach (serum TSH measurement) offers better sensitivity for detecting thyroid dysfunction than does FT4 testing. There are two reasons for using a TSH-centered strategy for ambulatory patients. As shown in Fig. 3.1, serum TSH and FT4 concentrations exhibit an inverse log/linear relationship such that small alterations in FT4 will produce a much larger response in serum TSH. Furthermore, twin studies have demonstrated that each individual has a genetically determined FT4 set-point. Any mild FT4 excess or deficiency will be sensed by the pituitary, relative to that individual’s FT4 set-point, and cause an amplified, inverse response in TSH secretion. It follows that in the early stages of developing thyroid dysfunction, a serum TSH abnormality will precede the development of an abnormal FT4 because TSH responds exponentially to subtle FT4 changes that are within the population reference limits. This is because population reference limits are broad, reflecting the different FT4 set-points of the individual members of the cohort of normal subjects studied.

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Serum FT4 measurement is a more reliable indicator of thyroid status than TSH when thyroid status is unstable, such as during the first 23 months of treatment for hypo- or hyperthyroidism. Patients with chronic, severe hypothyroidism may develop pituitary thyrotroph hyperplasia that can mimic a pituitary adenoma but resolves after several months of levothyroxine (L-T4) replacement therapy. In hypothyroid patients suspected of intermittent or noncompliance with L-T4 replacement therapy, both TSH and FT4 should be used for monitoring. Noncompliant patients may exhibit discordant serum TSH and FT4 values (high TSH/high FT4) because of persistent disequilibrium between FT4 and TSH. Currently, measurement of the serum TSH concentration is the most reliable indicator of thyroid status at the tissue level. Studies of mild (subclinical) thyroid hormone excess or deficiency (abnormal TSH/normal range FT4 and FT3) find abnormalities in markers of thyroid hormone action in a variety of tissues (heart, brain, bone, liver, and kidney). These abnormalities typically reverse when treatment to normalize serum TSH is initiated. It is important to recognize the clinical situations where serum TSH or FT4 levels may be diagnostically misleading. These include abnormalities in hypothalamic or pituitary function, including TSH-producing pituitary tumors. Also, serum TSH values are diagnostically misleading during transition periods of unstable thyroid status, such as occurs in the early phase of treating hyper- or hypothyroidism or changing the dose of L-T4. Specifically, it takes 612 weeks for pituitary TSH secretion to reequilibrate to the new thyroid hormone status. These periods of unstable thyroid status may also occur following an episode of thyroiditis, including postpartum thyroiditis when discordant TSH and FT4 values may also be encountered. Drugs that influence pituitary TSH secretion (i.e., dopamine and glucocorticoids) or thyroid hormone binding to plasma proteins may also cause discordant TSH values.

Effects of chronological age on thyroid test reference ranges Despite studies showing minor differences between older and younger subjects, adult age-adjusted reference ranges for thyroid hormones and TSH are not deemed necessary. In children, the hypothalamicpituitarythyroid axis undergoes progressive maturation and modulation. Specifically, there is a continuous decrease in the TSH/FT4 ratio from the time of midgestation until after the completion of puberty. As a result, higher TSH concentrations are typically seen in children. This maturation process dictates the use of agespecific reference limits. Lower serum total and FT3 concentrations (measured by most methods) are seen with pregnancy, during the neonatal period, in the elderly, and during caloric deprivation. Furthermore, higher total and FT3 concentrations are typically seen in euthyroid children. This suggests that the upper T3 limit for young patients (younger than 20 years of age) should be established as a

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gradient: between 6.7 pmol/L (0.44 ng/dL) for adults; up to 8.3 pmol/L (0.54 ng/dL) for children under 3 years of age.

Pregnancy Pregnancy is a physiological state that affects the relationship among thyroid hormones in a complex, but predictable, fashion. During pregnancy, estrogen production increases progressively elevating the mean TBG concentration. TBG levels plateau at 23 times the prepregnancy level by 20 weeks of gestation. This rise in TBG results in a shift in the total T4 (TT4) and total T3 (TT3) reference range to approximately 1.5 times the nonpregnant level by 16 weeks of gestation. These changes are associated with a fall in serum TSH during the first trimester, such that subnormal serum TSH may be seen in approximately 20% of normal pregnancies. This decrease in TSH is attributed to the thyroidstimulating activity of human chorionic gonadotropin (hCG) that has structural homology with pituitary TSH. The peak rise in hCG and the nadir in serum TSH occur together at about 1012 weeks of gestation. In approximately 10% of such cases (i.e., 2% of all pregnancies), the increase in FT4 reaches supranormal values and, when prolonged, may lead to a syndrome entitled “gestational transient thyrotoxicosis” that is characterized by more or less pronounced symptoms and signs of thyrotoxicosis. This condition is frequently associated with hyperemesis in the first trimester of pregnancy. The fall in TSH during the first trimester of pregnancy is associated with a modest increase in FT4. Thereafter in the second and third trimesters, there is now consensus that serum FT4 and FT3 concentrations decrease to approximately 20%40% below the normal mean, a decrease in free hormone that is further amplified when the iodide nutrition status of the mother is restricted or deficient. In some patients, FT4 may fall below the lower reference limit for nonpregnant patients. Serum Tg concentrations typically rise during normal pregnancy. Patients with differentiated thyroid carcinomas (DTCs) with thyroid tissue still present typically show a twofold rise in serum Tg with a return to baseline by 68 weeks postpartum. Mounting evidence suggests that hypothyroidism during early pregnancy has a detrimental effect on fetal outcome (fetal wastage and lower infant IQ). Thus prepregnancy or first-trimester screening for thyroid dysfunction using serum TSH and thyroperoxidase antibody (TPOAb) measurements is important both for detecting mild thyroid insufficiency (TSH .4.0 mIU/L) and for assessing risk for postpartum thyroiditis (elevated TPOAb). Initiation of L-T4 therapy should be considered if the serum TSH level is .4.0 mIU/L in the first trimester of pregnancy and if elevated serum TPOAb concentrations are identified ( as per the recent AACE guidelines for hypothyroidism). A high serum TPOAb concentration during the first trimester is a risk factor for postpartum thyroiditis. Serum TSH should be used to assess thyroid status during each trimester when pregnant patients are taking L-T4 therapy, with more frequent measurement if L-T4 dosage is changed.

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Pathological variables Medications Medications can cause both in vivo and in vitro effects on thyroid tests. Serum TSH concentration is affected less by medications than thyroid hormone concentrations. For example, estrogen-induced TBG elevations raise serum TT4 levels but do not affect the serum TSH concentration because pituitary TSH secretion is controlled by the FT4 independent of binding protein effects. Glucocorticoids in large doses can lower the serum T3 level and inhibit TSH secretion. Dopamine also inhibits TSH secretion and may even mask the raised TSH level of primary hypothyroidism in sick, hospitalized patients. Propranolol is sometimes used to treat manifestations of thyrotoxicosis and has an inhibitory effect on T4 to T3 conversion. Iodide, contained in solutions used to sterilize the skin and radiopaque dyes and contrast media used in coronary angiography and computed tomography scans, can cause both hyper- and hypothyroidism in susceptible individuals. Amiodarone used to treat heart patients has complex effects on thyroid gland function that can induce either hypothyroidism or hyperthyroidism in susceptible patients with positive TPOAb. Lithium can cause hypo- or hyperthyroidism in as many as 10% of lithium-treated patients, especially those with a positive TPOAb titer. Some therapeutic and diagnostic agents (i.e., phenytoin, carbamazepine, or furosemide/frusemide) may competitively inhibit thyroid hormone binding to serum proteins in the specimen and acutely increase FT4, resulting in a reduction in serum TT4 values through a feedback mechanism. Intravenous heparin administration, through in vitro stimulation of lipoprotein lipase, can liberate FFAs, which inhibit T4 binding to serum proteins and falsely elevate FT4.

Nonthyroidal illness Patients who are seriously ill often have abnormalities in their thyroid tests but usually do not have thyroid dysfunction. These abnormalities are seen with both acute and chronic critical illnesses and thought to arise from a maladjusted central inhibition of hypothalamic-releasing hormones, including TRH. The terms nonthyroidal illness (NTI) as well as “euthyroid sick” and “low-T4 syndrome” are often used to describe this subset of patients. As shown in Fig. 3.2, the spectrum of changes in thyroid tests relates both to the severity and stage of illness, as well as to technical factors that affect the methods and in some cases the medications given to these patients. Most hospitalized patients have low serum TT3 and FT3 concentrations, as measured by most methods. As the severity of the illness increases, serum TT4 typically falls because of a disruption of binding protein affinities, possibly caused by T4-binding inhibitors in the circulation. If a low TT4 is not associated with an elevated serum TSH ( . 20 mIU/L), and the patient is not

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Recovery

Illness

Well

Well

SEVERITY

Mortality rT3 FT4 Normal range

Normal range

TSH

Low T3 Low T3 –T4

Low T3

FIGURE 3.2 Changes in thyroid tests during the course of NTI.

profoundly sick, a diagnosis of central hypothyroidism secondary to pituitary or hypothalamic deficiency should be considered. TSH in the absence of dopamine or glucocorticoid administration is the more reliable test for NTI patients. Estimates of free or total T4 in NTI should be interpreted with caution in conjunction with a serum TSH measurement. Both T4 1 TSH measurements are the most reliable way for distinguishing true primary thyroid dysfunction (concordant T4/TSH abnormalities) from transient abnormalities resulting from NTI per se (discordant T4/TSH abnormalities). An abnormal FT4 result in a hospitalized patient should be confirmed by a reflex TT4 measurement. If both TT4 and FT4 are abnormal (in the same direction), a thyroid condition may be present. When TT4 and FT4 are discordant, the FT4 abnormality is unlikely due to thyroid dysfunction and more likely a result of the illness, medications, or an artifact of the test. A raised total or FT3 is a useful indicator of hyperthyroidism in a hospitalized patient, but a normal or low T3 does not rule it out. rT3 testing is rarely helpful in the hospital setting because paradoxically normal or low values can result from impaired renal function and low binding protein concentrations. Furthermore, the test is not readily available in most laboratories. It is clear that the diagnosis and treatment of thyroid dysfunction in the presence of a severe NTI are not simple and are best done with the help of an endocrinologist. Although the diagnostic specificity of TSH is reduced in the presence of somatic illnesses, a detectable serum TSH value in the 0.0220 mIU/L range, when measured by an assay with a functional sensitivity # 0.02 mIU/L, usually rules out significant thyroid dysfunction, provided that hypothalamicpituitary function is intact and the patient is not receiving

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medications that affect pituitary TSH secretion. However, in general, it is best to avoid routine thyroid testing in hospitalized patients if at all possible.

Specimen variables A few studies have examined the effects of storing blood samples on serum concentrations of total and free thyroid hormones, TSH and Tg. In general, these studies suggest that thyroid hormones are relatively stable whether stored at room temperature, refrigerated, or frozen. Hemolysis, lipemia, and hyperbilirubinemia do not produce significant interference in immunoassays, in general. However, FFAs can displace T4 from serum-binding proteins, which may partly explain the low TT4 values often seen in NTI. Heterophilic antibodies may be encountered in patient sera. Heterophilic antibodies fall into two classes. They can be human anti-mouse antibodies (HAMAs) or other specific human anti-animal immunoglobulins (HAAAs) such as human anti-rabbit antibodies. Either HAMAs or HAAAs affects immunometric assays (IMAs) more than competitive immunoassays by forming a bridge between the capture and signal antibodies, thereby creating a false signal and resulting in an inappropriately high value. Approaches to eliminate interference from HAMAs and HAAAs include the use of chimeric antibody combinations and blocking agents to neutralize the effects of these heterophilic antibodies on their methods.

Disease-specific variation Caloric deprivation Acute caloric deprivation lowers total and FT3 within the first 24 h reaching a nadir in 12 weeks. rT3 increases during this period. Total T4 is unchanged. FT4 levels are normal or slightly increased. TSH, after a slight downward shift, returns to baseline within 4 days. These effects are reversed by as little as 50 g of glucose or protein, but not by fat.

Hepatic disorders Patients with hepatic cirrhosis have low serum T3 levels due to decreased production. T4 levels are either normal or low (due to decreased albumin and TTR). Serum FT4 and FT3 are generally within the normal range. TSH levels are within normal limits but may occasionally be minimally elevated. Increased T4 and T3 levels are seen in acute infectious hepatitis, chronic active hepatitis, and biliary cirrhosis. This increase is due to increased production and release of TBG.

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Renal failure Patients with chronic renal failure have low T3, but normal rather than elevated rT3. T4 levels are low, but FT4 and TSH levels are normal. The thyroid hormone blood levels in nephrotic syndrome are similar, although patients lose T4, T3, and TBG through the kidney.

Acquired immunodeficiency syndrome and acquired immunodeficiency syndrome—related complex The circulating thyroid hormone blood concentrations in patients infected with human immunodeficiency virus (HIV) are somewhat different from patients with other NTIs. An unexplained rise in serum TBG produces high circulating T4 levels. FT4 is normal. T3 is also normal rather than decreased. rT3 is normal or low. The serum TBG levels continue to increase as the patient moves from an asymptomatic stage to acquired immunodeficiency syndrome (AIDS)-related complex and finally to AIDS. The serum TSH concentrations are normal or may be slightly high. Ultimately, circulating T4 and T3 concentrations decline signifying a preterminal stage. Thyroid function in patients with HIV who are treated with highly active antiretroviral therapy (HAART) appears to be variable. Elevated or suppressed TSH with normal T4 as well as isolated low FT4 concentrations have all been reported. Development of Graves’ thyrotoxicosis during immune cell reconstitution after HAART initiation, with increased T4 and T3, low TSH, and detectable anti-TSH receptor antibodies, has also been reported.

Psychiatric disorders A minority of patients with acute psychiatric illnesses may have elevated T4, FT4, and free thyroid index (FTI) at the time of admission. Serum T3 levels are generally normal or minimally elevated. rT3 is increased. Serum TSH levels may be elevated. TSH response to TRH may be blunted. Rapid reversal of these parameters, however, takes place.

Helpful clues to distinguish thyroid disease from nonthyroidal causes of abnormal hormone levels The following clues are helpful when confronted with thyroid parameters from a patient with NTI. G

In hyperthyroidism, TSH is below 0.04 mU/L, and FT4 may be high-normal or high. This degree of TSH suppression is extremely unlikely in NTI alone.

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G

G

G

G

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In moderate to severe primary hypothyroidism, TSH will be unquestionably high and FT4 low. Such a combination of TSH and FT4 levels is uncommon in NTI. Confusion arises when FT4 is low and TSH is either low-normal or TSH is minimally high. These combinations of parameters are compatible with NTI, mild primary hypothyroidism, or pituitary disease. FTI, calculated from T4 and T3 resin uptake ratio (RUR), may provide a clue. Although T4 is low in NTI, primary hypothyroidism, and pituitary disease, RUR is almost always elevated or at the upper limit of normal in NTI and is almost always low-normal or lower than normal in primary hypothyroidism and hypopituitarism. rT3 is rarely needed and is available in few laboratories. rT3 is high in NTI except in some liver disorders, renal failure, or HIV-related disorders and low in hypothyroidism. However, equilibrium, dialysis followed by mass spec assessment of free hormone is the best indicator. When faced with an ill patient with confusing thyroid hormone values that suggest the diagnosis of hypopituitarism, measurement of serum cortisol values may be of help. In severe illness, plasma cortisol levels are generally high. This assessment, however, cannot be accomplished if the patient is receiving exogenous glucocorticoids. Finally, assessment of antithyroid antibodies may provide a clue to the presence of autoimmune thyroid disease. Concomitant thyroid disease and NTI can confuse the picture. Occasionally, NTI will normalize thyroid indices and mask thyroid disease. The most common example is the normalization of minimally elevated TSH of mild primary hypothyroidism. Mildly elevated T4 and T3 concentrations of hyperthyroidism may also become normal with superimposed NTI.

Thyroid disorders Hyperthyroidism Hyperthyroidism is a state in which the body is exposed to an excessive amount of thyroid hormone. Common causes include Graves’ disease, toxic nodule, toxic multinodular goiter, and thyroiditis. Clinical hyperthyroidism can also be produced by ingestion of excessive amounts of thyroid hormones, T4 or T3. Except for thyroiditis, the change from euthyroidism to a hyperthyroid state and progression of hyperthyroidism is usually a slow and gradual process. In fully developed hyperthyroidism, TSH levels are below 0.04 mU/L, and FT4 concentrations are elevated. However, both these parameters do not become abnormal simultaneously. Usually, TSH concentrations fall to less than 0.04 mU/L before FT4 levels exceed the normal range. The patient at this stage may be asymptomatic, and a state of “subclinical hyperthyroidism” is said to exist. Additionally,

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TABLE 3.1 Progressive changes in thyroid hormones in hyperthyroidism. Hormone

Subclinical

Intermediate

Clinical disease

TSH

Low

Suppressed

Suppressed

T4

Normal

Normal

High

T3

Normal

High

High

FT4

Normal

Normal

High

in Plummer’s disease and early Graves’ disease, the T3 levels rise before the increase in T4. When the TSH is suppressed to less than 0.04 mU/L with elevated T3 concentrations and normal FT4 concentrations, the patient is said to have “T3 toxicosis.” It is important to appreciate that TSH concentrations may decline below the population normal range before T4 or FT4 concentrations become elevated (Table 3.1).

Iatrogenic hyperthyroidism Iatrogenic hyperthyroidism can be produced when patients are treated with T4 or T3. When the hyperthyroidism is produced by exogenous administration of T3, the TSH concentrations as well as FT4 will be low. T3 concentrations will be elevated but may not be tested unless the ingestion of T3 is known.

Transient hyperthyroidism Thyroiditis is an inflammatory disorder of the thyroid of viral or autoimmune etiology. Inflammation releases thyroid stores from the gland, followed by loss of function of thyroid cells. Ultimately, recovery and restoration of normal function take place. In thyroiditis, a spectrum of thyroid indices may be observed. In the early phase, the inflammation of the thyroid releases the thyroid hormone stores into circulation. Elevation in thyroid hormone concentrations suppresses TSH. The combined effects of low TSH and inflammation of the thyroid stop the synthesis of new thyroid hormones by the damaged cells. With the passage of time, circulating thyroid hormone levels decline to normal and then further to concentrations below normal. The TSH levels then start to rise. If the cells have regained the capacity to synthesize thyroid hormones, production will resume and both free thyroid hormones and total thyroid hormones, and then TSH will return to normal in succession. It is obvious that early in the course, FT4 concentrations will be normal or high and TSH below normal. With progression, TSH declines further as

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FT4 concentrations increase. This is followed by normal FT4 concentrations and low TSH concentrations, normal FT4, and normal TSH concentrations followed by low FT4 and high TSH concentrations, normal FT4, and high TSH concentrations with ultimate return to normal FT4 and TSH concentrations and euthyroidism. The time course of the disease varies. It may take as long as 6 months to 1 year for the disorder to complete the cycle. Continually changing thyroid hormone parameters are a clue to the underlying disorders. The term “transient hyperthyroidism,” although true, is not a comprehensive description of this disorder. The hyperthyroidism is followed in succession by a “transient euthyroid state,” followed by “transient hypothyroidism,” and followed usually by euthyroidism. This is the usual course of events; however, occasionally mild persistent hypothyroidism is the sequela. If the disorder is mild, and the early hyperthyroid phase is missed, the underlying disorder may not be appreciated, and continually changing thyroid hormone concentrations may be confounding.

High thyroid-stimulating hormone with hyperthyroidism TSH concentrations are almost always low in hyperthyroidism. Occasionally, a hyperthyroid state exists with a high TSH and high FT4 levels. TSHsecreting pituitary tumor and thyroid hormone resistance (THR) confined to the pituitary gland will produce such a picture. TSH response to TRH is particularly helpful to identify and to differentiate between the two disorders. In TSH-secreting pituitary tumor, the administration of exogenous TRH fails to significantly alter the elevated TSH levels observed prior to administration of TRH. In contrast, exogenous TRH produces a further increase in TSH observed in hyperthyroxinemia of THR. In THR due to receptor aberrations, the thyrotroph responds to a higher quantity than usual of free thyroid hormones in the negative feedback loop. As with the pituitary tumor, TSH and FT4 are both elevated, or TSH may be within the normal range with FT4 elevated.

Hypothyroidism Primary hypothyroidism In primary hypothyroidism, as FT4 concentrations start to decline, TSH concentrations increase. In fully developed hypothyroidism, FT4 values are low, and TSH concentrations are abundantly high. As in early hyperthyroidism, patients frequently pass through a stage of subclinical or “compensated” hypothyroidism when FT4 concentrations are normal, and TSH is minimally elevated (Table 3.2).

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TABLE 3.2 Progressive changes in thyroid hormones in hypothyroidism. Test

Mild/subclinical

Moderate

Clinical

TSH

Elevated

High

High

T4

Normal

Low

Low

T3

Normal

Normal

Low

FT4

Normal

Low

Low

Central hypothyroidism In central hypothyroidism, effective TSH stimulation of thyroid is diminished. TSH and FT4 concentrations are expected to be low. This occurs in advanced and total pituitary failure or, rarely, in hypothalamic dysfunction. Hypopituitarism is usually partial. FT4 concentrations may be accompanied by low, normal, or even high (minimal increase) TSH concentrations. The inappropriate TSH concentrations result from the measurement of biologically inactive TSH molecules.

Hypothyroidism after radioactive iodine therapy After radioactive iodine therapy for hyperthyroidism, FT4 concentrations decline before suppressed TSH concentrations return to normal. At this stage, FT4 and TSH concentrations are below normal and may be confused with central hypothyroidism. The patient may be clinically euthyroid or even complain of symptoms of hypothyroidism. If left untreated, and if the failure of the thyroid gland is progressive, TSH concentrations will ultimately rise above normal.

Treated hypothyroidism Circulating thyroid hormone levels in patients treated with exogenous thyroid hormone depend on the agent used. If L-T4 is used to attain euthyroidism, both FT4 and TSH levels are within the normal range. If T3 (Cytomel) is used to bring TSH into the normal range, FT4 concentrations will be low, but T3 levels are high.

Neonatal hypothyroidism In primary neonatal or juvenile hypothyroidism, TSH concentrations are elevated, whereas T4, T3, and FT4 levels are low or low-normal for age. In central hypothyroidism, all thyroid hormone-related measurements are low.

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Effects of binding protein aberrations on thyroid function tests G

G

G

In addition to diseases of the liver, the capacity of thyroid hormonebinding proteins may be abnormal either due to inherited abnormal levels of binding proteins or due to increased affinity for binding hormones. When TBG concentration is low, the resulting low T4 concentration may be confused with hypothyroidism. However, RUR is high, pointing to binding protein deficiency. Similarly, when TBG is high, circulating T4 is high, but RUR is low. Thankfully, FT4 and TSH concentrations are normal in this circumstance. An inherited abnormality of thyroid hormone binding by albumin that binds T4 with increased affinity will produce high T4 levels (dysalbuminemic hyperthyroxinemia). T3 levels are normal. RUR is not low in this condition because this test does not measure albumin. Thus RUR is normal, and FTI is high. TSH and FT4 are both normal, and the patient is euthyroid. Rarely, in autoimmune disorders of the thyroid, patients may develop antibodies to T4 and/or T3. In such circumstances, T4 and/or T3 concentrations are elevated and RUR decreased, providing a clue to binding protein problems. If such individuals are hypothyroid and receiving hormone supplements, confusion may arise. Because of increased protein-binding capacity for thyroid hormones, FT4 will decrease, and TSH will increase. An increase in the dose of exogenous thyroid hormone becomes necessary.

Screening for subclinical thyroid disease Who should be tested beyond those with clinical suspicion of thyroid disease? The previous discussion related to the interpretation of hormone concentrations obtained in patients with thyroidal or NTI. However, there is no consensus for testing asymptomatic populations. Most groups do not recommend screening asymptomatic populations. The focus has been the detection of overt disease in the general, unscreened population. A pragmatic substitute for routine screening is the “recommendation for clinicians to maintain a low threshold for suspecting hypothyroidism and to reserve testing for these patients.” The American College of Physicians has published clinical guidelines for screening for thyroid disease that take into account published data on prevalence, risk for complications, and appropriateness of treatment of subclinical disease. They recommend screening women older than 50 years of age for unsuspected but symptomatic thyroid disease using sensitive TSH. FT4 should be done if TSH is undetectable or .10 mU/L. Patients discovered by this screening protocol to have overt hypothyroidism (TSH . 10 mU/L and low FT4) or overt hyperthyroidism (undetectable TSH and high FT4) are likely to benefit from treatment. Subclinical hyperthyroidism and subclinical

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hypothyroidism as defined by these guidelines are patients whose TSH is undetectable or .10 mU/L but who have normal FT4. Data were not sufficient for this group to recommend treatment of subclinical disease. However, this remains controversial and awaits more definitive studies. In the target screening population, patients with normal TSH should be screened every 25 years. Some authors recommend routine testing of “high-risk” populations, including: G G G G G G

neonates; those with a higher prevalence of disease, such as individuals .60 years; women; those with dyslipidemia; those with a family history of thyroid dysfunction; and those taking drugs which affect thyroid function, for example, lithium and amiodarone.

Populations not recommended for screening include asymptomatic adults ,35 years of age and acutely ill patients unless there is a strong suspicion that thyroid disease is contributing to the current illness. The association of subclinical hypothyroidism with future progression to overt hypothyroidism, hypercholesterolemia and associated risk for cardiovascular disease, as well as the availability of effective treatment, stimulated an analysis of the cost-effectiveness of periodic screening for subclinical hypothyroidism. TSH and cholesterol were included in a health examination every 5 years beginning at age 35. Their model suggests that the screening for hypothyroidism is favorable, especially in older women, and is costeffective. No clinical experience with this approach has yet been reported.

Laboratory tests used in the assessment of thyroid function Thyroid-stimulating hormone (thyrotropin) TSH plays a central role in the regulation of the thyroid economy. As illustrated in Fig. 3.1, TSH and FT4 are inversely related. This relationship is log-linear, a twofold change in FT4 causing a 50-fold change in TSH. Thus TSH is the earliest hormone to respond to changes in thyroid gland function, and it is not uncommon to observe TSH outside the normal range, whereas the FT4 is within the population normal range. Conversely, with certain exceptions such as pituitary disease, a normal TSH level is consistent with the euthyroid state. This has led to use of a TSH-first strategy in the evaluation of a patient for possible thyroid disease. Modern-day TSH methods, with their enhanced sensitivity, are also capable of detecting the low TSH values typical of hyperthyroidism. These new methods are often based on nonisotopic IMA principles and are available on

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a variety of automated immunoassay analyzer platforms. Most of the current methods are capable of achieving a functional sensitivity of 0.02 mIU/L or less, which is a necessary detection limit for the full range of TSH values observed between hypo- and hyperthyroidism. With this level of sensitivity, it is possible to distinguish the profound TSH suppression typical of severe Graves’ thyrotoxicosis (TSH , 0.01 mIU/L) from the TSH suppression (0.010.1 mIU/L) observed with mild (subclinical) hyperthyroidism and in some patients with an NTI.

Specificity A discordant TSH result in an ambulatory patient with stable thyroid status may be a technical error. Specificity loss can result from laboratory error, interfering substances (i.e., heterophilic antibodies), or the presence of an unusual TSH isoform. When TSH is unexpectedly high, the specimen should be measured diluted, preferably in thyrotoxic serum, to check for parallelism. The laboratory should analyze the specimen by a different manufacturer’s method (send to a different laboratory if necessary). If the between-method variability for a sample is .50%, an interfering substance may be present. Once a technical problem has been excluded, biologic checks may be useful: A TRH stimulation test should be used for investigating a discordant low TSH result; a twofold ($4.0 mIU/L increment) response in TSH is expected in normal individuals. A thyroid hormone suppression test should be performed to verify a discordant high TSH concentration. Normal response to 1 mg of L-T4 or 200 μg of L-T3 administered per day a suppressed serum TSH of more than 90% by 48 h.

Sensitivity Historically, the “quality” of a serum TSH method has been determined from a clinical benchmark: the assay’s ability to discriminate euthyroid concentrations (B0.44.0 mIU/L) from the profoundly low (,0.01 mIU/L) TSH concentration typical of overt Graves’ thyrotoxicosis. Most TSH methods now claim a detection limit of 0.02 mIU/L or less (“3rd generation”) assays. Functional sensitivity is calculated from the 20% between-run coefficient of variation for the method and is used to establish the lowest reportable limit for the test.

Thyroid-stimulating hormone reference intervals Serum TSH levels exhibit a diurnal variation, with the peak occurring during the night and the nadir, which approximates to 50% of the peak value, occurring between 1000 and 1600 h. This biologic variation does not influence the diagnostic interpretation of the test result because most clinical TSH

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measurements are performed on ambulatory patients between 0800 and 1800 h, and TSH reference intervals are more commonly established from specimens collected during this time period. Serum TSH reference intervals should be established using specimens from TPOAb-negative, ambulatory, euthyroid subjects who have no personal or family history of thyroid dysfunction and no visible goiter. The variation in the reference intervals for different methods reflects differences in antibody epitope recognition by the different kit reagents and the rigor applied to the selection of appropriate normal subjects.

Thyroid-stimulating hormone upper reference limits Over the last two decades, the upper reference limit for TSH has steadily declined from B10 to approximately B4.04.5 mIU/L. This decrease reflects a number of factors including the improved sensitivity and specificity of current monoclonal antibody-based IMAs, the recognition that normal TSH values are log-distributed and, importantly, improvements in the sensitivity and specificity of the thyroid antibody tests that are used to prescreen subjects. The recent follow-up study of the Whickham cohort has found that individuals with a serum TSH .2.0 mIU/L at their primary evaluation had an increased odds ratio of developing hypothyroidism over the next 20 years, especially if thyroid antibodies were elevated. An increased odds ratio for hypothyroidism was even seen in antibodynegative subjects. It is likely that such subjects had low concentrations of thyroid antibodies that could not be detected by the insensitive microsomal antibody agglutination tests used in the initial study. Even the current sensitive TPOAb immunoassays may not identify all individuals with occult thyroid insufficiency. In the future, it is likely that the upper limit of the serum TSH euthyroid reference range will be reduced to 2.5 mIU/L because .95% of rigorously screened normal euthyroid volunteers have serum TSH values between 0.4 and 2.5 mIU/L. Thyroid-stimulating hormone lower reference limits Before the IMA era, TSH methods were too insensitive to detect values in the lower end of the reference range. Current methods, however, are capable of measuring TSH at the lower end and now cite lower reference limits between 0.2 and 0.4 mIU/L. As the sensitivity of the methods has improved, there has been an increased interest in defining the true lower limit of normal to better determine the presence of mild (subclinical) hyperthyroidism. Current studies suggest that TSH values in the 0.10.4 mIU/L range may represent thyroid hormone excess and in elderly patients might be associated with an increased risk of atrial fibrillation and cardiovascular mortality. It is therefore important to carefully exclude subjects with a goiter and any illness or stress in the normal cohort selected for reference range study.

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Clinical use of serum thyroid-stimulating hormone measurements Screening for thyroid dysfunction in ambulatory patients Most professional societies recommend that TSH be used for case finding or screening for thyroid dysfunction in ambulatory patients, provided that the TSH assay used has a functional sensitivity at or below 0.02 mIU/L. The log/linear relationship between TSH and FT4 dictates that serum TSH is the preferred test because only TSH can detect mild (subclinical) degrees of thyroid hormone excess or deficiency (Fig. 3.1). However, the TSH-first testing has two limitations. First, it assumes that hypothalamicpituitary function is intact and normal. Second, it assumes that the patient’s thyroid status is stable, that is, the patient has had no recent therapy for hypo- or hyperthyroidism, and if either of these criteria is not met, serum TSH results can be diagnostically misleading. It is important to confirm any TSH abnormality in a fresh specimen drawn after B3 weeks before assigning a diagnosis of mild (subclinical) thyroid dysfunction as the cause of an isolated TSH abnormality. After confirming a high TSH abnormality, a TPOAb measurement is a useful test for establishing the presence of thyroid autoimmunity as the cause of mild (subclinical) hypothyroidism. The higher the TPOAb concentration, the more rapid is the development of thyroid failure. After confirming a low TSH abnormality, it can be difficult to unequivocally establish a diagnosis of mild (subclinical) hyperthyroidism, especially if the patient is elderly and not receiving L-T4 therapy. If a multinodular goiter is present, thyroid autonomy is the likely cause of mild (subclinical) hyperthyroidism.

Elderly patients Most studies support screening for thyroid dysfunction in the elderly because the prevalence of both a low and a high TSH (associated with normal FT4) is increased in the elderly compared with younger patients. Hashimoto thyroiditis, associated with a high TSH and detectable TPOAb, is encountered with increasing prevalence as patients age. The incidence of a low TSH is also increased in the elderly. This could be due to a change in the FT4/TSH setpoint, a change in TSH bioactivity, or mild thyroid hormone excess. Multinodular goiter should be ruled out as the cause, especially in areas of iodide deficiency. Medication history should be thoroughly reviewed (including over-the-counter preparations, some of which contain T3). If a goiter is absent and the medication history negative, a serum TSH should be rechecked together with TPOAb measurements after 46 weeks. If the TSH is still low, and TPOAb is positive, the possibility of autoimmune thyroid dysfunction should be considered. Treatment of low TSH should be made on a case-by-case basis.

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L-T4 replacement therapy Hypothyroid patients have serum FT4 values in the upper third of the reference interval when the L-T4 replacement dose is titered to bring the serum TSH into the therapeutic target range (0.52.0 mIU/L). A euthyroid state is usually achieved in adults with a L-T4 dose averaging 1.6 μg/kg body weight/day. A serum TSH result between 0.5 and 2.0 mIU/L is generally considered the therapeutic target for a standard L-T4 replacement dose for primary hypothyroidism. A serum FT4 concentration in the upper third of the reference interval is the therapeutic target for L-T4 replacement therapy when patients have central hypothyroidism due to pituitary and/or hypothalamic dysfunction. A typical schedule for gradually titrating to a full replacement dose involves giving L-T4 in 25-μg increments each 68 weeks until the full replacement dose is achieved (serum TSH, 0.52.0 mIU/L). Both FT4 and TSH should be used for monitoring hypothyroid patients suspected of intermittent or noncompliance with their L-T4 therapy. The paradoxical association of a high FT4 1 high TSH is often an indication that compliance may be an issue. Specifically, acute ingestion of missed L-T4 doses before a clinic visit will raise the FT4 but fail to normalize the serum TSH because of the “lag effect” (Fig. 3.3). Annual TSH testing of patients receiving a stable dose of L-T4 is recommended.

FIGURE 3.3 The serum TSH/FT4 relationship typical of different clinical conditions.

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L-T4 suppression therapy L-T4 administration designed to suppress serum TSH concentrations to subnormal values is typically reserved for patients with well-DTC for which thyrotropin is considered a trophic factor. It is important to individualize the degree of TSH suppression by weighing patient factors such as age, clinical status including cardiac factors, and DTC recurrence risk against the potentially deleterious effects of iatrogenic mild (subclinical) hyperthyroidism on the heart and bone. Many physicians use a serum TSH target of 0.050.1 mIU/L for low-risk patients and a TSH of ,0.01 mIU/L for highrisk patients. Serum thyroid-stimulating hormone measurement in hospitalized patients with nonthyroidal illness Although most hospitalized patients with NTI have normal serum TSH concentrations, transient TSH abnormalities in the 0.0220 mIU/L range are commonly encountered in the absence of thyroid dysfunction (see Fig. 3.2). Central hypothyroidism The log/linear relationship between TSH and FT4 dictates that patients with primary hypothyroidism and a subnormal FT4 should have a serum TSH value of .10 mIU/L (Fig. 3.1). When the degree of TSH elevation in response to a low thyroid hormone concentration appears inappropriately low, pituitary insufficiency should be excluded. A diagnosis of central hypothyroidism will usually be missed using a “TSH-first” strategy. Inappropriate thyroid-stimulating hormone secretion syndromes Binding protein abnormalities or assay technical problems are the most common causes for a discordant FT4/TSH relationship. Specimens that display a discordant TSH/FT4 relationship are increasingly being identified now that sensitive TSH assays that can reliably detect subnormal TSH concentrations are available. It is critical to first exclude likely causes of a discordant TSH/ FT4 ratio, that is, technical interference and/or binding protein abnormality. This confirmatory testing should be performed on a fresh specimen by measuring TSH together with free and total thyroid hormone with a different manufacturer’s method. Only after the more common causes of discordance have been eliminated should rare conditions such as a TSH-secreting pituitary tumor or THR be considered. When the abnormal biochemical profile has been confirmed, the possibility that a TSH-secreting pituitary tumor is the cause of the paradoxical TSH should first be eliminated before assigning the diagnosis of THR. TSHsecreting pituitary tumors have similar biochemical profiles to THR but can be distinguished from THR by TSH-α-subunit testing and radiographic

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imaging. Additionally, TRH stimulation testing may occasionally be useful in developing the differential diagnosis. Specifically, a blunted TRH stimulation test and T3 suppression test is characteristic of most TSH-secreting pituitary tumors, whereas a normal response is seen in most cases of THR.

Thyroid-stimulating hormonesecreting pituitary tumors Pituitary tumors that hypersecrete TSH are rare, representing ,1% of cases of inappropriate TSH secretion. These tumors often present as a macroadenoma with symptoms of hyperthyroidism associated with a nonsuppressed serum TSH and magnetic resonance imaging (MRI) evidence of a pituitary mass. After excluding a technical reason for the paradoxically elevated TSH concentration (i.e., HAMAs), the diagnosis of TSH-secreting pituitary tumor is usually made on the basis of: G G G G

a lack of TSH response to TRH stimulation; an elevated serum TSH-α subunit; a high α-subunit/TSH ratio; and the demonstration of a pituitary mass on MRI.

Thyroid hormone resistance THR is usually caused by a mutation of the TR, TR-β receptor gene that occurs in 1 of 50,000 live births. Serum FT4 and FT3 are typically elevated (from a minimal degree to a two to threefold elevation above the upper normal limit) and associated with a normal or slightly elevated serum TSH that responds to TRH stimulation. THR patients typically have a goiter as a result of chronic hypersecretion of a hybrid TSH isoform that has increased biologic potency. The distinctive features of THR are the presence of a nonsuppressed TSH, together with an appropriate response to TRH despite elevated thyroid hormone levels. Thyrotropin-releasing hormone test The TRH test consists of measurements of TSH before and after administration of TRH. Administration of exogenous TRH normally elicits a rise in TSH concentration that reaches a peak at around 1520 min and returns to baseline in 60 min. (The extent of rise depends on the dose of TRH used.) Normal elderly men may have initially low-normal TSH concentrations and a somewhat blunted rise in serum TSH after TRH administration. In primary hyperthyroidism, the TSH concentrations are low and fail to rise further after TRH administration. In primary hypothyroidism, TSH concentrations are elevated and rise briskly after TRH administration. In hypothalamicpituitary disease, the pattern of response is altered; the peak rise is blunted, delayed, and returns to baseline after 60 min. With the availability of sensitive TSH,

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this test has been abandoned for most evaluations of hyperthyroidism, but it is still useful in TSH-secreting pituitary tumors and THR.

FT4 FT4 is used as a second-line test in evaluation of thyroid function in the TSH-first screening strategy. Circulating FT4 concentrations are normally higher than FT3 concentrations, so despite the greater biological activity of FT3, FT4 is the preferred analyte. FT4 is also preferred over total T4 measurements, as FT4 represents the bioactive fraction of T4 and thus is a more accurate indicator of thyroid function. Additionally, the circulating concentration of FT4 is not as strongly affected by alterations in binding protein concentrations as the total T4 concentration. Multiple methods are used in the measurement of FT4, but it is most commonly measured by competitive immunoassays or IMAs. Most methods can accurately distinguish hyperthyroidism and hypothyroidism from euthyroidism, but levels of FT4 are best interpreted in conjunction with TSH. Thyroid hormones in serum exist in equilibrium between free hormone and hormone bound to binding proteins, and consequently, the measured concentration of FT4 is dependent on this equilibrium. Physiological concentrations of FT4 may be affected by any factor that impacts the hormone-binding equilibrium, including a variety of drugs, nonthyroidal diseases, pituitary disease, and normal conditions such as pregnancy. Additionally, factors intrinsic to any given measurement method can affect the physiological binding equilibrium and thus the measured concentration of FT4. Consequently, reference ranges for FT4 are method-dependent, and it is important to select measurement methods for FT4 that minimize nonphysiological effects on measured FT4 concentration.

Influence of methods of analysis on test results G

G

G

G

FT4 measured by equilibrium dialysis is the “gold standard,” but the method is slow, labor-intensive, and not suitable for routine evaluations. It is used only for research or very unusual cases. FT4 measured by immunoassay is most widely performed. A variety of formats including a one-step, analog tracer format or a two-step, sequential assay format are available on many automated instruments. FT4 measured by ultrafiltration performs similarly to equilibrium dialysis with a shorter analysis time, but is not widely available and has poorer analytical sensitivity when compared with dialysis methods. Mass spectrometry methods (such as ultrafiltration coupled to liquid chromatography-tandem mass spectrometry) have been developed for the measurement of FT4 and other thyroid hormones. However, mass spectrometry is technically demanding.

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Interferences/considerations for interpretation of results Most contemporary immunoassays minimize interferences from varying concentrations of binding proteins in the patient sample. However, immunoassay methods, particularly one-step assays, can yield falsely decreased FT4 when serum albumin concentrations are low due to the expected binding of tracers to albumin in the patient sample. Immunoassays are also susceptible to interference from heterophilic antibodies, from anti-T4 antibodies, and from rheumatoid factor. Any method that involves sample dilution, including some two-step immunoassays and equilibrium dialysis, can yield falsely low FT4 values if any compounds that compete for hormone-binding sites (such as drugs) are present. Additionally, in patients receiving heparin therapy, falsely elevated FT4 values may be observed due to in vitro activation of lipoprotein lipase, which yields elevated FFAs in the sample and displacement of T4 from binding proteins. Ideally, FT4 methods should show good performance across the full range of binding protein concentrations, but methods are not standardized and the relationship between FT4 and log TSH is not always well correlated using automated immunoassay methods. In patients where these measurements are discordant or are expected to be discordant because of variation in albumin concentrations, an alternate method (such as equilibrium dialysis or mass spectrometry) should be employed for measurement of FT4. Where available, mass spectrometry methods are preferable, as they offer better analytical specificity, fewer interferences, and better correlation between FT4 and log TSH than do immunoassays.

Normal range Adult: 0.891.9 ng/dL (varies slightly with method); second and third trimesters of pregnancy: 0.651.2 ng/dL.

T4 T4 is the thyroid hormone circulating in highest concentration. T4 (free and protein-bound) is measured by competitive immunoassays that are generally reliable. Because circulating concentrations are strongly influenced by binding proteins and a variety of clinical conditions and drugs, the test has been largely replaced by FT4 or FTI.

Normal range Adult: 4.611.6 μg/dL (varies slightly with method); pediatric: cord blood, 6.617.5 μg/dL. After 1 month, same as adult range.

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Resin uptake ratio RUR, also called T3 resin uptake, is used to assess whether alterations in total T4 are the result of increased or decreased binding proteins or due to alterations in thyroid function. RUR is inversely proportional to unsaturated thyroid hormone-binding sites, predominately TBG, and is expressed as a ratio of the patient response to the normal response. RUR decreases (RUR , 1) when binding proteins increase (e.g., in pregnancy) or are relatively unsaturated with bound T4 as in hypothyroidism. RUR increases (RUR . 1) when binding proteins decrease from normal or are relatively saturated with bound T4 as in hyperthyroidism. RUR is used in conjunction with T4 to calculate FTI. A number of variations of “uptake” tests are commercially available, all of which provide a measure of the effect of thyroid hormone-binding proteins. The normal ranges and the calculations of FTI may vary with the individual laboratory.

Normal range Adult: 0.81.2.

Free thyroid index This index is calculated as total T4 3 RUR. This index is proportional to FT4. FTI correlates well with FT4 and inversely with TSH and is an acceptable substitute for free thyroid hormone measurement in most populations. In some cases, such as severe NTI, RUR does not fully compensate and does not adjust the T4 into the normal range. Because measured FT4 may be low in NTI, the elevated RUR may provide a clue to the mechanism of the low T4 and low FT4 observed in these conditions. In the evaluation of some complex or ambiguous cases, some endocrinologists still prefer to see separately the measurement of total T4 and the RUR as an independent measure of binding proteins. With the advent of tandem mass spectrometry, the FTI test has become obsolete.

Normal range FTI, adult: 5.510.0 μg/dL (varies slightly with method). Note that the units of FTI are the same as total T4. The normal range of FTI, calculated individually from normal subjects, is somewhat narrower than the T4 population normal range. This observation supports the hypothesis that individuals have an individual pituitary “set-point” or narrow range for free thyroid hormones at which point TSH begins to respond.

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Interpretation G Hyperthyroid. RUR is .1; T4 is elevated. Thus FTI (T4 3 RUR) is elevated, and FTI is .T4, confirming the hyperthyroid state. G Hypothyroid. RUR is ,1; T4 is low. Thus FTI is relatively decreased, and FTI is ,T4, accentuating the impression of hypothyroidism. G Euthyroid. Increased or decreased binding proteins result in elevated or decreased total T4 concentration. RUR changes will be opposite of T4. Thus FTI will move toward the middle of the normal range, confirming euthyroidism. For example, in pregnancy, TBG increases, resulting in increased T4. In this condition, RUR is ,1. FTI will be ,T4 (that is, T4 3 a factor ,1). RUR “normalizes” T4.

T3 T3 in blood is derived predominantly from peripheral conversion (deiodination) of T4. Thyroid secretion contributes only up to 20% in normal circumstances. The contribution of the thyroid to circulating T3 increases, however, in states of thyroid overactivity. Like T4, T3 circulates predominantly bound to thyroid-binding proteins, TBG, albumin, and prealbumin. T3 reflects the hormone stores in blood. Like T4, the T3 levels are high in fully developed hyperthyroidism. However, in very early hyperthyroidism, T3 concentrations are high, whereas T4 levels are still normal. The main clinical utility of the test is to confirm early hyperthyroidism when TSH is low, but FT4 is in the normal range (T3 thyrotoxicosis). The test is not needed to confirm advanced hyperthyroidism with suppressed TSH and elevated FT4. The test is also not necessary in the diagnosis of hypothyroidism.

Normal range 80200 ng/dL (varies slightly with method).

Free T3 FT3 is the bioactive thyroid hormone and is measured independent of binding proteins by equilibrium dialysis and, more commonly, analog tracer competitive binding immunoassay. Because of the analytical difficulties relating to the very low concentration of FT3 in serum, FT4 is the more reliable and preferred test to assess thyroid function. FT3 is useful to confirm early hyperthyroidism in a patient with altered binding proteins (estrogens) and who has suppressed TSH, but normal FT4, that is, T3 thyrotoxicosis, a rare condition. Because of the influence of binding proteins and drugs on total T3 concentrations (similar to T4), FT3 is preferred over T3.

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Normal range Adult: 200500 pg/dL (varies slightly with method).

Reverse T3 rT3 is elevated in severe NTI. Theoretically, rT3 could help differentiate between the low FT4 levels of systemic disease from the low FT4 concentrations due to thyroid or pituitary failure. However, TSH serves this purpose in most circumstances (high in hypothyroidism, low or normal in severe illness). In newborns with suspiciously high TSH and concomitant severe illness, early diagnosis and treatment of hypothyroidism are essential. The situation may be confusing because newborns normally have high TSH, which varies with age. An elevated rT3 may clarify the diagnosis by confirming the impact of the illness on thyroid hormones. rT3 has also been reported to be elevated in anorexia nervosa but is rarely needed because an elevated cortisol is a signal of anorexia nervosa. The measurement is not available in most laboratories and usually is not necessary.

Tg Tg is the thyroid protein on which thyroid hormones are synthesized. Serum concentrations of Tg are generally proportional to thyroid mass and increase with tissue damage or increased thyroid activity. Although Tg is not a tumor marker, it is useful in following the course of patients with treated thyroid carcinoma. In a patient who has been treated for DTC, Tg should be almost nondetectable. Increasing serum levels of Tg suggests persistence or growth of thyroid cancer.

Analytical methods Tg is measured by immunoassays in various formats, all of which suffer from lack of sensitivity and variable analytical specificity. Use of an international reference preparation (Community Bureau of Reference of the Commission of the European Communities) is hoped to improve intermethod comparison. Interference in Tg assays may occur when patients’ sera contain antibodies to Tg. This is a common source of interference, as the prevalence of anti-Tg antibodies is high in both the general population and in patients with thyroid cancers. High-dose “hook” effects have been reported (inappropriately low results measured in patients whose actual concentration is very high). Current guidelines indicate that testing for anti-Tg antibodies should be performed concurrently with Tg immunoassay measurements.

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Tg may also be measured by liquid chromatography-tandem mass spectrometry. In these methods, proteolytic digestion of Tg is used to release Tg peptides, which are detectable without interference from anti-Tg. The analytical sensitivity of these methods can be improved by immunoaffinity enrichment strategies, which may permit method sensitivity comparable to that of immunoassays with improved analytical specificity. Mass spectrometry methods are not automated and not widely available, but they are preferred in patients with anti-Tg antibodies due to the interference produced in Tg immunoassays.

Normal range Normal range depends on the assay method and sensitivity of the assay. Serial samples using the same method over a period of time is the preferred application of this test.

Thyroid autoantibodies Several diseases of the thyroid gland such as Graves’ disease, Hashimoto disease, and postpartum thyroiditis are caused by autoimmune processes. These conditions are associated with one or more antibodies to various proteins of the thyroid gland, including anti-Tg, anti-TPO (formerly thyroid microsomal antibody), and anti-TSH receptor (anti-TR). Anti-TR may act by either stimulating the TSH receptor or blocking the receptor. Despite the association with autoimmune thyroid disease, thyroid antibodies offer little additional diagnostic value. Anti-TPO alone is recommended as a cost-effective and sensitive assessment of autoimmune etiology. The presence of anti-TPO has been used by some to support the diagnosis of autoimmune disease; however, positive anti-TPO does not exclude the presence of malignancy. Anti-Tg assayed in conjunction with the Tg immunoassay is used to rule out potential assay interference. The recommended use of anti-TR is to assist in the diagnosis of Graves’ disease in a pregnant patient or in the management of a pregnant patient with current or previous history of Graves’ disease. The latter use is to assess the risk of fetal or neonatal thyrotoxicosis because some maternal anti-TR crosses the placenta.

Analytical methods Thyroid autoantibodies are detected by qualitative or quantitative immunoassays in varying formats. Results are reported as units per milliliter or as a titer. Unfortunately, results among methods vary widely because of different specificities of the antibodies, different, sometimes complex, antigens used to detect the antibodies, and a lack of consensus standards. Normal ranges are variable depending on the specificity and precision of the assay.

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Utilization of laboratory tests in the diagnosis and monitoring of thyroid disease: recommendations for testing For diagnosis and monitoring treatment of thyroid disease, either direct measurement of FT4 or another estimate of FT4 such as FTI can be used. The choice depends primarily on availability and cost. G

G G

G G G G

A TSH-first algorithm, which reflexes to FT4, is a medically efficient and cost-effective initial evaluation for most suspected thyroid disease. Do not use this algorithm to assess patients with possible pituitary disease. Recent guidelines published by the American Association of Clinical Endocrinologists indicate that choosing an upper limit of 2.5 mIU/L for normal TSH values will increase sensitivity and specificity of diagnosing subtle hypothyroidism. To confirm the diagnosis of thyroid disease, FT4 is preferred over FT3. If T3 thyrotoxicosis is suspected (low TSH with normal FT4), FT3 is preferred over total T3, if available. Monitor thyroid hormone replacement therapy with TSH. Monitor suppression therapy for thyroid cancer with sensitive TSH. Follow thyroid ablation for hyperthyroidism with FT4. A TSH-first algorithm is effective to screen asymptomatic individuals in special or high-risk populations, for example, the elderly, women over 60 years of age, neonates, individuals taking drugs known to affect the thyroid, and individuals with a personal or family history of thyroid disease.

Interpretive hints when thyroid-stimulating hormone and FT4 results seem discrepant G G

G

G

G

G

G

When TSH is low and FT4 normal: FT3 may confirm T3 thyrotoxicosis. TSH and FT4 both increased: suspect thyroid resistance or pituitary tumor (TSH may be in the upper normal range with FT4 increased in THR. This would not be apparent if an algorithm that reflexes only when TSH is abnormal were used for initial testing). TSH and FT4 both low-normal: NTI or suspect central hypothyroidism (hypothalamic or hypopituitary). TSH/T4 are hormones in dynamic equilibrium in each individual. In treated hypothyroidism, the blood levels may not reach steady state for 46 weeks. Wait 8 weeks after thyroid hormone dose adjustment to assess the effect. In patients being treated with thyroid hormone replacement, low TSH with normal FT4 may suggest overreplacement. Consider patient compliance, change in drug formulation, drug absorption, and assay interference. Noncompliance is common. Consider the impact of drugs, systemic illness, or pregnancy in the interpretation of TSH and FT4 before initiating extensive follow-up or esoteric diagnoses.

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Further reading American Association of Clinical Endocrinologists, American Association of Clinical Endocrinologists medical guidelines for clinical practice for the evaluation and treatment of hyperthyroidism and hypothyroidism, Endocr. Pract. 8 (2002) 457469. G. Azizi, J.M. Keller, M. Lewis, et al., Association of Hashimoto’s thyroiditis with thyroid cancer, Endocr. Relat. Cancer. 21 (6) (2014) 845852. R.S. Bahn Chair, H.B. Burch, D.S. Cooper, et al., Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists [published correction appears in Thyroid. 2011 Oct;21(10):1169] [published correction appears in Thyroid. 2012 Nov;22(11):1195]. Thyroid 21 (6) (2011) 593646. H.B. Burch, Drug effects on the thyroid, N. Engl. J. Med. 381 (2019) 749761. G. Brent, J. Hershman, Thyroxine therapy in patients with severe non-thyroidal illness and low serum thyroxine, J. Clin. Endocrinol. Metab. 63 (1986) 18. G. Burrow, D. Fisher, P. Larsen, Maternal and fetal thyroid function, N. Engl. J. Med. 331 (1994) 10721078. M. Danese, N. Powe, C. Sawin, P. Ladenson, Screening for mild thyroid failure at the periodic health examination: a decision and cost-effectiveness analysis, JAMA 276 (1996) 285292. L.M. Demers, C.A. Spencer (Eds.), Laboratory Medicine Practice Guidelines: Laboratory Support for the Diagnosis and Monitoring of Thyroid Disease. National Academy of Clinical Biochemistry, Washington, DC, 2002, 125 pp. ,http://www.aacc.org/members/ nacb/LMPG/OnlineGuide/PublishedGuidelines/ThyroidDisease/Pages/ThyroidDiseasePDF. aspx. (accessed 13.05.08). J.D. Faix, W.G. Miller, Progress in standardizing and harmonizing thyroid function tests, Am. J. Clin. Nutr. 104 (Suppl 3) (2016) 913S917S. J. Favresse, M.C. Burlacu, D. Maiter, D. Gruson, Interferences with thyroid function immunoassays: clinical implications and detection algorithm, Endocr. Rev. 39 (5) (2018) 830850. C. Feldkamp, J. Carey, An algorithmic approach to thyroid function testing in a managed care setting: 3-year experience, Am. J. Clin. Pathol. 105 (1996) 1116. C. Feldkamp, M. Zafar, Evaluation of thyroid status during pregnancy, Diag. Endocrinol. Metab. 15 (1997) 105113. U. Feldt-Rasmussen, Analytical and clinical performance goals for testing autoantibodies to thyroperoxidase, thyroglobulin, and thyrotropin receptor, Clin. Chem. 42 (1996) 160163. D. Fisher, Maternal-fetal thyroid function in pregnancy, Clin. Perinatol. 10 (1983) 615626. J.R. Garber, R.H. Cobin, H. Gharib, et al., Clinical practice guidelines for hypothyroidism in adults: cosponsored by the American Association of Clinical Endocrinologists and the American Thyroid Association [published correction appears in Endocr Pract. 2013 JanFeb;19(1):175]. Endocr. Pract. 18 (6) (2012) 9881028. A. Hoofnagle, M. Roth, Improving the measurement of serum thyroglobulin with mass spectrometry, J. Clin. Endocrinol. Metab. 98 (4) (2013) 13431352. M.S. Jayasuriya, K.W. Choy, L.K. Chin, et al., Reference intervals for neonatal thyroid function tests in the first 7 days of life, J. Pediatr. Endocrinol. Metab. 31 (10) (2018) 11131116. L.A. Kaplan, C.T. Sawin (Eds.), Laboratory Medicine Practice Guidelines: Laboratory Support for the Diagnosis and Monitoring of Thyroid Disease. National Academy of Clinical Biochemistry, Washington, DC, 1996, 64 pp. A.J. Krause, B. Cines, E. Pogrebniak, et al., Associations between adiposity and indicators of thyroid status in children and adolescents, Pediatr. Obes. 11 (6) (2016) 551558.

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M. Lambert, F. Zech, P. DeNayer, J. Jamez, B. Vandercam, Thyroxine-binding globulin (but not cortisol-binding globulin and sex hormone-binding globulin) associated with the progression of human immunodeficiency virus infection, Am. J. Med. 89 (1990) 748751. Z. L´ımanov´a, Thyroid disease in the elderly, Onemocnˇen´ı sˇt´ıtne´ zˇ l´azy v seniorske´m vˇeku. Vnitr. Lek. 64 (11) (2018) 9931002. J. LoPresti, J. Fried, C. Spencer, J. Nicoloff, Unique alteration of thyroid hormone indices in the acquired immunodeficiency syndrome (AIDS), Ann. Intern. Med. 110 (1989) 970975. A. Parsa, A. Bhangoo, HIV and thyroid dysfunction, Rev. Endocr. Metab. Disord. 14 (2013) 127131. S.B. Soh, T.C. Aw, Laboratory testing in thyroid conditions - pitfalls and clinical utility, Ann. Lab. Med. 39 (1) (2019) 314. C.A. Spencer, Clinical utility and cost-effectiveness of sensitive thyrotropin assays in ambulatory and hospitalized patients, Mayo Clin. Proc. 63 (1988) 12141222. C.A. Spencer, J.S.L. Presti, A. Patel, R.B. Guttler, A. Eigen, D. Shen, et al., Applications of a new chemiluminometric thyrotropin assay to subnormal measurement, J. Clin. Endocrinol. Metab. 70 (1990) 453460. R. Styra, R. Joffe, W. Singer, Hyperthyroxinemia in major affective disorders, Acta Psychiatr. Scand. 83 (1991) 6163. M.I. Surks, I.J. Chopra, C.N. Mariash, J.T. Nicoloff, D.H. Solomon, American Thyroid Association: guidelines for use of laboratory tests in thyroid disorders, JAMA 263 (1990) 15291532. S. Valde´s, C. Maldonado-Araque, A. Lago-Sampedro, et al., Reference values for TSH may be inadequate to define hypothyroidism in persons with morbid obesity: [email protected] study, Obesity (Silver Spring) 25 (4) (2017) 788793. H. Van Deventer, S. Soldin, The expanding role of tandem mass spectrometry in optimizing diagnosis and treatment of thyroid disease, Adv. Clin. Chem. 61 (2013) 127152. M. Vanderpump, J. Ahlquist, J. Franklyn, R. Clayton, Consensus statement for good practice and audit measures in the management of hypothyroidism and hyperthyroidism, Br. Med. J. 313 (1996) 539544. L. Wartofsky, K. Burman, Alterations in thyroid function in patients with systemic illness: the “euthyroid sick syndrome, Endocr. Rev. 3 (1982) 164217. A. Weetman, Hypothyroidism: screening and subclinical disease, Br. Med. J. 314 (1997) 11751178.

Chapter 4

Disorders of the adrenal gland Roger L. Bertholf Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, TX, United States

The adrenal gland produces glucocorticoids, mineralocorticoids, sex hormones, and catecholamines. Deficiency or excess of any of these hormones results in clinical disease. Aldosterone, a mineralocorticoid that regulates sodium and water balance, is produced in the outermost of three layers that comprise the adrenal cortex, the zona glomerulosa. Cortisol, a glucocorticoid that regulates numerous metabolic processes, is produced in the middle layer of the adrenal cortex, the zona fasciculata. The innermost layer of the adrenal cortex, the zona reticularis, is primarily responsible for synthesizing and secreting androgenic steroid hormones that regulate sexual development. The central region of the adrenal gland, the medulla, produces catecholamines, which stimulate the sympathetic nervous system.

Adrenocortical insufficiency Deficiency of adrenocortical hormones causes serious, and often life-threatening, disease. Cortisol stimulates glucose production through hepatic gluconeogenesis and inhibits the action of insulin, resulting in hyperglycemia. Cortisol also helps maintain vascular tone and lysosomal integrity in cells, acting as an antistress or antishock hormone. Aldosterone promotes sodium reabsorption and potassium and hydrogen ion excretion by the renal tubules. The adrenal cortex shares with the gonads certain metabolic pathways for androgenic steroid hormone biosynthesis. The adrenocortical biosynthetic pathways to aldosterone, cortisol, and the adrenal androgens are shown in Fig. 4.1. The Leydig cells of the testes synthesize testosterone from cholesterol by way of the 17α-hydroxylase pathway to dehydroepiandrosterone (DHEA), the same as in the adrenal cortex. (Fig. 4.2 shows the predominant pathway to testosterone.). In the ovarian thecal cells, cholesterol is converted to androstenedione, which is taken up by the granulosa cells where it is converted to estrone and then estradiol (Fig. 4.3). Adrenocortical 

This chapter is an updated version of the same chapter included in the second edition of this book, authored by Drs. William E. Winter and Neil S. Harris. Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00004-2 © 2021 Elsevier Inc. All rights reserved.

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FIGURE 4.1 Biosynthesis of adrenocortical hormones. Cholesterol is converted to pregnenolone by the action of cholesterol side-chain cleavage enzyme, also named 20,22-lyase or desmolase, encoded by CYP11A. 17α-hydroxylase converts pregnenolone to 17-hydroxypregnenolone and progesterone to 17hydroxyprogesterone, forming the only common pathways to aldosterone and cortisol synthesis. 3βhydroxysteroid dehydrogenase (the only non-P450 family enzyme involved in adrenocortical steroid synthesis) converts pregnenolone to progesterone and 17-hydroxypregnenolone to 17-hydroxyprogesterone. Aldosterone production follows from progesterone by 3 sequential hydroxylations (21-hydroxylase and aldosterone synthase), to produce 18-hydroxycorticosterone, which aldosterone synthase oxidizes to aldosterone. The pathway from 17-hydroxyprogesterone to cortisol has only one intermediate, 11-deoxycortisol (21-hydroxylase), which is converted to cortisol by 11β-hydroxylase. Androgens dehydroepiandrosterone (DHEA) and androstenedione are synthesized by 17α-hydroxylase action on 17hydroxypregnenolone and 17-hydroxyprogesterone, respectively. DHEA can be converted to androstenedione by 3β-hydroxysteroid dehydrogenase. Aldosterone biosynthesis occurs exclusively in the zona glomerulosa, whereas cortisol is produced only in the zona fasciculata. DHEA and androstenedione can be produced in both the zona reticularis and, to a lesser degree, the zona fasciculata.

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HO

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17-OH Pregnenolone

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Dehydroepiandrosterone

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CSCE: Cholesterol side-chain cleavage enzyme (CYP11A) 17AH: 17a-Hydroxylase (CYP17) 17KSR: 17-Ketosteroid reductase (17BHSD3) 3BHD: 3b-Hydroxysteroid dehydrogenase (HSD3B2)

O

Testosterone

FIGURE 4.2 The testes and the adrenal gland share many steroidogenic pathways. In the testes, the predominant route to the production of testosterone is conversion of androstenedione or androstenedione (produced by the same pathway as in the adrenal gland) to testosterone by 3βhydroxysteroid dehydrogenase. There are other, minor pathways to testosterone production.

HO

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O

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Dehydroepiandrosterone

AR

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CSCE: Cholesterol side-chain cleavage enzyme (CYP11A) 17AH: 17a-Hydroxylase (CYP17) 3BHD: 3b-Hydroxysteroid dehydrogenase (HSD3B2) AR: Aromatase (CYP19A1) 17BHSD1: 17b-Hydroxysteroid dehydrogenase (HSD17B1)

17BHSD1

HO

HO

Estradiol

Estrone

FIGURE 4.3 In the ovary, androstenedione is produced in the thecal cells by the same biosynthetic pathway as in the testes. Androstenedione then migrates to the granulosa cells where it is converted to estrone by estrogen synthase (also called aromatase), and then estradiol by 11-β-hydroxysteroid dehydrogenase.

failure can be manifested in cortisol deficiency, aldosterone deficiency, or combined deficiencies of both hormones. Combined adrenocortical failure is the clinical definition of Addison disease [1].

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Detecting cortisol deficiency Circulating cortisol concentrations are influenced by the presence of cortisolbinding globulin (CBG), also known as transcortin or serpin A6, an α-globulin encoded by the SERPINA6 gene [2]. If hepatic synthesis of CBG is deficient due to malnutrition, malabsorption, or liver disease, or from protein loss due to nephrotic syndrome, total cortisol concentrations may decline below the reference interval, yet the biologically active free cortisol concentration is adequate. Conversely, elevated CBG concentrations can raise total cortisol levels without increasing free cortisol activity [3]. Normally, about 70% of circulating cortisol is bound to CBG, 20% is bound to albumin, and 10% of total cortisol is unbound and biologically active [4]. Many hormones are highly protein-bound, either to specific hormone-binding proteins or albumin, or most commonly, a combination of both. The proteins buffer the concentration of free, active hormone, preventing abrupt changes in hormonal activity that could have unpleasant, or even life-threatening physiological consequences. There is great interest in measuring free cortisol [5]. In intensive care settings, where patients are frequently malnourished, low CBG has the potential to prompt the overdiagnosis of Addison disease [6]. Measuring CBG allows calculation of a free cortisol index, but there is disagreement over the reliability of calculated free cortisol estimates [7]. Direct measurement of free cortisol concentration by equilibrium dialysis or ultrafiltration is cumbersome and rarely done. However, two alternatives exist for estimating the free cortisol concentration: salivary cortisol and urinary free cortisol (UFC). Salivary cortisol reflects free cortisol concentrations in the blood [8]. The saliva specimen is typically collected on a sponge and expressed into a test tube before filtration and analysis by either immunoassay or mass spectrometry [9]. In addition to cortisol, there is interest in measuring other steroids in saliva. A recent report describes the measurement of classical and 11-oxygenated androgens in human saliva using liquid chromatography-tandem mass spectrometry (LC-MS/MS) [10]. Steroid profiling by LC-MS/MS, at one time limited mostly to laboratories testing athletes for performance-enhancing drugs, is becoming increasingly common for clinical laboratory assessment of endocrine disorders.

Clinical symptoms and signs of adrenocortical insufficiency Deficient cortisol production causes vascular instability, hypotension, and hypoglycemia, especially at times of severe stress [11]. Patients feel weak with general malaise, loss of energy, and may display nausea, vomiting, or diarrhea. Primary adrenocortical failure is named after the 19th century English physician Thomas Addison, who described the disease in 1855. The clinical features of Addison disease include weakness, lethargy, increased fatigability, anorexia, nausea, vomiting, diarrhea (sometimes alternating with constipation), weight loss, abdominal pain, salt craving, muscle and joint

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pain, and postural dizziness. The clinical signs of Addison disease are mottled and increased skin pigmentation, decreased hair growth, hypotension, dehydration, tachycardia, and pallor. Laboratory findings in primary adrenal insufficiency include hypoglycemia, a normocytic normochromic anemia, modest eosinophilia, and relative lymphocytosis. Hyponatremia, hyperkalemia, hyperchloremia, decreased bicarbonate, mild systemic acidosis, relatively alkaline urine, and azotemia result from mineralocorticoid deficiency. Mineralocorticoid deficiency produces type IV renal tubular acidosis (RTA) when the failure to excrete hydrogen ions leads to systemic acidosis. The failure to excrete potassium leads to hyperkalemia, whereas enhanced loss of sodium produces hyponatremia. In contrast, types I and II RTA are characterized by hypokalemia due to potassium loss in the urine. RTA type I results from defective secretion of hydrogen ions in the distal convoluted tubule, which impairs the production of bicarbonate. RTA type II is caused by defective reabsorption of bicarbonate ions in the proximal convoluted tubule, leading to excessive bicarbonate loss in the urine. RTA type III is a combination of proximal and distal RTA. When bicarbonate is either not produced (RTA type II) or is lost in the urine (RTA types I and IV), sodium is reabsorbed in an attempt to reverse hyponatremia, which elevates the cotransported chloride. Thus as bicarbonate declines and chloride rises, the patient experiences a hyperchloremic, normal anion gap acidosis. Relative or absolute hyperchloremia may be masked by electrolyte depletion and hyponatremia. Psychiatric symptoms of organic brain syndrome (impaired memory, confusion), depression, and psychosis may affect up to 40% of RTA patients. In primary adrenal insufficiency, hyperpigmentation is a result of chronically elevated adrenocorticotropic hormone (ACTH) concentrations. Hyperpigmentation can also be observed in ectopic ACTH secretion causing Cushing syndrome, the POEMS syndrome (peripheral neuropathy, organomegaly, endocrine dysfunction, monoclonal gammopathy, and skin pigmentation), and uncommonly in Cushing disease. ACTH shares its N-terminal 13 amino acid sequence with one of the melanocyte-stimulating hormones, α-MSH, giving ACTH intrinsic melanotropic activity. In the intermediate lobe of the fetal pituitary gland, ACTH is enzymatically cleaved by prohormone convertase 2 to α-MSH and corticotropin-like intermediate lobe peptide. The intermediate lobe of the pituitary is not present in adults. Patients with secondary or tertiary adrenal insufficiency from, respectively, ACTH or corticotropin-releasing hormone (CRH) deficiency, do not exhibit hyperpigmentation. Also, patients solely deficient in ACTH or CRH do not manifest severe dehydration, hypotension, hyperkalemia, or acidosis because aldosterone production and release are primarily regulated by angiotensin II; ACTH is only a minor regulator of mineralocorticoid production and release. Symptoms associated with ACTH or CRH deficiency are usually mild compared with Addison disease (primary adrenocortical failure) and

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may display hyponatremia, but not hyperkalemia. Hyponatremia can develop in the setting of cortisol deficiency because of a reduced ability to excrete a free water load. Inability to increase glucocorticoid or mineralocorticoid production in response to acute stress precipitates “Addisonian crisis,” in which patients present with profound shock, hypotension, hypoglycemia, acidosis, hyponatremia, and hyperkalemia [12]. Addisonian crisis is much more typical of primary adrenal failure than isolated ACTH deficiency. Untreated, Addisonian crisis can be fatal.

Causes of adrenocortical insufficiency A list of some of the causes of adrenocortical insufficiency appears in Table 4.1. Autoimmune destruction of the adrenal gland is the most common cause of primary adrenocortical failure in the industrialized world [13]. Less commonly, the adrenal cortex can be destroyed by hemorrhage, infection

TABLE 4.1 Causes of adrenocortical insufficiency. Endogenous causes Autoimmune polyglandular syndrome types 1 and 2 Congenital adrenal hyperplasia Congenital adrenal hypoplasia Adrenoleukodystrophy Intra-adrenal hemorrhage Neoplastic infiltration Exogenous causes Infection (granulomatous disease, TB, sarcoidosis, histoplasmosis, blastomycosis, sporotrichosis, cryptococcosis, coccidioidomycosis) Drugs that block steroid synthesis Etomidate (inhibits 11β-hydroxylase) Mitotane (inhibits cholesterol side-chain cleavage enzyme, 11β-hydroxylase, 18hydroxylase, and 3β-hydroxysteroid dehydrogenase) Aminoglutethimide (inhibits cholesterol side-chain cleavage enzyme and aromatase) Ketoconazole (inhibits cholesterol side-chain cleavage enzyme, 17α-hydroxylase, 17,20-lyase, and 11β-hydroxylase) Metyrapone (inhibits 11β-hydroxylase) Benzodiazepines (inhibit 17- and 21-hydroxylase, and possibly 11β-hydroxylase) Osilodrostat (inhibits aldosterone synthase and 11-β-hydroxylase)

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[tuberculosis (TB) is the most common], trauma, adrenalectomy, and neoplastic disease. Adrenal dysgenesis, inborn errors of adrenocortical steroidogenesis, adrenoleukodystrophy (ALD), familial glucocorticoid deficiency, congenital adrenocortical hypoplasia, and certain drugs (e.g., the blockers of steroid synthesis: mitotane, aminoglutethimide, trilostane, ketoconazole, metyrapone, and the glucocorticoid receptor (GR) blocker RU-486), are rare causes of adrenocortical insufficiency. Familial glucocorticoid deficiency is a heterogeneous group of predominantly autosomal recessive disorders (but X-linked inheritance has also been described), with defects in the ACTH receptor gene located on chromosome 18p11.2 [14]. Degeneration of the fasciculata and reticularis layers of the adrenal cortex is observed. A Reye-like syndrome involving hepatic encephalopathy may also be observed [15]. ACTH deficiency, causing secondary adrenocortical insufficiency, can result from suppression of the hypothalamic-pituitary-adrenal (HPA) axis with exogenous glucocorticoids, destructive or compressive pituitary tumors and their treatment, hypothalamic tumors and their treatment (leading to CRH deficiency), cerebral trauma, irradiation, infection, bleeding, congenital malformations of the pituitary gland or hypothalamus, and idiopathic hypopituitarism. Several transcription factor mutations are now recognized as causes of ACTH deficiency [16]. In some cases, there are multiple anterior pituitary hormone deficiencies. Molecular testing is available for POU1F1 (Pit-1) [cytogenetic location 3p11.2, codes for POU transcription factor 1 (Pit1, growth hormone factor 1)] and PROP 1 (location 5q35.3, codes for paired like homeodomain factor 1) mutations. Isolated mineralocorticoid deficiency is rare, although during the development of Addison disease symptoms of mineralocorticoid deficiency can be seen before glucocorticoid deficiency becomes evident. The most common cause of isolated mineralocorticoid deficiency is inadequate renin production due to interstitial nephropathy or diabetic nephropathy. Rarely, aldosterone deficiency results from deficiency in aldosterone synthase (CYP11B2) [17]. This enzyme converts corticosterone into aldosterone via 18-hydroxylation to 18-hydroxycorticosterone, followed by 18-oxidation to aldosterone (Fig. 4.4). Patients with primary adrenocortical hypofunction usually experience cortisol deficiency or combined deficiencies of cortisol and aldosterone (primary adrenocortical failure). In patients with primary adrenocortical failure, glucocorticoid deficiency may precede mineralocorticoid deficiency (or rarely, vice versa). Isolated cortisol deficiency results from insufficient ACTH (secondary adrenocortical insufficiency) or CRH (tertiary adrenocortical insufficiency). Resistance to glucocorticoids and mineralocorticoids has been reported [18,19]. Glucocorticoid resistance syndrome (GRS) is caused by loss of function mutations in the NR3C1 gene, located on chromosome 5q31, which encodes the glucocorticoid receptor (GR). Similar to other forms of primary adrenal insufficiency, ACTH is elevated in GRS with consequently increased

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AS

O OH

HO

OH

AS

O

O

Corticosterone

O

18-hydroxycorticosterone

Aldosterone

Key: AS: Aldosterone synthase (CYP11B2)

FIGURE 4.4 Adosterone synthase converts corticosterone to 18-hydroxycorticosterone, and then aldosterone. There are rare genetic deficiencies in the aldosterone synthase enzyme, coded by CYP11B2. Aldosterone synthase is related to 11β-hydroxylase, which is coded by CYP11B1.

levels of adrenal androgens causing hirsutism, oligomenorrhea, infertility, and precocious puberty. Elevated ACTH also leads to 11-deoxycortisol (11-DOC) overproduction, causing hypokalemic hypertension. It is unclear whether increased circulating cortisol concentrations totally compensate for glucocorticoid resistance. In addition to elevations in ACTH and cortisol, there is increased UFC excretion and increased urinary excretion of 17-hydroxycorticosteroids. Mutations in the mineralocorticoid receptor gene, NR3C2, located on chromosome 4q31, or one of the three genes encoding the subunits of the epithelial sodium channel ENaC (SCNN1A, SCNN1B, and SCNN1G), located on chromosome 12p13.31, cause pseudohypoaldosteronism (PHA) Type 1 [20]. There are two subtypes of PHA Type 1: In the autosomal dominant form, associated with mutations in the NR3C2 gene, the kidney is selectively resistant and the clinical presentation is of early-onset salt-wasting that resolves within a few years of birth. More severe is the autosomal recessive, multiorgan (or generalized) form of PHA type 1, caused by ENaC mutations, that involves the sweat glands, the salivary glands, and colon in addition to the kidney. This disorder does not improve with advancing age. PHA type 2 does not cause salt-wasting. Although not usually predisposing to Addisonian crisis from severe adrenal suppression, nasal steroids can nevertheless affect the HPA axis [21]. Symptomatic adrenal insufficiency from withdrawal of inhaled steroids in asthma has been reported [22] as well as adrenal crisis in children receiving high doses of inhaled steroids [23 25]. Intracranial hypertension can also result from inhaled steroids [26]. Bone density is another safety concern when high-dose inhaled steroids are used in children [27], but not in adults [28]. Kannisto et al. reported that 25% of children treated with inhaled steroids displayed mild adrenal insufficiency when challenged with a 0.5 μg/ 1.73 m2 dose of ACTH [29]. The inhaled glucocorticoids fluticasone and ciclesonide, compared with beclomethasone and budesonide, may produce less adrenal suppression [30].

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Laboratory investigation of adrenocortical insufficiency Plasma cortisol levels of ,5 μg/dL in the morning or during times of stress are presumptive evidence of glucocorticoid (cortisol) deficiency. Levels $ 18 20 μg/dL essentially exclude cortisol deficiency. If Addison disease is suspected and the patient is in crisis, a plasma cortisol level should be requested and the patient should be administered saline and glucocorticoids in pharmacologic doses (4 mg dexamethasone per m2 of body surface area; dexamethasone is preferred because it has higher glucocorticoid activity than cortisol and does not interfere with cortisol measurements) [31]. The patient should be treated for presumed Addisonian crisis until the cortisol concentration is determined, but the cortisol level is not urgent because dexamethasone treatment has little short-term risk. ACTH can be measured at a later time if desired to confirm the diagnosis of primary adrenocortical insufficiency. A flow diagram for the investigation of suspected glucocorticoid deficiency appears in Fig. 4.5.

Patients presenting with Addisonian crisis When a patient presents with hypotension, hypoglycemia, hyperpigmentation, hyponatremia, hyperkalemia, and systemic acidosis that are not explained by history, the diagnosis of Addison disease (primary adrenocortical failure) is strongly suggested. The diagnosis is confirmed by a plasma cortisol concentration of ,5 μg/dL at the time of stress. A concomitant elevation in plasma ACTH supports the diagnosis of Addison disease. Additional confirmation of the diagnosis can be obtained with a 1-h ACTH stimulation test, or an insulin tolerance test (ITT). In the ITT, insulin is administered to induce hypoglycemia, which is a powerful central stimulant for the release of CRH, inducing ACTH and subsequent cortisol secretion. Some authorities state that the ITT is indicated only when recent ACTH or CRH deficiency is suspected, and the 1-h ACTH stimulation test could be falsely normal. Otherwise, the 1-h ACTH stimulation test provides similar information to the ITT. In patients with a clinical history that suggests potential ACTH deficiency, the ITT can be performed to concurrently evaluate the cortisol and growth hormone (GH) response to hypoglycemia. The 1-h ACTH stimulation test is much easier, safer, and cheaper to perform than the ITT. Because neither of these tests differentiates primary adrenocortical failure from ACTH deficiency, a 2-day (or longer) ACTH stimulation test can be performed instead. Despite prolonged stimulation from exogenous ACTH in the 2-day or longer ACTH stimulation test, in Addison disease the cortisol levels remain ,5 μg/dL, confirming primary adrenocortical failure. In the 1-h ACTH stimulation test, the traditional dose is 250 μg. In multiday ACTH stimulation tests, the dose is 250 μg/day. Because 250 μg of ACTH is a suprapharmacologic dose, investigators have studied the

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FIGURE 4.5 Flow chart for evaluation of glucocorticoid deficiency when suspected on clinical grounds. In patients who present in Addisonian crisis with supporting electrolyte changes and low cortisol, the diagnosis of Addison disease is established. In patients with suspected adrenocortical insufficiency, stress or morning cortisols greater than 18 20 μg/dL virtually exclude glucocorticoid insufficiency. However, further testing can involve either a 1-h ACTH (Cortrosynt) stimulation test or an insulin tolerance test (ITT). At least one of these tests is required in patients with suspected glucocorticoid insufficiency when stress or morning cortisol levels are less than 18 20 μg/dL. After ACTH injection (250 μg) or insulin-induced hypoglycemia, cortisol levels greater than 18 20 μg/dL with a change from baseline greater than 7 10 μg/dL rules out glucocorticoid insufficiency. Abnormal responses are consistent with glucocorticoid insufficiency. The 2-day ACTH stimulation test is helpful in differentiating primary (Addison disease) from central (ACTH deficiency) adrenal insufficiency. With Addison disease, mineralocorticoid function must be evaluated by measuring electrolytes, aldosterone, and renin (not shown). Although not commonly used, the CRH stimulation test can assist in differentiating primary ACTH deficiency from CRH deficiency (secondary ACTH deficiency).

usefulness of giving only 1 μg in hopes of detecting subtler degrees of adrenal insufficiency [32]. Indeed, there are patients who have normal responses to 250 μg of ACTH yet inadequate responses to 1 μg of ACTH. This remains a point of controversy, and the 1 μg ACTH dose has not been accepted as universally superior to the 250-μg dose, particularly in children [33]. A lower cortisol response to 250 μg of ACTH may not predict better outcomes in patients with septic shock who are treated with glucocorticoids [34]. An ACTH dose of 0.5 μg has been investigated in premature infants [35]. Very preterm infants requiring ventilation had lower cortisol responses to stimulation than less ill, nonventilated, preterm infants. The need to rapidly detect glucocorticoid deficiency remains clinically important in the intensive care setting where the use of glucocorticoids in sepsis remains controversial [36]. If the patient can be shown to be glucocorticoid deficient, administration of glucocorticoids is justified.

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Patients with suspected glucocorticoid deficiency who do not present acutely ill If a patient presents with signs or symptoms suggestive of, but not diagnostic for, glucocorticoid deficiency, a laboratory investigation should be pursued before glucocorticoid replacement therapy is initiated. A patient ill enough to require acute glucocorticoid therapy should be evaluated the same as patients in Addisonian crisis. Fasting plasma cortisol levels should be obtained on at least two separate days between 0600 and 0800 h, preferably at the same time each day. Stress or morning fasting cortisol concentrations ,5 μg/dL are presumptive evidence of glucocorticoid deficiency (Fig. 4.5). Stress or morning fasting cortisol concentrations 5 18 μg/dL are potentially abnormal. Stress or morning fasting cortisol values $ 18 20 μg/dL essentially exclude glucocorticoid insufficiency, and further workup is not usually indicated. However, if a stress or morning cortisol less than 18 20 μg/dL raises the suspicion of glucocorticoid deficiency, a 1-h ACTH stimulation test or an ITT should be performed. Following ACTH stimulation, a peak cortisol of ,18 20 μg/dL and a change in cortisol over baseline of ,7 10 μg/dL (Δcortisol) are abnormal and are diagnostic for glucocorticoid deficiency. Further evaluation may or may not be indicated depending upon the clinical circumstances. For example, if the ACTH level is elevated at the time that the cortisol is low, primary adrenocortical insufficiency is highly likely, and further testing usually is not needed. If adrenal autoantibodies are detected, and the finding is associated with low stress or morning cortisol levels and an abnormal 1-h ACTH stimulation test or ITT, further testing should not be pursued because these results confirm autoimmune Addison disease. Once a patient has been confirmed as having glucocorticoid deficiency, the multiday ACTH stimulation test is useful for differentiating primary glucocorticoid deficiency (primary adrenocortical failure, Addison disease) from secondary (or central) glucocorticoid deficiency due to inadequate ACTH. When the adrenal gland is exposed to sustained exogenous ACTH stimulation, in cases of endogenous ACTH deficiency, the previously atrophied adrenal gland will begin to produce cortisol. However, in primary adrenocortical insufficiency, even with supraphysiologic exogenous ACTH stimulation, the adrenal gland is unable to increase cortisol production. Thus an abnormal multiday ACTH stimulation test confirms primary glucocorticoid deficiency because there is a deficient cortisol response to ACTH even when maximal doses of ACTH are applied. In cases of Addison disease or ACTH deficiency, further confirmation of a defect in the HPA axis can be achieved by a metyrapone (Metopirone) stimulation test. Metyrapone is a pyridine derivative that inhibits 11βhydroxylase, the enzyme that converts 11-deoxycortisol to cortisol in the zona glomerulosa of the adrenal cortex (see Fig. 4.1). Hence, metyrapone inhibits cortisol production. A typical protocol involves administering 30 mg

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metyrapone per kg of body weight at midnight and then measurement of plasma cortisol, ACTH, and 11-deoxycortisol at 0800 the next morning. A normal response is a decrease in cortisol, and an increase in ACTH ( . 150 pg/mL) and 11-deoxycortisol ( . 7 μg/dL). Some investigators believe that metyrapone testing is the most sensitive approach to determining adrenal insufficiency [37]. Patients may have normal basal concentrations of cortisol and ACTH, normal responses to ACTH stimulation, and normal responses to insulin-induced hypoglycemia, yet have deficient responses to metyrapone. Presently, metyrapone is not available for clinical use in the United States. Measuring morning ACTH levels may also assist in differentiating primary from secondary glucocorticoid deficiency. In primary glucocorticoid deficiency, ACTH is elevated, whereas in secondary glucocorticoid deficiency, ACTH is either low or inappropriately normal (when cortisol is low). However, ACTH is labile, and its concentration is highly variable over the course of a day because of the normal diurnal variation that is observed in the operation of the HPA axis. Therefore ACTH is not as reliable a marker for glucocorticoid deficiency as, for example, thyrotropin (TSH) is for hypothyroidism. ACTH deficiency is diagnosed when the multiday ACTH stimulation test is normal, yet the 1-h ACTH stimulation test or ITT is abnormal. ACTH deficiency can be primary (due to pituitary disease) or secondary (due to hypothalamic disease). As noted earlier, an inappropriately normal or low concentration of ACTH concurrent with a depressed cortisol level in the morning or at a time of stress also supports the diagnosis of ACTH deficiency. Because the differentiation between hypothalamic and pituitary disease is usually accomplished radiographically, the CRH test is only rarely used to differentiate primary from secondary ACTH deficiency. If primary glucocorticoid deficiency (primary adrenocortical failure) is confirmed, evaluation of mineralocorticoid status is indicated. Minimal evaluations include serum electrolytes and plasma renin concentration or activity (plasma renin activity; PRA) to define whether there is: (1) normal mineralocorticoid function (normal electrolytes, normal renin), (2) compensated mineralocorticoid deficiency (normal electrolytes, elevated renin), or (3) uncompensated mineralocorticoid deficiency (hyponatremia, hyperkalemia, and elevated renin).

Tests of adrenal function Adrenocorticotropic hormone stimulation tests The 1-h ACTH stimulation test determines whether or not the adrenal cortex can respond to a single, acute pharmacologic dose of ACTH with an increase in cortisol production. Although pig-derived corticotropin (ACTH) is available, it is not FDA-approved for diagnostic use. For diagnostic testing, a

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synthetic oligopeptide containing the active N-terminal 24 amino acids of corticotropin (ACTH has 39 amino acids) is used. The 24-amino acid analog is tetracosactide, also known as cosyntropin and by the trade names Cortrosyn (Amphastar Pharmaceuticals Rancho Cucamonga, CA), and Synacthen (Mallinckrodt, Staines-upon-Thames, United Kingdom—not available in the United States). Cosyntropin is available in generic form, as well. If there is primary gland failure or gland atrophy from ACTH deficiency, peak cortisol following a 250 μg dose of Cortrosyn is typically ,18 20 μg/ dL and the change from baseline is ,7 10 μg/dL. The absolute cortisol level is a more sensitive measure of adrenocortical response than the change in cortisol from baseline. The 1-μg Cortrosyn stimulation test has been proposed as an alternative to the 250-μg test because it is closer to physiological ACTH concentrations and may reveal a subtle degree of adrenal insufficiency not evident with the suprapharmacologic 250-μg Cortrosyn dose. However, a recent metaanalysis of studies comparing the two protocols concluded that both approaches have similar diagnostic accuracy [38]. The detection of subtle cortisol deficiency is believed to be very important in the intensive care setting [7,39]. Prolonged Cortrosyn administration (8-h, 2-day, and 3- to 5-day ACTH stimulation tests) provides a higher level of sustained corticotropin stimulation to overcome adrenocortical atrophy that may have occurred from chronic ACTH deficiency. In primary adrenal failure, prolonged ACTH stimulation does not produce cortisol concentrations at or above the 18 20 μg/ dL threshold regardless of the dose or duration of the Cortrosyn challenge. When hypocortisolism is the result of ACTH or CRH deficiency, prolonged and sustained exogenous cosyntropin will stimulate the atrophied gland to redevelop and produce a normal cortisol response. In acute ACTH or CRH deficiency, the serum cortisol response to stress is low whereas the response to exogenous cosyntropin can be normal. Several weeks or months of ACTH or CRH deficiency are required to induce atrophy of the adrenal cortex and a correspondingly deficient response to exogenous ACTH stimulation. If there is an abnormal serum cortisol level in response to stress and a normal Cortrosyn stimulation test when acute ACTH or CRH deficiency is a clinical possibility, an ITT or metyrapone test may be indicated. There is evidence that cosyntropin may interfere with some methods used to measure ACTH, although the stimulation test does not require measurement of ACTH.

The 1-h adrenocorticotropic hormone stimulation test After a specimen is collected for baseline plasma cortisol, Cosyntropin (Cortrosyn) is administered intravenously or intramuscularly (25 units, or 250 μg) as a bolus. Cortisol is then measured in samples collected 30 and 60 min after administration. Some protocols specify collection of only one poststimulation sample at 45 min. If the adrenal cortex is functioning

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normally, the stimulated cortisol is .18 20 μg/dL, and the change from baseline is typically .7 10 μg/dL. Between the two criteria, the absolute cortisol level is more diagnostic. If the basal cortisol is .18 20 μg/dL, ACTH administration may produce little further elevation in serum cortisol because the patient may already be maximally stimulated at baseline. Since plasma cortisol levels can be influenced by the concentration of CBG, it has been suggested that salivary cortisol after 250 μg cosyntropin stimulation may provide additional diagnostic value in patients predisposed to CBG alterations or patients with borderline plasma cortisol responses [40].

8-h, 2-day, and 3- to 5-day adrenocorticotropic hormone stimulation tests When ACTH or CRH deficiency is suspected, and the physician wants to prove that the adrenal gland otherwise functions normally, a prolonged ACTH stimulation test may be performed. There are several variations intended to provide sufficient ACTH stimulation to reverse any atrophy that has developed from chronic ACTH or CRH inadequacy. In the 8-h test, a baseline 24-h urine sample for UFC is measured. Assessment of UFC provides an integrated 24 h measurement of cortisol production. On day 2, a 24-h urine collection is begun, and Cortrosyn is delivered intravenously (25 units, or 250 μg) over 8 h. To demonstrate adequate adrenal function, the plasma cortisol at 60 min should be .18 20 μg/dL and at 8 h, .25 μg/dL, and UFC on day 2 should be approximately threefold greater compared with the baseline collection. With endogenous ACTH or CRH deficiency, the 8-h test may not be long enough to elicit a normal cortisol response. The 8-h ACTH stimulation test is rarely performed. The 2-day and 3- to 5-day tests deliver Cortrosyn every day (an infusion over 8 h or via intramuscular injection or a continuous infusion of ACTH) with similar expected increases in cortisol and UFC. Presently, the 2-day test is the most popular prolonged ACTH stimulation test (1 day of baseline urine collection and 2 days of continuous ACTH administration). After maximal exogenous ACTH stimulation, if cortisol does not increase appropriately ( . 18 20 μg/dL), primary adrenal insufficiency is confirmed. Finding an elevated basal plasma ACTH concentration is consistent with a primary failure of cortisol production.

Corticotropin-releasing hormone stimulation test In central glucocorticoid deficiency, ACTH versus CRH deficiency can be differentiated using the CRH stimulation test. In this test, if ACTH rises after CRH administration, glucocorticoid deficiency is due to CRH deficiency. However, if there is no increase in ACTH after CRH administration, there is evidence for pituitary failure to secrete ACTH. In the CRH stimulation test,

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ovine CRH (corticorelin ovine triflutate, trade name Acthrel; Ferring Pharmaceuticals, Parsippany, NJ) is administered by intravenous push in a dose of 1 μg/kg up to a maximum of 100 μg/dose [41]. ACTH can be measured at baseline and at 15, 30, and 60 min. The 60-min sample is the most important observation.

Insulin-induced hypoglycemia test Hypoglycemia is a strong stimulus of the HPA axis. In the insulin-induced hypoglycemia test (or ITT), 0.1 units of insulin per kg body weight are administered by intravenous push. Cortisol is measured at baseline and at 10, 20, 40, and 60 min. In obese individuals, or in individuals with other causes of suspected insulin resistance, the insulin dose should be increased to 0.15 U/kg. The ITT is a potentially dangerous test that can induce severe hypoglycemia and seizures, especially in children who are GH and/or cortisoldeficient. The ITT must be supervised continuously by a physician or other health care provider who is trained in the recognition of hypoglycemia and its treatment. Adequate intravenous access must be maintained during the test, and the blood glucose must be measured with every blood draw. If the patient develops signs or symptoms of hypoglycemia during the test, blood glucose should be measured immediately with a bedside monitor. If the blood glucose is ,60 mg/dL, a sample should be sent to the central laboratory for stat blood glucose measurement. If the blood glucose is confirmed to be ,40 mg/dL, and hypoglycemic symptoms have not improved, the test should be terminated after a final sample for cortisol measurement is obtained. Intravenous glucose or glucagon (1 mg intravenously) can then be administered, and the patient can be fed. If a child suffers a seizure during the ITT, the test should be immediately terminated with the intravenous administration of 1 mL/kg 50% dextrose (D50). Because of the high osmolality of D50, it is best administered rapidly into a large central vein or with great care more slowly into a smaller peripheral vein.

Glucagon stimulation test Glucagon stimulates ACTH release from the pituitary, and the glucagon stimulation test (GST) has been proposed as an alternative to the ITT for diagnosis of GH deficiency and secondary adrenal insufficiency [42]. Compared with the ITT, glucagon stimulation was equally sensitive for the detection of cortisol deficiency. The safety and simplicity of the GST is a considerable advantage over the ITT. In a typical protocol, 1 1.5 mg of glucagon is administered subcutaneously or intramuscularly. Peak plasma cortisol concentrations .18 μg/dL following glucagon stimulation are consistent with secondary adrenal insufficiency [43]. The GST is most useful for assessing GH deficiency in adults.

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Mineralocorticoid assessment Aldosterone concentrations vary with salt intake and body position. Salt restriction can raise aldosterone levels approximately eightfold. To account for these variables, a 24-h urine sodium should be measured when plasma aldosterone is measured. Aldosterone levels can then be interpreted in relation to salt intake. Prior to the measurement of basal plasma aldosterone concentrations, patients should receive their normal salt intake for 24 48 h, and be in the supine position for 30 min prior to collection of the blood specimen. Upright posture can double the plasma aldosterone concentration. Alternatively, a 24h urine can be collected for aldosterone excretion to assess the daily aldosterone production. When aldosterone production and secretion are insufficient, normally there is an increase in plasma renin or PRA. On normal sodium intake, aldosterone excretion is 3 10 μg/day. Values of 20 50 μg/day are achieved with a low-salt diet. If the diagnosis of mineralocorticoid deficiency is in question, plasma aldosterone and PRA should be obtained after 3 h of upright posture or after a day or more of salt restriction. Normally with such measures, aldosterone and PRA rise. Salt restriction should be undertaken with care so as not to precipitate Addisonian crisis. Low serum aldosterone, low urinary excretion of aldosterone, and elevated PRA are strong evidence of primary adrenal insufficiency. If electrolytes are normal, these tests of mineralocorticoid function may be performed in patients being evaluated for primary adrenocortical failure. However, if the patient history suggests mineralocorticoid deficiency and includes hyponatremia, hyperkalemia, and a normal anion gap metabolic acidosis, there is no additional benefit from aldosterone measurement. Aldosterone deficiency is treated with fludrocortisone (9α-fluorohydrocortisone, brand name Florinef, Monarch Pharmaceuticals, Inc., Bristol, TN). Patients with mineralocorticoid deficiency who are undertreated can display electrolyte abnormalities and hypertension from elevated renin and angiotensin II that cause vasoconstriction. In severe untreated mineralocorticoid deficiency, hypotension can quickly develop, followed by shock and/or death during times of severe stress.

Evaluation for specific causes of primary adrenocortical insufficiency Autoimmune Addison disease In the western world, autoimmune destruction of the adrenal glands is responsible for approximately 85% of Addison disease, excluding cases of congenital adrenal hyperplasia (CAH) [44]. Prior to the discovery of antibiotics, Addison disease most commonly resulted from TB. The diagnosis of autoimmune Addison disease is established by identification of

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autoantibodies to 21-hydroxylase [44,45], typically measured using radioimmunoassay or chemiluminescent-labeled 21-hydroxylase and immunoprecipitation. An enzyme-linked immunosorbent assay (ELISA) has also been described for measuring 21-hydroxylase autoantibodies [46]. Alternatively, the concentration of the 21-hydroxylase substrate, 17-OHP, can be measured, and results .1000 ng/dL are diagnostic for 21-hydroxylase deficiency, although the test does not discriminate between autoimmune and congenital causes [45]. Based on a European Expert Consensus Statement for diagnosis, treatment, and follow-up of primary adrenal insufficiency [44], when a patient with suspected primary adrenal insufficiency is negative for 21-hydroxylase autoantibodies, imaging studies are recommended, and if the patient is male, very long-chain fatty acids (VLCFA) to investigate the possibility of ALD. In children and adolescents presenting with primary adrenal insufficiency, the diagnosis of autoimmune polyendocrine syndrome type 1 (APS-1) should be considered. APS-1 is a rare autosomal recessive disorder presenting in childhood, caused by mutations in the autoimmune regulator (AIRE) gene on chromosome 21 [47]. The AIRE encodes a transcription factor that appears to modulate self-antigen expression in the thymus, which is important during T-cell ontogeny. The diagnosis of APS-1 is based on the finding of two of the following three disorders: autoimmune Addison disease, mucocutaneous candidiasis, and hypoparathyroidism. Other frequent manifestations of APS1 include enamel hypoplasia and enteropathy. Females with APS-1 often experience ovarian insufficiency. Less common complications include bilateral keratitis, periodic fever and rash, hepatitis, pneumonitis, nephritis, exocrine pancreatitis, and functional asplenia. Rarely, retinitis, metaphyseal dysplasia, red cell aplasia, and polyarthritis occur [48]. Autoimmune polyendocrine (or polyglandular) syndrome type 2 (APS-2) is a polygenic disorder that presents in later childhood or adulthood. APS-2 is far more common than APS-1 and occurs more frequently in women than in men. The diagnosis of APS-2 is based on the presence of two of three endocrinopathies: autoimmune Addison disease, type 1 diabetes, or autoimmune thyroid disease (AITD; Hashimoto thyroiditis, atrophic thyroiditis, or Graves disease). Serologic markers of type 1 diabetes include islet cell cytoplasmic autoantibodies, glutamic acid decarboxylase autoantibodies, IA-2 autoantibodies, and, in children, insulin autoantibodies. Markers of AITD are discussed in the chapter on thyroid disorders. The concurrence of Addison disease and AITD has been termed Schmidt syndrome [49], whereas the addition of type 1 diabetes identifies Carpenter syndrome [50]. Because of the association of type 1 diabetes with human leukocyte antigen-DR3 and -DR4, APS-2 is associated with these same human leukocyte antigens [51]. Although adrenal autoantibodies have been reported in the syndrome of immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX), there are no reports of Addison disease in this disorder. IPEX is a rare, autosomal recessive disorder associated with mutations in the FoxP3 transcription

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factor, a protein that influences the development and function of regulatory T cells [52]. In asymptomatic subjects positive for adrenal cortex autoantibodies (ACA) and/or autoantibodies to 21-hydroxylase, yearly cosyntropin stimulation testing can be used to detect incipient autoimmune adrenal insufficiency. Deficient cortisol responses to cosyntropin predict the development of frank primary adrenal insufficiency in such autoantibody-positive individuals [53]. In these cases, anticipatory, long-term hormone replacement can be provided to prevent the development of serious illness, such as Addisonian crisis.

Congenital adrenal hyperplasia CAH is a group of autosomal recessive disorders caused by mutations in the genes encoding enzymes involved in the biosynthesis of cortisol. The most common cause of CAH (95% of cases) is a mutation in CYP21A2, which codes for 21-hydroxylase, the enzyme that converts progesterone or 17-hydroxyprogesterone (17-OHP) to deoxycorticosterone (DOC) or 11-deoxycortisol (11-DOC), respectively (see Fig. 4.1). Deficiency in 21-hydroxylase is characterized by impaired adrenal production of aldosterone and cortisol. CYP21A2 overlaps another gene, TNXB, which encodes Tenascin-X, an extracellular matrix protein. Mutations affecting Tenascin-X cause an autosomal recessive form of Ehlers Danlos Syndrome (EDS), and up to 10% of patients with saltwasting CAH display some of the clinical features of EDS [54]. The remaining 5% of CAH cases are caused by deficient 11β-hydroxylase, 17-hydroxylase, 3β-hydroxysteroid dehydrogenase type 2, P45 oxidoreductase, and P450 cholesterol side-chain cleavage enzyme deficiencies, and all but 11β-hydroxylase deficiency are exceedingly rare. CAH is the most common cause of adrenocortical insufficiency in newborns. Rarely, adrenal failure occurs in newborns from traumatic adrenal hemorrhage or adrenal hemorrhage from sepsis. Because of the large fetal adrenal gland relative to body size, mechanical trauma to the gland is more likely at birth than at any other time during life. Neonatal screening for 21-hydroxylase deficiency by measurement of its substrate, 17-OHP, is relatively common in developed countries, but has a low positive predictive value. Measurement of 17-OHP by LC-MS/MS decreases the rate of false-positive results [55]. Measurement of 21deoxycortisol, a side-product formed by hydroxylation of 17-OHP by 11βhydroxylase, has been suggested as a better screening test because it is only formed in significant amounts when there is excess 17-OHP [56]. Older tests used to assess defects in adrenal steroid metabolism focused on urinary products of steroid metabolism; these include pregnanetriol, tetrahydrodeoxycortisol, 17-ketogenic steroids, 17-ketosteroids, and 17hydroxycorticosteroids. All of these are hepatic metabolites of circulating steroids, but these tests are no longer in common use.

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In response to cortisol deficiency, CAH patients overproduce ACTH, as well as the adrenal androgens DHEA and androstenedione (due to the accumulation of their precursors, 17-hydroxypregnenolone and 17-OHP). In females, overproduction of the adrenal androgens induces in utero virilization of the external genitalia, which can vary from mild fusion of the labia, to marked labial fusion with clitoromegaly, to complete masculinization with male external genitalia but with bilaterally undescended “testes,” a misnomer because they are in fact ovaries in their normal intra-abdominal location. Untreated, females will continue to virilize postnatally. The more profound the defect in cortisol biosynthesis, the greater the elevation in ACTH and the greater the degree of virilization in the female fetus. In males, adrenal androgen overproduction causes precocious puberty and some degree of hyperpigmentation of the male genitalia. As with virilization of females, the more profound the defect in cortisol biosynthesis, the greater the degree of precocious puberty in males [57]. In CAH, glucocorticoid insufficiency leads to malaise, failure to thrive, hypoglycemia, and vascular instability. In approximately one-half of infants affected with 21-hydroxylase deficiency, both cortisol and aldosterone production are impaired, the latter causing hyponatremia, hyperkalemia, acidosis, dehydration, and hypotension. In its most severe form, this type of “saltwasting” 21-hydroxylase deficiency clinically presents at 10 14 days of age with Addisonian crisis. Because salt-wasting 21-hydroxylase deficiency is a more severe expression of 21-hydroxylase deficiency, it is associated with greater degrees of virilization in males and females. Untreated, mortality is very high in salt-wasting 21-hydroxylase deficiency CAH. The nonsaltwasting form of the disease is referred to as “simple virilizing” 21hydroxylase deficiency CAH. In 11β-hydroxylase deficiency, the second most common cause of CAH, mineralocorticoid deficiency does not occur. Therefore although females with 11β-hydroxylase deficiency present with ambiguous genitalia, and males display precocious puberty, salt-wasting does not occur. Thus electrolyte and acidbase balance is normal, and Addisonian crisis does not develop in the newborn period or later. However, later in childhood, hypertension, with or without hypokalemia, can develop [58]. This is discussed in more detail as follows. The diagnosis of 21-hydroxylase and 11β-hydroxylase deficiencies depends upon elevated concentrations of 17-OHP in plasma or serum. Affected infants typically have random plasma 17-OHP concentrations .5000 ng/dL ( . 150 nmol/L; reference range ,130 ng/dL, ,4.2 nmol/L). 21-Hydroxylase deficiency and 11β-hydroxylase deficiency CAH can be differentiated by measuring 11-deoxycortisol. In 21-hydroxylase deficiency, both 11-deoxycortisol and cortisol are low, whereas in 11β-hydroxylase deficiency, deoxycortisol is elevated, and cortisol is low. Table 4.2 compares clinical and biochemical features of 21-hydroxylase and 11β-hydroxylase deficiency CAH.

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TABLE 4.2 Clinical and biochemical features of 21-hydroxylase and 11βhydroxylase deficiency CAH. Enzyme deficiency 21-Hydroxylase

11β-Hydroxylase

Percent of CAH cases

90% 95%

B5%

Gene

CYP21

CYP11B1

Plasma 17-OHP

Elevated

Elevated

Plasma 11-DOC

Depressed

Elevated

Plasma corticosterone

Depressed

Depressed

Plasma 11deoxycortisol

Depressed

Elevated

Plasma cortisola

Depressed

Depressed

Ambiguous genitalia

In females

In females

Precocious puberty

In males

In males

Salt loss

Approximately half of patients

Does not occur

Hypertension

No

Late childhood/ adolescence

a

In mild forms of CAH, cortisol may be low-normal due to the compensatory increase in ACTH.

A “late-onset” or “attenuated” (nonclassical) form of 21-hydroxylase deficiency can present in female adolescents as hirsutism or virilization. Late-onset 21-hydroxylase-deficient CAH is much milder than the classical (“virilizing”) form of 21-hydroxylase deficiency of prenatal onset. Although baseline and post-ACTH stimulation 17-OHP levels in attenuated 21-hydroxylase-deficient CAH are higher than in controls, the values are not as high as those seen in classical 21-hydroxylase deficiency. Graphs are available in the literature that allow basal 17-OHP to be plotted against postACTH 17-OHP in diagnosing CAH gene carriers versus attenuated 21-hydroxylase deficiency versus classical 21-hydroxylase [59]. In mild forms of CAH, cortisol may be low-normal from a compensatory elevation in ACTH. Gene carriers usually have normal basal levels, but elevated 17-OHP in response to ACTH stimulation. 21-Hydroxylase and 11β-hydroxylase deficiency CAH patients can be followed clinically during glucocorticoid replacement therapy by measuring an early morning 17-OHP or by measuring androstenedione at any time of the day [60]. The treatment of both forms of CAH is cortisol replacement. In 21-hydroxylase deficiency CAH, aldosterone is replaced with fludrocortisone.

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With adequate glucocorticoid replacement, 17-OHP or androstenedione should not be elevated above the upper limit of the reference interval [57]. Insufficient glucocorticoid replacement can allow excess adrenal androgen production and postnatal virilization, accelerated growth, premature closure of the epiphyses, and short stature [60]. Overtreatment with glucocorticoids can produce poor growth from iatrogenic Cushing syndrome. Hypertension, with or without hypokalemia, may also develop in children with 11β-hydroxylase deficiency [58]. Hypokalemic hypertension results from elevated concentrations of 11-DOC, but the cause of 11-DOC elevations is not well understood. 11-DOC accumulation is not due to the inborn 11β-hydroxylase error since expression of the CYP11B1 mutation is limited to the zona fasciculata; CYP11B2 converts 11-DOC to corticosterone in the zona glomerulosa and is not expressed in the fasciculata. Theoretically, 11-DOC is elevated in cases of CYP11B1 mutations because of excessive ACTH stimulation of the glomerulosa; conversion of progesterone to 11-DOC by 21hydroxylase is ACTH-dependent. 11-Deoxycortisol has neither substantial glucocorticoid nor mineralocorticoid activity, therefore elevations in 11deoxycortisol cannot explain hypertension in 11β-hydroxylase deficiency. A rare cause of CAH is a deficiency in 17α-hydroxylase, which converts pregnenolone to 17-hydroxypregnenolone and progesterone to 17hydroxyprogesterone. In 17α-hydroxylase deficiency CAH, production of cortisol is deficient but mineralocorticoids are overproduced due to accumulation of pregnenolone, causing salt and water retention and consequently hypertension. Because the gonads share many steroidogenic pathways with the adrenal glands, males with 17α-hydroxylase deficiency fail to virilize because concurrent gonadal androgen production is insufficient. Deficient estrogen production impairs normal puberty in females with 17α-hydroxylase deficiency. Since both 11β-hydroxylase deficiency CAH and 17α-hydroxylase deficiency CAH can produce hypertension, their properties are compared in Table 4.3. The synthesis of testosterone from cholesterol is illustrated in Fig. 4.2; 17α-hydroxylase converts pregnenolone and progesterone to DHEA and androstenedione, respectively (through hydroxylated intermediates). The external (and internal) genitalia of females (46,XX individuals) with 17αhydroxylase deficiency are normal because there is no exposure to excess adrenal androgens, in contrast to 21-hydroxylase, 11β-hydroxylase, and 3βhydroxysteroid dehydrogenase (3β-HSD, see below) deficiencies where virilization occurs. However, 17α-hydroxylase-deficient females do not feminize during puberty because estradiol production is insufficient due to the impaired ability to convert pregnenolone to 17-hydroxypregnenolone and DHEA (Fig. 4.3). The laboratory diagnosis of 17α-hydroxylase deficiency is established by increased ratios of pregnenolone to 17-OHP, progesterone to 17-OHP, and no elevations of DHEA and androstenedione [61].

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TABLE 4.3 Clinical and biochemical features of 17α-hydroxylase and 11βhydroxylase deficiency CAH. Enzyme deficiency

Percent of CAH cases

17α-hydroxylase

11β-hydroxylase

B1%

B5%

Gene

CYP17

CYP11B1

Plasma 17-OHP

Depressed

Elevated

Plasma 11-DOC

Elevated

Elevated

Plasma corticosterone

Depressed

Depressed

Plasma deoxycortisol

Depressed

Elevated

Plasma cortisola

Depressed

Depressed

Ambiguous genitalia

46,XY and 46,XX; female phenotype

In females

Precocious puberty

No

In males

Salt loss

No

No

Hypertension

Late childhood/adolescence

Late childhood/ adolescence

a

May be low-normal due to compensatory increase in ACTH.

Another rare cause of CAH is 3β-hydroxysteroid dehydrogenase (3βHSD) deficiency. 3β-HSD is the only enzyme in adrenocortical steroid synthesis pathways that is not from the cytochrome P450 family; it is also found in the ovary, testes, and placenta. Two isoenzymes of 3β-HSD exist in humans, encoded by HSD3B1 and HSD3B2 genes; the former is expressed primarily in skin and placenta, and the latter is expressed in the adrenal gland and gonads. 3β-HSD oxidizes Δ5-3β-hydroxysteroids to the Δ4-3-keto configuration, a necessary step in the biosynthesis of progesterone, 17-OHP, and androstenedione (Fig. 4.6). Because the gonads share these pathways with the adrenal glands, male fetuses are undervirilized because of deficient testosterone production, and female fetuses are overvirilized because of excessive DHEA production, which in turn results in excessive androstenedione. The least common, but most fatal, form of CAH is not an enzyme deficiency, but instead is a disorder involving the steroidogenic acute regulatory protein (StAR). StAR protein mediates the transport of cholesterol from the outer mitochondrial membrane to the inner membrane where the cytochrome

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FIGURE 4.6 The action of 3β-hydroxysteroid dehydrogenase (full name, 3β-hydroxysteroid dehydrogenase/Δ5,4 isomerase) converts pregnenolone to progesterone, 17α-hydroxypregnenolone to 17α-hydroxyprogesterone, and dehydroepiandosterone (DHEA) to androstenedione. Referencing the conventional steroid numbering system, the hydroxyl group at position 3 (stereochemically in the β orientation) is oxidized to a carbonyl, and the double bond connecting carbons 5 and 6 (Δ5) is transferred to carbons 4 and 5 (Δ4) through protonation carbon 6 and deprotonation of carbon 4.

P450 enzymes required for adrenocortical steroids reside. A deficiency in StAR protein causes congenital lipoid adrenal hyperplasia, so named because of the excessive accumulation of cholesterol and cholesterol esters within cells. Loss of functional StAR protein results in almost complete cessation of adrenal and gonadal steroid biosynthesis, causing severe mineralocorticoid, glucocorticoid, and sex hormone deficiency. Affected 46,XY males develop female external genitalia due to the absence of testosterone production. Since StAR protein is not essential for fetal steroidogenesis, affected infants may be born with adequate glucocorticoid and mineralocorticoid activity, but saltwasting usually develops after a few months [62].

Adrenoleukodystrophy and adrenomyeloneuropathy ALD is a progressive neurodegenerative disorder characterized by adrenal insufficiency and progressive dementia, spastic paralysis, and other neurologic and intellectual defects. The disease results from a deficiency of the ATP-binding cassette subfamily D (ALDP (adrenoleukodystrophy protein)), encoded by the ABCD1 gene on the long arm of the X chromosome (Xq28), and has a variable presentation that differs in some respects in men compared to women [63]. For example, ALD in males nearly always includes adrenal insufficiency, whereas women affected with the disease rarely develop this complication. Men with ALD are also more prone to cerebral demyelination than women with ALD, although the incidence of spinal cord disease is high in both sexes. The neurological problems in ALD result from demyelination. ALDP deficiency impairs β-oxidation of saturated VLCFA, so ALD is diagnosed by elevated levels of VLCFA in plasma. Childhood and neonatal forms of ALD have been described. The childhood form (also known as Schilder disease) with onset between ages 4 and 8

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years occurs in boys exclusively because the disorder is inherited as an Xlinked recessive trait. Manifestations of a central neuropathy include cortical blindness, dementia, coma, and quadriparesis. Adrenal failure, a late manifestation of the disease, has a prevalence greater than 80% in adult males with ALD [64]. Neonatal ALD is inherited as an autosomal recessive trait. Microscopically, there are decreased numbers and size of peroxisomes. This disorder can be considered to be a less severe form of the Zellweger cerebrohepatorenal syndrome (see below). Affected children may live up to several years. Clinical findings include hypotonia, seizures, hyporeflexia, retinopathy, and hepatic dysfunction. Overt adrenal insufficiency is uncommon in neonatal ALD [65]. Adrenomyeloneuropathy (AMN) is a key clinical syndrome associated with X-linked ALD. AMN is a progressive neuropathy that affects the ascending sensory and descending motor pathways, leading to stiffness, weakness, sphincter disturbances, and impotence. This slowly progressive disease develops over decades. The average of onset in men is 28 years, and about two-thirds present with adrenal failure. An AMN-like syndrome develops in about 65% of heterozygous females. In females, the age of onset is typically between the 5th and 6th decades, and adrenal insufficiency is rare. However, 10% 15% of female carriers will display overt neurologic disturbances, and 50% will exhibit mild neurologic abnormalities [66].

Zellweger spectrum disorder Zellweger spectrum disorder (ZSD) is a major subgroup of peroxisomal biogenesis disorders that involves defects in one or more of the 13 PEX genes that code for multiple forms of the protein peroxin, which is found in peroxisomes. In ZSD there is defective development of peroxisomes, organelles that contain digestive enzymes such as catalase that are involved in the catabolism of fatty acids, D-amino acids, and polyamines. Peroxisomes exist in all eukaryotic cells, and one of the digestion products they produce is hydrogen peroxide, from whence the organelle’s name is derived. ZSD can be divided into three subgroups, based on the age of onset: childhood, adolescent, and adult forms of the disorder occur [67]. The neonatal-infantile presentation of ZSD includes high forehead, a flat facial appearance, microcephaly, hypotonia, enlarged liver with jaundice, renal cysts, and psychomotor retardation. Up to 30% of ZSD patients may also present with primary adrenal insufficiency [68].

Congenital adrenal hypoplasia Congenital adrenal hypoplasia (adrenal hypoplasia congenita, AHC) is an uncommon cause of primary adrenal insufficiency in children, accounting for

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only 20 of 434 cases in a recent review of cases in China [69]. AHC results from loss of function mutations in the NR0B1 gene (chromosome Xp21) that encodes DAX-1,1 a nuclear transcription factor that is essential for proper development of the adrenal and pituitary glands, and the gonads. Since AHC is X-linked, the disease typically affects males and the onset of adrenal insufficiency occurs in infancy or early childhood [70]. Because DAX-1 is also expressed in the pituitary gland and gonads, other endocrine disorders are possible, such as hypogonadotropic hypogonadism. AHC is associated with other genetic disorders that are mapped to the short arm of the X chromosome (Xp21), including glycerol kinase (GK) deficiency and Duchenne (pseudohypertrophic) muscular dystrophy (DMD). Therefore the metabolic and phenotypic features of GK deficiency and DMD may be present in AHC [71]. GK deficiency is manifested as episodic vomiting, acidemia, central nervous system depression, hypotonia, a possible Reye-like illness, and pseudohypertriglyceridemia.2

Steroidogenic factor-1 Mutations in steroidogenic factor-1 (SF-1, or NR5A1) disrupt gonadal and adrenal development. In animal models, deletion of the NR5A1 gene results in adrenal agenesis and gonadal dysgenesis [72], but this is a rare phenotype in humans [71]. Mutations in NR5A1 are among the most common genetic causes of gonadal dysgenesis [73].

Wolman disease Wolman disease (WD) is a rare (less than 1/100,000 births) variant of the approximately 50 lysosomal enzyme or protein defects and is caused by a deficiency in lysosomal acid lipase (LAL) due to mutations in the LIPA gene (for Lipase A) at locus 10q23.31 on the long arm of chromosome 10. WD is

1. The name, DAX-1, is an acronym for the unwieldy “Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome 1,” and is officially known as NR0B1. 2. Triglycerides (more strictly, triacylglycerides) are glycerol molecules esterified with three fatty acids. Most laboratories measure triglycerides using a lipase/glycerol kinase (GK) method, in which the glycerol-fatty acid esters are enzymatically hydrolyzed by lipase, leaving glycerol as the product. The total glycerol is measured by its reaction with GK. Endogenous glycerol is typically too low in concentration to significantly affect the measurement, unless the patient has a GK deficiency. In GK deficiency, glycerol accumulates in the blood, and the triglyceride measurement is therefore falsely elevated due to the high concentration of free glycerol that is present, since the assay does not distinguish between free glycerol and glycerol generated by hydrolysis of triglycerides. The falsely elevated triglyceride concentration is termed “pseudohypertriglyceridemia”.

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characterized by the complete absence of LAL activity necessary for the degradation of cholesterol and triglycerides in lysosomes. Fifteen (mostly nonsense) mutations with deletions or insertions have been identified for WD; these mutations halt the coding of LAL [74,75]. Cholesterol ester storage disease (CESD) is a less severe form of LAL deficiency. Individuals with WD have hepatosplenomegaly, severe malabsorption, failure to thrive, and adrenal calcification. The normal action of acid lipase is to catalyze the conversion of cholesterol ester to cholesterol plus free fatty acids, and triglycerides to glycerol plus free fatty acids. In WD and CESD, esterified lipids accumulate in cells causing generalized xanthomatosis. Enlarged adrenal glands are typically evident by 6 months of age, and WD is usually fatal within the first year. WD patients typically present with elevated serum transaminase (ALT, AST) enzymes, and liver fibrosis, cirrhosis, and hepatomegaly. Serum LDL cholesterol, triglycerides, and total cholesterol are also elevated. Measurement of tissue LAL activity distinguishes between WD (no activity) and CESD (1% 12% of normal), but genetic testing can confirm the diagnosis, as well. Calcification of the adrenal glands is only rarely seen in CESD [75].

Mitochondrial causes of Addison disease Oxidative phosphorylation, the most efficient source of cellular energy, takes place in the mitochondria. Therefore mitochondrial disorders characterized by decreased ATP production most severely affect tissues and organs with high energy requirements such as the nervous system, skeletal and cardiac muscle, and the endocrine system. Mitochondrial disorders are often associated with lactic acidosis, myopathy, cataracts, and nerve deafness. Kearns Sayre syndrome (KSS) is a type of progressive external ophthalmoplegia, characterized by onset before age 20 and pigmentary retinopathy. KSS usually results from a 1.1 to 10 kilobase deletion of mitochondrial DNA. Endocrine disturbances, including adrenal insufficiency, are observed in up to two-thirds of cases [76].

Smith Lemli Opitz syndrome Smith Lemli Optiz syndrome (SLOS) is a disorder of cholesterol production caused by deficient 7-dehydrocholesterol reductase (7-DCR; also known as δ-sterol reductase or 3β-hydroxysteroid-Δ-7-reductase; EC 1.3.1.21) activity, due to mutations in the DHCR7 gene that codes for the enzyme. DHCR7 resides at chromosome 11q12 13, and mutations in this gene are inherited in an autosomal recessive pattern. 7-DCR converts 7-dehydrocholesterol to cholesterol in the final step in cholesterol biosynthesis, so a deficiency in this enzyme results in accumulation of 7-dehydrocholesterol and its isomer, 8-dehydrocholesterol.

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Although the clinical presentation of SLOS can be widely variable, it is often characterized by growth retardation, intellectual disability, learning and behavioral problems, and multiple anatomic malformations, including syndactyly, polydactyly, microcephaly, and distinctive facial features that include cleft palate, high broad forehead, narrow temples, drooping eyelids, and a broad nasal bridge [77]. Metabolically, SLOS often presents with adrenal insufficiency and undervirilization in males due to dihydrotestosterone deficiency in utero [78].

Adrenocortical excess Clinical symptoms of glucocorticoid excess (Cushing syndrome) Cushing syndrome is a disorder associated with chronic glucocorticoid (cortisol) excess. The disorder has a variety of signs and symptoms, many of which are nonspecific and often seen in other diseases. Therefore diagnosis of Cushing syndrome can be complicated by the presence of other disorders that have similar clinical presentations, such as diabetes and obesity, the global prevalence of which has been increasing for several decades. Additionally, pharmaceutical use of glucocorticoids is increasing, and overuse of these medications can produce Cushing-like symptoms [79]. The symptoms of Cushing syndrome include (roughly, from most to least common) weight gain, menstrual irregularities and sexual dysfunction, hirsutism, striae and atropic skin, weakness and proximal myopathy, and psychiatric disturbances. Signs of Cushing syndrome include “moonshaped” facies, acne, thoracic kyphosis, supraclavicular fat pads, hypertrichosis, purplish-blue striae, psychosis, easy bruising, hypertension, buffalo hump, and centripetal obesity. Excess cortisol may produce glucose intolerance or frank Type 2 diabetes. Severe myopathy can be manifested as proximal muscle weakness. Women can experience hirsutism or virilization if adrenal androgens are concomitantly overproduced. Biochemical and hematological abnormalities may include alkalosis, hypokalemia, elevated hematocrit, and eosinopenia, which may be associated with hyperglycemia [80]. If hypercortisolism is due to chronic elevations in corticotropin (ACTH), hyperpigmentation results from the melanotropin stimulating hormone-like activity of ACTH. Prolonged, marked cortisol excess may also lead to sodium and water retention similar to aldosterone excess. The potent antiinflammatory activity of glucocorticoids suppresses immune response, predisposing the patient to infection. Osteopenia in adults and poor linear growth in children result from excess cortisol. Pathologic elevations in cortisol levels produce immunosuppression, catabolism of protein (as substrate for hepatic gluconeogenesis), central redistribution of fat, and insulin resistance. Insulin resistance contributes to dysglycemia and

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dyslipidemia. Therefore several features of Cushing syndrome are shared with the metabolic syndrome [81]. Excessive cortisol secretion produces mineralocorticoid activity similar to that seen in hyperaldosteronism, including hypokalemia, metabolic alkalosis (hypochloremia with high bicarbonate concentration), and hypertension from sodium and water retention and expansion of circulating blood volume. Although the normal circulating concentration of cortisol is approximately 1000 times higher (10 20 μg/dL) than the normal circulating concentration of aldosterone (,10 ng/dL), the renal mineralocorticoid receptor is protected from the effects of cortisol by the action of 11β-hydroxysteroid dehydrogenase-2 (11-HSD2), which converts cortisol to cortisone, an inactive metabolite [82]. However, when present in high enough concentrations, cortisol can stimulate the mineralocorticoid receptor.

The differential diagnosis of glucocorticoid excess The most common cause of hypercortisolism is overuse of pharmacological glucocorticoids for their immunosuppressive and antiinflammatory effects, resulting in “exogenous” Cushing syndrome. When the cause of hypercortisolism is a disease of the hypothalamus, pituitary, or adrenal gland, the resulting Cushing syndrome is “endogenous.” The possibility of exogenous Cushing syndrome should be ruled out before any further biochemical investigation of Cushing-like symptoms is undertaken. Endogenous Cushing syndrome may be either (1) ACTH-independent, when hypercortisolism is due to autonomous overproduction of cortisol by the adrenal gland due to adrenocortical adenoma or carcinoma, micronodular dysplasia, or macronodular hyperplasia, or (2) ACTH-dependent, when inappropriate or excessive ACTH secretion overstimulates the adrenal cortex to secrete cortisol. ACTH-dependent hypercortisolism can result from a pituitary adenoma (Cushing disease), or from a nonpituitary tumor that produces ACTH (ectopic ACTH syndrome) or CRH (ectopic CRH syndrome). The distinctions among adrenocortical adenoma, carcinoma, micronodular dysplasia, and macronodular hyperplasia are clinical (evidence of metastases) and histological, based on gross and microscopic examination of surgical pathology specimens [83]. Grossly and microscopically, a glucocorticoid-producing adrenal adenoma looks identical to an aldosterone-producing adenoma: both are usually unilateral, circumscribed, brown-yellow, homogeneous in consistency, less than 30 g in weight, and with a histologically normal cellular appearance [84]. In these cases, the clinical history of Cushing syndrome is typically longer than 6 months. Adrenocortical carcinomas are rare and usually larger (100 140 mm, .100 g) than adenomas (usually ,60 mm and ,50 g),

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solid, lobulated, may show necrosis, and rarely are cystic [83]. About half of adrenocortical carcinomas are nonsecretory; therefore steroid hormone levels are normal [85]. The clinical history of Cushing syndrome associated with adrenocortical carcinoma is typically shorter compared to adenoma. Immunophenotypic and genetic tests can be used to distinguish between several subtypes of adrenocortical carcinomas [86]. Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism that is diagnosed on the basis of clinical Cushing syndrome, elevated 24-h UFC, elevated AM cortisol .2 μg/dL ( . 50 nmol/L) after overnight 1 mg dexamethasone suppression, and pigmented adrenocortical micronodules ,10 mm in diameter. PPNAD is most frequently diagnosed in children and young adults, and results from germline defects in genes involved in cAMPPKA signaling, including PRKAR1A, PDE11A, PDE8B, or PRKACA. PPNAD is most often associated with Carney complex, which is an inherited autosomal dominant disorder caused by mutations in the tumor suppressor gene, PRKAR1A, mapped to the long arm of chromosome 17 (17q22 24), predisposing affected individuals to adrenal tumors. Another loci associated with Carney complex is on the short arm of chromosome 2 (2q16), a region that codes for proopiomelanocortin and a DNA-mismatch repair gene, MSH2. Both mutations express the same phenotype [87]. Primary bilateral macronodular hyperplasia (PBMAH) typically occurs in adults aged .50 years and is identified by the presence of Cushing syndrome and nodules of 0.2 cm to more than 4 cm diameter with the gland potentially weighing more than 100 g. Like PPNAD, PBMAH involves defects in the cAMP-PKA signaling system [88]. Rarely, PBMAH can be associated with multiple endocrine neoplasia type 1 (MEN-1), familial adenomatous polyposis, or hereditary leiomyomatosis and renal cell carcinoma. There have also been cases of PBMAH that were characterized by hyperaldosteronism without hypercortisolism [88]. PBMAH is associated with aberrant expression of G-protein-coupled receptors by subpopulations of adrenocortical cells. These abnormal receptors respond to a variety of hormones, including vasopressin, catecholamines, luteinizing hormone, serotonin, and glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide, GIP), a hormone secreted from the gastrointestinal tract in response to meals [88,89]. If a tumor expresses GIP receptors, meals may stimulate a transient hypersecretion of cortisol. PBMAH is very rare. Li Fraumeni syndrome is characterized by sarcomas, brain tumors, leukemia, lymphomas, and early onset (,45-years old) breast cancer caused by p53 mutations. The syndrome is caused by mutations in the TP53 gene coding for a tumor protein 53, a tumor suppressor factor [90].

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Cortisol resistance and ectopic adrenocorticotropic hormone Cushing syndrome The GR gene (NR3C1) is on the short arm of chromosome 5 (5q31Y32), and several variants have been identified. Various mutations in the GR gene can result in either hypersensitivity or resistance of the GR to glucocorticoid stimulation. The most extensively studied polymorphisms in the GR gene are Bc1I, N363S (both cause GR hypersensitivity), and Er22/23EK, which causes GR resistance [91]. Elevated ACTH in cortisol resistance and ectopic ACTH Cushing syndrome also lead to overproduction of 11-DOC, salt retention, and hypertension.

The biochemical diagnosis of glucocorticoid excess The terminology associated with hypercortisolism can be confusing and has evolved over time. Cushing syndrome refers to the clinical manifestations of glucocorticoid excess, regardless of the cause. Cushing disease is a specific cause of Cushing syndrome, resulting from a pituitary corticotroph adenoma. There can be other sources of excess ACTH, such as ectopic ACTH-secreting tumors or certain physiological conditions that also result in elevated ACTH. These states are collectively called “ACTH-dependent” hypercortisolism, since the excess cortisol results from ACTH stimulation of the adrenal cortex. Glucocorticoid excess can also result from a cortisol-secreting adrenal tumor—or, very rarely, an ectopic cortisol-secreting tumor [92,93]—in which case the Cushing syndrome is “ACTH-independent” since cortisol production does not require ACTH. Distinction can also be made between hypercortisolism resulting from a tumor (neoplastic or pathologic Cushing syndrome) or from physiological causes (nonneoplastic hypercortisolism, or NNH). Finally, Cushing syndrome can occur from pharmacological administration of glucocorticoids, which is sometimes referred to as “exogenous Cushing syndrome,” in contrast to neoplastic and physiological causes, which are “endogenous” (refer to Table 4.1). The most common endogenous cause of hypercortisolism is an ACTHsecreting pituitary adenoma, which somewhat confusingly bears the name Cushing disease, as opposed to Cushing syndrome. Overall, ACTHdependent Cushing syndrome is far more common (70% 80%) than ACTHindependent disease (also called adrenal Cushing’s). The laboratory approach to diagnosis in patients with symptoms of Cushing syndrome typically involves: (1) exclusion of exogenous glucocorticoids and pseudo-Cushing syndrome, (2) confirmation of excessive cortisol secretion, (3) determination of whether the disease is ACTH-dependent or independent, and (4) identification of the specific lesion [94].

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The differentiation between Cushing disease and ectopic ACTH is of critical importance in patient management because treatment of Cushing disease requires transsphenoidal surgery for removal of the pituitary adenoma. In transsphenoidal surgery, the nasal septum is temporarily removed, and access to the pituitary is gained through the sphenoidal sinus that is in front of the sella turcica (the bone at the base of the brain that forms a pocket around the pituitary). Ectopic ACTH requires oncologic evaluation to identify the location of the tumor. Often, patients with ectopic ACTH first seek medical attention not because of Cushing syndrome but because of cancer-related symptoms, such as inanition, malaise, and anemia. Physiologic hypercortisolism/NNH, also known as “pseudo-Cushing syndrome,” is a form of ACTH-dependent hypercortisolism unrelated to an ACTH-secreting tumor. In NNH there is clinical and biochemical evidence of hypercortisolism, including abnormal midnight salivary cortisol, overnight dexamethasone suppression, and UFC. NNH has many causes, including neuropsychiatric disorders, alcohol abuse, insulin-resistant obesity, polycystic ovary syndrome, and end-stage renal disease [95]. Rare causes of NNH include glucocorticoid resistance and primary adrenal disease associated with incomplete suppression of ACTH. The differential diagnosis of NNH versus ACTH-dependent hypercortisolism resulting from mild to moderate Cushing disease can be difficult, because the clinical and biochemical findings are often very similar. A modification of the dexamethasone suppression test (DST) and a test for response to vasopressin can be helpful to distinguish between Cushing syndrome, Cushing disease, and pseudo-Cushing syndrome. Screening tests for hypercortisolism include late-night plasma or salivary cortisol, 24-h UFC, 1 mg overnight DST, and low dose (0.5 mg every 6 h for a total of eight doses) DST. Two or more positive screening tests suggest a high probability of disease. Additional studies may include radiography, plasma ACTH, CRH stimulation, and inferior petrosal venous sinus sampling.

Plasma and salivary cortisol Cortisol is highest in the morning and lowest near midnight in people who have a normal nighttime sleep cycle; midnight cortisol concentration is normally ,50% of peak values in the morning. Two abnormal patterns of plasma cortisol may be observed in Cushing syndrome: (1) elevated morning and evening cortisol (with or without loss of diurnal variation), or (2) normal morning but elevated evening concentrations (loss of diurnal variation). In Cushing syndrome, random cortisol levels are rarely less than 7 μg/dL (190 nmol/L), and plasma cortisol is typically 15 35 μg/dL (415 965 nmol/L), regardless of the time of the day.

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Hypersecretion of cortisol can be episodic, which presents a significant diagnostic challenge. Measuring plasma cortisol when patients are symptomatic is a reasonable strategy, although symptoms of hypercortisolism do not always correlate with elevated plasma cortisol. Normal plasma cortisol levels alternating with hypercortisolism suggest a “cyclic” Cushing syndrome, whereas the diagnosis of “episodic” Cushing syndrome may require a long period of observation and testing. Evening cortisol concentration is much more informative than morning values if loss of diurnal variation is suspected. For the sake of convenience, and because it is unaffected by changes in CBG concentration, salivary cortisol is recommended. Elevated evening salivary cortisol has 91% 100% sensitivity and 93% 100% specificity for the diagnosis of Cushing syndrome [96]. LC-MS/MS is the preferred method for measurement of salivary cortisol, although automated immunoassays also are available [5]. Over half of patients with Cushing syndrome display a loss of diurnal variation in plasma cortisol concentrations. The optimal time to measure the evening cortisol value is between midnight and 0100 h (or 1 h after retiring to bed) on at least two occasions. Morning plasma cortisol should be collected between 0600 and 0800 h. Midnight salivary cortisol is normally ,145 ng/dL (4 nmol/L). Typical reference ranges for plasma cortisol are 7 25 μg/dL (195 690 nmol/L) in the morning and 2 9 μg/dL (55 250 nmol/L) in the evening (2000 2200 h).

Urinary free cortisol measurements Measurement of UFC is another useful screening test for Cushing syndrome, and its use has been advocated since the 1970s. The test name is somewhat redundant because only the unbound fraction of cortisol is filtered in the glomerulus, so only the unbound (free) fraction of cortisol appears in the urine. Confounders in the UFC test include incomplete or overcollected 24-h urine specimens, low glomerular filtration rate, cyclic hypercortisolism, and excessive fluid intake [79]. The finding of an elevated UFC in a 24-h urine specimen indicates overproduction of cortisol over a 24 h period. At least two baseline 24-h urine collections should be obtained since there is day-to-day variation in cortisol secretion. A UFC greater than 62 μg/24 h (170 nmol/ 24 h) is consistent with Cushing syndrome, although UFC results can be variable if samples are obtained during periods of high stress, such as trauma or surgery, and intra-individual variability can be significant. One study reported 50% intra-individual variability in UFC [97].

Overnight dexamethasone suppression test After evaluation of late-night salivary cortisol and UFC, if the biochemical diagnosis of Cushing syndrome is still unclear, an overnight DST is

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indicated. In the overnight DST, 1 mg of dexamethasone (or 0.3 mg/m2 in children) is administered orally at 2200 h. At 0800 h the following morning, a fasting cortisol is measured. A normal response is a plasma cortisol concentration less than 5 μg/dL (140 nmol/L), demonstrating that the HPA axis has been appropriately suppressed by an exogenous glucocorticoid. Dexamethasone is used because it does not cross-react in the typical cortisol immunoassay and has approximately 100 times the glucocorticoid activity of cortisol, so the dose required to suppress ACTH secretion is very low. PostDST plasma cortisol levels of 5 9.9 μg/dL (140 275 nmol/L) are equivocal, and plasma cortisol greater than 10 μg/dL (275 nmol/L) at 0800 h is consistent with Cushing syndrome. The overnight DST has a 95% sensitivity for detecting Cushing syndrome. However, there is a 10% 15% false-positive rate (failure of suppression in the absence of Cushing syndrome) due to failure to take the dexamethasone dose, increased dexamethasone metabolism, or episodic ACTH secretion. False-negative results are unusual but can result from delayed metabolism of dexamethasone. The standard dose of 1 mg dexamethasone for overnight suppression of cortisol has been criticized for poor sensitivity, since the dose is high enough to suppress cortisol in patients with mild or episodic Cushing syndrome. A modification of the standard overnight DST replaces the 1 mg dose of dexamethasone with a 0.25 mg dose, which causes less suppression of ACTH release and therefore is more sensitive for mild Cushing syndrome. A morning plasma cortisol greater than 7.6 μg/dL (210 nmol/L) is consistent with Cushing syndrome [98].

Low-dose dexamethasone-corticotropin-releasing hormone test As screening tests, midnight salivary cortisol and UFC both suffer from false-positive results. A modification of the overnight DST involves a low dose of dexamethasone (0.5 mg orally) administered every 6 h over 2 3 days, followed by morning administration of CRH; plasma ACTH and cortisol are measured at baseline, 15 min, and 30 min. Peak plasma cortisol greater than 1.4 μg/dL (38 nmol/L) is highly specific for Cushing syndrome [99]. Healthy individuals and individuals with pseudo-Cushing syndrome should not display a rise in their cortisol concentration in response to CRH after 2 days of low-dose dexamethasone. However, a patient with true Cushing disease could exhibit a cortisol response to CRH because the pituitary set-point for negative feedback is raised in Cushing disease. A disadvantage of the low-dose DST is that it may require hospitalization, which can make the test prohibitively expensive. There is controversy over the optimal dexamethasone dose and the interpretation of results. Some have suggested that the plasma dexamethasone concentration should be measured along with cortisol to correct for variable gastrointestinal absorption of the drug. In 2014 Lindholm [100] reviewed the

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available literature on dexamethasone suppression testing and concluded that variabilities in the reported sensitivities and specificities of the test for Cushing disease were considerable. A consistent finding was a correlation between basal and postdexamethasone concentrations of cortisol in plasma, leading to the conclusion that the notion of an absence of suppression in Cushing disease due to adrenal fatigue is not supported by data.

DDAVP stimulation test Corticotroph adenomas may express receptors for the posterior pituitary hormone vasopressin (antidiuretic hormone). Administration of desmopressin acetate, a synthetic vasopressin analog, typically stimulates ACTH secretion in patients with Cushing disease, whereas patients with ACTH-independent hypercortisolism and healthy patients have little or no response. The DDAVP (a trade name for desmopressin) test involves morning administration of 10 μg of DDAVP intravenously, followed by measurement of plasma ACTH and cortisol 15, 30, and 60 min after administration. Peak plasma ACTH greater than 27 pg/mL (6 pmol/L) has a 75% 87% sensitivity and 90% 91% specificity for ACTH-dependent hypercortisolism [99]. However, desmopressin-stimulated cortisol measurements do not separate pseudoCushing states from cases of an ectopic tumor Cushing syndrome. One way to increase pituitary stimulation to differentiate Cushing disease from nonpituitary Cushing syndrome involves combined pituitary stimulation with desmopressin and CRH [101].

Adrenocorticotropic hormone measurements Plasma ACTH measurements are helpful to differentiate ACTH-dependent from ACTH-independent Cushing syndrome, but accurate measurement of the hormone is challenging. ACTH is unstable, so strict specimen handling precautions are necessary for accurate results [102]. In addition, ACTH displays diurnal variations the same as cortisol and has a very short plasma half-life of 15 min. Therefore multiple plasma ACTH measurements are usually required to provide a reliable assessment of ACTH secretion. Moreover, there is considerable variability between different methods used to measure ACTH [103]. A plasma ACTH less than 5 pg/mL (1.1 pmol/L) at its nadir around midnight effectively rules out ACTH-dependent Cushing syndrome [104]. When high-dose dexamethasone fails to suppress UFC excretion, adrenal tumor and ectopic ACTH/CRH syndrome remain diagnostic possibilities. If low basal ACTH measurements were obtained during initial evaluation of the patient, it is probable that pituitary ACTH is suppressed by autonomous cortisol emanating from an adrenal tumor. In contrast, ACTH is normal or elevated in Cushing disease or ectopic ACTH/CRH syndrome. If plasma

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ACTH concentration is below the reference range, and a high-dose dexamethasone fails to suppress UFC, radiographic imaging studies for an adrenal tumor should be pursued. If plasma ACTH is normal or elevated and high-dose dexamethasone fails to suppress UFC, ectopic ACTH/CRH syndrome should be suspected; likely causes would be oat cell carcinoma of the lung or carcinoid.

Bilateral inferior petrosal venous sinus sampling and corticotropinreleasing hormone stimulation testing Biochemical evidence of Cushing disease should prompt radiographic evaluation of the pituitary by computed tomography (CT) or magnetic resonance imaging (MRI) to confirm the presence of a tumor. If a pituitary tumor cannot be identified radiographically, the source of ACTH is uncertain, and a CRH stimulation test should be performed with simultaneous bilateral inferior petrosal venous sinus sampling (BIPSS). In the BIPSS procedure, the patient typically undergoes bilateral femoral venous catheterization with line placement in each of the inferior petrosal venous sinuses; a third line is placed in the inferior vena cava for peripheral measurements. Samples at all three sites are obtained at baseline and following intravenous bolus administration of 1 μg of CRH per kg body weight (or 100 μg, in some protocols). Both ovine and recombinant human forms of CRH are available. In a typical protocol, specimens are collected from each catheter 1, 3, 5, 10, and 15 min after CRH administration. An alternative protocol uses desmopressin (DDAVP) to stimulate ACTH secretion [105]. If a pituitary microadenoma is present, CRH will elicit an increase in ACTH, whereas in ectopic ACTH syndrome, pituitary ACTH will be suppressed and will be no higher than the peripheral venous ACTH concentration. A central to peripheral ACTH ratio $ 2 in the basal specimens, or $ 3 at any time following CRH stimulation specimens strongly suggests the pituitary is the source of ACTH. When the central to peripheral ACTH ratio is $ 3, the sensitivity of BIPSS for an adrenal adenoma has been reported as high as 100%, whereas a ratio of ,2 in basal specimens or ,2.3 after stimulation suggests an ectopic source of ACTH [106]. Contrary to early reports, BIPSS does not reliably lateralize the lesion within the pituitary [105].

Evaluation for adrenal tumors If biochemical evaluation is consistent with an adrenal tumor as the source of excess and autonomous cortisol secretion (ACTH-independent hypercortisolism), adrenal CT/MRI studies should be used to help identify the tumor. Although certain features of the radiographic image (e.g., size, homogeneity) of an adrenal mass can sometimes suggest whether it is cortisol-secreting, histological examination of the tumor is usually

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necessary to confirm its secretory activity. The adrenal glands often display bilateral enlargement and may have perceptible nodules, in severe ACTH-dependent hypercortisolism [107]. Recently it has been suggested that adrenal vein sampling, a procedure normally used to localize mineralocorticoid-secreting tumors, may also help establish the bilaterality of cortisol-producing adrenal adenomas [108]. Adrenal vein sampling studies should confirm whether only one of the two adrenal glands is the source of excess cortisol. In cases of bilateral micronodular or macronodular hyperplasia, adrenal vein cortisol concentrations would both be elevated compared with the peripheral cortisol concentration. Asymptomatic adrenal masses detected by imaging studies performed for purposes other than to investigate suspected adrenal disease are, somewhat amusingly, called incidentalomas. These lesions are relatively common, affecting at up to 9% of the general population; their frequency increases with age [109]. The incidental adrenal mass may be an adenoma or hyperplastic adrenal tissue, and it may hypersecrete cortisol, aldosterone, or catecholamines. The American Association of Clinical Endocrinologists and the American Association of Endocrine Surgeons issued guidelines in 2009 for the medical management of adrenal incidentalomas [110]. For the biochemical assessment of adrenal incidentalomas, they recommend plasma aldosterone or renin activity, free metanephrines and normetanephrines, and the overnight 1 mg DST. Incidentalomas larger than 4 cm in diameter should be resected after hormonal testing, but smaller incidentalomas with normal hormonal tests may be followed for progression of the tumor. In 2016 the European Society of Endocrinology issued similar practice guidelines for management of adrenal incidentalomas [109]. Hormonal testing does not differentiate between benign and malignant lesions; histologic examination is needed to assess capsular or blood vessel invasion as evidence of malignancy. Also, if radiologic investigation demonstrates metastases, the tumor is malignant.

Mineralocorticoid excess Adrenal tumors Adrenal tumors may overproduce mineralocorticoids (aldosterone) without producing excess glucocorticoids. Conn syndrome results from an aldosterone-secreting adrenal tumor that causes hypertension from salt and water retention. In Conn syndrome, glucocorticoid excess and clinical symptoms of Cushing syndrome are absent. Alkalosis and hypokalemia accompany hypertension when mineralocorticoids are in excess, and the patient should be evaluated by measuring serum and/or urine aldosterone and plasma renin or PRA. With autonomous overproduction of mineralocorticoid, serum and urine aldosterone are elevated whereas renin is suppressed.

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Glucocorticoid-remediable aldosteronism Glucocorticoid-remediable aldosteronism (GRA) is the result of an autosomal dominant trait that involves a chimeric gene duplication causing cells in the zona fasciculata to express aldosterone synthase (CYP11B2), which hydroxylates corticosterone to produce aldosterone (see Fig. 4.1) [111]. In the chimeric duplication, the 5’ ACTH-responsive promoter region of the 11β-hydroxylase (CYP11B1) gene fuses with the CYP11B2 gene, resulting in ectopic aldosterone synthase activity in the zona fasciculata under the regulation of ACTH. GRA is the most common monogenic (or Mendelian) form of hypertension [112] and accounts for about 1% of primary aldosteronism cases. In GRA, normal levels of ACTH produce hyperaldosteronism, early-onset hypertension, hypokalemia, and an increased risk for stroke. Suppression of ACTH with physiologic doses of glucocorticoids lowers aldosterone secretion and normalizes blood pressure; conventional antihypertensive therapies are contraindicated in these patients due to the risk of hypokalemia. Genetic tests exist for GRA, but suppression of aldosterone by dexamethasone (0.5 mg every 6 h) is an alternative biochemical assessment.

Apparent mineralocorticoid excess A rare autosomal recessive mutation in the HSD11B2 gene results in deficient activity of 11β-hydroxysteroid dehydrogenase Type 2 (11BHS2), an oxidoreductase enzyme found in aldosterone-sensitive tissues. 11BHS2 converts cortisol to the inactive metabolite cortisone, protecting mineralocorticoid receptors from responding to cortisol, the concentration of which is normally 1000 times the concentration of aldosterone. Therefore a deficiency in 11BHS2 activity causes overstimulation of aldosterone receptors by cortisol and an apparent mineralocorticoid excess. The same phenomenon can be observed in overexposure to glycyrrhetinic acid or its synthetic analog, carbenoxolone, which are competitive inhibitors of 11BHS2 [113]. The biochemical profile of the disease typically features low aldosterone and renin, along with a metabolic alkalosis and severe hypokalemia. The ratio of urinary cortisol to cortisone may be useful in establishing the diagnosis.

Biochemical evaluation of primary aldosteronism Primary aldosteronism causes 4% 10% of cases of hypertension. Initial screening for primary aldosteronism typically involves measurement of the plasma aldosterone/renin ratio, followed by CT imaging studies, if the results indicate hyperaldosteronism. Historically, plasma renin has been measured by its activity, and the ratio therefore was aldosterone concentration divided

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by renin activity. This has caused some confusion, because contemporary renin methods directly measure the concentration of the hormone. Therefore the decision limits depend on the specific assays used in the laboratory, as well as the units of concentration or activity in which aldosterone and renin are expressed [114]. However, the interpretation rests on whether the aldosterone concentration is inappropriately high in the absence of renin hyperstimulation. A variety of challenge tests have been proposed to investigate primary aldosteronism, but none of them are considered to be a “gold standard” [115]. Proposed tests include the saline infusion test, oral sodium loading, captopril challenge test, and fludrocortisone suppression test. Choice of an appropriate test largely depends on availability and convenience. In patients with confirmed primary aldosteronism, adrenal vein sampling can be used to localize an aldosterone-secreting adenoma. Catheters are fed into the left and right adrenal veins and the inferior vena cava (for peripheral circulation measurements). After collection of baseline specimens from all three locations, a bolus 250 μg dose of ACTH (as Cosyntropin) is administered intravenously, and specimens are collected 5, 10, and 15 min after ACTH administration [116]. Measurement of aldosterone (and sometimes cortisol) in the specimens establishes whether aldosterone is being produced unilaterally or bilaterally, which is essential to inform any decision on possible surgical intervention since imaging studies do not distinguish between active and inactive adrenal tumors [105,117].

Pheochromocytoma and paraganglioma Clinical overview Pheochromocytoma is neuroendocrine tumor that originates predominantly (80% 85%) in the adrenal medulla. Pheochromocytomas are histologically and functionally indistinguishable from paragangliomas, which are extraadrenal and comprise 15% 20% of catecholamine-secreting neuroendocrine tumors [118]. Pheochromocytoma and paraganglioma are differentiated by location only and “PPGL” (pheochromocytoma/paraganglioma) is commonly used in reference to both tumors. PPGL is a rare tumor, with an estimated incidence of 0.6 cases per 100,000 [119]. The clinical presentation of PPGL is nonspecific, but cardinal symptoms include severe headache, diaphoresis, palpitations, and hypertension. Other symptoms may include hypertensive retinopathy, nausea and vomiting, fasting hyperglycemia, orthostatic hypotension, and weight loss. PPGL should be considered in patients with a family history of the disease, evidence of a multiple endocrine neoplasia type 2, an incidental suprarenal mass, or any history of an adverse cardiovascular response to anesthesia or medications such as opiates, histamine, ACTH, saralasin, or glucagon, all of which have been reported to precipitate symptoms in patients with PPGL.

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Isolated and familial pheochromocytoma/paraganglioma PPGL can occur as an isolated tumor, but in as many as 25% of cases, it is part of a familial cancer syndrome. Such autosomal dominant familial cancer syndromes include multiple endocrine neoplasia types 2A and 2B caused by rearranged during transfection (RET) proto-oncogene mutations, familial PPGL syndrome, neurofibromatosis type 1, von Hippel Lindau type II syndrome, tuberous sclerosis, and Sturge Weber syndrome [118]. Bilateral PPGL is more common in genetic syndromes because the underlying somatic mutation occurs in all cells. Although only 10% of adults with PPGL exhibit bilateral tumors, 35% of children with PPGL display bilateral involvement. PPGL is very rare in children; most cases occur in the 4th and 5th decades of life. PPGL is present in 4% 7% of patients with adrenal incidentalomas [114].

Neurofibromatosis Type 1 Between 1% and 5% of patients with neurofibromatosis Type 1 develop a PPGL. Neurofibromatosis (also known as von Recklinghausen’s syndrome) is an autosomal dominant disorder that results from a loss of function mutation in the NF1 gene located on chromosome 17q11.2; it is among the most common inherited disorders of the nervous system, with an estimated prevalence of one case per 3000 [120]. The protein product of the NF1 gene is a 250 kDa protein with over 2800 residues, neurofibromin, which is expressed in a wide variety of tissues. Loss of function mutations in NF1 predisposes affected individuals to neural crest tumors. Neurofibromin has multiple functional domains, the best studied of which is a 360 residue domain that is similar to the catalytic domain of GTPase-activating protein that downregulates ras, a cellular proto-oncogene [121]. Hence, loss of neurofibromin regulatory activity enhances ras-associated tumorigenic potential.

Von Hippel Lindau disease Von Hippel Lindau disease is an autosomal dominant disorder that results from a loss of function mutation in the VHL tumor suppressor gene, and is associated with the development of cerebellar and retinal hemangioblastomas, renal cell carcinomas, pancreatic neuroendocrine tumors and serous cystadenomas, endolymphatic sac tumors, and PPGL. The VHL gene is located at chromosome 3p25 26 and codes for the pVHL, which is part of a multiprotein complex that degrades hypoxia-inducible factors. In the absence of pVHL activity, hypoxia-inducible factors accumulate, increasing the transcription and expression of vascular endothelial growth factor and plateletderived growth factor, which promote tumorigenesis. About one-fifth of patients with von Hippel Lindau syndrome develop a PPGL, nearly always of the nonadrenergic phenotype (see below) [122].

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Laboratory diagnosis of the pheochromocytoma/paraganglioma The adrenal medulla produces the neurotransmitters epinephrine, norepinephrine, and dopamine. Depending on the phenotype, PPGL may secrete all, none, or any combination of the three catecholamines. Biochemically, the diagnosis of PPGL is ordinarily based on elevated concentrations of the catecholamine metabolites (metanephrines) in the plasma or urine. Because plasma catecholamines have short half-lives, and their secretion may be episodic, 24-h urine collections are generally recommended for quantitation of catecholamines and metanephrines. The U.S. Endocrine Society recommends measuring plasma free metanephrine or urinary fractionated metanephrines as the initial screening test for PPGL [123]. The biochemical pathways for catecholamine synthesis in the adrenal medulla are shown in Fig. 4.7. Note that dopamine is a precursor to epinephrine and norepinephrine, and there are reports of rare PPGLs that secrete only dopamine. It has been reported that metanephrines may be normal in some cases of dopamine-secreting PPGL (see Fig. 4.8) [124]. Approximately 3% 7% of the general population display nonspecific anatomic adrenal abnormalities; therefore radiologic screening is not

FIGURE 4.7 Catecholamines (epinephrine, norepinephrine, and dopamine) are biosynthesized in the adrenal medulla by hydroxylation of l-tyrosine to produce l-dihydroxyphenylalanine (L-DOPA). DOPA decarboxylase removes the carboxylate group to generate dopamine. Hydroxylation of dopamine generates norepinephrine, which is methylated to produce epinephrine.

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FIGURE 4.8 In post-synaptic nerve terminals, catecholamines are converted into inactive metabolites by the activity of monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) enzymes. The primary metabolic products from methylation of epinephrine and norepinephrine by COMT are metanephrine and normetanephrine, respectively. Other catecholamine metabolites have been measured in urine, but plasma metanephrines are the most sensitive biomarkers of secreting neuroendocrine tumor of the adrenal medulla (pheochromocytoma) or paraganglioma.

advisable unless biochemical screening tests reveal abnormal catecholamine release [125].

Urine catecholamines and metanephrines Older tests for detecting PPGL include urinary catecholamines and vanillylmandelic acid (a metabolite of both epinephrine and norepinephrine; see Fig. 4.8). However, urinary metanephrines have been established to have superior sensitivity for the disease, and areas under receiver-operating characteristic curves for plasma and urinary metanephrines range from 0.91 to 0.95 [126,127]. Twenty-four-hour urine collections should be acidified to stabilize catecholamines and metanephrines. A recent study suggested that measurement of the normetanephrine/creatinine ratio in randomly collected urine specimens has

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greater than 98% specificity for PPGL [128]. Immunoassays exist for catecholamines and metanephrines, but a more specific method such as high-pressure (or performance) liquid chromotography (HPLC) combined with mass spectrometry is recommended for measuring these compounds.

Plasma catecholamines and metanephrines Catecholamines (epinephrine, norepinephrine, and dopamine)3 are metabolized through multiple pathways, one of which is methylation by the enzyme catechol-O-methyltransferase (COMT). COMT exists in two isoforms: a membrane-bound “long” form (MB-COMT), and a soluble “short” form (S-COMT), although these designations are slightly misleading since neither form exists extracellularly; S-COMT is primarily associated with the cytoplasm and nuclei, whereas MB-COMT resides in the rough endoplasmic reticulum [129]. The products of COMT action on epinephrine, norepinephrine, and dopamine are metanephrine, normetanephrine (collectively called “metanephrines”; Fig. 4.8), and 3-methoxytyramine (Fig. 4.9), respectively. The adrenal medulla preferentially expresses MB-COMT, which has a higher affinity for catecholamines compared to the soluble form of COMT [130]. As a result, 90% of plasma metanephrine and 24% 40% of plasma normetanephrine are produced in the adrenal gland [131]. The relative specificity of these metabolites for

FIGURE 4.9 Monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) enzymes interconvert dopamine and its metabolites, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid.

3. Chemically and physiologically, epinephrine, norepinephrine, dopamine, and serotonin are the monoamine neurotransmitters. The former three all contain a catechol (1,2-dihydroxybenzene) group and therefore are called catecholamines. Serotonin (5-hydroxytryptamine) is also a monoamine neurotransmitter, but it is produced by enterochromaffin cells in the gastrointestinal tract, and does not contain the catechol group.

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adrenal tissue enhances their value as circulating biochemical markers for PPGL. Sensitivities of total plasma metanephrine and normetanephrine for detecting PPGL have been reported to be as high as 86% and 100%, respectively [131]. Measurement of plasma metanephrines is desirable because it avoids the need for a 24-h urine collection and the hazard of exposure to the acid required to stabilize catecholamines and metanephrines in urine. All catecholamines and their metabolites (except for vanillylmandelic acid, VMA) eventually are sulfated by the action of a specific sulfotransferase isoenzyme encoded by the SULT1A3 gene. In plasma, concentrations of the sulfated (or “conjugated”) metanephrine fractions exceed the free metabolites by more than 10-fold. Older methods for measuring plasma and urinary metanephrines lacked the analytical sensitivity to measure the unconjugated forms, so acid hydrolysis was used to release the sulfate group from the conjugated metanephrines; the resulting total concentration was often referred to as “deconjugated” metanephrines. However, modern mass spectrometric methods have the analytical sensitivity to measure the unconjugated metanephrines in plasma, which have superior diagnostic performance for detecting PPGL since they are products of the adrenal chromaffin cells; sulfation occurs peripherally [132]. Since modern methods are also capable of distinguishing between the various metabolites, test panels that measure and report the concentrations of multiple catecholamine metabolites are often referred to as “fractionated metanephrines.” For plasma catecholamine and metanephrine measurement, most guidelines recommend that the patient rest in a supine position for at least 30 min prior to collection of the blood specimen for analysis, although recent studies suggest that plasma metanephrines can also be measured in patients without the 30-min resting period and in the sitting position, with similar sensitivity and specificity of the test [133,134].

Clonidine suppression test Although plasma and urinary free metanephrines have very high sensitivities for PPGL, their specificities are lower, and false-positive rates of 10% 20% have been reported [135]. The false-positive rate is likely due to the episodic nature of catecholamine secretion, and other preanalytical variables that can affect the test. Therefore a confirmatory test for PPGL has been sought, and a clonidine suppression test may be used to improve the diagnostic specificity. Clonidine [N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine] is an α-2 agonist used to treat hypertension. By binding to α-2A adrenergic receptors, clonidine inhibits normal neurogenically mediated norepinephrine release, but autonomous norepinephrine release by PPGL is not inhibited. In one reported urine clonidine suppression test protocol, 4.3 μg of clonidine per kg body weight (up to a maximum dose of 0.3 mg) was administered orally after a baseline 12-h urine collection. Urine was collected for the

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following 12 h, and the urinary catecholamine concentrations (as a ratio to creatinine concentration) in the pre- and postchallenge collections were compared. A normal result was .50% reduction in norepinephrine and .40% reduction in normetanephrine [135]. A plasma clonidine suppression test protocol was described involving suspension of all antihypertensive medications 48 h prior to an overnight fast. The patient rested in the supine position for 30 min prior to collection of two baseline blood specimens 5 min apart from a venous cannula. Clonidine (0.3) was administered orally, and blood was collected hourly for 3 h. Diagnostic criteria for PPGL were any of the following: (1) plasma noradrenaline .2.96 nmol/L (500 ng/L) 3 h postclonidine; (2) plasma noradrenaline plus adrenaline .2.96 nmol/L (500 ng/L) 3 h postclonidine [or baseline adrenaline plus noradrenaline .11.82 nmol/L (2000 ng/L)]; or (3) plasma noradrenaline .2.96 nmol/L (500 ng/L) 3 h postclonidine and ,50% decrease in noradrenaline compared to baseline. The specificity of the protocol for PPGL was reported to be 95% [136].

Chromogranin A Chromogranin A (also called parathyroid secretory protein 1, coded on CHGA) is a neuroendocrine secretory protein that has been suggested as useful marker for PPGL. However, the protein is produced in several tissues outside of the adrenal medulla, and its diagnostic performance does not support its use as a diagnostic marker for PPGL [137]. Neuroblastoma, ganglioneuroma, and ganglioneuroblastoma Neuroblastoma is a malignant tumor that most often develops in the adrenal gland, and 90% of cases are in infants and children ,5 years of age; the median age at diagnosis is 15 months [138]. Neuroblastoma is associated with elevated levels of catecholamines and metabolites. Measurements of urinary VMA and homovanillic acid (HVA) have been used as biochemical markers for neuroblastoma, but have relatively poor sensitivity for the disease. An expanded profile of eight urinary catecholamine metabolites has been suggested, reportedly improving the sensitivity of the test to 95% [139]. Ganglioneuromas are rare, benign, well-differentiated neural crest tumors that can occur anywhere in the sympathetic nervous system, but occasionally arise in the adrenal gland. The tumors are typically asymptomatic. These tumors do not usually secrete hormones, but there have been rare cases of catecholamine-producing adrenal ganglioneuromas [140]. Ganglioneuroblastomas display an intermediate phenotype between neuroblastoma and ganglioneuroma. Because these tumors are biologically more primitive than PPGL, laboratory detection of these tumors involves measurement of urinary HVA and VMA. However, one study reported that only 5 of

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26 (19%) of patients with ganglioneuroblastoma had abnormal urinary VMA, compared to 180 of 186 (97%) of patients with neuroblastoma [141].

List of abbreviations 7-DCR: 11-DOC: 17-OHP: ACA: ACTH: ADH: AHC: AIRE: AITD: ALD: ALDP: AMN: APS: BIPSS: CAH: CBG: CESD: COMT: CRH: CT: DAX-1: DHEA: DMD: DOC: DST: EDS: ELISA: FDA: GH: GIP: GK: GR: GRA: GRS: GST: HPA: ITT: KSS: LAL: LC-MS/MS: MAO:

7-Dehydrocholesterol reductase 11-Deoxycortisol 17-Hydroxyprogesterone Adrenal cortex autoantibodies Adrenocorticotropic hormone (corticotropin) Antidiuretic hormone Adrenal hypoplasia congenita Autoimmune regulator Autoimmune thyroid disease Adrenoleukodystrophy Adrenoleukodystrophy protein Adrenomyeloneuropathy Autoimmune polyendocrine syndrome Bilateral inferior petrosal sinus sampling Congenital adrenal hyperplasia Cortisol-binding globulin Cholesterol ester storage disease Catechol-O-methyltransferase Corticotropin-releasing hormone Computerized tomography Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) Dehydroepiandrosterone Duchenne muscular dystrophy Deoxycorticosterone Dexamethasone suppression test Ehlers-Danlos syndrome Enzyme-linked immunosorbent assay (U.S.) Food and Drug Administration Growth hormone Gastric inhibitory peptide Glycerol kinase Glucocorticoid receptor Glucocorticoid-remediable aldosteronism Glucocorticoid resistance syndrome Glucagon stimulation test Hypothalamic-pituitary-adrenal Insulin tolerance test (insulin-induced hypoglycemia test) Kearns-Sayre syndrome Lysosomal acid lipase Liquid chromatography-tandem mass spectrometry Monoamine oxidase

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MEN: MRI: MSH: NNH: PBMAH: PHA: PPGL: PPNAD: PRA: RTA: SLOS: StAR: TSH: UFC: VLCFA: VMA: WD: ZSD:

Multiple endocrine neoplasia Magnetic resonance imaging Melanocyte-stimulating hormone Non-neoplastic hypercortisolism Primary bilateral macronodular hyperplasia Pseudohypoaldosteronism Pheochromocytoma/paraganglioma Primary pigmented nodular adrenocortical disease Plasma renin activity Renal tubular acidosis Smith-Lemli-Opitz syndrome Steroidogenic acute regulatory protein Thyroid-stimulating hormone (thyrotropin) Urinary free cortisol Very long-chain fatty acids Vanillylmandelic acid Wolman disease Zellweger spectrum disorder

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Chapter 5

Endocrine disorders of the reproductive system Angela M. Ferguson1 and Mark A. Cervinski2,3 1

Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, University of Missouri-Kansas City School of Medicine, Kansas City, MO, United States, 2Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Health Systems, Lebanon, NH, United States, 3The Geisel School of Medicine at Dartmouth, Hanover, NH, United States

Hypothalamic-pituitary-gonadal axis Reproductive endocrinology includes the hormones of the hypothalamicpituitary-gonadal axis and the adrenal glands. The hormones necessary for proper reproductive function include gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and various sex steroids. Steroids that feminize are classified as estrogens, and those that masculinize are known as androgens. These steroids are produced by the ovaries, testes, and adrenal glands.

Female reproductive disorders The complexity of the female reproductive system allows the opportunity for many abnormalities to occur (Table 5.1). Included here are prepubertal disorders, hirsutism, irregular menses, and polycystic ovarian syndrome (PCOS). All of these disorders can affect fertility, and that will be discussed here in a separate infertility section.

Prepubertal disorders Puberty occurs in a predictable sequence of events, with breast development appearing first, followed by growth of pubic hair, a linear growth spurt, and finally menarche [1]. Puberty is initiated by the release of GnRH by the hypothalamus, which stimulates secretion of LH and FSH by the pituitary. The normal age for onset of puberty for females is between 8 and 13 years [1]. Precocious puberty is the development of secondary sexual characteristics before the age of Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00005-4 © 2021 Elsevier Inc. All rights reserved.

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TABLE 5.1 Female reproductive disorders. Hypogonadotropic hypogonadism Pituitary Necrosis Adenoma Inflammatory disorders Hypothalamus GnRH defect (chronic stress, malnutrition, Kallmann syndrome, strenuous exercise) Tumors Infection Hypergonadotropic hypogonadism Premature ovarian failure Chronic anovulation (PCOS, adrenal disease, thyroid disease, ovarian tumor) Gonadal agenesis Gonadal dysgenesis Steroidogenesis deficiency (17-hydroxylase deficiency, 17,20-lyase deficiency)

eight and is 10 times more common in girls than boys. Nonpathologic variants of precocious puberty include premature thelarche (premature breast development) and premature adrenarche (premature sexual hair development). When these signs are present individually and not in conjunction with increased rates of bone growth, they are usually not indicative of precocious puberty, and further investigation may not be warranted [2]. Pathologic precocious puberty is either GnRH-dependent, known as central precocious puberty (CPP), or GnRH-independent, which is referred to as peripheral precocious puberty (PPP). The majority of cases of CPP in girls are idiopathic. Known causes of CPP include central nervous system tumors such as hypothalamic hamartomas, hydrocephalus, neurofibromas, neoplasms, and trauma. The gold standard for the diagnosis of CPP is still the GnRH stimulation test, even with the increases in sensitivity and accuracy with LH testing [3]. A peak serum LH concentration between 5 and 8 IU/L after GnRH administration is considered diagnostic for precocious puberty [3,4]. It is also useful to compare the ratio of FSH concentration with LH concentration after stimulation, and a ratio of greater than 0.66 is indicative of a pubertal response [3,4]. Treatment of CPP includes the use of GnRH agonists to inhibit gonadotropin release and delay pubertal progression [2]. In girls with PPP, the secretion of sex steroids is independent of pituitary gonadotropin release; the response to the GnRH stimulation test is repressed,

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and concentrations of LH and FSH are low. The increase in sex steroids could be caused by a gonadal or adrenal tumor, hepatoblastoma, hypothyroidism, McCune Albright syndrome (MAS), or congenital adrenal hyperplasia (CAH). Of the cases of CAH, 95% are due to 21-hydroxylase (21-OH) deficiency, which results in cortisol deficiency and androgen excess [5]. Classic cases of CAH present with virilization, growth acceleration, and accelerated bone maturation. Basal, early morning 17-hydroxyprogesterone concentrations measured via liquid chromatography-mass spectrometry can be used to screen for 21-OH-deficient CAH. 17-Hydroxyprogesterone is the substrate of 21-OH, and high concentrations will be present when 21-OH is nonfunctional. The gold standard for diagnosis of this disorder is the adrenocorticotropic hormone (ACTH) stimulation test, with a diagnostic cutoff for 17-hydroxyprogesterone of .1000 ng/dL (30 nmol/L) poststimulation [6]. Delayed puberty is defined as no signs of breast development by age 13 [7]. Constitutional delay of growth and puberty is the most common cause of delayed puberty, and the majority of patients have a family history of a parent with delayed puberty. Constitutional delay of puberty is a diagnosis of exclusion. Other causes of delayed puberty can be diagnosed by measuring serum LH, FSH, and estradiol. Patients with hypergonadotropic hypogonadism have increased concentrations of LH and FSH compared to the pubertal range. Hypergonadotropic hypogonadism may be congenital or acquired, and is more commonly found in girls as opposed to boys. Hypogonadotropic hypogonadism results in low levels of LH and FSH as compared to the pubertal range and can be classified as functional or persistent. Patients with the persistent form will need hormonal treatment to induce puberty [7].

Hirsutism Hirsutism is defined as the excessive growth of terminal hair in women and children in a male-like pattern [8 10] and is found in 70% 80% of patients with hyperandrogenism [8]. True hirsutism (thick and coarse terminal hair growth in androgen-responsive areas) must be distinguished from hypertrichosis (excessive growth of nonandrogen-responsive hairs). Women with androgen-responsive hirsutism either have excess androgen or have an increased sensitivity to normal concentrations of androgens. Not all hirsute patients have evidence of androgen excess or endocrine imbalance; this is termed idiopathic hirsutism and can be due to their ethnic background or an increase in 5α-reductase activity in the skin [10]. PCOS is the most common cause of androgen hypersecretion, and 70% 80% of hirsute women also have PCOS [11]. Nonclassical congenital adrenal hyperplasia (NCCAH), acromegaly, hyperprolactinemia, menopause, and ACTH-dependent Cushing syndrome can also be causes of hirsutism. The 2018 clinical practice guideline on evaluation and treatment of hirsutism by the Endocrine Society suggests testing for elevated androgen concentrations in all women with an abnormal Ferriman Gallwey hirsutism score or in women

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with patient-important hirsutism and menstrual irregularities [12]. Serum total testosterone should be measured, followed by serum-free testosterone, in women with moderate to severe hirsutism who have normal total testosterone. Measurement of testosterone in women with automated laboratory immunoassays is problematic as these assays lack sufficient sensitivity to accurately and precisely quantify the low concentrations found in women. Measurement of serum testosterone in women is currently best assessed by liquid chromatography-tandem mass spectrometry or other technology that is sufficiently precise at these low concentrations. Women who are hyperandrogenemic should also be screened for NCCAH by measuring 17-hydroxyprogesterone. Women with a high risk of NCCAH should also be screened, even if they have normal total and free testosterone concentrations [12]. The laboratory evaluation for hirsutism may also include measurement of dehydroepiandrosterone sulfate (DHEA-S) [9,10]. If DHEA-S concentrations are elevated, it suggests an adrenal source of androgens, whereas an increase in testosterone suggests either an ovarian or an adrenal source. If circulating concentrations of testosterone are greater than 200 ng/dL, and DHEA-S concentrations are greater than 700 μg/dL, the possibility of an androgen-producing tumor should be evaluated. In the majority of cases, the most likely causes of hyperandrogenemia in women are PCOS or hyperthecosis [13]. Oral contraceptives are recommended as first-line therapy for hirsutism, with an antiandrogen added after 6 months of treatment if patient-important hirsutism remains [12]. Insulin-lowering drugs are not recommended for the treatment of hirsutism when hirsutism is the only clinical indication. Idiopathic hirsutism is a diagnosis of exclusion in women with mild hirsutism, but normal ovulatory function and normal androgen concentrations [10].

Irregular menses Menstruation occurs, on average, every 28 days in a normal ovulatory menstrual cycle. Amenorrhea, the absence of menstrual bleeding, can be characterized as either primary (never having a menstrual period) or secondary (menstruation is present and then ceases) (Table 5.2). Pregnancy should always be ruled out first when dealing with both primary and secondary amenorrhea. Along with pregnancy, all conditions that can cause secondary amenorrhea can also cause primary amenorrhea, but the reverse is not the case [14]. Primary amenorrhea is defined as a failure to establish spontaneous periodic menstruation by the age of 15 or within 5 years of breast development if breast development occurs before the age of 10 [15]. There is some controversy about this age limit because some studies suggest that it should be lowered [14]. A physical examination, patient history, and laboratory evaluation of thyroidstimulating hormone (TSH), FSH, and prolactin can identify the most common causes of amenorrhea [15]. If there are high concentrations of FSH and LH in conjunction with low Tanner stages, this can be a sign of delayed puberty and hypergonadotropic hypogonadism. There are numerous causes of primary

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TABLE 5.2 Causes of amenorrhea. Primary amenorrhea Pituitary-hypothalamic disorders . Turner syndrome Hypopituitarism Kallmann syndrome Nutritional disorders Thyroid disorders Hypothyroidism Adrenal disorders Congenital adrenal hyperplasia Lower tract defects Vaginal aplasia Ovarian disorders Testicular feminization Polycystic ovarian syndrome Uterine disorders Endometritis Mu¨llerian agenesis Secondary amenorrhea Pregnancy/lactation Adrenal disorders Cushing syndrome Ovarian disorders Ovarian tumors Polycystic ovarian syndrome Premature ovarian failure Uterine disorders Asherman syndrome Hypothalamic disorders Tumors Excessive exercise (Continued )

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TABLE 5.2 (Continued) Nutritional disorders Stress Pituitary disorders Sheehan syndrome Thyroid disorders Hypothyroidism Hyperthyroidism Adrenal disorders Nonclassical congenital adrenal hyperplasia Cushing syndrome Tumors Iatrogenic Antidepressants Drugs with estrogenic activity

amenorrhea including Turner syndrome, androgen insensitivity syndrome, Mu¨llerian agenesis, and hypogonadotropic hypogonadism, among others [15]. The most common cause for hypergonadotropic hypogonadism in females is Turner syndrome, which is defined as a 45,X karyotype and female phenotype [14,15]. Moderately high concentrations of FSH and LH are also found in cases of androgen insensitivity syndrome, in which the patient has a 46, XY karyotype but is phenotypically female. Androgen insensitivity is characterized by the development of a vagina, absence of the ovaries and uterus, and presence of testes in the abdominal cavity. Approximately 10% of cases of primary amenorrhea are due to Mu¨llerian agenesis. In Mu¨llerian agenesis the Mu¨llerian ducts fail to develop and there is a consequent absence of the uterus and variable hypoplasia of the upper vagina. In addition to physical differences, Mu¨llerian agenesis can be distinguished from androgen insensitivity by evaluation of testosterone concentrations. In androgen insensitivity, testosterone will be in the male reference interval or higher, whereas in Mu¨llerian agenesis, testosterone will be in the female reference interval [15]. Patients with hypogonadotropic hypogonadism present with low concentrations of FSH, LH, and estradiol Kallmann syndrome (KS) is one example of hypogonadotropic hypogonadism and is discussed further in the section on male reproductive disorders. Secondary amenorrhea is defined as the absence of three or more consecutive menstrual periods at some point after the first menstrual period.

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Oligomenorrhea is infrequent menstruation, occurring less than nine times a year [16]. When investigating secondary amenorrhea, the laboratory testing should be guided by the presence or absence of signs of hyperandrogenism such as hirsutism, acne, and rapid weight gain. Concentrations of testosterone and DHEA-S higher than 200 and 700 μg/dL, respectively, are suggestive of adrenal or ovarian tumors [14]. Slightly lower concentrations are suggestive of PCOS, which is discussed in the next section. In patients without signs of hyperandrogenism, elevated concentrations of FSH and LH could indicate ovarian failure. Premature ovarian failure is the failure of estrogen production in the setting of high gonadotropin concentrations when the patient is younger than 40 years of age [15]. Ovarian failure can be confirmed by an FSH concentration persistently in the menopausal range. An MRI to assess the presence of a pituitary tumor should be performed after hypothyroidism is ruled out [15]. Elevated concentrations of prolactin can also cause amenorrhea, and menses can resume once the prolactin concentrations are reduced by the preferred treatment of dopamine agonists, both in the presence or absence of a pituitary tumor [15]. Other disorders of the uterus, pituitary, and hypothalamus can also cause amenorrhea. If the cause of amenorrhea has not been elucidated, serum estradiol should be measured, or a progesterone challenge can be done to determine relative estrogen status. Treatment with progesterone will stimulate menstrual bleeding in women with an estrogen-primed uterus. This so-called “withdrawal bleeding” (called this because bleeding begins when progesterone treatment is ceased) will occur as long as the estrogen concentration is adequate, and there is no outflow obstruction. The progesterone challenge test is preferred to measuring serum estradiol due to the daily fluctuations in estradiol concentrations, but can have a false-positive rate of about 20% [15].

Polycystic ovarian syndrome PCOS is the most common endocrine disorder of reproductive-aged women, affecting approximately 10% of this population [17]. PCOS is an endocrine disorder that presents with a spectrum of symptoms that may include hyperandrogenism, ovulatory dysfunction, the presence of polycystic ovarian morphology on ultrasound, menstrual cycle disturbances, anovulatory infertility, insulin resistance, and obesity [18]. PCOS is also associated with an increased risk of endometrial cancer [18]. The term “polycystic ovary” is actually a misnomer as the ovaries are not cyst filled, but rather are studded with numerous ovarian follicles arrested in the small antral follicle stage of development. The diagnosis of PCOS is currently made based on the Rotterdam diagnostic criteria that require that for adult women two of the following three criteria be met: (1) The patient must demonstrate oligo- or anovulation; (2) there must be clinical and/or biochemical signs of

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hyperandrogenism; (3) demonstration of polycystic ovaries via ultrasound. Importantly, other causes of these features such as hyperprolactinemia, thyroid disease, and others must also be absent [18]. For adolescents (,20 years of age) the diagnostic criteria are slightly different in that the use of ultrasound is not recommended and a minimum of 2 years must have passed since menarche [18]. The assessment of androgen excess does not rely on laboratory testing and be done via the observation of hirsutism and female pattern hair loss. Laboratory assessment of the androgen excess may include evaluation of serum testosterone, androstenedione, LH, and FSH or the calculation of the free androgen index [18,19]. Testosterone and androstenedione are frequently elevated in PCOS; however, there is a marked overlap in the distribution of values between women with and without PCOS. In PCOS, the concentrations of LH are often elevated, while FSH can be low-to-low-normal, for this reason some have proposed the use of an elevated LH/FSH ratio to indicate PCOS. For all these proposed serum markers there is considerable overlap in the distributions between women with and without PCOS. Consequently, the area under the curve (AUC) from receiver operator characteristic analyses are quite low with the AUC of testosterone equal to 0.75, androstenedione 0.66, and LH/FSH ratio equal to 0.72 [19]. It is also important to recall that while PCOS is a disorder characterized by hyperandrogenism that patients with PCOS will also typically present with high estradiol due to the peripheral aromatization of testosterone. Recently there have been a number of publications that have explored the use of anti-Mu¨llerian hormone (AMH) as a diagnostic marker of PCOS. In women, AMH is produced primarily by the small antral follicles of the ovary, which is the stage at which follicles are arrested in PCOS. Consequently, serum AMH concentrations in women with PCOS are higher than in non-PCOS women [20] and AMH concentration correlates better with the antral follicle count than do other serum biomarkers such as FSH, LH, estradiol, or inhibin B [21]. AMH as of yet has not been adopted into the diagnostic criteria for PCOS, partially because of the need for assay harmonization [18]. Other challenges to the use of AMH that will need to be addressed is the need for age-specific thresholds as AMH normally decreases with age beginning at approximately the age of 25. AMH concentrations are affected by body mass and smoking with lower concentrations observed in overweight and obese women, as well as in women who smoke [22 24].

Infertility Infertility is defined as the inability to conceive after 1 year of unprotected intercourse. Age is one of the main factors in a woman’s ability to conceive, and beyond the age of 35, the chances of conceiving naturally as well as the successful outcome of treatment strategies decrease dramatically [25]. If a couple has not conceived after 1 year of unprotected sexual intercourse

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History, cervical smear

Normal

Regular cycle

Abnormal

Irregular cycle

Further evaluation and treatment

BBT, LH, midluteal progesterone

Progestin challenge

Anovulatory

Ovulatory

Assess tubal patency and uterine cavity

Normal

Abnormal

Progestin challenge

Normal

Abnormal

Normal

Continue intercourse 6 months to 1 year, consult specialist

Laparoscopy or surgery, IVF

Ovulation induction

Abnormal

TSH, prolactin, testosterone, FSH/LH ratio

FIGURE 5.1 Algorithm for evaluating female infertility. BBT 5 basal body temperature. Modified from A. Taylor, Abc of subfertility. Making a diagnosis, BMJ 327, 2003, 494 497; and R.D. Nerenz, E. Jungheim, A.M. Gronowski, Reproductive endocrinology and related disorders, in: N. Rifai, A.R. Horvath, C.T. Whittwer (Eds.), Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, sixth ed., Elsevier, St. Louis, 2018, pp. 1617 1654.

(6 months if the woman is over 35), testing should be undertaken to try to determine the nature of the problem. One approach to the evaluation of female infertility is shown in Fig. 5.1. Factors that can contribute to female infertility include ovulatory dysfunction, ovarian reserve, and structural factors. Female infertility evaluation History Duration of infertility Menstrual history Previous methods of contraception Physical exam Weight and BMI Thyroid nodules Signs of androgen excess Ovulatory function Progesterone measurements Ovarian reserve AMH FSH and estradiol Uterine abnormalities Tubal patency Source: Modified from D.A. Klein, J.E. Emerick, J.E. Sylvester, K.S. Vogt, Disorders of puberty: an approach to diagnosis and management, Am. Fam. Physician 96 (2017) 590 599 and Practice Committee of the American Society for Reproductive Medicine, Diagnostic evaluation of the infertile female: a committee opinion, Fertil. Steril. 103 (2015) e44 e50.

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Ovulatory dysfunction Disorders of ovulation account for up to 30% of infertility and can manifest as either oligomenorrhea or amenorrhea [27]. PCOS is the most common cause of anovulation and has been discussed earlier in this chapter. Obesity, hypothalamic and pituitary dysfunction, hyperprolactinemia, and thyroid disorders can also cause ovulatory dysfunction. Current laboratory tests do not directly confirm ovulation, but the measurement of serum progesterone serves as a good indicator. The increase in progesterone that occurs during the midluteal phase (days 21 23) indicates that a corpus luteum has been formed but cannot confirm that an egg was released. If ovulation does not occur, the corpus luteum does not form normally, and the rise in progesterone may be subnormal. As menstrual cycles vary in length, collection of a specimen for progesterone testing should be done approximately 1 week prior to the expected start of menses rather than a specific cycle day [26]. Due to the wide fluctuations in progesterone concentrations, a progesterone concentration of .3 ng/mL is evidence of ovulation. Another indicator of ovulation is an increase in basal body temperature. Ovulation is associated with a rapid increase in body temperature by 0.5 F due to the increased progesterone concentration. Basal body temperature thermometers are inexpensive and simple to use, but they cannot be used to time intercourse because they indicate ovulation retrospectively. To prospectively indicate when ovulation should occur, serum or urine LH concentration can be measured. LH is detectable in the urine just after the serum LH surge and 24 36 h before ovulation. Serum concentrations of LH during the midcycle peak range from 21.9 to 56.6 IU/L. An alternative method to determine the LH surge is the use of home urine LH devices. These lateral flow qualitative immunoassays indicate the LH surge and become positive with urine LH concentrations generally .30 mIU/mL [28].

Ovarian reserve Women in their mid to late 30s and early 40s with infertility constitute the largest portion of the total infertility population. These women also have a higher incidence of pregnancy loss. The decrease in fertility is due to a diminished ovarian reserve because of follicular depletion and decline in oocyte quality. As the number of follicles decreases with age, FSH concentrations in the follicular phase gradually increase. This increase in FSH is thought to be associated with the decreased production of inhibin B by the developing small antral follicles [29]. Assessment of ovarian reserve via FSH and estradiol is performed by measuring both compounds on day 3 of the menstrual cycle. Day 3 FSH concentrations .5 IU/L are considered elevated and associated with poor reproductive outcome, as are basal estradiol concentrations .75 pg/mL [29,30].

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Inhibin B has been proposed as a more direct measurement of ovarian reserve as it is directly produced by the developing follicles, and because it is produced by gonadal tissue, it is thought to be a more direct marker of ovarian reserve than pituitary hormones. Day 3 inhibin B concentrations are decreased in women with diminished ovarian reserve, and this inhibin B decrease can be detected prior to observation of an increase in FSH concentrations [31]. Recently, anti-Mu¨llerian hormone, a hormone produced predominantly by the large antral follicles of the ovary, has been examined as an indicator of ovarian reserve, and with a cutoff of 0.25 pg/mL, it provides higher sensitivity and specificity than age, FSH, estradiol, and antral follicle count [32]. To date, studies examining the relationship between AMH and achieving successful pregnancy have not shown correlation between AMH concentrations and successful pregnancy [33]. AMH concentrations have however been found to be useful in the tailoring of ovarian stimulation protocols to reduce the risk of ovarian hyperstimulation syndrome (OHSS). OHSS is a potential complication of the use of exogenous gonadotropin-mediated stimulation of the ovaries for the purposes of oocyte retrieval as part of in vitro fertilization (IVF) treatment. This potentially life-threatening side effect of ovarian stimulation is due to the recruitment and maintenance of an unusually large number of follicles that evolve into luteal bodies following oocyte retrieval. The luteal bodies in addition to producing large concentrations of progesterone also produce vascular endothelial growth factor, which increases vascular permeability. OHSS is characterized by increased vascular permeability leading to fluid shifts from the vascular compartment to extravascular spaces. If sufficiently severe OHSS can cause edema, renal impairment, pericardial and pulmonary effusions, and ascites [34].

Structural factors Evaluation of tubal patency should be done in a normal cycling woman to rule out structural abnormalities in the Fallopian tubes [35]. Hysterosalpingography or hysterosalpingo-contrast ultrasonography can indicate whether there is a tubal blockage and give information about the shape of the uterine cavity. Hysterosalpingocontrast ultrasonography has the added benefit of an ultrasound scan of the pelvis, which can identify fibroids or polycystic ovaries [25]. Sonohysterography can identify abnormalities in the uterine cavity such as intrauterine adhesions, endometrial polyps, and submucosal fibroids [36].

Treatments Patients dealing with infertility have many options to aid them when trying to conceive. Standard treatment for a woman with anovulation is ovulation induction with clomiphene citrate or gonadotropins followed by timed

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intercourse or intrauterine insemination. In the case of tubal sterility, diminished ovarian reserve, or the failure of standard treatments, IVF can be performed. The process of IVF includes ovarian stimulation, retrieval of the ova, fertilization, and then transfer of the fertilized ova into the uterus. A slightly different procedure called gamete intra-Fallopian transfer places the unfertilized ova and sperm into the Fallopian tube, whereas zygote intra-Fallopian transfer places a fertilized zygote into the Fallopian tube. Fertilization of the ova can also take place by direct intracytoplasmic sperm injection (ICSI) [37]. Laboratory monitoring throughout all the assisted reproduction treatments discussed is necessary to determine not only the dose of therapy but also its duration and efficacy [35,38]. For more indepth information on this topic, the following reviews are recommended [37,39].

Male reproductive disorders There are a number of abnormalities that can affect the male reproductive system throughout life (Table 5.3). These include hypogonadotropic hypogonadism, hypergonadotropic hypogonadism, Klinefelter syndrome, defects in androgen action, erectile dysfunction, and gynecomastia. These abnormalities can also affect fertility, which will be discussed here in a separate infertility section.

TABLE 5.3 Male reproductive disorders. Hypogonadotropic hypogonadism Panhypopituitarism Hypothalamic syndrome (tumors, infection) Hyperprolactinemia GnRH deficiency (stress, malnutrition, strenuous exercise, Kallmann syndrome) Hypergonadotropic hypogonadism Gonadal failure Chromosomal defects (Klinefelter syndrome) Defective androgen biosynthesis (CAH) Acute and chronic disease Defects in androgen action Testicular feminization Incomplete androgen sensitivity

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Prepubertal disorders Precocious puberty is less common in male children than female children but still does occur with a frequency of 1 2 per 10,000 male children, a rate approximately 10 20-fold lower than females [40]. Precocious puberty for males is the onset of pubertal changes before 9 years of age [41]. True puberty is characterized by the increase in the pulsatility of GnRH release from the hypothalamus, which in turn increases the secretion of LH and FSH from the pituitary and the consequent effects on the Leydig and Sertoli cells of the testes. The production of testosterone from the Leydig cells promotes the development of male secondary sexual characteristics including penile enlargement, increase in testicular volume and growth of pubic hair. The onset of puberty must be differentiated adrenarche, which is characterized by an increase in the production of the weak androgen, DHEA-S by the adrenal glands. In young males adrenarche is characterized clinically by the development of axial (underarm) odor and in the absence of accelerated linear bone growth or penile enlargement additional testing is not warranted [41]. As with precocious puberty in females, male precocious puberty can be either GnRH-dependent or CPP, or GnRH-independent, also known as peripheral precocity (PP). In males, CPP is less commonly found to be idiopathic than in females, with idiopathic CPP in males reported from various sources to be between 26% and 74% [42 44]. Causes of CPP include central nervous system tumors such as hypothalamic hamartomas, hydrocephalus, neurofibromas, neoplasms, and trauma [45]. The gold standard for the diagnosis of CPP is the GnRH stimulation test, a discussion of which is included previously in this chapter. In boys with PP, the testosterone secretion is independent of FSH and LH concentration and these gonadotropins are appropriately suppressed. Leydig cell tumors of the testes are typically nonmetastatic, unilateral, and readily produce testosterone independent of LH secretion [46]. PP may also present due to the production of human chorionic gonadotropin (hCG) by germ cell tumors occurring in gonadal and extragonadal tissues including the liver, brain, anterior mediastinum, and retroperitoneum [47]. hCG shares significant homology with LH and consequently can bind and activate the LH receptors of Leydig cells to stimulate testosterone production [34]. Other causes of PP in males are similar to the causes of female PP and include MAS, NCCAH, and severe hypothyroidism. Although MAS occurs with similar frequency in males and females, male patients are less likely to develop peripheral precocity. Severe or profound hypothyroidism may produce PP via high FSH concentrations due to TRH stimulation as well as by stimulation of the FSH receptor by high circulating concentrations of TSH. Hypothyroidism, however, is not associated with an increase in androgen production.

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Hypogonadotropic hypogonadism Male hypogonadism is characterized by decreased testicular function and can be classified as either hypogonadotropic or hypergonadotropic, depending on the cause of the decreased function. Hypogonadotropic hypogonadism occurs when the testes do not receive appropriate gonadotropin hormone stimulation due to defects in the hypothalamus or pituitary. Hypogonadotropic hypogonadism is therefore characterized by low concentrations of both testosterone and the gonadotropic hormones, LH and FSH.

Hypothalamic causes The hypothalamic causes of hypogonadotropic hypogonadism are all associated with GnRH deficiency and lack of normal hypothalamic stimulation of the pituitary. The most common of the hypothalamic disorders associated with hypogonadotropic hypogonadism is isolated GnRH deficiency (IGD). KS, the combination of IGD and anosmia (impaired olfaction), is a subset of IGD and accounts for approximately 60% of IGD. The inheritance of IGD is complex with approximately 25 known associated genes. Pathologic variants in these known genes are transmitted in autosomal recessive, autosomal dominant, and X-linked manners. Regardless of the manner in which IGD is inherited the key defect is the failure of GnRH-expressing neurons migration into the hypothalamus during development [48]. Male patients with IGD typically present with a prepubertal body habitus with Tanner stage I or II testicular volume. Laboratory testing demonstrates a low serum testosterone (,100 ng/dL) with low or inappropriately normal LH and FSH in the setting of a low testosterone concentration. Induction of secondary sexual characteristics of males with IGD can be accomplished either with testosterone or induction of testosterone secretion via hCG. Neither hCG nor testosterone treatments will, however, not treat the associated infertility due to the lack of appropriate LH and FSH stimulation. For patients wishing to achieve fertility pulsatile treatment with GnRH will restore pituitary gonadotropin release [49,50]. As GnRH treatment is, however, not widely available, treatment with the combination of hCG and FSH may also be used [48].

Hypergonadotropic hypogonadism Hypergonadotropic hypogonadism arises from primary gonadal failure. This results in a loss of the negative feedback control of the hypothalamic-pituitary axis, which is still intact and unregulated increases in gonadotropin production. Patients with primary testicular failure have elevated concentrations of LH and FSH ( . 120 mIU/mL) and decreased concentrations of testosterone (,200 ng/ dL). Primary hypogonadism can be acquired or congenital in nature. Acquired

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hypogonadism can occur due to testicular damage from trauma, irradiation chemotherapy, and other disease conditions. Congenital causes include chromosomal defects; inherited gonadotropin variants, or inherited variants to gonadotropin receptors; defects in enzymes involved in androgen synthesis; testicular agenesis; and seminiferous tubular disease, among others. Laboratory tests to diagnose hypogonadism are discussed in the section on infertility.

Klinefelter syndrome The most common cause of congenital hypergonadotropic hypogonadism is Klinefelter syndrome. Klinefelter syndrome is the most common sex chromosome disorder, affecting 1 in B660 men. Patients have one Y chromosome and two or more X chromosomes, with 47,XXY occurring in 90% of karyotypes [51]. Klinefelter syndrome is associated with dysgenesis of the seminiferous tubules, a lack of sperm production, and marked elevation of serum FSH and LH concentrations. Hypogonadism is the hallmark of this disorder, but most patients have testosterone concentrations just below the lower limit of the reference interval. Klinefelter syndrome has a wide phenotypic spectrum of presentation; consequently delayed diagnosis and underdiagnosis are problems in Klinefelter syndrome. Adults are usually diagnosed after presenting with infertility and hypogonadism, whereas those patients who present as children tend to have a more severe phenotype. Early diagnosis can be key in preventing some of the consequences of gonadal insufficiency. Diagnosis should be made based on clinical and laboratory findings as well as a cytogenetic analysis [51].

Defects in androgen action The most common defect in androgen action is androgen resistance [52]. Androgen resistance can be described as complete androgen insensitivity syndrome or partial androgen insensitivity syndrome (PAIS) and results from mutations in the androgen receptor. In the complete form, also called testicular feminization syndrome, there is sex reversal, with a genotypic XY patient with the phenotype of a female. Features include a female habitus with normal breast development; however, the vagina ends in a blind pouch, no uterus or Fallopian tubes are present, and male testes are found. In a continued attempt to stimulate the mutant androgen receptor, the testes produce testosterone at concentrations equal to or greater than a normal adult male. The excess testosterone is converted to estrogen, and together with the unresponsive testosterone receptor, results in feminization. LH concentrations are also increased, and FSH is either normal or slightly elevated. In PAIS, there is some response to androgens, leading to some male genital development. This commonly results in ambiguous external genitalia at birth, making a sex assignment very difficult. 5α-Reductase deficiency is

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one cause of PAIS [53]. In this disorder, there can be considerable variation in the extent of masculinization of the genitalia. At the onset of puberty, patients have an increase in muscle mass, voice changes, enlargement of the phallus, and growth spurts similar to unaffected siblings. If the diagnosis is made in infancy, most newborns are raised as females, but discovery of the disorder at puberty necessitates careful psychological evaluation of the patient to aid in gender selection. To help in the diagnosis of androgen insensitivity when ambiguous genitalia are present, serum testosterone can be measured after hCG stimulation. If the concentration of testosterone is .300 ng/dL, this indicates normal function of the Leydig cells. Androgen insensitivity commonly results in concentrations of testosterone .1000 ng/dL. In the absence of descended testes, measurement of Mu¨llerian-inhibiting substance and inhibin B have also been used to confirm the presence of testicular tissue [45].

Erectile dysfunction Erectile dysfunction (ED), also termed impotence is defined as the consistent or recurrent inability to attain or retain an erection sufficient for sexual activity, with the condition present for at least 3 months [54]. The prevalence of erectile dysfunction is between 2% and 9% in males between 40 and 49 years of age and increases with age so that by age 60 69 the prevalence is between 20% and 40% [55]. Erectile dysfunction can arise from psychogenic, physiologic (organic), or a mixed psychogenic and physiologic causes. Physiologic causes of erectile dysfunction are diverse and include neurogenic causes (damage to spinal cord or afferent and efferent neuropathy), vasculogenic, drug-induced (antiandrogens, antidepressants, recreational drugs), damage to the cavernous tissue of the penis, or endocrine factors. Endocrine causes of erectile dysfunction include diabetes mellitus, hypogonadism, and hyperprolactinemia. In regards to diabetes and the association with ED, it is not currently clear how diabetes increased ED risk. As diabetes is a disease associated with microvascular and macrovascular complications, hypertension and in the case of type II diabetes, obesity, the association between diabetes and ED is likely multifactorial. Hyperprolactinemia and the associated inhibition of GnRH and the consequent hypogonadism may lead to erectile dysfunction. Androgen concentrations are important in penile erection as testosterone has been demonstrated to upregulate the expression and activity of nitric oxide synthase and the expression of phosphodiesterase type 5 (PDE5). The production of nitric oxide (NO) is necessary for the penile smooth muscle relaxation associated with the production of an erection. The testosterone associated increased PDE5 expression would appear to be a counterintuitive response as inhibitors of PDE5 (PDE5I) such as sildenafil (Viagra) role in erectogenesis is to degrade cyclic guanosine monophosphate, an important mediator in

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promoting smooth muscle relaxation [55]. Despite this apparent contradiction the addition of testosterone supplementation to PDE5I treatment is an effective option for men unresponsive to PDE5I alone [55]. If hypogonadism or hyperprolactinemia is suspected in the etiology of a patient’s ED, measurements of morning serum testosterone, sex hormonebinding globulin, LH, FSH, Prolactin, and TSH are recommended [55].

Gynecomastia Benign growth of male breast glandular tissue is termed gynecomastia and is associated with an increase in the ratio of estrogen to androgen concentrations [56]. Gynecomastia is common in the neonatal and pubertal periods with a third peak of prevalence occurring in middle-aged men. Transient neonatal gynecomastia is quite common with greater than 60% of neonates demonstrating the transient growth of glandular tissue. Transient neonatal gynecomastia arises from the in utero exposure of the fetus to high concentrations of placental estrogens whose concentrations peak near the end of a full-term pregnancy. During puberty, the concentration of testosterone in males will increase approximately 30-fold, while estradiol will only increase by approximately threefold. Estradiol concentrations in pubertal males will reach their adult ranges before testosterone leading to a transient disturbance of the estrogen to androgen ratio. This imbalance of estrogens to androgens produces the physiologic gynecomastia that typically resolves in 6 18 months. Gynecomastia occurring during early puberty before significant concentrations of testosterone and estradiol are synthesized by the testes is the result of the peripheral conversion of androstenedione to estrone by aromatase and the subsequent conversion of estrone to estradiol by 17-beta-hydroxysteroid dehydrogenase. In later puberty although the testes will produce both estradiol and estrone in addition to testosterone, the majority of circulating estradiol (85%) will arise from the peripheral aromatization of testosterone [57]. A third peak in the incidence of gynecomastia can also appear in adult men between above the age of 50. Idiopathic gynecomastia in the adult male may be produced by a number of factors including decreased testosterone production associated with normal aging and increased body fat percentage, which is associated with the increased aromatization of testosterone to estradiol due to the expression of aromatase in adipose tissue. In adolescent and adult males, causes of gynecomastia include hyperthyroidism, side effects of certain medications, neoplasms, hypogonadism, and cirrhosis. Hyperthyroidism may induce gynecomastia from the hepatic upregulation of sex hormone-binding globulin (SHBG) expression. As SHBG has a higher affinity for testosterone than estradiol, the increase in SHBG perturbs the balance favoring glandular growth [58]. In cirrhotic males, testosterone concentrations are typically low and SHBG concentrations elevated. There is also an increased aromatization of

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testosterone to estradiol in cirrhosis; as a result, gynecomastia is a common finding in cirrhotic males [59]. A few medications are also associated with gynecomastia including leuprolide used in the treatment of androgenresponsive prostate cancer, as well as the potassium-sparing diuretic spironolactone. While the association of leuprolide on the suppression of androgens and development of gynecomastia is relatively straightforward, the effect of spironolactone is slightly more complex. The effect of spironolactone is twofold, causing both decreased testicular production of testosterone and increased peripheral aromatization to estradiol [60]. Estrogen mimics such as tea tree and lavender oils are emerging causes of gynecomastia with multiple case reports in literature demonstrating a temporal association between the topical use of these oils and the onset and regression of gynecomastia in prepubertal males. Other causes of gynecomastia include germinal cell or nonendocrine tumors that produce hCG and estrogen-producing tumors of the adrenal glands, testes, or liver. To determine the causative factor in gynecomastia, in cases where no specific disorder is indicated, measurement of hCG, plasma estradiol, testosterone, and LH is recommended.

Infertility Multiple factors make determining the prevalence of male infertility challenging leading to wide variations in the reported prevalence. In the United States, 15% 20% of couples are estimated to experience infertility, with approximately 30% of the causes due to male infertility alone, 35% to female causes alone, and 20% due to a combination of male and female factors [29]. The remaining 15% of infertility is unexplained and cannot be assigned to either partner. Factors that contribute to male infertility include primary testicular failure or other endocrine disorders, genital tract obstruction, hypothalamic-pituitary disease, and abnormal spermatogenesis. Only 20% of infertile men will have a defined cause of their infertility; the other 80% will not. Among men with no identifiable cause of infertility, the majority will have a decreased sperm count, abnormal morphology or motility, again the cause of which will be unknown. This group of men is categorized as having idiopathic dysspermatogenesis or idiopathic oligoasthenoteratozoospermia spermatozoa [61]. Because there are many potential causes of dysspermatogenesis and infertility, a complete diagnostic evaluation is needed. The laboratory evaluation is separated into two components: the semen analysis and endocrine parameters. One approach to the evaluation of male infertility is shown in Fig. 5.2.

Semen analysis Semen analysis measures ejaculate volume, pH, sperm count, motility, and forward progression. To properly perform semen analysis samples should be analyzed within 1 h of collection. These measurements are compared with

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History, physical exam Abnormal

Normal

Further evaluation and treatment

Semen analysis

Normal

Abnormal

No further testing indicated

Repeat

Abnormal

Normal

Testosterone, FSH, LH

↓FSH, ↓ LH, ↓testosterone

All normal

↑FSH, ↑ LH, ↓ or normal testosterone

Hypothalamic or pituitary failure, hypogonadotropic hypogonadism

Idiopathic

Gonadal failure, hypergonadotropic hypogonadism

FIGURE 5.2 Algorithm for evaluating male infertility. Modified from R.D. Nerenz, E. Jungheim, A.M. Gronowski, Reproductive endocrinology and related disorders, in: N. Rifai, A.R. Horvath, C.T. Whittwer (Eds.), Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, sixth ed., Elsevier, St. Louis, 2018, pp. 1617 1654.

TABLE 5.4 WHO reference values of semen variables. Parameter

Reference value

Ejaculate volume

$ 1.5 mL

Total sperm number

$ 39 million/ejaculate

Sperm concentration

$ 15 million/mL

Total motility

$ 40%

Progressive motility

$ 32%

Normal forms

$ 4%

Vitality

$ 58% live (i.e., excluding dye)

pH

$ 7.2

White blood cells

# 1 million/mL

Source: Modified from T.G. Cooper, E. Noonan, S. von Eckardstein, J. Auger, H.W. Baker, H.M. Behre, et al., World Health Organization reference values for human semen characteristics. Hum. Reprod. Update 16 (2010) 231 245.

the values that have been established by the World Health Organization to comprise a “normal” semen profile (Table 5.4) [63]. If the sample is abnormal, a second sample should be analyzed 6 weeks later [61]. The presence of abnormal semen analysis findings can be informative as to potential causes of male infertility; however, semen analysis does not provide any information about the ability of the sperm to function. A male whose semen analysis

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parameters are all within the WHO reference intervals may still experience infertility and there are no tests that can unequivocally predict the fertilizing capacity of spermatozoa. If there is a complete absence of spermatozoa in the ejaculate (azoospermia), an obstructive process is one potential cause that should be evaluated. In obstructive azoospermia, spermatogenesis is normal, but there is a blockage in the epididymis or the vas deferens. Low semen concentrations of glucosidase in the presence of testes of normal size, normal semen volume, and normal serum FSH are also indicative of an obstruction or congenital bilateral absence of the vas deferens (CBAVD), which only accounts for small proportion of male infertility. As CBAVD is commonly associated with mutations in the cystic fibrosis gene in approximately 70% of the cases all men with this diagnosis should be the screen for cystic fibrosis [25,35]. The postcoital test, which historically has been important in the evaluation of an infertile couple, is not considered reliable and is no longer routinely used [25,38].

Endocrine parameters In addition to semen analysis, the assessment of male infertility should include measurements of serum testosterone, FSH, LH, and prolactin. Testosterone is essential for sperm development, and due to the circadian rhythm of its daily secretion, serum should be collected in the morning. Expected testosterone concentrations range from 264 to 916 ng/dL [64]. Males with borderline or suppressed total testosterone concentrations can be evaluated with an hCG stimulation test [45]. hCG binds to LH receptors and, like LH, stimulates testosterone production by the Leydig cells. After collection of a basal sample, 5000 units of hCG are administered intramuscularly, and a serum testosterone concentration measured 3 days later. A doubling of baseline testosterone concentration is consistent with normal Leydig cell function. Failure to elevate testosterone by at least 150 ng/dL is indicative of testicular failure and primary hypogonadism [62]. FSH should be measured in men with sperm counts lower than 5 million/mL because the majority of these patients will have elevated FSH concentrations in conjunction with normal LH and testosterone concentrations [35]. Concentrations of FSH .120 mIU/mL along with decreased testosterone are indicative of primary testicular failure, and decreased concentrations of FSH and testosterone suggest hypogonadotropic hypogonadism. To distinguish between hypothalamic and pituitary dysfunction, exogenous GnRH can be administered. For proper response to GnRH, patients with long-standing functional hypogonadism must first be given exogenous testosterone for at least 1 week for appropriate gonadotropin secretion. Patients are given 100 μg of GnRH intravenously, and blood is collected at baseline and 30, 60, 120, and 180 min after injection. An increase in serum gonadotropins of at least 10 mIU/mL from baseline is a normal result. Patients with

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pituitary disease will demonstrate little or no rise in gonadotropins. Patients with hypothalamic disease should demonstrate a delayed increase of at least 7 mIU/mL by 180 min.

Treatment Only a small percentage of infertile males have treatment options available to them. In patients whom infertility is secondary to hypogonadism, treatment with pulsatile GnRH can be effective in increasing the sperm quality enough to be able to achieve a pregnancy [35]. As GnRH treatment is, however, not widely available treatment with the combination of hCG and FSH may also be used. Patients with low sperm counts can use techniques such as intrauterine insemination or IVF to increase their odds of fertilization by bringing the sperm into closer contact with an egg. The emergence of ICSI has revolutionized treatment for infertile males [65]. In ICSI, one sperm is inserted directly into the oocyte cytoplasm, and this has allowed patients with reduced sperm numbers (oligozoospermia), increased abnormal forms of sperm (teratozoospermia), and/or reduced sperm motility (asthenozoospermia) to conceive [37,39].

References [1] W.A. Marshall, J.M. Tanner, Variations in pattern of pubertal changes in girls, Arch. Dis. Child. 44 (1969) 291 303. [2] T.D. Nebesio, E.A. Eugster, Current concepts in normal and abnormal puberty, Curr. Probl. Pediatr. Adolesc. Health Care 37 (2007) 50 72. [3] L. Soriano-Guillen, J. Argente, Central precocious puberty, functional and tumor-related, Best. Pract. Res. Clin. Endocrinol. Metab. (2019) 101262. [4] R.S. Aguirre, E.A. Eugster, Central precocious puberty: from genetics to treatment, Best Pract. Res. Clin. Endocrinol. Metab. 32 (2018) 343 354. [5] D.P. Merke, S.R. Bornstein, Congenital adrenal hyperplasia, Lancet 365 (2005) 2125 2136. [6] P.W. Speiser, W. Arlt, R.J. Auchus, L.S. Baskin, G.S. Conway, D.P. Merke, et al., Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an endocrine society clinical practice guideline, J. Clin. Endocrinol. Metab. 103 (2018) 4043 4088. [7] D.A. Klein, J.E. Emerick, J.E. Sylvester, K.S. Vogt, Disorders of puberty: an approach to diagnosis and management, Am. Fam. Physician 96 (2017) 590 599. [8] B.O. Yildiz, Diagnosis of hyperandrogenism: clinical criteria, Best Pract. Res. Clin. Endocrinol. Metab. 20 (2006) 167 176. [9] R.L. Rosenfield, Clinical practice. Hirsutism, N. Engl. J. Med. 353 (2005) 2578 2588. [10] R. Azziz, The evaluation and management of hirsutism, Obstet. Gynecol. 101 (2003) 995 1007. [11] L.A. Sanchez, E.S. Knochenhauer, R. Gatlin, C. Moran, R. Azziz, Differential diagnosis of clinically evident hyperandrogenism: experience with over 1000 consecutive patients, Fertil. Steril. 76 (2001) S111.

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[12] K.A. Martin, R.R. Anderson, R.J. Chang, D.A. Ehrmann, R.A. Lobo, M.H. Murad, et al., Evaluation and treatment of hirsutism in premenopausal women: an Endocrine Society clinical practice guideline, J. Clin. Endocrinol. Metab. 103 (2018) 1233 1257. [13] J. Mihailidis, R. Dermesropian, P. Taxel, P. Luthra, J.M. Grant-Kels, Endocrine evaluation of hirsutism, Int. J. Women Dermatol. 3 (2017) S6 S10. [14] G.B. Slap, Menstrual disorders in adolescence, Best Pract. Res. Clin. Obstet. Gynaecol. 17 (2003) 75 92. [15] Practice Committee of American Society for Reproductive Medicine, Current evaluation of amenorrhea, Fertil. Steril. 90 (2008) S219 S225. [16] R.L. Rosenfield, R.B. Barnes, Menstrual disorders in adolescence, Endocrinol. Metab. Clin. North Am. 22 (1993) 491 505. [17] G. Bozdag, S. Mumusoglu, D. Zengin, E. Karabulut, B.O. Yildiz, The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis, Hum. Reprod. 31 (2016) 2841 2855. [18] H.J. Teede, M.L. Misso, M.F. Costello, A. Dokras, J. Laven, L. Moran, et al., Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome, Hum. Reprod. 33 (2018) 1602 1618. [19] J.H. Barth, H.P. Field, E. Yasmin, A.H. Balen, Defining hyperandrogenism in polycystic ovary syndrome: measurement of testosterone and androstenedione by liquid chromatography-tandem mass spectrometry and analysis by receiver operator characteristic plots, Eur. J. Endocrinol. 162 (2010) 611 615. [20] D. Dewailly, H. Gronier, E. Poncelet, G. Robin, M. Leroy, P. Pigny, et al., Diagnosis of polycystic ovary syndrome (PCOS): revisiting the threshold values of follicle count on ultrasound and of the serum AMH level for the definition of polycystic ovaries, Hum. Reprod. 26 (2011) 3123 3129. [21] R. Fanchin, L.M. Schonauer, C. Righini, J. Guibourdenche, R. Frydman, J. Taieb, Serum anti-Mullerian hormone is more strongly related to ovarian follicular status than serum inhibin B, estradiol, FSH and LH on day 3, Hum. Reprod. 18 (2003) 323 327. [22] A. Piouka, D. Farmakiotis, I. Katsikis, D. Macut, S. Gerou, D. Panidis, Anti-Mullerian hormone levels reflect severity of PCOS but are negatively influenced by obesity: relationship with increased luteinizing hormone levels, Am. J. Physiol. Endocrinol. Metab. 296 (2009) E238 E243. [23] V. Moy, S. Jindal, H. Lieman, E. Buyuk, Obesity adversely affects serum anti-Mullerian hormone (AMH) levels in Caucasian women, J. Assist. Reprod. Genet. 32 (2015) 1305 1311. [24] M. Dolleman, W.M. Verschuren, M.J. Eijkemans, M.E. Dolle, E.H. Jansen, F.J. Broekmans, et al., Reproductive and lifestyle determinants of anti-Mullerian hormone in a large population-based study, J. Clin. Endocrinol. Metab. 98 (2013) 2106 2115. [25] A. Taylor, Abc of subfertility. Making a diagnosis, BMJ 327 (2003) 494 497. [26] Practice Committee of the American Society for Reproductive Medicine, Diagnostic evaluation of the infertile female: a committee opinion, Fertil. Steril. 103 (2015) e44 e50. [27] D. Hamilton-Fairley, A. Taylor, Anovulation, BMJ 327 (2003) 546 549. [28] When the test really counts. Consumer Reports, February 2003, pp. 45 50. [29] N.S. Macklon, B.C. Fauser, Ovarian reserve, Semin. Reprod. Med. 23 (2005) 248 256. [30] F.L. Licciardi, H.C. Liu, Z. Rosenwaks, Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization, Fertil. Steril. 64 (1995) 991 994.

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[31] D.B. Seifer, R.T. Scott Jr., P.A. Bergh, L.K. Abrogast, C.I. Friedman, C.K. Mack, et al., Women with declining ovarian reserve may demonstrate a decrease in day 3 serum inhibin B before a rise in day 3 follicle-stimulating hormone, Fertil. Steril. 72 (1999) 63 65. [32] C. Ficicioglu, T. Kutlu, E. Baglam, Z. Bakacak, Early follicular antimullerian hormone as an indicator of ovarian reserve, Fertil. Steril. 85 (2006) 592 596. [33] L. Hawkins Bressler, A. Steiner, Anti-mullerian hormone as a predictor of reproductive potential, Curr. Opin. Endocrinol. Diabetes Obes. 25 (2018) 385 390. [34] R. Fleming, F. Broekmans, C. Calhaz-Jorge, L. Dracea, H. Alexander, A. Nyboe Andersen, et al., Can anti-mullerian hormone concentrations be used to determine gonadotropin dose and treatment protocol for ovarian stimulation? Reprod. Biomed Online 26 (2013) 431 439. [35] G. Forti, C. Krausz, Clinical review 100: evaluation and treatment of the infertile couple, J. Clin. Endocrinol. Metab. 83 (1998) 4177 4188. [36] Infertility workup for the women’s health specialist, ACOG Committee opinion, number 781, Obstet. Gynecol. 133 (2019) e377 e384. [37] L. Thurston, A. Abbara, W.S. Dhillo, Investigation and management of subfertility, J. Clin. Pathol. 72 (2019) 579 587. [38] K.A. Frey, K.S. Patel, Initial evaluation and management of infertility by the primary care physician, Mayo Clin. Proc. 79 (2004) 1439 1443. quiz 43. [39] R. Wang, N.A. Danhof, R.I. Tjon-Kon-Fat, M.J. Eijkemans, P.M. Bossuyt, M.H. Mochtar, et al., Interventions for unexplained infertility: a systematic review and network metaanalysis, Cochrane Database Syst. Rev. 9 (2019) CD012692. [40] G. Teilmann, C.B. Pedersen, T.K. Jensen, N.E. Skakkebaek, A. Juul, Prevalence and incidence of precocious pubertal development in Denmark: an epidemiologic study based on national registries, Pediatrics 116 (2005) 1323 1328. [41] P. Kaplowitz, C. Bloch, Section on Endocrinology AAoP. Evaluation and referral of children with signs of early puberty, Pediatrics (2016) 137. [42] K.H. Choi, S.J. Chung, M.J. Kang, J.Y. Yoon, J.E. Lee, Y.A. Lee, et al., Boys with precocious or early puberty: incidence of pathological brain magnetic resonance imaging findings and factors related to newly developed brain lesions, Ann. Pediatr. Endocrinol. Metab. 18 (2013) 183 190. [43] L.S. Topor, K. Bowerman, J.T. Machan, C.L. Gilbert, T. Kangarloo, N.D. Shaw, Central precocious puberty in Boston boys: a 10-year single center experience, PLoS One 13 (2018) e0199019. [44] A. Alikasifoglu, D. Vuralli, E.N. Gonc, A. Ozon, N. Kandemir, Changing etiological trends in male precocious puberty: evaluation of 100 cases with central precocious puberty over the last decade, Horm. Res. Paediatr. 83 (2015) 340 344. [45] L.S. Nield, N. Cakan, D. Kamat, A practical approach to precocious puberty, Clin. Pediatr. (Phila.) 46 (2007) 299 306. [46] A.A. Gheorghisan-Galateanu, Leydig cell tumors of the testis: a case report, BMC Res. Notes 7 (2014) 656. [47] A. Bravo-Balado, L. Torres Castellanos, A. Carrillo Rodriguez, D. Gomez Zapata, J.J. Lammoglia Hoyos, R. Andrade, et al., Primary mediastinal pure seminomatous germ cell tumor (germinoma) as a rare cause of precocious puberty in a 9-year-old patient, Urology 110 (2017) 216 219. [48] R. Balasubramanian, W.F. Crowley Jr., Isolated gonadotropin-releasing hormone (GnRH) deficiencyVol. Seattle (WA) in: M.P. Adam, H.H. Ardinger, R.A. Pagon, S.E. Wallace, L.J.H. Bean, K. Stephens, A. Amemiya (Eds.), GeneReviews, 1993.

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[49] N. Pitteloud, F.J. Hayes, A. Dwyer, P.A. Boepple, H. Lee, W.F. Crowley Jr., Predictors of outcome of long-term GnRH therapy in men with idiopathic hypogonadotropic hypogonadism, J. Clin. Endocrinol. Metab. 87 (2002) 4128 4136. [50] P.S. Tsai, J.C. Gill, Mechanisms of disease: insights into X-linked and autosomaldominant Kallmann syndrome, Nat. Clin. Pract. Endocrinol. Metab. 2 (2006) 160 171. [51] A. Bojesen, C.H. Gravholt, Klinefelter syndrome in clinical practice, Nat. Clin. Pract. Urol. 4 (2007) 192 204. [52] I.A. Hughes, A. Deeb, Androgen resistance, Best Pract. Res. Clin. Endocrinol. Metab. 20 (2006) 577 598. [53] C. Sultan, S. Lumbroso, F. Paris, C. Jeandel, B. Terouanne, C. Belon, et al., Disorders of androgen action, Semin. Reprod. Med. 20 (2002) 217 228. [54] S. Sivalingam, H. Hashim, H. Schwaibold, An overview of the diagnosis and treatment of erectile dysfunction, Drugs 66 (2006) 2339 2355. [55] R. Shamloul, H. Ghanem, Erectile dysfunction, Lancet 381 (2013) 153 165. [56] D.E. Greydanus, L. Matytsina, M. Gains, Breast disorders in children and adolescents, Prim. Care 33 (2006) 455 502. [57] G.D. Braunstein, Clinical practice. Gynecomastia, N. Engl. J. Med. 357 (2007) 1229 1237. [58] J.F. Dunn, B.C. Nisula, D. Rodbard, Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma, J. Clin. Endocrinol. Metab. 53 (1981) 58 68. [59] M. Sinclair, M. Grossmann, P.J. Gow, P.W. Angus, Testosterone in men with advanced liver disease: abnormalities and implications, J. Gastroenterol. Hepatol. 30 (2015) 244 251. [60] L.I. Rose, R.H. Underwood, S.R. Newmark, E.S. Kisch, G.H. Williams, Pathophysiology of spironolactone-induced gynecomastia, Ann. Intern. Med. 87 (1977) 398 403. [61] A. Hirsh, Male subfertility, BMJ 327 (2003) 669 672. [62] R.D. Nerenz, E. Jungheim, A.M. Gronowski, Reproductive endocrinology and related disorders, in: N. Rifai, A.R. Horvath, C.T. Whittwer (Eds.), Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, sixth ed, Elsevier, St. Louis, 2018, pp. 1617 1654. [63] T.G. Cooper, E. Noonan, S. von Eckardstein, J. Auger, H.W. Baker, H.M. Behre, et al., World Health Organization reference values for human semen characteristics, Hum. Reprod. Update 16 (2010) 231 245. [64] T.G. Travison, H.W. Vesper, E. Orwoll, F. Wu, J.M. Kaufman, Y. Wang, et al., Harmonized reference ranges for circulating testosterone levels in men of four cohort studies in the United States and Europe, J. Clin. Endocrinol. Metab. 102 (2017) 1161 1173. [65] D.M. De Kretser, H.W. Baker, Infertility in men: recent advances and continuing controversies, J. Clin. Endocrinol. Metab. 84 (1999) 3443 3450.

Chapter 6

Gastroenteropancreatic neuroendocrine tumors Neeraj Ramakrishnan1, Seong Hyun Ahn1 and Ishwarlal Jialal2,3 1

California Northstate University, College of Medicine, CA, United States, 2Sacramento VA Medical Center, CA, United States, 3Retired Distinguished Professor, UCDAVIS, CA, United States

Neuroendocrine tumors (NETs) of the gastrointestinal (GI) tract can be broadly classified as gastrointestinal NETs (GI-NETs) also referred to as previously and more commonly as carcinoid tumors and pancreatic NETs (PNETs). The NETs can be functional (secreting peptides and amines) or nonfunctional and are classified based on histology, mitotic counts, and Ki67 staining into low, intermediate, and high grade, that is, Grades 1, 2, and 3, respectively. In this chapter, we briefly review the physiology and biochemistry of the hormones resulting in clinical syndromes and then discuss the clinical presentation and diagnosis of the more common syndromes. Whilst we discuss the clinical syndromes such as PNETs and GI-NETs, it is important to emphasize that some of these tumors can emanate from both the pancreas and intestinal cells (e.g., gastrinomas). It appears that up to 25% are associated with inherited disorders including multiple endocrine neoplasia type 1 (MEN1), von Hippel Lindau disease, neurofibromatosis 1 (NF-1), and tuberous sclerosis complex. It appears that the commonest type of NET is a nonfunctional PNET. In fact, loss of heterozygosity at the MEN1 locus on chromosome 11q13 is seen in 93% of sporadic PNETs [1].

Biochemistry and physiology of the more common gastrointestinal hormones Enteroendocrine cells of the GI tract release numerous hormones that orchestrate the complex digestive functions of the stomach, intestines, and pancreas. Even before the consumption of food has begun, the anticipation of food triggers gastric secretions that will prime the GI lining for pending food ingestion, a process commonly referred to as the cephalic phase. Gastric and intestinal phases follow once the food reaches the stomach lumen, during Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00006-6 © 2021 Elsevier Inc. All rights reserved.

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which the GI hormones regulate digestion, absorption, and gut motility. In addition to their roles in digestion, the GI hormones aid in the growth and maintenance of gut mucosa, and in excess, they are implicated in the pathogenesis of NETs of the GI tract, as will be discussed in the following sections of this chapter. There have been over 50 gut hormones that have been discovered, only a fraction of which has been heavily investigated in the medical science community. Among the well-established and understood gut hormones are gastrin, cholecystokinin (CCK), secretin, somatostatin, ghrelin, motilin, and vasoactive intestinal peptide (VIP). While all of these are peptide hormones, they are varied in their chemical make-up and function [2].

Gastrin Gastrin is a linear peptide hormone released from G cells in the gastric antrum in response to stomach distension, increased pH, and vagal stimulation. It is the primary hormone for gastric acid secretion, as it stimulates parietal cells to release hydrochloric acid (HCl). The mechanism of action is via one of two ways: a direct pathway and an indirect pathway. Gastrin can exert its effects on parietal cells by directly binding gastrin receptors on parietal cells or by stimulating enterochromaffin-like cells (ECL) to release histamine, which then binds its receptors on parietal cells. Gastrin also stimulates the growth of gastric mucosa and is a known contributor to the pathophysiology of gastrinoma. The precursor peptide, preprogastrin, could be cleaved into three forms: gastrin-34 (big gastrin), gastrin-17 (little gastrin), and gastrin-14 (minigastrin). Gastric acid secretion can be terminated by low levels of pH and somatostatin, both of which signal fasting [2].

Somatostatin Somatostatin is a peptide hormone released by D cells throughout the GI tract. It naturally occurs in two forms: 14 and 28 amino acids. Additionally, there is a widely used 8-amino acid synthetic analog, which has a much longer half-life. Its release is triggered by low levels of gastric pH, and subsequently it acts to inhibit most GI hormones, including insulin, glucagon, and gastrin. This essentially blocks the release of HCl from parietal cells and their powerful downstream stimulatory pathway. It inhibits growth hormone release from the hypothalamus and most GI hormones such as insulin, glucagon, and gastrin. Inhibition of CCK release and the resultant reduction in gallbladder contractility lead to cholelithiasis in patients with somatostatinomas. Pancreatic insufficiency and associated fat malabsorption may result in diarrhea and steatorrhea. Decreased gastrin secretion can also present as gastric hypochlorhydria. Measurement of somatostatin is inherently difficult. Because somatostatin is labile in vitro, it requires special handling and rapid

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separation. Somatostatin presents with a 20% diurnal variation: it peaks at midnight and is at its nadir at 8 a.m. [1 3].

Secretin Secretin prepares for the intestinal phase of digestion by promoting secretions from the pancreas and gallbladder and simultaneously inhibiting gastrin and halting acid secretions from parietal cells. It is secreted by duodenal S cells, and it acts to induce bicarbonate release from pancreatic duct cells, effectively raising the pH in the small intestines. Clinically, secretin is used as a diagnostic test for gastrinomas, as it increases the release of gastrin in patients with gastrinoma [2].

Cholecystokinin CCK, in Greek for “gallbladder move,” promotes contraction of gallbladder and relaxation of sphincter of Oddi for the secretion of pancreatic enzymes. In line with its role in aiding in fat digestion in the intestines, CCK also acts to suppress gastric emptying. CCK is released from intestinal I cells by the presence of fatty acids and monoglycerides, as well as amino acids in the intestines [2].

Pancreatic polypeptide Pancreatic polypeptide (PP) is a 36-amino acid peptide, which is predominantly produced by F cells in the pancreas. Besides its function of stimulating pancreatic enzyme secretion and contraction of the bladder, little else is known about its actions [4]. The World Health Organization classified PP tumors as well-differentiated NETs with features such as solid, trabecular, gyriform, or glandular pattern, uniform nuclei, and finely granular cytoplasm The head of the pancreas has the greatest density of PP cells, which produce PP. PP secreting tumors are highly vascular and can have the ability to invade the wall of the duodenum or cause thrombosis of the splenic or portal vein [5]. In Table 6.1 summarized the physiology of the GI hormones.

Pancreatic neuroendocrine tumors Pancreatic NETs (PNETs) are a group malignancies of pancreatic islet cells. Pathologies of this category are insulinoma, Zollinger Ellison syndrome (ZES) (gastrinoma), α-cell tumors (glucagonomas), δ-cell tumors (somatostatinomas), VIPoma, and pancreatic carcinoid tumors. Making up merely 2% of all pancreatic tumors, NETs are rare in comparison to those of pancreatic exocrine counterparts. Presentations vary from single to multiple lesions, benign to malignant, nonfunctional to hyperfunctional neoplasms. Malignancy is

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TABLE 6.1 GI hormones. Hormone

Cells of production (section of intestine)

Major actions

Cholecystokinin

CCK (duodenum, jejunum)

Gallbladder contraction, amylase secretion by pancreas, decreases appetite

Somatostatin

D (diffuse)

Inhibits glucagon and insulin production, intestinal motility, gastric acid secretion

Serotonin

Enterochromaffin-like cells (stomach)

Stimulates gastric acid secretion

Gastrin

G (stomach)

Stimulates gastric acid secretion

Ghrelin

P/D1 (stomach, duodenum)

Increases food intake, decreases insulin

GIP

GIP (duodenum, jejunum)

Increases glucose-mediated insulin release; inhibits gastrin production

GLP-1, PYY

L (jejunum, ileum, colon)

(both) Decreases appetite; (GLP-1) increases insulin, slows gastric emptying

Glucagon

A

Increases blood glucose

Insulin

B

Decreases blood glucose

Pancreatic polypeptide

PP

Stimulates bile flow and exocrine pancreatic secretion

determined by the presence and progression of metastases, vascular invasion, and local infiltration [6].

Glucagonoma Glucagon, a hormone secreted by pancreatic alpha cells, is primarily responsible for breaking down glycogen to glucose in the liver. Glucagonoma (α-cell tumor) is a very rare malignancy of pancreatic islet cells with an approximate annual incidence of 0.01 0.1 per 100,000 [7]. Most glucagonomas are sporadic, and ,10% of cases present in association with MEN1 syndrome. They are usually solitary tumors and located in the body and tail of the pancreas and .50% are metastatic at diagnosis [8]. Glucagonomas are functional neoplasms and are thus associated with increased serum levels of glucagon resulting in a clinical syndrome including mild diabetes mellitus, a characteristic skin rash necrolytic

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migratory erythema (NME), and anemia. They present more frequently in women than men, and particularly in perimenopausal and postmenopausal women. The development of symptoms in glucagonomas is driven by the supraphysiologic levels of circulating serum glucagon. Glucagon increases hepatic catabolic reactions through amino acid oxidation and gluconeogenesis, which leads to the characteristic weight loss. The cause of NME is still not elucidated, but hyponutrition with low zinc and amino acid levels could be important. Diarrhea may be present due to excess glucagon and cosecretion of other digestive hormones such as gastrin, vasoactive intestinal peptide, serotonin, or calcitonin. Hypersecretion of the hormone glucagon causes a combination of symptoms, easily summarized as the 4Ds: Dermatosis, Diabetes, Deep Vein Thrombosis, and Depression. Present in up to 90% of patients, the dermatosis associated with glucagonoma syndrome is referred to as NME. It is a red, blistering rash that is itchy and painful. NME can commonly affect the genitals, buttocks, groin, and the extremities, and severity may fluctuate. Mucosal abnormalities include glossitis, stomatitis, and cheilitis. The NME initially starts out as erythematous papules or plaques and may enlarge and develop into bullae in severe forms. In a subset of patients, hair loss and nail dystrophy may also be present. The mechanism of NME is likely due to malnutrition and amino acid deficiency [9]. Glucose intolerance affects 75% 95% of patients, with elevated fasting glucose levels. Of note, hyperglycemia due to glucagonoma rarely develops into diabetic ketoacidosis, since beta cells are preserved and able to secrete insulin in response to circulating levels of glucagon [10]. Up to 50% patients can present with deep vein thrombosis including pulmonary embolism. The high incidence of thromboembolism in glucagonoma patients is still poorly understood. Unexplained deep vein thrombosis in patients with NET should alert the possibility of glucagonoma [11]. The mechanistic explanation for depression in glucagonoma patients is poorly studied, but has traditionally been attributed to other associated symptoms such as hair loss and NME. There are many other neuropsychiatric manifestations reported in glucagonoma patients, including dementia, psychosis, agitation, hyperreflexia, ataxia, paranoid delusions, and proximal muscle weakness, which may suggest underlying physiologic or biochemical mechanisms driving such family of symptoms [8,12]. In patients with new onset of diabetes, rash, and weight loss, glucagonoma should be suspected. Diagnosis of glucagonoma can be made with an elevated fasting plasma glucagon level ( . 500 pg/mL), usually 10 20 times the reference range. Glucagon level above 1000 pg/mL can be pathognomonic. Increased glucagon levels can be due to other conditions including acute trauma, diabetes, burns, cirrhosis, etc., but levels do not exceed 500 pg/mL in

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these contexts. Mahvash disease, which is due to a homozygous inactivating mutation in the glucagon receptor gene, GCGR, similarly results in hyperglucagonemia, alpha cell hyperplasia, and adenomas, but without clinical features of glucagonoma [8,13]. Since all these tumors are rare, the required assays are usually performed in reference laboratories and require careful sample preparation such as addition of a protease inhibitor, using chilled tubes and freezing the sample at below 20 C immediately [3]. Working with the reference laboratory will be most helpful to obtain a valid result. Glucagon assays from different vendors show variable cross reactivity with different isoforms of glucagon. It is also prudent to check both zinc and amino acid levels as part of the work-up [14]. As is the case with other NETs, imaging studies are necessary for accurate localization and staging of primary and metastatic glucagonomas. Somatostatin receptor scintigraphy, in combination with either Cat Scan (CT) or magnetic resonance imaging (MRI), is invaluable in the work-up of a patient with glucagonoma. Treatment of glucagonoma should focus on the management of glucose intolerance and nutritional support for counterbalancing the catabolic effects of glucagon. In advanced, metastatic disease, surgical resection of the liver or hepatic artery embolization with or without chemotherapy may be used not for curative, but palliative intent [15]. Somatostatin analog is the current treatment of choice, especially for metastatic disease, as these tumors have abundant somatostatin receptors. In addition to decreasing the level of secreted hormones, somatostatin analog have been shown to have some cytostatic activity [16,17].

Somatostatinoma Somatostatinomas are tumors of pancreatic D cells that secrete increased amounts of somatostatin. These tumors are extremely rare, with the annual incidence rate estimated at 1 in 40 million. Somatostatinomas are the least likely to be present in patients with MEN1 syndrome. The tumors are typically present in the pancreas, and less frequently in the duodenum and jejunum. Symptoms are more common with pancreatic than intestinal tumors. Tumors are usually solitary and large and liver metastases are present in over 69% of cases [18]. Patients with this neoplasm typically present with diabetes mellitus, cholelithiasis, steatorrhea, and hypochlorhydria due to the inhibitory effects of somatostatin on cellular function. Nonfunctional somatostatinomas are also seen frequently in 0% 10% of patients with neurofibromatosis [19]. Somatostatinomas are functional neoplasms and secrete high levels of somatostatins. Unlike other functional endocrine tumors of the GI tract, however, somatostatinoma less frequently presents with symptoms in

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relation to excess somatostatin secretion. As somatostatin is a well-known inhibitor of all the GI hormones that have been studied, excess secretion of it could lead to an increase in GI transit time and decrease in intestinal motility and nutrient absorption. Appropriately, the most common presenting symptoms in patients with somatostatinoma are abdominal pain and weight loss. In a smaller percentage of patients, somatostatinomas present with diabetes mellitus, cholelithiasis, and diarrhea/steatorrhea, the triad termed somatostatinoma syndrome. This syndrome is more commonly reported in patients with pancreatic somatostatinomas than duodenal somatostatinomas [20]. Somatostatinomas are usually found during exploratory laparotomy or upper GI radiographic studies with CT or MRI for patients with unexplained abdominal pain, nausea, vomiting, melena, hematemesis, persistent diarrhea, weight loss, fatigue, and anemia. Patients presenting with the triad of diabetes mellitus, cholelithiasis, and diarrhea/steatorrhea should be suspected of having somatostatinomas and evaluated for fasting plasma levels of somatostatin. Hormone level greater than 30 pg/mL can establish diagnosis. Other methods of diagnosis include evaluating tissue concentrations of somatostatin by immunohistochemistry or, when applicable, histopathology of surgical specimen that demonstrates positivity for somatostatin on immunohistochemistry [21]. Imaging studies with OctreoScan, CT, and MRI can also be considered for localization and staging of advanced disease, as these malignancies express somatostatin receptors on their cell surface. While somatostatinomas are highly associated with metastatic disease, the survival rate is considerably high. Thus aggressive management and attempts to surgically remove these tumors, in the case that they are primary, are important for curative intent. As most somatostatinomas are present in the head of the pancreas or duodenum, surgical resection via a pancreaticoduodenectomy is the treatment of choice. Patients with metastatic disease, or inoperable tumors, however, should be treated medically. Therapy with somatostatin analog that inhibits the secretion of somatostatin is the first line of treatment and symptom relief in patients with unresectable tumors [22]. However, its role in antitumor activity has not been demonstrated. In this regard, other lines of treatment can be considered, such as with molecularly targeted therapy (e.g., everolimus, sunitinib, etc.), cytotoxic chemotherapy, or peptide receptor radioligand therapy [21].

VIPoma Vasoactive intestinal peptide tumors are usually solitary NETs, which secrete the hormone VIP in an uncontrolled manner. They were first described in 1958 by Verner and Morrison as a pancreatic tumor resulting in watery diarrhea with hypokalemia that was resistant to treatment. Because of the severity of its course and similarity to Vibrio cholerae infection, it was later

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nicknamed pancreatic cholera. It is also referred to as the WDHA syndrome since there is Watery Diarrhea, Hypokalemia, and Achlorhydria. In 1973, Bloom, Polak, and Pearse identified the VIP hormone as the cause of this syndrome [23]. Moreover, they are extremely rare tumors with an incidence between 0.05% and 2% that present in both adults and children. In 95% of cases the tumor is intrapancreatic and most commonly occurs between the ages of 30 50 years [24]. VIPomas are part of MEN1 in around 5% of cases and .50% have metastasized by the time of diagnosis. The 28-amino acid VIP is posttranslationally modified from its 170-amino acid precursor prepro VIP. VIP is a neuropeptide present in both central and peripheral nervous systems that very strongly stimulates intestinal secretion of water and electrolytes. It is a member of the secretin-glucagon family. In addition, this neuropeptide has an inhibitory effect on gastrin secretion and is a vasodilator and promotes blood flow in the GI tract mainly. It modulates these actions through its stimulation of intestinal cyclic adenosine monophosphate (cAMP) production. VIPoma is caused by excess secretion of this neuropeptide. Through continuous stimulation of cAMP, VIPoma presents with enormous secretion of water and electrolytes. The common presentation of hypochlorhydria occurs from the direct inhibitory effect of VIP on gastrin-17 [25]. Seventy-five percent of VIPomas initially present in the body and tail of the pancreas, while the other 25% originate in the head of the pancreas. VIPoma classically presents with greater than 3.0 L of watery stool per day, hypokalemia, and achlorhydria [26]. The hypokalemia and hyperchloremic metabolic acidosis are due to the copious diarrhea and substantial loss of bicarbonate. The hypercalcemia could be due to dehydration on part of the MEN syndrome. In this early stage, this tumor predominantly presents with episodic and intermittent diarrhea. As the tumor progresses and enlarges, this diarrhea becomes continuous with presentation of severe electrolyte abnormalities. Unfortunately, at the time of presentation, 70% of patients will already have metastases of the tumor [27]. Hyperglycemia, hypercalcemia, and flushing are common accompanying presentations as well. Especially in the context of features such as hypercalcemia, VIPoma may be associated with a MEN1 syndrome. Other important features that can present include tetany, from hypomagnesemia, myopathy, and cardiac arrhythmias from hypokalemia. In the pediatric population, VIPoma may present as a failure to thrive. Other mimickers of VIPoma that must be ruled out include ZES, chronic laxative abuse, AIDS, and celiac sprue [28]. Patients with excessive tea colored diarrhea along with electrolyte abnormalities and other possible features such as flushing should be suspected of having a VIPoma. The diarrhea is secretory; that is, it persists after 48 h of fasting. Also the diarrhea has a low osmotic gap below 50 mOsm /Kg. The diagnosis of VIPoma is made with secretory diarrhea greater than 3 L/day with a VIP level of 250 500 pg/mL (normal range: 0 190 pg/mL).

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Biochemical detection of VIP levels is achieved through measurement of VIP by immunoassay. The serum sample needs to be added to tubes containing the protease inhibitor, aprotinin, centrifuged and frozen below 20 C [3]. It is critical to repeat this test, as a single plasma VIP level value cannot diagnose or exclude VIPoma. Between bouts of diarrhea, the VIPoma endocrine tumor may not actively be secreting VIP. Moreover, it is most accurate to obtain VIP levels from the patient when he or she is actively having diarrhea [25]. Localization of the tumor itself is initiated through contrast-enhanced CT scan. This imaging modality has greater than 80% sensitivity for detecting intrapancreatic NETs. It should be noted that endoscopic ultrasound has been shown to have a higher sensitivity for detecting NETs, compared to CT, but is not as commonly used [29]. Intravenous (IV) contrast can also be utilized for detection of smaller tumors that may not be possible by contrast CT. In scenarios of identifying extrapancreatic VIPomas, such as metastases, somatostatin receptor scintigraphy can be used. Treatment of VIPomas is focused on a combination of medical and surgical management. Because of the profuse diarrhea that patients present with, initial treatment must always focus on aggressive replacement of fluids and electrolytes. Isotonic fluid should be used and electrolyte replacement can prevent dangerous sequelae such as cardiac arrhythmias and neurovascular defects. Somatostatin analogs such as octreotride and lanreotide can be used for symptomatic control by inhibiting secretion of VIP. Octreotide doses of 50 100 μg administered subcutaneously every 8 h or intramuscular longacting formulation of octreotide and Sandostatin LAR can be used. Glucocorticoids and interferon-alpha have been used in patients refractory to the above treatments [25]. Surgical resection is the treatment of choice in regard to removal of the VIPoma tumor itself. If complete resection is not possible, surgical debulking may also be done for palliative purposes. Surgical removal has been shown to be very effective in treating symptoms of VIPoma. When the tumor has metastasized, other approaches must be pursued [25]. For instance, when the tumor has metastasized to the liver, resection of the liver is often times merited. Radiofrequency ablation and cryoablation have been effective treatment modalities for metastases less than 3 cm [30]. Unfortunately, systemic chemotherapy has not been proven to be effective in cases of extrahepatic metastases or large, bulky tumors. Currently, newer agents such as sunitinib, a tyrosine kinase inhibitor, and everolimus, an mTOR inhibitor, have been approved in the United States for treatment of advanced and welldifferentiated PNETs such as VIPomas. These modalities have proven to be effective with Everolimus treatment resulting in stable disease in 67.8% of patients with VIPoma, for example [25]. Median survival is 96 months in patients with VIPomas. Prognostic factors depend on tumor stage, grade, and surgical resectability. Postresection follow-

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up includes history and physical exam, imaging such as multiphasic CT, and measurement of serum VIP in the initial 3 12 months. After 1 year, it is recommended to follow these guidelines every 6 12 months [24]. Insulinomas are detailed in the chapter on hypoglycemia. Suffice it is to say is that the majority are benign tumors that present with fasting or exercise induced hypoglycemia, which can be confirmed with a 72-h fast under medical supervision. Also both C-peptide and proinsulin levels can be increased and helpful in the differential diagnosis of fasting hypoglycemia

Gastrointestinal neuroendocrine tumors Gastrinomas are neuroendocrine neoplasms resulting in excessive secretion of gastrin resulting in severe acid-related peptic ulceration and diarrhea. The clinical syndrome accompanying hypergastrinemia is referred to as the ZES [18]. Sporadic gastrinomas are frequently found in the duodenum (50% 90%), followed by the pancreas (10% 40%). Gastrinomas can either be sporadic or as a part of multiple endocrine neoplasia type 1 syndrome [6,18]. Gastrin is the key promoter of gastric acid release from parietal cells, an important mechanism for macronutrient breakdown in the stomach. In the context of uncontrolled growth, gastrinomas cause excessive stimulation of parietal cells, resulting in inappropriate acid secretion, hypertrophy/hyperplasia of gastric mucosa, and ulceration. These clinical symptoms are collectively coined the ZES. In ZES, hypergastrinemia provokes the hypersecretion of gastric acid, leading to multiple peptic ulcerations in unusual locations, such as the jejunum. Interestingly, these ulcers are typically unresponsive to treatment with proton pump inhibitors, in contrast to ulcers that occur independently of gastrinomas. The major symptoms are related to peptic ulcer disease (PUD) or severe gastroesophageal reflux disease with or without diarrhea [18]. PUD is usually in the duodenum but multiple ulceration, refractory to medical therapy, and PUD associated with prominent gastric folds should increase suspicion for ZES. ZES can present as diarrhea in 50% of the patients, and in 30% of patients, it is the presenting symptom. Unfortunately, more than half of gastrin-secreting tumors are already metastatic at the time of diagnosis. Twenty-five percent of gastrinoma patients also copresent with other NETs, which is then identified as MEN1 syndrome [31]. As with any other functional GI-NETs, gastrinomas are diagnosed based on inappropriate circulating levels of the hormone, gastrin. As the initial presentation of ZES is very similar to PUD, physicians must use clinical judgment in deciding which patients with PUD need to be worked up for ZES, based on family history of endocrinopathy, diarrhea, and hypercalcemia. In general, patients with peptic ulcers that are refractory to proton pump inhibitor treatment should be suspected of having a gastrinoma and be followed up as such [32].

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Initial evaluation is through a fasting serum gastrin level. Gastrin circulates in at least three forms: G34, G17, and G14. Hence, immunoassays report different levels depending on the epitopes targeted by the respective antibodies. Patients need to be fasted prior to the study since circulating levels increase after a meal. Given the instability of the hormone, the separated sample should preferably be frozen to below 270 C immediately [32]. Fasting serum gastrin level of 1000 pg/mL, a 10-fold excess of the normal range, is pathognomonic of gastrinoma. Higher levels of serum gastrin are correlated with tumors of pancreatic origin, larger tumor size, and metastases. ZES patients also present with less than 10 times the normal range of fasting serum gastrin 60% of the time. In order to confirm the diagnosis in these patients, they should be given the secretin stimulation test, which remains the most sensitive and accurate test of ZES. Secretin which normally is inhibitory of G cells and gastrin secretion paradoxically stimulates gastrinomas to secrete gastrin, which serves as the mechanistic basis of the secretin stimulation test. This study involves IV administration of 2 μg/kg secretin and monitoring serum levels of gastrin at 2, 5, 10, 20, and 30 min. Greater than 200 pg/mL increase in gastrin level strongly suggests ZES [12,31,33]. As there are other possible causes of hypergastrinemia, clinicians must ensure to exclude these following factors before diagnosing ZES: postvagotomy state, atrophic gastritis, retained antrum syndrome, primary G-cell hyperplasia, and achlorhydria primary or secondary to proton pump inhibitor therapy [32]. The diagnosis is enhanced if the hypergastrinemia is associated with a gastric pH ,2.0 and the basal acid output is .15 mEq/h [18]. MEN1 syndrome must be excluded from patients with ZES. If a patient with ZES has a family history of a similar endocrinopathy or hypercalcemia at the time of diagnosis, then serum PTH, prolactin GH, and insulin-like growth factor-1 should also be assayed. Imaging studies may also be pursued for assessing tumor burden and localizing and staging the metastases of gastrinomas. As somatostatin receptors are ubiquitously expressed on the cell surfaces of NETs, recent radiologic developments have progressed to use fluorescence-tagged somatostatin receptor ligands for NET imaging, called OctreoScan. In modern practice, images taken from OctreoScan are combined with SPECT or CT for their advantages in anatomical resolution [34]. Gastrinomas can be managed medically or treated surgically. Medical therapy is the treatment of choice for most patients with ZES as a manifestation of MEN1 syndrome. Surgical therapy can be considered for patients with sporadic ZES. The goal of management in ZES is to manage the symptoms and complications of PUD. This can be achieved via high-dose proton pump inhibitors, which bind and inhibit hydrogen/potassium ATPase on the luminal surface of parietal cells, or in refractory cases, with octreotide, a somatostatin analog. Patients with ZES should be started on a high dose of a proton pump

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inhibitor (omeprazole 60 mg daily, esomeprazole 120 mg daily, lansoprazole 45 mg daily, rabeprazole 60 mg daily, or pantoprazole 120 mg daily) [35 37]. In patients with no signs of metastatic spread or MEN1 syndrome, an attempt can be made at exploratory laparotomy and resection with curative intent [38,39].

Carcinoid tumor NETs are malignant growths that stem from neuroendocrine cells [40]. NETs arising from the intestine were formerly known as carcinoid tumors, though this term is still widely used by physicians today [41]. In 1888 Otto Lubarsch was credited with documenting the presence of ileal carcinoid tumors in two patients at autopsy. In 1953 Lembeck identified serotonin in an ileal carcinoid tumor and as the predominant hormone responsible for carcinoid syndrome [40]. Forty-eight percent of NETs arise in the GI tract, while 25% arise in the lungs and 9% arise in the pancreas. NETs are extremely rare and compromise less than 2% of all malignancies in the United States. Moreover, in the United States, there is a prevalence of less than 200,000 cases. Detection of these tumors has recently been on the rise, most likely due to incidental findings on increasing utilization of imaging. The hallmark of NETs is their ability to produce the hormone serotonin, which leads to symptoms such as flushing and diarrhea [40]. Carcinoid tumors are thought to originate from Kulchitsky cells, enterochromaffin cells, in the crypts of Lieberkuhn of the gut. These cells secrete a variety of substances such as 5-hydroxytryptamine (5-HT), 5-hydroxytryptophan (5-HTT), histamine, kallikrein, and prostaglandins [41]. Carcinoid syndrome, which classically presents with watery diarrhea, bronchospasm, flushing, hypotension, and right-sided heart disease, is caused by the release of these various substances and appears to occur in ,10% of patients with carcinoids. The Kulchitsky cells use the majority of tryptophan stores to metabolize into serotonin. In contrast, a healthy individual without serotonin syndrome only has 1% of total tryptophan stores metabolized into serotonin. This serotonin is eventually excreted in the urine as 5-hydroxyindoleacetic acid (5-HIAA). Carcinoid syndrome presents only when the tumor metastasizes to the liver or bypasses the portal circulation. This is because serotonin, as well as other biologically active amines and peptides, is typically inactivated in the liver before reaching systemic circulation [28,42 45]. In most scenarios, carcinoid tumors are asymptomatic until advanced stages. When these tumors metastasize to liver and or bypass the portal circulation, the manifestation of carcinoid syndrome presents with distinct characteristics. Midgut carcinoids account for around 60% of all carcinoid syndromes. Biologically active amines, peptides, and prostaglandins produce a variety of vasodilatory effects such as flushing and diarrhea (secretory),

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which occur in around 70% of patients at onset [18]. In fact, flushing is the most common clinical feature, with 85% of patients presenting with this symptom. The flushing can be precipitated by stress, certain foods, and medications. This flushing is very distinct, with a salmon pink to dark discoloration of skin in the upper body. Because of serotonin’s effect of increased motility and secretion of the GI tract, patients often presents with diarrhea and malabsorption. The diarrhea is watery and explosive, occurring up to 30 times a day. Patients can also present with steatorrhea. Valvular disease secondary to plaques on the endocardium has been described in up to 60% 70% of patients. The valvular defects are tricuspid regurgitation, tricuspid stenosis, and pulmonary stenosis in order of frequency. Signs of right-sided heart failure are seen. Just as carcinoid syndrome can cause valvular fibrosis, it can also cause mesenteric and small bowel fibrosis. Secretion of serotonin, along with tachykinins and growth factors, is involved in the pathogenesis of this fibrosis through stimulation of fibroblasts. Additionally, with the conversion of most tryptophan stores in the body to serotonin, there is niacin deficiency with the patients presenting with diarrhea, dermatitis, and dementia, a constellation of pellagra. Late stages of carcinoid syndrome can present with a purple rash due to prolonged vasodilation. A feared complication of carcinoid syndrome is carcinoid crisis, a life threatening presentation of flushing, bronchospasm, and rapidly fluctuating blood pressure precipitated by anesthetic or tumor manipulation [40,43,46]. Diagnosis of carcinoid tumor is inherently difficult. This is because of low incidence, small size of the tumor with high metastatic potential, and varied clinical presentation. Moreover, early diagnosis requires highly sensitive and specific biomarkers. Twenty-four hour measurement of 5-HIAA urinary secretion is the initial gold standard screening test, as it has a 90% sensitivity and specificity for diagnosis of intestinal carcinoid. The urine should be collected in acidified containers. Patients cannot eat serotonin rich foods for 3 days before this test to prevent false positive results. Recent studies have also validated plasma HIAA levels to aid in diagnosis. Plasma levels are not affected by eating or time of day but are increased with renal impairment (Estimated glomerular filtration rate ,60 mL/min) [45]. Since the majority of serotonin resides in platelets, whole blood serotonin is the best measure but the sample has to be taken in a tube containing a reducing agent such as ascorbate [47]. Concentration of chromogranin A (CgA), a glycoprotein secreted by carcinoid tumors, can also be measured. However, this biomarker is better suited for follow-up rather than diagnosis due to its poor specificity and is a measure of tumor burden. Issues with the measurement of CgA are detailed under nonfunctional NETs [48]. After laboratory testing is completing, imaging must be pursued to localize and stage the tumor. CT/MRI imaging and functional imaging are the two modalities that are generally utilized. Because of its wide availability, CT with contrast is most commonly used. It is recommended that all patients

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with a suspected carcinoid tumor should receive CT of chest, abdomen, and pelvis. Disadvantages of CT include a low specificity, difficulty in detecting tumors less than 1 cm, and difficulty differentiating between colorectal adenocarcinomas and colorectal carcinoid tumors. MRI is pursued if there is suspicion of liver metastases. Functional imaging, such as the octreotide scan, can be used to detect metastasis beyond the abdominal region. Octreotide scan is used with positron emission tomography scanning to detect octreotride uptake in the abdominal region. This modality has a sensitivity in asymptomatic patients of 80% 90%. However, its utility in surveillance of carcinoid syndrome is questionable. Newer modalities such as the Gallium-68 positron emitter have also emerged as highly sensitive tests for detecting carcinoid tumors [40,41,43]. Similar to other endocrine tumors of the GI tract, treatment resolves around both medical and surgical management. Medical management is preferred for patients with carcinoid syndrome and carcinoid tumors that are unresectable. On the other hand, surgical intervention is most important for cure of carcinoid tumor. Octreotride and lanreotide, somatostatin analogs, are used to inhibit release of biogenic amines that cause classical symptoms of serotonin syndrome such as flushing and diarrhea. This is effective as 80% of NETs have somatostatin receptors. These somatostatin analogs have been shown to provide relief in 50% 70% of patients. Shortacting octreotide subcutaneous injection is preferred for patients with severe symptoms. A common side effect of these analogs is pancreatic malabsorption, with pancreatic enzyme supplementation often provided for patients. Surgical resection with negative margins is the main treatment for nonmetastatic carcinoid tumors confined to the intestine. Because tumors in the small intestine have a high chance of metastasis, the involved area of the small bowel along with small bowel mesentery must be resected. On the other hand, colorectal carcinoid tumors tend to be greater than 2 cm and more invasive. These tumors must be treated with partial colectomy and lymphadenectomy. Cytoreductive surgical resection is preferred for metastatic tumors along with concomitant medical therapy. Patients with hepatic metastases are candidates for percutaneous hepatic transarterial embolization. Complications from carcinoid syndrome such as valvular failure are treated with tricuspid valve replacement. For this reason, it is critical to perform echocardiograms in patients with significantly elevated levels of 5-HIAA and/or symptoms of carcinoid syndrome. The complication of carcinoid crisis is treated with a 500 1000 mg IV bolus of octreotide followed by continuous infusion at 50 200 μg/h. Treatment options for refractory symptoms include interferon-alpha, Telotristat, an oral tryptophan hydroxylase inhibitor, and systemic chemotherapy such as everolimus [40,41,43,49].

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Nonfunctioning neuroendocrine tumor (pancreatic neuroendocrine tumors) The WHO classification of neoplasms of the neuroendocrine pancreas includes the category of nonfunctioning NETs. These tumors are generally pancreatic neoplasms that have no functional clinical syndrome, and symptoms are largely due to the tumor mass itself [18,28]. Nonfunctioning PNETs are now considered to represent .60% of all PNETs. Although these tumors are not associated with a clear clinical syndrome, they secrete numerous peptides, the most relevant of which are PP, CgA, and neuron-specific enolase (NSE), amongst others. These nonfunctional tumors are also frequently seen in systemic diseases such as MEN1, von Hippel Lindau disease, and tuberous sclerosis [18,28]. These tumors present with the nonspecific features of a tumor mass and this includes abdominal pain, jaundice, weight loss, bleeding, and an abdominal mass [19,39]. Furthermore, because of late presentation the tumors are large (around 5 cm), invasive, and around 50% have liver metastases. Imaging is very useful and the modalities discussed above for functioning tumors including CT, MRI, Endoscopic Ultrasound, and somatostatin receptor scintigraphy are extremely useful. The diagnosis requires a biopsy and measurement of CgA, PP, and NSE. In well-differentiated functional and nonfunctional tumors, immunohistochemistry confirms staining for CgA, synaptophysin, NSE, PP and cluster of differentiation 56 (CD56-neural cell adhesion molecule) [50]. CgA, PP, NSE, and pancreastatin have been found to be the best biomarkers for diagnosis of nonfunctioning tumors. PP serum levels are measured by immunoassay using Ethylenediamine tetraacetic acid plasma. PP levels are increased in nonfunctioning and functioning tumors. Since levels increase with exercise and eating, fasting levels are desired to diagnose and monitor therapy. Also levels are not altered by proton pump inhibitor therapy [47]. CgA is widely distributed in neuroendocrine cells and could serve as a useful biomarker for both nonfunctioning and functioning tumors, as well as it correlate with tumor burden. It is not specific for malignancy. However, the assay has several limitations in addition to the well-known effects of heterophile antibody interference and the hook effect [3]. It has a large biological variation, significant differences between plasma and serum samples, increases with food intake, large assay-to-assay variation possibly due to antibodies directed to different epitopes, and increases with impaired renal function [47]. Levels are also increased with gastric achlorhydria (e.g., pernicious anemia and proton pump inhibitor therapy). Despite these issues it generally serves as a reliable biomarker for the diagnosis, monitoring and prognosis of neuroendocrine tumors both functional and nonfunctional. NSE can also serve as a tumor marker for gastroenteropancreatic NETs. It appears to be a more valid marker of prognosis and is increased with poorly

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differentiated, rapid growing tumors [18,47]. Caveats include its biological variability and the major interference from hemolysis due to the abundance of NSE in erythrocytes [48]. Pancreastatin has shown utility in monitoring effects of treatment and progression of these tumors [51]. Staging is performed through CT of the abdomen, pelvis, and thorax. MRI is performed to identify liver metastases, while upper digestive endoscopy with ultrasound allows for detection of small lesions. The combination of 111-labeled octreotide scintigraphy with CT/MRI imaging has improved detection of both primary and metastatic lesions. Functional staging through the use of plasma CgA further guides staging and treatment of these tumors [5]. Because most patients at the time of diagnosis have metastatic disease, curative surgical resection can only be done in about 10% of cases. In cases of isolated metastases to the liver, liver resection can lead to both symptomatic relief and increased survival. Hepatic artery embolization poses as another option in patients with predominantly hepatic involvement. For candidates where curative surgery is not possible, debulking can be considered to reduce hormonal production by the tumor, symptoms, and tumor mass. Similar to other GI endocrine tumors, somatostatin analog of octreotide and lanreotide can be utilized as medical therapy when surgery is not possible or ineffective. Systemic chemotherapy involving agents such as streptozocin, doxorubicin, and cyclophosphamide can be used in progressive metastatic disease. Streptozocin and doxorubicin is the current combination of choice. Even with chemotherapy, PP secreting tumors exhibit a high rate of recurrence. As a result, tyrosine kinase inhibitors such as sunitinib have been explored to improve survival [5]. In Table 6.2 is summarized the neuroendocrine tumor syndromes.

Conclusion Since the previous edition of this textbook, there have been many advancements in the diagnosis and treatment of NETs. Diseases such as carcinoid syndrome have been found to have greater incidence in the population than thought. Also, functional imaging has evolved to the point where NETs can be more accurately diagnosed and staged. Gallium-68 positron emission tomography/CT scan has emerged as an effective modality for detecting PNETs with high sensitivity, reduced radiation exposure, and increased convenience for patients. Treatment modalities have also expanded considerably. However, with respect to laboratory measurement of all of the above hormones and biomarkers they have multiple issues the most important being standardization. Given the rarity of these syndromes it is understandable why the progress with regard to standardization, harmonization, and availably of these assays other than insulin still remains a major issue.

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TABLE 6.2 Neuroendocrine tumors. NET clinical syndrome

Main biomarker

Most common location

Clinical feature

Glucagonoma

Glucagon

Alpha cells in pancreas

G G G

G

Somatostatinoma

VIPoma

Somatostatin

VIP

Delta cells in pancreas head

G

Pancreas body and tail

G

G G

G G G

Gastrinoma

Gastrin

Duodenum— most frequent site

G G

G

G

Carcinoid tumor

Serotonin

Midgut

G

G G

G

G

Nonfunction NET

Pancreatic polypeptide

Pancreas head

Unintended weight loss New-onset diabetes Necrolytic migratory erythema: red blistering, itchy, and painful rash Glossitis, stomatitis, and cheilitis Abdominal pain Weight loss Diabetes mellitus, cholelithiasis, and diarrhea/ steatorrhea Greater than 3.0 L of watery stool daily, hypokalemia, and achlorhydria Hyperglycemia Hypercalcemia Flushing Peptic ulcer disease Severe gastroesophageal reflux disease Symptoms refractory to proton pump inhibitor therapy Diarrhea Watery and explosive diarrhea/steatorrhea Flushing Right-sided valvular diseases such as tricuspid stenosis and tricuspid regurgitation Pellagra: diarrhea, dermatitis, and dementia Carcinoid crisis: flushing, bronchospasm, and rapidly fluctuating blood pressure

Nonspecific symptoms: G Abdominal pain G Jaundice G Abdominal mass

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[18] J.L. Jameson, A.S. Fauci, D.L. Kasper, S.L. Hauser, D.L. Longo, J. Loscalzo, et al., Harrison’s Principles of Internal Medicine, 19th ed., McGraw Hill Education, New York, 2018. [19] N. Garbrecht, M. Anlauf, A. Schmitt, T. Henopp, B. Sipos, A. Raffel, et al., Somatostatinproducing neuroendocrine tumors of the duodenum and pancreas: incidence, types, biological behavior, association with inherited syndromes, and functional activity, Endocr. Relat. Cancer 15 (1) (2008) 229 241. Available from: https://doi.org/10.1677/erc-07-0157. [20] A. Vinik, Somatostatinoma. Retrieved from ,https://www.ncbi.nlm.nih.gov/books/ NBK279034/., 2017. [21] A. Vinik, K. Pacak, E. Feliberti, R.R. Perry, in: K.R. Feingold, B. Anawalt, A. Boyce, G. Chrousos, K. Dungan, A. Grossman, et al. (Eds.), Glucagonoma Syndrome, 2017. Retrieved from ,https://www.ncbi.nlm.nih.gov/pubmed/25905270.. [22] G.M. Doherty, Rare endocrine tumours of the GI tract, Best Pract. Res. Clin. Gastroenterol. 19 (5) (2005) 807 817. Available from: https://doi.org/10.1016/j. bpg.2005.05.004. [23] W.W. Herder, J.F. Rehfeld, M. Kidd, I.M. Modlin, A short history of neuroendocrine tumours and their peptide hormones, Best Pract. Res. Clin. Endocrinol. Metab. 30 (1) (2016) 3 17. Available from: https://doi.org/10.1016/j.beem.2015.10.004. [24] S. Sandhu, I. Jialal, VIPoma. Retrieved from ,https://www.ncbi.nlm.nih.gov/books/ NBK507698/., 2019 [25] A. Vinik, Vasoactive intestinal polypeptide (VIPoma), Endotext (Internet). Retrieved from ,https://www.ncbi.nlm.nih.gov/books/NBK278960/., 2017. [26] D. Farina, K. Krogh, J. Boike, Chronic diarrhea secondary to newly diagnosed VIPoma, Case Rep. Gastroenterol. 13 (1) (2019) 225 229. Retrieved from ,https://www.ncbi.nlm. nih.gov/pmc/articles/PMC6514521/., 2019. [27] Y. Chen, D. Shi, F. Dong, S. Han, Z. Qian, L. Yang, et al., Multiple-phase spiral CT findings of pancreatic vasoactive intestinal peptide-secreting tumor: a case report, Oncol. Lett. 10 (4) (2015) 2351 2354. Available from: https://doi.org/10.3892/ol.2015.3615. [28] T. Ito, H. Igarashi, R. Jensen, Pancreatic neuroendocrine tumors: clinical features, diagnosis and medical treatment: advances, Best Pract. Res. Clin. Gastroenterol. 26 (6) (2012) 737 753 Retrieved from ,https://www.sciencedirect.com/science/article/pii/S1521691813000292.. [29] M.A. Khashab, E. Yong, A.M. Lennon, E.J. Shin, S. Amateau, R.H. Hruban, et al., EUS is still superior to multidetector computerized tomography for detection of pancreatic neuroendocrine tumors, Gastrointest. Endosc. 73 (4) (2011) 691 696. Available from: https:// doi.org/10.1016/j.gie.2010.08.030. [30] S.J. Moug, E. Leen, P.G. Horgan, C.W. Imrie, Radiofrequency ablation has a valuable therapeutic role in metastatic VIPoma, Pancreatology 6 (1 2) (2006) 155 159. Available from: https://doi.org/10.1159/000090257. [31] M.J. Berna, K.M. Hoffmann, S.H. Long, J. Serrano, F. Gibril, R.T. Jensen, Serum gastrin in Zollinger-Ellison syndrome, Medicine 85 (6) (2006) 331 364. Available from: https:// doi.org/10.1097/md.0b013e31802b518c. [32] S.L. Orloff, H.T. Debas, Advances in the management of patients with Zollinger-Ellison syndrome, Surg. Clin. N. Am. 75 (3) (1995) 511 524. Available from: https://doi.org/ 10.1016/s0039-6109(16)46637-5. [33] E. Feliberti, Gastrinoma. Retrieved from ,https://www.ncbi.nlm.nih.gov/books/ NBK279075/., 2017. [34] J.E. Maxwell, J.R. Howe, Imaging in neuroendocrine tumors: an update for the clinician, Int. J. Endocr. Oncol. 2 (2) (2015) 159 168. Available from: https://doi.org/10.2217/ije.14.40.

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[35] R.T. Jensen, Zollinger-Ellison syndrome, JAMA 271 (18) (1994) 1429. Available from: https://doi.org/10.1001/jama.1994.03510420061035. [36] B.I. Hirschowitz, J. Simmons, J. Mohnen, Clinical outcome using lansoprazole in acid hypersecretors with and without Zollinger-Ellison syndrome: a 13-year prospective study, Clin. Gastroenterol. Hepatol. 3 (1) (2005) 39 48. Available from: https://doi.org/10.1016/ s1542-3565(04)00606-8. [37] D.C. Metz, G.M. Comer, E. Soffer, C.E. Forsmark, B. Cryer, W. Chey, et al., Three-year oral pantoprazole administration is effective for patients with Zollinger-Ellison syndrome and other hypersecretory conditions, Aliment. Pharmacol. Ther. 23 (3) (2006) 437 444. Available from: https://doi.org/10.1111/j.1365-2036.2006.02762.x. [38] J.A. Norton, D.L. Fraker, H.R. Alexander, R.T. Jensen, Value of surgery in patients with negative imaging and sporadic Zollinger-Ellison syndrome, Ann. Surg. 256 (3) (2012) 509 517. Available from: https://doi.org/10.1097/sla.0b013e318265f08d. [39] J.A. Norton, D.L. Fraker, H.R. Alexander, F. Gibril, D.J. Liewehr, D.J. Venzon, et al., Surgery increases survival in patients with gastrinoma, Ann. Surg. 124 (2006) 76 85. Available from: https://doi.org/10.1097/01.sla.0000234802.44320.a5. [40] M. Raphael, D. Chan, C. Law, S. Singh, Principles of diagnosis and management of neuroendocrine tumours, CMAJ 189 (10) (2017) E398 E404. Available from: https://doi.org/ 10.1503/cmaj.160771. [41] M. Krishnan, F. Tuma, Cancer, intestinal carcinoid, Statpearls (Internet), 2018. [42] B. Oronsky, P.C. Ma, D. Morgensztern, C.A. Carter, Nothing but NET: a review of neuroendocrine tumors and carcinomas, Neoplasia 19 (12) (2017) 991 1002. Available from: https://doi.org/10.1016/j.neo.2017.09.002. [43] S. Pandit, K. Bhusal, Carcinoid syndrome. Retrieved from ,https://www.ncbi.nlm.nih. gov/books/NBK448096/., 2019. [44] S. Lee, S. Tomoyoshi, K. Haga, H. Sasaki, C. Ogata, O. Nomura, et al., Multiple carcinoid tumors of the small intestine preoperatively diagnosed by double-balloon endoscopy, Med. Sci. Monit. 18 (12) (2012). Available from: https://doi.org/10.12659/msm.883588. [45] T. Ito, L. Lee, R.T. Jensen, Carcinoid-syndrome: recent advances, current status and controversies, Curr. Opin. Endocrinol. Diabetes Obes. 25 (2018) 22 35. Available from: https://doi.org/10.1097/med.0000000000000376. [46] I.M. Modlin, M.D. Shapiro, M. Kidd, Carcinoid tumors and fibrosis: an association with no explanation, Am. J. Gastroenterol. 99 (12) (2004) 2466 2478. Available from: https:// doi.org/10.1111/j.1572-0241.2004.40507.x. [47] N. Rifai, Tietz Textbook of Clinical Chemistry and Molecular Diagnostics E-Book, Elsevier Health Sciences, St. Louis, 2017. [48] V. Aluri, J. Dillon, Biochemical testing in neuroendocrine tumors, Endocrinol. Metab. Clin. N. Am. 46 (3) (2017) 669 677. Available from: https://doi.org/10.1016/j.ecl.2017.04.004. [49] S.N. Pinchot, K. Holen, R.S. Sippel, H. Chen, Carcinoid tumors, Oncologist 13 (12) (2008) 1255 1269. Available from: https://doi.org/10.1634/theoncologist.2008-0207. [50] M. Cives, J. Strosberg, Gastroenteropancreatic neuroendocrine tumors, CA Cancer J. Clin. 68 (2018) 471 487. Available from: https://doi.org/10.3322/caac.21493. [51] V. S´anchez-Margalet, C. Gonz´alez-Yanes, S. Najib, Pancreastatin, a chromogranin A-derived peptide, inhibits DNA and protein synthesis by producing nitric oxide in HTC rat hepatoma cells, J. Hepatol. 35 (1) (2001) 80 85. Available from: https://doi.org/ 10.1016/s0168-8278(01)00071-x.

Chapter 7

Evaluation of hypoglycemia William E. Winter1 and Neil S. Harris2 1

Departments of Pathology, Immunology & Laboratory Medicine, Pediatrics, and Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States, 2Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States

Introduction The body must be protected from hypoglycemia because severe acute hypoglycemia can cause seizures, coma, and even death [1]. On the other hand, chronic hyperglycemia is toxic to blood vessels, basement membranes, and proteins that become glycated [2]. Therefore, the body must maintain the level of energy currency (glucose) in the bloodstream within narrow limits. This is a challenging task because the biochemical demands for glucose throughout the body can vary tremendously. Although there are four hormones specifically designed to raise glucose concentrations in the bloodstream [epinephrine, glucagon, cortisol, and growth hormone (GH)], there is only one hormone designed to specifically lower glucose concentrations: insulin. This chapter addresses the etiologies and diagnostic evaluations for hypoglycemia.

Clinical symptoms of hypoglycemia In routine medical practice, hypoglycemia (low blood glucose) is commonly encountered in the immediate neonatal period [especially in premature infants and infants born to diabetic mothers (IDM)] and in insulin-treated diabetic patients who take excessive doses of insulin, skip meals, and/or exercise more than usual [3]. Acute hypoglycemia produces adrenergic symptoms (e.g., tachycardia, palpitations, pounding heart, sweating, tremulousness, anxiety, nervousness, feeling cold, and sweating) and neuroglycopenia [low blood glucose affecting the brain: decreased consciousness, tiredness or drowsiness, faintness, confusion, blurred or double vision, hemiparesis, behavioral changes, dizziness, paresthesias (pins and needles feelings), incoordination, slurred speech, hunger, headache, seizures, coma, and even death]. Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00007-8 © 2021 Elsevier Inc. All rights reserved.

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Fasting hypoglycemia versus reactive hypoglycemia Fasting (postabsorptive) hypoglycemia occurs most commonly in the morning following an overnight fast, between meals, or with exercise. Fasting hypoglycemia usually implies a serious medical condition that requires thorough evaluation. Reactive hypoglycemia is also referred to as “postprandial” or “stimulative” hypoglycemia. This form of reputed hypoglycemia usually does not imply a serious disorder [47]. The hypothesis behind “reactive” (or foodstimulated) hypoglycemia is that excessive release of insulin follows a large glucose load and causes hypoglycemia several hours after eating. Many patients with symptoms of reactive hypoglycemia regularly miss meals and/ or do not eat a balanced diet (e.g., B55%60% carbohydrate, B15% protein, # 30% fat). The diagnosis of reactive hypoglycemia is sometimes inappropriately pursued by performing a 5-h oral glucose tolerance test (OGTT). When patients with hypoglycemic-like symptoms manifest a glucose of ,40 mg/dL in the later stages of the OGTT, reactive hypoglycemia is diagnosed. However, $ 10% of adults display glucose values of ,50 mg/dL at 3 h or more after an oral glucose challenge. In such general population studies, there has been no correlation between the adrenergic symptoms of reactive hypoglycemia and glucose values ,50 mg/dL 35 h after the oral glucose load. For this reason, most endocrinologists do not consider reactive hypoglycemia to be a disease. Clinicians should therefore be discouraged from performing 5-h OGTTs. Successful treatment of the symptoms of reactive hypoglycemia involves achieving healthy eating patterns and regular meals. The only hypoglycemic disorder that is truly “reactive” involves the “dumping” syndrome, where food (often hyperosmolar) rapidly transits from the stomach into the small intestine and large amounts of glucose are rapidly absorbed [8]. Subsequently, there is a surge of glucose-driven and incretindriven insulin release that does, in fact, lead to true “reactive” hypoglycemia [9]. This form of “reactive” hypoglycemia can be treated with smaller, more frequent meals. Hyperinsulinism following gastric bypass surgery is another cause of reactive hypoglycemia [10,11].

Diagnosis of hypoglycemia The diagnosis of hypoglycemia is established when three criteria (“Whipple’s triad”) are documented: (1) symptoms compatible with hypoglycemia are substantiated; (2) there is laboratory confirmation of a low blood glucose concentration at the time of symptoms (serum or plasma glucose ,45 mg/dL); and (3) the symptoms are relieved promptly with the rapid administration of glucose [12]. Because self-blood glucose monitoring systems are usually not accurate at values ,4060 mg/dL, the glucose

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determination must be performed on a sample obtained by venipuncture using instrumentation with an accurate and precise lower limit of detection, ideally, of 10 mg/dL or less.

Biochemical definition of hypoglycemia Hypoglycemia is biochemically defined as a venous whole-blood glucose of ,40 mg/dL or a venous plasma or serum glucose of ,45 mg/dL (note: whole-blood glucose is B15% lower than plasma or serum glucose because of the nonaqueous constituents of whole blood). Some references set a cutoff for hypoglycemia as a serum or plasma glucose ,40 mg/dL. Other experts judge any serum or plasma glucose concentration of ,50 mg/dL as representing hypoglycemia. Because glucose concentrations in plasma or serum are not affected by changes in the hematocrit, plasma or serum is the preferred specimen for glucose analysis. Although newborns, especially premature infants, commonly display glucose values of 2040 mg/dL or less, these values are considered to be potentially harmful [1316]. Therefore, the definition of hypoglycemia described above should apply to individuals of all ages. Pseudohypoglycemia (artifactual hypoglycemia) occurs most often when the measured blood glucose is artifactually depressed because of delayed analysis [17]. Because cellular elements of the bloodstream (e.g., white blood cells and red blood cells) consume glucose, unless samples are to be assayed within 1 h of phlebotomy, blood should be drawn into gray-top tubes containing NaF to inhibit glycolysis (which takes B1 h for complete inhibition). Potassium chromate serves as an anticoagulant in gray-top tubes. Without the addition of NaF, serum or plasma glucose will continue to fall by B2%3% h after the first-hour postphlebotomy. Samples placed in NaF tubes will be stable at room temperature for 24 h. High white blood cell counts (e.g., chronic myelocytic leukemia) can produce more rapid rates of decline in blood glucose [18]. Artifactual hypoglycemia has also been described in persons with polycythemia vera [19]. Factitious hypoglycemia is self-induced hypoglycemia (e.g., a person with diabetes intentionally takes an excessive dose of insulin with the intention of producing hypoglycemia).

Causes of hypoglycemia In the absence of a recognized cause of hypoglycemia (e.g., an IDM or a diabetic patient who took a known overdose of insulin), the initial challenge is to determine whether the patient has hyperinsulinemic hypoglycemia or nonhyperinsulinemic hypoglycemia. Assuming that hypoglycemia has been biochemically confirmed, the age of the patient has a major influence on the likely causes of hypoglycemia (Table 7.1). Therefore, the age of the patient should influence the laboratory tests that are ordered and performed. For

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TABLE 7.1 Causes of hypoglycemia by age: relative frequency as causes of hypoglycemia. Neonate

Child

Adult

Hyperinsulinemia

11

11

11 1

Drugs

1

11 1

11 1

Hormone deficiency

11

11

1

Inborn errors

11

1

2

Liver disease

11

11

11

Renal disease

2

2

2

Extrapancreatic neoplasm

2

2

1

Critical or severe illness

11 1

11

1

Neonatal hypoglycemia

11 1

N/A

N/A

Ketotic hypoglycemia

N/A

11

N/A

Severe malnutrition

1

1

1



N/A, Not applicable;  , major cause of persistent neonatal hypoglycemia; scale: 11 1 , very common; 11, common; 1 , possible; 2 , uncommon.

example, in individuals who first develop hypoglycemia after the ages of 4 or 5 years, studies in search of inborn errors of metabolism should not generally be pursued.

Diagnostic workup for hypoglycemia Evaluation of acute hypoglycemia Should the patient suffer an acute episode of hypoglycemia (e.g., the patient arrives in the emergency room in coma or postictal and is found to be hypoglycemic), Table 7.2 lists the studies that should be considered. If possible, more than one blood sample should be obtained at this time [e.g., the initial sample and a sample drawn just before intravenous (IV) glucose or glucagon are administered] [20]. Tests for the evaluation of hypoglycemia can be divided into routine versus optional, and those tests that should be obtained at the time of acute hypoglycemia versus those tests that need not be obtained at the time of acute hypoglycemia. The optional tests are dependent upon the patient’s age, history, physical examination, and likely causes of hypoglycemia depending upon the specific clinical situation. For example, if in the initial evaluation, the patient is found to have liver failure, other causes of hypoglycemia are less likely and need not immediately be pursued. These guidelines for obtaining samples also apply to prolonged fasts.

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TABLE 7.2 Biochemical studies in the evaluation of hypoglycemia. Test

Indication

Routine measurements in adults and children at the time of acute hypoglycemia Plasma glucose

Measured to diagnose hypoglycemia; decreased value is necessary for the diagnosis of hypoglycemia (gray-top tube is the preferred sample)

βHB (and/or) urine ketones

Assessment of ketosis; hyperinsulinism can suppress ketosis

Insulin

Measured to rule out hyperinsulinism (consider proinsulin and C-peptide measurements)

Routine measurements in infants and young children at the time of acute hypoglycemia Lactic acid

Assessment for lactic acidosis; measurement in adults is optional (unless otherwise measured, as in sepsis)

Urine-reducing substances

Measured to exclude certain inborn errors of carbohydrate metabolism; measure in all infants (e.g., # 2 years): measure at a time when the infant is on their normal diet

Optional measurements at the time of acute hypoglycemia Drug screen/ ethanol

Performed to rule out intoxication; perform blood/urine screening when intoxication cannot be otherwise excluded; screen for sulfonylureas and meglitinides when indicated

Cortisola

Measured to rule out hypocortisolism; hold sample for cortisol measurement if indicated

Growth hormone (GH)

Measured to rule out GH deficiency; hold sample for GH measurement if indicated

C-peptide

Measured to confirm that hyperinsulinism is endogenous; Cpeptide is elevated in insulinoma and sulfonylurea/meglitinide toxicity, with exogenous insulin administration, C-peptide is suppressed

Branched-chain amino acids

Measured to document the biochemical effects of hyperinsulinism; rarely needed; helpful in confirming “functional” hyperinsulinism when insulin levels are low but other agents cause hypoglycemia (e.g., IGF-II excess or agonistic antiinsulin receptor autoantibodies)

Free fatty acids (FFAs)

Measured to document the biochemical effects of hyperinsulinism or suspected fatty acid oxidation disorders (FAOD)

Urine dicarboxylic acids

Measured when an aminoacidopathy or FAOD are suspected

Arterial blood gasesb

Measured to assess acid/base balance; acidosis is common in many disorders that cause hypoglycemia (Continued )

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SECTION | 2 Analytes

TABLE 7.2 (Continued) Test HCO2 3,

Indication 2

Cl , urine

pH

Measured to assess acid/base balance; acidosis is common in many disorders that cause hypoglycemia

Routine measurements not required at the time of acute hypoglycemia Liver functionc d

Renal function

Measured to rule out liver disease Measured to rule out kidney disease

Optional measurements not required at the time of acute hypoglycemia Thyroid studiese

Measured to rule out thyroid dysfunction; measure when thyroid disease is clinically suspected

Serum/plasma amino acids

Measured to rule out an aminoacidopathy

Uric acid

Measured when inborn errors of metabolism are a consideration; often elevated when lactate is elevated

Cholesterol/ triglycerides

Measured when inborn errors of metabolism are a consideration; elevated in certain glycogen storage diseases

Creatine kinase (CK)

Measured when myopathy is suspected; elevated in certain glycogen storage diseases

Carnitine

Measured when FAOD is suspected; low in cases of carnitine deficiency

a

Rule out hypopituitarism. pH, pO2, pCO2. c ALT, alkaline phosphatase, total bilirubin, direct bilirubin, albumin, total protein, consider prothrombin time (PT), and AST. d Creatinine, estimated glomerular filtration rate, blood urea nitrogen (BUN), urinalysis. e TSH and free T4. b

In the absence of an acute hypoglycemic episode, the best time to test for hypoglycemia is in the morning after an overnight fast. Plasma glucose values of ,45 mg/dL are consistent with hypoglycemia, whereas plasma glucose concentrations of 4560 mg/dL are suggestive of hypoglycemia. In either case, in persons presenting with symptoms suggestive of hypoglycemia, further evaluation when the glucose is # 60 mg/dL is indicated (e.g., a prolonged fast should be undertaken). When hypoglycemia is a serious clinical consideration, several fasting blood glucose determinations are warranted before hypoglycemia is excluded. Plasma glucose concentrations .60 mg/dL are normal, when present on several occasions (in the absence of any values # 60 mg/dL) rule against fasting hypoglycemia. This discussion of plasma glucose cut points exposes areas that demand clarification. The typical “normal range” for fasting plasma glucose is

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7099 mg/dL. Yet, we have defined a plasma glucose of .60 mg/dL as being “normal” and a plasma glucose of ,45 mg/dL as being pathologically low. Therefore, we need a better understanding of, and a term for, this borderline range of plasma glucose values that are of from 45 to 60 or 69 mg/dL [analogous to the description of “prediabetes” (see “Evaluation of hyperglycemia” in Chapter: 8)].

Prolonged fasts in the evaluation of hypoglycemia A prolonged fast lasting up to 72 h in adults, or at least 2436 h in children, may be required to determine whether or not hypoglycemia is actually present. In adults with proven insulinoma, fasting for 48 h is usually sufficient to elicit hypoglycemia [21]. In children with ketotic hypoglycemia, fasting for as little as 8 h may precipitate hypoglycemia [22]. “Ketotic hypoglycemia” likely reflects the limited biochemical capacities of young children to maintain normoglycemia because of their proportionately smaller muscle mass (as a source of gluconeogenic precursors) and higher ratio of brain size to liver size [e.g., the ratio of the glucose consumer (the brain) to the glucose producer (the liver) compared to adults]. Normal children do not become hypoglycemic with short-term fasting [23]. Prolonged fasts are performed in the hospital, so that purported symptoms and signs of hypoglycemia can be carefully recorded. As well, plasma glucose concentrations can be regularly measured, blood samples can be obtained for analysis, IV glucose is readily available for emergency administration, and IV glucagon can be administered at the termination of the fast to assess the degree of glucose reserve [e.g., the amount of glycogen present in the liver that can be liberated by glucagon injection at the end of the fast (see below)]. During the planned fast, the subject is given nothing by mouth except for free access to water or a glucose-free electrolyte solution. At the beginning of the prolonged fast (before retiring for sleep), an IV line is introduced into a vein of sufficient size, so that blood can be periodically withdrawn for analysis. The line is flushed with a heparinized saline solution (e.g., 100 units/mL) and clamped. Prior to drawing subsequent samples, 35 mL of blood is withdrawn to clear the line of the flush solution (e.g., drawing a “blank”). Unless the patient is anemic, the “blank” blood sample can be discarded. Blood must be drawn at the beginning of the fast, whose analysis provides a baseline plasma glucose value. Because adrenergic symptoms of hypoglycemia can become attenuated in some individuals who suffer chronic and recurrent hypoglycemia, during the fast, the plasma glucose must be measured regularly (e.g., every 4 h) by the central laboratory. Between these 4-h measurements, finger stick capillary glucose can be measured with a meter to monitor the course of the fast. If any of the

210

SECTION | 2 Analytes

capillary glucose values show a significant decline (e.g., decrease of 10 mg/dL or more), a confirmatory venous plasma glucose should be drawn from the indwelling catheter. Recall that capillary glucose values require correction to serum levels (capillary glucose 3 1.15 B 5 serum glucose) because capillary and whole-blood glucose values are B15% lower than venous plasma glucose values. Alternatively, if the point-of-care testing device reports the capillary glucose levels as plasma values, no adjustment is required. During the fast, all urine samples are tested for ketones. Ketonuria is a normal response to fasting over prolonged periods of time (e.g., $ 1824 h of fasting). Once the plasma glucose drops are below 70 mg/dL, venous draws can be increased to every 2 h. The fast is continued until frank symptoms of hypoglycemia are present or the venous plasma glucose is persistently ,45 mg/dL (e.g., for at least 30 min). If this does not occur by the conclusion of the fast, fasting hypoglycemia is excluded, and further studies are not indicated. During the fast, once symptoms of hypoglycemia initially occur or the venous plasma glucose initially falls below 45 mg/dL, blood should be drawn for clinical studies (Table 7.2). This is a critical time for sampling when the laboratory results are most informative. Blood should be drawn again B30 min later and the plasma glucose measurement is repeated. Because the inclusion or exclusion of hyperinsulinism is key to the differential diagnosis of hypoglycemia, insulin should essentially always be measured at these critical times (see below). At the termination of the fast, if biochemical hypoglycemia is documented, IV glucagon (1 mg or 0.03 mg/kg, minimum dose in children is usually 0.5 mg) is administered. Blood for glucose measurement is drawn approximately every 1015 min thereafter for up to 45 min, and blood is again stored for future analyses as described above. Interpretation of the plasma glucose postglucagon is discussed below. Obviously, the tests actually run depend upon the age of the patient, clinical history, physical examination, and clinical suspicion. The laboratory should be able to process and hold specimens until the physician decides which studies are required. Physicians should be encouraged to consult their clinical pathologists or clinical chemists about such matters.

Testing strategy for evaluation of hypoglycemia In the differential diagnosis of hypoglycemia, the first diagnostic assessment concerns whether hyperinsulinism is present or absent at the time of hypoglycemia. This divides the etiologies of hypoglycemia into hyperinsulinemic hypoglycemia (Fig. 7.1) and nonhyperinsulinemic hypoglycemia (Fig. 7.2).

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211

Hyperinsulinemic hypoglycemia

Ketones, FFA, branched chain amino acids: suppressed C-peptide Elevated

Suppressed Insulin antibodies

Sulfonylurea meglitinide Present

Absent

Present

Sulfonylurea induced hyperinsulinism

Insulinoma versus islet cell dysplasia

Exogenous insulin administration versus autoimmune hypoglycemia

Absent

Consider: insulinomimetic autoantibodies

FIGURE 7.1 If the insulin (or proinsulin) concentration is elevated at the time of documented hypoglycemia, we anticipate suppressed concentrations of alternative fuels such as ketones, FFAs, and branched-chain amino acids. The branched-chain amino acids are substrates for gluconeogenesis. The next challenge is to differentiate endogenous from exogenous hyperinsulinism by measuring C-peptide. An elevated C-peptide indicates that the hyperinsulinism is pancreatic in origin. Next, sulfonylureas and meglitinides must be excluded. If C-peptide is suppressed, exogenous insulin injection is the major etiologic consideration. Autoimmune hypoglycemia and insulinomimetic autoantibodies are very rare causes of hypoglycemia in North America.

Hypoglycemia syndromes with hyperinsulinism To determine whether hyperinsulinemic hypoglycemia is present, the clinician must assess two aspects of the patient’s presentation: (1) the insulin concentration relative to the glucose concentration and (2) whether other biochemical findings [e.g., urine ketones, β-hydroxybutyrate (βHB), free fatty acids (FFAs), branched-chain amino acids] are consistent with hyperinsulinism. Even when the insulin concentration is below the lower limit of the reference interval for an individual in the fasting state, the absolute insulin concentration may still be inappropriately elevated for the patient’s plasma glucose concentration. Some references suggest that measurements of C-peptide or proinsulin may provide better information than insulin measurements in determining whether hyperinsulinism is present [24]. “Functional” hyperinsulinism (e.g., increased insulin action without an inappropriate elevation in the insulin concentration) can also occur when there is an insulin surrogate such as an elevated insulin-like growth factor

212

SECTION | 2 Analytes

Nonhyperinsulinemic hypoglycemia FFA elevated

Ketones Elevated

Depressed or absent

Salicylates ethanol propranolol Absent cortisol, GH

Cortisol def. GH def.

Present

Galactosemia hereditary fructose intolerance

Present NL response

Deficient

Fatty acid oxidation disorder

Urine reducing substances

Confirmation: low carnitine organic aciduria

Drug-induced hypoglycemia

Absent Organic aciduria Absent

Present Msud Mma isovaleric acidemia 3-methylglutaconic aciduria

Lactic acidosis Present

Absent Rule out:

GSD deficiency of gluconeogenic enzymes

Liver / renal disease limited substrate/ incr. utilization

FIGURE 7.2 In cases of nonhyperinsulinemic hypoglycemia, elevated FFA levels are expected because of insulin suppression. Ketosis is a normal physiologic response to hypoglycemia with a suppressed insulin level. If ketones are depressed or absent, a fatty acid oxidation disorder must be excluded. Because drugs are a common cause of hypoglycemia, drug toxicities must be sought by review of the medical history and laboratory testing as indicated. If drugs are excluded as a cause of hypoglycemia, endocrine disorders should be sought. If cortisol and GH responses are appropriately elevated at the time of hypoglycemia, liver disease and inborn errors must next be considered, remembering that inborn errors most commonly present in infancy. GH, Growth hormone; NL, normal; MSUD, Maple syrup urine disease; MMA, methylmalonic aciduria.

(IGF) II concentration from a tumor [25] or an agonistic antiinsulin receptor autoantibody (insulinomimetic autoantibody) [26]. Some clinicians find that calculating the insulin/glucose ratio is helpful in diagnosing hyperinsulinism. If the insulin/glucose ratio is .0.250.30 (units: 100 uIU/mg), pathologic hyperinsulinism is diagnosed (to convert uIU/mL to pmol/L, multiply uIU/mL by 6.0). Other endocrinologists state that any measurable insulin in the setting of hypoglycemia is inappropriately elevated. However, this may not be appropriate as the lower limit of detection for insulin immunoassays is declining with advances in electrochemiluminescent technologies. The laboratorian and clinician must also understand the specificity of the lab’s insulin assay. There are many recombinant DNA-derived insulins with altered sequences and compositions that are available for patient use. Not all of these insulins react on a 1 to 1 molar basis relative to human insulin in

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various insulin immunoassays. Certain insulin immunoassays only detect human insulin. Even if this is the case, the assay does not discriminate endogenous human insulin from injected human insulin. To further complicate the situation is the finding that insulin antibodies (IA) develop in more than 90% of insulin-treated patients regardless of the nature of the injected insulin [2730]. These IA act as circulating binding proteins and raise the total insulin concentration. However, since most of this insulin is bound in the plasma to the IA, only the “free” (unbound) insulin is bioactive. Also problematic is that there is no reliable way to measure free insulin (those assays are not robust and are poorly documented in the literature) [3134]. In the setting of IA, because plasma insulin measurements do not reflect the level of bioactive insulin, insulin should not be measured in insulin-treated patients. IA can appear within 2 wk of beginning insulin treatment. An elevated proinsulin level or proinsulin/total insulin ratio in the face of hypoglycemia can also suggest hyperinsulinism. However, this requires an immunoassay selective for proinsulin. An amended insulin/glucose ratio was proposed in the 1970s: ([insulin] 3 100)/(glucose—30 mg/dL) (abnormal, .50). However, some authors believe that the best assessment for hyperinsulinism is the measurement of absolute insulin concentration during hypoglycemia (as noted above). Current generation automated electrochemiluminescent (ECL) insulin assays have very low to lower limits of detection for insulin. With hyperinsulinism, the following laboratory findings are common because of the suppression of lipolysis, ketogenesis, and proteolysis by hyperinsulinism: G G G G

urine negative for ketones; normal βHB (βHB is not elevated); normal FFA concentrations (FFAs are not elevated); and normal branched-chain amino acids [35] (branched-chain amino acids are not elevated).

These findings reflect the suppressive effect of hyperinsulinism on the generation of alternative fuels (e.g., ketones) and the suppressive effect of hyperinsulinism on the liberation of amino acids from muscle (e.g., branched-chain amino acids) as substrate for gluconeogenesis [36]. FFA and branched-chain amino acid determinations will usually be available only at reference laboratories. The absence of urine ketones and normal βHB in the face of hypoglycemia should alert the clinician and the laboratorian to the possibility of hyperinsulinism. However, by itself, the presence of urine ketones does not exclude hyperinsulinism. Lactic acidosis or elevated lactate does not occur as part of the hyperinsulinism syndromes. As will be discussed, lactic acidosis is seen with certain inborn errors of metabolism involving, for example, defective gluconeogenesis or glycogenolysis (e.g., glycogen storage disease [GSD]).

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SECTION | 2 Analytes

Another clinical aid in making the diagnosis of hyperinsulinism is the plasma glucose response to glucagon administration. This is usually done at the completion of a period of fasting when hypoglycemia has been confirmed or with acute hypoglycemia if glucagon is available for emergency administration (and the glucagon also serves as potential treatment for hypoglycemia). When hyperinsulinism is present, exogenous glucagon administered in a pharmacologic dose (e.g., 1 mg intramuscularly or IV) reverses hyperinsulinemic inhibition of glycogenolysis (as well as gluconeogenesis) and promotes rapid glycogenolysis, raising the plasma glucose of $ 3040 mg/dL within B45 min. With nonhyperinsulinemic hypoglycemia, the plasma glucose response to glucagon is ,3040 mg/dL and can be delayed or absent.

Hyperinsulinism with elevated C-peptide If hyperinsulinism is diagnosed, the source of the insulin must be identified. Was the insulin released from the β cells of the islets of Langerhans, or was the insulin injected? When the pancreatic β cell is the source of hyperinsulinism (e.g., elevated C-peptide and insulin), the β cell may be functioning autonomously (e.g., insulinoma) or the β cell may be stimulated to oversecrete insulin by drugs [37], or, rarely, by β cell agonist autoantibodies [38]. A recent paper reported the abuse of sulfonylureas to induce a euphoric “hypoglycemic rush” [39]. The differential diagnosis of autonomous β cell hyperfunction in neonates includes transient hyperinsulinism in small for gestational age (SGA) infants [40] and transient hyperinsulinism secondary to exposure to maternal hyperglycemia (e.g., IDM) or erythroblastosis fetalis [41]. Hypoglycemia in newborns limited to the first day of life usually represents “immaturity of fasting adaptation” due to a transient delay in the liver’s ability to carry out gluconeogenesis and ketogenesis [42]. Guidelines on the management of neonatal hypoglycemia have been published by the American Academy of Pediatrics and the Pediatric Endocrine Society. Hypoglycemia that persists over the first few days of life can result from maternal factors such as maternal diabetes, IV glucose administration to the mother during labor (that elicits hyperinsulinism), terbutaline (a β2 agonist) use, or propranolol (a beta-blocker) use [43]. Hypoglycemia that lasts more than 2 d (but is not permanent and usually resolves by the age of 23 months) can be due to perinatal stress that causes transient hyperinsulinism (e.g., birth asphyxia [44], toxemia, erythroblastosis fetalis [45], SGA infant, or premature infant), hypopituitarism, or the BeckwithWiedemann syndrome.

Neonatal hyperinsulinism When hypoglycemia is persistent and hyperinsulinism is confirmed, several inborn errors must be considered, including loss-of-function mutations where

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the β cell potassium channel (KATP channel) is closed inducing persistent β cell depolarization and insulin release [46] and gain-of-function mutations in glutamine dehydrogenase (GDH) [47] or glucokinase [48]. KATP channel mutations can be inherited as autosomal dominant or recessive traits. The KATP channel is composed of four potassium channel subunits (Kir6.2) surrounded by four regulatory subunits [sulfonylurea receptor (SUR) 1]. Kir6.2 is encoded by the gene KCNJ11 (potassium inwardly rectifying channel, subfamily J, number 11). SUR1 is encoded by the gene ABCC8 (ATP binding cassette subfamily C member 8). KATP channel mutations can cause diffuse β cell hyperplasia (requiring near-total pancreatectomy) or focal β cell hyperplasia (that can be managed through resection of a focal lesion) [49]. GDH is encoded by GLUD1. Many new and rare causes of hyperinsulinemic hypoglycemia in infants have been described in the last 10 years (Table 7.3) [55]. Molecular testing is available for many of the genetic hyperinsulinism disorders. As well, there are many genetic syndromes with reported hyperinsulinism and hypoglycemia including AKT3 (v-akt murine thymoma viral oncogene homolog 3) [56], BeckwithWiedmann [57], calcium voltagegated channel subunit alpha1 D (CACNAID), Costello [58], Kabuki [59], Ondine [60], Patau (trisomy 13), Perlman, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA), SimpsonGolabi [61], Solos [62], Timothy [63], TRNA methyltransferase 10A (TRMT10A), Turner (45, X), and Usher [64].

Hyperinsulinism in children and adults Excluding infancy, endogenous hyperinsulinism in children can be caused by islet cell dysplasia or, uncommonly, by insulinoma. Familial hyperinsulinism due to a mutation in the insulin receptor has been reported [65] and can cause neonatal hypoglycemia (see above). Excluding drug-induced hyperinsulinism, endogenous hyperinsulinism in adults is caused by insulinoma [66,67]. Even in adults, insulinomas are uncommon, with a yearly incidence reported at 1/1,000,000 to 1/1,250,000. There is no marked sex bias (60/40; female/male ratio). Most patients with insulinoma are 4060 years old. Ten percent of the insulinomas occur in patients younger than 20, and 10% of cases occur in patients older than 60. In B4% of cases, insulinoma is associated with the multiple endocrine neoplasia (MEN) syndrome type I (i.e., tumors of the pituitary, pancreatic islets, and parathyroid glands). With MEN type I, insulinoma may be multifocal. Multiple insulinomas are found in 6%10% of the affected patients. The most common islet tumor in MEN type I is a gastrinoma causing ZollingerEllison syndrome. Much less common islet tumors include glucagonomas, pancreatic polypeptide-producing tumors, VIPomas, and somatostatinomas.

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SECTION | 2 Analytes

TABLE 7.3 Causes of hyperinsulinemic hypoglycemia in newborns. Protein

Gene

Comment

Kir6.2

KCNJ11

Diffuse β cell involvement: AR or AD Focal β cell involvement: parentally inherited KCNJ11 mutation (1) somatic maternal allele LOH

SUR1

ABCC8

Diffuse β cell involvement: AR or ADFocal β cell involvement: parentally inherited ABCC8 mutation(1) somatic maternal allele LOH

Glutamate dehydrogenase 1

GLUD1

AD (gain-of-function); hyperammonemia

Glucokinase

GCK

AD (gain-of-function)

Hydroxyacyl-CoA

HADH

AR; protein intake elicits hyperinsulinism [50]

Dehydrogenase

(SCHAD)

Solute carrier family 16 member 1

SLC16A1

AD; monocarboxylate transporter 1; postexercise hypoglycemia

Uncoupling protein 2

UCP2

AD; inactivating mutations, increased ATP production (theoretical) [51]

Adenosine kinase

ADK

AR; function: transfer of ATP gammaphosphate to adenosine; mechanism of hyperinsulinism: unclear [52]

Channelopathies

Enzymopathies

Various carbohydratedeficient glycoprotein disorders

AR; congenital disorders of glycosylation; Includes defects in: ALG3, ALG6, MP1, PGMI, PMM2; mechanism of hyperinsulinism: unclear; abnormal transferrin IEF

Transcription factor disorders HNF-1α

HNF1A

AD; activating mutation; loss-of-function produces MODY3 [53]

HNF-4α

HNF4A

AD; activating mutation; loss-of-function produces MODY1

INSR

AD or AR (unclear): one patient each; mechanism: unclear [54]

Receptoropathies Insulin receptor

AD, Autosomal dominant; AR, autosomal recessive; LOH, loss of heterozygosity of the in a restricted group of β cells.

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Ten percent of the insulinomas are malignant as identified by the presence of metastases to peripancreatic lymph nodes and/or the liver. Only 2% of the insulinomas are extrapancreatic (e.g., located in the duodenal wall or porta hepatis). Patients with insulinoma may have symptoms for 2 wk to 20 years before the diagnosis is made. When hyperinsulinism is present and the C-peptide is elevated, drug-induced hyperinsulinism should also be considered [68]. The most common types of drugs to produce hyperinsulinism are the sulfonylureas and meglitinides. Meglitinides (e.g., repaglinide and nateglinide) are short-acting oral hypoglycemic agents. Sulfonylureas and meglitinides bind to the β cell SUR1. SUR1 is the regulatory subunit of the β cell potassium channel (KATP). When these drugs bind to SUR1, the potassium channel closes. Next, the cell depolarizes, the calcium channel opens, and the β cell secretes insulin-containing granules. To a far lesser extent, quinine and pentamidine have reportedly caused hypoglycemia. It is important to also recognize that pentamidine can cause glucose intolerance. When sulfonylurea/meglitinide use cannot be excluded, toxicological testing of the urine for sulfonylureas is indicated. Drugs that stimulate insulin secretion by β cell are fairly common causes of hypoglycemia in the adult population. The laboratorian should be aware of which sulfonylureas are detectable in their assay or in their reference laboratory’s assay. Older methods do not necessarily detect all of the current generations of sulfonylureas that are in clinical use. Referral of the sample to a reference laboratory for further sulfonylurea testing may be necessary. Very rarely, the β cell may be stimulated to oversecrete insulin by spontaneous anti-β cell agonist autoantibodies (e.g., “Graves disease of the β cell”) [69]. Such autoantibodies bind to and activate the insulin receptor.

Hyperinsulinism with suppressed C-peptide Assuming that hyperinsulinism is present, measuring C-peptide at the time of hypoglycemia will determine whether the source of the insulin is endogenous (e.g., from the pancreatic β cells: C-peptide elevated) or exogenous [e.g., from insulin injection or IA (autoimmune hypoglycemia): C-peptide low or absent] [70]. With surreptitious insulin injection in otherwise noninsulin-treated patients, hyperinsulinism is present at the time of hypoglycemia, yet C-peptide is low or not detectable because C-peptide is absent from commercial insulin preparations. If a recombinant DNA form of insulin was injected that has an altered amino acid sequence or other modifications compared with native human insulin, the laboratorian must be aware of the specificity of their insulin immunoassay (as discussed above). For example, a current ECL assay for insulin does not detect glargine. Therefore, the clinician who suspects hyperinsulinism by injection finds a negative insulin assay result needs to consider whether such an altered form of insulin might have been administered. At our institution, we are aware of two cases of parents

218

SECTION | 2 Analytes

administering insulin to their children for financial and/or other gain (e.g., Mu¨nchausen syndrome by proxy). Publications have highlighted the selfadministration of excess insulin causing hypoglycemia as a means of gaining social attention [71]. Rare in the United States but common in Japan is the autoimmune hypoglycemia syndrome [7274]. Similar to clandestine administration of exogenous insulin, with autoimmune hypoglycemia, hyperinsulinism is present, yet C-peptide is not elevated. In this disorder, spontaneous autoantibodies to insulin develop and bind large quantities of insulin in the circulation. These can be termed insulin autoantibodies (IAA). At certain times, glucose intolerance can be present when free insulin concentrations are deficient. At other times, insulin will be released from the insulin autoantibodies at a rate inappropriate for metabolic needs, causing hypoglycemia. Thus, the key to recognizing exogenous insulin administration and autoimmune hypoglycemia is biochemical evidence of hyperinsulinism (absent ketones, low βHB, low FFAs, and reduced concentrations of branched-chain amino acids), absent or low C-peptide, and the finding of antibodies to insulin (assuming that insulin was not injected chronically eliciting IA). With endogenous hyperinsulinism (and in the absence of IA or IAA that interfere with the measurement of insulin), the molar insulin/C-peptide ratio is ,1 in peripheral blood. The peripheral blood molar ratio is less than 1 because B50% of insulin secreted by the pancreas is cleared on its first pass through the liver while little C-peptide is cleared on the first pass. However with exogenous (injected) hyperinsulinism, the molar insulin/C-peptide ratio is $ 1 as the β cell’s release of insulin and C-peptide is suppressed by the hypoglycemia produced by the exogenous insulin.

Hyperinsulinism: biochemical findings compatible with hyperinsulinism in the absence of measured hyperinsulinism In the face of confirmed hypoglycemia when ketogenesis is absent (negative urine ketones, normal βHB), branched-chain amino acids and FFAs are normal, and hyperinsulinism is absent (by laboratory measurement), insulin surrogates as causes of hypoglycemia must be considered. Note that insulin surrogates (other than insulinomimetic autoantibodies) are not included in Fig. 7.1 but can, nonetheless, be diagnosed based upon this present discussion. A variety of tumors that can rarely secrete IGF-II that should be considered in such circumstances [75,76]. The IGF-II may be abnormally large and not normally bound to IGF-binding proteins [77]. Thus, the combination of a high IGF-II concentration and a greater proportion of “free” IGF-II can produce substantial insulin-like effect. At high concentrations, IGF-II binds to the insulin receptor, causing hypoglycemia, which, in turn, suppresses pancreatic insulin secretion. High IGF-II also suppresses GH, which in turn leads to low concentrations of circulating IGF-I.

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Insulinomimetic autoantibodies have been described above [78]. Because of autoantibody binding to the insulin receptor, insulinomimetic autoantibodies may actually impede the clearance of endogenously secreted insulin, which may occasionally elevate insulin concentrations. Insulinomimetic autoantibody measurements are available only on a research basis.

Nonhyperinsulinemic hypoglycemia If hyperinsulinism is excluded, the results of other tests drawn at the time of hypoglycemia (or during the workup) are reviewed in evaluating the other potential etiologies of hypoglycemia (Fig. 7.2). It should be emphasized that sufficient sample volume and types of specimens of blood and urine should be obtained at the time of acute hypoglycemia to perform all of the needed studies. The causes of nonhyperinsulinemic hypoglycemia (Table 7.4) can be divided into those conditions where ketones and FFAs are both elevated

TABLE 7.4 Causes of nonhyperinsulinemic hypoglycemia. Elevated ketones and FFAs Drugs Ethanol Salicylates Propranolol Hormone deficiency Growth hormone deficiency Cortisol deficiency Hypopituitarism Thyroid dysfunction Inborn errors of metabolism Carbohydrate disorders Aminoacidopathies Liver disease Renal disease Substrate limited/increased utilization Critical or severe illness Neonatal hypoglycemia Severe malnutrition Non-IGF-II secreting extrapancreatic neoplasms Ketotic hypoglycemia Suppressed ketones and elevated or normal FFAs Disorders of fatty acid metabolism Systemic carnitine deficiency Carnitine palmityl transferase (CPT) deficiency Acyl-CoA dehydrogenase deficiency Glutaric aciduria type II HMG CoA lyase deficiency

220

SECTION | 2 Analytes

versus those conditions where ketones are depressed, and FFAs alone are normal or elevated (i.e., disorders of fatty acid oxidation) [79,80]. This contrasts with hyperinsulinism discussed previously, in which ketones (e.g., urine ketones and βHB) and FFAs are both suppressed. The etiologies listed in Table 7.3 can be directly assessed by laboratory analysis.

Drugs as causes of hypoglycemia Certainly insulin, sulfonylureas, and meglitinides, either directly or through the release of insulin, commonly cause hypoglycemia. Alternatively, many drugs can cause hypoglycemia without producing hyperinsulinism. A clinical history of drug ingestion must always be sought in cases of hypoglycemia (Fig. 7.2) [81,82]. If ethanol ingestion or frank poisoning is a clinical consideration [83], the blood ethanol (alcohol) concentration should be measured at the time the patient is hypoglycemic. If there are large quantities of ethanol to burn, theoretically, nicotinamide adenine dinucleotide (NAD1) can become depleted. Next with insufficient NAD1 to convert lactate to pyruvate, gluconeogenesis is impaired. However, the validity of ethanol as a cause of hypoglycemia in adults has been questioned [84]. Ethanol ingestion has been implicated as cause of hypoglycemia in children following an overnight fast. Salicylate poisoning can produce hypoglycemia presumably from liver dysfunction. Salicylates can be tested for in the urine, or a serum salicylate level, can be measured. Propranolol, as detected by thin-layer chromatography, can produce hypoglycemia by blocking the metabolic effects of epinephrine.

Endocrinopathies as causes of hypoglycemia Adrenocorticotropin (ACTH) and GH deficiency can be isolated pituitary deficiencies, or, they can coexist as components of panhypopituitarism. Primary adrenocortical insufficiency is recognized clinically by the findings of hypoglycemia, hyponatremia, hyperkalemia, acidosis, and shock (e.g., Addisonian crisis) (see Chapter: 4). Cortisol concentrations of ,1820 mcg/dL at the time of hypoglycemia are suggestive of ACTH deficiency or primary adrenal insufficiency. GH deficiency in children is suggested by a GH concentration of ,7 ng/mL (depending upon the specific GH immunoassay) during a hypoglycemic episode (see Chapter: 147). In adults, GH deficiency is suggested by a GH concentration of ,5 ng/mL after stimulation. Thyroid dysfunction (e.g., hypothyroidism) is a rare cause of hypoglycemia and should be pursued only when there is a clinical suspicion of thyroid disease. Excluding individuals with diabetes mellitus, epinephrine deficiency, and glucagon deficiency are extremely rare. However, in long-standing diabetes mellitus, epinephrine and/or glucagon deficiency can develop as a consequence of presumed autonomic neuropathy [85].

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Carbohydrate and amino acid inborn errors of metabolism causing hypoglycemia These rare disorders present almost exclusively in newborns and infants (Fig. 7.2) [86]. Although hypoglycemia may be an important clinical aspect of these diseases, other clinical findings such as seizures, acidosis, lethargy, or liver disease are the usual initial complaints. Rare conditions exist, such as FanconiBickel syndrome [mutations in glucose transporter 2 (GLUT2) [87]] and GSD type 0 (glycogen synthase deficiency) where there is fasting hypoglycemia and postprandial hyperglycemia due to. A further description of the FanconiBickel syndrome can be found in the Chapter: 8 on hyperglycemia.

Disorders of carbohydrate metabolism Galactosemia and hereditary fructose intolerance These two categories of disorders are grouped together because nonglucose carbohydrate excretion in the urine produces a positive urine test for reducing substances but a negative result on urine glucose dipstick testing (e.g., glucose oxidase method) (Fig. 7.2). In the case of suspected galactosemia, urine-reducing substances may be negative if galactose has not been consumed recently. Note that testing the urine of young children for reducing substances is no longer recommended. Galactosemia is not a single disease entity. Three inborn errors that are inherited in an autosomal recessive pattern, galactokinase (GALK) deficiency, galactose-1-phosphate uridyltransferase (GALT) deficiency, and uridine diphosphate galactose-4-epimerase (GALE) deficiency, can cause galactosemia (Fig. 7.3). Galactose is absorbed from the gut but is not

Galactokinase

Galactose-1-phosphate uridyl transferase

Galactose ---------> Galactose-1-phosphate -------------------------> Glucose-1-phosphate

ATP

ADP

UDPglucose

UDPgalactose

Epimerase

FIGURE 7.3 Normal galactose metabolism is depicted. Following meals, galactose is absorbed by the intestine into the bloodstream. Within cells, galactose is initially phosphorylated to galactose 1-phosphate by galactokinase (GALK). Catalyzed by galactose-1-phosphate uridyltransferase (GALT), glucose 1-phosphate is released from uridine 50 -diphosphate (UDP)-glucose, as galactose-1-phosphate is metabolized to UDP-galactose. UDP-galactose is then converted to UDP-glucose through the action of an epimerase (GALE). ATP, Adenosine 50 -triphosphate; ADP, adenosine 50 -diphosphate.

222

SECTION | 2 Analytes

otherwise metabolized in GALK deficiency. This accounts cause for galactosuria, galactosemia, and cataracts and the lack of other clinical disorders. Galactose 1-phosphate uridyltransferase deficiency and uridine diphosphate galactose 4-epimerase deficiency elevate galactose 1-phosphate concentrations because of its metabolism to glucose-1-phosphate is impaired. In turn, galactose 1-phosphate is toxic to hepatocytes, kidney cells, and other cells in the body, causing early onset cataracts, hepatomegaly, liver dysfunction (as evidenced in hyperbilirubinemia) or failure, severely delayed development (when untreated), and failure to thrive [88]. The liver dysfunction appears to be responsible for hypoglycemia, although the presenting complaints are usually failure to thrive and cataracts [89]. Galactose-1-phosphate uridyltransferase and uridine diphosphate galactose-4-epimerase can be measured in red blood cells, and testing is available in reference laboratories. Infants with galactosemia are at increased risk for Escherichia coli sepsis possibly because of hepatic phagocytic (Kupffer cell) dysfunction [90,91]. Hereditary fructose intolerance (aldolase B deficiency, a.k.a.—a deficiency or fructose-bisphosphate B, or fructose-1-phosphate aldolase; ALDOB gene) (Fig. 7.4) is manifested when fructose [part of sucrose (table sugar)] is first introduced into the diet at B36 months of age [92]. This differs from galactosemia, where disease onset occurs shortly after birth. Hypoglycemia and metabolic derangements following fructose ingestion are short lived. However, without fructose restriction, hepatic and kidney failure may develop, leading to death. Infants can display poor feeding, vomiting, failure to thrive, hepatomegaly, jaundice (due to liver dysfunction), or hemorrhage (due to decreased clotting factor production).

The glycogen storage diseases Several forms of GSD can cause hypoglycemia [93]. In GSD types I, III, VI, and IX, there is a defect in the breakdown of glycogen to glucose, leading to increased hepatic glycogen stores, subsequent hepatomegaly, and hypoglycemia [94]. Insulin secretion is suppressed, glucagon concentrations are elevated, hyperlipidemia is common (e.g., increases in cholesterol and/or triglycerides), and growth retardation results when hypoglycemia and lactic Fructokinase

Aldolase B

Fructose ---------------------> Fructose-1-phosphate -----------> Dihydroxyacetonephosphate + Glyceraldehyde ATP ADP FIGURE 7.4 Normal fructose metabolism is depicted. Following meals, fructose is absorbed by the intestine into the bloodstream. Within cells, fructose is initially phosphorylated to fructose 1-phosphate by fructokinase (ketohexokinase; KHK). Catalyzed by aldolase B, fructose 1-phosphate is converted to dihydroxyacetone phosphate and glyceraldehyde. ATP, Adenosine 50 -triphosphate; ADP, adenosine 50 -diphosphate.

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acidosis are not prevented. The initial complaint is usually hepatomegaly with or without hypoglycemia. In GSD type 0 (glycogen synthetase 2 deficiency, a.k.a., liver glycogen synthetase; GYS2 gene) failure to synthesize adequate glycogen leads to fasting hypoglycemia. As well, the failure to synthesize glycogen after a meal can produce postprandial hypoglycemia. Because insulin levels are normally suppressed during hypoglycemia, GSD type 0 leads to ketotic hypoglycemia. Hepatomegaly is absent in this disorder [95]. Glycogen synthetase 1 (GYS1) in muscle is normal. GSD type XI is the FanconiBickel syndrome, which was discussed above and is discussed further in the hyperglycemic chapter. During the early evaluation of a patient with a suspected GSD, because of the possibility of myopathy, creatine kinase (CK) should be measured. The most severe form of GSD is von Gierke disease (GSD type I) (Fig. 7.5). Two forms of GSD type I are recognized: GSD type Ia—glucose6-phosphatase deficiency (the most common form of GSD I; G6PC gene) and GSD type Ib—glucose-6-phosphate translocase deficiency (G6PT; T1 transporter deficiency; solute carrier family 37 member 4; SLC37A4 gene). Neutropenia leading to serious infections can occur in GSD type Ib. Characteristic laboratory findings include fasting hypoglycemia, lactic Glycogenolysis and/or gluconeogenesis

G-6-P

G-6-P

GSD Ia

ER Membrane

GSD Ib G-6-P transporter (G6PT; SLC37A4)

Cytoplasm cellular space Extra-

Glucose-6-phosphatase (G6PC)

GLUT2 (SLC2A2)

Er membrane

+ Phosphate

Glucose

Glucose

Phosphate NPT4 (SLC17A3)

Plasma membrane

Cytoplasm

Glucose

GLUT2

Endoplasmic reticulum (ER)

FIGURE 7.5 Glucose-6-phosphate (G-6-P) is converted to glucose plus inorganic phosphate within the endoplasmic reticulum (ER) of hepatocytes. Initially, G6PT (G-6-P transporter) translocates G-6-P into the ER lumen. G6PC (glucose-6-phosphatase) then converts G-6-P to glucose plus phosphate. Glucose exits the ER lumen into the cytosol via the facilitative glucose transporter GLUT2 (SLC2A2). NPT4 (Na 1 /inorganic phosphate cotransporter 4; SLC17A3) transports phosphate into the cytosol. In the case of the liver, glucose exits the hepatocyte via GLUT2.

224

SECTION | 2 Analytes

acidosis, hyperuricemia, hypertriglyceridemia, and hypercholesterolemia. There is no increase in the blood glucose concentration in response to glucagon injection because of the impaired conversion of glucose-6-phosphate to glucose. Relevant to GSD type III, the enzyme amylo-1,6-glucosidase (amyloalpha-1, 6-glucosidase, 4-alpha-glucanotransferase; AGL) transfers three to four glucose units in 1 $ 4 linkages from the branched chain to the main chain of glycogen, leaving a single glucose residue in the branched chain in a 1 $ 6 linkage. Subsequently, free glucose is released by the action of amylo-1,6-glucosidase upon the 1 $ 6 linkage (Fig. 7.6). Without normal amylo-1,6-glucosidase activity, after some hours of fasting, glycogen can no longer be degraded, and hypoglycemia may ensue. This problem causes GSD type III (Cori disease), which is the only type of GSD that affects both muscle and the liver. In addition to hepatomegaly (which may resolve after puberty), there is muscular weakness in adulthood. The biochemical findings are similar to, although less severe than, GSD type I. CK may be elevated because of myopathy. For some hours after eating, blood glucose concentrations will increase after glucagon injection in persons with GSD type III. Glycogen breakdown, in part, is regulated by epinephrine and glucagon. These hormones ultimately activate the liver’s inactive glycogen phosphorylase (phosphorylase b) to active glycogen phosphorylase (phosphorylase a). GSD type VI (Hers disease) results from a deficiency of the liver’s glycogen phosphorylase (glycogen phosphorylase L; PYGL gene), which is necessary for glycogen degradation to G-1-P (Fig. 7.7). Similar to GSD type I, muscle

4

OH

Debrancher enzyme: amylo[1 -> 6]-glucosidase

Glucose monomer

6 CH2OH O

1

OH

OH OH

CH2OH OH

CH2OH O

OH

CH2OH O

-O

O OH

OH

CH2 O

O

O-

-O

-O OH

OH

OH

OH

1 ---> 4 FIGURE 7.6 The normal action of the debrancher enzyme is to move glucose moieties from a 1 $ 6 linkage to a 1 $ 4 linkage. In glycogenolysis, glucose 1-phosphate is released from 1 $ 4 linkages via the action of liver glycogen phosphorylase a. The numbering of carbons in the glucose monomer (upper left) illustrates carbons 1, 4, and 6.

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Epinephrine glucagon Multiple steps

Glycogen phosphorylase L (inactive) (phosphorylase b) Phosphorylase b kinase

Protein phosphatase-1 Glycogen phosphorylase L (active) (phosphorylase a)

Glycogen (N + 1) ------------------> Glycogen (N) + G-1-P Phosphoglucomutase

G-6-P FIGURE 7.7 Epinephrine and glucagon via their cell surface receptors, Gs proteins, and adenyl cyclase activate phosphorylase kinase. Active phosphorylase kinase converts liver glycogen phosphorylase b to active liver glycogen phosphorylase a. Active liver glycogen phosphorylase a releases one glucose-1-phosphate unit from glycogen. Glucose-1-phosphate is isomerized to G-6P via phosphoglucomutase. Active liver glycogen phosphorylase a is converted back to inactive liver glycogen phosphorylase b via the action of protein phosphatase-1.

is not affected in GSD type VI. Hepatomegaly is recognized. Only mild hypoglycemia may be observed. Regarding GSD’s types VI and IX, phosphorylation of liver phosphorylase (phosphorylase b) leads to its activation (phosphorylase a). Phosphorylase b kinase carries out this activation activity. Phosphorylase b kinase is made up of 16 subunits: four each of (1) the phosphorylase kinase regulatory subunit alpha 1 (PHKA1 gene); (2) phosphorylase kinase regulatory subunit alpha 2 (PHKA2 gene); (3) phosphorylase kinase regulatory subunit beta (PHKB gene); and (4) phosphorylase kinase catalytic subunit gamma 2 (PHKG2 gene). Defects in PHKA2, PHKB, or PKHG2 cause, respectively, GSD-type IXa, GSD-type IXb, and GSD-type IXc. For this reason, GSD-type IX is similar to GSD-type VI. Regarding phosphorylase kinase catalytic subunit gamma 1 (PHKG1), PHKG1 is expressed in muscle. All forms of GSD are autosomal recessives except for GSD-type IXa because PHKA2 is encoded on the X chromosome. The diagnosis of the specific GSD is usually made through gene sequencing [96].

Defects in gluconeogenic enzymes In the Cori cycle, the liver produces glucose. Exercising muscle anaerobically burns glucose to lactate. Lactate is then taken up by the liver and is reconverted to glucose through gluconeogenesis. In addition to G-6-Pase (G6PC gene; the cause of GSD type I), pyruvate carboxylase (PC gene)

226

SECTION | 2 Analytes

[97], phosphoenolpyruvate carboxykinase (PEPCK; PCK2 gene), and fructose-1,6-bisphosphatase (a.k.a.—fructose bisphosphatase 1, FBP1 gene) are required for gluconeogenesis (Fig. 7.8). With a deficiency of any of these enzymes, glucose cannot be sufficiently regenerated from lactate causing hypoglycemia and lactic acidosis (i.e., the Cori cycle is interrupted). Similar to the GSDs, the diagnosis of a gluconeogenic defect is predominantly through genetic studies [98].

Aminoacidopathies with associated hypoglycemia Similar to many of the defects described under disorders of fatty acid oxidation and gluconeogenic enzyme deficiencies, aminoacidopathies often precipitate overwhelming illness in the first days of life, frequently noted as acidosis and organic aciduria (Fig. 7.2) [99,100]. Although hypoglycemia may be present in many of these disorders (e.g., maple syrup urine disease, methylmalonic aciduria, isovaleric acidemia, 3-methylglutaconic aciduria), hypoglycemia is not the sole initial complaint, as hypoglycemia is just one Exercising muscle

Lactate

Glucose GLUT2 (SLC2A2)

MCT1 (SLC16A1)

Glucose G-6-Pase; G6PC Glucose-6-P

Lactate

Fructose-6-P

LD

Fructose-1,6-bisPase; FBP1 Pyruvate

Fructose-1,6-bisP Several steps

PEP

Pyruvate PC

PEPCK; PCK2 Oxaloacetate

Oxaloacetate Malate

Malate

Cytoplasm

FIGURE 7.8 This figure illustrates the basic steps in the Cori cycle. There are four rate-limiting enzymes in gluconeogenesis: pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK; PCK2), fructose-1,6-bisphosphatase (F-1,6-bisPase; FBP1), and glucose-6-phosphatase (G-6-Pase;G6PC). LD, Lactate dehydrogenase; PEP, phosphoenolpyruvate.

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aspect of these systemic, metabolic disorders. Diagnosis can require assessment of plasma amino acids and urine organic acids. Various molecular genetic studies are available [101].

Liver and renal disorders as causes of hypoglycemia Hepatic or renal diseases as contributors to hypoglycemia are easily assessed (Table 7.2) by the liver and kidney function tests mentioned earlier. Significant liver disease can cause hypoglycemia because the liver is the major site of gluconeogenesis, glycogen storage, and glycogenolysis [102]. Hepatocellular carcinoma is an example of such a condition causing hypoglycemia. The kidney has about 10% of the liver’s capacity for gluconeogenesis. Hepatic and especially renal disorders as causes of hypoglycemia should be considered diagnoses of exclusion (e.g., other causes of hypoglycemia must be excluded before hypoglycemia can be attributed strictly to liver or renal disease) [103].

Limited substrate/increased utilization as causes of hypoglycemia The common theme in these disorders is increased demand for glucose (often because of critical illness) and/or decreased hepatic ability to produce glucose (Fig. 7.2) [104]. Sepsis can produce hypoglycemia [105,106]. Treatment of childhood acute lymphocytic leukemia is commonly associated with hypoglycemia [107,108]. The mechanism of hypoglycemia in cyanotic congenital heart disease is unclear [109]. Newborn infants, especially premature infants, are at high risk for hypoglycemia based on liver immaturity, high caloric demands (e.g., countering cold stress), limited or no caloric intake immediately following birth, limited amino acid substrate for gluconeogenesis (e.g., minimal muscle mass), limited ability to generate ketones, and an increased proportion of brain size to liver size, further increasing glucose demands [110]. With severe malnutrition (i.e., kwashiorkor), there is depletion of all energy resources needed for glucose generation (e.g., very low muscle mass) and ketone body generation (e.g., exhausted supply of triglycerides from adipose tissue). The non-IGF-II-secreting extrapancreatic neoplasms are theorized to anaerobically consume glucose at high rates, producing hypoglycemia and elevating lactate concentrations. Ketotic hypoglycemia is a clinical diagnosis based upon the development of hypoglycemia, ketonuria, and vomiting after only modest durations of fasting (e.g., overnight) in otherwise healthy children between ages 18 months and 6 years. There are no inborn errors of metabolism in this condition. These children are often male and were SGA at birth. This is a variant of normal. Children, in general, have less tolerance for fasting than adults (e.g., during a fast, some lean, young children may develop hypoglycemia, whereas healthy adults do not develop hypoglycemia during a fast).

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Disorders of fatty acid oxidation Similar to aminoacidopathies and carbohydrate disorders that cause hypoglycemia, disorders of fatty acid oxidation present in the newborn period or during infancy [79]. These disorders lead to a deficiency of ketone body production [111]. With ketone body deficiency, the sole source of energy during fasting becomes glucose. In comparison, with starvation and normal ketogenesis, up to two-thirds of the brain’s energy requirements can be met through ketone body oxidation. In fatty acid oxidation disorders, with increased glucose demands during fasting (because alternative fuels are not available), glucose production does not keep pace with glucose demand, and hypoglycemia ensues [112]. When ketone production is low (e.g., absent urine ketones, normal βHB) and FFAs are elevated, a disorder of fatty acid oxidation is suggested. Carnitine is critical to fatty acid β-oxidation because carnitine is involved in the transport of FFAs into the mitochondrion (Fig. 7.9). Carnitine should be measured because carnitine concentrations are frequently low in these syndromes [113]. When fatty acids cannot be oxidized by β-oxidation, ω-oxidation occurs, producing dicarboxylic acids that are excreted in the urine. Such acids produce an anion gap metabolic acidosis.

Summary of the evaluation of hypoglycemia The potential causes of hypoglycemia are legion [114]. The differential diagnosis and laboratory evaluation of hypoglycemia should be assessed FFA

Acyl-CoA synthetase

Cytosol

Acyl-CoA Carnitine

CoA-SH

ATP

Carnitine

AMP + PPi

CoA-SH

CPT I

Mitochondrial outer membrane

Transporter

Transporter Mitochondrial inner membrane

CPT II Acyl-carnitine

CoASH

Mitochondrial matrix Carnitine

Acyl-CoA

Beta oxidation

FIGURE 7.9 Oxidation of FFAs requires that the FFAs be “activated” through binding to coenzyme A (CoA) and converted to acyl-carnitines by carnitine palmitoyltransferase I (CPT I). Acyl-carnitines enter the mitochondrial matrix via an inner membrane transporter. Once within the mitochondrial matrix, acyl-carnitines are converted back to acyl-CoA via carnitine palmitoyltransferase II (CPT II). Carnitine is recycled back to the cytoplasm. In summary, carnitine is responsible for transferring FFA into the mitochondrion. AMP, Adenosine 50 -monophosphate; PPi, pyrophosphate ion; CPT, carnitine palmitoyltransferase.

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according to the patient’s age, history, and physical examination. Figs. 7.1 and 7.2 provide a reasonable approach to these disorders. A specific diagnosis must always be sought, so that proper therapy can be pursued. Genetic analyses for specific inborn errors have become a diagnostic cornerstone.

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[109] K.H. Lundell, K.G. Sabel, B.O. Eriksson, G. Mellgren, Glucose metabolism and insulin secretion in children with cyanotic congenital heart disease, Acta Paediatr. 86 (10) (1997) 10821084. [110] C.A. Stanley, Hypoglycemia in the neonate, Pediatr. Endocrinol. Rev. (Suppl. 1)(2006) 7681. [111] J.O. Sass, Inborn errors of ketogenesis and ketone body utilization, J. Inherit. Metab. Dis. 35 (1) (2012) 2328. [112] T. Fukao, G. Mitchell, J.O. Sass, T. Hori, K. Orii, Y. Aoyama, Ketone body metabolism and its defects, J. Inherit. Metab. Dis. 37 (4) (2014) 541551. [113] C.A. Stanley, Carnitine deficiency disorders in children, Ann. N.Y. Acad. Sci. 1033 (2004) 4251. [114] F.J. Service, Clinical review 42: hypoglycemias, J. Clin. Endocrinol. Metab. 76 (1993) 269272.

Chapter 8

Evaluation of hyperglycemia William E. Winter1, David L. Pittman2, Sridevi Devaraj3, Danni Li4 and Neil S. Harris5 1

Departments of Pathology, Immunology & Laboratory Medicine, Pediatrics, and Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States, 2Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States, 3Texas Children’s Hospital, Houston, TX, United States, 4 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, United States, 5Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States

Hyperglycemia: acute versus chronic Hyperglycemia is defined as an elevated plasma glucose (PG) concentration. Hyperglycemia can be classified as transient (acute) or sustained (chronic). Chronic hyperglycemia constitutes diabetes mellitus (which will be referred to as “diabetes” in the rest of the chapter; other than Wolfram syndrome, diabetes insipidus will not be discussed). Specific PG cutpoints defining diabetes mellitus are discussed below. Causes of transient hyperglycemia are listed in Table 8.1. Diabetes is a family of metabolic diseases characterized by sustained (chronic) hyperglycemia [1]. In some cases, prolonged use of a medication such as glucocorticoids [2] or an atypical antipsychotic medication [3] causes diabetes. However, discontinuation of these medications may lead to a reversal of the diabetic state. Pathophysiologically, diabetes results from a decline in insulin action. Insulin action is the approximate multiplicative product of the absolute insulin concentration and the insulin sensitivity of the tissues. The three major targets for insulin action are the liver, muscle, and adipose tissue. In the liver, insulin stimulates glycolysis, sparing free fatty acids for triglyceride synthesis. Gluconeogenesis is suppressed, making amino acids available for protein synthesis. Insulin promotes energy storage through stimulation of glycogen synthesis. The net effect of insulin on the liver is increased glucose utilization and decreased hepatic glucose output. Recall that during the fasting state, the source of circulating glucose is the liver. During fasting, Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00008-X © 2021 Elsevier Inc. All rights reserved.

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TABLE 8.1 Causes of transient hyperglycemia. Significant degrees of metabolic stress (secondary to elevations in the antiinsulin stress hormones: glucagon, epinephrine, cortisol, and growth hormone) Myocardial infarction Burns Acute, severe illness Medications used for short durations of time (e.g., only days to weeks) Glucocorticoids (antiinsulin stress hormones) Catecholamines (antiinsulin stress hormones) Growth hormone (an antiinsulin stress hormone) Oral contraceptives (causes insulin resistance) Thiazides (causes insulin resistance) Furosemide (causes potassium depletion) Iatrogenic etiologies (excessive rates of parenteral glucose infusion) Intravenous glucose infusion Hyperalimentation Acute pancreatitis (transient pancreatic islet-cell dysfunction) Head injury (dysfunctional autonomic control of the pancreatic islets)

glucose is initially provided through glycogenolysis, whereas some 12 18 h later, significant gluconeogenesis is occurring. In muscle and adipose tissue, insulin stimulates the migration of insulinresponsive glucose transporters (GLUT4; SLC2A4; solute carrier family 2 member 4; chromosome 17p13.1) to the cell membrane, permitting increased rates of glucose uptake by these tissues. GLUT4 is a nonenergy-requiring facilitative GLUT. Adipose tissue burns glucose to acetate (e.g., acetyl coenzyme A) that is substrate for fatty acid synthesis and subsequent triglyceride synthesis. While burning glucose for energy, muscle will also store glucose as muscle glycogen to a limited degree. All three tissues increase glucose clearance from the circulation with a resultant fall in PG concentrations following meals.

Clinical symptoms of diabetes When the PG concentration exceeds the renal threshold for glucose (e.g., more glucose is filtered by the glomerulus than can be reabsorbed by the renal tubules), glycosuria results. Because of the resulting elevated urine

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osmolality, a diuresis ensues that is clinically evident as polyuria (increased urination). Electrolytes lost during this osmotic diuresis include sodium, potassium, magnesium, and phosphate. Hyperglycemic patients will frequently begin to void at night (nocturia), whereas children may experience bed-wetting (enuresis). With increased urinary fluid loss, thirst is stimulated, causing increased drinking (polydipsia). Because of diminished insulin action, adipose tissue lipolysis increases. As well, protein catabolism accelerates to provide amino acid carbon skeletons for gluconeogenesis by the liver. The clinical consequence of this catabolic state is weight loss in patients with severe insulin deficiency. Fat breakdown fueling ketogenesis also contributes to weight loss. Some patients experience increased appetite (e.g., polyphagia) because large amounts of calories are lost in the urine. Blurred vision may occur resulting from fluid shifts involving the lens of the eye secondary to hyperglycemia. Children who are in very poor diabetic control may grow poorly (e.g., Mauriac syndrome). However, poor growth in diabetic children is the exception and not the rule. Usually, diabetic children grow well even if they are in poor glycemic control. Chronic hyperglycemia affects the skin and connective tissues. The skin over the backs of the hands becomes palpably thickened with reduced flexibility [4]. Joints become stiff with reduced range of motion. If the patient is asked to oppose the fingers and palms of the hands, because of limited joint mobility, the fingers do not approximate one another at the proximal or distal interphalangeal joints (e.g., the “prayer” sign). The wrists, elbows, and neck can be affected. These changes are believed to reflect a nonenzymatic glycation of connective tissue proteins. Hyperglycemia clearly impairs phagocyte function [5]. When impaired innate (e.g., intrinsic or natural immunity) immunity is combined with the nutrient-rich tissue and body fluid conditions provided by hyperglycemia, increased susceptibility to bacterial and fungal infections often occurs in patients with poorly controlled diabetes. Diabetic subjects are predisposed to soft tissue infections, balanitis (infection of the space between the foreskin and glans), vaginitis, and urinary tract infections. Severe acute insulin deficiency in patients with type 1 diabetes can precipitate diabetic ketoacidosis (DKA). Less commonly, DKA develops in severely stressed patients with type 2 diabetes. In DKA, the rate of ketone body production exceeds the rate of ketone body consumption. The accumulation of acetoacetic acid and β-hydroxybutyric acid leads to an anion-gap, normochloremic acidosis. Hyperglycemia leads to acute dehydration through an excessive osmotic diuresis. Because ketosis can cause nausea and vomiting, patients are less likely to drink further, which increases the degree of dehydration. With poor tissue perfusion, lactic acidosis may additionally develop. Untreated, profound acidosis can lead to cardiovascular failure and death. Children are at risk of developing cerebral edema, which can cause death. DKA is fatal in 10% 15% of adults and 0.5% of children who

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experience this condition. When severe hyperglycemia occurs in patients with type 2 diabetes who then suffer profound dehydration, the hyperglycemic hyperosmolar state [HHS; previously termed: hyperglycemic nonketotic coma (HNKC)] can ensue, with a consequent high mortality rate. Patients with HHS have minimal or no ketosis and minimal or no acidosis. For patients who have had diabetes for 10 or more years, there is an increasing likelihood that they will experience diabetic complications. These problems include macrovascular, microvascular, and neuropathic diseases. Macrovascular disease may present as angina, heart attack (myocardial infarction), heart failure (chronic coronary artery disease), stroke (cerebrovascular disease), or claudication (peripheral vascular disease). Claudication is the fatigue and pain that patients experience in their legs when walking when there is inadequate blood supply to match oxygen demand. Microvascular disease produces retinopathy and nephropathy. Retinopathy can lead to blindness, whereas nephropathy can lead to renal failure. Retinopathy is classified as background (nonproliferative) or proliferative (e.g., there is new blood vessel formation over the surface of the retina). Peripheral neuropathy can produce loss of sensation in the feet and hands. Painful peripheral neuropathy can be incapacitating. Autonomic neuropathy may be evidenced in gastroparesis (decreased gastrointestinal tract motility), impotence, decreased sexual response in women, postural hypotension (low blood pressure upon arising from the sitting or supine position often accompanied by light-headedness or fainting), and loss of the normal beat-to-beat variation in heart rate that occurs with breathing. Autonomic neuropathy may play a role in the development of hypoglycemia in persons with diabetes.

Classification of diabetes Chronic (sustained) hyperglycemia may be permanent (e.g., type 1 or type 2 diabetes) or time-limited [e.g., gestational diabetes mellitus (GDM) (diabetes first recognized during the second or third trimester of pregnancy that resolves following delivery)]. Diabetes is classified into four major types (Table 8.2). The classification of diabetes is based upon etiology and not the

TABLE 8.2 Classification of diabetes. Type 1 diabetes Type 2 diabetes Other specific types of diabetes Gestational diabetes mellitus (GDM)

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type of therapy that a patient receives. The nomenclature “insulin-dependent diabetes,” “type I (Roman numeral) diabetes,” “noninsulin-dependent diabetes,” and “type II (Roman numeral) diabetes” is replaced, respectively, by the terms type 1 (Arabic) and type 2 (Arabic) diabetes.

Type 1 diabetes Type 1 diabetes results from absolute insulinopenia (low insulin concentration in the bloodstream). Insulinopenia most commonly results from autoimmune destruction of the insulin-producing β cells. This condition is frequently accompanied by the expression of several islet autoantibodies that can be recognized at or before the time of diagnosis of type 1 diabetes: islet cell cytoplasmic autoantibodies (ICAs), insulin autoantibodies (IAAs), glutamic acid decarboxylase autoantibodies (GADAs), insulinomaassociated-2 autoantibodies (IA-2As), and zinc transporter 8 autoantibodies (ZnT8A) [6 8]. ICAs are detected by indirect immunofluorescence, whereas IAAs, GADAs, IA-2As, and ZnT8A are detected by a variety of immunoassays [immunoprecipitation, ELISA with a spacer for the autoantigen, luciferase immunoprecipitation system, electrochemiluminescence, etc.]. The presence of any of these autoantibodies indicates an autoimmune etiology for diabetes and classification as type 1A diabetes. IAA must be measured prior to insulin administration because after B14 days exogenous insulin injection can induce insulin antibodies that cannot be distinguished analytically from IAA. If the clinical differentiation between type 1 and type 2 diabetes is problematic in a patient, measurement of islet autoantibodies should strongly be considered. Such instances include obese patients presenting with DKA or lean patients previously diagnosed with type 2 diabetes that progress to insulin dependence. When the cause of the insulinopenia is unidentified in type 1 diabetes, diabetes is classified as type 1B diabetes. Type 1B diabetes is rare and may be caused by viral infection [9]. Viruses proposed to trigger type 1 diabetes include coxsackie A and B viruses, cytomegalovirus, ECHO virus [10], Epstein Barr virus, rubella virus [11], mumps virus, and retroviruses. Because type 1A diabetes is far more common than type 1B diabetes, type 1A diabetes will hereafter be referred to simply as “type 1 diabetes.” If a specific nonautoimmune etiology for the insulinopenia is identified [e.g., β cell destruction from Vacor (rat poison)], the diabetes is then classified under the heading of “other specific types of diabetes” (see below).

Type 2 diabetes Type 2 diabetes results from a combination of tissue insulin resistance and relative β cell failure (e.g., relative insulinopenia) [12]. Relative

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TABLE 8.3 Features of the metabolic syndrome. Centripetal obesity Insulin resistance Hyperinsulinemia Hypertriglyceridemia Hypoalphalipoproteinemia Elevated apolipoprotein B Dense LDL particles Type 2 diabetes Nonalcoholic fatty liver Nonalcoholic steatohepatitis Elevated ferritin Elevated C-reactive protein (CRP) Elevated fibrinogen Hyperuricemia Gout Polycystic ovarian syndrome Male menopause (andropause) (under investigation) Acanthosis nigricans

insulinopenia infers that even if the absolute concentration of insulin in the circulation is greater than normal, the supranormal insulin concentration is still insufficient to correct the patient’s hyperglycemia. Insulin resistance is most often postreceptor; for example, the insulin receptor is structurally normal; however, there is deficient generation of second messengers within the target cell, leading to decreased insulin action. Accompanying most forms of diabetes is hyperglucagonemia. Type 2 diabetes is commonly part of the metabolic syndrome (see below). There is no consensus definition of the metabolic syndrome among professionals [13]. Because of the impact of the metabolic syndrome on health, life expectancy in the United States may decline in the 21st century [14]. Features of the metabolic syndrome are listed in Table 8.3 [15].

Metabolic syndrome The metabolic syndrome is a group of reproducible, recognizable characteristics that relate insulin resistance and central obesity to increased

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cardiovascular risk, as well as several other health problems [16]. Central obesity is defined as an increased abdominal circumference where there is an increased waste-to-hip ratio. The distribution of fat in central obesity is both subcutaneous and intra-abdominal; however, it is the intra-abdominal (e.g., visceral or omental fat) that is most strongly correlated with insulin resistance and adverse outcomes [17]. Several terms have been applied to the metabolic syndrome including the insulin resistance syndrome, syndrome X, the diabesity syndrome, the morbesity syndrome, the cardiometabolic syndrome, and the dysmetabolic syndrome X (ICD-10 code: E88.81). The metabolic syndrome has been defined by the NIH as “. . . a group of risk factors linked to overweight and obesity that increase the patient’s chance for heart disease and other health problems such as diabetes and stroke” [18]. There are at least four major hypotheses linking obesity and insulin resistance. (1) Free fatty acid levels in the blood are elevated in the metabolic syndrome [19]. Elevated free fatty acid levels in obesity can lead to triglyceride deposition in the liver, skeletal muscle, and beta cells [20]. Such fat deposition in the liver and skeletal muscle interferes with insulin signaling reducing insulin’s effect on these tissues. From the uptake of free fatty acids by tissues, the resulting diacyl glycerol may affect second messenger signaling of the insulin receptor. Fat deposition damages the beta cells contributing to relative insulinopenia in type 2 diabetes. (2) As muscle is “marbled” (a.k.a.—infiltrated) with increased numbers of fat cells that accompany obesity, these adipocytes locally produce tumor necrosis factor-alpha (TNF-α) inducing skeletal muscle insulin resistance [21]. It is worth noting that skeletal is quantitatively the major site of glucose clearance. (3) Adipose tissue is not a passive receptor or distributor of fat and fatty acids. Adipose tissue regulates systemic insulin sensitivity through the production of many adipokines including adiponectin. Deficient adiponectin in the metabolic syndrome leads to decrease insulin sensitivity and produces a pro-inflammatory and a pro-atherogenic state [22]. (4) With the conversion of circulating steroids to cortisol in adipose via the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), an element of hypercortisolism may be present in the metabolic syndrome [23]. Despite the above evidence, there is a growing body of evidence that proposes that beta-cell hyperresponsiveness may drive the metabolic syndrome [24]. Pathophysiologically, the consequences of the metabolic syndrome can be divided into (1) those consequences that result from hyperinsulinism that is an attempted compensation for insulin resistance, and (2) those consequences that result from inadequate insulinization despite elevated circulating insulin levels, or, later in the course of the metabolic syndrome, declining beta-cell insulin secretion [25].

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Metabolic syndrome consequences that result from hyperinsulinism Hyperinsulinism has been associated with atherosclerosis [26]. Certainly insulin serves as a growth factor. It is controversial if atherogenesis is caused by hyperinsulinism, or whether atherogenesis is merely associated with hyperinsulinism. There is a strong association between hyperinsulinism and hypertension [27]. Hyperinsulinism causes sodium and water retention that could increase circulating blood volume (at least transiently). Hyperinsulinism increases sympathetic tone causing vasoconstriction. Next, hyperinsulinism appears to induce vascular hypertrophy causing vasoconstriction. Lastly, hyperinsulinism increases the activity of the Na1/K1 ATPase pump causing vasoconstriction. These factors contribute to the development of hypertension in persons affected with the metabolic syndrome. Hyperinsulinism causes hypercoagulability which is pro-atherogenic. Insulin stimulates the production of plasminogen activator inhibitor-1 (PAI1) [28]. Normally tissue plasminogen activator (tPA) stimulates the conversion of plasminogen to plasmin with plasmin-causing fibrinolysis. However, PAI-1 inhibits tPA creating a pro-thrombotic state. Because the metabolic syndrome is a pro-inflammatory state, additionally levels of factor VIII and fibrinogen are elevated. Hyperinsulinism contributes to hyperuricemia and gout through increased uric acid reabsorption [29]. Urinary sodium and urate reabsorption are linked: both are increased by insulin. Increased plasma uric acid predisposes to gout, which occurs commonly in persons with the metabolic syndrome. There is intriguing data that uric acid may be pro-atherogenic [30 32]. Hyperandrogenism in women is linked to hyperinsulinism [33]. A serious clinical manifestation of hyperandrogenism is the polycystic ovarian syndrome [34]. Pituitary FSH is suppressed and LH is stimulated by elevated insulin levels. Together with elevated LH, high insulin levels stimulate ovarian overproduction of androgens. Hyperinsulinism lowers hepatic sex hormone binding globulin (SHBG) production. Reduced SHBG and elevated total testosterone levels raise free testosterone levels causing hirsutism, menstrual irregularities, and even infertility in women [35]. By causing salt retention, which raises blood pressure, increasing low-density lipoproteins (LDL)-cholesterol, and lowering high-density lipoproteins-cholesterol (HDLC), hyperandrogenism fosters a pro-atherogenic state. Acanthosis nigricans is a cutaneous manifestation of hyperinsulinism [36]. Acanthosis nigricans is characterized by velvety, warty benign growths and hyperpigmentation localized in areas of skin friction: the axillae, neck, anogenital area, and groin [37]. The differential diagnosis of acanthosis nigricans additionally includes malignancy, endocrine disorders, and obesity.

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Metabolic syndrome consequences that result from inadequate insulinization Within insufficient insulin action despite elevations in the absolute circulating insulin concentration, three major consequences can result: (1) dysglycemia (abnormal glucose metabolism manifested as prediabetes or frank diabetes), (2) dyslipidemia, and (3) nonalcoholic fatty liver (NAFL) disease. The pathophysiology of dysglycemia is the pathophysiology of type 2 diabetes as discussed elsewhere in this chapter [38]. Normal glucose tolerance can progress to postmeal-challenge glucose intolerance, to fasting hyperglycemia, to frank type 2 diabetes. Type 2 diabetes is a rather late manifestation of the metabolic syndrome. High glucose and low levels of insulin action foster a pro-inflammatory state. Consequently, the metabolic syndrome is recognized as a state of heightened inflammation with elevated levels of ferritin, C-reactive protein, serum amyloid A, and fibrinogen [39]. Increased glucose concentrations increase the transcription factors NFkappaB, activator protein-1 (AP-1), I kappa B kinase-alpha and IKK B, p47phox (a cytoplasmic NADPH oxidase subunit), the cytokines IL-1, IL-6, and TNF-α, expression of ICAM-1 and Eselectins, matrix metalloproteinases, and reactive oxygen species. Increased glucose decreases cytoplasmic I kappa B which regulates NFkappaB. On the other hand, increased insulin action decreases the transcription factors NFkappaB (and may decrease VGEF, TNF-α, and IL-6), AP-1 (that regulates metalloproteinases), and early growth response-1 (Egr-1; that regulates PAI-1 and tissue factor expression), ICAM-1, MCP-1, CRP, reactive oxygen species, and p47phox. Increased insulin action increases cytoplasmic I kappaB. In terms of lipids, insulin resistance is manifested as hypertriglyceridemia, low HDL-C, increased apo-B concentrations, and dense LDL particles [40]. Please see Chapter 9 for a formal discussion of such dyslipidemias. With increased levels of free fatty acids coursing through the liver, fatty infiltration of the liver is an increasingly common finding in the metabolic syndrome [41]. Such “nonalcoholic fatty liver” (NAFL) can progress through inflammation to “nonalcoholic steatohepatitis” (NASH). NASH can progress to cirrhosis or liver failure and predisposes to hepatocellular carcinoma. Elevated alanine aminotransferase (ALT) levels are not particularly sensitive markers of NAFL and related disorders. Abnormal ultrasound findings do correlate more closely with fatty infiltration [42]. The reader is referred to several recent reviews on the topic of the metabolic syndrome [43 45].

Other specific types of diabetes Other specific types of diabetes are classified into eight subtypes (Table 8.4).

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TABLE 8.4 Subtypes of “other specific types of diabetes.” Genetic defects of β cell function Genetic defects in insulin action Diseases of the exocrine pancreas Endocrinopathies Drug- or chemical-induced diabetes Infections Uncommon forms of immune-mediated diabetes Other genetic syndromes associated with diabetes

Genetic defects of β cell function Genetic defects of β cell function include inborn errors causing insulinopenic diabetes. Examples include: (1) maturity-onset diabetes of youth (MODY) (Tables 8.5 and 8.6), (2) neonatal diabetes due to several inborn errors, (3) mitochondrial diabetes, (4) familial hyperproinsulinemia, and (5) insulinopathies. For individuals 35 years of age and younger, the likelihood of MODY can be calculated on-line [61]. Maturity-onset diabetes of youth MODY is defined as nonketotic, initially noninsulin-requiring diabetes with onset before age 25 that is inherited in an autosomal dominant (single gene) pattern [62] (Table 8.5). Presently 13 types of MODY have been described [63 68]. Most forms of MODY appear to result from β cell defects. Several forms of MODY can present in infancy or, more commonly, childhood, adolescence, or early adulthood (Table 8.6). Islet-1 (chromosome 5q11-q13) is a Lim domain homeobox gene that encodes a transcription factor that regulates insulin expression [69]. While being associated with type 2 diabetes in a few families, islet-1 has not been associated (so far) with MODY [70]. Gene sequencing has become the most effective means to diagnose MODY (and neonatal diabetes, see below) [71]. As with any diagnosis based on a gene sequence, the significance of novel mutations requires careful consideration [72]. The case for routine DNA sequencing in infants and children with type 1 diabetes or neonatal diabetes has been made [73]. Neonatal diabetes Strictly speaking, neonatal diabetes mellitus (NDM) is diabetes diagnosed within the first 28 days of life. However, the window of time for the

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TABLE 8.5 Causes of maturity-onset diabetes of youth. Disorder

Mutated protein

Abbreviation

MODY1

Hepatocyte nuclear factor 4α

HNF-4α

MODY2

Glucokinase

GCK

MODY3

Hepatocyte nuclear factor 1α (a.k.a.—transcription factor 1, TCF-1)

HNF-1α

MODY4

Pancreatic and duodenal homeobox protein 1 (a.k.a.—insulin promoter factor 1, IPF-1)

PDX1

MODY5

Hepatocyte nuclear factor 1β

HNF-1β

MODY6

Neuro D/beta 2



MODY7

Kru¨ppel-like factor 11

KLF11

MODY8

Carboxyl ester lipase

CEL

MODY9

Paired box gene 4

PAX4

MODY10

Insulin

INS

MODY11

B-lymphocyte kinase

BLK

MODY12

Sulfonylurea receptor 1

SUR1

MODY13

A beta-cell potassium channel

Kir6.2

diagnosis of diabetes is extended by some experts to 6 or even 12 months of age. NDM is a rare condition with prevalence estimates of 1 in 200,000 to 1 in 800,000 newborns, with the median estimate of 1 in 400,000. NDM can be permanent (PNDM) or transient (TNDM). Furthermore, later in childhood permanent diabetes can occur in individuals with initially TNDM. Genetic mutations causing neonatal diabetes are listed in Table 8.7 [74]. Imprinting abnormalities of genes on chromosome 6 (6q24) are the most commonly recognized cause of TNDM [75]. Besides neonatal diabetes, usually diagnosed in the first week of life, characteristic features of these disorders include severe intrauterine growth retardation, resolution of hyperglycemia by age 18 months (but usually by 12 weeks of age), fluid depletion, and lack of ketoacidosis. Intrauterine growth retardation results from insulin deficiency in utero. In utero, insulin is extremely important for growth. Other findings can include umbilical hernia and macroglossia. Diabetes recurs in approximately 50% of cases during childhood. Initially, the diabetes may be responsive to oral hypoglycemic agents, although insulin is often required with recurrence of the diabetes later in life. Within the 6q24 differentially methylated region of chromosome 6q24, relative hypomethylation leads to increased expression of the PLAGL1 (PLAG1 like zinc finger 1; chromosome 6q24.2) and HYMAI (hydatidiform

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TABLE 8.6 Notable clinical features of the maturity-onset diabetes of youth syndromes. Disorder

Comment

MODY1

Progressive insulinopenia; management with sulfonylureas for decades; complications can occur; possible: decreased apolipoprotein (apo) C2, apo C3, lipoprotein(a), and triglycerides; increased birth weight; possible neonatal hyperinsulinism; B80% penetrance by age 40 [46].

MODY2

Nonprogressive insulinopenia; management without drugs is common; complications are uncommon; decreased birth weight; B50% penetrance by age 40; common cause of MODY [47].

MODY3

Progressive insulinopenia; complications can occur; decreased birth weight; B80% penetrance by age 40; common cause of MODY [48].

MODY4

Homozygosity causes pancreatic agenesis; affected individuals can present with GDM-like or a type 2 diabetes-like phenotype [49].

MODY5

Possible: renal cysts, vaginal/uterine malformations, abnormal liver function, nondiabetic renal disease [50].

MODY6

Low penetrance; can cause neurological abnormalities (e.g., intellectual disability) [51].

MODY7

Rare cause of MODY [52,53].

MODY8

Exocrine pancreatic dysfunction [54,55].

MODY9

Severe diabetic complications [56].

MODY10

Described in a Chinese man presenting with “type 2 diabetes” at age 31 years; INS mutations can cause neonatal diabetes [35].

MODY11

BLK modulates insulin synthesis and secretion [57].

MODY 12

Also a common cause of neonatal diabetes; gene name: ATP-binding cassette, subfamily C, member 8 (ABCC8) [58].

MODY 13

Also a common cause of neonatal diabetes; gene name: potassium channel, inwardly rectifying, subfamily J, member 11 (KCNJ11) [59,60].

mole-associated and imprinted transcript; chromosome 6q24.2) which are imprinted genes. Other genetic mechanisms have also been described (e.g., paternal gene duplication of 6q24 and chromosome 6 paternal uniparental disomy). PLAGL1 (a.k.a.—ZAC; zinc-finger protein that regulates apoptosis and cell cycle arrest) is the “pleomorphic adenoma of the salivary gland gene like 1” and is a tumor suppressor gene. This DNA-binding protein is widely expressed and regulates apoptosis. Mutations in ZFP57, a transcription factor (ZFP57 zinc finger protein; chromosome 6p22.1) needed for normal methylation maintenance during embryonic development, commonly also occur [76].

TABLE 8.7 Monogenic causes of neonatal diabetes. Permanent diabetes (PNDM)

Transient diabetes (TNDM)

a

X

Kir6.2 mutations

X

X

SUR1 mutations

X

X

Chromosomal Imprinting anomalies on chromosome 6q24 KATP channel

Transcription factor HNF-1α (MODY3)

X

PDX1 (IPF1; MODY4)

X

HNF-1β (MODY5)

X

IPEX syndrome

X

PTF1A

X

RFX6

X

GATA4

X

GATA6

X

GLIS3

X

NEUROG3

X

NEUROD1 (MODY6)

X

PAX6

X

NKX2-2

X

MNX1

X

X

Enzymes GCK

X

EIF2AK3

X

Transporters SLC19A2

X

SLC2A2

X

Transmembrane endoplasmic reticulum protein WFS1

Xb

Insulin INS a

X

X

Hypoglycemia is possible in infancy or childhood. Due to the rare autosomal dominant form of Wolfran syndrome, autosomal recessive Wolfran syndrome presents in childhood. b

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Gain-of-function mutations in the β cell inwardly rectified potassium channel (KATP) can cause permanent or transient neonatal diabetes [77,78]. Such mutations (where KATP does not close) inhibit β cell depolarization, causing insulinopenia. Four Kir6.2 subunits constitute the actual potassium channel, whereas four sulfonylurea receptor-1 (SUR1) subunits regulate Kir6.2. These are the most common causes of PNDM. Kir6.2 is encoded by the gene KCNJ11 (potassium inwardly rectifying channel subfamily J member 11; chromosome 11p15.1), and SUR1 is encoded by ABCC8 (ATP-binding cassette subfamily C-branch member 8; chromosome 11p15.1). Developmental delay and epilepsy have been recognized in individuals with severe KCNJ11 mutations. Such findings together with NDM are termed the DEND (developmental delay, epilepsy, neonatal diabetes) syndrome. SUR1 is expressed in pancreatic β cells and neurons, whereas SUR2A is expressed in the heart, and SUR2B is expressed in smooth muscle. Molecular analysis of KCNJ11 and ABCC8 is available commercially in the United States. Both disorders respond to sulfonylureas. This strengthens the case for molecular testing to establish such diagnoses where insulin injections are not the treatment of choice. Various transcription factor mutations can lead to insulinopenia and possibly a disturbance in pancreatic or islet neogenesis in utero. HNF stands for hepatocyte nuclear factor. PDX1 (chromosome 13q12.2) stands for “Pancreatic and Duodenal Homeobox 1,” whereas IPF stands for “insulin promotor factor.” HNF-1α (MODY3; HNF1 homeobox A; chromosome 12q24.31) or HNF-1β (MODY5; HNF1 homeobox B; chromosome 17q12) mutations can cause NDM. Except for HNF1β mutations that can cause transient or permanent diabetes, the other transcription factor mutations cause permanent diabetes [79]. Homozygosity or compound heterozygosity for mutations in PDX1 (IPF1; MODY4) is one cause of pancreatic agenesis [80]. The rare immunodysregulation, polyendocrinopathy, enteropathy, Xlinked (IPEX) syndrome can cause neonatal diabetes [81 83]. The mutation in this syndrome involves the forked-head transcription factor FOXP3 (forkhead box P3; chromosome Xp11.23). FOXP3 expression is a feature of CD4 1 CD25 1 regulatory T cells. This is not a disorder of intrinsic β cell development or insulin gene regulation. IPEX is an autoimmune disease. PTF1A is “pancreas transcription factor 1A” (chromosome 10p12.2). In the rare condition of homozygous PTF1A mutations, PNDM results that can be accompanied by pancreatic and cerebellar agenesis [84]. Another autosomal recessive transcription factor disorder results from mutations in RFX6 (regulatory factor X6; chromosome 6q22.1). RFX6 is necessary for normal β- cell development [85]. Homozygous RFX6 mutations have been reported in Mitchell Riley syndrome, which is characterized by NDM, severe intrauterine growth retardation, agenesis of the gallbladder, cholestasis, intestinal malrotation, annular pancreas, and chronic diarrhea [86]. GATA4 (GATA binding protein 4; chromosome 8p23.1) encodes a zincfinger transcription factor that regulates several genes involved in

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embryogenesis (including pancreatic development). Neonatal and childhoodonset diabetes can result from mutations in GATA4 [87]. Congenital heart defects are frequent in persons with mutations of GATA4. Haploinsufficiency of GATA6 (GATA binding protein 6; chromosome 18q11.2) has caused pancreatic agenesis with neonatal diabetes [88,89]. GLIS3 (GLIS family zinc finger 3; chromosome 9p24.2) codes for a zincfinger protein transcription factor. Mutations can cause neonatal diabetes and congenital hypothyroidism [90]. Discovery of the cause of neonatal diabetes is more than academic; specific therapies are under development in addition to the targeted use of sulfonylureas in MODY1 and MODY3 [91]. NEUROG3 (neurogenin 3; chromosome 10q22.1) codes for a basic helixloop-helix transcription factor that plays a role in neurogenesis. Defects in the expressed protein result in malabsorptive diarrhea from early in life. Neonatal diabetes may or may not be present [92]. NEUROG3 is being investigated in reprogramming cells into β cells [93]. NEUROD1 (neuronal differentiation 1; chromosome 2q31.3) mutations are recognized causes of MODY (i.e., MODY6). A report indicates that at least one NEUROD1 mutation can cause neonatal diabetes with neurological problems [94]. This emphasizes the concept that neonatal diabetes and MODY form a continuum of types of monogenic diabetes [95]. A patient with Down syndrome and neonatal diabetes, hypopituitarism, microphthalmia, and a brain malformation has been reported with compound heterozygous PAX6 mutations [96]. PAX6 (paired box 6; chromosome 11p13) is involved in eye development, as well as, the development of other tissues. NKX2-2 (NK2 homeobox 2; chromosome 20p11.22) is a homeobox domain transcription factor. NKX2-2 mutations cause neonatal diabetes. Three affected children were small for gestation age (consistent with in utero insulin deficiency), displayed normal exocrine pancreatic function, exhibited developmental delay and short stature [97]. This same paper reports reduced birth weight and neonatal diabetes with normal exocrine pancreatic function associated with MNX1 mutations. MNX1 (motor neuron and pancreas homeobox 1; chromosome 7q36.3) encodes a homeobox domain transcription factor. The linkage between the site of transcription factor action and its effect on diabetogenesis and pancreatic exocrine and endocrine organogenesis is displayed in Table 8.8 [98,99]. In the table, the arrows (i.e., “-” and “2”) indicate which cell gives rise to the next cell in development. Note that the tip and trunk progenitors can interconvert. In the table, transcription factors are in bold. In this model the pancreatic endoderm gives rise to the pancreatic progenitors. Tip-trunk progenitors are derived from the pancreatic progenitors. The tip progenitors give rise to acinar cells, whereas the trunk progenitors give rise to duct cells and islet cell precursors that develop into α or β cells. As suggested from studies in mice, there is also a hierarchy of associated transcription factors that regulate, in part, insulin (INS) gene expression (Table 8.9) [100]. In the table, the arrows (i.e., “-”) indicate which

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TABLE 8.8 Genes causing monogenic forms of diabetes (MODY and/or neonatal diabetes) and their role in pancreatic organogenesis. Cell type

Transcription factors expressed that can cause monogenic diabetes

Pancreatic endoderm

PDX1, PTF1A, GATA4, MNX1, RFX6

v v Pancreatic progenitors

PDX1, PTF1A, GATA4, GATA6, HNF1B, GLIS3, MNX1

v v Tip-trunk progenitors Tip

PDX1, PTF1A, GATA4

Acinar cell

PTF1A, GATA4

PDX1, HNF1B

- Duct cell

PDX1, HNF1B

-Islet precursor

NKX2.2

z Trunk

-β cell

PDX1, NKX2.2, RFX6, PAX6, NEUROD1, MNX1, INS

-α cell

NKX2.2, PAX6

transcription factor controls the next transcription factor. In this model, HNF-1α and PDX1 most proximally control insulin (INS) gene expression. Mutations in two enzymes can produce neonatal diabetes: eukaryotic translation initiation factor 2-α kinase 3 (EIF2AK3; EIF2A; chromosome 3q25.1) [101] and homozygous or compound heterozygous loss-of-function mutations in glucokinase (GCK; chromosome 7p13) [102]. Mutations in both EIF2A copies cause Wolcott Rallison syndrome. In addition to NDM, this rare autosomal recessive disorder displays skeletal dysplasia, retarded growth, and liver disease [103]. EIF2AK3 is required for initiation of translation by facilitating binding of the initiating methronyl transfer tRNA fMet to the 30S ribosomal subunit. GCK serves as the glucose sensor of the β cell [104,105]. Loss-of-function mutations in GCK cause mildly decreased β cell

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TABLE 8.9 Hierarchy of selected transcription factors that regulate insulin (INS) gene expression. HNF-1β (MODY5) v v HNF-4α (MODY1) v v HNF-1α

-

PDX1

(MODY3)

(MODY4)

v

v

v

v

INS gene

INS gene

detection of glucose and subsequent insulinopenia [106]. Women with GCK mutations may exhibit GDM. When both GCK genes are defective, PNDM is the outcome [107]. GCK gene analysis is available commercially. Novel insulin gene mutations that cause insulin misfolding are detected by the unfolded protein response triggering β cell apoptosis [108]. Because the “abnormal” insulin is not secreted, it is not detected in assays of circulating insulin as are the insulinopathies and the hyperproinsulinemias (see below). The Akita mouse serves as an animal model for this pathogenic mechanism [109]. Defective β cell development may also be a cause (if not the main cause) of “β-cell failure” [110]. Defects in GLUT2 (encoded by SLC2A2, solute carrier family 2 member 2; chromosome 3q26.2) cause the Fanconi Bickel syndrome (FBS) [111]. FBS is manifested as fasting hypoglycemia (because of inadequate release of glucose from hepatocytes) and postprandial hyperglycemia (because of inadequate uptake of glucose by β cells causing insulinopenia and, possibly, reduced hepatic uptake of glucose following a meal) and transient neonatal diabetes [112]. Glycogen accumulated in the renal tubules causes proximal renal tubular dysfunction (i.e., Fanconi syndrome of the tubules) [113]. Glycogen accumulation in the intestinal wall causes epithelial dysfunction potentially manifested as malabsorption and diarrhea. SLC19A2 (solute carrier family 19 member 2; chromosome 1q24.2) encodes the thiamin transporter protein. Autosomal recessive defects in

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SLC19A2 cause thiamin-responsive megaloblastic anemia, sensory-neural hearing loss, and diabetes of possible neonatal onset [114,115]. Thiamin plays a role in several aspects of glucose and intermediary metabolism [86]. The literature does not clearly define why thiamin inborn errors cause diabetes; however, it may relate, in part, to the effect of thiamine pyrophosphate in activating pyruvate decarboxylation via the pyruvate dehydrogenase complex (pyruvate dehydrogenase) [116]. Wolfram syndrome (WFS; a.k.a.—DIDMOAD: diabetes insipidus; diabetes mellitus, optic atrophy, deafness) results from mutations in WFS1 (Wolframin ER transmembrane glycoprotein; chromosome 4p16.1) [117]. WFS1 helps maintain the endoplasmic reticulum and collaborates in cellular calcium regulation [118]. Diabetes in autosomal recessive WFS1 does not occur in infancy but an autosomal dominant WFS1 form of WFS causes permanent neonatal diabetes [119]. A variant form of WFS (i.e., WFS2) is caused by CISD2 [cysteine (C), aspartic acid (D), glycine (G), serine/alanine/threonine (S/A/T), histidine (H) (CDGSH); iron-sulfur domain 2; chromosome 4q24] mutations. CDGSH also localizes to the ER. Similarly, CDGSH attaches to an iron/sulfur cluster and, like WFS1, may affect calcium homeostasis. WFS due to WFS2 mutations causes diabetes in childhood but not in neonates [120]. Mitochondrial diabetes Mitochondrial mutations (e.g., predominantly the A3243G mutation in tRNALeu(UUR)) have been associated with diabetes and deafness (maternally inherited diabetes and deafness, MIDD) as well as more complex syndromes such as MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like syndrome) and MERRF (myoclonic epilepsy and ragged-red fibers) (Table 8.10) [124,125]. Many mitochondrial diseases are inherited from the mother. The conceptus’ mitochondria are provided by the ovum and not the sperm [126]. However, most mitochondrial proteins are encoded by nuclear DNA genes. Because the mitochondrion is the powerhouse of the cell, mitochondrial disease can affect the nervous system, cardiac or skeletal muscle, and the endocrine system (e.g., tissues that all have high energy demands). Molecular testing is available for mitochondrial mutations. MIDD results from mutations in MT-TL1 and, rarely, MT-TK or MT-TE [127]. MT-TL1 [mitochondrially encoded TRNA-Leu (UUA/G) 1] encodes the tRNA for leucine (UUA/G). This gene is affected by the A3243G mutation. MT-TK [mitochondrially encoded TRNA-Lys (AAA/G)] codes for the tRNA for lysine (AAA/G). MT-TE [mitochondrially encoded TRNA-Glu (GAA/G)] encodes the tRNA for glutamic acid (GAA/G). These mutations impair the addition of amino acids to proteins undergoing synthesis because of the tRNA mutations.

TABLE 8.10 Mitochondrial disorders with possible diabetes. Disorder

Abbreviation

mtDNA/ nDNA

Involved gene/mutation

Comment

Chronic progressive external ophthalmoplegia with myopathy

CPEO 1

mtDNA (or) nDNA

Deletions (or) mutations

Deletions: Sporadic or maternal inheritance Mutations in: POLG or RRM2B PLOG: polymerase gamma (AD) [121] RRM2B: ribonucleotide reductase regulatory TP53 inducible subunit M2B (autosome)

Kearns Sayre syndrome

KSS

mtDNA

Deletion

Usually sporadic

Leber hereditary optic neuropathy

LHON

mtDNA

Various mutations

Maternal inheritance [122]

Maternally inherited diabetes and deafness

MIDD

mtDNA

MT-TL1 (A3243G)

Most common cause of mitochondrial diabetes; maternal inheritance; rare mutations: MT-TK A8296G and MT-TE T14709C

Mitochondrial neurogastrointestinal encephalopathy

MNGIE

nDNA

TYMP

Autosomal recessive inheritance; thymidine phosphorylase; an angiogenic factor [123]

Myopathy, encephalopathy, lactic acidosis, and stroke-like syndrome

MELAS

mtDNA

MT-TL1

Maternal inheritance; MT-TLI: most common gene involved; other mutation: T3271C

Myoclonic epilepsy and ragged-red fibers

MERRF

mtDNA

MT-TK

Maternal inheritance

or

(A8344G)

nDNA

PLOG

AR inheritance

mtDNA

Deletion

Usually sporadic

Pearson syndrome



AD, autosomal dominant; AR, autosomal recessive; mtDNA, mitochondrial DNA; nDNA, nuclear DNA.

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Insulinopathies and hyperproinsulinopathies Insulinopathies (missense mutations in insulin) and hyperproinsulinopathies (e.g., incomplete cleavage of proinsulin to insulin and C-peptide) are rare causes of diabetes. At least six such INS mutations have been described [128]. These mutations are different from the INS mutations that cause neonatal diabetes (see earlier in this chapter). These disorders are inherited as autosomal dominant traits. No homozygous mutations have been described. Individuals with these mutations may develop clinical diabetes only when other risk factors for diabetes are present (e.g., obesity with insulin resistance). At least three insulinopathies have been described [129]: G G G

A chain amino acid 3, valine-leucine, insulin Tokyo/Wakayama B chain amino acid 24, phenylalanine-serine, insulin Los Angeles B chain amino acid 25, phenylalanine-leucine, insulin Chicago

Other specific types of diabetes: considerations The β cell defects discussed above can be conceptually linked by the finding that these defects variously interfere with β cell development, the production of insulin (and insulin that is functionally normal), glucose sensing, conversion of glucose sensing to increased ATP, or closure of the β cell potassium channel. Fig. 8.1 depicts the β cell in its basal state when exposed to low levels of interstitial glucose. β cell development and/or INS gene expression requires a plethora of transcription factors [HNF-1α (MODY3), PDX1 (IPF1; MODY4), HNF-1β (MODY5), PTF1A, RFX6, GATA4, GATA6, GLIS3, NEUROG3, NEUROD1 (MODY6), PAX6, NKX2-2, and MNX1] many of which act in hierarchies. Normal protein synthesis must also be preserved (e.g., EIF2AK3 defects). Fig. 8.2 depicts the β cell after exposure to rising concentrations of interstitial glucose. In this latter case, glucose enters the β cell via GLUT2 (defective in the Fanconi Bickel syndrome) and is converted to glucose6-phosphate (G-6-P) via glucokinase (GCK, MODY2). Entry of the pyruvate into the mitochondrion leads to increased ATP generation (mitochondrial mutations impair ATP generation; as well, SLC19A2 may play a role in the PDH complex). ATP binds to SUR1 closing the KATP (defective in SUR1 or Kir6.2 mutations). In turn, the β cell depolarizes and the calcium channel opens (which may be related to WFS1 mutations). Entry of calcium into the cytoplasm of the β cell triggers the fusion of insulincontaining granules with the plasma membrane affording insulin release into the interstitium to be absorbed into the circulation for distribution. If the insulin triggers an unfolded protein response, the β cell is injured or dies. On the other hand, insulinopathies and hyperproinsulinemias can impair normal insulin action.

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Transcription factors GLUT2

Glucose

INSgene

GCK

G-6-P

Glucose ATP

mRNA

ADP Pyruvate

Preproinsulin ER Golgi

Mitochondrion

ATP Kir6.2

KATP

Insulin-containing granules

SUR1

Ca++ channel

K+

Ca ++

FIGURE 8.1 Glucose enters the beta cell via the facilitative glucose transporter GLUT2. Glucose is converted to glucose-6-phosphate (G-6-P) by glucokinase (GCK). The rate of production of G-6-P is dependent on the extracellular glucose concentration because GCK is a high Km hexokinase. As glycolysis proceeds, pyruvate is formed that enters the mitochondrion once it is converted to acetyl-CoA (not shown). Within the mitochondrion, the Krebs citric acid cycle commences with ATP produced via oxidative phosphorylation (not shown). In the setting of a low ATP to ADP ratio, the beta-cell potassium channel (KATP) remains closed. Normally ATP regulates the activity of the four sulfonylurea receptor-1 (SUR1) subunits that regulate the innermost portion of KATP, the four Kir6.2 subunits. In absence of beta-cell stimulation, the calcium channel remains closed. The insulin (INS) gene and beta-cell development are regulated by a variety of transcription factors. Transcription yields a preproinsulin mRNA whose translation in the cytoplasm produces preproinsulin. Upon entry of preproinsulin into the endoplasmic reticulum (ER), the presequence is cleaved producing proinsulin. Processing of proinsulin in the Golgi apparatus yields insulin plus C-peptide. Insulin-containing granules bud from the Golgi apparatus and are stored in the beta cell until the beta cell is stimulated by glucose, amino acids, fatty acids, or incretins to degranulate.

Genetic defects in insulin action Genetic defects in insulin action can result from insulin receptor mutations [130]. Many insulin receptor mutations have been described that involve the α- or β-chains. However, these mutations are rare in the general population of patients with type 2 diabetes. Conditions with insulin receptor mutations include type A insulin resistance [131], Rabson Mendenhall syndrome [132], and leprechaunism (Donohue syndrome) [133]. Patients with lipoatrophic diabetes are highly insulin-resistant. The mechanism appears to be leptin deficiency [134]. Lipodystrophy can be genetic,

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GLUT2

Glucose

GCK

G-6-P

Glucose ATP

ADP

Pyruvate Mitochondrion

ATP KATP

Insulin-containing granules

K+

SUR1 Ca++ channel Kir6.2 Ca++

FIGURE 8.2 The image depicts the basic mechanics of how beta cells monitor glucose [GCK, the generation of glucose-6-phosphate (G-6-P) and ATP production from glycolysis and oxidative phosphorylation in the mitochondrion], transmission of this “glucose detection signal” into closing the KATP channel, beta-cell depolarization, opening of the calcium channel, and exocytosis of insulin from the beta cell as insulin-containing granules fuse with the plasma membrane.

or more commonly, acquired (e.g., a consequence of highly active antiretroviral therapy for HIV infection [135]). The familial lipodystrophies display autosomal dominant (“AD” in the Table 8.11) or autosomal recessive (“AR” in Table 8.11) inheritance. There are several excellent reviews that the reader is referred to [136,137]. Table 8.11 provides a summary of genetic lipodystrophies [138]. The table points out that a single clinical phenotype can be caused by mutations in a variety of genes (e.g., congenital generalized lipodystrophy). As well, different mutations in the same gene can produce a variety of clinical phenotypes (e.g., lamin A/C mutations can cause familial partial lipodystrophy, Hutchinson Gilford progeria syndrome or the atypical progeroid syndrome). This later observation should not be unexpected as mutations in dystrophin produce more than one type of disease (e.g., Duchenne muscular dystrophy or Becker muscular dystrophy) as do mutations in the fibroblast growth factor receptor 3 (FGFR3) [causing either achondroplasia, hypochondroplasia, severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN), or thanatophoric dysplasia]. Diseases of the exocrine pancreas The most common diseases of the exocrine pancreas to produce diabetes are cystic fibrosis [139] and chronic pancreatitis. With increasing longevity in

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TABLE 8.11 Genetic causes of lipodystrophy. Gene

Full name

Manifestation (inheritance)

LMNA

Lamin A/C

Familial partial lipodystrophy (AD)

PPARG

Peroxisome proliferatoractivated receptor gamma

Familial partial lipodystrophy (AD)

AKT2

AKT serine/threonine kinase 2

Familial partial lipodystrophy (AD)

PLIN1

Perilipin 1

Familial partial lipodystrophy (AD)

LMNA

Lamin A/C

Hutchinson Gilford progeria syndrome (AD)

LMNA

Lamin A/C

Atypical progeroid syndrome (AD)

FBN1

Fibrillin 1

Neonatal progeroid syndrome (AD)

CAV1

Caveolin 1

Neonatal progeroid syndrome (AD)

POLD1

DNA polymerase delta 1, catalytic subunit

Mandibular hypoplasia, deafness, progeroid features (MDP), syndrome (AD)

PIK3R1

Phosphoinositide-3-kinase regulatory subunit 1

SHORT syndrome associated with lipodystrophy (AD)

KCNJ6

Potassium voltage-gated channel Subfamily J Member 6

Keppen Lubinsky syndrome associated with lipodystrophy (AD)

AGPAT2

1-Acylglycerol-3phosphate Oacyltransferase 2

Berardinelli Seip syndrome (congenital generalized lipodystrophy) (AR)

BSCL2

BSCL2 lipid droplet biogenesis associated, seipin

Berardinelli Seip syndrome (congenital generalized lipodystrophy) (AR)

CAV1

Caveolin 1

Berardinelli Seip syndrome (congenital generalized lipodystrophy) (AR)

CAVIN1

Caveolae-associated protein 1

Berardinelli Seip syndrome (congenital generalized lipodystrophy) (AR)

LMNA

Lamin A/C

Mandibuloacral dysplasia (AR)

ZMPSTE24

Zinc metallopeptidase STE24

Mandibuloacral dysplasia (AR)

CIDEC

Cell death-inducing DFFAlike effector C

Familial partial lipodystrophy (AR)

Abbreviation

(Continued )

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TABLE 8.11 (Continued) Gene

Full name

Manifestation (inheritance)

Lipase E, hormonesensitive type

Familial partial lipodystrophy (AR)

WRN

WRN RecQ like helicase

Familial partial lipodystrophy (AR)

PCYT1A

Phosphate cytidylyltransferase 1, choline, alpha

Familial partial lipodystrophy (AR)

PSMB8

Proteasome subunit beta 8

Autoinflammatory lipodystrophy (AR)

PPARG

Peroxisome proliferatoractivated receptor gamma

Congenital generalized lipodystrophy-like phenotype (AR)

FOS

Fos proto-oncogene, AP-1 transcription factor subunit

Congenital generalized lipodystrophy-like phenotype (AR)

Abbreviation LIPE

AD, autosomal dominant; AR, autosomal recessive.

cystic fibrosis patients due to better pulmonary management, diabetes is becoming more common. Other pancreatic disorders causing diabetes include trauma, pancreatectomy, neoplasia, hemochromatosis, and fibrocalculous pancreatopathy [140]. The development of diabetes can precede the clinical presentation of pancreatic cancer by several years. How pancreatic cancer causes diabetes is unknown. A paraneoplastic mechanism has been proposed by some experts. Endocrinopathies causing diabetes Numerous endocrinopathies cause diabetes including Cushing syndrome, acromegaly (growth hormone excess), pheochromocytoma, glucagonoma, hyperthyroidism, somatostatinoma, and aldosteronoma. These conditions are discussed in other chapters. Drug-induced diabetes Chronic use of various drugs such as glucocorticoids, growth hormone, catecholamines, high-dose nicotinic acid, excess thyroid hormone, diazoxide, statins, and atypical antipsychotics (e.g., olanzapine, clozapine, risperidone, quetiapine, ziprasidone, and aripiprazole) can lead to diabetes [141]. Diabetes secondary to pentamidine has been observed in acquired immunodeficiency syndrome patients who have been treated for Pneumocystis pneumonia [142]. In such cases, pentamidine caused apparent β-cell necrosis. The

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rat poison Vacor has caused diabetes [143]. Reverse transcriptase inhibitors and protease inhibitors used in the treatment of human immunodeficiency virus infection are associated with lipodystrophy and diabetes [144]. Infection and diabetes Through their effects on increasing the concentrations of the anti-insulin hormones, infection may produce a diabetic state; however, this state usually remits with resolution of the infection. β-cell destruction and type 1B diabetes can result from various viruses as noted above. Uncommon forms of immune-mediated diabetes Uncommon forms of immune-mediated diabetes include anti-insulin receptor autoantibodies that can produce profound insulin resistance (e.g., type B insulin resistance) [145]. Although autoimmune hypoglycemia from antiinsulin autoantibodies is best known for its hypoglycemic symptoms, glucose intolerance can also be observed in this rare autoimmune condition. Stiffperson syndrome produces an autoimmune insulinopenic form of diabetes associated with high-titer GADAs and neuromuscular stiffness [146]. Genetic syndromes and diabetes Many genetic syndromes are associated with diabetes such as Down syndrome, Klinefelter syndrome, Turner syndrome, Wolfram syndrome (see above), Cockayne syndrome, Werner syndrome, McCune Albright syndrome, porphyria, Bardet Biedl syndrome (a.k.a.—Lawrence Moon Biedl syndrome), Prader Willi syndrome, Friedreich ataxia, Huntington chorea, and myotonic dystrophy [147]. Gestational diabetes mellitus According to the American Diabetes Association (ADA), GDM is “diabetes diagnosed in the second or third trimester of pregnancy that was not clearly overt diabetes prior to gestation” [148]. GDM usually remits following delivery; however, women with GDM are at increased risk of developing type 2 diabetes later in life. The risk for eventually developing type 2 diabetes can approach 25% 50%. There are serious adverse fetal health consequences when maternal diabetes is unrecognized or is untreated [149]. These complications include macrosomia (large size) that can lead to difficult delivery (e.g., dystocia), often requiring cesarean section, and neonatal hypoglycemia or hypocalcemia immediately following birth. Infants born to diabetic mothers are at increased risk for respiratory distress syndrome and hyperbilirubinemia than age-matched infants. Infants born to mothers with preexisting diabetes (e.g., type 1 diabetes) are at an increased threefold risk for congenital malformations. GDM produces an increased risk of caudal regression syndrome (e.g.,

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SECTION | 2 Analytes

reduced development of the lower half of the body) in the infant of the diabetic mother. Optimal preconception glycemic control of maternal diabetes is necessary to avoid such increased risks for birth defects.

Diagnosis of diabetes Nonpregnant adults and children Diabetes is diagnosed when a patient presents with (1) classic symptoms of diabetes (e.g., polyuria, polydipsia, blurry vision, weight loss, etc.) and hyperglycemia, or (2) hyperglycemic crisis (e.g., DKA or HHS). If these conditions are not met, diabetes is diagnosed when (1) hyperglycemia is detected on two different days (preferably using the same type of measurement), or (2) hyperglycemia is detected by two different metrics on the same day [an elevated fasting PG (FPG) plus elevated 2-h PG as part of an OGTT; elevated FPG plus elevated hemoglobin A1c (HbA1c); or elevated 2-h PG as part of an OGTT plus an elevated HbA1c]. Excluding acutely ill patients who present with diabetic symptoms and a PG of 200 mg/dL or greater, or those patients that present in hyperglycemic crisis, acute illness, or hospitalization within the previous 6 8 weeks are relative contraindications to testing for diabetes because severe acute illness can cause transient hyperglycemia. As well, patients recovering from surgery, especially intra-abdominal surgery, may be poorly nourished and may be glucose intolerant because of inadequate caloric intake. There are three criteria for hyperglycemia in the diagnosis of diabetes: (1) elevated FPG, (2) elevated 2-hour (2-h) PG during an oral glucose tolerance test (OGTT), and (3) elevated HbA1c. Those cutpoints are listed in Table 8.12. Note that Table 8.2 refers to “normal” and not reference intervals. This is because these cutpoints were defined relative to the risk of developing microvascular diabetic complications. Therefore, a statistically-defined reference interval (i.e., the mean 6 2 standard deviations) is not relevant. Also note that there are gray zones between “normal” and “hyperglycemia.” These gray zones define the state of prediabetes which indicates an increased risk for the development of type 2 diabetes. When the FPG is between 100 and 125 mg/dL, a state of impaired fasting glucose exists. During an OGTT when

TABLE 8.12 Plasma glucose (PG; mg/dL) and HbA1c (%) cutpoints. Normal

Hyperglycemia

Fasting PG

70 99

$ 126

2-h PG, OGTT

,140

$ 200

HbA1c

,5.7

$ 6.5

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the 2-h value is between 140 and 199 mg/dL, a state of impaired glucose tolerance exists. HbA1c levels between 5.7% and 6.4% are also intermediate. Prediabetes is not a disease or a diagnosis unto itself. Prediabetes is a descriptive term. If the FPG was initially measured and was less than 126 mg/dL, and if diabetes was a serious clinical consideration, an OGTT should next be performed or the HbA1c should be measured. The 2-h PG measurement during an OGTT is the most sensitive assessment in search of hyperglycemia; however, OGTTs are cumbersome for the patient. The FPG is not as sensitive a test for diabetes as the 2-h PG during the OGTT. While not as sensitive as either the FPG or 2-h PG during the OGTT, the measurement of the HbA1c is the most convenient test because patients do not need to be fasting.

Choice of the blood specimen for measuring glucose Glucose concentrations can be measured in [1] plasma, [2] serum, [3] whole blood (obtained by venipuncture), or [4] capillary samples (whole blood obtained by lanceting a finger, or with some systems, the forearm). The preferred sample is plasma collected in a gray-top tube (see below). Plasma and serum glucose levels are essentially identical. Whole blood and capillary glucose concentrations are lower than plasma or serum by B10% 15% because whole blood contains cells and cells contain more solids than plasma and cells are metabolizing glucose. Manufacturers of point-of-care devices that measure capillary glucose concentrations and report PG concentrations assume that whole blood glucose concentrations are lower than PG concentrations by B10% 15% because of the differences between whole and plasma described above. At the extremes of hematocrit (hematocrit ,40% and .50%), the relationship between the whole blood glucose concentration and the PG concentration is perturbed. As the hematocrit declines (especially ,15% 20%), whole blood glucose concentrations progressively rise toward the PG concentration as more and more of the whole blood volume is plasma. On the other hand as the hematocrit rises (especially $ 60%), whole blood glucose concentrations decline to a greater extent, relative to PG, because less and less of the whole blood volume is plasma. Therefore if absolute accuracy of the circulating glucose concentration is sought, neither whole blood nor capillary samples are satisfactory. Other preanalytical considerations are the potential differences in the glucose concentrations between arterial samples and venous samples depending upon whether the patient is fasting or the patient is prandial (e.g., eating and absorbing glucose from the intestine). In the fasting state, arterial samples are 3 5 mg/dL higher than venous samples. With good capillary blood flow (e.g., an “arterialized” capillary sample where the digit has been modestly warmed prior to being punctured so that the rate of capillary flow is high

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SECTION | 2 Analytes

and is reflective of arterial blood), the capillary glucose concentration is essentially equivalent to the arterial glucose concentration. With eating, the arterial glucose concentration can exceed the venous glucose concentration by B20 mg/dL. With eating the higher arterial glucose concentration reflects the absorption of glucose from the gut, whereas glucose then diffuses out of the circulation into the interstitium with glucose being cleared from the interstitium by the tissues (especially the muscles, adipose tissue, and the liver) displayed as a lower venous glucose concentration.

Choice of tube type for phlebotomy Unless the blood specimen is rapidly analyzed (e.g., the plasma is separated from the cells in ,60 min, or the chemical analysis is performed in ,60 min), sodium fluoride (NaF)-containing gray-top tubes should be used (10 30 mg/5 mL whole blood). NaF poisons glycolysis, after the first hour, inhibiting the usually observed B2% 3%/h fall in serum glucose that results from red blood cell metabolism of glucose. Typically, gray-top tubes also contain potassium oxalate as an anticoagulant to produce plasma, although NaF is also a weak anticoagulant. A partially anticoagulated sample is technically undesirable because fibrin clots commonly obstruct instrumentation pipettes, pumps, ports, and/or tubing.

The oral glucose tolerance test In children and nonpregnant adults, the standard OGTT involves fasting overnight followed by a PG measurement (zero time), administration of the glucose challenge, ending with the measurement of the PG 2 h ( 6 10 min) after the patient began to drink the glucose beverage. For fasting samples (with or without being part of the OGTT), patients should be nil per os (nothing my mouth) (other than water) overnight for at least 8 h but not longer than 16 h. As well, patients must not eat, smoke, vap, or take medications prior to or during the OGTT. Therefore the OGTT in nonpregnant adults and children includes two points: an FPG sample and a 2-h PG sample. The exception to this “2 1 2 OGTT rule” (two samples-2 h) is OGTT testing during pregnancy, as will be described below. Similar to FPG determinations, preanalytic considerations are important in OGTT testing: a regular diet of B100 150 g carbohydrate/day for at least 3 days prior to testing, no acute severe illnesses, no recent hospitalizations, and unrestricted activity prior to testing are necessary. An afebrile upper respiratory tract infection is not a contraindication to testing; however, gastroenteritis with vomiting and/or diarrhea [e.g., the common “flu” (not influenza)] would be a contraindication. Excluding pregnancy, where 100 g of glucose is administered, adults receive 75 g of anhydrous glucose dissolved in a flavored drink (e.g.,

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“Glucola”). In children, 1.75 g/kg glucose is administered to a maximum dose of 75 g. To reduce the likelihood of nausea, the drink can be chilled and sipped over the entire 5 min. In the drink, the maximum glucose concentration is 25 g/dL. Laboratories may consider delaying or canceling an OGTT when the subject’s glucose measured using a point-of-care testing (POCT) device is $ 165 mg/dL. This suggestion is arrived at as follows: If the cutpoint for hyperglycemia is 126 mg/dL, and the POCT device has B20% negative bias, the POCT device reading of 151 mg/dL actually represents 126 mg/dL. To be sure that there is not an underestimate of the true glucose (leading to an inadvertent cancellation of the OGTT), this 151 mg/dL cutoff can be raised by 10%, giving a POCT cutoff of B165 mg/dL.

Measuring insulin or C-peptide Although measurements of insulin, C-peptide, and/or proinsulin are critical to many metabolic research studies of diabetes, none of these measurements are used routinely for diagnostic purposes to determine whether or not a patient has diabetes. In special circumstances, the clinician may want to measure insulin (in a nontreated patient) or C-peptide (in an insulin-treated patient) to assess β cell function that might help differentiate, for example, type 1 diabetes from type 2 diabetes (type 1, low β cell function; type 2, elevated insulin or C-peptide). A standardized mixed liquid meal [Boost; Socie´te´ des Produits Nestle´ S.A., Vevey, Switzerland] can be used as a C-peptide stimulant. In the Diabetes Control and Complications Trial (DCCT), Sustacal (an earlier name for Boost) was administered orally at a dose of 6 mL/kg to a maximum dose of 360 mL. Blood for C-peptide is usually drawn at 30-min intervals after baseline for 90 120 min. Alternatively, glucagon can be given as a β cell stimulant at the dose of 1 mg given intravenously. Although a number of studies demonstrate that C-peptide measurements can differentiate with good (but not excellent) accuracy type 1 and 2 diabetes, routine testing is not recommended [150 153]. The interpretation of C-peptide or insulin levels must be tempered by many factors such as hyperglycemic β cell toxicity. In an acutely hyperglycemic patient, elevated blood glucose can temporarily suppress β cell function [154]. With correction of hyperglycemia through administration of exogenous insulin, within 50 h, insulin secretion can recover. Once exogenous insulin is administered for several days, the development of insulin antibodies will interfere with the measurement of endogenous insulin, and in such circumstances, C-peptide must be measured. Normally, less than 5% of secreted insulin has not been cleaved and is released as proinsulin. However, with β cell failure of any cause, the proportion of insulin released as proinsulin rises. Concerning insulin measurements, one major challenge is a lack of comparability among current insulin immunoassays.

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Testing for gestational diabetes mellitus If diabetes is diagnosed at the first prenatal visit in the first trimester using the diabetes criteria previously discussed, the term “GDM” is not applied. Recognizing the increasing prevalence of obesity throughout all ages, asymptomatic type 2 diabetes can be present in young women. GDM is diagnosed; therefore only in the second or third trimesters. A single set of diagnostic criteria for GDM is lacking. Physicians can elect to initially screen pregnant women at 24 and 28 weeks gestation with a nonfasting 50-g, 1-h OGTT or proceed directly to a 75 g, 2-h pregnancy OGTT (Table 8.13) (note: this is a different test than the 75 g, 2-h OGTT performed in children and nonpregnant adults where there are only two time points and different cutpoints). For the 75 g, 2-h pregnancy OGTTs, GDM is diagnosed when one or more cutpoints are met or exceeded. The 50 g, 1-h OGTT consists of a 50-g glucose load with measurement of a 1-h postload PG. The patient need not be fasting. Concerning this test, if the 1h PG level meets or exceeds 140 mg/dL, a 100 g, 3-h OGTT is then performed. The sensitivity of the nonfasting 1-h, 50-g test can be increased by reducing the cutoff to 130 mg/dL or 135 mg/dL. The cutpoints for the 100 g, 3-h pregnancy OGTT are given in Table 8.14. For the 100 g, 3-h OGTT, GDM is

TABLE 8.13 Cutpoint values defining hyperglycemia during a 75-g, 2-h pregnancy OGTT. Time

Cutpoint (mg/dL)

0

92

1

180

2

153

TABLE 8.14 Cutpoint values defining hyperglycemia during 100-g, 3-h pregnancy OGTTs. Time

Cutpoint (mg/dL) Carpenter/Coustan

NDDG

0

95

105

1

180

190

2

155

165

3

140

145

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diagnosed when two or more cutpoints are met or exceeded. For the 100 g, 3-h pregnancy OGTT, in the past either Carpenter/Coustan or National Diabetes Data Group (NDDG) criteria could be used. The most recent year 2000 guidelines from the American Diabetes Association declare that the Carpenter/ Coustan criteria for GDM should be used because more women will be diagnosed with GDM. The ADA also recommends that women with GDM undergo a 75-g, 2-h OGTT at 4 12 weeks postpartum in search of glucose intolerance or frank diabetes. Thereafter every 3 years, women with a history of GDM should be screened for diabetes.

Screening strategies for diabetes mellitus Adults The ADA recommends that all nondiabetic adults ages 45 and older be screened for prediabetes and type 2 diabetes via an FPG, 75 g, 2-h OGTT, or HbA1c measurement every 3 years. This is because the frequency of diabetes rises sixfold between ages 40 and 45. Furthermore, many type 2 diabetes patients already have complications at the time of clinical diagnosis (e.g., between 10% and 15% of newly diagnosed patients with type 2 diabetes have microvascular disease). This indicates that by otherwise waiting for the blatant clinical presentation of type 2 diabetes (e.g., polyuria, polydipsia, etc.), the biochemical diagnosis of type 2 diabetes is significantly delayed. Therefore biochemical screening is warranted. Subjects with prediabetes should be screened yearly. Indications to test for diabetes before age 45 are outlined in Table 8.15.

TABLE 8.15 Indications to consider diabetes screening before age 45 in persons with an elevated body mass index ($25 kg/m2 or $ 23 kg/m2 in Asian Americans). First-degree relative to diabetes High-risk ethnicity (e.g., African American, Hispanic American, Native American, Asian American, Pacific Islander) History of cardiovascular disease Hypertension ($140/90 mmHg or on antihypertensive therapy for hypertension) HDL cholesterol level ,35 mg/dL and/or triglycerides .250 mg/dL Women with polycystic ovary syndrome Physical inactivity Other clinical conditions associated with insulin resistance (e.g., severe obesity, acanthosis nigricans)

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Children Children with a body mass index at or above the 85th percentile for age and sex should be screened for type 2 diabetes in certain circumstances through the same methods as described for adults. Screening would begin at age or 10 years or when the child has entered into puberty (whichever occurs first). Indications to test such children for diabetes are outlined in Table 8.16.

Biochemical monitoring in diabetes The goal of therapy in caring for individuals with diabetes is: (1) elimination of symptoms of hyper or hypoglycemia; and (2) control of the PG, lipids, blood pressure, and avoidance of microalbuminuria to delay or prevent diabetic complications. In children with diabetes, a further goal of diabetes care is to ensure normal growth and development. Routine laboratory assessments are vital to the monitoring of diabetes in optimizing clinical management. These assessments fall into four major categories: (1) self-monitoring of blood glucose (SMBG), (2) long-term assessment of diabetic glycemic control, (3) evaluation of lipid status, and (4) renal evaluation.

Self-monitoring of blood glucose and point-of-care testing SMBG is the cornerstone of daily diabetes management for insulin-treated patients [155 158]. Recent developments include continuous subcutaneous glucose measurements (CGM) and closed-loop insulin delivery systems [159]. It is controversial whether SMBG improves outcome in noninsulintreated patients [160]. “Time in range” (between 70 and 180 mg/dL) is a recent goal based on the availability of CGM glucose results. With SMBG, the patient can measure their capillary glucose any time of the day or night to make informed choices about diet, exercise, and insulin or other diabetic medication administration. In insulin-treated patients,

TABLE 8.16 Indications to consider diabetes screening in children Maternal history of diabetes or GDM while in utero First- or second-degree relative with type 2 diabetes High-risk ethnicity (e.g., African American, Hispanic American, Native American, Asian American, Pacific Islander) Signs of insulin resistance or insulin-resistant associated conditions (small-forgestational-age birth weight, hypertension, dyslipidemia, acanthosis nigricans, or polycystic ovarian syndrome)

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SMBG should be performed at least 3 4 times per day and more often as required. CGM can provide interstitial glucose measurements reported up to every 5 min. Treatment algorithms can assist in making informed changes in insulin dosing. The reader is referred to the second edition of this handbook for a technical discussion of SMBG and glucose measurements [161]. Regarding glycemic control in ICUs and in critically ill patients [162], the benefits of tight glycemic control shown in early studies [163] have not been borne out in later studies [164]. Nevertheless, severe hyperglycemia is to be avoided in the patient setting. Hyperglycemia can increase the prevalence of dehydration and impair innate immunologic responses to infections. Phagocyte function can be impaired when the PG is B250 mg/dL or greater.

Assessment of diabetic control The ADA-recommended glycemic goals are summarized in Table 8.17 [165]. Such goals should be personalized and therefore may be more or less stringent. If HbA1C goals are not achieved despite reaching the premeal capillary PG target, postmeal PG can be assessed. Circumstances where lower HbA1c goals might be sought include people with shorter durations of diabetes, noninsulin-treated type 2 diabetes, persons with expected longevity, and nonsignificant cardiovascular disease. Circumstances where higher HbA1c goals (e.g., $ 8%) might be tolerated include a history of severe hypoglycemia, reduced life expectancy, advanced vascular complications (either micro or macrovascular disease), significant comorbidities, and failure to achieve lower HbA1c targets despite good-faith attempts. Glycated hemoglobin results from the nonenzymatic addition of glucose to the hemoglobin molecules inside red blood cells. Biochemically, glucose first forms a reversible (labile) product with amine groups of hemoglobin forming an aldimine (Schiff base). A nonreversible Amadori rearrangement then occurs to form a ketoamine. Since the average red blood cell life span is 120 days, glycated hemoglobin measurements represent the mean PG of

TABLE 8.17 American Diabetes Association-recommended goals for persons with diabetes. Parameter

Goal

Premeal capillary PG

80 130 mg/dL

1 2-h postmeal capillary PG

,180 mg/dL

A1C

,7.0

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the previous 2 3 months. Therefore the glycated hemoglobin concentration is proportional to the elevation and duration of elevation in the PG level. HbA1c levels do not reflect wide fluctuations in PG levels—only mean PG levels. Indeed the correlation between HbA1c and average glucose is linear but displays wide interindividual variability. Glycated hemoglobin is not one molecular species. Glycated hemoglobin [a.k.a.—hemoglobin A1 (HbA1)] includes three forms of hemoglobin A: hemoglobin A1a (HbA1a), hemoglobin A1b (HbA1b), and HbA1c. Hemoglobin A1a is composed of two subfractions: HbA1a1 (fructose 1,6diphosphate bound to the N-terminal valine of the β-chain) and HbA1a2 (glucose-6-phosphate bound to the β-chain). HbA1b represents the addition of pyruvic acid to the β-chain N-terminal valine. In HbA1c, glucose is added to the N-terminal β-chain valine. On average, HbA1c represents B75% 80% of glycated hemoglobin. Total glycated hemoglobin and HbA1c are expressed as a percentage of the total hemoglobin (hemoglobin A0 plus glycated hemoglobin) where hemoglobin A0 is nonglycated hemoglobin A. Clinical situations where red blood cell survival is shortened will falsely depress glycated hemoglobin measurements. Such situations include hemolysis of any cause, hereditary spherocytosis, and hypersplenism. Likewise, prolonged red blood cell survival as seen with polycythemia or following splenectomy can falsely raise glycated hemoglobin concentrations. Iron deficiency also raises HbA1c by B1.5% although the reason for this observation is not well understood [166]. Glycated hemoglobin species can be measured by a wide variety of techniques including ion-exchange or affinity column chromatography, electrophoresis, ion-exchange high-performance liquid chromatography (HPLC), capillary electrophoresis, enzymatic assay, and immunoassay. A glycated hemoglobin assay should be chosen that is traceable to the DCCT [167] reference method (HPLC). Assay certification for manufacturers is supplied by the National Glycohemoglobin Standardization Program [168]. Calibration is linked to the definitive IFCC method [169,170]. The second edition of this handbook reviews the IFCC calibration and its relationship to HPLC calibration in detail. The ADA recommends that HbA1c be monitored at the time of initial clinical assessment to document the degree of glycemic control and regularly thereafter. Unless the patient is in good glycemic control (HbA1c ,7%) when HbA1c can be measured as infrequently as every 6 months, monitoring HbA1c every 3 months is appropriate. Although the target HbA1c in most adults is ,7% (and ,6% may be achievable), the target HbA1c in children is ,7.5% [171]. A major clinical challenge is that as the HbA1c declines with more intensive therapy, the frequency of hypoglycemia increases. Short-term, yet severe, hypoglycemia can be life-threatening and is a significant worry to all patients

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who strive for tight glycemic control. Closed-loop insulin delivery systems can achieve improved glycemic control without an increased risk of hypoglycemia. HbA1c testing at the point of care should be encouraged. Immediate access to the HbA1c measurements at the time of the visit can improve patient management and lower subsequent HbA1c measurements. Urine glucose testing is discouraged as part of the daily diabetes management of patients. Urine glucose testing has been replaced by SMBG and CGM. At present, the measurement of 1,5-anhydroglucitol (GlycoMark) as a marker of short-term control has garnered little interest from the clinical or laboratory communities [172 174].

Fructosamine In pregnant patients and in patients with certain hemoglobinopathies (e.g., SS, SC, or CC where there is no HbA0 to be glycated to HbA1c), glycated serum proteins can be monitored [175]. Total glycated serum protein, or as is more often measured, glycated albumin, reflects B10 days of glycemic control. The glycated albumin assay detects fructosamine (a 5-member ring with a fructose-like structure); thus fructosamine is the test usually ordered for glycated albumin. Because this test only displays short-term control, other than in the clinical conditions outlined earlier, HbA1c measurements are preferred over glycated albumin measurements [176]. If there are accelerated rates of plasma protein turnover, fructosamine measurements could be falsely decreased relative to the patient’s glycemic status. Note: this is not a negative analytic bias; the measurement is correct but the fructosamine concentration is reduced because of a reduced plasma protein half-life. The basis for testing for fructosamine during pregnancy is that the pregnant woman needs to determine her intermediate-term glycemic control more often than can be assessed by HbA1c measurements. Infants born to women who were diabetic prior to their pregnancy who were not in good glycemic control suffer increased rates of birth defects as discussed previously. However, if diabetes is well controlled prior to conception and throughout the first trimester, the risk for having an infant born with a birth defect is no higher than in nondiabetic pregnancies. Thus excellent glycemic control in the diabetic woman who is sexually active who is not using birth control is highly desirable. In pregnancies where the woman has preexisting diabetes or develops diabetes during pregnancy (e.g., gestation diabetes mellitus), poor glycemic control in mid- and late gestation results in macrosomia and metabolic complications in the infant of the diabetic mother as discussed earlier.

Evaluation of lipid status Macrovascular disease is the leading cause of death in both type 1 and 2 diabetes. Therefore fasting lipid profile measurements (total cholesterol,

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triglycerides, HDL-C, and calculated LDL-cholesterol) play a major role in the routine laboratory evaluation of patients with diabetes. In adults not on lipid-lowering medications, the ADA recommends that a lipid profile be obtained at diagnosis, with initial therapy and then every 5 years if under age 40 [177]. If lipid medications are initiated, a lipid profile is repeated yearly. The current guidelines just cited describe in detail the therapeutic lipid targets. Further discussion of lipid targets goes beyond the scope of this chapter.

Renal evaluation In patients with type 1 diabetes, albuminuria (a.k.a.—minimal albumin excretion, elevated urinary albumin excretion, or microalbuminuria) is sought yearly beginning 5 years after diagnosis [178]. Albuminuria is sought at the time of diagnosis of type 2 diabetes and then yearly thereafter. The more aggressive testing for albuminuria in type 2 diabetes is because diabetic complications can already be present at the time of the diagnosis of type 2 diabetes. Estimated glomerular filtration rate based upon a serum or plasma creatinine measurement is also indicated for monitoring purposes. One approach to albuminuria testing is to perform a standard urine dipstick test for protein. If the dipstick is positive, “macroproteinuria” (e.g., clinical proteinuria or “dipstick” proteinuria) is present (e.g., $ 300 μg albumin/mg creatinine) and a 24-h urine protein excretion should be obtained. If the dipstick is negative, then albuminuria is sought by immunoassay. Patients with fever, dehydration, hematuria, or urinary tract infection should delay testing until such conditions have resolved. Albuminuria can be measured in a 24-h collection, in a timed urine collection of 24 h, or in a spot urine sample. In all situations, urinary creatinine is measured. In the timed collections, urinary creatinine permits an assessment of the completeness of collection. For spot urine samples, the albumin to creatinine ratio is calculated. The diagnosis of persistent albuminuria requires that 2 of 2 or 2 of 3 urines demonstrate elevated albumin excretion over a span of no more than 3 6 months. Although cutoffs are laboratory specific, general interpretative guidelines are outlined in Table 8.18. For

TABLE 8.18 Cutpoints defining various degrees of proteinuria. Normal

Albuminuria

Clinical albuminuria

Spot urine (μg/mg creatinine)

,30

30 300

.300

Timed sample (μg/min)

,20

20 200

.200

24-h sample (mg/24 h)

,30

30 300

.300

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standardization, some institutions (e.g., the VA Health System) have chosen to limit albuminuria testing to spot albumin to creatinine ratios. There are POCT devices for albuminuria. Creatinine with estimation of the glomerular filtration rate (e.g., eGFR using the MDRD equation) is recommended yearly to detect early renal impairment. The National Kidney Foundation (http:// www.kidney.org) guidelines for the description of chronic kidney disease can be followed.

Ketone testing When patients are ill, or moderately to markedly hyperglycemic (e.g., PG $ 240 300 mg/dL), urine ketones should be measured. Insulinopenia, either absolute or relative, can cause ketosis. Therefore if ketones are detected, assuming that the patient has not fasted extensively, insulinopenic is likely present that should immediately demand review by the patient, and possibly, a member of the health care team should ketosis be persistent or severe, or if accompanied by nausea or vomiting. Trace urine ketones indicate the need to increase oral fluid intake. By dipstick, ketones of 1 1 or greater indicate the need for an additional short-acting insulin injection. Blood ketone measurements (e.g., β-hydroxybutyrate) can be helpful in the management of DKA. However, many physicians can manage DKA adequately through periodic measurements of the serum total carbon dioxide concentration as an assessment of acid/base balance [179]. It is worthwhile mentioning that ketosis can falsely elevate the creatinine when the creatinine is measured using the Jaffe picric acid method.

Other testing Because of the high frequency of autoimmune conditions associated with type 1 diabetes, screening is recommended for autoimmune thyroid disease, pernicious anemia, and celiac disease [180]. Tests for autoimmune thyroid disease begin with testing for thyroperoxidase autoantibodies. If these are negative, thyroglobulin autoantibodies can be sought but they are less common than thyroperoxidase autoantibodies. In individuals positive for a thyroid autoantibody, yearly thyroid-stimulating hormone (TSH) testing is recommended. Testing for pernicious anemia susceptibility is accomplished by measuring gastric parietal cell autoantibodies. If positive, yearly measurements of vitamin B12 and ferritin are indicated. Screening for thyroid and gastric autoimmunity can be accomplished at the time of diagnosis of type 1 diabetes. In persons with type 1 diabetes, the ADA now recommends routine testing for celiac disease via IgA tissue transglutaminase (tTG) autoantibodies. Negative patients should be tested for IgA deficiency. If IgA deficient, either IgG tTG autoantibodies or deamidated gliadin autoantibodies can be sought.

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Because of the high frequency of hepatic steatosis in insulin-resistant patients and patients with type 2 diabetes [181], alanine aminotransferase (ALT) testing could be considered although there are no recommendations from the ADA to screen for liver disease in persons with diabetes [182]. If the ALT were elevated, and other causes of hepatic disease were excluded (e.g., hepatitis B, hepatitis C, hemochromatosis, α1-antitrypsin deficiency, Wilson disease, etc.) the working diagnosis should be NAFL. NAFL can progress to steatohepatitis (NASH), cirrhosis, liver failure, or even hepatocarcinoma. Ultrasound of the steatotic liver displays a characteristic appearance and may be more sensitive for the detection of fatty liver than ALT measurements. The definitive diagnostic tool is liver biopsy.

Conclusion Diabetes is a state of chronic hyperglycemia with many adverse consequences for the individual and society. Desirable glycemic control decreases the risk of complications in type 1 diabetes [183] and type 2 diabetes [184]. The frequency of type 2 diabetes in the general population is rising and is most likely due to the high frequency of obesity and inactivity. Type 2 diabetes has permeated widely even into the pediatric minority population. The present ADA guidelines simplify the diagnosis of diabetes. Screening for diabetes should be carried out under rigorous, controlled conditions to ensure accuracy. The routine clinical management of patients with diabetes includes a number of laboratory assessments and monitors (Table 8.19). An approach to the diagnosis of the type of diabetes affecting an individual could begin by answering the question: “How did the patient clinically present?” If the patient presented with a clinical syndrome (e.g., other specific types of diabetes genetic syndromes), diabetes is not likely to be the presenting complaint. Likewise, in cases of exocrine pancreatic disease, endocrinopathies, drug- or chemical-induced diabetes, or diabetes due to infection, diabetes is unlikely to be the presenting complaint. When diabetes is the presenting complaint, the diagnosis of type 1 diabetes is favored by leanness, acute-onset diabetes, ketosis, DKA, or positivity for islet autoantibodies (e.g., ICA, GADA, IAA, IA-2A, or ZnT8A). In the absence of these findings, type 2 diabetes is suggested by an increased body mass index, lack of ketosis or DKA, evidence of insulin resistance (e.g., hypertension, dyslipidemia, acanthosis nigricans, hirsutism, PCOS, etc.) or a family history of a similar diabetes phenotype. If the patient does not display these features, then a monogenic form of diabetes should be considered recognizing that many of these disorders are potentially inherited as dominant Mendelian traits or maternally inherited (e.g., mitochondrial disorders) (Fig. 8.3).

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TABLE 8.19 Laboratory testing employed in the management of persons diagnosed with diabetes. Test Application Glucose

FPG or OGTT assessments

HbA1c

Assessment of glycemic control over 2 3 months

Lipid profile

Assessment of glycemic control and cardiovascular risk

Creatinine

Estimation of glomerular filtration rate

Albuminuria

Assessment of renal function

Urine ketones

Detection of urine ketones indicates a significant deficiency in insulin action.

Thyroperoxidase (or) thyroglobulin autoantibodies

Screening for thyroid autoimmunity in type 1 diabetes patients

Gastric parietal cell autoantibodies

Screening for gastric parietal cell autoimmunity in type 1 diabetes patients

IgA transglutaminase (or if IgA deficient) deaminated gliadin autoantibodies

Screening for celiac disease in type 1 diabetes patients

Optional tests Fructosamine

Assessment of glycemic control over 2 3 weeks (other than pregnancy and hemoglobinopathies, use of this test is controversial)

C-reactive protein

Assesses risk for atherosclerosis

ALT

Elevations can be indicative of fatty liver disease associated with the metabolic syndrome

TSH

Testing for thyroid dysfunction in patients with serological evidence of thyroid autoimmunity

Vitamin B12

Testing for vitamin B12 deficiency in patients with gastric parietal cell autoimmunity

Ferritin

Testing for iron deficiency in patients with gastric parietal cell autoimmunity

Adrenal autoantibodies

Testing for adrenal autoimmunity in persons with type 1 diabetes who express thyroperoxidase or gastric parietal cell autoantibodies

276

SECTION | 2 Analytes Lean, acute-onset, ketotic, DKA, islet autoantibody (+)

Yes

Type 1 diabetes likely Yes Type 2 diabetes likely

No

Incr. BMI, non– ketotic, insulin resistant, Fhx (+)

No

Consider a monogenic diabetes syndrome

FIGURE 8.3 This is a simple (but practical) algorithm helping define the type of diabetes present in children and nonpregnant adults. Persons who are lean, experienced acute-onset diabetes, were ketotic or were affected with DKA, that express one or more islet autoantibodies, are diagnosed with type 1 diabetes. If those features are lacking and the patient has an elevated body mass index (usually with centripetal obesity), is nonketotic, displays features of insulin resistance (e.g., hypertension, hypertriglyceridemia, low HDL-cholesterol, gout, polycystic ovarian syndrome, hirsutism, acanthosis nigricans, etc.), and has a family history of a similar type of diabetes, type 2 diabetes is diagnosed. If the type of diabetes cannot otherwise be characterized, monogenic diabetes should be considered assuming other causes of “other specific types of diabetes” have been excluded.

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[152] L.E. Katz, A.F. Jawad, J. Ganesh, M. Abraham, K. Murphy, T.H. Lipman, Fasting cpeptide and insulin-like growth factor-binding protein-1 levels help to distinguish childhood type 1 and type 2 diabetes at diagnosis, Pediatr. Diabetes. 8 (2) (2007) 53 59. [153] T.H. Park, M.S. Kim, D.-Y. Lee, Clinical and laboratory characteristics of childhood diabetes mellitus: a single-center study from 2000 to 2013, Chonnam Med. J. 52 (2016) 64 69. [154] B. Giri, S. Dey, T. Das, M. Sarkar, J. Banerjee, S.K. Dash, Chronic hyperglycemia mediated physiological alteration and metabolic distortion leads to organ dysfunction, infection, cancer progression and other pathophysiological consequences: an update on glucose toxicity, Biomed. Pharmacother. 107 (2018) 306 328. [155] G. Geffken, W.E. Winter, Hardware and software in diabetes mellitus: performance characteristics of hand-held glucose testing devices and the application of glycemic testing to patients’ daily diabetes management, Clin. Chem. 47 (2001) 11 12. [156] W.E. Winter, Point of care testing in the management of diabetes (Chapter 10), in: J.H. Nichols (Ed.), Point of Care Testing: Performance Improvement and Evidence Based Outcomes, Marcel Dekker, New York, 2002, pp. 235 292. [157] W.E. Winter, N.S. Harris, Self-monitoring of blood glucose: needless expense or a vital glycemic monitor? Point Care Test. J. Near Patient Test. Technol. 2 (2003) 62 68. [158] W.E. Winter, Rosetta stone for insulin treatment: self-monitoring of blood glucose, Clin. Chem. 50 (2004) 985 987. [159] W. Majeed, H. Thabit, Closed-loop insulin delivery: current status of diabetes technologies and future prospects, Expert Rev. Med. Devices 15 (8) (2018) 579 590. [160] J. Speight, J.L. Browne, J. Furler, Challenging evidence and assumptions: is there a role for self-monitoring of blood glucose in people with type 2 diabetes not using insulin? Curr. Med. Res. Opin. 29 (3) (2013) 161 168. [161] W.E. Winter, L.J. Sokoll, I. Jialal (Eds.), Handbook of Diagnostic Endocrinology, second ed., American Association of Clinical Chemistry Inc., Washington, DC., 2008. [162] G. van den Berghe, P. Wouters, F. Weekers, C. Verwaest, F. Bruyninckx, M. Schetz, et al., Intensive insulin therapy in critically ill patients, N. Engl. J. Med. 354 (2001) 1359 1367. [163] G. Van den Berghe, A. Wilmer, G. Hermans, et al., Intensive insulin therapy in the medical ICU, N. Engl. J. Med. 354 (2006) 449 461. [164] The NICE-SUGAR Study Investigators, Intensive versus conventional glucose control in critically ill patients, N. Engl. J. Med. 360 (2009) 1283 1297. [165] American Diabetes Association, Glycemic targets: standards of medical care in diabetes 2019, Diabetes Care 42 (Suppl. 1) (2019) S61 S70. [166] R.C. Sundaram, N. Selvaraj, G. Vijayan, et al., Increased plasma malondialdehyde and fructosamine in iron deficiency anemia: effect of treatment, Biomed. Pharmacother. 61 (2007) 682 685. [167] The Diabetes Control and Complications Trial Research Group, The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus, N. Engl. J. Med. 329 (1993) 977 986. [168] R.R. Little, Glycated hemoglobin standardization: National Glycohemoglobin Standardization Program (NGSP) perspective, Clin. Chem. Lab. Med. 41 (2003) 1191 1198. [169] G.S. Dhatt, M.M. Agarwal, B. Bishawi, HbA1c: a comparison of NGSP with IFCC transformed values, Clin. Chim. Acta 358 (2005) 81 86.

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[170] R.R. Little, C.L. Rohlfing, The long and winding road to optimal HbA1c measurement, Clin. Chim. Acta 418 (2013) 63 71. [171] American Diabetes Association, Children and adolescents: standards of medical care in diabetes 2019, Diabetes Care 42 (Suppl. 1) (2019) S148 S164. [172] T. Yamanouchi, T. Inoue, E. Ogata, A. Kashiwabara, N. Ogata, N. Sekino, et al., Postload glucose measurements in oral glucose tolerance tests correlate well with 1,5-anhydroglucitol, an indicator of overall glycaemic state, in subjects with impaired glucose tolerance, Clin. Sci. (Lond.) 101 (2001) 227 233. [173] J.B. Buse, J.L. Freeman, S.V. Edelman, L. Jovanovic, J.B. McGill, Serum 1,5-anhydroglucitol (GlycoMark): a short-term glycemic marker, Diabetes Technol. Ther. 5 (2003) 355 363. [174] C.M. Parrinello, E. Selvin, Beyond HbA1c and glucose: the role of nontraditional glycemic markers in diabetes diagnosis, prognosis, and management, Curr. Diabetes Rep. 14 (11) (2014) 548. [175] D.A. Armbruster, Fructosamine: structure, analysis, and clinical usefulness, Clin. Chem. 33 (1987) 2153 2163. [176] R.T. Ribeiro, M.P. Macedo, J.F. Raposo, HbA1c, fructosamine, and glycated albumin in the detection of dysglycaemic conditions, Curr. Diabetes Rev. 12 (1) (2016) 14 19. [177] American Diabetes Association, Cardiovascular disease and risk management: standards of medical care in diabetes 2019, Diabetes Care 42 (Suppl. 1) (2019) S103 S123. [178] American Diabetes Association, Microvascular complications and foot care: standards of medical care in diabetes 2019, Diabetes Care 42 (Suppl. 1) (2019) S124 S138. [179] W.H. Porter, H.H. Yao, D.G. Karounos, Laboratory and clinical evaluation of assays for beta-hydroxybutyrate, Am. J. Clin. Pathol. 107 (3) (1997) 353 358. [180] W.E. Winter, Autoimmune endocrinopathies, in: F. Lifshitz (Ed.), Pediatric Endocrinology, 5th ed., Informa Healthcare USA, New York, 2007, pp. 595 616. [181] B.A. Neuschwander-Tetri, Fatty liver and the metabolic syndrome, Curr. Opin. Gastroenterol. 23 (2007) 193 198. [182] American Diabetes Association, Comprehensive medical evaluation and assessment of comorbidities: standards of medical care in diabetes 2019, Diabetes Care 42 (Suppl. 1) (2019) S34 S45. [183] The Diabetes Control and Complications Trial Research Group, The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus, N. Engl. J. Med. 329 (1993) 977 986. [184] Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33): UK Prospective Diabetes Study (UKPDS) Group, Lancet 352 (1998) 837 853.

Chapter 9

Lipoproteins Anna Wolska and Alan T. Remaley Lipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, United States

Background Two of the most abundant lipid components in plasma are cholesterol and triglycerides (TG); however, both are virtually insoluble in water. To be transported to various tissues, these lipids must be packaged into a lipoprotein particle, the core of which is composed of cholesteryl esters and TG, and the surface is composed of phospholipids, free cholesterol, and apolipoproteins [1]. Lipoprotein particles are freely water soluble and along with albumin are the main transporters of hydrophobic substances in plasma. In man and most other mammals, there are four major lipoprotein classes (Table 9.1) [1]. Chylomicrons (CM) and very low-density lipoproteins (VLDL) are the main TG-carrying particles. Low-density lipoproteins (LDL), which are produced after the lipolysis of TG on VLDL, are cholesterol-rich lipoproteins. High-density lipoproteins (HDL) are also cholesterol-enriched and besides apolipoprotein (apo) A-I (apoA-I), its main structural protein, they transport a wide variety of proteins such as apoA-II or apoC-II. It is important to note that all the different lipoproteins contain the same lipids but only in different proportions (Table 9.1) and they are commonly classified by either their density or electrophoretic mobility [origin (CM), pre-β (VLDL), β (LDL), and α (HDL)]. As will be discussed in more detail later, disturbances in lipoprotein metabolism have two major pathophysiologic consequences. Elevated LDLcholesterol (LDL-C) can predispose to premature atherosclerosis [2], whereas very high concentrations of plasma TG ( . 1000 mg/dL) carried by CM and VLDL can precipitate pancreatitis [3]. It is unclear how HDL relates to the pathogenesis of atherosclerosis, but they are at the very least a useful negative biomarker for coronary heart disease (CHD) risk. Overall, lipoprotein metabolism can be viewed as three interconnected pathways (Fig. 9.1), namely the exogenous, endogenous, and reverse cholesterol transport pathways [1]. The exogenous pathway (Fig. 9.1, pathway I) is involved in the absorption of dietary fat and the delivery of TG to peripheral Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00009-1 © 2021 Elsevier Inc. All rights reserved.

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TABLE 9.1 Composition and properties of major lipoproteins. CM

VLDL

LDL

HDL

Origin

Gut

Liver

VLDL

Liver, intestine

Density (g/mL)

0.94

0.941.006

1.0191.063

1.0631.21

Electrophoretic mobility

Origin

Pre-β

β

α

Major proteins

apoB-48, C, E

apoB-100, C, E

apoB-100

apoA-I, A-II, C, E

% Protein

1%

10%

20%

50%

Major lipids

TG (90%)

TG (55%)

Cholesterol (50%)

Phospholipids (25%), cholesterol (20%)

Main function

Transport dietary TG

Transport endogenous TG

Transport cholesterol esters

Reverse cholesterol transport

cells and the liver. In this pathway, dietary fat is first packaged in the intestine as a CM particle, which contains apoB-48 and is initially secreted into lymphatics, which eventually drain into the circulatory system. TG in CM are hydrolyzed by a pivotal enzyme in lipoprotein metabolism, lipoprotein lipase (LPL), which is bound to the vessel wall and requires apoC-II as its cofactor. The hydrolysis of TG results in the release of fatty acids, which are avidly taken up by peripheral tissues and can be re-esterified into TG for energy storage or can undergo β-oxidation for energy production. The CM remnants that are formed as a consequence of lipolysis by LPL are rapidly removed by the liver by LDL receptor-related protein (LRP) and by proteoglycans in the hepatic sinusoidal spaces. The endogenous pathway (Fig. 9.1, pathway II) is responsible for the delivery of hepatic-derived TG as a source of energy to peripheral cells, during fasting. In this pathway, VLDL, which contain apoB-100 and are enriched in TG, are secreted by the liver. Upon entry into the circulation, VLDL have the same fate as CM in that they are acted upon by LPL, resulting in the formation of VLDL remnants, which are also called intermediatedensity lipoproteins (IDL). VLDL also become enriched in cholesteryl esters, because of cholesteryl ester transfer protein (CETP)-mediated exchange of lipids between VLDL and HDL. VLDL remnants can be directly removed by the liver by LDL receptors (LDLR), LRP and proteoglycans or can continue to lose additional TG by LPL-dependent lipolysis to generate LDL. LDL can be removed from the circulation by the LDLR, which are on many cell types, but it is the level of LDLR on the liver that primarily regulates plasma levels of LDL. LDL are atherogenic most likely because of their

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FIGURE 9.1 Lipoprotein metabolism pathways: I (red oval)—exogenous pathway, CM deliver dietary lipids to the liver; II (blue oval) —endogenous pathway, VLDL mediate the transport of endogenous lipids to peripheral tissues; and III (green oval) —reverse cholesterol transport pathway, HDL transport cholesterol from peripheral tissues back to the liver. A-I 5 apolipoprotein A-I; ABCA1 5 ATP-binding cassette transporter A1; B48 5 apolipoprotein B-48; B100 5 apolipoprotein B-100; C-II 5 apolipoprotein C-II; CETP 5 cholesteryl ester transfer protein; CM 5 chylomicrons; E 5 apolipoprotein E; FAA 5 free fatty acids; HDL 5 high-density lipoproteins; LCAT 5 lecithin cholesterol acyltransferase; LDL 5 low-density lipoproteins; LDLR 5 low-density lipoprotein receptor; LRP 5 low-density lipoprotein receptor-related protein; LPL 5 lipoprotein lipase; SR-B1 5 scavenger receptor class B type 1; TG 5 triglycerides; VLDL 5 very low-density lipoproteins.

propensity to be oxidized and to be taken up by macrophages in the vessel wall, which results in the formation of foam cells and fatty streaks, one of the earliest hallmarks of atherosclerosis [4]. In contrast to the apoB-containing lipoproteins, which primarily promote the delivery of TG to peripheral tissues, HDL participate in the reverse cholesterol transport pathway [1] (Fig. 9.1, pathway III). This is the pathway by which cells maintain cholesterol homeostasis, although it is most important in cells like macrophages that take up large amounts of exogenous cholesterol by various scavenger receptors, for example, a scavenger receptor class B type 1 (SR-B1) and by the phagocytosis of cholesterol-rich membranes from other cell types. HDL, which contain apoA-I as their main protein, mediate the removal of excess cholesterol from peripheral cells and return it to the liver, where it can be

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SECTION | 2 Analytes

directly excreted into the bile or converted into a bile salt. Cholesterol is also thought to be directly excreted by the intestine by the transintestinal cholesterol efflux pathway, but the details of this pathway are not fully understood. Because most cells besides hepatocytes cannot catabolize cholesterol, the reverse cholesterol transport pathway has been proposed to be their main atheroprotective function. HDL can be formed as a consequence of lipolysis of VLDL and CM, which causes the release of apoA-I and surface lipids that can then spontaneously reorganize as HDL particles. Most HDL, however, are probably formed by the liver and intestine when the ATP-binding cassette transporter A1 (ABCA1) on the plasma membranes adds phospholipids to newly secreted apoA-I, which contains a tandem array of amphipathic helices and acts like a protein detergent. In other tissues, ABCA1 also promotes the removal of excess cholesterol from peripheral cells to HDL and in this way helps cells maintain cholesterol homeostasis. Lecithin cholesterol acyltransferase (LCAT) is a key enzyme in the reverse cholesterol transport pathway, because it converts cholesterol to cholesteryl esters, thus trapping it in the core of HDL until it can be transported to the liver and taken up by the hepatic SR-B1. HDL, however, have many other potentially beneficial antiatherogenic properties, which are not fully understood, such as their antioxidant and antiinflammatory properties, that probably relate to their other protein and lipid components [5]. Finally, CETP interconnects the reverse cholesterol transport pathway with the exogenous and endogenous pathways by exchanging cholesteryl esters on HDL with TG from apoB-containing lipoproteins.

Classification of dyslipidemias Lipoprotein disorders can be classified as either being primary due to a genetic etiology (Table 9.2) or secondary due to a metabolic or nutritional disorder or sometimes can be classified as iatrogenic as a consequence of a drug treatment (Table 9.3). A common practical approach for classifying the various causes for dyslipidemia is to first determine which of the major lipids or lipoproteins are present in abnormal amounts, either high or low. Thus the various dyslipidemias will be discussed in this chapter under the headings hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia (high TG and total cholesterol, TC), hypolipidemia with hypoalphalipoproteinemia (low HDL-cholesterol, HDL-C), and hypotriglyceridemia. In addition, the original phenotypic classification by Fredrickson, D.S., which was based on agarose gel electrophoretic separation of lipoproteins [6], is also shown in Table 9.2

Hypercholesterolemia Because much of the risk of high cholesterol is when it is in LDL, LDL-C is usually the main cholesterol parameter to classify patients at risk for CHD. According to the 2018 American Heart Association/American Association of Cardiology (AHA/ACC) guidelines an optimal LDL-C in adults is ,100 mg/dL,

TABLE 9.2 Familial hyperlipoproteinemias. Genetic disorder

Phenotype

Biochemical defect

Clinical presentation

TC

TG

Familial LPL deficiency

I

Absence of LPL activity

Eruptive xanthoma; hepatosplenomegaly; pancreatitis

Increased 1

Increased 111

Familial apoC-II deficiency

I or V

Absence or abnormal structure of apoC-II

Pancreatitis

Increased 1

Increased 111

Familial hypercholesterolemia

IIa or IIb

Deficiency of LDL receptors

Tendinous and tuberous xanthoma; premature atherosclerosis

Increased 111

Normal (IIa) or increased 1 (IIb)

Familial dysbetalipoproteinemia

III

Abnormal apoE and defect in TG-rich lipoprotein metabolism

Tuberoeruptive and planar xanthoma; premature atherosclerosis

Increased 11

Increased 11

Familial combined hyperlipidemia

IIa, IIb, or IV

Increased apoB production

Premature atherosclerosis

Increased or normal

Increased or normal

Familial hypertriglyceridemias

IV and V

Unknown

Type V: eruptive xanthoma; hepatosplenomegaly; pancreatitis

Normal (IV) Increased 1 (V)

Increased 1 (IV) Increased 111 (V)

1 is for mildly increased; 11 is for moderately increased; 111 is for severely increased

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TABLE 9.3 Causes of secondary dyslipidemia. Predominantly hypercholesterolemia Hypothyroidism Cholestasis Nephrotic syndrome Dysglobulinemias Diuretics Cyclosporine Predominantly hypertriglyceridemia Obesity Diabetes mellitus Alcoholism Renal failure Lipodystrophy Dysglobulinemias Estrogen therapy Steroid therapy Beta-blocker therapy Isotretinoin therapy Hypoalphalipoproteinemia Obesity Diabetes Physical inactivity Smoking High-carbohydrate diet Anabolic steroids Beta-blockers Liver disease

near optimum 100129 mg/dL, borderline high is 130159 mg/dL, high 160189 mg/dL, and very high is $ 190 mg/dL [7]. Based on a wide variety of experimental and clinical studies, LDL-C is known to be causally related to the development of CHD. Furthermore, numerous studies have shown that lowering

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LDL-C, either by diet or in combination with a drug therapy, can reduce cardiovascular morbidity and mortality and even total mortality [2]. Having established that a patient has hypercholesterolemia with an elevated concentration of LDL-C, it is first important to exclude any secondary cause of hypercholesterolemia (Table 9.3), which is best treated by addressing any underlying disorder. For example, hypothyroidism can present with elevated LDL-C and TG. It is more effective to treat it with thyroid hormone replacement therapy rather than a lipid-lowering drug, which will also address the other clinical consequences of the thyroid disease. A prototypical genetic disorder that results in hypercholesterolemia is familial hypercholesterolemia, which is fairly frequent, with an incidence between 1/200 and 1/300 in the heterozygous state [2]. This is typically an autosomal dominant disorder characterized by increased levels of LDL-C, usually due to a genetic defect in the LDLR. Patients that are homozygous for defects in the LDLR typically present in pediatric practice, and these children have few or no functional LDLRs, resulting in marked elevations in LDL-C, xanthoma formation on the skin and tendons, premature atherosclerosis, aortic valve disease, and myocardial infarction as early as the second decade of life. The more common variety, which presents in adults, is the heterozygote in whom there is about a 50% reduction in LDLR activity, resulting in about a twofold increase in LDL-C. They can also present with tendon xanthomas and premature atherosclerosis, usually manifesting in early to middle adulthood. To date, numerous mutations of the LDLR gene have been described that affect either its synthesis, correct orientation on the cell, or its internalization by endocytosis. It has been shown that approximately 18% carriers and noncarriers of a mutation for LDLR are being misdiagnosed when using only cholesterol levels for their diagnosis [8,9]. Recently, defects in two auxiliary proteins important in the function of the LDLR, namely LDL receptor adaptor protein 1 (LDLRAP1) and proprotein convertase subtilisin/kexin type 9 (PCSK9), have also been described as a rare cause of familial hypercholesterolemia [2]. LDLRAP1 is an adaptor protein important in the endocytosis of the LDLR and mutations in it are only apparent when both alleles are mutated, and thus it is an autosomal recessive form of hypercholesterolemia. PCSK9 is a secretory protein produced mainly by the liver that promotes the internalization and degradation of the LDLR. Loss-offunction mutations in PCSK9 are associated with low LDL-C and patients have reduced CHD risk, whereas patients with gain-of-function mutations have high LDL-C and have increased CHD risk. Recently a new therapy based on monoclonal antibodies against PCSK9 has been developed for markedly lowering LDL-C when given in conjunction with a statin [10]. Mutations in the receptor-binding region of apoB-100, at position 3500 and rarely elsewhere on apoB-100, have also been shown to result in a defective apoB-100 with decreased affinity to the LDLR, resulting in an elevated LDL-C. Clinically, this type of dyslipidemia is usually not as severe as familial

294

SECTION | 2 Analytes

hypercholesterolemia and has been named familial defective apoB. Familial hypercholesterolemia can also be subclassified into two types, depending on plasma TG. In the type IIa disorder, plasma TG are normal, whereas in the type IIb disorder, plasma TG are increased due to elevated levels of VLDL (Table 9.2) and these patients may be at even more risk for CHD than type IIa. Patients with elevated lipoprotein(a), Lp(a), can also present with hypercholesterolemia [11]. Lp(a) is like LDL in structure but has an additional highly polymorphic protein that differs in size called apo(a), which is covalently attached to apoB-100 by a disulfide bond. Apo(a) resembles plasminogen in structure and may interfere with clot lysis, which is possibly the reason that Lp(a) is so proatherogenic. In fact, polymorphisms that affect its synthesis and the size polymorphisms of apo(a) are one of the strongest genetic risk factors for not only CHD but also aortic valve calcification. It may also be a cause for thrombotic stroke. There are currently no effective therapies for lowering Lp(a), but individuals who are at risk because of high levels of Lp(a) should be aggressively treated for other risk factors. About quarter of the population have elevated Lp(a) levels that put them at risk for CHD and not all these patients will have hypercholesterolemia, so patients that have a strong family history of CHD and/or do not respond well to statin therapy should ideally be tested by an immunoassay for Lp(a) [12]. Most patients who present with hypercholesterolemia have only modest increases in LDL-C when compared with familial hypercholesterolemia. In these cases, it is likely a consequence of a complex interaction of common genetic polymorphisms that affect lipid metabolism and environmental factors and has been referred to as polygenetic hypercholesterolemia. In contrast to familial hypercholesterolemia, these patients often show a good response to drug therapy and are also more likely to respond to lifestyle changes and thus have a better prognosis. According to AHA/ACC 2018 guidelines [7], it is recommended that all adults 20 years or older be screened with a lipid panel (TC, TG, HDL-C, and a calculated or measured LDL-C) every 5 years. Treatment goals and drug approaches are primarily based on LDL-C levels and vary depending on the risk for CHD (Fig. 9.2). Risk is determined for age 4075 years, using a calculation based on the presence of clinically existing CHD or risk equivalents (diabetes mellitus, chronic kidney disease); on laboratory tests, such as LDL-C and TG concentrations (Table 9.5); and risk-enhancing factors for CHD (Table 9.4). Those patients at greatest risk should be treated more aggressively, which usually involves lipid-lowering drug treatment, most often a statin (Fig. 9.2). Statins by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol biosynthesis, decrease the regulatory pools of cholesterol in hepatocytes. Statins then cause an upregulation of the LDLR and the lowering of plasma LDL. In contrast to previous recommendations, a nonfasting sample can be used as a screening test. If nonfasting TG is greater than 400 mg/dL, a fasting sample should be tested. Besides a lipid panel, for those patients at intermediate risk other laboratory tests, which are referred to as

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295

FIGURE 9.2 Primary prevention of CHD in adults. CAC 5 coronary artery calcium score; CHD 5 coronary heart disease; DM 5 diabetes mellitus; LDL-C 5 low-density lipoproteins cholesterol. Modified from 2018 American Heart Association/American College of Cardiology Guideline on the Management of Blood Cholesterol: Executive Summary [7].

TABLE 9.4 CHD risk-enhancing factors. Family history of premature CHD Persistently elevated LDL-C $ 160 mg/dL Chronic kidney disease (eGFR 1559/min/1.73 m2) Metabolic syndrome (TG . 175 mg/dL, elevated blood pressure, elevated glucose, HDL-C , 40 mg/dL in men; ,50 mg/dL in women) Conditions specific to women (e.g., preeclampsia, premature menopause) Inflammatory diseases (e.g., rheumatoid arthritis, psoriasis, HIV) Ethnicity (e.g., South Asian ancestry) Lipid biomarkers (e.g., TG $ 175 mg/dL, hsCRP $ 2 mg/dL, Lp(a) $ 50 mg/dL, apoB $ 130 mg/dL, ABI , 0.9) ABI, ankle-brachial index; apoB, apolipoprotein B; CHD, coronary heart disease; eGFR, estimated glomerular filtration rate; HDL-C, high-density lipoproteins cholesterol; HIV, human immunodeficiency; hsCRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoproteins cholesterol; Lp(a), lipoprotein(a); TG, triglycerides. Modified from 2018 American Heart Association/American College of Cardiology Guideline on the Management of Blood Cholesterol: Executive Summary [7].

risk enhancers (Table 9.4), can be used to decide therapy, such as apoB, Lp(a), and high-sensitivity C-reactive protein (hsCRP), a marker of inflammation. For those at the highest intermediate risk, cardiovascular imaging for coronary vessel calcification may also be helpful. For those age 2039 years, they should be counseled to follow a healthy lifestyle and statins can be considered if there is a strong family history and LDL-C is greater than 160 mg/dL.

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FIGURE 9.3 Dyslipidemia algorithm in children and adolescents: target LDL cholesterol. CHD 5 coronary heart disease; CHILD 5 cardiovascular health integrated lifestyle diet; FHx 5 family history; HDL-C 5 high-density lipoproteins cholesterol; LDL-C 5 low-density lipoproteins cholesterol; non-HDL-C 5 non-high-density lipoproteins cholesterol; RF 5 risk factor; TC 5 total cholesterol; TG 5 triglycerides. Modified from 2011 Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents: Summary [13].

The pediatric guidelines for children and adolescents [13] are similar to adults but there are key differences in terms of the recommended age for lipid testing, decision cut-points, as well as in the recommended treatments (Fig. 9.3). Another important distinction is that the identification of dyslipidemia in a parent or sibling can often trigger an investigation of the extended family for identifying other family members at risk, particularly in the case of familial hypercholesterolemia. A nonfasting sample can also be used in the initial screen but should be followed by a fasting sample if abnormal. Recommendations also exist for other age intervals not shown in the figure but mostly do not relate to lipids.

Hypertriglyceridemia A classification of hypertriglyceridemia in adults based on plasma TG levels, according to the new AHA/ACC guidelines [7], is as follows: normal, ,150 mg/dL; borderline high, 150199 mg/dL; high, 200499 mg/dL; and very high, $ 500 mg/dL. The relationship between TG and CHD appears to be complex [3] and may be explained, in part, by its association with low

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concentrations of HDL and abnormal forms of LDL (e.g., small dense LDL) and elevated remnant lipoproteins concentrations. Based on recent genetic studies, there is now, however, a consensus that hypertriglyceridemia is causally related to atherosclerosis [19]. The effectiveness, however, of treating TG to lower CHD risk is unclear, but treatment of patients with high TG with eicosapentaenoic acid, a component of fish oil, has recently been shown to substantially reduce CHD when added on top of statins [20]. Treatments, such as low-fat diets, fibrates, and fish oils, are currently recommended for patients with TG $ 500 mg/dL, because such patients can develop chylomicronemia syndrome and a life-threatening pancreatitis. The mechanism of pancreatitis with hypertriglyceridemia remains to be elucidated. One hypothesis is that the hydrolysis of TG and phospholipids results in excess lysolecithin and free fatty acids in the pancreas, where they trigger inflammation and acute pancreatitis. Some of the secondary causes of hypertriglyceridemia, such as obesity, diabetes, alcoholism, and renal failure, are listed in Table 9.3. Like for hypercholesterolemia, secondary causes should always first be ruled out as the cause for hypertriglyceridemia. When it is secondary to obesity, many of these patients can show dramatic improvement in response to lifestyle changes, such as by reducing caloric intake, increased exercise, and weight loss. There are several genetic causes of hypertriglyceridemia (Table 9.2). Type IV hyperlipoproteinemia (primary endogenous hypertriglyceridemia) is an inherited autosomal dominant form of familial hypertriglyceridemia and results in moderately elevated TG concentrations due to mostly the accumulation of VLDL. This is a relatively common disorder that manifests mainly in adulthood and does not appear to have many unique biochemical features. There is no direct evidence that these patients are more prone to CHD from their increased VLDL. It is thought that the VLDL particles that accumulate in this disorder are too large to enter the vessel wall; however, many of these patients will also have other risk factors that may put them at risk for CHD. These patients generally are obese and can have abnormal glucose tolerance and/or hypertension. TG concentrations in these patients are generally between 250 and 600 mg/dL; LDL-C concentrations are usually normal or only slightly elevated. There are several rare causes of very severe hypertriglyceridemia that result in chylomicronemia syndrome. In children and young adults, the chylomicronemia syndrome can be due to LPL deficiency (type I hyperlipoproteinemia; Table 9.2), which is an autosomal recessive disorder. LPL deficiency typically presents in childhood with pancreatitis, eruptive xanthoma, hepatosplenomegaly, and lipemia retinalis. These patients are probably not at high risk for premature atherosclerosis but are particularly predisposed to developing pancreatitis. Deficiency or mutation in the cofactor for LPL, apoC-II [21], and in other genes related to lipolysis can also cause

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chylomicronemia syndrome. The most common type of chylomicronemia syndrome in adults is type V hypertriglyceridemia (Table 9.2). It often occurs in patients previously diagnosed with familial hypertriglyceridemia (type IV), and then some secondary factors, such as obesity, diabetes, and alcoholism, trigger these patients to form excess CM along with VLDL. Although the familial nature of type V hypertriglyceridemia is well recognized, the pattern of inheritance is not clear, and no specific molecular defect has been identified. Like patients with the type I hyperlipoproteinemia, patients with type V hypertriglyceridemia can also present with eruptive xanthoma, hepatosplenomegaly, lipemia retinalis, and pancreatitis but typically it is not as severe as type I (Table 9.2).

Combined hyperlipidemia A relatively common disorder with elevations of both TG and TC is familial combined hyperlipidemia, which is also referred to as familial multiple lipoprotein-type hyperlipidemia. Patients and their affected first-degree relatives may at various times develop hypercholesterolemia, hypertriglyceridemia, or combined abnormalities and have a type IIa, IIb, or type IV lipoprotein patterns (Table 9.2). It also appears to be mostly inherited in an autosomal dominant mode, but the underlying molecular defect is not known. There often appears to be an overproduction of apoB-containing lipoproteins by the liver, which results in increased levels of VLDL, IDL, and LDL. Patients usually present with hyperlipidemia in the third or fourth decades of life and have an increased propensity for premature atherosclerosis. This disorder may also be accompanied by obesity and abnormal glucose tolerance. HDL-C concentrations are usually low in these patients. The diagnosis depends on the demonstration of multiple abnormal lipoprotein phenotypes in first-degree relatives of the patient. Plasma apoB-100 concentrations are invariably elevated in these subjects and are often used as part of the diagnosis [22]. Familial dysbetalipoproteinemia or type III hyperlipoproteinemia (Table 9.2), which is sometimes referred to as remnant disease or broad-β disease, is a rare disorder that can also result in a combined hyperlipidemia. These patients usually have an abnormality in apoE, which is a ligand for removal of VLDL and IDL, leading to the accumulation of these particles. ApoE has three major alleles, namely apoE3, apoE2, and apoE4 [23]. The majority of the population is homozygous for apoE3. Patients who usually develop type III disorder are homozygous for apoE2 or have a rare mutation in apoE. However, this is not sufficient to develop the disorder, because the frequency of E2 homozygosity in the general population is 1/100, whereas the frequency of type III dyslipidemia is only 1/50001/10,000. A secondary factor that results in overproduction of VLDL and IDL, such as obesity, diabetes, or hypothyroidism, is usually needed for this disorder to be fully manifested. The accumulation of IDL and other remnant particles

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because of their small size is possibly the reason for the high risk of CHD in this disorder. Characteristically, the level of TG and TC is elevated to a similar degree in type III hyperlipoproteinemia. Electrophoresis of their serum can reveal a broad β-band in about half of patients. To confirm the diagnosis, ultracentrifugation is sometimes used to measure the ratio of VLDL-cholesterol to total plasma TG, which is typically $ 0.30 when expressed in mg/dL units. Often these patients develop by mid-adulthood with two different types of xanthoma. Xanthomas can form in the creases of their palms and fingers, where they impart a yellowish discoloration and are pathognomonic for type III hyperlipidemia. In addition, they can also develop the same type of tubero-eruptive xanthomas, which occur at the elbows, knees, and buttocks, as in other forms of hypercholesterolemia. In addition to CHD, they can also develop peripheral vascular disease presumably also due to the deposition of small IDL and other remnant particles in other vessels besides the coronary arteries.

Hypolipidemia Hypoalphalipoproteinemia An HDL-C level below 40 mg/dL is defined as low and constitutes an independent positive risk factor for CHD and is used in most algorithms for calculating CHD risk (Table 9.4) [14]. Like hypercholesterolemia, secondary disorders (Table 9.3), such as obesity and liver disease, commonly account for low HDL and should first be considered before relatively rare genetic disorders. The classic genetic disorder of low HDL is Tangier disease, which is due to defects in the ABCA1 transporter, which transfers excess cellular cholesterol to HDL (Fig. 9.1) [15]. Mutations in LCAT also result in low HDL-C and are associated with the formation of an abnormal lipoprotein particle called Lipoprotein-X (LpX), which can cause renal disease [16]. LpX can also be formed in cholestatic liver disease. Mutations in apoA-I, the main HDL protein, can also cause low HDL, and some mutations can also cause amyloidosis [17]. Interestingly, unlike familial hypercholesterolemia many patients with primary genetic disorders of HDL such as Tangier disease do not have a marked increased risk for CHD, possibly because it is often also associated with low LDL-C. Much of the cholesterol on LDL comes from VLDL that get cholesterol transferred from HDL by CETP (Figure 9.1), so in general when HDL-C is low and so is LDL-C. Nevertheless, identifying patients with low HDL-C is an important step in determining CHD risk and in the clinical management of patients’ dyslipidemia. There are few drugs that can raise HDL-C like niacin and CETPinhibitors, but they have not been proven to reduce CHD [14]. In addition, high levels of HDL-C have recently been associated with increased CHD and a wide variety of other diseases, and therefore high HDL-C is no longer considered to be protective [18].

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Hypotriglyceridemia Hypotriglyceridemia can occur in patients with what is caused familial combined hypolipidemia. These patients usually have total TG levels below 50 mg/dL and also have low LDL-C and HDL-C. It is due to homozygous mutations in an angiopoietin-like protein 3 (ANGPTL3), which inhibits LPL [24]. Because of their low LDL, they appear to be protected against CHD and otherwise appear healthy. Two other closely related causes of hypotriglyceridemia that are important to diagnose are abetalipoproteinemia and familial hypobetalipoproteinemia [25]. Patients usually have markedly reduced levels of TG that are sometimes not detectable and usually have severe fat malabsorption from a young age. Abetalipoproteinemia is due to mutations in a microsomal transfer protein, MTP, which is necessary for the secretion of CM and VLDL. As a consequence, patients can develop severe fat-soluble vitamin deficiencies and have to be treated with large doses of Vitamin E and A to prevent peripheral neuropathy and blindness. Familial hypobetalipoproteinemia is usually due to truncation mutations in apoB, leading to its rapid catabolism and inability to transport fat-soluble vitamins in the circulation. Like in abetalipoproteinemia patients with familial hypobetalipoproteinemia also should be treated with Vitamin A and E. In both of these disorders, plasma apoB levels are markedly decreased.

Laboratory assessment of dyslipidemia In addition to a careful history and clinical examination, laboratory testing is a critical step in the diagnosis of dyslipidemia and for ruling out secondary causes [26]. Laboratory testing is also important for the monitoring of lipidlowering therapy. Either plasma or serum can be used for lipid testing. Ethylenediaminetetraacetic acid (EDTA) plasma is often preferred because samples can be processed more rapidly, and the addition of EDTA inhibits lipid peroxidation and proteolysis. Values obtained with plasma for both TC and TG are, however, about 3% lower than in serum, because of dilution from the anticoagulant [27]. It is important that at least two lipid tests, preferably a week apart, were done before an individual is diagnosed as having a dyslipidemia and placed on a drug therapy, because plasma lipids can show significant biological variations from day to day. Reported biologic variability for TC, TG, and LDL-C and HDL-C are 6.1%, 22.6%, 9.5%, and 7.4%, respectively [28] (Please read more about how to calculate CVi and CVg in Chapter 1). In addition, samples for lipid testing should be collected from patients when they are in a stable metabolic state and do not have concurrent illnesses, which can markedly alter lipid and lipoprotein levels. Other precautions that should be undertaken prior to blood sampling include the following: avoiding alcohol on the evening prior to blood collection; maintaining a habitual weight for at least 23 weeks;

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lipid analysis should be deferred for 23 weeks after minor illness and for 3 months after a major illness, surgery, or trauma; collection of the sample after the patient has been seated for 510 min; and finally, if possible, one should wait at least 3 weeks after any type of dietary or drug change in lipid-lowering therapy. According to the most recent AHA/ACC guidelines [7], the initial evaluation of CHD risk from lipids in adults should begin with the measurement from a nonfasting sample a complete lipid panel (Table 9.5), which consists of tests for TC, TG, HDL-C, and a measured or calculated LDL-C. Until recently most guidelines only recommended the use of fasting samples because as described below elevated TG after a meal can interfere with the calculation of LDL-C. Most patients, however, have only a modest change in their TG after a meal, which does not significantly impact LDL-C estimation. Furthermore, several studies have shown that postprandial lipid and lipoprotein levels may be more predictive for CHD risk than from a fasting sample. If, however, a patient’s nonfasting TG level is greater than 400 mg/dL, it is recommended that a fasting sample be used to investigate the cause for the hypertriglyceridemia. TC and TG are routinely measured using automated coupled enzymatic assays. These assays are analytically very robust and well standardized. Both

TABLE 9.5 Laboratory assessment of dyslipidemia and CHD.

apoB, apolipoprotein B; apoE, apolipoprotein E; CHD, coronary heart disease; HDL-C, highdensity lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoproteins cholesterol; Lp(a), Lipoprotein(a); NMR, nuclear magnetic resonance spectroscopy; TC, total cholesterol; TG, triglycerides. a Obtain LDL-C if TC $ 240 mg/dL, if TC between 200 and 239 mg/dL with two CHD risk factors, or HDL-C , 35 mg/dL, or if TC , 200 mg/dL with HDL-C , 35 mg/dL.

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values can be used for calculating LDL-C, which is an important first step in classifying patients at risk for CHD. Plasma TG levels are also used for CHD risk calculation and for identifying patients at risk for pancreatitis. Traditionally, HDL-C was measured on a fasting sample by performing a cholesterol test on a supernatant after manually precipitating VLDL and LDL with a polyanion, such as dextran sulfate in the presence of a divalent cation [29]. Because they do not require any sample preparation, such as centrifugation, direct or homogenous assays for HDL-C are fully automated and have become the primary method for this test [29]. There are several different types of direct assays for HDL-C, but they all depend upon masking or enzymatically consuming cholesterol from non-HDL fractions, so that it is not detected when doing the cholesterol test for HDL. These assays work reasonably well, but for some patients with dyslipidemia or rare genetic disorders they can yield discordant results from the classic precipitationbased method [30]. The prime purpose of the HDL-C is to determine CHD risk status by using one of many different types of algorithms, such as the 2013-pooled cohort equations [31] used by the most recent ACC/AHA guidelines [7]. Even though it may not be causally related to the process of atherosclerosis, low HDL-C has a strong inverse association with CHD. This may relate to its inverse relationship with TG, which is not a good biomarker because of its very large biological variability. HDL-C is also used in the calculation of LDL-C. Like HDL-C there are also direct tests for measuring LDL-C. These tests, however, are not as widely used as direct HDL-C tests, because of the ability to calculate LDL-C from the other lipid values in a lipid panel. Also, direct LDL-C tests can sometimes yield erroneous results in patients with dyslipidemia [30], but they are less adversely affected than calculated LDL-C by high TG samples. Although there are many different equations for calculating LDL-C, the first method, the Friedewald equation [32], is still the most widely used. LDL-C is computed as follows: LDL-C 5 TC 2 HDL-C 2

TG ½mg=dL 5

The Friedewald equation, however, is not reliable if the TG are .400 mg/dL or in the case of type III hyperlipoproteinemia, because the TG/5 term is an estimate for VLDL-cholesterol and is invalid under these conditions. Recently, Martin et al. [33] have described a new equation that is similar to the Friedewald equation, but instead of using a fixed factor of 5 for dividing TG, different values of the adjustable factor are used, which depend on TG and non-HDL-C levels: LDL-C 5 TC 2 HDL-C 2

TG ½mg=dL Adjustable factor

It has been shown that the Martin equation is more accurate, particularly on low LDL-C samples, which are now much more common with the advent of anti-PCSK9 monoclonal antibody therapy.

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Regardless of the specific type of lipoprotein test that is done, it is important that it complies with the National Cholesterol Education Program (NCEP) goals for total error, which for TC, TG, HDL-C, and LDL-C are # 8.9%, # 15%, # 13%, and # 12%, respectively [34]. Otherwise, errors in the lab measurements of lipids and lipoproteins can lead to the substantial misclassification of CHD risk, particularly because of the many different lipid cut-points that are used that fall in the middle of the population distribution. Although lipoprotein electrophoresis, once a standard diagnostic test, is no longer recommended for the initial investigation of hyperlipidemia, it still has several useful applications. It can be used to identify the broad β-band in patients with type III hyperlipoproteinemia and can be used to differentiate these patients from the more common type IIb variety, which displays more discrete β and pre-β bands. Also, lipoprotein electrophoresis may be useful to confirm the presence of CM, which remain trapped at the origin on electrophoresis gels because of their large size. In subjects who have high fasting TG . 500 mg/dL, CM can also be seen as a creamy layer on top of a sample stored at 4 C for at least 18 h. Finally, the presence of a backward running band on the gel is usually indicative of the presence of LpX, which can occur in familial LCAT deficiency and more commonly in several types of cholestatic diseases [16]. Unlike a normal lipoprotein, LpX has a vesicular structure with an aqueous core and is primarily composed of phospholipids and free cholesterol. It has very little proteins associated with it. Some patients with cholestasis can have the majority of their plasma cholesterol associated with LpX. Besides causing renal damage, it can reach high enough levels to cause hyperviscosity syndrome and result in laboratory testing artifacts, such as pseudohyponatremia. Other tests, such as assays for apoB and apoA-I, may also be helpful in examining the cause for dyslipidemia. Increased levels of apoB, decreased levels of apoA-I, and an increased apoB/apoA-I ratio have been shown to be good discriminators in identifying patients at risk for CHD [35]. Based on discordant analysis, apoB may even be a better predictor than LDL-C for CHD [36]. As pointed out previously, the apoB assay can be very helpful in patients with combined hyperlipidemia, when the suspected diagnosis is familial combined hyperlipidemia. ApoB was also recently recommended by the AHA/ACC guideline for more carefully assessing CHD risk in patients who are at intermediate risk based on the standard lipid panel [7]. Rarely, β-quantification, an ultracentrifugation procedure for removing VLDL, is required to confirm type III hyperlipoproteinemia. The VLDL-cholesterol/TG ratio is generally $ 0.3 in this disorder and the VLDL exhibits β mobility on electrophoresis [23]. Also in these patients, apoE phenotyping by electrophoresis or now more commonly done by genotyping may be useful for determining whether a patient is homozygous for apoE2. Only two enzyme tests are sometimes measured for lipid disorders, although they are usually only done by specialty laboratories. The first is postheparin LPL activity test to establish the diagnosis of type I

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hyperlipoproteinemia. LPL is normally bound to the vessel wall but can be released into plasma, if a patient is treated with a heparin. Collecting two sets of plasma, before and after an intravenous heparin administration (60 U/kg body weight) determines LPL enzyme activity. The other enzyme test is for LCAT for the diagnosis of familial LCAT deficiency, which can be measured based on its ability to esterify cholesterol. In many cases, however, these two diseases can be diagnosed by the other lab tests and clinical features or by gene sequencing. In addition to the above laboratory tests, there are many other specialty or research laboratory tests (Table 9.5) that may be used to predict CHD risk, such as CRP, Lp(a), HDL subfractions, LDL subfractions, oxidized LDL, remnant lipoproteins, homocysteine, and secretory phospholipase A2 [3,3739]. There is great interest in all of these tests, which are often referred to as emerging risk factor tests. These tests are all being actively investigated, because it is widely recognized that our current laboratory tests are inadequate and do not identify over half of all patients who develop CHD. At this time, however, most of these tests are not incorporated into our current national guidelines except for CRP and Lp(a). CRP is a stable biomarker of inflammation, an important part of the atherosclerosis process and has been shown in multivariant analyses to independently predict cardiovascular events [40]. The most recent AHA/ACC guidelines [7] have recommended the use of CRP in patients at intermediate risk. Using hsCRP concentrations ,1 mg/L are usually considered low risk, 13 mg/L average risk, and .3 mg/L high risk. It is critical to obtain two measurements at least a month apart to ensure that the person does not have any acute inflammatory response, which can also raise CRP. hsCRP assays are now available on many standard immunoassay and chemistry analyzers. Like CRP, the recent AHA/ACC guidelines [7] have also endorsed the use of Lp(a) testing for patients at intermediate risk. This test is usually only performed once, since it does not change much in response to nutritional or drug therapy. There are new drugs such as synthetic oligonucleotides targeting apoB and apo(a) that lowers Lp(a) by up to 50% [41], however, in latestage clinical trials, so it may be necessary to do repeat testing in the future to monitor therapy. Because of problems related to the differential binding of assay antibodies to the different size isoforms of apo(a), this assay is not well standardized at the current time and thus care should be taken when comparing values from different assays. Two different types of units are used for this assay, thus further complicating its use. The role of lipoprotein subfractions for both LDL and HDL for predicting CHD risk is also an active area of investigation, but many of these tests are not well standardized and often require instrumentation that is now widely available [42]. A nuclear magnetic resonance-based assay that detects the methyl groups on lipoproteins has recently been approved by the FDA. This test can measure most of the lipids in the standard lipid panel but also can

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measure the particle number of LDL, which is a better risk marker than LDL-C, particularly in obese individuals and diabetics. Recently, a fully automated assay for cholesterol of small dense LDL particles has also been approved by the FDA and was shown to also be a better predictor of CHD than LDL-C [38,43]. Finally, non-HDL-C, which is really not a test but simply a calculation (TC minus HDL-C) has been recommended as a target goal for treatment of patients with metabolic syndrome and hypertriglyceridemia by many guidelines [44]. Clinical laboratory testing is also important for ruling out the secondary causes of hyperlipidemias. Besides a careful history and physical examination, tests for urea, creatinine, thyroid-stimulating hormone, glucose, albumin, plasma protein electrophoresis (to identify paraproteins), bilirubin, alkaline phosphatase (cholestasis), and a urinalysis for protein and glucose may all be useful for investigating secondary causes of hyperlipidemia. In addition, transaminases (alanine aminotransferase and aspartate aminotransferase) are often measured to monitor hepatic toxicity from various lipid-lowering medications.

Conclusion Given the widespread prevalence of CHD and the availability of effective therapies, the laboratory assessment of lipoprotein disorders is a rare example of a valuable laboratory screening test. Furthermore, most of the current routine lipid and lipoprotein tests are now relatively easy to perform, robust, inexpensive, and because of great efforts over many years, relatively well standardized and accurate. Our current laboratory tests, however, still do not have ideal specificity and sensitivity for predicting CHD risk. Besides differences in the level of circulating lipoproteins, differences in the biologic response of cells involved in atherosclerosis, such as macrophages and endothelial cells, to proatherogenic factors in the plasma may account for our inability to accurately assess CHD risk. Many of these cellular response differences may not be tractable by our current plasma-based tests, but it is likely that such differences could have an underlying genetic cause, which could be tested in the future by the genotyping of blood samples. In addition, the recent experimental findings of the importance of inflammation and oxidation in the pathogenesis of atherosclerosis have raised expectations that in the future some of the emerging risk factor tests will further improve the laboratory assessment of CHD.

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Chapter 10

Disorders of calcium metabolism William E. Winter1 and Neil S. Harris2 1

Department of Pathology, Immunology & Laboratory Medicine, Pediatrics, and Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States, 2 Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States

Introduction Calcium is vital to many aspects of human biology [1]. For example, calcium is involved in intracellular and intercellular signal transmissions, muscular contraction, coagulation, and bone biology. The body has developed intricate regulatory pathways to ensure a narrow range of concentrations of ionized calcium in the plasma, free calcium in the cytoplasm, calcium incorporation into bone, and calcium absorption by the gastrointestinal (GI) tract. Tightly linked to calcium homeostasis are the regulation of plasma phosphate and the incorporation of phosphate into bone together with calcium [2]. From a biodynamic viewpoint, phosphate-containing compounds and their breakdown are critical in facilitating nearly all energy-dependent cellular processes. Likewise, magnesium (e.g., hypomagnesemia) can impact calcium biology [3]. Magnesium is necessary for normal parathyroid hormone (PTH) release and response to PTH. After providing an introductory discussion of calcium biology, this chapter deals with three major topics: (1) hypocalcemia, (2) hypercalcemia, and (3) monitoring of bone turnover.

Calcium distribution in the body The majority of calcium in the body exists in the bone (B98% 2 99%) [4]. One percent of calcium is in the exchangeable pools. The slowly exchangeable pool includes calcium in atheroma or cartilage (e.g., dystrophic [abnormal] calcification) and calcium in the subcellular pool (e.g., mitochondria and endoplasmic reticulum). The rapidly exchangeable pool is calcium in the plasma, interstitial space, cytosol, and surface calcium in recently deposited bone. Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00010-8 © 2021 Elsevier Inc. All rights reserved.

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Cytoplasmic calcium concentrations are tightly controlled and are approximately 10,000 that of extracellular calcium concentrations. For example, elevations in cytoplasmic calcium concentrations trigger muscular contraction.

Circulating calcium Calcium exists in the circulation: (1) in a free (ionized) fraction that is biologically active; (2) complexed with anions (e.g., bicarbonate, phosphate, citrate, and lactate); and (3) bound to plasma proteins, predominantly albumin. Approximately 40%50% of total calcium in the plasma is ionized. The ionized and complexed forms of calcium are filtered by the glomerulus. Proteinbound calcium is not filtered unless there is glomerular disease with loss of pathological amounts of protein. Because total calcium is significantly influenced by the albumin concentration, if hypocalcemia is identified, albumin should be determined, or, preferably, ionized calcium should be measured to assess to what degree a decline in calcium might reflect hypoalbuminemia and not true hypocalcemia. As an estimate, for each decline in albumin of 1 g/dL, total calcium declines by 0.8 mg/dL, yet ionized calcium will be maintained in the reference interval through the actions of PTH and vitamin D [5]. The differential diagnosis of hypoalbuminemia includes (1) decreased synthesis from liver disease or liver damage or decreased substrate (e.g., malnutrition including malabsorption); (2) chronic inflammatory conditions, infections, or cancer; (3) albumin loss (e.g., protein-losing enteropathy, nephrotic syndrome, burns, and eczematous lesions); (4) dilutional hypoalbuminemia (e.g., posthemorrhage hemodilution or the syndrome of inappropriate antidiuretic hormone); (5) increased catabolism (e.g., thyrotoxicosis, pregnancy, infection, and malignancies); and (6) acute inflammation (albumin is a negative acute phase reactant). Hemoconcentration from dehydration and prolonged tourniquet time can elevate total calcium. Because renal function has a profound effect on calcium metabolism, at the minimum, creatinine must always be measured when assessing calcium and phosphate and certainly when the calcium or phosphate concentrations are outside either of their reference intervals. It can be argued that a blood urea nitrogen measurement and urinalysis should also be included, at a minimum, in a basic renal evaluation as well as calculation of the estimated glomerular filtration rate (eGFR).

Calcium measurements Clinical laboratories measure ionized calcium using ion-selective electrodes (ISEs), whereas total calcium is measured with dye-binding assays (e.g., arsenazo III, o-cresolphthalein complexone) or by ISE after release of calcium into the free state by the addition of acid. In an ISE, an electrical potential is developed across a membrane that is selectively permeable to a

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311

specific ion. For spectrophotometric total calcium measurements, the sample is acidified to release calcium from protein. The o-cresolphthalein complexone-calcium complex then forms at an alkaline pH. If plasma calcium is measured, the anticoagulant should be heparin (either sodium or lithium). Ethylenediaminetetraacetic acid (EDTA) (e.g., a purple or lavender top tube) binds calcium and prevents its binding to ocresolphthalein complexone and arsenazo III lowering the measured calcium level. Calcium-EDTA complexes are soluble and can be measured by atomic absorption, although this is rarely performed. Certain forms of gadolinium (used as a radiological contrast agent; specifically gadodiamide and gadoversetamide; gadoteridol, gadopentetate dimeglumine, gadobutrol, and gadoxetate disodium do not reduce measured total calcium) can bind calcium and lower measured calcium without altering the patient’s ionized calcium level. Therefore, some types of gadolinium can cause a “pseudohypocalcemia” [6,7]. Ionized calcium can be measured in whole blood (e.g., heparinized blood, either sodium or lithium heparin) using an ISE; however, serum measurements are an alternative. Proper sample handling to avoid stability problems is important. For the measurement of whole blood ionized calcium, the sample should be delivered to the lab on wet ice within 10 min of being drawn. It should be kept refrigerated and measured within 4 h. For serum-ionized calcium, once the blood has coagulated, the sample should be quickly centrifuged (e.g., within 1 h) following the phlebotomy. The sample must be maintained anaerobically until the assay is completed because loss of carbon dioxide from the sample will alkalinize the sample and lower the ionized calcium concentration because pH changes can affect the balance of ionized versus bound calcium. Alkalosis increases the bound fraction, whereas acidosis increases the ionized fraction. Such changes can also occur in vivo; a hyperventilating patient can develop tetany from an acute decline in the ionized calcium concentration. Specimens must also be promptly analyzed or separated from the cells to prevent the change in pH associated with metabolism. The desirable imprecision for calcium based on biological variation is 0.9%, which is much less than that mandated by the Clinical Laboratory Improvement Amendments (acceptable imprecision is 6 1 mg/dL) [8]. It may be necessary to determine urinary calcium (and phosphate or cyclic adenosine monophosphate [cAMP]) excretion in a 24-h urine collection. The 24-h urine excretion of cAMP can be determined as the cAMP urine concentration multiplied by the urine volume. cAMP can also be expressed as ratio of its concentration relative to the creatinine concentration. Standard procedures are used for such measurements. The upper limit of the reference interval for calcium excretion in men is 300 mg/day and in women it is 250 mg/day. Adults daily excrete about 4 mg of calcium/kg of body weight. To assess the completeness of collection, creatinine should also be measured on a 24-h urine sample. Normal adults

312

SECTION | 2 Analytes

excrete 12 g of creatinine in the urine daily. The upper limit for the calcium to creatinine ratio in adults is 0.14 mg/mg of creatinine. In children, a calcium to creatinine ratio of $ 0.20 mg/mg is considered to be elevated [9]. In this reference, the correlation of the calcium to creatinine ratio to the 24-h urinary calcium excretion was significant (P , .001); however, the strength of correlation was modest (r 5 0.548). Also of interest, 34 year old children had higher calcium excretion (calcium to creatinine ratio: mean 0.16) than children of 59 years old (calcium to creatinine ratio: mean 0.10). The tubular reabsorption of calcium (TRCa) can be calculated as 1 minus the fractional excretion of calcium (U 5 urine; P 5 plasma):    TRCa 5 1  UCa11 =PCa11 3 PCr =UCr

Phosphate measurements 22 Phosphate in serum exists as H2 PO2 4 and HPO4 . This inorganic phosphate is measured by these methods: upon reaction of H3PO4 with (NH4)6Mo7O24 under acidic conditions, (NH4)3[PO4(MoO3)12] is formed that absorbs at 340 nm. Under reducing conditions, molybdenum blue is formed that absorbs at 600700 nm. Xylidyl blue (magnon) or formazan dye can also be used in complexing magnesium. Plasma phosphate concentrations are reported as the plasma phosphorus (the element) concentrations (31 mg/L elemental phosphorus 5 1 mmol/L phosphate). The tubular reabsorption of phosphate (TRP) can be calculated as 1 minus the fractional excretion of phosphate:    TRP 5 1  UPhosphate =PPhosphate 3 PCr =UCr

Clinical indications to measure calcium, phosphate, and related analytes Because calcium is involved in many processes, there are many reasons to measure plasma or serum total calcium. Examples of such indications include central nervous system (CNS) problems (e.g., neurotransmission), GI problems (e.g., motility disorders), muscular problems (e.g., muscle contraction), bone or soft tissue disorders (e.g., calcium is a structural component of bone and can be secondarily deposited in injured soft tissue), cardiac arrhythmias (e.g., signal conduction), renal disease (e.g., calcium abnormalities as a cause or consequence), and cases of acute severe illness where acquired (yet transient) hypoparathyroidism is common. Table 10.1 lists examples of these problems. Calcium should be measured when any therapy is used that can alter calcium (e.g., high-dose vitamin D or vitamin D analogs, PTH, bisphosphates, cinacalcet, or calcitonin [CT]).

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TABLE 10.1 Examples of indications to measure calcium. G

G

G

G

G G

CNS problems: coma, lethargy, decreased mentation, depression, headache, seizures, ataxia GI problems: vomiting, nausea, anorexia, diarrhea, constipation, weight loss, failure to thrive, malnutrition Muscular/join problems: hypotonia, weakness, arthralgias, tetany, muscle cramps, muscle pain Bone/soft tissue disease: rickets, osteomalacia, osteopenia, pathological fractures, soft tissue calcification, any bony abnormality Cardiac arrhythmias: increased or decreased QT interval on EKG Renal disease: any type/any stage; stones, hematuria; decreased urinaryconcentrating ability: unexplained polyuria, polydipsia

Phosphate should be measured even if the calcium is normal in patients with unexplained weakness, renal disease, malnutrition, refeeding following malnutrition, and when drugs are used that can affect phosphate levels (e.g., vitamin D) [10]. Hypophosphatemia can produce respiratory insufficiency from diaphragmatic failure [11]. Cardiomyopathy from hypophosphatemia has been reported [12]. Dietary phosphate restriction and phosphate binders can decrease plasma phosphate concentrations. If the calcium and/or phosphate are abnormal, measurements of alkaline phosphatase and PTH are indicated. Alkaline phosphatase is produced by osteoblasts in response to PTH, by the biliary tract, by the intestine, and by the placenta during the third trimester of pregnancy. In cases of unexplained hypocalcemia, magnesium should also be measured. Chronic hypomagnesemia causes impaired PTH release and impaired PTH responses that can cause hypocalcemia [13,14]. Causes of chronic hypomagnesemia include dietary deficiency, gastrointesntial disease (e.g., malabsorption, pancreatitis), increased urinary excretion (e.g., diuretics, diabetes or use of drugs such as cyclosporine), alcoholism, certain drugs (e.g., proton-pump inhibitors), and a variety of endocrine and metabolic disorders (e.g., hyper or hypothyroidism, hyper or hypoparathyroidism, primary hyperaldosteronism, and hungry bone syndrome) [15]. Congenital magnesium malabsorption is an extremely rare disorder. Acute pancreatitis or massive transfusion with citrated blood can cause a transient hypomagnesemia. Measurement of urinary calcium excretion can be clinically important. For example, measurement of urinary calcium excretion is required to establish the diagnosis of familial hypocalciuric hypercalcemia (FHH) [16]. On the other hand, in primary hyperparathyroidism, if a sufficient degree of hypercalcemia is present, hypercalciuria will be present. This presents a risk for nephrocalcinosis and nephrolithiasis. As noted above, calcium excretion can be expressed as the calcium/creatinine ratio obtained on a spot urine collection, or calcium excretion can be

314

SECTION | 2 Analytes

expressed per 24 h when a 24-h urine collection is obtained. It may be necessary to assess vitamin D stores (i.e., 25-hydroxyvitamin D [25-OHD], calcidiol), the most active vitamin D metabolite (i.e., 1,25-dihydroxyvitamin D [1,25-OH2D], calcitriol), or markers of bone turnover (e.g., bone alkaline phosphatase [BAP], deoxypyridinoline, N-telopeptides, C-telopeptides, and osteocalcin). It can be argued that 1,25-OH2D measurements should only be ordered by endocrinologists as these levels require expertise in interpretation. To properly interprete 25-OHD levels, PTH should be simultaneously measured. For example, if vitamin D intake is deficient, and there is deficient calcium absorption, PTH should be elevated. However, if vitamin D intake is excessive, causing hypercalcemia, PTH should be suppressed. In pathologic conditions of PTH excess, PTH stimulates increased conversion of 25-OHD to 1,25-OH2D. However some patients with hyperparathyroidism do not display frank elevations in 1,25-OH2D presumably due to an ultrashort feedback loop where 1,25-OH2D suppresses its own production. Similarly, reference intervals for 25-OHD and PTH cannot be established without excluding individuals with mild vitamin D deficiency (and subsequent mild secondary hyperparathyroidism due to vitamin D deficiency), which is common in apparently healthy populations.

Calcium and phosphate physiology To understand the appropriate selection and interpretation of laboratory tests, a systematic review of basic physiology is necessary. Total body calcium is controlled primarily through the regulation of calcium absorption from the GI tract (through 1,25-OH2D). On the other hand, the major mechanism controlling total body phosphate is the regulation of phosphate excretion by the kidney (regulated by PTH, vitamin D, and fibroblast growth factor-23).

Parathyroid glands and parathyroid hormone The chief cells of the parathyroid glands secrete PTH. The function of mitochondria-rich parathyroid oxyphilic cells (which are derived from chief cells) is unknown [17]. Normally, there are four parathyroid glands in the neck, but the number of glands can vary between three and five, with ectopic glands located even in the mediastinum [18]. The parathyroid gland senses interstitial ionized calcium through its cell membrane calcium-sensing receptor (CaSR; chromosome 3q13.33-q21.1) (Fig. 10.1) [19]. Interstitial ionized calcium reflects plasma-ionized calcium. The CaSR is a 1078-amino acid transmembrane protein with a large extracellular domain that interacts with ionized calcium. There are seven transmembrane domains and a cytoplasmic tail. The CaSR is a member of the G-protein-coupled receptor family [20]. The factors that regulate PTH synthesis and secretion include (1) plasma-ionized calcium; (2) plasma phosphate; (3) 1,25-dihydroxyvitamin D

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315

Ionized Ca++ NH2

Parathyroid chief cell

CaSR COOH

Rough endoplasmic reticulum

Secretory granule

Pre-pro PTH [115 AA] V -25 AA Pro-PTH [90 AA] V -6 AA PTH(1–84) [84 AA]

PTH(1–84)

PTH(7–84)

PTH(1–84)

PTH(7–84)

Other PTH fragments FIGURE 10.1 Interstitial ionized calcium (reflective of plasma-ionized calcium) is monitored by the calcium-sensing receptor (CaSR) located on the cell surfaces of the parathyroid gland chief cells and renal tubular cells. In the parathyroid chief cells parathyroid hormone (PTH) is synthesized from prepro-PTH (115 amino acids) that is first converted to pro-PTH (90 amino acids) upon entry into the lumen of the rough endoplasmic reticulum and finally PTH(184) (i.e., 84 amino acids). Presumably conversion of PTH(184) to PTH(784) occurs in a secretory granule and will occur after secretion. Extracellularly PTH(184) can also be metabolized to various PTH fragments.

(1,25-OH2D), and (4) fibroblast growth factor-23 (FGF23). Their actions are summarized in Table 10.2. A decline in ionized calcium results in signaling through CaSR with the release of PTH. Elevated phosphate can also release PTH (note: PTH has a phosphaturic effect on the renal tubules). Vitamin D inhibits PTH synthesis. The 50 regulatory region of the PTH gene has response elements for vitamins D and A. When 1,25-OH2D binds to the vitamin D receptor (VDR; chromosome 12q13.11), this ligandreceptor complex heterodimerizes with the

316

SECTION | 2 Analytes

TABLE 10.2 Regulation of PTH secretion. Factor

Effect on PTH release

Increased ionized calcium

Decreased PTH

Increased phosphate

Increased PTH

Increased 1,25-OH2D

Decreased PTH

Increased FGF23

Decreased PTH

retinoic acid X receptor that serves as a transcription factor binding to the 50 regulatory region of the PTH gene. Finally, FGF23 serves to lower plasma phosphate and suppresses PTH [21]. This action of FGF23 may relate to the effect of PTH on increasing the formation of 1,25-OH2D, which will raise phosphate through increased GI tract absorption. FGF23 also stimulates 24-hydroxylase (see below) and inhibits the conversion of 25-OHD to 1, 25-OH2D. The actions of FGF23 are mediated by its binding to FGFR1 (fibroblast growth factor receptor 1; chromosome 8p11.23) and to, a lesser degree, FGFR4 (fibroblast growth factor receptor 4; chromosome 5q35.2). Klotho (KL; chromosome 13q13.1) is a coreceptor for FGF23 [22]. PTH is initially synthesized within the cell as a 115-amino acid preproPTH molecule (Fig. 10.1). As prepro-PTH enters into the endoplasmic reticulum, the pre- (or leader) 25-amino acid sequence is cleaved. Within the endoplasmic reticulum, pro-PTH (90 amino acids) is cleaved to intact PTH (84 amino acids; PTH[184]). Once secreted, PTH(184) has a half-life of only B4 min. Within the parathyroid cell and after secretion, PTH is metabolized to PTH(784), N-terminal-truncated PTH fragments of 33 or 36 amino acids, as well as carboxyl (C)-terminal fragments [23,24]. The concentration of C-terminal fragments is much higher than that of intact or N-terminal-truncated fragments. C-terminal PTH is cleared via the kidney. PTH(784), the most prevalent N-terminal-truncated PTH form, appears to have the opposite biologic effect to that of PTH, whereas PTH(184) raises calcium, PTH(784) lowers calcium and is antiresorptive [25]. PTH(784) binds to a C-terminal PTH (C-PTH) receptor [26]. The bioactivity of PTH is contained in its first 34 amino acids. In the circulation, the ratio of PTH(184) to PTH(784) varies in health and disease. Usually only 20% of PTH immunoreactivity is PTH(184) with the remaining immunoreactivity due to C-terminal fragments [27]. PTH (784) is increased in concentration relative to PTH(184) in hypercalcemia and chronic renal failure, and is decreased in hypocalcemia. Table 10.3 summarized the actions of PTH and PTH fragments [28]. PTH(184) normally binds to the PTH receptor-1 (PTHR1), whose gene is located on chromosome 3p21.123p21.31. Because PTH-related

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TABLE 10.3 Actions of PTH and PTH fragments. PTH (intact or fragment)

Effect on plasma calcium

Receptor

184

Increases

PTH/PTHrP receptor (PTHR1)

134

Increases

PTH/PTHrP receptor (PTHR1)

784

Decreases

C-PTH receptor

peptide (PTHrP; see below) also binds to the PTHR1, PTHR1 can also be referred to as the PTH/PTHrP receptor. PTHR1 is a member of the group II subgroup of the G-protein-linked receptor superfamily. The receptor has a large extracellular domain, seven transmembrane domains (e.g., heptahelical), and a cytoplasmic tail similar to the calcium-sensing and thyroid-stimulating hormone receptors. Other examples of this receptor family include receptors for glucagon, glucagon-like peptide I, CT, growth hormone-releasing hormone, secretin, and vasoactive intestinal polypeptide. PTHrP is produced by some cancers and can cause hypercalcemia through its PTH-like actions. This type of hypercalcemia is termed “humoral hypercalcemia of malignancy.” However, PTHrP is not detected in PTH immunoassays, and separate immunoassays for PTHrP are available from reference laboratories. A second PTH receptor (PTHR2; encoded on chromosome 2q33) binds PTH. Sites of expression of PTHR2 include in the brain, pancreas, testis, and placenta. PTHR2 does not appear to be involved in calcium/phosphate homeostasis [29]. When PTH binds to its receptor, PTHR1 changes conformation and is able to interact with a variety of G-proteins including the guaninestimulatory (Gs) complex and Gq/11 [30] (Fig. 10.2). In the basal state, Gs includes Gsα, Gsβ, and Gsϒ. Guanosine diphosphate (GDP) is bound to Gsα in this basal state. The interaction of PTHR1 with Gs leads to the release of Gsβ and Gsϒ from Gsα, whereas Gsα releases GDP and binds guanosine triphosphate (GTP). Gsα-GTP next activates adenylate cyclase, converting adenosine triphosphate (ATP) to cAMP. cAMP then carries forward the PTH signal within the target cell. Gsα-GTP is self-regulatory: Gsα-GTP expresses GTPase activity cleaving a phosphate from GTP yielding GDP. The resulting Gsα-GDP recombines with Gsβ and Gsϒ, and Gs in its basal state is reconstituted, and continued stimulation of adenylate cyclase is terminated. PTHR1 activation via PTH also stimulates phospholipase C activity via Gq/11. Other G-proteins include G12/13 and Gi.

318

SECTION | 2 Analytes

Gα PTH

GDP GTP

GDP

NH2

Gα PTHR1

COOH

GαβΥ

GTP

GβΥ

GDP

GTPase

Adenylate cyclase (inactive) ATP Adenylate cyclase (active)

cAMP

Gα GDP

Pi

FIGURE 10.2 When PTH binds to the PTHR1, interaction with the GS (s 5 stimulatory) protein complex stimulates Gαβϒ to separate into Gα and Gβϒ. GDP is attached to Gα (Gα-GDP) in the basal complex. GDP is then replaced by GTP activating Gα (Gα-GTP). Gα-GTP activates adenylate cyclase which converts ATP to cAMP. In addition to activating adenylate cyclase, Gα-GTP acquires GTPase activity. This GTPase activity cleaves the GTP of Gα-GTP to Gα-GDP. This turns off the process and Gα-GDP reassociates with Gβϒ to reconstitute the basal G protein Gαβϒ.

Calcium and the renal tubules Normally 70% of filtered calcium is reabsorbed in the proximal convoluted tubule (PCT) (Fig. 10.3). The thick ascending loop of Henle (TAL) reabsorbs 20% of filtered calcium with 9% reabsorbed in the distal convoluted tubule (DCT). One percent of filtered calcium is excreted in the urine. In the DCT calcium reabsorption is increased by PTH and 1,25-OH2D. In the PCT and TAL, calcium reabsorption parallels sodium reabsorption. The clinical correlate is that volume depletion will increase renal tubule sodium and calcium reabsorption. In contrast, administering IV saline will encourage calciuria potentially lowering plasma calcium. This is why IV fluids, in part, can help lower plasma calcium concentrations. As well, diluting plasma albumin (with expanded plasma volume from IV fluid infusion) will lower plasma calcium. However, IV fluids would not lower ionized calcium by this mechanism if PTH or vitamin D were in excess. Loop diuretics, such as furosemide that inhibit NKCC2 (the Na-K-2Cl Cotransporter; also known as SLC12A1 [solute carrier family 12 member 1]; chromosome 15q21.1), lower plasma calcium by increasing calciuria which is secondary to the natriuretic effect of the diuretic. In contrast to loop diuretics, thiazide diuretics that inhibit NCC (Na-Cl Cotransporter; also known as SLC12A3 [solute carrier family 12 member 3; chromosome 16q13]) cause increased calcium reabsorption as DCT sodium wasting elicits increased sodium reabsorption by the PCT and TAL (which increase sodium reabsorption and calcium reabsorption is thus increased).

Disorders of calcium metabolism Chapter | 10 ~9%

PCT

Glomerulus

319

DCT

TAL ~70% ~20%

Collecting duct Loop of Henle ~1% excreted FIGURE 10.3 Approximately 70% of filtered calcium is reabsorbed in the proximal convoluted tubule (PCT). Approximately 20% of filtered calcium is reabsorbed in the thick ascending loop of Henle (TAL). Approximately 9% of filtered calcium is reabsorbed in the distal convoluted tubule (DCT). Approximately 1% of filtered calcium is excreted.

Cells expressing PTHR1 include renal tubule cells and osteoblasts. In the renal tubular cells, there is increased calcium reabsorption from the renal tubular fluid in the TAL and DCT in response to PTH and 1,25-OH2D. Physiologically, hypercalciuria is to be avoided as hypercalciuria can ultimately cause renal failure from nephrocalcinosis and/or obstruction from nephrolithiasis. Similar to the GI tract, calcium is reabsorbed from renal tubular fluid through paracellular (between cells) and transcellular (across cells) routes [31]. Paracellular calcium absorption is driven by charge differences through transmembrane tight junctions involving claudins 2 (CLDN2; chromosome Xq22.3), 12 (CLDN12; chromosome 7q21.13), and 15 (CLDN15; 7q22.1) [32]. In the PCT, 20% of calcium reabsorption is transcellular utilizing an apical calcium channel and a basal calcium-ATPase pump (Fig. 10.4). Eighty percent of PCT calcium reabsorption is paracellular and is driven by charge. In the TAL, there are both transcellular and paracellular calcium reabsorptions (Fig. 10.5). The transcellular calcium reabsorption of the calcium in the DCT utilizes CaT2 (TRPV5: transient receptor potential cation channel subfamily V member 5; chromosome 7q34) (Fig. 10.6) [33]. The CaSR is involved in the regulation of urinary calcium reabsorption. The CaSR is expressed in the PCT, TAL, and DCT [34]. The CaSR is

320

SECTION | 2 Analytes

Na+ Na+/K+

Ca++

Interstitium

Ca++-ATPase

ATPase pump

K+ Transcellular

PCT cell

PCT Paracellular cell

20%

80%

Ca++ channel

NHE3 Tubular lumen

Na+ H+

Ca++

(filtered)

Cl–

Ca++

FIGURE 10.4 In the proximal convoluted tubule (PCT), 80% of filtered calcium (Ca11) reabsorption is paracellular (driven a 11 to 13 mV charge). Twenty percent of filtered calcium reabsorption is transcellular. Calcium reabsorption follows sodium (Na1) reabsorption. Transcellular calcium reabsorption is accomplished by a calcium (Ca11) channel. Sodium reabsorption is accomplished by the sodium/hydrogen exchanger NHE3 (SLC9A3; solute carrier family 9 member A3). Sodium exits the cell into the interstitium via the sodium/potassium (Na1/K1) ATPase pump. Intracellular calcium exits the cell via calcium (Ca11) ATPase to enter the interstitium.

Na+/K+ ATPase pump

Na+

Basolateral Cl channel

Ca++

Interstitium

Ca++-ATPase

TAL cell

Barttin

K+

TAL cell

Transcellular ROMK

NKCC1

Paracellular

Ca++ channel

Tubular lumen

Na+ K+ Cl–

K+

Ca++ (filtered)

Ca++

FIGURE 10.5 In the thick ascending loop of Henle (TAL), paracellular reabsorption of calcium (Ca11) is charge driven similar to the PCT (11 to 13 mV). Calcium reabsorption follows sodium (Na1) reabsorption. Transcellular calcium reabsorption is accomplished by a calcium (Ca11) channel. Sodium reabsorption results from the action of the sodiumpotassiumchloride carrier [NKCC1; SLC12A2; solute carrier family 12 member 2]. Reabsorbed potassium (K1) can leave the cell via ROMK (renal outer medullary K1 channel; KCNJ1; potassium voltage-gated channel subfamily J member 1). Chloride (Cl2) exits the cell via the basolateral chloride channel that is associated with barttin [BSND (Barttin CLCNK Type Accessory Beta Subunit); gene located on chromosome 1p32.3]. Intracellular calcium leaves the cell via calcium (Ca11) ATPase to enter the interstitium.

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321

Na+/Ca++ exchanger

Na+/K+ ATPase pump

K+

DCT cell

Na+

Ca++

Ca++

Interstitium

Ca++-ATPase

Na+

Transcellular

DCT cell

TRPV5

NCC Tubular lumen

Na+ Cl–

Ca++ (filtered)

FIGURE 10.6 In the distal convoluted tubule (DCT), transcellular calcium reabsorption is accomplished by the calcium channel TRPV5 (transient receptor potential cation channel subfamily V member 5). Sodium reabsorption results from the action of the sodium chloride carrier (NCC: Na-Cl cotransporter; SLC12A3, solute carrier family 12 member 3). Intracellular calcium leaves the cell via a calcium (Ca11) ATPase and via a sodiumcalcium (Na1/Ca11) exchanger to enter the interstitium.

most highly expressed in the TAL [35]. When lower plasma calcium concentrations are present, there is reduced stimulation of the CaSR and this leads to increased calcium reabsorption [36]. On the other hand, when plasma calcium concentrations are higher, there is increased stimulation of the CaSR causing calciuria. Therefore, the renal tubular expression of the CaSR provides a mechanism, in part, to regulate plasma calcium independent of the parathyroid glands, C-cells, vitamin D, and FGF23.

Phosphate and the renal tubules Of the filtered phosphate, B80% is reabsorbed by the PCT, whereas B10% is reabsorbed in the DCT and the remaining 10% is excreted (Fig. 10.7). PCT TRP is regulated by PTH, 1,25-OH2D, and FGF23. 1,25-OH2D increases phosphate reabsorption similar to its actions on the intestine. PTH and FGF23 cause increased phosphaturia. These hormones regulate the sodium-dependent cotransporter Npt2c (NaPi-IIa; SLC34A3: sodium-linked channel family 34 member 3; chromosome 9q34.3) and the sodiumphosphate cotransporter Npt2a (NaPi-IIa; SLC34A1: sodium-linked channel family 34 member 1; chromosome 5q35.3) (Fig. 10.8). In humans, Npt2c is more important in phosphate reabsorption than Npt2a [37]. The role of PiT2

322

SECTION | 2 Analytes ~10%

PCT

Glomerulus

DCT

TAL ~80%

Collecting duct Loop of Henle ~10% excreted FIGURE 10.7 Approximately 80% of filtered phosphate is reabsorbed in the proximal convoluted tubule (PCT). Approximately 10% of filtered phosphate is reabsorbed in distal convoluted tubule (DCT). Approximately 10% of filtered phosphate is excreted.

K+ Na+ HPO4–

Na+/K+ ATPase pump

Interstitium

Transcellular Sodium phosphate cotransporter IIa PCT cell

Npt2a

Sodium phosphate cotransporter IIc Npt2c

PiT2

PCT cell

Filtered – > PO4– Tubular lumen

3 Na+ HPO4– 2 Na+ HPO4– 2 Na+ H2PO4–

FIGURE 10.8 Three transporters reabsorb filtered phosphate in the proximal convoluted tubule (PCT): the sodium-phosphate cotransporter IIa (Npt2a; SLC34A1, solute carrier family 34 member 1), the sodium-phosphate cotransporter IIc (Npt2c; SLC34A3, solute carrier family 34 member 3), and PiT2 (SLC20A2, solute carrier family 20 member 2).

(SLC20A2; solute carrier family 20 member 2; chromosome 8p11.21) on phosphate reabsorption is controversial. Several other genes that relate to phosphate homeostasis will be discussed in the section on phosphatemic rickets including PHEX, DMP1, ENPP1, and CLCN5.

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323

Parathyroid hormone and bone PTH stimulates osteoblasts to build new bone. Stimulated osteoblasts secrete alkaline phosphatase, which is a marker of bone activity. Alkaline phosphatase in growing children is normally two to three times higher than in adults. Adults with healing fractures will have increased alkaline phosphatase levels in the blood. Osteoblasts that become “encased” in ossified bone become osteocytes. Stimulation of osteoblasts will subsequently cause increased osteoclast activity as osteoblast and osteoclast activities are coupled together. Osteoclasts break down the bone. Normal levels of PTH are necessary for healthy bone turnover. However, excessive PTH leads to increased bone breakdown, with a relative abundance of osteoclastic activity compared with osteoblastic activity. Bone is a dynamic biomaterial that in health displays beneficial compromises between lightness and strength, and stiffness and flexibility [38]. Osteopenia, even in the absence of frank osteoporosis, is a major health problem in the elderly that is associated with fractures [39]. Normal intermittent pulses of PTH stimulate the development of osteoblasts from stromal cells (Fig. 10.9). Stimulated osteoblasts secrete Stromal (osteoprogenitor) cell

Intermittent PTH Osteoblast

M-CSF OPG RANκ

RANκL

Osteoclast

Precursor fusion Macrophage

Osteoclast precursor

FIGURE 10.9 Under intermittent exposure to PTH, a stromal osteoprogenitor cell becomes an osteoblast. Via the secretion of macrophage colony-stimulating factor (M-CSF) from osteoblasts, macrophages become osteoclast precursor cells. Through the interaction of receptor activator of nuclear factor NFκB ligand (RANκL) on osteoblasts and RANK on osteoclast precursors, the osteoclast precursors are stimulated and fuse one to the other forming multinucleated osteoclasts. Osteoblasts produce osteoprotegerin (OPG) which is a soluble decoy receptor for RANκL. In this way, osteoblasts regulate the stimulation of osteoclast precursors as OPG blocks the RANκLRANκ interaction.

324

SECTION | 2 Analytes

macrophage colony-stimulating factor that stimulates the replication of macrophages into osteoclast precursors. Osteoblasts express the receptor activator of NFκB ligand (RANκL) that binds to receptor activator of NFκB (RANκ) on osteoclast precursors. This stimulates osteoclast precursor differentiation and fusion into osteoclasts. RANκL is also known as osteoclast-differentiating factor. Osteoblasts also secrete osteoprotegerin (OPG) serves as a soluble decoy receptor for RANκL. This binds to RANκL antagonizing the action of osteoblasts in otherwise stimulating osteoclasts. OPG is clinically important as mutations in OPG can cause juvenile osteoporosis. RANκL is clinically important as the monoclonal antibody denosumab can be used to treat osteoporosis.

Calcitonin and bone The CT gene family includes five members (Table 10.4). The major clinically relevant gene products are CT (323 amino acids) and pro-CT (PCT; 116 amino acids). CT is secreted by the parafollicular clear (C) cells within the interfollicular areas of the thyroid gland. PCT is produced by many tissues; its secretion in inflammatory conditions may be from adipose tissue [40]. CT-related polypeptide-I (CGRP-I) is found within the CNS where it may act as a neurotransmitter [41]. Katacalcin lowers calcium [42]. The identification of katacalcin is elusive: one reference identifies katacalcin as amino acids 96 to 106 of PCT that would be within CGRP-I [43]. CGRP-II induces vasodilation of the coronary, cerebral, and systemic blood vessels. Its presence in the CNS also suggests a role as a neurotransmitter or neuromodulator [44] Adrenomedullin is also a vasodilator [45].

TABLE 10.4 Calcitonin gene family. Gene

Protein product(s)

Chromosome location

CALCA; calcitonin-related polypeptide alpha

CT, CT generelated peptide-I (CGRP-I) and katacalcin

11p15.2

CALCB; calcitonin-related polypeptide beta

CT generelated peptide-II (CGRP-II)

11p15.2

CALCP; calcitonin pseudogene

Pseudogene

11p15.2

AD; adrenomedullin

Adrenomedullin

11p15.4

IAPP; islet-associated polypeptide

IAPP (also known as amylin)

12p12.1

CT, calcitonin; IAA, islet-associated polypeptide.

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Islet amyloid polypeptide (IAPP; also known as amylin) is produced by pancreatic islet β cells and is cosecreted with insulin. Amylin forms the amyloid deposits seen in and around the islets in about half of the patients with type 2 diabetes who come to autopsy. Amylin injections are available for use therapeutically in the treatment of type 2 diabetes. Pharmacologically, pramlintide, a synthetic analog of amylin, delays gastric emptying and thus can attenuate rises in blood glucose following meals. Pramlintide also suppresses glucagon secretion and centrally regulates food intake. Concerning CT, osteoclasts are inhibited and plasma calcium is lowered by pharmacological levels of CT. Although CT is a very important hormone in saltwater fish because saltwater contains high levels of calcium, CT in humans has modest physiological importance. For example, if patients receive adequate thyroid hormone replacement following surgical thyroidectomy, bone density will be normal despite CT deficiency. CT deficiency does not cause hypercalcemia. Serum CT measurements are important tumor markers for the monitoring of residual medullary thyroid carcinoma (MTC) following thyroidectomy [46]. MTC can occur sporadically and predominantly in older individuals. MTC in younger individuals frequently occurs as part of the multiple endocrine neoplasia type 2 (MEN2) [47], whereas B100% of persons with MEN2 will develop MTC, 50% will develop pheochromocytoma. Lesser numbers of individuals affected with MEN2 will exhibit hyperparathyroidism. In search of residual MTC following thyroidectomy, provocative testing with calcium or pentagastrin is more sensitive and specific than the measurement of basal CT levels [48]. Familial (autosomal dominant) MTC independent of MEN2 does occur [49]. Familial MTC can be caused by RET proto-oncogene mutations (see below). As an aside, a drug administered by injection or as a nasal spray, salmon CT (Miacalcin) is Food and Drug Administration (FDA) approved for the treatment of osteoporosis in women who have been menopausal for at least 5 years.

Procalcitonin—a marker of bacterial infection As noted above, the precursor of CT is PCT. PCT has been proposed as an inflammatory marker indicative of serious bacterial infections [50]. Studies in tissue culture demonstrate that interleukin-1β stimulates PCT production, whereas interferon gamma reduces PCT production [51]. PCT increases within 68 h of the onset of fever, plateaus at 12 h, and decreases with antibiotic administration. Thus, PCT is an acute phase reactant; however, its biologic role in inflammation is unclear. It displays a higher sensitivity and specificity for sepsis than C-reactive protein (CRP). Elevations in PCT appear to predict bacterial infection versus a nonbacterial source of infection.

326

SECTION | 2 Analytes

PCT also predicts localized infection better than CRP. Elevations in PCT are associated with multiple-organ dysfunction and increased mortality. The concept for the clinical use of this marker is that lower PCT concentrations suggest a nonbacterial infection not requiring antibiotic therapy improving antibiotic stewardship [52]. However, the value of such testing is controversial [53,54]. A recent study in more than 1600 patients with lower respiratory tract infections found that PCT testing did not reduce antibiotic use [55].

Vitamin D physiology Vitamin D metabolism and actions are critical to calcium and phosphate homeostasis including bone health. The intestine is where total body calcium is regulated and net calcium gain occurs. Vitamin D controls the absorption of dietary calcium and phosphate. Dietary (e.g., plant) ergosterol is a provitamin D2 compound. Ergosterol is converted by light exposure to previtamin D2 and then isomerized to vitamin D2 (ergocalciferol) (Fig. 10.10). Endogenous 7-dehydrocholesterol in skin is a provitamin D3. Light converts provitamin D3 to previtamin D3, which isomerizes to vitamin D3 (cholecalciferol) (Fig. 10.11). Because vitamin D is a fat-soluble vitamin, the liver is responsible, in part, for dietary vitamin D absorption. The liver is also the site of conversion of vitamin D to 25-OHD through vitamin D 25-hydroxylase (CYP2R1; cytochrome P450 family 2 subfamily R member 1; chromosome 11p15.2) (Fig. 10.12). Vitamin D stores, measured as 25-OHD, are dependent on ultraviolet light exposure and dietary vitamin D intake. The conversion of 25-OHD to 1,25-OH2D by 25-hydroxyvitamin D, 1αhydroxylase (CYP27B1; chromosome 12q14.1) is tightly controlled in the renal tubule. The conversion is stimulated by PTH, whereas the conversion is inhibited by elevated phosphate concentrations, elevated concentrations of 1,25-OH2D (i.e., an ultrashort-negative feedback loop), FGF23, elevated Provitamin D2

Previtamin D2

ergosterol

Vitamin D2 ergocalciferol

OH

HO

UV light 290–315 nm

Thermal isomerization HO

CH2

FIGURE 10.10 Provitamin D2 (ergosterol) is transformed to previtamin D2 by UV light at 290315 nm. Previtamin D2 then thermally isomerizes to vitamin D2 (ergocalciferol).

Provitamin D3

Previtamin D3

Vitamin D3

7-dehydrocholesterol (present in epidermis)

Cholecalciferol a.k.a., calciol

OH

HO

UV light 290–315 nm

Thermal

CH2

isomerization HO

FIGURE 10.11 Provitamin D3 (7-dehydrocholesterol present in the epidermis) is transformed to previtamin D3 by UV light at 290315 nm. Previtamin D3 then thermally isomerizes to vitamin D3 (cholecalciferol, also known as calciol).

OH

CH2 HO

Liver

CH2 HO

Vitamin D3

OH

Renal tubule

CH2 HO

25-OHD3

OH

1,25-OH2D3

FIGURE 10.12 Illustrated for vitamin D3 is its 25-hydroxylation to 25-hydroxyvitamin D3 (25-OHD3) by 25-hydroxylase in the liver. Subsequently, 25hydroxyvitamin D3 (25-OHD3) is converted to 1,25-dihydroxyvitamin D3 (1,25-OH2D3) by 1α-hydroxylase in the renal tubule. These metabolic conversions are similar for vitamin D2.

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calcium, and a high-calcium diet. In addition to increasing the absorption of calcium and phosphate from the gut, 1,25-OH2D is also necessary for normal bone remodeling. Some endocrinologists consider 1,25-OH2D as the active vitamin D “hormone,” whereas 25-OHD is a “prohormone.” 1,25-OH2D is approximately 1000 times more potent than 25-OHD; however, the concentration of 25-OHD is 1000-fold greater than 1,25-OH2D. A typical adult reference interval for 25-OHD is 954 ng/mL versus 1560 pg/mL for 1,25-OH2D. Despite this typical laboratory reference interval for 25-OHD, many experts define vitamin D deficiency as a 25-OHD of ,20 ng/mL, relative insufficiency as 2029 ng/mL, and vitamin D sufficiency as 30 ng/mL or more [56]. However, what constitutes a “healthy” range for 25-OHD is very controversial [57]. Vitamin D toxicity is only observed when levels of 25-OHD exceed B100 ng/mL. From a pharmacologic perspective, extremely high-dose oral vitamin D (e.g., 4000 units/day to 10,000 units/day) reduces bone mineral density (BMD) [58]. Although there has been enthusiasm for a role for vitamin D in immunity [59], as of 2011, the Institute of Medicine’s (IOM) literature review did not support a role for vitamin D in immunity [60]. The IOM concluded that the only proven physiological role for vitamin D is in bone health. Needless to say, this is frequently challenged in the literature [61].

25-OHD and 1,25-OH2D metabolism An emerging area of interest is the measurement of the metabolites of 25-OHD and 1,25-OH2D (Fig. 10.13). 25-OHD can be converted to 24,25-dihydroxyvitamin D (24,25-OH2D) by the action of 24-hydroxylase (CYP24A1; cytochrome P450 family 24 subfamily A member 1; chromosome 20q13.2). 24,25-OH2D does not have substantial vitamin D activity. However, a recent study suggests that 24,25-OH2D assists in fracture healing involving the transmembrane receptor FAM57B2 (family with sequence similarity 57, member B) [62]. 24-hydroxyase also acts on 1,25-OH2D converting 1,25-OH2D to 1,24,25-trihydroxyvitamin D (1,24,25,-OH3D), which is inactive similar to 24,25-OH2D (e.g., Babsent binding to the VDR). This is referred to as the C24 oxidation pathway. Therefore we have two pathways that oppose one another: 25-OHD, 1α-hydroxylase increases 1,25-OH2D; whereas, 24hydroxylase reduces 1,25-OH2D concentrations. While increasing the activity of 25-OHD, 1α-hydroxylase, PTH also suppresses 24-hydroxylase [63]. Similar are the actions of phosphate deficiency in raising 1,25-OH2D concentrations and reducing 24-hydroxylase activity. The actions of FGF23 and 1,25-OH2D are opposite to those of PTH (e.g., reduced conversion of 25-OHD to 1,25-OH2D and increased 24-hydroxylase activity inactivating 25-OHD [to 24,25-OH2D] and 1,25-OHD [to 1,24,25OH3D]). Important regulators are the ultrashort-negative feedback loop with

330

SECTION | 2 Analytes Vitamin D (D2 or D3) v 25-hydroxylase (liver) v 25-OHD v 1α-hydroxylase (kidney)

PTH (–) FGF23 (+) 1,25-OH2D (+)

24-hydroxylase

24,25-OH2D

PTH (+) FGF23 (–) PO 43– (–) 1,25-OH2D (–)

v v (–)

v

1,25-OH2D

24-hydroxylase

PTH (–) FGF23 (+) 1,25-OH2D (+)

1,24,25-OH3D FIGURE 10.13 The metabolism of 25-hydroxyvitamin D (25-OHD) to 24,25-dihydroxyvitamin D (24,25-OH2D) via 24-hydroxylase is illustrated. The metabolism of 1,25-dihydroxyvitamin D (1,25-OH2D) to 1,24,25-trihydroxyvitamin D (1,24,25-OH3D) via 24-hydroxylase is also illustrated. 24-Hydroxylase is stimulated by FGF23 and 1,25-OH2D and is suppressed by PTH. On the other hand, 1α-hydroxylase is stimulated by PTH and is suppressed by FGF23, phosphate (PO32 4 ) and 1,25-OH2D.

1,25-OH2D suppressing its own form formation and the conversion of 1, 25-OH2D to 1,24,25-OH3D through 24-OHylase. There is an evidence for a 23 lactone pathway where 1,25-OH2D is converted to 1,23,25-trihydroxyvitamin D (1,23,25-OH3D; also known as 1α, 25-(OH)2D-26,23-lactone) through 24-hydroxylase [64]. However, this pathway may not be physiologically important. Because of the tight regulation of 1,25-OH2D concentrations, because of various compensations, 1,25-OH2D is usually within the reference interval even in deficiency states. One can then conclude that there are few clinical situations where 1,25-OH2D should be measured. Possibly the best use of 1,25-OH2D measurements is in the evaluation of persons with suspected vitamin Ddependent rickets (VDDR) (see below).

Calcium and phosphate absorption from the gut Transcellular dietary calcium absorption involves three steps (Fig. 10.14). First, calcium enters the intestinal brush border cells through a calcium

Disorders of calcium metabolism Chapter | 10

Ca++

Ca++

Ca++

CaT2

Ca++

Ca++

Calbindin D

Na+

Ca++

Ca++-ATPase

Na+/Ca++ exchanger

Cav1.3

TRPV6 Ca++

Ca++

331

Ca++

Ca++

FIGURE 10.14 Calcium in the intestinal lumen enters the cytoplasm of enterocytes via CaT2 (SLC7A2, solute carrier family 7 member 2), TRPV6 (transient receptor potential cation channel subfamily V member 6) and Cav1.3 (CACNA1D, calcium voltage-gated channel subunit α1 D). Calbindin D (CALB1, calbindin 1) binds absorbed cytoplasmic calcium during its passage to the basal membrane for cellular export via a calcium ATPase and via a sodiumcalcium exchanger. Calbindin D maintains a low concentration of ionized calcium within the cytoplasm of the enterocytes.

transporter (CaT1; also known as TRPV6: transient receptor potential cation channel subfamily V member 6; chromosome 7q34). To a lesser extent, CaT2 (SLC7A2; solute carrier family 7 member 2; chromosome 8p22) is responsible for transcellular calcium absorption [65]. Cav1.3 (CACNA1D; calcium voltage-gated channel subunit α1 D; chromosome 3p21.1), an apical L-type calcium channel, may also provide for calcium absorption by the intestine [32,66] Next, the 9-kDa cytosolic calcium-binding protein calbindin D (CALB1; calbindin 1; chromosome 8q21.3) mediates calcium diffusion within the cell. This allows the cytoplasmic calcium concentration to remain physiologically low. Finally, calcium is pumped out of the cell by a basolateral membrane calcium ATPase (ATP2B1; ATPase plasma membrane Ca21 transporting 1;

332

SECTION | 2 Analytes

chromosome 12q21.33) and a Na1/Ca21 exchanger (SLC8A1: solute carrier family 8 member A1; chromosome 2p22.1). The first two steps are rate limiting in the process of calcium absorption; TRPV6 (CaT1) synthesis is B90% dependent on 1,25-OH2D, whereas calbindin D synthesis is completely dependent on 1,25-OH2D. Usually, about 70% of dietary phosphate is absorbed. This is dependent upon the actions of 1,25-OH2D on the gut. There is a sodium-phosphate transporter expressed in the gut apical membrane: NaPi2b (SLC34A2; see discussion of hypophosphatemic rickets below).

Integrating parathyroid hormone and vitamin D actions In summary, elevated PTH enhances bone resorption and increases calcium and phosphate concentrations in the blood due to bone resorption. Increased absorption of calcium and phosphate from the intestine stimulated by 1,25OH2D (consequent to an elevated PTH action) also leads to increased calcium and phosphate in plasma. PTH does increase renal tubular calcium reabsorption to increase calcium concentration. However, PTH causes phosphaturia that is of sufficient magnitude to reduce circulating phosphate concentrations despite its effects on the intestine and bone in raising phosphate. Thus, the overall effect of PTH is to raise calcium and decrease phosphate concentrations (Fig. 10.15). From this basic physiology, it can be readily predicted that PTH excess can cause hypercalcemia, hypophosphatemia, and an elevated alkaline phosphatase concentration, whereas PTH deficiency causes hypocalcemia and hyperphosphatemia. If the plasma calcium and phosphate concentrations are both elevated to the higher reference intervals or above, ectopic calcification can occur. As a rule of thumb, if the calcium concentration (in mg/dL) is multiplied by the phosphate concentration (in mg/dL) and exceeds B70, ectopic calcification is possible. Sites of calcium deposition can include the renal tubules, skin, and vasculature. The mechanisms of ectopic calcification are still debated but may involve matrix vesicle secretion [67,68].

Parathyroid hormone assays The measurement of PTH deserves special attention because of its diagnostic and prognostic importance. In chronic kidney disease, higher PTH levels correlate with greater degrees of metabolic bone disease. First-generation PTH assays of the 1960s and 1970s were polyclonal competitive radioimmunoassays with wide specificity for PTH and PTHrelated fragments. Second- and third-generation PTH immunoassays are two-site, noncompetitive (“sandwich”) assays. These methods have not been harmonized [6971]. The two-site assays were originally termed “intact PTH” assays (vs the older mid-region, C-terminal, or N-terminal PTH

Disorders of calcium metabolism Chapter | 10

Urine PO4 –

Ca++ Bone resorption

Renal tubule

Ca++

333

Stool PO4 – GI tract

1,25-OH2D

Ca++

PO4



Ca++

PO4



Ca++ PO4–

Ca++ PO4



Interstitium/plasma FIGURE 10.15 The effects of PTH on target tissues are illustrated: elevated PTH stimulates osteoclasts to breakdown bone liberating calcium (Ca11) and phosphate (PO32 4 ). PTH increases renal tubular absorption of calcium and increases phosphaturia serving to lower plasma phosphate. PTH stimulates the conversion of 25-OHD to 1,25-OH2D in the kidney. 1,25-OH2D stimulates increased absorption of calcium and phosphate from the GI tract. Overall, PTH raises plasma calcium concentrations and lowers plasma phosphate concentrations.

competitive radioimmunoassays) before it was appreciated that they measure both intact hormone (PTH[184]) and N-terminal-truncated fragments such as PTH(784). Detection of PTH(184) and PTH(784) results from an N-terminal-detecting antibody that does not require the presence of the first 6 N-terminal amino acids in the PTH molecule. These two-site assays that detect (PTH[184]) and predominantly PTH(784) constitute the secondgeneration PTH assays developed in the 1980s. Several diagnostics companies have since developed immunoassays that measure intact PTH(184) but not PTH(784). These third-generation PTH assays have also been termed “biointact,” “whole,” “bioactive,” or “cyclaseactivating” PTH (CA-PTH) PTH assays. Cyclase activating refers to the ability of PTH(184) to stimulate adenylate cyclase. There is evidence that a larger “intact” PTH fragment is also detected by these assays [72]. There was concern that because the intact PTH assays measure PTH (784) and other N-terminal-truncated forms in addition to PTH(184) that these immunoassays could overestimate PTH concentrations in patients with renal failure and secondary hyperparathyroidism (see below). The worry was then that if the PTH was overestimated, vitamin D treatment (e.g., calcitriol) of secondary hyperparathyroidism would be overly aggressive, possibly

334

SECTION | 2 Analytes

leading to vitamin D toxicity. If one compares the concentrations of PTH measured by the intact PTH assays and the biointact PTH assays, although the results are significantly lower (slope 5 0.52) with biointact PTH assays, the two methods are highly correlated (r 5 0.985) [73]. This emphasizes the importance of establishing separate target ranges for intact PTH versus biointact PTH concentrations that are method-specific because of the lack of PTH measurement harmonization. That being said, intact PTH and biointact PTH are highly correlated whether the patients are on dialysis, are uremic, or have primary hyperparathyroidism [74]. With these results in hand, the biointact PTH assay has yet to significantly alter clinical practice [75]. In one informative study, biointact PTH results did not correlate better with markers of bone turnover than intact PTH results [76]. Also, if the ratio of PTH(184) to PTH(784) is calculated, this ratio does not correlate with the rate of bone formation (24). In receiveroperator characteristic curve evaluation of the ability of PTH to predict bone turnover, intact PTH and biointact PTH give similar areas under the curve (AUCs), whereas the ratio of biointact PTH to intact PTH did not perform as well as either marker alone. This is also true in uremic patients where the ratio assessment performs even more poorly [77]. Individual second-generation assays exhibit good PTH reproducibility (e.g., coefficients of variation of ,10%). Because of the lack of PTH assay harmonization, differences between methods usually exceed 20%. The International Federation of Clinical Chemistry (IFCC) is working to harmonize PTH measurements [72]. A 2005 study reported a reference interval for intact PTH measuring PTH(184) and PTH(784)] of 1065 pg/mL for “normal” subjects and 1046 pg/mL for vitamin D-sufficient subjects [78].

Clinical manifestations of disordered calcium or phosphate metabolism Disorders of calcium metabolism clinically present as hypocalcemia, hypercalcemia, bone disease (e.g., rickets), and/or renal disease (e.g., impaired renal function). Patients with hypocalcemia can experience muscle cramps, muscle spasms, paresthesias, tetany, or seizures. Electrophysiologically, hypocalcemia affects the sodium channel by lowering its threshold for depolarization. Hypocalcemia can prolong the patient’s QT interval on electrocardiographic (EKG) testing, even causing arrhythmias with severe hypocalcemia. The spectrum of inherited bone disorders is very wide [79]. Many of these disorders involve mutations in collagen [80], various receptors [81], SHOX (short stature homeobox; chromosome Xp22.33) [82], and the ALPL gene (alkaline phosphatase, biomineralization associated; chromosome 1p36.12) [83] as prime examples. The 11th International Skeletal Dysplasia

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335

Society describes 42 unique forms of skeletal dysplasias resulting from mutations in 346 genes [84]! Supportive clinical evidence of hypocalcemia includes demonstration of the Chvostek sign and the Trousseau sign. The Chvostek sign is ipsilateral (e.g., same side) facial contraction when the facial nerve is lightly tapped with the examiner’s fingers. The Trousseau sign is forearm muscle contract with brachial artery occlusion for 3 min using a blood pressure cuff (e.g., the cuff pressure is 10 mmHg above the systolic pressure). Complications of chronic hypocalcemia include basal ganglia calcification and cataract formation. Mild hypercalcemia (e.g., total calcium ,12 mg/dL) may be asymptomatic. Modest hypercalcemia can produce vague complaints of fatigue, malaise, weakness, depression, apathy, and an inability to concentrate. Chronic hypercalcemia can impair the concentrating ability of the kidney (e.g., nephrogenic diabetes insipidus). Patients with hyperparathyroidism classically complain of psychic disorders, renal stones (nephrolithiasis [85]) or nephrocalcinosis (i.e., in the kidney parenchyma the ectopic deposition of calcium salts [86]), GI disorders, and bone pain (e.g., osteopenia, cystic bone lesions, pathologic fractures). Subperiosteal bone resorptions noted in the fingers and around the teeth are classic radiologic findings in hyperparathyroidism.

Hypocalcemia A reduced level of total calcium in the blood requires confirmation by measurement of either albumin (in search of hypoalbuminemia) or ionized calcium (the better approach). If the reduction in calcium is not due to reduced carrier protein concentrations (e.g., hypoalbuminemia), “true” hypocalcemia is present. There are numerous causes for hypocalcemia (Table 10.5) [87]. Clinically, the most prevalent chronic calcium disorders occur in patients with renal failure. Renal failure will be discussed below.

Decreased parathyroid hormone action Reduced PTH activity can result from hypoparathyroidism or PTH resistance (e.g., pseudohypoparathyroidism). Reduced PTH activity is manifested as hypocalcemia and hyperphosphatemia. Alkaline phosphatase concentrations are not elevated because PTH action is deficient. In hypoparathyroidism, the PTH level is inappropriately low for the ionized calcium concentration. The PTH concentration may be below the reference interval or within the reference interval (which is still inappropriate in a hypocalcemic patient). In cases of PTH resistance, the PTH is elevated because of PTH resistance, and results in hypocalcemia and hyperphosphatemia. The symptoms of hypocalcemia are as described above.

336

SECTION | 2 Analytes

TABLE 10.5 Causes of hypocalcemia. Decreased parathyroid hormone action Parathyroid hormone deficiency Acquired hypoparathyroidism (see Table 10.6) Genetic causes of hypoparathyroidism (see Table 10.7) Parathyroid hormone resistance (see Table 10.8) Parathyroid hormone receptor defects: PTHR1 mutations Postparathyroid hormone receptor (PTHR1) signaling defects (Gsα/cAMP/PKA pathway) GNAS defects Defects distal to GNAS Vitamin D disorders—reduced vitamin D activity (see Table 10.9) Dietary deficiency Inborn errors in vitamin D metabolism (see Table 10.10) Hyperphosphaturia (see Tables 10.11 and 10.12) Other causes of hypocalcemia Calcium deposition into necrotic tissue (saponification) Healing phase of bone disease Dietary calcium deficiency Calcium malabsorption Hyperphosphatemia Miscellaneous causes of hypocalcemia

Parathyroid hormone deficiency—acquired hypoparathyroidism Hypoparathyroidism may exist as an isolated disorder or may be associated with other endocrine or nonendocrine disorders. Isolated hypoparathyroidism can be idiopathic or autoimmune. Rarely, the parathyroid glands are destroyed by cancer metastases, iron deposition (e.g., hemochromatosis) [88], or copper deposition (e.g., Wilson disease) [89]. Acquired causes of hypoparathyroidism are listed in Table 10.6. Autoimmune hypoparathyroidism can occur sporadically or as part of a genetic autoimmune polyglandular syndrome (APS) (see below) [90]. Currently, there are no clinically available tests for parathyroid autoantibodies. However, in research studies, autoantibodies to NAIP, CIITA, HET-E, and TP1 (NACHT) leucine-rich-repeat protein 5 (NALP5) [91] and the CaSR [92] have been described. CaSR-specific cytotoxic CD8 T cells have been reported in hypoparathyroidism [93]. Immune checkpoint inhibitor therapy has caused antibody-mediated hypoparathyroidism [94]. During thyroidectomy, unintended parathyroidectomy may occur [95]. Irradiation can damage the parathyroid glands [96]. Transient hypoparathyroidism in the setting of acute severe illness is common in infants, children, and adults. This is especially true in premature infants, sick term newborns, and infants of diabetic mothers [97]. Vitamin D deficiency and hypomagnesemia can contribute to hypocalcemia in newborns [98]. Maternal hypercalcemia or

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337

TABLE 10.6 Acquired causes of hypoparathyroidism. Autoimmune Parathyroidectomy Thyroid irradiation Transient hypoparathyroidism associated with acute severe illness Transient neonatal hypoparathyroidism Prematurity Sick newborns (e.g., sepsis, birth asphyxia, infant of a diabetic mother) Maternal hypercalcemia Maternal normocalcemic hyperparathyroidism Magnesium deficiency

maternal normocalcemic hyperparathyroidism can transiently suppress the parathyroid glands of their newborns (because of presumed in utero hypercalcemia), leading to transient hypocalcemia [99].

Parathyroid hormone deficiency—genetic causes of hypoparathyroidism Hypoparathyroidism is observed in several genetic conditions as listed in Table 10.7 [100]. If there is a gain-of-function mutation in the CaSR (a type of “receptoropathy”), the parathyroid glands are overly sensitive to calcium, and there is decreased PTH release causing hypocalcemia and hyperphosphatemia [101]. This disorder is also known as “autosomal-dominant hypocalcemia with hypercalciuria type 1 (ADH1)” [102]. Autosomal-dominant hypocalcemia with hypercalciuria type 2 (ADH2) is a consequence of GNA11 (G protein subunit α 11; chromosome 19p13.3) gain-of-function mutations [103,104]. GNA11 is a G-protein subunit α involved in CaSR signaling in the parathyroid chief cells. The gain-of-function mutation is similar to the CaSR gain-of-function mutation where the parathyroid gland senses increased calcium concentrations and lowering PTH secretion despite the fact that the plasma calcium concentration is really not elevated. There are a variety of non-CaSR/non-GNA11 genetic causes of familial hypoparathyroidism displaying autosomal-dominant inheritance [105] due to PTH mutations [106] and glial cells missing transcription factor 2 (GCM2; chromosome 6p24.2) inactivating mutations [107109]. The PTH mutation caused the accumulation of uncleaved prepro-PTH within the parathyroid chief cells with subsequent impaired PTH secretion. Also reported are X-linked-recessive inheritance of hypoparathyroidism due to FHL1 missense mutations (four and a half Lin-11, isl-1, and mec-3 (LIM) domains 1; chromosome Xq26.3; mechanism of hypoparathyroidism is unclear) [110] and deletions near SOX3 [111]. At least two of three conditions must be presented to diagnose APS type 1 (also known as autoimmune polyendocrinopathycandidiasisectodermal

338

SECTION | 2 Analytes

TABLE 10.7 Genetic causes of hypoparathyroidism (genetic causes of hypomagnesemia are not included). Subtype Gene/genetic aberration

Inheritance

Comments

CaSR

AD

Gain-of-function mutation; autosomaldominant hypocalcemia with hypercalciuria type 1 (ADH1)

GNA11

AD

Gain-of-function mutation; autosomaldominant AD hypocalcemia with hypercalciuria type 2 (ADH2)

PTH

AD

Loss-of-function mutation; familial isolated hypoparathyroidism, autosomal dominant

GCM2

AD

Loss-of-function mutation; familial isolated hypoparathyroidism, autosomal dominant

FHL1

XLR

Missense mutations; familial isolated hypoparathyroidism, X-linked recessive

SOX3

XLR

Deletion near SOX3; familial isolated hypoparathyroidism, X-linked recessive

AIRE

AR

Loss-of-function; autoimmune polyglandular syndrome type 1 (APS type 1) or isolated hypoparathyroidism

Polygenic

Polygenic

Autoimmune polyglandular syndrome type 2 (APS type 2)

FOXP3

XLR

Immunodysregulation, polyendocrinopathy, and enteropathy, X-linked (IPEX)

GATA3

AD

Decreased expression of functional transcripts; hypoparathyroidism with sensorineural deafness and renal dysplasia secondary

TBCE

AR

Amino acid deletions; hypoparathyroidism associated with mental retardation and a dysmorphic appearance (hypoparathyroidismretardation-dysmorphism [HRD])

Parathyroid defects

Autoimmune defects

Syndromic disorders

(Continued )

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339

TABLE 10.7 (Continued) Subtype Gene/genetic aberration

Inheritance

Comments

TBCE

AR

Kenny-Caffey syndrome type 1 (KCS1)

FAM111A

AD

Missense mutation; Kenny-Caffey syndrome type 2 (KCS2)

FAM111A

AD

Gracile bone dysplasia

22q11.2

AD or sporadic

Microdeletion; DiGeorge syndrome (DGS1)

10p monosomy

Sporadic (?)

Microdeletion; DiGeorge syndrome (DGS2)

Ring chromosome 16

Sporadic

Reported as single case

Ring chromosome 18

Sporadic

Reported as single case

HADHB

AR

Missense mutation; trifunctional protein deficiency (affects mitochondria)

mtDNA deletions

Sporadic; rarely maternal inheritance

Various deletions; KearnsSayre syndrome (KSS)

ACADM

AR

Missense mutation; medium-chain acyl-CoA dehydrogenase deficiency

MT-ND1, MT-ND5, MT-TH, MT-TL1, and MT-TV

Maternal inheritance

MELAS

CHD7

Sporadic or AD

Decreased protein half-life; CHARGE syndrome

DHCR7

AR

Missense mutation; SmithLemliOpitz syndrome (SLOS)

Chromosomal anomalies

Mitochondrial defects

Other disorders

AD, autosomal dominant; AR, autosomal recessive; mtDNA, mitochondrial DNA; XLR, X-linked recessive.

dystrophy [APECED] syndrome): hypoparathyroidism; mucocutaneous candidiasis; and Addison’s disease or adrenal autoantibodies [112]. Affecting boys and girls equally, APS type 1 is an autosomal-recessive disorder of early onset caused by mutations in the transcription factor, autoimmune regulatory (AIRE)

340

SECTION | 2 Analytes

gene (chromosome 21q22.3). The biological activity of AIRE concerns the expression of self-peptides in the thymus. Other disorders observed in APS type 1 include autoimmune hepatitis, gonaditis (producing pubertal failure or infertility in women), autoimmune thyroid disease, vitiligo, alopecia, fat malabsorption, IgA deficiency, pernicious anemia, type 1 diabetes mellitus, red cell aplasia, and progressive myopathy. Recently biallelic AIRE mutations were also reported in familial cases of autosomal-recessive hypoparathyroidism [113]. Polygenic in origin affecting women more than men and presenting in childhood or later in life, APS type 2 is recognized by the presence of Addison’s disease or adrenal autoantibodies and autoimmune thyroid disease (also known as Schmidt syndrome) and/or type 1 diabetes [114]. Addison’s disease, autoimmune thyroid disease, and type 1 diabetes are known collectively as Carpenter syndrome [115]. Infrequently hypocalcemia has been recognized in the immunodysregulation, polyendocrinopathy, and enteropathy, X-linked (IPEX) syndrome (immunodysregulation, polyendocrinopathy, and enteropathy, X-linked) caused by FOXP3 mutations (forkhead box P3; chromosome Xp11.23) [116]. Hypoparathyroidism can be associated with sensorineural deafness and renal dysplasia secondary to GATA3 gene mutations that is inherited as an autosomal-dominant trait [117]. GATA3 (GATA-binding protein 3; chromosome 10p14) is a transcription factor. Autosomal-recessive hypoparathyroidism associated with mental retardation and a dysmorphic appearance (hypoparathyroidism-retardation-dysmorphism [HRD]) results from tubulin folding cofactor E (TBCE; chromosome 1q42.3) deletion mutations [118]. The pathway leading to correctly folded beta-tubulin that involves cofactor E (one of four such cofactors). Kenny-Caffey syndrome is characterized by hypoparathyroidism, dysmorphic features, and growth retardation [119]. The autosomal-recessive type 1 variant (KCS1) is caused by TBCE mutations just as HRD is caused by TBCE mutations. The even more rare autosomal-dominant type 2 variant (KCS2) results from mutations in FAM111A (family with sequence similarity 111 member A; chromosome 11q12.1) [120]. FAM111A is cell-cycle regulated and is located in the nucleus. It contains a proliferating cell nuclear antigen-interacting peptide box. Also caused by FAM111A mutations, gracile bone dysplasia is a perinatally fatal disorder that displays thin bones, premature closure of basal cranial sutures, and microphthalmia [121]. Gracile bone dysplasia may represent a more severe form of KCS2. DiGeorge syndrome (DGS) results from a sporadic or inherited developmental defect in the third and fourth pharyngeal pouches that causes thymic gland aplasia or hypoplasia, parathyroid aplasia or hypoplasia, abnormalities of the aortic arch, and facial variations [122]. Congenital heart disease and/or immunodeficiency can be fatal in patients affected with DGS [123]. A microdeletion of the long arm of chromosome 22 (i.e., 22q11.2) is the

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most common cause of DGS1 [124]. This may be observed by karyotype or fluorescence in situ hybridization studies. DGS patients lacking the chromosome 22q11.2 microdeletion harbor a chromosome 10p microdeletion (DGS2) [125,126]. Sharing some features with DGS (including hypoparathyroidism) is the coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear anomalies (CHARGE) syndrome [127]. The CHARGE syndrome results from CHD7 (cadherin 7; chromosome 18q22.1) mutations that reduce its halflife [128]. CHD7 regulates chromatin remodeling that affects gene expression. Most cases of CHARGE syndrome are sporadic; however, once the CHD7 mutation occurs, offspring of the affected patient has a 50:50 chance of inheriting the defective gene consistent with autosomal-dominant inheritance [129]. Isolated cases of ring chromosome 16 [130] and ring chromosome 18 [131] have been reported in association with hypoparathyroidism. Hypoparathyroidism has rarely been reporting in mitochondrial disorders [132,133]. There are specific reports of hypoparathyroidism in the autosomalrecessive trifunctional protein deficiency [134,135], KearnsSayre syndrome (from mitochondrial DNA deletions) [136,137], the autosomal-recessive medium-chain acyl-CoA dehydrogenase deficiency [138], and MELAS (from possible mutations in several mitochondrial DNA genes including MT-ND1, MT-ND5, MT-TH, MT-TL1, and MT-TV) [139]. The common thread in mitochondrial disorders causing endocrinopathies is that endocrine organs have high energy requirements, and reduced energy production from mitochondrial dysfunction is then exhibited as endocrine deficiencies [140]. Hypocalcemia has been reported in SmithLemliOpitz syndrome [141]. This autosomalrecessive disorder results from DHCR7 (7-dehydrocholesterol reductase; chromosome 11q13.4) mutations. DHCR7 is required for cholesterol synthesis.

Parathyroid hormone resistance PTH resistance is a family of disorders caused by defects in the PTH receptor (PTHR1) or defects in post-PTHR1 receptor signaling (e.g., G protein signaling: Gsα/cAMP/PKA pathway; PKA, protein kinase A) (Table 10.8) [142]. The phenotypes of PTHR1 mutations and post-PTHR1 receptor signaling are very different as described below.

Parathyroid hormone receptor defects: parathyroid hormone receptor-1 mutations Defects in PTHR1 cause several bony dysplasias: (1) chondrodysplasia, Blomstrand type (BOCD); (2) Jansen metaphyseal chondrodysplasia (JMC); (3) enchondromatosis; or (4) Eiken syndrome [143]. Defects in post-PTHR1 receptor signaling present as pseudohypoparathyroidism.

TABLE 10.8 Genetic causes of decreased PTH action. Gene/genetic aberration

Inheritance

Number of mutated alleles

Mutations

Comments

PTHR1

AR

2

LoF

Chondrodysplasia, Blomstrand type (BOCD)

PTHR1

Sporadic or AD

1

GoF

Jansen metaphyseal chondrodysplasia (JMC)

PTHR1

Sporadic

1

LoF

Ollier disease (most cases caused by IDH1 or IDH2 mutations)

PTHR1

AR

2

LoF

Eiken syndrome

GNAS

AD; mat. chr.

1

LoF

Pseudohypoparathyroidism type 1a (PHP1a)

GNAS

Sporadic

1

LoI of mat.chr. DMRs or chr. 20 parental UPD

Pseudohypoparathyroidism type 1b (PHP1b)

STX16

AD; mat. chr.

1

Deletion

Pseudohypoparathyroidism type 1b (PHP1b) (causes loss of methylation at exon A/B DMR of maternal GNAS complex locus)

NESP55

AD; mat. chr.

1

Deletion

Pseudohypoparathyroidism type 1b (PHP1b) (causes loss of methylation at exon A/B DMR of maternal GNAS complex locus)

AS

AD; mat. chr.

1

Deletion

Pseudohypoparathyroidism type 1b (PHP1b) (causes loss of methylation at exon A/B DMR of maternal GNAS complex locus)

GNAS

?

1

See text

Pseudohypoparathyroidism type 1c (PHP1c; causes of most cases are unknown)

GNAS

AD; pat.chr.

1

LoF

Pseudopseudohypoparathyroidism (PPHP)

GNAS

AD; pat.chr.

1

LoF

Progressive osseous heteroplasia (POH)

PRKAR1A

Sporadic or AD

1

LoF

Acrodysostosis type 1 (ACRODYS1)

PDE4D

Sporadic or AD

1

LoF

Acrodysostosis type 2 (ACRODYS2)

Unknown

?

?

?

Pseudohypoparathyroidism 2 (PHP2)

AD, autosomal dominant; AR, autosomal recessive; DMR, differentially methylated region; LoF, loss-of-function; LoI, Loss of imprinting; Mat.chr., maternal chromosome; Pat.chr., parental chromosome; UPD, uniparental disomy.

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SECTION | 2 Analytes

BOCD is a lethal disorder that results from autosomal-recessive loss-offunction mutations in PTHR1 [144146]. In BOCD, there is abnormal bone development with very short limbs and disproportionate short stature (see Chapter 14: Laboratory Evaluation of Short Stature in Children). Nonlethal (sometimes referred to as “benign”) JMC results from activating PTHR1 mutations [147]. However, JMC is progressive as the metaphyses enlarge during life. Other features include abnormal regulation of endochondral bone formation, increased bone turnover, and short-limbed disproportionate short stature. Some cases of JMC display autosomal-dominant inheritance. The PTHR1 gainof-function mutations in JMC can explain the laboratory findings of elevated plasma calcium, low plasma phosphate, reduced tubular TRP, elevated urinary excretion of cAMP, and elevated plasma 1,25-OH2D [148]. BOCD may be more clinically severe than JMC because two PTHR1 mutations are present in BOCD (i.e., both PTHR1 genes display germline mutations), whereas JMC results from activating mutations (not loss-of-function mutations as in bio-organic-mineral complex (BOMC)) in only one PTHR1 gene [149]. Enchondromas are benign cartilaginous growths within the bones. Many disorders can cause enchondromas including Ollier disease, Maffucci syndrome, metachondromatosis, genochondromatosis, spondyloenchondrodysplasia, dysspondyloenchondromatosis, and cheirospondyloenchondromatosis [150]. Maffucci syndrome is a sporadic disease also caused by somatic mutations in isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) [151]. The phenotype of Maffucci syndrome includes multiple enchondromas and hemangiomas. Maffucci syndrome is not a disorder of PTH resistance. In enchondromatosis (also known as Ollier disease) multiple enchondromas (benign cartilaginous growths within the bones) develop. However enchondromas can become malignant (i.e., chondrosarcomas). Ollier disease most commonly results from somatic mutations in IDH1 or IDH2. Uncommonly, heterozygous PTHR1 mutations have been described in Ollier disease [152]. In two cases, PTHR1 mutations were found only in the enchondromas whereas one patient had mutations in enchondromas and leukocytes. Eiken syndrome is an autosomal-recessive disorder resulting from biallelic missense (presumed loss-of-function) mutations in PTHR1 [153]. The phenotype of Eiken syndrome includes multiple epiphyseal dysplasia, delayed ossification, abnormal bone modeling, abnormal pelvis cartilage persistence, and reduced stature. It is reported that plasma calcium and phosphate were normal in Eiken syndrome [154].

Postparathyroid hormone receptor-1 signaling Post-PTHR1 resistance causes similar biochemical changes to hypoparathyroidism except that PTH levels are elevated. Vitamin D is not deficient and

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magnesium concentrations are normal. Because the PTH is paradoxically increased above the reference interval in the face of biochemical hypoparathyroidism (e.g., hypocalcemia and hyperphosphatemia), PTH resistance causes “pseudohypoparathyroidism” [155]. PTH resistance can be diagnosed using injected PTH and observing the response of urinary cAMP (e.g., the EllsworthHoward test or related tests) [156]. However, such testing is not commonly pursued.

Guanine nucleotide-binding protein (G protein), alpha guanine nucleotide-binding protein, α stimulating defects Post-PTHR1 receptor signaling defects can result from guanine nucleotidebinding protein, α stimulating (GNAS), PRKAR1A, PDE4D, or PDE3A mutations (the Gα/cAMP/PKA pathway includes cAMP degradation by phosphodiesterases) (Fig. 10.16). The GNAS gene (GNAS complex locus, chromosome 20q13.32) encodes the Gsα subunit. After PTH binds to PTHR1, Gαβϒ dissociates into Gα and Gβϒ. GDP is attached to Gα. Replacement of GDP by GTP yielding Gα-GTP produces the active form of Gα. Gα-GTP then activates adenyl cyclase converting ATP to cAMP. In turn cAMP activates PRKAR1A (protein kinase cAMP-dependent type I regulatory subunit; chromosome 17q24.2). cAMP is degraded by PDE4D (phosphodiesterase 4D; chromosome 5q11.2-q12.1). PDE3A (phosphodiesterase 3A; chromosome 12p12.2) is also a phosphodiesterase. The biology of the GNAS locus is complex because of variations in gene imprinting leading to biallelically, paternally, or maternally expressed mRNAs [157]. This locus has a highly complex imprinted expression pattern. In their 50 exons, some mRNAs include a differentially methylated region (DMR). 30 exons can be alternative spliced leading to variable forms of Gsα. Mutations in GNAS gene result in (1) pseudohypoparathyroidism type 1a (PHP 1a); (2) pseudohypoparathyroidism type 1b (PHP 1b); (3) pseudopseudohypoparathyroidism; (4) progressive osseous heteroplasia; (5) McCuneAlbright syndrome (including polyostotic fibrous dysplasia of bone); and (6) some pituitary tumors. There is no GNAS mutation in pseudohypoparathyroidism type 1c (PHP 1c). One functional GNAS gene is compatible with life but is insufficient to provide normal Gsα subunit activities. This can be termed “haploinsufficiency.” Because Gsα is utilized by several hormone receptor systems, other hypofunctionalities can be exhibited, including hypogonadism and hypothyroidism. McCuneAlbright syndrome will be discussed with the hypercalcemic disorders. PHP 1a is inherited as an autosomal-dominant trait. GNAS point mutations in exons 1 to 13 cause loss-function mutations in the maternally inherited GNAS allele (also known as heterozygous inactivating mutations) [158]. Besides PTH resistance in PHP1a, there is resistance to other hormones (e.g., thyroid stimulating hormone (TSH)) that utilize cAMP as a second

346

SECTION | 2 Analytes PTH NH2 PTHR1

COOH

Gα (GNAS)

cAMP

PRKAR1A

PDE4D

AMP FIGURE 10.16 PTH acts by binding to the PTHR1 which activates G-proteins (specifically Gα encoded by GNAS). Activation of adenylate cyclase (not shown) increases cAMP levels in target cells. cAMP then serves to activate PRKAR1A to continue this secondary signal pathway. cAMP is degraded by PDE4D to AMP terminating the stimulatory process.

messenger. The “Albright hereditary osteodystrophy” (AHO) phenotype is present. The AHO phenotype includes mental retardation, short stature, obesity, round- and heart-shaped facies, and short fourth and/or fifth metacarpals. Plasma PTH concentrations are elevated with increased phosphate concentrations followed later by hypocalcemia [159]. In the setting of hypocalcemia, urine calcium is reduced. 1,25-OH2D concentrations can be normal or reduced.

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PHP 1b is perhaps the most perplexing of all calcium disorders and involves disorders of genomic imprinting. The AHO phenotype is usually absent in PHP 1b although TSH resistance and other hormonal resistances can occur. To understand the pathobiology of PHP 1b, we should examine the GNAS gene and the GNAS complex. From 50 to 30 , the GNAS complex is composed of (1) an neuroendocrine secretory protein (NESP) (also known as NESP55) antisense region (GNAS-AS1) that includes the alternative first exon NESP; (2) another alternative first exon XL (also known as XLα or XLαs; s 5 stimulatory); (3) the third alternative first exon A/B (also referred to as 1A or 10 ); and (4) the 13-exon GNAS gene [160]. Upstream of the GNAS complex is the 8-exon STX16 gene (syntaxin 16; chromosome 20q13.32), which is normally responsible for methylation of the GNAS A/B: transcription start site (TSS)DMR. The three DMR regions of the GNAS complex are (1) the NESP region (paternally methylated); (2) NESP antisense (NESPAS)/XLαs region (maternally methylated); and (3) exon A/B (maternally methylated). All individuals with PHP1b exhibit a loss of methylation at the exon A/B DMR of the maternal GNAS complex locus [161]. PHP1b can occur sporadically or within families inherited as an autosomal-dominant trait. There are two causes of sporadic PHP1b: there can be a loss of imprinting of the maternal GNAS DMRs or paternal uniparenteral disomy of chromosome 20 (e.g., both copies of chromosome 20 are from the father meaning that the maternal copy of the GNAS complex is absent) [158]. The autosomal-dominant familial form of PHP1b can result from a deletion of the maternal copy of the STX16 gene. Alternative causes of autosomal-dominant PHP 1b are deletions of the maternal copies of NESP55 (neuroendocrine secretory protein 55) or AS (antisense script) that are involved in the imprinting of GNAS DMRs [162]. STX6 mutations are not found in sporadic cases of PHP1b. As originally described, although the kidney was resistant to PTH effects (e.g., urinary cAMP did not increase after exogenous PTH administration), there was bone sensitivity to PTH (e.g., urinary phosphate did increase after exogenous PTH administration) in “pseudohypohyperparathyroidism” [163]. Therefore, although there were an elevated PTH, hypocalcemia, and hyperphosphatemia occurred due to renal resistance, but the bone displayed pathologic changes (e.g., demineralization) consequent to the effects of elevated PTH concentrations (e.g., osteitis fibrosa cystica). Seeing that the typical bony changes of AHO are absent in PHP1b, PHP1b and pseudohypohyperparathyroidism may be the same disorder [164]. This condition may result from normal expression of Gsα in bone with subnormal expression of Gsα in the renal tubules. Individuals with all of the clinical features of PHP 1a who lack exons 1 to 13 GNAS mutations are classified as having PHP1c. GNAS function

348

SECTION | 2 Analytes

does not appear to be normal in some patients with PHP1c; however, the causes of most PHP1c cases are unknown. In vitro studies of GNAS mutations in a few PHP1c patients with GNAS mutations in the extreme Cterminus of the protein showed disrupted receptor-mediated activation, but normal receptor-independent cholera toxin-induced cAMP accumulation [165]. Another group has demonstrated abnormal methylation patterns of GNAS exons A/B, AS, XL, and NESP [166]. Therefore, a variety of mutations can apparently cause PHP1c. Paternal loss-of-function point mutations or deletions of the GNAS gene with an AHO phenotype who display normal biochemistries are diagnosed with pseudopseudohypoparathyroidism [158]. Inherited as an autosomal-dominant, progressive osseous heteroplasia is recognized by the pathologic progressive ectopic calcification of cutaneous and skeletal muscle. The underlying cause is similar to pseudopseudohypoparathyroidism (PPHP). PHP2 is little mentioned in many current review articles. Possibly this is because its differentiation from PHP1 is difficult and requires the measurement of urinary cAMP following exogenous PTH injection [167]. The cause of PHP2 is unknown [168]. In the classic EllsworthHoward test, in PHP1, increased urinary cAMP does not follow PTH injection. However, in PHP2, urinary cAMP does rise following PTH injection suggesting a defect distal to cAMP generation. In neither PHP1 nor PHP2 does phosphaturia follow PTH injection. Two review articles suggested that PHP2 and acrodysostosis types 1 and 2 (see below) could be the same disorders [159,169].

Defects distal to guanine nucleotide-binding protein, α stimulating Characteristics of acrodysostosis include very short fingers and toes, underdeveloped facial bones, a small nose, short stature and, not uncommonly, delayed developmental and intellectual disabilities. This clinical phenotype results from mutations in PRKAR1A (causing acrodysostosis type 1; ACRDYS1) or PDE4D (causing acrodysostosis type 2; ACRDYS2) [170]. Both conditions are caused by loss-of-function mutations [171]. Sporadic and familial occurrences (AD) of acrodysostosis types 1 and 2 have been reported [142,172]. PRKAR1A is activated by cAMP to continue the signaling cascade initiated when PTH binds to PTHR1. PDE4D degrades cAMP. PRKAR1A mutations appear to result in resistance to cAMP activation [173]. PDE4D mutations appear to be gain-of-function mutations that would increase cAMP clearance [174]. Hormone resistance in addition to PTH resistance can be observed in both ACRODYS1 and ACRODYS2 although hormone resistance is uncommon in ACRODYS2. Autosomal-dominant hypertension with brachydactyly syndrome results from gain-of-function PDE3A mutations [175]. The consequence of this

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mutation would be reduced cAMP concentrations. Because this disorder of the Gsα/cAMP/PKA pathway does not affect calcium, this disorder is not included in Table 10.8 [142].

Vitamin D disorders—reduced vitamin D activity A variety of problems that concern vitamin D action can lower the serum calcium concentration [176]. The pathophysiology of deficient vitamin D action begins with inadequate absorption of calcium and phosphate from the GI tract. Deficient calcium absorption leads to hypocalcemia. Hypocalcemia is detected by the parathyroid glands, and in turn PTH is released, which returns plasma calcium to the lower reference interval. As a consequence of this secondary hyperparathyroidism, excessive urinary excretion of phosphate occurs, producing hypophosphatemia. The effects of low plasma phosphate concentrations and secondary hyperparathyroidism produce the bony findings of rickets in growing infants and children. In adults where the growth plates have fused, the disease is termed osteomalacia. The biochemical hallmarks of rickets and osteomalacia, therefore, include low normal plasma calcium, low plasma phosphate, and a greatly elevated alkaline phosphatase concentration as a consequence of secondary (compensatory) hyperparathyroidism. Causes of inadequate vitamin D action are presented in Table 10.9 [56].

Vitamin D-deficient rickets/osteomalacia Dietary deficiency of vitamin D is supported by a clinical history of inadequate vitamin D intake often coupled with limited exposure to sunlight [177,178]. More highly pigmented skin, sunscreen use, aging, the winter season, and skin grafts for treatment of burns are risk factors for vitamin D

TABLE 10.9 Causes of reduced vitamin D activity. G G

G

G

G

G

G

Dietary vitamin D deficiency (e.g., vitamin D-deficient rickets or osteomalacia) Deficient conversion of 25-OHD to 1,25-OHD (e.g., vitamin Ddependent rickets [VDDR] type IA or vitamin Ddependent osteomalacia) Deficient conversion of vitamin D to 25-OHD (e.g., VDDR type IB or vitamin Ddependent osteomalacia) Resistance to the effects of vitamin D due to defects in the vitamin D receptor (VDR) (e.g., VDDR type IIA or vitamin Ddependent osteomalacia) Overproduction of a vitamin D hormone-responsive element-binding protein (e.g., VDDR type IIB) Liver disease with vitamin D malabsorption and (to a lesser extent) decreased conversion of vitamin D to 25-OHD (e.g., hepatic rickets or osteomalacia) Toxic hepatic hydroxylation (e.g., increased metabolism of vitamin D).

350

SECTION | 2 Analytes

deficiency [179]. Vitamin D can even be lost in the urine in cases of nephrotic syndrome. 25-OHD levels are low in cases of vitamin D deficiency [180]. In health, the desired target range for 25-OHD levels is above 30 ng/mL although controversy surrounds the definition of vitamin D adequacy [181]. Maximal absorption of dietary calcium occurs when 25-OHD levels are near 30 ng/mL. In vitamin D deficiency, the 1,25-OH2D levels have been reported to vary from subnormal to supranormal. In part, the elevated 1,25-OH2D levels may reflect the increased conversion of 25-OHD to 1,25-OH2D because of hypophosphatemia and hyperparathyroidism. The elevation in PTH is termed secondary hyperparathyroidism where an increased PTH level is a physiologic response to hypocalcemia from inadequate intestinal absorption of calcium.

Vitamin Ddependent rickets/osteomalacia The term VDDR is applied to conditions where rickets is not cured with physiological doses of vitamin D (e.g., 400 IU/day); however, supraphysiological doses (e.g., 2000 IU/day) do cure the rickets (Table 10.10). VDDR results from one of four possible problems: (1) an autosomal-recessive inherited deficiency of renal 25-hydroxyvitamin D, 1α-hydroxylase impairing the formation of 1,25-OH2D from 25-OHD (VDDR type IA) [182]; (2) an autosomal-recessive inherited deficiency of vitamin D 25-hydroxylase (CYP2R1) impairing the formation of 25-OHD from vitamin D (VDDR type IB) [183]; (3) VDR resistance to 1,25-OH2D (e.g., an end-organ impaired responsiveness; VDDR type II) also inherited as an autosomal-recessive trait [184]; and (4) VDDR type IIB where an abnormal nuclear-binding protein (competitive response element-binding protein [REBiP] hnRNP C1/C2; HNRNPC; heterogeneous nuclear ribonucleoprotein C [C1/C2]; chromosome 14q11.2) prevents the action of 1,25-OH2D and produces target cell resistance and increased levels of 1,25-OH2D [185].

TABLE 10.10 Features of vitamin Ddependent rickets (VDDR). VDDR

Inheritance

25-OHD

1,25-OH2D

VDR mutation

IA

AR

Nl

Decr.

Absent

IB

AR

Decr.

Incr., Nl or decr.

Absent

IIA

AR

Nl

Incr.

Present

IIB

Sporadic

Nl

Incr.

Absent

AR, autosomal recessive; Decr., decreased; Incr., increased; Nl, normal (within the reference interval); VDR, vitamin D receptor.

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In VDDR type IA, 1,25-OH2D levels are low, and 25-OHD levels are usually normal [186]. This disorder may also be termed “vitamin D pseudodeficiency (pseudodeficient D-resistant rickets).” In VDDR type IB, 25-OHD concentrations are low [187]. 1,25-OH2D concentrations have been reported as elevated, normal, and decreased [187,188]. Because VDDR type II results from vitamin D resistance, these disorders have been renamed as hereditary resistance to vitamin D. In VDDR type IIA and IIB, 1,25-OH2D levels are elevated. In VDDR type IIA, 25-OHD is normal [189]. Alopecia may be present in persons with VDDR IIA [190]. 25-OHD is also normal in VDDR type IIB [191]. These latter two conditions can also be referred to as “hereditary 1,25-OH2D-resistant rickets.”

Hepatic rickets With inadequate bile salt production by the liver, fat-soluble vitamin malabsorption may occur. This can affect vitamin D as well as vitamins A, E, and K. Infants with biliary atresia can suffer from rickets if they do not receive water-soluble formulations of vitamin D or its analogs parenterally [192]. To a lesser extent, chronic liver disease may impair the conversion of vitamin D to 25-OHD.

Accelerated vitamin D metabolism “Toxic hepatic hydroxylation” (also known as drug-induced vitamin D deficiency) is a term that applies to altered vitamin D metabolism as a consequence of drugs that activate the P450 system. For example, children treated with diphenylhydantoin or phenobarbital may develop rickets if there is concurrent deficient exposure to sunlight [193]. Other agents that increase vitamin D catabolism include glucocorticoids and highly active antiretroviral therapy as used in the treatment of human immunodeficiency virus (HIV) infection [194].

Hyperphosphaturia causing hypophosphatemia In addition to disordered vitamin D physiology, rickets and osteomalacia can also be caused by hypophosphatemia due to primary hyperphosphaturia [195]. Without adequate phosphate levels in the blood, ossification of bone is impaired. There are several conditions that can lead to hypophosphatemic rickets or hypophosphatemic osteomalacia (Table 10.11).

Renal tubular disorders: primary and secondary conditions Hyperphosphaturia can be part of pattern of renal tubular dysfunction also affecting the reabsorption of electrolytes, bicarbonate, glucose, and amino

352

SECTION | 2 Analytes

TABLE 10.11 Causes of hyperphosphaturia causing hypophosphatemia. G G G G

Primary renal tubular disorders (e.g., Fanconi syndrome) Secondary renal tubular disorders (e.g., renal tubular dysfunction in Wilson disease) Renal phosphate wasting tumor-induced osteomalacia Hereditary forms of hypophosphatemic rickets/hypophosphatemic osteomalacia G X-linked hypophosphatemic rickets G Autosomal-dominant hypophosphatemic rickets G Autosomal-recessive hypophosphatemic rickets G Hereditary hypophosphatemic rickets with hypercalciuria

acids (e.g., a “primary” Fanconi syndrome) [196]. A functional-Fanconi-like syndrome (a “secondary” Fanconi syndrome) can result from renal tubular injury from toxins or heavy metals [e.g., Wilson disease that results from loss of functions in ATPase copper-transporting beta (ATP7B) located on chromosome 13q14.3] [197].

Renal phosphate wasting tumor-induced osteomalacia The paraneoplastic secretion of FGF23 by various tumors (typically small, benign mesenchymal tumors) can produce tumor-induced osteomalacia (TIO) [198]. When this condition occurs in a child, tumor-induced rickets develops [199]. Recall that the normal action of FGF23 is to produce phosphaturia.

Hereditary forms of hypophosphatemic rickets/osteomalacia Vitamin D-resistant rickets (VDRR) is named as such because exogenous vitamin D is unable to heal the rickets. These conditions are treated by the oral ingestion of phosphate up to six times per day. Because of elevations in FGF23 in most cases of hereditary VDRR, decreased production of 1,25OH2D occurs. For this reason, patients are also treated by 1,25-OH2D. Recall that FGF23 normally suppresses the conversion of 25-OHD to 1.25OH2D and enhances the activity of the 24-hydroxylase that metabolizes 25OHD to 24,25-OH2D and 1,25-OH2D to 1,24,25-OH3D. X-linked hypophosphatemic rickets (XLHR), autosomal-dominant hypophosphatemic rickets (ADHR), autosomal-recessive hypophosphatemic rickets (ARHR), and hereditary hypophosphatemic rickets with hypercalciuria (HHRH) are genetic forms of VDRR (Table 10.12; Fig. 10.17). Hyperphosphaturia can be detected by measuring the urinary phosphate excretion in a timed urine sample or by calculating the fractional excretion of phosphate that is elevated in hypophosphatemic rickets.

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TABLE 10.12 Hereditary forms of hypophosphatemic rickets/ hypophosphatemic osteomalacia. Mode of inheritance

XLHR XLD

ADHR AD

ARHR AR

HHRH AR

1,25-OH2D concentrations

Normal or decreased

Decreased

Normal or decreased

Elevated

Urinary calcium

Not elevated

Not elevated

Not elevated

Elevated

Mechanism of hyperphosphaturia

Increased FGF23

Decreased FGF23 clearance

Increased FGF23

Phosphate transporter loss-offunction mutation

Genetic cause

PHEX

FGF23

DMP1 ENPP1

SLC34A3a

AD, autosomal dominant; AR, autosomal recessive; XLD, X-linked dominant.

X-linked hypophosphatemic rickets Phosphotonins describe proteins that produce phosphaturia [200]. Phosphotonins include FGF7, FGF23, secreted frizzled-related protein 4 (sFRP-4), and matrix extracellular phosphoglycoprotein. Normally phosphate-regulating endopeptidase homolog, X-linked (PHEX; chromosome Xp22.11) reduces phosphotonin expression [201]. PHEX loss-of-function mutations cause XLHR (also known as familial hypophosphatemic rickets; an X-linked-dominant condition). These mutations elevate FGF23 causing hyperphosphaturia. Elevated FGF23 also inhibits renal 25-OHD, 1-α hydroxylase impairing the conversion of 25-OHD to 1,25-OH2D. Consequently 1,25-OH2D concentrations in VDRR may be low, which is inappropriate in the setting of hypophosphatemia [202]. For this reason, vitamin D or its analogs are prescribed together with oral phosphate supplementation. Despite its role as an endopeptidase homolog, PHEX does not degrade FGF23. Specifically how PHEX regulates FGF23 levels is still under investigation. Hemizygous males are clinically more severely affected than heterozygous females. A new treatment for XLHR is burosumab, which is a monoclonal antibody directed against FGF23 [203]. Autosomal-dominant hypophosphatemic rickets ADHR results from a heterozygous gain-of-function mutation in FGF23. The mutation impairs the degradation of FGF23 [204]. This mutation is reminiscent of mutations in clotting factor 5 (i.e., factor V Leiden) that cause hypercoagulability because of the resistance of factor 5 to degradation by

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Osteocyte FGF23 mutation; ADHR PHEX mutation (XLHR)

Incr. FGF23 FGFR1 (lesser importance: FGFR4)

DMP1 mutation (ARHR type I)

DNA transcription of Npt-IIc & Npt-IIa Decr. synthesis Npt-2c mutations (HHRH)

Na+ PO43–

Renal tubular cell

ENPP1 mutation (ARHR type II)

(–)

Phosphaturia

FIGURE 10.17 Hereditary forms of vitamin Dresistant rickets result from increased concentrations of FGF23 in the plasma or loss-of-function mutations in Npt2c (HHRH). FGF23 is produced by osteocytes. FGF23 can be elevated because of mutations in (1) PHEX (XLHR); (2) FGF23 (ADHR); (3) DMP1 (ARHR type I); or (4) ENPP1 (ARHR, type II). PHEX, DMP1, and ENPP1 are expressed in osteocytes. Increased concentrations of FGF23 reduce the expression of the phosphate transporter Npt2c causing hyperphosphaturia and consequent hypophosphatemia and rickets. Not shown is that FGF23 suppresses the conversion of 25-OHD to 1,25-OH2D.

activated protein C [201]. Similar to XLHR, 1,25-OH2D concentrations are low or within the reference interval presumably due to the suppressive effects of FGF23 on the renal 25-OHD, 1-α hydroxylase [205].

Autosomal-recessive hypophosphatemic rickets ARHR can be caused by mutations in two genes. Dentin matrix acidic phosphoprotein (DMP1; chromosome 4q22.1) mutations cause ARHR type I [206,207], whereas ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1; chromosome 6q23.2) mutations cause ARHR type II [208,209]. DMP1 is an extracellular matrix protein that is important for correct mineralization of dentin and bone being present in various cells of the teeth and bone. DMP1 mutations raise FGF23 concentrations, thus explaining hypophosphatemia. ENPP1 cleaves pyrophosphate (PPi) from ATP. ENPP1 mutations also

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raise FGF23 concentrations. Similar to PHEX mutations, the mechanism whereby DMP1 or ENPP1 mutations increase FGF23 is unknown.

Hereditary hypophosphatemic rickets with hypercalciuria HHRH results from a loss-of-function mutation in Npt2c (SLC34A3) causing hypophosphatemia [210,211]. In this case, the resulting hypophosphatemia leads to increased renal 25-OHD, 1-α hydroxylase activity with elevated 1,25-OH2D concentrations causing hyperabsorption of calcium, hypercalcemia, and hypercalciuria [212,213]. The elevated calcium suppresses PTH leading to further decreases in urinary calcium reabsorption and increased calciuria, thus HHRH can result in nephrocalcinosis and/or nephrolithiasis. HHRH is therefore not treated with vitamin D (or 1,25-OH2D). Other rare genetic disorders that can manifest as rickets include hypophosphatemic nephrolithiasis/osteoporosis type 1 (caused by Npt2a [SLC34A1] mutations), hypophosphatemic nephrolithiasis/osteoporosis type 2 (caused by SLC9A3R1; solute carrier family 9 member A3 regulator 1) mutations, Dent disease (caused by CLCN5 [chloride voltage-gated channel 5] or OCRL [oculocerebrorenal syndrome of Lowe inositol polyphosphate-5-phosphatase] mutations), and hypophosphatemic rickets with hyperparathyroidism (HRH) caused by KL mutations. Other causes of hypocalcemia Calcium deposition in necrotic tissue If fat necrosis occurs during pancreatitis, plasma calcium can bind to the released fatty acids in a process termed “saponification.” The deposition of calcium into the necrotic tissue causes hypocalcemia (and this can also result in hypomagnesemia) [214]. Subcutaneous fat necrosis in infants can also cause hypocalcemia [215]. Healing phase of bone disease When osteopenic bone begins to heal, calcium uptake can then depress plasma calcium concentrations. This process can be termed “hungry bone syndrome.” Treatment of hyperthyroidism can induce such subsequent hypocalcemia [216]. Hungry bone syndrome most commonly follows parathyroidectomy for hyperparathyroidism [217]. Hungry bone syndrome has also been reported in cases of prostate cancer with osteoblastic metastases [218].

Dietary calcium deficiency Although uncommon in developed countries, dietary calcium deficiency can lead to hypocalcemia [219,220]. Malabsorption from intestinal disease can

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also cause hypocalcemia [221]. Magnesium malabsorption can lead to hypocalcemia as a consequence of hypomagnesemia [222].

Hyperphosphatemia Hyperphosphatemia can lower calcium as observed in the tumor lysis syndrome, rhabdomyolysis, or high-phosphate formulas given to infants [223]. Hypocalcemia resulting from excessive dietary phosphate causes “neonatal tetany” [224]. This can occur when cows’ milk is fed instead of breast milk or baby formula. Cows’ milk has a much higher phosphate content than these other nutritional sources.

Miscellaneous causes of hypocalcemia Drugs that can lower calcium include CT [225], bisphosphonates [226], furosemide [227], various antineoplastic drugs [228], and massive blood transfusion with citrated blood [229].

Laboratory approach to hypocalcemia When hypocalcemia is confirmed, PTH should be measured (Fig. 10.18) [230]. If the PTH level is inappropriately within or below the reference interval, hypoparathyroidism is present. An elevated phosphate concentration and normal alkaline phosphatase concentration support this diagnosis. Hypomagnesemia should be excluded [231]. Although there are no clinically available tests for the detection of autoantibodies against the parathyroid gland, associated autoantibodies could be sought (e.g., adrenal cytoplasmic autoantibodies or 21-hydroxylase autoantibodies) in cases where an APS is being considered. In hypocalcemic patients with elevated PTH levels, measurement of phosphate can be very helpful diagnostically. Low phosphate is consistent with deficient vitamin D activity or a primary hyperphosphaturia. Due to secondary hyperparathyroidism in states of vitamin D deficiency, alkaline phosphatase is elevated. Other causes of elevated alkaline phosphatase include bone fractures, biliary tract disease, and pregnancy (i.e., the third trimester). Infants can rarely exhibit an idiopathic, familial form of hyperalkaline phosphatasemia where there are no recognizable disorders of the hepatobiliary system, bone, calcium, or phosphate metabolism [232]. The alkaline phosphatase concentration can be in the thousands of units per milliliter in such cases of idiopathic hyperalkaline phosphatasemia. This condition usually remits spontaneously as the infant ages. Increased PTH together with decreased calcium and increased phosphate concentrations is consistent with pseudohypoparathyroidism (PHP; e.g., PTH resistance). After administration of exogenous PTH, an increase in urinary

Disorders of calcium metabolism Chapter | 10

Normal or decreased Hypoparathyroidism

Yes

Hypocalcemia confirmed

PTH

Increased

Increased

Phosphate

Pseudohypoparathyroidism (PHP)

No

Incr. cAMP post-PTH injection

Pseudohypoparathyroidism type 1 (PHP1)

357

No

No further action

Decreased

Deficient vitamin D action or primary hyperphosphaturia (see Figure 10.19) Yes

Pseudohypoparathyroidism type 2 (PHP2)

Genetic (GNAS/chromosomal) analysis

PHP1a

PHP1b

PHP1c

FIGURE 10.18 An approach to the evaluation of hypocalcemia is provided. Details are provided in Table 10.8 and in the text.

cAMP excretion is consistent with PHP2 whose cause is unknown. Failure of urinary cAMP excretion to increase following PTH injection is consistent with PHP1. The subtypes of PHP1 are most readily differentiated by genetic testing involving studies of the GNAS gene complex and STX16 and possibly karyotypic analysis. Fig. 10.19 outlines an approach to a child who represents with rickets or an adult who presents with osteomalacia. The absence of a family history of a similar disorder suggests a sporadic inborn error or acquired vitamin D deficiency (e.g., vitamin D-deficient rickets, hepatic rickets, accelerated vitamin D metabolism, or primary and secondary renal tubular disorders (excluding hereditary vitamin D disorders). When a family history of rickets is present [233], an X-linked-dominant pattern of inheritance is consistent with XLHR. An autosomal-dominant pattern of inheritance is consistent with ADHR. FGF23 can then be measured to sort through the various autosomalrecessive and sporadic disorders. If urinary calcium is increased, then the HHRH is diagnosed. 25-OHD can then be measured if hypercalciuria is absent. Decreased 25-OHD (in the absence of nutritional vitamin D deficiency) is consistent with VDDR type IA. If the 1,25-OH2D concentration is elevated, VDDRIIA and VDDR IIB can be differentiated by molecular testing of the VDR. Some reference laboratories offer gene panels that include testing for “hypocalcemia, autosomal-dominant (ADH),” “hypophosphatemic rickets,” and “VDDR.” Genetic studies can be helpful in examining the CaSR, GNAS, etc. Some commercial companies offer whole-genome sequencing.

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A child represents with rickets (or an adult presents with osteomalacia) Yes

Family history of rickets

Mode of inheritance

XLD

AD

XLHR

ADHR

Increased

Vitamin D deficient rickets Hepatic rickets Accelerated vitamin D metabolism Primary and secondary renal tubular disorders (excluding hereditary vitamin D disorders)

AR or sporadic

FGF23

ARHR

No

Not increased Urinary calcium Increased

Not increased

HHRH Decr. VDDR IB

25-OHD Decr.

VDDR IA

Normal 1,25-OHD Mutated VDDR IIA

Increased VDR

Not mutated VDDR IIB

FIGURE 10.19 An approach to the evaluation of patients with rickets or osteomalacia is provided. Details are provided in the text. Also see Tables 10.1010.12.

Hypercalcemia The most common causes of hypercalcemia are hyperparathyroidism and cancer (Table 10.13) [234,235]. Together, they account for approximately 90% of cases of hypercalcemia in adults. If hypercalcemia is detected, hemoconcentration and elevations in albumin must be excluded [236]. Assuming that the hypercalcemia is confirmed on a second phlebotomy at least 1 week later (assuming that the patient is not severely hypercalcemic and symptomatic requiring immediate therapy) [237], a ready approach to the classification of hypercalcemic begins with the measurement of phosphate, alkaline phosphatase, and PTH [238]. A normal or elevated PTH in the setting of hypercalcemia is inappropriate and most commonly defines autonomous (primary) hyperparathyroidism. If the PTH is suppressed below the reference interval in the setting of hypercalcemia, other causes of hypercalcemia must be pursued (e.g., non-PTHdependent hypercalcemia). Malignancy must be excluded in cases of nonPTH-dependent hypercalcemia.

Hyperparathyroidism Neoplastic parathyroid causes of hyperparathyroidism include parathyoid hyperplasia, adenoma, and carcinoma [239]. Seventy percent or more of

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cases of primary hyperparathyroidism are due to parathyroid adenomas, 15%20% result from parathyroid hyperplasia and 1% or less are due to parathyroid carcinoma. Primary hyperparathyroidism increases in frequency with advancing age. Such parathyroid disorders may exist autonomously, or they can exist in association with a familial cancer syndrome (e.g., MEN) or another endocrinopathy (e.g., acromegaly [240], or ZollingerEllison syndrome as part of MEN I). Some papers propose that elevated concentrations of 1,25-OH2D are more important causes of hypercalcemia in acromegaly than elevated PTH concentrations [241]. In children, primary hyperparathyroidism is rare. Non-PTH-dependent hypercalcemia is more common in children than PTH-dependent hypercalcemia [242]. Rarely hyperparathyroidism is observed in cases of McCuneAlbright syndrome [243].

TABLE 10.13 Causes of hypercalcemia. PTH-dependent hypercalcemia Hyperparathyroidism Neoplastic Adenoma Hyperplasia Carcinoma Paraneoplastic secretion of PTH Associated with other endocrine disorders (see Table 10.14) MEN (including ZollingerEllison syndrome in MEN1) Acromegaly Medication-induced (increased set point for PTH secretion) Lithium-induced hyperparathyroidism Thiazide-induced hyperparathyroidism Other conditions Autoimmune hypercalcemia Tertiary hyperparathyroidism Genetic forms of hyperparathyroidism (see Table 10.14) PTH-independent hypercalcemia Malignancy Endocrine disorders (excluding hyperparathyroidism) Hypothyroidism and hyperthyroidism Addison disease Pheochromocytoma William syndrome Vitamin D and/or A toxicity Granulomatous diseases Immobilization Ketogenic diet Milk-alkali syndrome Medications and miscellaneous causes

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With a parathyroid adenoma, there is a gross enlargement of one gland (e.g., normal parathyroids weigh 4050 mg and adenomas on average weigh B550 mg). Microscopically, hypercellularity of the gland is observed, and there is an increased chief cell to fat ratio (note: chief cells secrete PTH). Various uncommon histologies have also been reported including oxyphilic adenomas and rare “water-clear” adenomas [244]. A capsule and compression of the surrounding tissue by the adenoma are typically recognized microscopically [245]. In cases of hyperplasia, all four parathyroid glands are enlarged, although enlargement may be unequal among the glands. The combined weights of the parathyroid glands in cases of hyperplasia usually range between 1 and 3 g. In hyperplasia, no capsules are observed microscopically. Carcinomas are recognized by gross and/or microscopic invasion of adjacent tissues and/or metastases. Preoperative localization of a single adenoma can allow the surgeon to possibly choose less invasive incisional approaches to adenoma removal highlighting the clinical importance of such localization [246,247]. Such studies include technetium-99m-methoxyisobutylisonitrile (99mTc-sestamibi or MIBI) scintigraphy, sestamibi-single photon emission computed tomography (SPECT or MIBI-SPECT), SPECT and computed tomographic (CT) fusion, subtraction (dual isotope) thyroid scan, ultrasound, four-dimensional computed tomography, magnetic resonance imaging or positron emission tomography, and CT. Localization of an adenoma using invasive methods includes selective venous sampling or selective arteriography. Rarely, a patient may have a parathyroid cyst, and analysis of the cyst fluid for PTH can confirm that the cyst is indeed parathyroid in origin [248]. However, it is problematic to validate the measurement of PTH on body fluids other than plasma or serum. Parathyroidectomy in the treatment of hyperparathyroidism is indicated (1) in all symptomatic patients; (2) when the plasma calcium level is greater than 1 mg/dL above the upper limit of the reference interval; (3) there is renal involvement evidenced by nephrolithiasis, nephrocalcinosis, hypercalciuria (24-h urine calcium level .400 mg/dL) with increased stone risk, or a glomerular filtration rate of less than 60 mL/min; (4) when there is osteoporosis, a fragility (pathologic) fracture, or evidence of a vertebral compression fracture; (5) if the patient is less than 50 years old; (6) if parathyroid cancer is diagnosed; and (7) when patients are unable or unwilling to comply with observation protocols [249]. Indications for parathyroidectomy in asymptomatic patients are similar [250]. Calcimimetics that sensitize the CaSR to plasma calcium do reduce plasma calcium and PTH levels in cases of primary hyperparathyroidism; however, such drugs do not improve bone health [251]. If hyperparathyroidism is clinically present and not otherwise explained in a person with a truly normal calcium level, the term “normocalcemic primary hyperparathyroidism” is applied [252]. Measuring ionized calcium may be helpful because ionized calcium can be elevated when total calcium

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is within the reference interval in some patients. Multiglandular disease may be more common in patients with normocalcemic primary hyperparathyroidism than hypercalcemic primary hyperparathyroidism. Over time, this condition can progress to frank hypercalcemia [253]. Medication-induced hyperparathyroidism includes lithium- and thiazide diuretic-induced hyperparathyroidism. Lithium can induce multiple-gland hyperparathyroidism in 10%15% of patients treated for bipolar disorder [254]. Chronic lithium therapy appears to raise the set point in the parathyroid gland for negative feedback eliciting elevated PTH levels that cause hypercalcemia. Because the renal tubules are also affected with decreased calcium sensitivity, increased urinary calcium reabsorption produces hypocalciuria. Hypocalciuria should minimize or prevent nephrolithiasis and nephrocalcinosis [255]. Therefore, the pathophysiology of lithium-induced hyperparathyroidism is similar to that of benign familial hypercalcemia. Increased PTH with resultant hypercalcemia can rarely be observed in thiazide-treated patients. Not uncommonly, many such patients eventually develop frank primary hyperparathyroidism over time. Nevertheless, thiazides must be withdrawn for 23 months to determine whether hypercalcemia is persistent (i.e., suggestive of hyperparathyroidism independent of thiazides). Similar to familial hypocalciuric hypercalcemia (FHH, also known as benign familial hypercalcemia; see below) and lithium-induced hyperparathyroidism, thiazides can produce hypocalciuria [256]. Autoimmune hypercalcemia results from the production of a CaSR autoantibody that appears to block the detection CaSR of calcium, resulting in hyperparathyroidism [257]. Because the CaSR in the renal tubules is similarly blocked by the CaSR autoantibodies, hypocalciuria develops [258]. Thus, the pathophysiology of autoimmune hypercalcemia is similar to that of benign familial hypercalcemia. Paraneoplastic secretion of PTH is possible and has been reported in cases of pheochromocytoma [259]. Otherwise, hypercalcemia in pheochromocytoma may result from the association of pheochromocytoma and hyperparathyroidism in MEN2A. Catecholamines may also stimulate PTH release from the parathyroid glands. Paraneoplastic PTH secretion is uncommon but has also been reported in the following tumors: ovarian neoplasias (smallcell and clear-cell carcinomas), lung neoplasias (oat cell and squamous cell carcinomas), thymoma, and a primitive neuroendocrine tumor [260]. Secondary hyperparathyroidism does not cause hypercalcemia. Secondary hyperparathyroidism is a compensatory response to vitamin D deficiency or chronic renal failure. In chronic renal failure, secondary hyperparathyroidism develops for several reasons. With renal insufficiency, there is a decreased conversion of 25OHD to 1,25-OH2D. Phosphate retention in renal failure also impairs the conversion of 25-OHD to 1,25-OH2D. Deficiency of 1,25-OH2D results in decreased calcium absorption and hypocalcemia. Furthermore, if phosphate

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is sufficiently elevated, calcium phosphate salts will precipitate out into soft tissues (e.g., ectopic calcification), worsening the patient’s hypocalcemia. In response to hypocalcemia, compensatory (secondary) hyperparathyroidism develops, returning the calcium to the low normal range at the price of the hyperparathyroidism. This secondary hyperparathyroidism causes renal osteodystrophy, which histologically appears as osteitis fibrosa cystica. With secondary hyperparathyroidism, the bones lose calcium, become weak, and are susceptible to fracture. Tertiary hyperparathyroidism is defined as continued and autonomous hyperparathyroidism despite seemingly adequate therapy of secondary hyperparathyroidism [261]. For example, after a patient receives a renal transplant for kidney failure, secondary hyperparathyroidism should subside. However, if hyperparathyroidism is persistent posttransplant, tertiary hyperparathyroidism has developed. PTH alone may be elevated, or hypercalcemia may also be present. One option for the treatment of secondary and tertiary hyperparathyroidisms is the use of a calcimimetic drug that can reduce elevated PTH levels [262,263]. Cinacalcet and etelcalcetide are such calcimimetic drugs. There are several genetic forms of hyperparathyroidism (Table 10.14) [264]. Approximately 10% of cases of primary hyperparathyroidism are hereditary [265]. MEN is the multiple endocrine neoplasia syndrome [266]. MEN type 1 (MEN1) results from loss-of-heterozygosity affecting the tumor-suppressor gene MEN1 (menin 1; chromosome 11q13.1). Functioning in epigenetic gene regulation and histone modification, menin is a scaffold protein. Scaffold proteins serve in assembling specific cellular molecular components [267]. MEN1 includes neoplasms of the pancreatic islets (e.g., gastrinomas or insulinomas), parathyroid glands (see below), and pituitary (e.g., prolactinomas). MEN2 is caused by mutations in the RET proto-oncogene (RET; chromosome 10q11.21). One hundred percent of MEN2 patients develop MTC. Approximately 50% of patients develop pheochromocytoma. MEN2B is clinically distinguished from MEN2A by MEN2B patients manifesting mucosal neuromas and a marfanoid habitus. The locations of the mutations in RET differ between MEN2A and MEN2B: in MEN2A, the RET mutations are extracellular allowing spontaneous dimerization in the absence of ligand, whereas the RET mutations in MEN2B are in the cytoplasmic tyrosine kinase domains achieving tyrosine kinase activity in the absence of RET dimerization. Some papers refer to MEN2A as MEN2 and MEN2B as MEN3. Multigland parathyroid disease is observed in MEN1 with a disease spectrum spanning hyperplasia to monoclonal or oligoclonal adenoma-like lesions [268]. Similarly, adenomas or hyperplasia can be recognized in MEN2A [268]. Hyperparathyroidism is rare in MEN2B. In individuals with an autosomal-dominant, MEN1-like clinical phenotype who are negative for menin mutations, mutations in CDKN1B (cyclin-dependent kinase inhibitor 1B; chromosome 12p13.1) have been recognized. This condition is described as MEN type 4 (MEN4) [269].

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TABLE 10.14 Genetic causes of hyperparathyroidism. Gene/genetic aberration

Inheritance

Comments

Menin

AD

MEN1 and FIPH

RET proto-oncogene

AD

MEN2A; MEN2B (hyperparathyroidism is rare)

CDKN1B

AD

MEN4

CasR

AD

FHH1; homozygous: NSHPT

GNA11

AD

FHH2

AP2S1

AD

FHH3

CDC73

AD

HPT-JT

GCM2

AD

FIPH

TBX1

AD

FIPH

AD, autosomal recessive; FHH, familial hypocalciuric hypercalcemia; FIHP, familial isolated PHPT; HPT-JT, hyperparathyroidism-jaw tumor; MEN, multiple endocrine neoplasia; NSHPT, neonatal severe hyperparathyroidism.

Benign familial hypercalcemia (also known as FHH) is a group of autosomal-dominant disorders that are generally benign [270]. In FHH1, a loss-of-function mutation in the CaSR causes parathyroid insensitivity to the detection of calcium causing a high normal or, less commonly, increased PTH level [271]. Subsequently the calcium is mildly elevated (but usually # 12 mg/dL), and there are no adverse clinical consequences. 1,25-OH2D and phosphate levels are normal [272]. Usually no treatment is indicated. Calcimimetics are reported to improve subject complaints (if present) and reduce PTH concentrations in FHH [273]. Recently, recognized novel causes of FHH include FHH2 that results from a loss-function mutation in the parathyroid Gα subunit protein GNA11 (G protein subunit α11; chromosome 19p13.3) [274] and FHH3 that results from a loss-function mutations in AP2S1 (adaptor-related protein complex 2 subunit σ 1; chromosome 19q13.32) [275]. AP2S1 functions in clathrinmediated endocytosis of the CaSR that is involved in calcium sensing. The adapter protein (AP2) itself is an α, β, μ, and σ subunit heterotetramer. Similar to CaSR loss-of-function mutations, reduced detection of plasma calcium resulting from FHH2 or FHH3 triggers mild hyperparathyroidism. In FHH, because the tubule’s CaSR aberrantly perceives a low calcium concentration (similar to the parathyroid gland) in FHH, tubular hyperabsorption of calcium develops, causing hypocalciuria. Although a single dose of the defective CaSR is benign, homozygosity for a loss-of-function mutation in the CaSR can produce life-threatening neonatal severe hypercalcemia

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(NSHPT) that may require emergency parathyroidectomy. Molecular methods (including DNA sequencing) can be used to differentiate FHH1, FHH2, and FHH3 [276]. Characterized by hyperparathyroidism, mandibular or maxillary ossifying fibromas, and renal cysts and tumors, hyperparathyroidism-jaw tumor syndrome (HPT-JT) is inherited as an autosomal-dominant trait [277]. Parathyroid adenomas or double adenomas occur in HPT-JT syndrome. Parathyroid carcinomas may occur in B15% of patients with the HPT-JT syndrome. CDC73 (cell division cycle 73; chromosome 1q31.2) mutations cause HPT-JT syndrome. An older name for CDC73 is “hyperparathyroidism 2 protein” (HRPT2). When hyperparathyroidism occurs in families in an autosomal-dominant mode of inheritance in the absence of other recognized disorders or causes of syndromic, autosomal-dominant inheritance (e.g., MEN), the term “familial isolated hyperparathyroidism” is applied. MEN1 [278], GCM2 [265], and TBX1 (T-box 1; chromosome 22q11.21) [279] mutations have been observed in a subset of such families. Loss-of-function mutations in GCM2 causing hereditary hypoparathyroidism were discussed above contrasted with activating mutations of GCM2 causing hyperparathyroidism [280].

Intraoperative parathyroid hormone measurements Intraoperative measurements of PTH have revolutionized the surgical management of hyperparathyroidism [281,282]. In cases of single adenomas, surgical success is confirmed when the PTH falls by 50% or more compared with the baseline PTH concentration approximately 10 min following parathyroidectomy. This occurs because PTH has a very short half-life (e.g., # 5 min). The baseline PTH is established after anesthesia is administered in the operating room. The patient’s neck should not be massaged because this can reportedly falsely raise the baseline PTH. Likewise, the baseline PTH must be drawn prior to the surgical incision to avoid any release of PTH that could likewise falsely raise the baseline PTH. If the PTH does not decline sufficiently after parathyroidectomy, there may be multiple parathyroid adenomas or parathyroid hyperplasia. This would require further exploration of the neck. There are varieties of criteria for successful parathyroidectomy that compete for the best sensitivity versus specificity [283]. A good criterion predicting postoperative normocalcemia was a 10-min postexcision PTH decline of more than 50% compared with the preincision PTH level. PTH measurements can be rapidly obtained using mobile instrumentation posted within or immediately adjacent to the operating room [284]. However, more hospitals actually measure the PTH in their central laboratories, where analyzers may provide on-instrument immunoassay times as short as 10 min or less [285]. If a parathyroid adenoma is localized preoperatively (see above), parathyroidectomy can be performed on an outpatient basis

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through a small lateral neck incision. One can also argue that if intraoperative PTH testing is performed, there should be no reason to perform frozen sections and save this expense. Fixed tissue can then solely be studied for microscopic pathologic examination. Outpatient surgery is clearly less expensive than inpatient surgery. Morbidity is also less when the patient has a smaller incision.

Nonparathyroid hormone-dependent causes of hypercalcemia There are many causes of hypercalcemia where the PTH level is appropriately suppressed below the reference interval (Table 10.13) [147,286]. A general classification of these disorders includes malignancy, endocrine disorders (excluding hyperparathyroidism), vitamin D or A excess, granulomatous disease, immobilization, ketogenic diet, milk-alkali syndrome, medications, and miscellaneous causes. There are four mechanisms whereby cancer can cause hypercalcemia [287,288]: 1. A metastasis to bone causes bone destruction and liberates calcium. This can be termed “local osteolytic hypercalcemia of malignancy.” These effects can be cytokine driven [289]. 2. Some cancers release PTHrP as a paraneoplastic product that causes systemic bone breakdown and consequent hypercalcemia. PTHrP does not react in PTH immunoassays, and a separate immunoassay is available in reference laboratories for PTHrP. This form of malignancy-induced hypercalcemia is titled “humoral hypercalcemia of malignancy” and is the most common etiology for malignancy-related hypercalcemia. 3. Overproduction of 1,25-OH2D has been reported in some lymphomas to cause hypercalcemia (see granulomatous disease). 4. Ectopic PTH secretion: Coexisting primary hyperparathyroidism and malignancy have been reported in approximately 5% of patients with malignancy and hypercalcemia. Obviously this is a type of PTHdependent hypercalcemia and will not be further discussed. As for nonparathyroid endocrine causes of hypercalcemia (which rarely cause severe hypercalcemia), thyroid aberrations can result in an elevated calcium level. It is theorized that hypothyroidism may increase PTH, decrease urinary calcium excretion, and decrease vitamin D metabolism, and therefore increase the effects of vitamin D [290,291]. Hyperthyroidism is believed to cause an accelerated bone breakdown, leading to an elevation in the calcium level [292,293]. Glucocorticoids normally inhibit bone turnover and glucocorticoid-induced protein catabolism degrades osteoid leading to osteopenia. A deficiency of glucocorticoids (i.e., Addison’s disease) may raise the calcium concentration but this is predominantly because of dehydration and hemoconcentration [294].

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Non-PTH-dependent hypercalcemia may be observed in patients with pheochromocytoma when catecholamines cause bone resorption or the tumor produces PTHrP. Persons with William (elfin facies) syndrome, a microdeletion syndrome involving chromosome 7q, occasionally display hypercalcemia [295]. These children are affected with supravalvular aortic stenosis. The etiology of the hypercalcemia is unclear, but it may result from hypersensitivity to vitamin D or overproduction of active vitamin D metabolites [296]. Vitamin D toxicity is recognized by the presence of hypercalcemia and hyperphosphatemia. This reflects the action of vitamin D on the intestine increasing both calcium and phosphate absorptions [297]. Hypercalcemia should suppress PTH that would further contribute to hyperphosphatemia through reduced phosphaturia. A rigorous inquiry for “health foods or supplements” is very important [298]. Loss-of-function mutations in CYP24A1 (the 24-hydroxylase discussed above that normally clears 25-OHD and 1,25OH2D through 24-hydroxylation) can raise 25-OHD and 1,25-OH2D causing an autosomal-recessive form of “infantile idiopathic hypercalcemia” [299]. In addition to hypercalcemia, this disorder causes vomiting, dehydration, failure to thrive, and nephrocalcinosis. Vitamin A toxicity causes increased bone turnover and possible hypercalcemia [300]. Vitamins A and D toxicity may be concurrent when fat-soluble vitamins are taken in excess. Manganese intoxication can cause hypercalcemia. Other drugs reported to elevate calcium include estrogens and antiestrogens, growth hormone, theophylline, and foscarnet (an anti-HIV drug). Any granulomatous disease can cause hypercalcemia. In these conditions, the activated macrophages aberrantly express 25-hydroxyvitamin D, 1αhydroxylase increasing 1,25-OH2D concentrations [301]. The pathophysiology is then similar to vitamin D excess. Examples of granulomatous diseases that can cause hypercalcemia include sarcoidosis, coccidioidomycosis, histoplasmosis, tuberculosis, silicosis, silicon injection, berylliosis, nocardiosis, candidiasis, cat-scratch fever, leprosy, Wegener granulomatosis, eosinophilic granuloma, Crohn disease, hepatic granulomatosis in chronic dialysis, talc granulomatosis, bacillus CalmetteGue´rin therapy, lipoid pneumonia, subcutaneous fat necrosis of the newborn, and reactions to silicone implants and paraffin injections [302,303]. Miscellaneous causes of hypercalcemia include immobilization [304], ketogenic diet [305], antibiotic-impregnated calcium sulfate beads [306], parenteral nutrition [307], the healing of muscle following rhabdomyolysis (e.g., calcium is pumped out of the healing tissues) [308], hyperalimentation (e.g., parenteral nutrition), and Paget disease. When the diet is high in calcium, and large amounts of alkali are taken orally (e.g., for treatment of peptic ulcer disease), hypercalcemia may result. This is termed the “milk-alkali” syndrome [309]. Rare causes of this disorder include oyster shell calcium ingestion, betel nut chewing, massive cheese ingestion, and overdose with buffered aspirin.

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A thorough review of the patient’s prescriptions and use of vitamins and supplements is necessary (e.g., intake of thiazides or lithium). Teriparatide (Forteo) is a recombinant DNA-produced 134-amino acid PTH fragment that is FDA approved for the treatment of osteoporosis. After injection, teriparatide fosters bone anabolism. However, it may increase the plasma calcium concentration, 1,25-OH2D, and urinary calcium excretion while lowering phosphate.

Laboratory approach to hypercalcemia Once hypercalcemia is confirmed, PTH should be measured (Fig. 10.20). If the PTH is within or above the reference interval, this is inappropriate in the setting of hypercalcemia and hyperparathyroidism is diagnosed. Phosphate and alkaline phosphatase should also be measured. The alkaline phosphatase will be elevated, and the phosphate may be low as a consequence of hyperphosphaturia. If both the calcium and phosphate are elevated, vitamin D toxicity should be investigated, beginning with a measurement of 25-OHD. If the PTH is suppressed, non-PTH-dependent causes of hypercalcemia should be sought such as malignancy (Table 10.13) [310]. In patients with hyperparathyroidism, urinary calcium excretion should next be determined. Decreased urinary calcium excretion limits the differential diagnosis list to FHH and nonfamilial autoimmune or lithium-induced Hypercalcemia confirmed

Yes

PTH

Decreased

Non-PTH-dependent hypercalcemia (see Table 13)

No

No further action

Normal or increased Hyperparathyroidsm

Urinary calcium Decreased

Increased

Family history of hypercalcemia

Family history of hypercalcemia

No Hyperparathyroidism - Adenoma - Hyperplasia - Carcinoma

Yes Hyperparathyroidism - Menin - RET proto-oncogene - CDKN1B - CDC73 - GCM2 - TBX1

No

Hyperparathyroidism - Autoimmune - Lithium-induced

Yes FHH

Genetic testing

FHH1 FHH2 FHH3

FIGURE 10.20 An approach to the evaluation of hypercalcemia is provided. Details are provided in the text and Tables 10.13 and 10.14.

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hyperparathyroidism. The various forms of FHH can be resolved by genetic testing. In cases of hyperparathyroidism with hypercalciuria, family history and molecular testing can help differentiate autosomal-dominant forms of hyperparathyroidism (Table 10.14) from the neoplastic causes of hyperparathyroidism: adenoma, hyperplasia, and carcinoma.

Laboratory monitoring of bone turnover Although not hormonal measurements, it is worthwhile overviewing the analytes that can be measured to assess bone turnover [311,312]. In the case of osteoporosis (where calcium, phosphate, alkaline phosphatase, and PTH are routinely normal), increased bone turnover can be documented by serum and/or urine measurements. There is only a modest association between bone turnover markers and fracture risk [313]. When type I collagen is degraded during bone resorption, characteristic peptides and pyridinium cross-links (deoxypyridinoline and pyridinolines) are released. Immunoassays are available for both N-terminal telopeptide QYDGKGVG (NTx) and C-terminal telopeptide EKAHDGGR (CTx; Crosslaps). NTx and CTx can be measured in serum or urine. The pyridinium cross-links, pyridinoline, and deoxypyridinoline are released with type I collagen degradation and can be assayed in urine using either a random specimen or a timed collection. Deoxypyridinoline is more frequently measured than pyridinoline because of its greater specificity for bone resorption. The concentration of these markers in serum and urine varies because of the diurnal variation of bone resorption and formation. Markers are highest in the early morning and lowest in the afternoon. Specimens should be collected at a specific time of day for monitoring and compared with reference intervals. For urine, a second morning void collected by 10:00 h is widely used. Using serum to measure telopeptides increases convenience, eliminates the need to measure urine creatinine, and reduces within-subject and day-to-day variation. Urinary hydroxyproline excretion is not used as a marker of bone turnover because hydroxyproline is not specific for bone. Noncollagenous markers of bone turnover include BAP and osteocalcin (BGLAP gene; bone gamma-carboxyglutamate protein; chromosome 1q22). Osteocalcin is a cytosolic calcium-binding protein. It is also released into the circulation. Produced by osteoblasts, (GLA)-rich osteocalcin is a major noncollagenous bone matrix peptide. Due to the action of vitamin K, glutamic acid residues in osteocalcin undergo ϒ carboxylation (e.g., GLA domains). Intact osteocalcin(149) is rapidly hydrolyzed in serum to osteocalcin (143). Consequently, using a method that measures both osteocalcin(149) and osteocalcin(143) simplifies specimen handling and improves the reliability of measurements. Urinary osteocalcin has been investigated as a marker of bone turnover but is not routinely measured [314]. Similarly, BAP

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is released from active osteoblasts. Immunoassays for BAP are available. These methods are superior to electrophoresis, fractionation of total alkaline phosphatase by heat denaturation, or wheat germ lectin inhibition. BAP and osteocalcin can be considered markers of bone formation. BAP measurements pose several advantages over osteocalcin measurements: (1) unlike osteocalcin, it does not exhibit diurnal variation because of its long half-life (13 days) and (2) BAP is more stable in vitro and does not require special specimen handling. However, BAP may be misleading in individuals with liver disease (a hepatic isoform of BAP cross reacts in the assay) or severe osteomalacia (increased BAP but there is failure to mineralize). The general concept is that increased bone turnover with increased collagen degradation will elevate these markers [315]. Preanalytical factors should be tightly controlled to reduce preanalytical variability [316]. In individuals past their 20 s, osteoclast activity usually outstrips osteoblast activity, and increased bone turnover leads to a net loss of bone. Therefore, effective therapies of osteoporosis should lower the serum concentrations or urinary excretion of these markers. There are little data showing that therapeutic modifications in managing osteoporotic patients based on these markers improve patient outcome [317319]. Peak BMD is achieved for men and women in their 20s. Thereafter BMD declines. Because men’s peak BMD is greater than that of women in their 20s, a decline in BMD that predisposes to pathological fractures occurs some 10 years later in men than in women in their elder years. Therefore, men are not immune from osteoporosis.

Other bone diseases with possible laboratory implications Paget disease of the bone results from disordered regulation of osteoclast and osteoblast activity [320]. There is excessive bone breakdown by osteoclasts followed by exuberant and disorganized new bone formation by osteoblasts. The resulting bone is structurally weak and predisposed to deformation, although bone mass may be increased. Alkaline phosphatase is elevated in Paget disease and is the bone marker of choice [321]. Although BAP is more sensitive in mild disease, total alkaline phosphatase is usually measured because of cost and convenience (alkaline phosphatase measurements are part of the comprehensive metabolic profile). A recent review found that procollagen type 1 amino-terminal propeptide (P1NP) was the best monitor of disease activity [322]. The diagnosis of Paget disease is clinical and radiologic in nature. Calcium and phosphate are usually normal in Paget disease. Osteogenesis imperfecta (brittle bone disease) is characterized by bones that are easily fractured because of osteopenia and reduced strength [323]. Dental disease and hearing loss are common. There are many clinical forms of osteogenesis imperfecta that result from mutations in the α1 or α2 procollagen chains that form type I collagen [324]. The autosomal-dominant forms

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are not as severe as the less common autosomal-recessive forms. Poor collagen formation can produce a thinned sclera with a blue appearance due to visualization of the choroidal venous system. Alkaline phosphatase can be elevated [325] and hypercalciuria [326] is recognized in severely affected children. Hypophosphatasia, caused by mutations in ALPL (alkaline phosphatase, biomineralization associated; chromosome 1p36.12), produces rickets, osteomalacia, and premature tooth loss [83]. Biochemically, the alkaline phosphatase concentration is below normal [327]. Laboratories should be attentive to presenting age-appropriate reference intervals for alkaline phosphatase. Urinary excretion of phosphoethanolamine is increased. Hypercalcemia and hypercalciuria have been reported [328]. Recombinant DNA ALPL is available for the treatment of hypophosphatasia [329].

Conclusions Understanding the basic biology of calcium, phosphate, PTH, related analytes, and target organs is the key to solving problems involving the parathyroid gland, end organs, and bone. Hopefully in the future, novel markers will be developed that will allow improved diagnostic and monitoring strategies for these disorders (particularly osteoporosis) [330].

Acknowledgment The authors would like to thank Dr. David Endres for his previous contributions.

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[307] P.H. Brener Dik, M.F. Galletti, L.T. Bacigalupo, S. Fern´andez Jonusas, G.L. Mariani, Hypercalcemia and hypophosphatemia among preterm infants receiving aggressive parenteral nutrition, Arch. Argent. Pediatr. 116 (3) (2018) e371e377. [308] L.A. Hechanova, S.A. Sadjadi, Severe hypercalcemia complicating recovery of acute kidney injury due to rhabdomyolysis, Am. J. Case Rep. 15 (2014) 393396. [309] B.I. Medarov, Milk-alkali syndrome, Mayo Clin. Proc. 84 (3) (2009) 261267. [310] W. Goldner, Cancer-related hypercalcemia, J. Oncol. Pract. 12 (5) (2016) 426432. [311] H.W. Woitge, M.J. Seibel, Markers of bone and cartilage turnover, Exp. Clin. Endocrinol. Diabetes 125 (7) (2017) 454469. [312] R. Eastell, T. Pigott, F. Gossiel, K.E. Naylor, J.S. Walsh, N.F.A. Peel, Diagnosis of endocrine disease: bone turnover markers: are they clinically useful? Eur. J. Endocrinol. 178 (1) (2018) R19R31. [313] A. Tian, J. Ma, K. Feng, Z. Liu, L. Chen, H. Jia, et al., Reference markers of bone turnover for prediction of fracture: a meta-analysis, J. Orthop. Surg. Res. 14 (1) (2019) 68. [314] K.K. Ivaska, S.-M. Kakonen, P. Gerdhem, K.J. Obrant, K. Pettersson, H.K. Vaananen, Urinary osteocalcin as a marker of bone metabolism, Clin. Chem. 51 (2005) 618628. [315] P. Szulc, Bone turnover: biology and assessment tools, Best. Pract. Res. Clin. Endocrinol. Metab. 32 (5) (2018) 725738. [316] P. Szulc, K. Naylor, M.E. Pickering, N. Hoyle, R. Eastell, E. Leary, Use of CTX-I and PINP as bone turnover markers: National Bone Health Alliance recommendations to standardize sample handling and patient preparation to reduce pre-analytical variability, Ann. Biol. Clin. (Paris.) 76 (4) (2018) 373391. [317] R. Eastell, P. Szulc, Use of bone turnover markers in postmenopausal osteoporosis, Lancet Diabetes Endocrinol. 5 (11) (2017) 908923. [318] P. Glendenning, S.A.P. Chubb, S. Vasikaran, Clinical utility of bone turnover markers in the management of common metabolic bone diseases in adults, Clin. Chim. Acta 481 (2018) 161170. [319] S. Jain, P. Camacho, Use of bone turnover markers in the management of osteoporosis, Curr. Opin. Endocrinol. Diabetes Obes. 25 (6) (2018) 366372. [320] R.K. Lalam, V.N. Cassar-Pullicino, N. Winn, Paget disease of bone, Semin. Musculoskelet. Radiol. 20 (3) (2016) 287299. [321] I. Kravets, Paget’s disease of bone: diagnosis and treatment, Am. J. Med. 131 (11) (2018) 12981303. [322] A.A. Al Nofal, O. Altayar, K. BenKhadra, O.Q. Qasim Agha, N. Asi, M. Nabhan, et al., Bone turnover markers in Paget’s disease of the bone: a systematic review and metaanalysis, Osteoporos. Int. 26 (7) (2015) 18751891. [323] T. Palomo, T. Vilac¸a, M. Lazaretti-Castro, Osteogenesis imperfecta: diagnosis and treatment, Curr. Opin. Endocrinol. Diabetes Obes. 24 (6) (2017) 381388. [324] F.S. Van Dijk, D.O. Sillence, Osteogenesis imperfecta: clinical diagnosis, nomenclature and severity assessment, Am. J. Med. Genet. A 164A (6) (2014) 14701481. [325] O. Marginean, R.C. Tamasanu, N. Mang, I. Mozos, G.F. Brad, Therapy with pamidronate in children with osteogenesis imperfecta, Drug. Des. Devel. Ther. 11 (2017) 25072515. [326] A. Ammenti, M. Nitsch, Hypercalciuria in osteogenesis imperfecta type I, Klin. Padiatr. 215 (5) (2003) 283285. [327] A. Deeb, A. Elfatih, Could alerting physicians for low alkaline phosphatase levels be helpful in early diagnosis of hypophosphatasia? J. Clin. Res. Pediatr. Endocrinol. 10 (1) (2018) 1924.

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[328] J.P. Barcia, C.F. Strife, C.B. Langman, Infantile hypophosphatasia: treatment options to control hypercalcemia, hypercalciuria, and chronic bone demineralization, J. Pediatr. 130 (5) (1997) 825828. [329] T. Rolvien, T. Schmidt, F.N. Schmidt, S. von Kroge, B. Busse, M. Amling, et al., Recovery of bone mineralization and quality during asfotase alfa treatment in an adult patient with infantile-onset hypophosphatasia, Bone 127 (2019) 6774. [330] E. Canalis, A. Giustina, J.P. Bilezikian, Mechanisms of anabolic therapies for osteoporosis, N. Engl. J. Med. 357 (2007) 905916.

Chapter 11

Laboratory evaluation of endocrine hypertension William E. Winter1 and Neil S. Harris2 1

Department of Pathology, Immunology & Laboratory Medicine, Pediatrics, and Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States, 2Department of Pathology, Immunology & Laboratory Medicine, University of Florida, Gainesville, FL, United States

Introduction Hypertension is common, silent, and often deadly when not successfully treated [1,2]. Hypertension’s initial presentation can include cardiovascular disease (CVD) or renal disease [3]. Although many sources associate hypertension and headaches, mild (140 to 159/90 to 99 mmHg) or moderate (160 to 179/100 to 109 mmHg) hypertension does not induce headaches [4]. To avoid or reduce the morbidity and mortality of hypertension, blood pressure (BP) measurements must be a routine element of care for individuals of all ages [5,6]. To address the laboratory evaluation of endocrine etiologies of elevated BP (i.e., hypertension), this chapter will provide an overview of the current definition of hypertension, risk stratification and treatment, and the general causes and the endocrine causes of hypertension. This chapter is a revision and update of the 2008 American Association for Clinical Chemistry publication by the authors [7]. Hypertension is of tremendous clinical importance because it has adverse effects on many systems of the body. If left untreated, hypertension produces significant morbidity and mortality [8]. The physiological control of BP is complex. The most basic concept is that BP equals the cardiac output (C.O.) multiplied by the peripheral vascular resistance (PVR) (BP  C.O. 3 PVR). If either C.O. or PVR increase, BP will rise. C.O. (mL/min or L/min; C.O. 5 stroke volume 3 heart rate) is dependent on preload (related to blood volume and the return of blood to the heart), contractility (also known as the ionotropic state of the heart), afterload, and heart rate. Afterload is determined by the diastolic BP and the resistance of the aortic valve to opening during ventricular systole. PVR (mmHgUmin/mL) is determined by the blood viscosity (η; which is usually Handbook of Diagnostic Endocrinology. DOI: https://doi.org/10.1016/B978-0-12-818277-2.00011-X © 2021 Elsevier Inc. All rights reserved.

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not an important variable unless polycythemia is present), the length of the resistance blood vessel (i.e., the small arteries and arterioles) which is fixed, and the radius (r) of the resistance blood vessel (i.e., the small arteries and arterioles) that is variable. Because resistance (PVR, or more simply “R”) is proportional to the inverse of r to the fourth power (r4), small changes in r produce large changes in R (resistance). Radius (r) is controlled by the contractile state of the vascular smooth muscle surrounded the small arteries and arterioles. Rα

η3L r4

Some biologic agents act directly on vascular smooth muscle (e.g., both catecholamines and vasopressin cause vasoconstriction). Epinephrine can be released systemically by the adrenal medulla or locally by the sympathetic nervous system. Some agents sensitize tissues to catecholamines (e.g., thyroid hormone increases tissues’ responsiveness to catecholamines). Certain agents act via the central nervous system in changing sympathetic tone (angiotensin II [9] and aldosterone [10]). Other agents control vascular smooth muscle tone indirectly via nitric oxide (NO) produced by the endothelium of the small arteries and arterioles. In turn NO relaxes the vascular smooth muscle reducing PVR. Histamine (from mast cells), serotonin (from platelets), bradykinin (from the plasma), and platelet-activating factor (from many types of cells) stimulate the endothelium to produce NO causing vasodilation. Prostaglandins (PGI2, prostacyclin) produced by the endothelium can also act as vasodilators. Blood volume has a major influence on BP. The sympathetic nervous system is regulated by low-pressure baroreceptors that sense blood volume in the atria and pulmonary vessels. Decreased blood volume elicits vasoconstriction via increased sympathetic nervous system discharge. High-pressure arterial baroreceptors in the aortic arch and carotid arteries sense arterial pressure. Increased BP elicits vasodilatation via reduced sympathetic nervous system discharge. The sympathetic nervous system also regulates renal perfusion that affects blood volume by regulating the glomerular filtration rate. The central manager of autonomic function (that anatomically includes the sympathetic nervous system) is the central nervous system that regulates BP by influencing sympathetic activity and fluid balance [11]. Because free water reabsorption by the kidney influences blood volume, vasopressin [also known as antidiuretic hormone (ADH)] is an important regulator of fluid status. Vasopressin regulates free water reabsorption predominantly in the collecting duct (CD). Via hypothalamic “osmoreceptors,” increased plasma osmolality elicits vasopressin secretion causing increased free water reabsorption. Sympathetic nervous system baroreceptors (as described earlier) also regulate vasopressin release. Decreased volume or BP elicit vasopressin release. Only at supra-physiologic concentrations of vasopressin regulate BP

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through its direct vasoconstrictor action. Not to be ignored, organs do autoregulate their own perfusion to maintain optimal organ perfusion over a wide range of systemic BPs.

Definition of hypertension The most recent hypertension guidelines from the American College of Cardiology, Clinical Policy Approval Committee and the American Heart Association, Science Advisory and Coordinating Committee were released in 2017 and published in 2018 [12]. This “2017 ACC/AHA/AAPA/ABC/ ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults” will be referred to as the “2017 ACC/AHA guideline” for simplicity. This 2017 ACC/AHA guideline updated the seventh report of the Joint National Committee (JNC 7) on the “Prevention, Detection, Evaluation, and Treatment of High Blood Pressure” that was released in August of 2004 [13]. JNC 7 had updated JNC 6 [14]. The 2017 ACC/AHA guideline addressed four questions concerning: (1) self-directed monitoring of BP and/ or ambulatory BP monitoring, (2) the optimal BP target for BP during therapy of hypertension, (3) antihypertensive drug classes and outcome, and (4) monotherapy versus initial multidrug therapy and outcome [12]. Of significance for laboratorians, there were changes in the cut points used to describe various BP levels and changes in the recommended laboratory evaluation (to be discussed later). Adapted from the 2017 ACC/AHA guideline is Table 11.1. Compared to the 2004 guideline, the category of “prehypertension” was removed and the category of “elevated BP” was substituted. The definition of normal BP was not changed (i.e., ,120 mmHg/ , 80 mmHg). In 2004 prehypertension was defined as 120139 mmHg/8090 mmHg. In 2017, “elevated” BP was defined as 120139 mmHg/ , 80 mmHg. The cut points for the definitions of stages 1 and 2 hypertension were lowered.

TABLE 11.1 Blood pressure classification in adults (2017 ACC/AHA guideline). BP classification

SBP (mmHg)

DBP (mmHg)

Normal

,120

and

,80

Elevated BP

120129

and

,80

Stage 1 hypertension

130139

or

8089

Stage 2 hypertension

$ 140

or

$ 90

BP, Blood pressure, DBP, diastolic BP; SBP, systolic BP.

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Before the diagnosis of hypertension can be established, BP must be measured two or more times on two or more visits. The 2017 ACC/AHA guideline states that ambulatory BP monitoring (ABPM) and home BP monitoring (HBPM) improve the opportunities for the diagnosis of hypertension and hypertension treatment monitoring. However, better understanding of ABPM and HBPM is required before these monitors are included in routine clinical practice. Older publications advised caution in recommending ABPM as a “gold standard” for the definition of hypertension [15]. The greatest elevation in BP, either systolic or diastolic, is used to grade the severity of the hypertension. Systolic hypertension is just as much a risk factor for an adverse outcome as diastolic hypertension. For example, in the Framingham study, systolic hypertension was as powerful a predictor of coronary heart disease as diastolic hypertension [16]. Before age 50, diastolic hypertension is more common than either systolic hypertension or combined systolic and diastolic hypertension. After age 50 with increasing age, systolic hypertension becomes predominant. For reference, in the general population [17] from age 30 and onward, systolic BP and pulse pressure (the difference between the systolic and diastolic BPs) progressively rise. Diastolic BP rises until age B50 and then declines thereafter. Therefore in our senior years, in the absence of hypertension, systolic BP is higher, diastolic is lower and pulse pressure is wider. Note: Treatment recommendations for hypertension are provided in the 2017 ACC/AHA guideline. A detailed discussion of these therapeutic guidelines is outside the scope of this chapter.

Definition of hypertension in children The current 2017 guidelines regarding hypertension in children [18] update the 2004 guidelines [19]. These guidelines were written in 2016 by the American Academy of Pediatrics. BP in children is assessed using tables based upon the child’s sex, age, and height [18]. The assessment of BP in children is illustrated in Table 11.2. BP cut points for children age 13 and older are now identical to current adult cut points.

Causes of hypertension Most cases of hypertension are without a recognized etiology, which is termed “primary” hypertension [20,21]. The older term “essential” hypertension is no longer in use. Primary hypertension usually presents clinically between ages 35 and 55 years. The genetic basis of essential hypertension is poorly understood [22]. Whereas hypertension is strongly familial, it is uncommon that individual gene alleles affect BP [23]. A reduction in glomeruli number is one factor proposed as a cause of essential hypertension [24]. It is estimated that 10% of cases of hypertension in adults have an underlying etiology [25,26]. The recognition of such cases of “secondary”

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TABLE 11.2 Definitions of blood pressure (BP; percentiles are for systolic and diastolic BP) in children age 1 to less than 13 years of age (2016 American Academy of Pediatrics guideline). BP classification

Criteria

Normal

,90th %tile

Elevated

$ 90th %tile to ,95th %tile [or] 120/80 mm Hg to ,95th %tile

Stage 1 hypertensiona

$ 95th %tile to ,95th %tile 1 12 mmHg [or] 130/80 mm Hg to 139/89 mm Hg

Stage 2 hypertensiona

$ 95th %tile plus 12 mm Hg [or] $ 140/ $ 90 mm Hg

a

%tile, percentile. a Whichever cut point is lower defines the applied cut point.

hypertension is important because treatment of the underlying cause of the hypertension may lead to a cure or marked improvement in the BP with a reduction in risk for CVD. A 2004 study from Japan reported a 9% frequency of secondary hypertension [27]. Hypertension in children is more often secondary to an underlying disorder than in adults [28,29]. A leading cause of secondary hypertension is renal disease. Renal disease appears to cause hypertension via: (1) activation of the reninangiotensin-aldosterone system (RAAS) and (2) sodium retention accompanied by fluid retention. In some cases, the adrenergic nervous system may also be stimulated causing vasoconstriction. It is beyond the scope of this chapter to examine all causes of secondary hypertension. The classification of hypertension by physiology can begin by separating hypertension into: (1) systolic hypertension (e.g., cases where the diastolic BP is normal) and (2) combined systolic and diastolic hypertension (Table 11.3) (early on, diastolic hypertension may be solely present). Systolic hypertension results from aortic stiffness or increased left ventricular contractility (e.g., increased thyroid hormone—hyperthyroidism) and is physiologic (but transient) during exercise. Diastolic hypertension results from increases in PVR. Consequently if diastolic hypertension occurs, systolic hypertension often follows.

Laboratory evaluation of hypertension in adults Routine evaluations recommended by the 2017 ACC/AHA guideline (Table 11.4) provide information about: (1) the etiologies of hypertension (e.g., glucose, urinalysis, creatinine, and potassium, etc.), (2) target organ

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TABLE 11.3 Differential diagnosis of hypertension. I) Systolic hypertension (usually with a wide pulse pressure) A) Decreased aortic compliance (also known as increased aortic rigidity): arteriosclerosis in the elderly (most commonly caused by atherosclerosis) B) Increased stroke volume 1) Primary increase in cardiac output a) Aortic regurgitation (valvular insufficiency) b) Thyrotoxicosis c) Hyperkinetic heart syndrome d) Fever e) Anemia 2) Primary decrease in peripheral vascular resistance a) Arteriovenous fistula b) Patent ductus arteriosus c) Paget disease of the bone d) Beriberi (thiamine deficiency) II) Combined systolic and diastolic hypertension A) Uncertain etiology 1) Primary hypertension B) Renal hypertension 1) Renal parenchymal diseases a) Chronic pyelonephritis b) Acute and chronic glomerulonephritis c) Polycystic renal disease d) Hydronephrosis 2) Renovascular hypertension (RVH) a) Renal artery obstruction i) Atherosclerotic ii) Fibromuscular dysplasia iii) Renal artery emboli b) Renal transplantation c) Necrotizing vasculitis d) Intravenous drug abuse e) Malignant hypertension f) Coarctation of the aorta g) Aortic dissection C) Endocrine hypertension 1) Estrogen-induced hypertension a) Pregnancy-induced hypertension b) Oral contraceptive-induced hypertension 2) Hyperadrenocorticalism: steroid elevation with mineralocorticoid action a) Adrenal origin (elevated aldosterone, suppressed renin, low potassium) i) Primary hyperaldosteronism 1) Aldosterone-producing adenoma (APA) 2) Bilateral adrenal hyperplasia (BAH, a.k.a. - idiopathic bilateral hyperplasia) 3) Other conditions (unilateral adrenal hyperplasia, etc.) ii) Cushing syndrome iii) Glucocorticoid-remediable aldosteronism (GRA) iv) Congenital adrenal hyperplasia (CAH) 1) 11β-Hydroxylase deficiency 2) 17α-Hydroxylase deficiency (Continued )

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TABLE 11.3 (Continued) b) Renal origin i) Renin-secreting tumors 1) JG cell tumor 2) Extrarenal renin-secreting tumors ii) 11β-Hydroxysteroid dehydrogenase type 2 deficiency (also a target cell defect) c) Target cell defects i) Cortisol resistance d) Drug-induced hypertension i) Glucocorticoids ii) Mineralocorticoids iii) 11β-Hydroxysteroid dehydrogenase type 2 blockers 1) Licorice 2) Chewing tobacco iv) Anabolic steroids 3) Adrenomedullary-induced hypertension a) Pheochromocytoma b) Other neural crest tumors (e.g., hypertension is uncommon in neuroblastoma) c) Drug-induced i) Sympathomimetics ii) Tyramine iii) MAO inhibitors iv) Appetite suppressants v) Nasal decongestants 4) Acromegaly 5) Hypercalcemia 6) Thyroid disease a) Hyperthyroidism b) Hypothyroidism 7) Vasopressin excess from SIADH 8) Diabetes mellitus 9) Obesity 10) Toxemia of pregnancy D) Neurogenic 1) Psychogenic a) Pain b) Hypoglycemia c) Alcohol withdrawal 2) Neurologic disease a) Acute increase in intracranial pressure b) Quadriplegia from acute spinal cord section c) Polyneuritis i) Acute porphyria ii) Lead poisoning iii) Guillain-Barre´ syndrome d) Familial dysautonomia (RileyDay syndrome) e) Diencephalic syndrome (Continued )

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TABLE 11.3 (Continued) E) Volume-mediated 1) Polycythemia F) Drugs and chemicals 1) Increased sympathetic nervous system (SNS) activity or action a) Sympathomimetics (e.g., methamphetamines, methylphenidate, and amphetamines) b) MAO inhibitors c) Selected appetite suppressants d) Nasal decongestants (e.g., phenylephrine and pseudoephedrine) e) Cocaine 2) Adrenocorticoids or drugs with similar effect a) Glucocorticoids b) Mineralocorticoids c) 11β-Hydroxysteroid dehydrogenase type 2 blockers i) Licorice (metabolite: glycyrrhetinic acid) ii) Chewing tobacco iii) Carbenoxolone d) Anabolic steroids (also endocrine) 3) Miscellaneous mechanisms a) Oral contraceptives (especially high-dose estrogen OCPs; also endocrine) b) Nonsteroidal antiinflammatory agents (especially with chronic use) c) Antidepressants (including TCAs and SSRIs) d) Antacids containing sodium e) Calcineurin inhibitors (e.g., cyclosporine and tacrolimus) f) Angiogenesis inhibitors (e.g., bevacizumab; inhibition of VEGF-mediated vasodilation). g) Tyrosine kinase inhibitors (e.g., sunitinib and sorafenib; VEGF signaling pathway inhibition) h) Erythropoietin (mechanism: vasoconstrictor and/or volume-mediated) i) Phenothiazines MAO, Monoamine oxidase; OCPs, oral contraceptive pills; SSRIs, selective serotonin reuptake inhibitors; TCAs, tricyclic antidepressants; VEGF, vascular endothelial growth factor.

disease (e.g., creatinine), and/or (3) the risk status of the patient for CVD (e.g., creatinine, glucose, and fasting lipid profile) are listed. Compared to 2004, the 2017 ACC/AHA guideline adds sodium and thyroid-stimulating hormone (TSH) as screening tests and replaces hematocrit (Hct) testing with the complete blood count (CBC). Uric acid is added as an optional test. While not a laboratory test, the 12-lead electrocardiogram (EKG) is again recommended as an assessment for left ventricular hypertrophy, ischemia, previous myocardial infarction, and arrhythmia. The echocardiogram is an optional test that can inform the physician regarding cardiac anatomy (e.g., LV wall thickness), contractility, dyskinesis, and valvular disease. The basic tests (Table 11.4) are accomplished in all patients when hypertension is first diagnosed. More comprehensive testing should be pursued

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TABLE 11.4 Investigations in adults with hypertension as recommended by the 2017 ACC/AHA guideline. Routine tests

Information provided (commentary form the authors)

Fasting plasma glucose

A screening test for diabetes mellitus. Not mentioned as a screening test for diabetes is hemoglobin A1c, which is one of three diabetes screening tests recommended by the American Diabetes Association in 2019.a Diabetes may also be observed in Cushing syndrome, acromegaly, and pheochromocytoma. Hypokalemia (potentially from hypermineralocorticoidism) can cause hyperglycemia from reduced insulin secretionb

CBC

Polycythemia can cause hypertension. Anemia is a finding in many chronic diseases including chronic renal insufficiency

Lipid profile

Assesses the patient’s risk for the development of atherosclerosis. Includes: total cholesterol, triglycerides, HDL cholesterol, and calculated LDL cholesterol. Should be drawn in the fasting state

Creatinine

Assessment of renal function. Creatinine can be used to estimate the glomerular filtration rate (eGFR) in persons age 18 years and older

Sodium

Useful in monitoring diuretic, ACE inhibitor, or ARB treatment of hypertension

Potassium

Elevated K1 is seen in advanced renal insufficiency. Low K1 is seen in some cases of hypermineralocorticoidism (hyperaldosteronism, hypercortisolism, or elevated DOC), K1wasting forms of chronic renal failure and as a side effect of potassium-wasting diuretics. A baseline K1 is required because diuretics can produce hypokalemia, whereas ACE inhibitors, ARBs, spironolactone, amiloride, and triamterene can cause hyperkalemia

Calcium

Elevated calcium can cause hypertension, for example, primary hyperparathyroidism. If hypercalcemia is detected, additional measurements should include ionized calcium, phosphate, alkaline phosphatase, and parathyroid hormone

TSH

Hypothyroidism and hyperthyroidism can both cause hypertension. If the TSH is elevated, free (unbound) thyroxine (FT4) should be measured. If the TSH is depressed, FT4 and triiodothyronine (T3) should be measured

Urinalysis

Assessment of renal function. Systemic illness can frequently produce an abnormal urinalysis

EKG

Screens for LVH, ischemia, previous myocardial infarction, and arrhythmias

Optional tests

Information provided (Continued )

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TABLE 11.4 (Continued) Routine tests

Information provided (commentary form the authors)

Echocardiogram

Cardiac anatomy (e.g., LV wall thickness), contractility, dyskinesis, and valvular disease

Urinary albumin to creatinine

Sensitive indicator of diabetic nephropathy and, in nondiabetic individuals, endothelial dysfunction. Increased urinary albumin excretion occurs in stage III diabetic nephropathy in type 1 diabetes prior to the development of ratio (ACR) dipstick-positive proteinuria

Uric acid

Frequently elevated in gout and the metabolic syndrome. Elevation in uric acid helps support the diagnosis of insulin resistance

ACE, Angiotensin-converting enzyme; ACR, albumin-to-creatinine ratio, ARB, angiotensin receptor blocker; LV, left ventricle; LVH, left ventricular hypertrophy; TSH, thyroid-stimulating hormone. a American Diabetes Association, Classification and Diagnosis of Diabetes: Standards of Medical Care in Diabetes—2019, Diabetes Care 42 (Supplement 1) (2019) S13S28. b J.H. Helderman, D. Elahi, D.K. Andersen, G.S. Raizes, J.D. Tobin, D. Shocken, R. Andres, Prevention of the glucose intolerance of thiazide diuretics by maintenance of body potassium, Diabetes 32 (2) (1983) 106111.

when: (1) the severity of hypertension worsens, (2) there is an inadequate reduction in BP using standard treatments, (3) there is more target organ damage than anticipated for the severity of the hypertension, or (4) a secondary cause is possible based upon historical or clinical suspicions.

Laboratory evaluation of hypertension in children The 2017 pediatric guidelines [18] state that: (1) if there is a family history of hypertension, (2) the child has a body mass index $ 85 percentile, or (3) the child lacks a history or physical findings indicative of possible secondary hypertension, an extended workup for secondary causes of hypertension is not necessary in children age 6 and older. For those hypertensive children that do warrant laboratory testing, Table 11.5 provides testing recommendations. A 2019 review [29] recommends that the bedside CKiD Schwartz equation be used to calculate a pediatric estimated glomerular filtration rate (eGFR) from the creatinine concentration [30]. The Modification of Diet in Renal Disease (MDRD) calculation of the eGFR is not recommended in children because this calculation has not been validated in children.

Endocrine hypertension and mechanisms In several endocrine conditions, the mechanisms of hypertension are unclear or multifactorial. Nevertheless, there are two major mechanisms that appear to cause hypertension of endocrine origin (Table 11.6). The major proposed

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TABLE 11.5 Investigations in children with persistent hypertension as recommended by the 2017 pediatric guidelines. Patient population

Screening tests

All children

Chemistry panel (Na1, K1, Cl2, HCO2 3 , BUN and creatinine) Lipid profile (fasting or nonfasting to include high-density lipoprotein and total cholesterol) Urinalysis Renal ultrasonography [children ,6 years old (or) abnormal urinalysis or renal function]

Obese childrena

Additional testing: Hemoglobin A1c (diabetes screening) Aspartate transaminase (AST) and alanine transaminase (ALT; screening for NAFL) Fasting lipid panel (screen for dyslipidemia)

Optional testsb

Complete blood count (assessment for anemia or other chronic illness) Fasting serum glucose (for children at high risk for diabetes mellitus) Thyroid-stimulating hormone Drug screen Sleep study (if loud snoring, daytime sleepiness, or reported history of apnea)

BUN, blood urea nitrogen, NAFL, Nonalcoholic fatty liver. a BMI (body mass index): $ 95th percentile for age and sex. b When indicated by history, physical examination, or initial studies.

mechanisms are [1] sodium retention and [2] vascular smooth muscle contraction (also known as vasoconstriction; with or without cardiac stimulation). These two mechanisms may interact [31]. Drugs can also perturb these systems [3234]. Drugs that activate the renin- RAAS include 9-α-fluorohydrocortisone (fludrocortisone), glucocorticoids, and sex steroids (e.g., androgens). Drugs with catecholamine activity (e.g., methamphetamines, methylphenidate, amphetamine, phenylephrine, and pseudoephedrine) can potentially cause hypertension. Such drugs should be sought by history and urine drug screening if there is an index of suspicion for catecholamine use.

Physiology Mechanistically, many disorders causing hypertension share as a common pathway ENaC (the epithelial sodium channel) activation. ENaC is nonvoltage-gated and amiloride-sensitive, and consists of α (SCNN1A gene; sodium channel

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TABLE 11.6 General mechanisms of endocrine hypertension and prototypic examples. Mechanisms of hypertension

Prototypic examples

(1) Sodium retention: (1a) Activation of ENaC: (1a1) ENaC activation via the reninangiotensin-aldosterone system (RAAS), or

Excess mineralocorticoid activity [e.g., excess aldosterone, desoxycorticosterone (DOC), cortisol, or sex steroids]

(1a2) ENaC activation via an end-organ disorder.

Apparent mineralocorticoid excess Mineralocorticoid receptor gain-offunction mutation (Geller syndrome) ENaC gain-of-function mutations (Liddle syndrome)

(1b) Hyperinsulinism

States of insulin resistance: obesity, type 2 diabetes, metabolic syndrome, acromegaly

(2) Vascular smooth muscle contraction (i.e., increased peripheral vascular resistance) and possible cardiac stimulation: (2a) Direct hormone effects on β1adrenergic myocardial receptors and β2adrenergic receptors (e.g., catecholamines, with or without central actions)

Pheochromocytoma

(2b) Increased sensitivity to catecholamines

Hyperthyroidism

(2b) Divalent hormone (e.g., calcium) effects on vascular smooth muscle

Hypercalcemia

(2c) Other direct effects on vascular smooth muscle or the endothelium

Mineralocorticoid excess Vasopressin excess Toxemia of pregnancy

ENaC, Epithelial sodium channel.

epithelial 1 subunit alpha, chromosome 12p13), β (SCNN1B gene; sodium channel epithelial 1 subunit beta, chromosome 16p12.2), and Y (SCNN1G gene; sodium channel epithelial 1 subunit gamma, chromosome 16p12.2) subunits in a 1:1:1 stoichiometry. Interestingly, each subunit has two transmembrane domains with the N-termini and C-termini being intracellular. ENaC controls fluid and sodium transport across epithelia in many organs.

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In essence, ENaC regulation fine-tunes renal sodium excretion. Sodium balance is critical to maintaining the distribution of fluid between the intracellular and extracellular spaces, as well as the reabsorption of filtered water via the renal tubules. Therefore we begin by examining the RAAS (Fig. 11.1). Renin is encoded by the REN gene on chromosome 1q32.1. Angiotensinogen is encoded by the AGT gene on chromosome 1q42.2. Transcription of the renin mRNA in juxtaglomerular granular cells produces preprorenin [35]. Upon entry into the endoplasmic reticulum (ER), the renin presequence is cleaved from preprorenin yielding prorenin in the lumen of the ER. Prorenin glycosylation occurs in the Golgi apparatus. For regulated secretion, prorenin is then cleaved into renin (340 amino acids) and is stored in secretory granules. A constitutive pathway of the juxtaglomerular

JG apparatus Decreased renal perfusion, low [Na+] in the ALH or β2 adrenergic stimulation.

Renin Angiotensinogen

Increased blood pressure and increased renal perfusion

Angiotensin I ACE

*

Angiotensin II

Aldosterone** (+)

(+)

(+) Adrenal

ACTH Incr. K+ ** also: also raises blood pressure.

* Incr. PCT Na+ reabsorption Vasoconstriction Incr. thirst Incr. catecholamine release Incr. vasopressin

FIGURE 11.1 The juxtaglomerular (JG) apparatus releases renin in response to decreased renal perfusion, low sodium concentration ([Na1]) in the ascending loop of Henle (ALH), or β2 adrenergic stimulation. Renin converts angiotensinogen (the renin substrate) to angiotensin I. Predominantly in the lungs, angiotensin-converting enzyme (ACE) transforms angiotensin I to II. Angiotensin II is the body’s most powerful endogenous vasoconstrictor, which acts to elevate blood pressure, therefore suppressing renin secretion. Acting on the zona glomerulosa, angiotensin II stimulates aldosterone synthesis and secretion. Other actions of angiotensin II include: increased proximal convoluted tubule (PCT) sodium (Na1) reabsorption, vasoconstriction, increased thirst, increased catecholamine release, and increased vasopressin release. Aldosterone acts on the distal convoluted tubule and collecting duct of the nephron to retain sodium in exchange for hydrogen ion and potassium ion that are excreted in the urine. Escalated sodium reabsorption causes increased water reabsorption, expanded circulating blood volume, which increases renal perfusion. Aldosterone also raises blood pressure directly. If renal blood flow is sufficiently reestablished, renin secretion is reduced to normal. Hyperkalemia and adrenocorticotrophic hormone (ACTH) are both minor stimulators of aldosterone secretion. Incr. 5 increased.

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granular cells releases prorenin. Renin/prorenin receptors exist raising the possibility that prorenin plays a physiologic activity of its own [36]. As well, although this chapter emphasizes the endocrine actions of the RAAS system, a tissue RAAS appears to exist [37]. There are three factors that regulate renin secretion. First, the juxtaglomerular apparatus (JGA) monitors the afferent arteriole perfusion that supplies the glomerular vasculature with blood. Reduced perfusion pressure (as detected by decreased stretch of baroreceptors in the granular cells) triggers renin release from the perivascular granular renin-producing cells. Second, renin release increases when there is a reduced sodium concentration in the lumen of the upper pole of the ascending loop of Henle [ALH; also known as thick ascending loop of Henle (TAL)]. Anatomically the loop of Henle rises from the renal medulla and courses back toward the renal cortex. The macula densa (which is part of the JGA) forms part of the wall of the ALH. Macula densa cells possess large nuclei, are closely grouped, from a plaque-like structure, and monitor ALH sodium concentration via the Na-K2Cl cotransporter (NKCC2; BSC1; SLC12A1 gene: solute carrier family 12 member 1) [38]. The macula densa cells are immediately adjacent to the renin-secreting cells of the afferent arteriole. In states of low ALH sodium concentration, the macula densa secretes increased levels of prostaglandins (PGE2) and decreased adenosine diphosphate (ADP) stimulating renin release from the nearby granular cells [39]. The beneficial physiology behind such a response is as follows: If there is hypovolemia, the renal tubules proximal to the ALH will be maximally reabsorbing sodium and chloride to preserve water and extracellular fluid volume. Therefore the ALH sodium concentration would be low in hypovolemic states or states of reduced renal perfusion (because of hypotension) where activation of the RAAS is desirable. The third factor regulating renin release is catecholamines where β2-adrenergic activity stimulates renin release. Such β2-adrenergic agonists can be locally secreted by the sympathetic nervous system or such agonists are circulating (e.g., epinephrine secreted by the adrenal medulla). Once renin is secreted, acting as an aspartyl protease, angiotensinogen (452 amino acids) is cleaved to angiotensin I (ANG I; 10 amino acids). ANG I is converted to angiotensin II (ANG II; 8 amino acids) in the lungs and other tissues via angiotensin-converting enzyme (ACE; a dipeptidyl carboxypeptidase; 170 kDa; ACE gene; chromosome 17q23.3). ACE has a single transmembrane domain and a short cytoplasmic tail. ACE has two homologous extracellular domains, each with a catalytic site and a Zn21-binding region. ACE also inactivates bradykinin but it does not degrade ANG II. ANG II binds to angiotensin II receptors (AGTR1 gene; angiotensin II receptor type 1; chromosome 3q24) that have seven transmembrane domains. The receptors are coupled by G proteins to phospholipase C. The type 1 receptor (AT1A receptor subtype) is found in blood vessel walls, the brain, and many tissues. The AT1B receptor subtype is found in the anterior pituitary and

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the adrenal cortex. The function of the AT2 receptors is not well studied. ANG II has several actions: (1) Aldosterone is released from the adrenal cortical glomerulosa layer, (2) ANG II has vasoconstrictor activity (via the central nervous system (CNS)) and direct action on blood vessel precapillary arterioles, (3) there is increased sympathetic discharge (a CNS effect) and subsequent catecholamine release from the adrenal medulla, (4) there is increased sodium uptake by the proximal convoluted tubule, (5) ANG II stimulates thirst, and (6) ANG II stimulates vasopressin release. The half-life of ANG II is very short (B12 min). Although the major secretagogue of aldosterone is ANG II, aldosterone is also released to lesser degrees by hyperkalemia and adrenocorticotrophic hormone (ACTH). The regulation of aldosterone synthesis by ANG II is enacted by regulation of depolarization of the zona glomerulosa cells that involve several ion channels [Kir3.4 channels (KCNJ5), potassium leak channels (TASK 1/3, two poredomain leak channels)], two pumps [Na1/K1 ATPase (ATP1A1) and the calcium ATPase (ATP2B3)], and a voltage-gated calcium channel (CACNA1D). Receptor binding events lead to an increased cytoplasmic calcium concentration derived from the ER. Ultimately, the production of the enzyme aldosterone synthase (CYP11B2 gene; cytochrome P450 family 11 subfamily B member 2; chromosome 8q24.3) increases. Aldosterone biosynthesis from cholesterol is outlined in Fig. 11.2. Aldosterone binds to the mineralocorticoid receptor (MR) in distal convoluted tubule (DCT) cells and luminal cells of the CD. Another name for the MR is the “type I glucocorticoid” receptor. The formal name of the MR is NR3C2 (nuclear receptor subfamily 3 group C member 2; chromosome 4q31). The present name for the MR emphasizes the fact that the MR is both a receptor and a transcription factor. For reference, the type II glucocorticoid receptor (NR3C1; nuclear receptor subfamily 3 group C member 1; chromosome 5q31.3) binds glucocorticoids. While the effects of aldosterone on mediating gene expression (via the MR as a transcription factor) are emphasized, like other steroid hormones, there are rapid, nongenomic effects of aldosterone that can be mediated by nontranscription factor receptors [40]. Aldosterone receptors expressed on vascular endothelial plasma membranes have been described. Aldosterone causes vasoconstriction mediated by the central nervous system increasing sympathetic outflow. Some experts assert that the major reason why hyperaldosteronism causes hypertension is this effect of aldosterone as contrasted with the salt and water retention caused by aldosterone [41]. However, salt retention is likely necessary for the vasoconstrictive activity of aldosterone to be effective [42]. Therefore these two mechanisms of aldosterone-induced hypertension may be complementary. Whereas aldosterone does cause salt and water retention, within days of increased aldosterone secretion, a spontaneous diuresis (e.g., “aldosterone escape”) is observed decreasing extracellular fluid volume toward normal

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Cholesterol HO

O

CYP11A1

Pregnenolone HO

Controlled by ACTHa

O

HSD3B2

Progesterone O

OH O

CYP21A2

Desoxycorticosterone (DOC) O OH CYP11B2

O HO

Corticosterone

O

Controlled by angiotensin II

CYP11B2

OH HO

18-Hydroxycorticosterone OH

O

O CYP11B2

O

HO

O

HO

Aldosterone O

FIGURE 11.2 Aldosterone is synthesized in the adrenal cortex zona glomerulosa from progesterone. CYP11A1 (cytochrome P450 family 11 subfamily A member 1), HSD3B2 (hydroxydelta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2; also known as 3 betahydroxysteroid dehydrogenase-delta4,5 isomerase), and CYP21A2 (cytochrome P450 family 21 subfamily A member 2) are all positivity controlled by ACTH. On the other hand, CYP11B2 (cytochrome P450 family 11 subfamily B member 2) is controlled by angiotensin II. aY. Xing, C.R. Parker, M. Edwards, W.E. Rainey, ACTH is a potent regulator of gene expression in human adrenal cells. J. Mol. Endocrinol. 45 (1) (2010) 5968.

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[43]. “Aldosterone escape” explains why edema does not occur in states of mineralocorticoid excess. Natriuretic hormone secretion may, in part, explain aldosterone escape [44]. Regarding salt and water retention, in the DCT aldosterone activates the Na-Cl cotransporter (NCC; SLC12A3: solute carrier family 12 member 3; chromosome 16q13). In the CD, aldosterone binds to a single unit of an MRheat shock protein (HSP) complex. The HSP dissociates and the ligandreceptor complex (aldosterone—MR) dimerizes. Upon interaction with a DNA hormone-response element, serum and glucocorticoid-inducible kinase 1 (SGK1; chromosome 6q23.2) is expressed. SGK1 regulates ENaC expression via inhibition of its degradation. Phosphorylation of Nedd4.2 (NEDD4L Gene; NEDD4-like E3 ubiquitin protein ligase; chromosome 18q21.31) by SGK1 impairs the ubiquitination of ENaC subunits. Ubiquitinated ENaC subunits are otherwise targeted for degradation. As well, the MR binding by aldosterone triggers increased activity of ENaC through induction of the ENaC alpha subunit expression and conversion of the ENaC gamma subunit from 85 to 70 kDa [45]. Additionally activation of the MR leads to increased ATP generation to allow increased activity of the basal membrane Na1/K1 ATPase pump to transport Na1 out of the CD luminal cell into the interstitium, while K1 is taken up into these cells. Increased ENaC activity results in increased Na1 uptake from the CD lumen into the CD tubular cell. Water retention initially accompanies sodium retention, producing expanded blood volume. However, aldosterone escape prevents edema formation in states of hyperaldosteronism. As Na1 is absorbed, hydrogen ion (H1) and potassium ion (K1) are excreted. Therefore the effect of aldosterone is to increase Na1 reabsorption (and subsequent water reabsorption) while excreting H1 and K1. Urinary potassium wasting is evident with a relatively elevated urinary potassium concentration of .30 meq/L. In contrast, the kidney will normally retain potassium in the event of systemic hypokalemia. Despite increased Na1 uptake, hypernatremia is not common in states of hyperaldosteronism because water is reabsorbed together with Na1 [46]. While the plasma sodium concentration may be in the higher range of normal, frank hyperkalemia only occurs in B10% of causes of hyperaldosteronism. Aldosterone is the major mineralocorticoid (i.e., steroids that retain Na1 in exchange for H1 and K1) in man. However, cortisol, desoxycorticosterone (DOC), and corticosterone all have mineralocorticoid activity [47]. Indeed, the MR binds cortisol and corticosterone with the same affinity, as the MR binds aldosterone [48]. Therefore the MR must be “protected” from cortisol because cortisol is in 1000-fold molar excess in the circulation compared to aldosterone. In mineralocorticoid-responsive tissues, cortisol is converted to cortisone by the action of 11-beta-hydroxysteroid dehydrogenase type 2 (HSD11B2; hydroxysteroid 11-beta dehydrogenase 2; chromosome 16q22.1). The other 11β-hydroxysteroid dehydrogenase, HSD11B1, normally

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transforms cortisone into cortisol, the reverse of HSD11B2. However, HSD11B1 is not expressed in mineralocorticoid-sensitive tissues. Overall in the setting of hypoperfusion of the kidney, the RAAS produces an integrated multisystem response designed to restore perfusion that is regulated in a negative feedback algorithm (Fig. 11.1). This provides many opportunities for disordered RAAS activity as well as many sites for pharmacologic intervention. When aldosterone is in excess, hypertension, hypochloremic metabolic alkalosis, and hypokalemia can result. Hypokalemia can be manifested as fatigue, weakness, muscle cramps or myalgia, impaired insulin secretion (causing worsening glycemic control in diabetes), palpitations, psychological symptoms (e.g., depression, delirium, hallucinations or psychosis), and, from nephrogenic diabetes insipidus, impaired renal concentrating ability producing polyuria (which is an undesirable chronic complication).

Laboratory notes Hypertension accompanied by hypokalemia and hypochloremic metabolic alkalosis suggests excess ENaC activation, most commonly, via hypermineralocorticoidism. From a diagnostic point of view, the recognition of persistent hypokalemia is extremely important as a clinical trigger for considering mineralocorticoid excess. However, many persons with mineralocorticoid excess do not display hypokalemia [49]. Therefore an additional indication to test for mineralocorticoid excess is early-onset, severe, and/or resistant hypertension regardless of the plasma potassium concentration. Of note, hypokalemia secondary to diuretics (e.g., thiazides or furosemide) must be excluded before an extensive evaluation for hypermineralocorticoidism is undertaken. If hypokalemia is present during the evaluation of a patient for hypertension, diuretics that cause renal potassium loss should be discontinued for B2 months before potassium is remeasured and reassessed clinically. Conversely, individuals being treated with antihypertensive drugs that interfere with the RAAS may display a falsely normal level of potassium. Thus in patients with baseline normal potassium values, consideration can be given to discontinuing RAAS inhibitors prior to “potassium” profiling. Performing a proper laboratory evaluation prior to beginning drug treatments for hypertension certainly reduces opportunities for the misinterpretation of potassium concentrations since some antihypertensive drugs raise potassium, whereas other antihypertensive drugs lower potassium. An analytical issue of concern is that inadvertent postphlebotomy cleavage of prorenin to renin will raise the measured renin concentration possibly perturbing the interpretation of the renin level [50]. Cooling a plasma sample prior to centrifugation will stimulate conversion of prorenin to renin. Therefore samples for renin testing should be rapidly processed. Once drawn, the sample should be kept at room temperature and quickly delivered to the lab [51,52]. Once centrifuged in refrigerated conditions, the resulting plasma

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can be frozen, which should impair the conversion of prorenin to renin. In terms of interpreting renin levels, drugs that ultimately reduce the action of the CD ENaC (e.g., ACE inhibitors [53]) can raise renin. Therefore the patient’s medication history should be reviewed before a renin level is interpreted. Also when measuring renin or aldosterone, the patient’s position at the time of phlebotomy and their history of salt intake do affect the concentrations of these analytes. Several commercial laboratories provide renin and aldosterone reference intervals that reflect the patient’s sex, age, salt-intake status, and their posture. When measuring renin and aldosterone, it is recommended that the patient be kept supine for 20 min prior to the phlebotomy. This provides a standardized patient condition for the analyses.

Mechanisms of sodium retention (mechanism 1) Sodium retention can cause hypertension (mechanism 1). Sodium retention can result from (1) ENaC activation (mechanism 1A of sodium retention) or (2) insulin resistance (mechanism 1B). ENaC can be stimulated via increased RAAS activity (mechanism 1A1) or via defects in the end organ (mechanism 1A2). Causes of ENaC activation via mineralocorticoids can be grouped as (1) excess aldosterone, (2) excess desoxycorticosterone (DOC), (3) excess cortisol, or (4) excess sex steroids. End-organ defects causing excessive ENaC activity include (1) apparent mineralocorticoid excess (AME), (2) states of MR gain-offunction mutations, and (3) ENaC gain-of-function mutations.

Hyperaldosteronism (mechanism 1a1) Hyperaldosteronism can be subclassified as (1) renin-dependent: elevated renin concentrations and elevated aldosterone concentrations (e.g., “secondary hyperaldosteronism” which is driven by renin excess), or (2) reninindependent: suppressed renin concentrations and elevated aldosterone concentrations. This later condition is termed “primary hyperaldosteronism.” Primary hyperaldosteronism has many potential causes as will be discussed later. At one time renin profiling of primary hypertension was a common practice [54,55]. However, because this approach had no tangible implications for treatment or outcomes, this practice is no longer undertaken [56]. The differentiation of renin-dependent versus renin-independent hyperaldosteronism is greatly aided by calculating the ratio of aldosterone to renin. The 2017 ACC/AHA guidelines recommend measuring plasma renin activity (PRA) although newer direct renin measurements are available [57,58]. Rossi et al. found that the direct renin measurement (e.g., renin mass) was diagnostically equivalent to the PRA measurement [59]. It is important to recognize that many factors besides the analytes that are measured can affect the ratio [60]. Aldosterone (in nanograms per deciliter)/renin (as an activity measurement, nanograms per milliliter per hour) ratios of

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.2025 [ng/dL]/[ng/mL per hour] signify hyperaldosteronism with relative suppression of renin (e.g., primary hyperaldosteronism). Ratios of $50 [ng/dL]/[ng/mL per hour] are essentially diagnostic for renin-independent primary hyperaldosteronism. Analytical problems with both the aldosterone [61,62] and the renin assays have been emphasized that affect the sensitivity and specificity of the ratio for the diagnosis of primary hyperaldosteronism [63,64]. One editorial cynically recommended that “the ratio should be repeated until it unmistakably is, or is not, raised. . .” [65]. Because the aldosterone to renin ratio is not diagnostically infallible, other tests differentiating primary hyperaldosteronism versus primary (essential) hypertension are available. These tests include (1) fludrocortisone suppression test, (2) oral salt loading or IV saline infusion, and (3) the captopril challenge test [66,67]. The treating physician can also consider these tests as confirmatory measures of primary hyperaldosteronism. Indeed the Endocrine Society’s 2016 guidelines recommend that one confirmatory test be performed to diagnose primary hyperaldosteronism when the aldosterone to renin ratio is elevated. These confirmatory tests are discussed in the next paragraphs.

Fludrocortisone suppression test The activity of fludrocortisone (9α-fluorohydrocortisone) as a mineralocorticoid should reduce aldosterone concentrations in normal individuals. Evidence of primary hyperaldosteronism would be that aldosterone is not suppressed after fludrocortisone is administered. In normal, nonhypertensive subjects who are not affected by hyperaldosteronism, urinary aldosterone excretion declines to ,5 μg/24 h following salt loading or administration of fludrocortisone. Oral salt loading or IV saline infusion In normal individuals, salt administration (PO or IV) should lower aldosterone concentrations. However if aldosterone concentrations do not decline following salt administration, primary hyperaldosteronism is possible. If the patient’s BP is extremely elevated, salt loading could be dangerous by further raising BP [68]. If the patient is volume expanded (e.g., “salt loaded”) via the infusion of 0.9% NaCl (normal saline) at the rate of 500 mL/h for 4 h (i.e., the saline infusion test) or is given 10 g/day of extra NaCl for 3 days, and the aldosterone is not suppressed, the diagnosis of primary hyperaldosteronism is supported. If the aldosterone is suppressed, the diagnosis is essential hypertension. During the oral salt-loading test, aldosterone can be measured in a 24-h urine collected following the 3 days of salt administration. If aldosterone excretion exceeds 1014 μg/day (2839 nmol/day) when the urinary sodium excretion is .250 mmol/day, the diagnosis of primary hyperaldosteronism is supported. During the intravenous

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salt-loading test, plasma aldosterone levels .10 ng/dL ( . 280 pmol/L) also support the diagnosis of primary hyperaldosteronism.

Captopril challenge test The normal action of the drug captopril as an ACE inhibitor is to decrease the conversion of ANG I to ANG II. Therefore aldosterone levels should fall. Failure of captopril to reduce aldosterone concentrations can be consistent with primary hyperaldosteronism with autonomous aldosterone secretion. Procedures for these tests are provided in the 2016 Endocrine Society guidelines [42]. Laboratory notes A recent Endocrine Society guideline recommends pursuit of the diagnosis of primary hyperaldosteronism in persons with hypertension when: (1) BP exceeds 150/100 mmHg on 3 separate days, (2) the hypertension is resistant (e.g., BP is .140/90 when the patient is treated with three conventional antihypertensive medications (including a diuretic), (3) hypertension is controlled on four or more medications, (4) hypokalemia is detected, (5) an adrenal mass is incidentally detected from an abdominal imaging study, (6) sleep apnea is present, (7) the family history is positive for early-onset hypertension, or (8) a first-degree relative was diagnosed with primary hyperaldosteronism [42]. The relationship between obstructive sleep apnea and hyperaldosteronism is curious: it may be that hyperaldosteronism causes fluid accumulation in the upper airway [69]. Of importance, hypokalemia is not a requirement to consider primary hyperaldosteronism, as not all patients with hyperaldosteronism are hypokalemic. The Endocrine Society guideline reports that only 9%37% of persons with primary hyperaldosteronism are hypokalemic. On the other hand, if hypokalemia is identified upon testing of a hypertensive individual, there is a high likelihood of hypermineralocorticoidism. The 2017 ACC/AHA guideline recommends that the plasma aldosterone: renin activity ratio be determined when hypertensive adults (1) suffer from resistant hypertension, (2) are hypokalemic, (3) manifest an adrenal incidentaloma (e.g., an incidentally recognized adrenal mass), (4) have a positive family history of early-onset hypertension, or (5) suffered a stroke before age 40. Regarding incidentalomas, several pathologies should be considered including adrenal adenomas and pheochromocytomas [70]. Renin-dependent hyperaldosteronism (also known as hyperreninemic hyperaldosteronism) (mechanism 1a1) In renin-dependent hyperaldosteronism (e.g., secondary hyperaldosteronism), hyperreninism drives hyperaldosteronism and aldosterone to renin ratio is 10 or less. Three disorders cause renin-dependent hyperaldosteronism: (1)

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primary hyperreninism (e.g., renin-secreting tumor, also known as reninoma), (2) renal artery stenosis, or (3) renal (parenchymal) disease. Renal artery stenosis causes renal hypoperfusion that elicits hyperreninism. Hyperreninism is a major cause of hypertension in renal (parenchymal) disease.

Renin-secreting tumors (mechanism 1a1) Reninomas (juxtaglomerular cell tumors) are rare tumors that are most commonly diagnosed in adolescents or young adults [71,72]. Usually there is a long history of headaches, and hypertension is recognized during the subsequent clinical evaluation. Hypokalemia and hypochloremic metabolic alkalosis then point the clinician to RAAS activation. Because aldosterone and renin are both elevated, the aldosterone to renin ratio will not be pathologically elevated. Rarely in a paraneoplastic fashion, extrarenal tumors may secrete renin [73]. Pathologic hyperreninism has been reported in rare cases of ovarian cancer, pancreatic adenocarcinoma, adenocarcinoma of the lung, small-cell tumor of the lung, adrenocortical carcinoma, and angiolymphoid hyperplasia with eosinophilia.

Renovascular hypertension (mechanism 1a1) Renovascular hypertension (RVH) is hypertension that results from impaired macro or microvascular flow to the kidney [74]. RVH has many causes as listed in Table 11.3. Evidence of renovascular disease should be sought when there is resistant hypertension, hypertension of abrupt onset, or worsening or increasingly difficult to control hypertension, severe, acute-onset pulmonary edema, or early-onset hypertension. Renovascular disease should also be considered when the physical examination reveals abdominal bruits (a sound heard with a stethoscope produced by turbulent blood flow through an artery). Renal ischemia then elicits elevated renin and subsequent elevations in ANG II and aldosterone. Secondary hyperaldosteronism can also be caused by nonvascular renal (parenchymal) disease. Renal ultrasound is a screening test for renal (parenchymal) disease in pediatrics. For renovascular disease, renal duplex Doppler ultrasound, magnetic resonance angiography, and abdominal computed tomography (CT) scanning are screening tests. Renal angiography (bilateral selective renal intraarterial angiography) is a confirmatory test for renovascular disease. If renal artery stenosis is diagnosed, pharmacologic antihypertensive therapy should be initiated. If such medical therapy fails or the stenosis is not due to atherosclerosis (e.g., fibromuscular dysplasia), percutaneous renal artery angioplasty and/or stent placement can be considered [75]. Surgical revascularization is not superior to medical therapy [76].

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Obviously, RVH is not a primary endocrine disorder. However, because the mechanism of hypertension is renin-induced activation of the RAAS, the topic is included for completeness.

Renin-independent hyperaldosteronism (mechanism 1a1) If the secretion of aldosterone is autonomous or is under the control of ACTH (e.g., glucocorticoid-remediable aldosteronism, GRA) and is excessive, hyperaldosteronism results. Hyperaldosteronism can cause vasoconstriction, and salt and water retention causing hypertension. In turn, elevated BP will suppress renin secretion. In the evaluation of hypertension, hypokalemia, alkalosis, an elevated aldosterone to renin ratio suggests primary hyperaldosteronism (e.g., renin-independent hyperaldosteronism, hyporeninemic hyperaldosteronism). As well, as noted earlier, hyperaldosteronism should be sought in cases of severe, resistant, or youth-onset hypertension not otherwise explained. Of note, a depressed renin level is also observed with hyporeninemic hypoaldosteronism in association with diabetes mellitus, following bilateral nephrectomy, with glycyrrhizic acid ingestion (formerly found in black licorice) or mineralocorticoid ingestion, and with administration of certain drugs [e.g., β-adrenergic blockers, clonidine, and DOC]. Renin-independent hyperaldosteronism (also known as primary hyperaldosteronism) can be further divided into sporadic disorders and inherited disorders (Table 11.7). The sporadic disorders are much more common than the genetic disorders. However, for those individuals and families with genetic disorders, proper diagnosis and treatment are extremely important.

Renin-independent hyperaldosteronism, sporadic causes (mechanism 1a1) The two most common causes of sporadic hyporeninemic hyperaldosteronism (primary hyperaldosteronism) are bilateral adrenal hyperplasia (BAH) and aldosterone-producing adenomas (APAs) [77]. Rarely, (1) hyperplasia can be unilateral [78], (2) there can be an aldosterone-producing adrenocortical carcinoma, (3) an ectopic aldosterone-producing adenoma or carcinoma is present, or (4) there are aldosterone-producing cell clusters (APCC). Such cell clusters can also be observed in normal adrenal glands [79]. There is a bimodal distribution of adrenal carcinomas with peaks in the first and seventh decades [80]. In addition to signs of hormone excess, functional adrenocortical carcinomas may present with flank pain or as an abdominal mass. Carcinomas producing aldosterone are almost always more than 4 cm in diameter. Aldosterone-producing adrenocortical carcinomas, ectopic aldosteroneproducing adenomas or carcinomas, and APCC are the providence of the

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TABLE 11.7 Sporadic and inherited causes of primary hyperaldosteronism (hyporeninemic hyperaldosteronism) and their distribution. Proportion of causes of primary hyperaldosteronisma Sporadic causes Aldosterone-producing adenoma

35%

Aldosterone-producing adrenocortical carcinoma

Rare

Bilateral adrenal hyperplasia

60%

Ectopic aldosterone-producing adenoma or carcinoma

,0.1%

Primary (unilateral) adrenal hyperplasia

2%

Aldosterone-producing cell clusters

Unknown

Inherited causes Glucocorticoid-remediable aldosteronism (familial hyperaldosteronism type I)

,1%

Familial aldosterone-producing adrenal adenomas or hyperplasia (familial hyperaldosteronism type II)

,2%

KCNJ5 mutation (familial hyperaldosteronism type III)

Rare

a R.M. Fagugli, C. Taglioni, Changes in the perceived epidemiology of primary hyperaldosteronism, Int. J. Hypertens. 2011 (2011) 162804.

radiologist (anatomic localization and spread) and the surgical pathologist who is examining the gross specimen and its microscopic sections in making the final anatomic diagnosis. Therefore we will focus on APAs, BAH, and their differentiation.

Aldosterone-producing adenoma Adrenocortical adenomas can be nonfunctional or they can secrete any variety of steroid hormones including aldosterone, desoxycorticosterone (DOC), cortisol, androgens, or estrogens [81]. If incidental adrenal masses detected by imaging studies (e.g., incidentalomas) are indeed identifying adrenal adenomas, adrenal adenomas may affect B2% or more of the adult population [82]. Adenomas are equally prevalent in males and females with a peak incidence in the fourth and fifth decades.

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An APA (also known as aldosteronoma) produces aldosterone in excess causing hypermineralocorticoidism. The consequences of mineralocorticoid excess are hypertension, hypokalemia, and alkalosis. It is estimated that up to 10% of cases of hypertension are due to primary hyperaldosteronism (either BAH or an APA) [83]. APAs are typically unilateral and solitary with a size of 0.52.5 cm but usually less than 2 cm in diameter. Finding an adrenal mass on CT or magnetic resonance imaging (MRI) studies does not guarantee that this anatomic finding is responsible for a patient’s hyperaldosteronism. In cases of primary hyperaldosteronism, where both adrenal glands contribute to hyperaldosteronism, a bilateral process is likely. Specifically, this describes BAH. Similar to other sporadic neoplasms, an increasing number of mutated genes are being recognized as contributors to the development of APAs [84]. Gene mutations have been reported in B50% of APAs to date [85]. KCNJ5 mutations were found in 36% of APAs by Choi et al. [86]. Other mutated genes in APAs include ATP1A1, ATP2B3, CACNA1D, CACNA1H, CTNNB1, and ARMC5 [87]. As opposed to inherited gene mutations causing genetic BAH, the above mutations are somatic but usually affect both adrenal glands.

Bilateral adrenal hyperplasia Because the etiology is not known in most cases of BAH, BAH can be termed “idiopathic hyperaldosteronism.” In the several familial autosomaldominant forms of BAH, the genetic etiology is known and these conditions are discussed in the section on “inherited causes of hyporeninemic hyperaldosteronism.” BAH is the most common cause of primary hyperaldosteronism representing B60% of cases. APA accounts for B35% of cases of primary hyperaldosteronism [83]. Once hyporeninemic hyperaldosteronism is diagnosed, imaging is performed seeking evidence of an adrenal mass lesion. Large masses (e.g., .4 cm in diameter), especially with invasion or metastasis, would suggest a carcinoma [88]. Because imaging does not define function, it is clinically important to biochemically differentiate unilateral disease (i.e., an APA that may be curable through adrenectomy) from bilateral disease (i.e., BAH, which is managed medically) [42]. The differentiation between unilateral and bilateral disease can be accomplished by renal vein cannulation with aldosterone measurements in each vein [89]. Adrenal vein cannulation is technically difficult and usually requires a highly skilled radiologist. Some protocols include cortisol measurements after corticotropin-releasing hormone administration. Other protocols employ ACTH stimulation of aldosterone [90]. Most commonly, APAs are unilateral and only rarely are they bilateral [91]. To define a unilateral source of the hyperaldosteronism, two criteria must be met: (1) The aldosterone concentration in one adrenal vein should exceed

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the aldosterone concentration in the opposite adrenal vein 2.5-fold or more. Some experts seek a ratio of .4 to diagnose an APA. In addition, (2) the aldosterone to cortisol ratio from the potentially unaffected side must be no higher than the aldosterone to cortisol ratio in the inferior vena cava or cubital fossa (e.g., the “peripheral” aldosterone concentration). This latter criterion (when met) demonstrates suppression of aldosterone production from the presumably normal adrenal gland. When these two criteria are met, unilateral adrenal removal (usually performed laparoscopically) should cure the hypertension or reduce the patient’s BP regardless of the nature of the unilateral lesion (i.e., adenoma or unilateral hyperplasia) [92]. Having emphasized the value of the aldosterone to renin ratio, the reproducibility of the ratio has been questioned [93]. There are at least four other approaches to differentiating APA and BAH. (1) A simple undertaking involves aldosterone’s response to upright posture. With BAH, aldosterone can rise with upright posture, whereas aldosterone in cases of APA should not rise with upright posture or may even decline with upright posture [42,94]. However, complicating the interpretation of this test is that in APA aldosterone will uncommonly rise with upright posture. In a sense, this negates much of the value of this test. (2) Production of a novel adrenal steroid, 18-hydroxycorticosterone, is elevated in APA ( . 50100 ng/mL) compared with BAH (,50 ng/mL) [95]. 18-Hydroxycorticosterone is the immediate precursor of aldosterone. Theoretically in APAs, the inefficiency of production of aldosterone explains the elevated 18-hydroxycorticosterone concentration. However, B40% of patients with primary hyperaldosteronism may not be differentiated using 18-hydroxycorticosterone measurements alone [42]. Commercial laboratory testing is available for 18-hydroxycorticosterone measurements. The diagnostic performance of (1) salt loading, (2) the captopril suppression test, and (3) furosemide stimulation testing on the differentiation of APA and idiopathic adrenal hyperplasia (IAH) is limited [96]. Captopril inhibits conversion of angiotensin I to angiotensin II, thereby physiologically decreasing aldosterone production in normal individuals. In the captopril suppression test, in patients with primary hyperaldosteronism, the plasma aldosterone levels are not suppressed by captopril because of the autonomy of the APA. However, the aldosterone to renin ratio postcaptopril was not different between APA and BAH as reported by Wu et al. [97]. In furosemide stimulation test, administration of this loop-diuretic should raise aldosterone levels. In APA, this response would not be seen. In summary, differentiating APA from BAH can be challenging.

Renin-independent hyperaldosteronism, inherited causes (mechanism 1a1) We now will address three autosomal-dominant genetic causes of inherited hyporeninemic hyperaldosteronism: (1) glucocorticoid-remediable hypertension (familial hyperaldosteronism type I), (2) familial aldosterone-producing adrenal

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adenomas or hyperplasia (CLCN5 mutations; familial hyperaldosteronism type II), and (3) KCNJ5 mutations (familial hyperaldosteronism type III). Such monogenic syndromes causing hypertension may be underdiagnosed because they may lack a unique clinical presentation [98].

Glucocorticoid-remediable aldosteronism (familial hyperaldosteronism type I) (mechanism 1a1) When hyperaldosteronism is present in the absence of an adrenal tumor and bilateral hyperplasia, the diagnosis of GRA should be entertained (Fig. 11.3) [99]. GRA may also be referred to as “dexamethasone-suppressible hypertension” or “familial hyperaldosteronism (FH) type I.” This is an autosomal-dominant inborn error that typically produces earlyonset hypertension. A cerebrovascular accident (i.e., stroke) in a young person raises the possibility of GRA. Somewhat surprisingly, normokalemia is observed in persons with GRA; however, a potassium-wasting diuretic can trigger hypokalemia in GRA patients. GRA may be the most common form of monogenic hypertension. Normally, the 11β-hydroxylase activity of the adrenal cortex’s glomerulosa and fasciculata is the result of independent genes and the enzymes that these genes encode. In the fasciculata, CYP11B1 (cytochrome P450 family 11 subfamily B member 1; chromosome 8q24.3) has 11β-hydroxylase activity that converts desoxycortisol to cortisol. Expression of CYP11B1 is controlled by pituitary ACTH. On the other hand, in the glomerulosa, CYP11B2 (cytochrome P450 family 11 subfamily B member 2; chromosome 8q24.3) has 11β-hydroxylase activity that converts DOC to corticosterone. CYP11B2 is controlled by potassium and ANG II (and by ACTH to much lesser extents). CYP11B2 encodes two other activities: 18-hydroxylase, which converts corticosterone to 18-hydroxycorticosterone, and 18-oxidase, which CYP11B2

CYP11B1

CYP11B2

Hybrid

CYP11B1

ACTH-responsive aldosterone synthase

FIGURE 11.3 Glucocorticoid-remediable aldosteronism (also known as dexamethasonesuppressible hypertension) results from a dominant mutation producing a CYP11B2/CYP11B1 fusion (hybrid) gene. As a result of an unequal crossover event, this hybrid aldosterone synthase gene is generated that responds to ACTH. Normally aldosterone synthase is controlled predominantly by angiotensin II.

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converts 18-hydroxycorticosterone to aldosterone. Collectively CYP11B2 has all three of these activities constituting “aldosterone synthase.” Thus aldosterone production is normally controlled by the RAAS, whereas cortisol production is controlled by pituitary ACTH. The CYP11B2 and CYP11B1 genes are normally 3040 kb apart. In GRA, unequal crossing over produces a fusion gene composed of the 50 portion of CYP11B1 (that includes an ACTH response element) and the 30 portion of CYP11B2 (encoding 11β-hydroxylase, 18-hydroxylase, and 18oxidase activities) [100]. This places the aldosterone synthesis activity of the chimeric gene errantly under the control of ACTH. With the fusion gene aberrantly under ACTH control, hyperaldosteronism results from normal levels of pituitary ACTH secretion [101]. In an understandable paradox, by administering exogenous glucocorticoids at physiologic concentrations to suppress endogenous pituitary ACTH, hypertension is alleviated because aldosterone production then declines. Because suppression was initially achieved using dexamethasone, an older term for GRA is “dexamethasone-suppressible hypertension.” Dexamethasone is a good choice for treatment of this disorder because dexamethasone lacks mineralocorticoid activity. If GRA is a clinical consideration, the diagnosis is supported by normalization of the BP after an approximate 2-week course of dexamethasone at physiological doses. Because of its genetic etiology, a family history of early-onset hypertension or cerebral hemorrhage before age 30 should raise the possibility of GRA. Other reasons to consider GRA include: the diagnosis of primary hyperaldosteronism in the absence of a tumor, hyporeninemic hypertension present in a child, low potassium develops after therapy with a potassium-wasting diuretic, and when hypertension is refractory (e.g., lack of response to ACE inhibitors or calcium channel blockers).

Laboratory notes Hyporeninemic hyperaldosteronism suggests many possible etiologies of disease in an individual patient. If GRA is possible, molecular testing should be considered (e.g., see ref. [102]). Measurement of urinary 18-hydroxy- and 18-oxocortisol is not available commercially and the genetic test should be much more definitive. Familial aldosterone-producing adrenal adenomas or hyperplasia (familial hyperaldosteronism type II) (mechanism 1a1) Familial hyperaldosteronism type II is an autosomal-dominant disorder that causes aldosterone-producing adrenal adenomas or hyperplasia. Recent studies report mutations in a chloride channel CLCN2 (chloride voltage-gated channel 2; chromosome 3q27.1) [103]. CLCN2 may increase aldosterone

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synthase activity (CYP11B2) [104]. This may occur when the mutation biases cells of the zona glomerulosa to depolarize triggering calcium influx via an activated voltage-gated calcium channel.

Laboratory notes It is hoped that commercial genetic testing will be developed as exists for GRA and other monogenic causes of ENaC activation. KCNJ5 mutation (familial hyperaldosteronism type III) (mechanism 1a1) KCNJ5 (potassium voltage-gated channel subfamily J member 5; chromosome 11q24.3) encodes GIRK4 [G protein-gated inwardly rectifying potassium (GIRK) channel]. The wild-type GIRK4 is a potassium channel allowing K1 to exit adrenocortical cells. This action of GIRK4 keeps a voltage-gated calcium channel closed and hyperaldosteronism is prevented. However, the mutant GIRK4 becomes permeable to Na1. When extracellular Na1 enters into the adrenocortical cell, the voltage-gated calcium channel opens triggering increased constitutive aldosterone production [105]. Thus germline KCNJ5 mutations cause an autosomal-dominant form of hyperaldosteronism (familial hyperaldosteronism type III) [106]. The specific KCNJ5 mutation may not predict the severity of the disease [107]. Sporadic mutations in KCNJ5 occur in B40% of APAs [108]. The features of APAs displaying KCNJ5 mutations comprise more severe hyperaldosteronism, younger aged patients, more women than men affected, and tumors of larger size [109].

Laboratory notes It is hoped that commercial testing for germline KCNJ5 mutations will play a role in evaluating patients with familial (autosomal dominant) hyperaldosteronism that lack a CYP11B1/2 chimeric gene (familial hyperaldosteronism type I) and lack CLCN2 mutations (familial hyperaldosteronism type II). Excess desoxycorticosterone (mechanism 1a1) DOC has salt-retaining properties and when pathologically elevated, hyporeninemic hypermineralocorticoidism results. Three conditions produce DOC-induced hypermineralocorticoidism: (1) 11β-hydroxylase deficiency (CYP11B1; cytochrome P450 family 11 subfamily B member 1) [110], (2) CYP17 deficiency [17α-hydroxylase 17,20-lyase (“17α-hydroxylase”); CYP17A1, cytochrome P450 family 17 subfamily A member 1; chromosome 10q24.32] [111], and (3) DOComas (i.e., DOC-secreting adrenal adenomas) [112]. 11β-Hydroxylase deficiency and 17α-hydroxylase deficiency are types of congenital adrenal hyperplasia (CAH). Their initial presentations may not

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involve hypertension. Data from the early 1990s suggested that DOC was more greatly elevated in ectopic ACTH syndrome causing Cushing syndrome than in other causes of Cushing syndrome [113]. Those data have not been well substantiated.

Desoxycorticosterone-secreting tumors (mechanism 1a1) Rarely, some adrenal adenomas will secrete DOC instead of aldosterone (e.g., DOComas) [114]. DOC has salt-retaining properties; thus DOC excess can cause hypokalemia, alkalosis, and hypertension. Similar to other conditions of DOC excess, aldosterone and renin are suppressed in DOComas [115].

11β-Hydroxylase deficiency (mechanism 1a1) Approximately 5% of cases of CAH result from 11β-hydroxylase deficiency (Fig. 11.4) [116]. As noted above, the 11β-hydroxylase of the fasciculata (CYP11B1) is encoded by the CYP11B1 gene. Normally this 11βhydroxylase converts 11-desoxycortisol to cortisol. If cortisol production is deficient because of decreased CYP11B1 activity, ACTH will rise. In turn ACTH increases the production of DOC, which has mineralocorticoid activity. Elevated DOC due to the CYP11B1 mutation produces hypertension that is usually of childhood onset. The effects of DOC causing salt and water retention will suppress renin secretion (Note: CYP11B2 of the zona glomerulosa has aldosterone synthase activity. CYP11B2 is controlled by angiotensin II). Because of the biochemical barrier to cortisol synthesis induced by CYP11B1 deficiency, under the influence of elevated ACTH concentrations, adrenal steroid production is shunted toward the manufacture of adrenal androgens. This is expressed as elevations in the concentrations of androstenedione and dehydroepiandrosterone (DHEA). Also frequently elevated is 17-hydroxyprogesterone, which is the immediate precursor of androstenedione. Thus a state of hyperandrogenism exists in utero. In 46XX fetuses, exposure to elevated concentrations of adrenal androgens produces virilization of the external genitalia manifested as ambiguous genitalia (a “46XX, disorder of sexual development”; see Chapter 16). If untreated, girls will further virilize during childhood. Male children affected by CYP11B1 mutations display precocious puberty. Infants with 11β-hydroxylase deficiency do not manifest salt-losing crises (e.g., Addisonian crisis) because salt wasting is prevented by elevated DOC levels. 11β-Hydroxylase deficiency leads to elevations in serum desoxycortisol (e.g., “compound S”) and increased urinary excretion of its metabolite tetrahydrodeoxycortisol (tetrahydro-compound S).

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Cholesterol HO

O

O

CYP11A1

CYP17A1

OH

17-OH Pregnenolone

Pregnenolone HO

HO O

HSD3B2

CYP17A1

O OH

HSD3B2

17-OH Progesterone

Progesterone O

O

OH

OH O

CYP21A2

O

CYP21A2

OH

Desoxycorticosterone (DOC)

Desoxycorticol O

O OH CYP11B2

CYP11B1

O HO

HO

OH HO

O

O

HO

18-Hydroxycorticosterone OH

O

O CYP11B2

OH

Cortisol

Corticosterone

O CYP11B2

OH

O

O

HO

Aldosterone O

FIGURE 11.4 11β-Hydroxylase deficiency (also known as CYP11B1 deficiency) leads to reduced concentrations of cortisol. This elicits increased ACTH release from the anterior pituitary. Enzymes responsive to ACTH (CYP11A1, HSD3B2, and CYP21A2) lead to increased concentrations of desoxycorticosterone (DOC). DOC has mineralocorticoid activity. A high level of DOC production suppresses aldosterone production because of renin suppression. Note: HSD3B2 (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2) is also known as 3 beta-hydroxysteroid dehydrogenase-delta4,5 isomerase.

CYP17 deficiency (mechanism 1a1) A rare cause of CAH is 17α-hydroxylase deficiency (CYP17A1; more formally: 17α-hydroxylase 17,20-lyase) (Fig. 11.5) [117]. Normally, CYP17 converts pregnenolone to 17-hydroxypregnenolone, and progesterone is converted to 17-

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Cholesterol HO

O

CYP11A1

O CYP17A1

OH

Pregnenolone

17-OH Pregnenolone

HO

HO O

HSD3B2

CYP17A1

O OH

HSD3B2

Progesterone O

17-OH Progesterone O

OH O

CYP21A2

OH O

CYP21A2

OH

Desoxycorticosterone (DOC) O

Desoxycorticol O

OH CYP11B2

O

OH CYP11B1

HO

Corticosterone

O CYP11B2

OH HO

OH

Cortisol

O

O

HO

18-Hydroxycorticosterone OH

O

O CYP11B2

O HO

O

HO

Aldosterone O

FIGURE 11.5 In 17β-hydroxylase deficiency (also known as CYP17A1) there is impaired conversion of pregnenolone to 17-hydroxypregnenolone and impaired conversion of progesterone to 17-hydroxyprogesterone (vertical hatched bar). Reduced cortisol production elicits increased ACTH release from the anterior pituitary. Enzymes responsive to ACTH (CYP11A1, HSD3B2, and CYP21A2) lead to increased concentrations of desoxycorticosterone (DOC). DOC has mineralocorticoid activity. A high level of DOC production suppresses aldosterone production because of renin suppression. Note: HSD3B2 (hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2) is also known as 3 betahydroxysteroid dehydrogenase-delta4,5 isomerase.

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hydroxyprogesterone. In the absence of normal CYP17 activity, there is severe impairment of cortisol biosynthesis. CYP17 mutations will also influence sex steroid production because this gene also encodes the 17,20-desmolase (lyase) activity. 17,20-Desmolase converts 17-hydroxypregnenolone to DHEA and 17-hydroxyprogesterone to androstenedione. Thus, in addition to interference in cortisol biosynthesis, CYP17 mutations preclude normal sex steroid production of both androgens and estrogens. In contrast, the aldosterone pathway remains intact and salt wasting does not occur. Analogous to 11β-hydroxylase deficiency, ACTH levels rise because of cortisol deficiency in CYP17 deficiency. Elevated ACTH elicits DOC overproduction in the zona glomerulosa. Salt and water retention by DOC suppresses renin and aldosterone synthesis and secretion. Because of the mineralocorticoid activity of DOC, salt wasting is not present in CYP17 deficiency; in fact, excessive salt and water retention from excessive DOC activity frequently produces hypertension presenting in childhood in affected individuals. Because testosterone is not produced in normal concentrations in utero in CYP17 deficiency, 46,XY males will appear as phenotypic females and present with delayed puberty (a “46XY, disorder of sexual development”; see Chapter ____). Uterus, fallopian tubes, and the upper one-third of the vagina are absent because of anti-Mu¨llerian hormone that is elaborated by the testicular Sertoli cells. There is no virilization of the external genitalia in utero because of the block in testosterone biosynthesis. In 46,XX females, the genitalia are not ambiguous; however, puberty and menses will not occur spontaneously because estrogens will not be produced.

Laboratory notes In a person with hyporeninemic, hypokalemic, alkalotic hypertension that lacks elevations in aldosterone and cortisol, DOC should be measured. The clinical features of adenoma versus CAH are helpful in distinguishing these disorders. Urine steroid testing [e.g., 17-ketogenic steroids (breakdown products of 17-hydroxyprogesterone, desoxycortisol, and cortisol), 17hydroxycorticosteroids (breakdown products desoxycortisol and cortisol), and 17-ketosteroids (breakdown products of adrenal androgens)] is no longer performed.

Excess cortisol (mechanism 1a1) If cortisol is sufficiently elevated, in mineralocorticoid-responsive tissues, cortisol can overcome the “inhibition” to raised intracellular cortisol normally afforded by HSD11B2. Hypercortisolism can result from Cushing syndrome or the rare entity termed “cortisol resistance.”

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Cushing syndrome (mechanism 1a1) Cushing syndrome is suggested by centripetal obesity, moon facies, buffalo hump, stria, easy bruisability, psychosis, osteoporosis, glucose intolerance, or frank diabetes mellitus in addition to hypertension. Hypertension in Cushing syndrome results from: (1) increased production of angiotensinogen (the renin substrate), (2) increased tissue responsiveness to catecholamines, and (3) increased mineralocorticoid activity because excess cortisol binds to the MR. Laboratory notes In order to diagnose Cushing syndrome, hypercortisolism must be confirmed. There are three approaches that can indicate hypercortisolism: (1) elevated 24-h urinary free cortisol (UFC), (2) elevated midnight salivary cortisol, or (3) lack of suppression of an 8 a.m. plasma cortisol level following a 10 p.m. dose of dexamethasone (1 mg, PO) taken the previous evening. Two such screening tests should be abnormal before the diagnosis of hypercortisolism is established. If one test is normal and one test is abnormal, a third “tie-breaker” test should be performed. Once hypercortisolism has been established, the cause of the hypercortisolism is most commonly pursued using one of two approaches: (1) the classic low-dosehigh-dose dexamethasone suppression test or (2) inferior petrosal venous sinus sampling. More details concerning the diagnosis of Cushing syndrome can be found in Chapter__.

Cortisol resistance (mechanism 1a1) Cortisol resistance is a very rare syndrome that results from a defect in the type II glucocorticoid receptor in the periphery, hypothalamus, and pituitary gland [118,119]. Because of the glucocorticoid receptor resistance, cortisol levels are perceived as low centrally, and ACTH levels then rise. The elevated ACTH concentrations drive increased cortisol and DOC production. The increased cortisol levels avert a clinically hypoglucocorticoid state and patients are not usually clinically Cushingoid because of their resistance to cortisol. However, the elevated cortisol levels can have mineralocorticoid activity causing hypermineralocorticoidism, hypertension, hypokalemia, and suppressed renin and aldosterone concentrations [120]. Laboratory notes Elevated UFC, elevated plasma cortisol, and hypertension in the absence of a Cushingoid appearance and other clinical features of Cushing syndrome differentiate this condition from Cushing syndrome. High ACTH concentrations in

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cortisol resistance can additionally stimulate excess adrenal androgen production, leading to hirsutism and precocious puberty [121].

Excess sex steroids (mechanism 1a1) If sufficiently elevated in concentration, sex steroids (e.g., androgens) could theoretically bind to the MR inducing salt and water retention. However, other non-RAAS mechanisms may be at work in cases of testosterone-induced hypertension [122,123]. Excessive levels of sex steroids do have salt-retaining capacities and pathological anabolic steroid excess can cause hypertension [124]. In one study of black South Africans, hypertensive men and women did have higher testosterone levels [125]. A study from Germany found that higher testosterone levels and higher BP were related [126]. On the other hand, there are data that reduced levels of testosterone may also be associated with hypertension [127,128]. A study in elderly men showed lower free testosterone levels in the hypertensive men [129]. GarciaCruz et al. reported that in men undergoing prostate biopsy, reduced testosterone concentrations were related to hypertension [130]. In the insulinresistant metabolic syndrome where hypertension is common, testosterone levels were also reduced [131]. The apparently conflicting data in the above two paragraphs can be resolved if there is a “U-shaped” relationship between testosterone and BP: Either deficient or excess androgens might elevate BP. Certainly there are other U-shaped relationships in biology. For example, risk of death and cholesterol levels display a U-shaped relationship. The effect of estrogen on BP is controversial [132,133]. Postmenopausal increases in BP may be related to estrogen deficiency in such women [134]. The role of progesterone in BP regulation is not well understood [135]. Progesterone can bind to the MR but at affinities much lower than aldosterone 136]. This suggests that progesterone can serve as an antagonist to aldosterone. In pregnancy, aldosterone is elevated 46-fold. Estrogen and progesterone may alter the set point for vasopressin release favoring water retention [137]. Oral contraceptive use accounts for 2%4% of hypertension in adults and is a common cause of endocrine-induced hypertension [138,139]. Oral contraceptive-induced hypertension likely results from activation of RAAS axis and increased production of angiotensinogen, which is the substrate for renin.

Laboratory notes There are no specific diseases in this category where elevations in androgens cause hypertension to the exclusion of any other findings (e.g., hirsutism or

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virilization in women). The authors would not recommend that testosterone, estrogen, or progesterone be measured as part of the workup for hypertension. However, clinicians should ask about sex steroid use as part of the patient’s drug-use history [140].

End-organ disorders causing ENaC activation: Apparent mineralocorticoid excess, mineralocorticoid receptor gain-offunction mutations, and ENaC gain-of-function mutations Apparent mineralocorticoid excess (mechanism 1a2) AME should be considered when alkalotic, hypokalemic, hyporeninemic hypertension is associated with depressed aldosterone and DOC concentrations and normal cortisol concentrations [141]. This uncommon autosomal recessive condition results from deficiency of the enzyme HSD11B2 (hydroxysteroid 11-beta dehydrogenase 2) (Fig. 11.6) [142]. HSD11B2 normally converts cortisol to cortisone in tissues that are normally mineralocorticoid responsive (e.g., the DCT and CD of the kidney). This biotransformation also takes place in the liver. The age of onset of AME can be in childhood. CH2-OH C=0

CH2-OH HO

OCH C = 0

O

CH2OH C=0

OH

HO

O

HSD11B2

O

Cortisol

Aldosterone

OH

O

Cortisone

Binds

Does not bind

Normal mineralocorticoid activity

Normal blood pressure

Mineralocorticoid (type I glucocorticoid) receptor (MR)

Binds

Binds CH2-OH

CH2-OH

HO

OCH C = 0

O

Aldosterone

HO

O

Cortisol

C=0 OH

HSD11B2 Deficiency

Excessive mineralocorticoid activity

Hypertension and hypokalemia and alkalosis

FIGURE 11.6 Normally the mineralocorticoid receptor (MR) is protected from the action of cortisol by the conversion of cortisol to cortisone catalyzed by HSD11B2 (top panel). In apparent mineralocorticoid excess (bottom panel), a deficiency of HSD11B2 allows the persistence of cortisol in mineralocorticoid-sensitive tissues. Therefore cortisol binds to the mineralocorticoid receptor and produces hypermineralocorticoid-induced hypertension.

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Pathophysiologically, with insufficient HSD11B2 activity, the conversion of cortisol to cortisone is impaired causing higher cortisol levels in mineralocorticoid-responsive tissues [143]. In turn excess cortisol is available to bind to the MR. The resulting mineralocorticoid action of cortisol elicits salt and water retention that produces hypokalemia, alkalosis, and hypertension. Salt retention and elevated BP suppress renin, and depressed renin subsequently suppresses aldosterone secretion. “Apparent mineralocorticoid excess” (AME) is an appropriate name for this condition as the condition clinically is consistent with hyperaldosteronism, yet there are no elevations in aldosterone, cortisol, or DOC. Spironolactone and dexamethasone have been used to treat HSD11B2 deficiency. Spironolactone is an MR blocker, whereas dexamethasone suppresses cortisol synthesis and dexamethasone lacks mineralocorticoid activity. An acquired form of AME can result from eating black licorice that contains glycyrrhetinic acid that can inhibit HSD11B2.

Laboratory notes An increased ratio of cortisol metabolites to cortisone metabolites [increased ratio of tetrahydrocortisol to tetrahydrocortisone (THF/THE ratio)] is consistent CH2-OH

Cortisol

Cortisone

C=0 HO

OH

CH2-OH C=0

O

HSD11B2

O

OH

O

Urinary metabolites

CH2-OH

Tetrahydrocortisol (THF) HO

H

C=0

Tetrahydrocortisone (THE) O

OH

CH2-OH C=0 OH

H

HO H

H

HO H

H

FIGURE 11.7 With a deficiency of HSD11B2 (causing apparent mineralocorticoid excess), cortisol is incompletely converted to cortisone, and, therefore, cortisol can bind to the mineralocorticoid receptor (MR). Because of deficient HSD11B2 activity, there is an increased ratio of the urinary cortisol metabolite THF (tetrahydrocortisol) to the urinary cortisone metabolite THE (tetrahydrocortisone).

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with the diagnosis of HSD11B2 deficiency (Fig. 11.7). Further biochemical evidence of AME can be found in the detection of two metabolites of cortisol: 18-hydroxycortisol and 18-oxocortisol that result from the 18-hydroxylase activity of CYP11B2 (Fig. 11.8). The urinary metabolite of 18-oxocortisol is tetrahydro-18-oxocortisol. In AME, the ratio of tetrahydro-18-oxocortisol to tetrahydroaldosterone is increased. Despite such elegant biochemistry, the esoteric urine tests are not commercially available. However, the diagnosis of AME can be established on a molecular basis and such genetic testing is commercially available.

Mineralocorticoid receptor gain-of-function mutations (mechanism 1a2) A gain-of-function mutation in the MR has been described as a cause of autosomal-dominant hypokalemic hypertension termed “Geller syndrome” (also known as pseudoaldosteronism type II) [144]. Experimental data suggest that progesterone can bind to this mutant MR, stimulating the receptor. Normally, progesterone does not activate the wild-type MR. During pregnancy when progesterone concentrations are sufficiently elevated, hyperaldosteronism and hypertension then develop. Hypertension is worse during the second and third trimesters of pregnancy when progesterone levels are highest. Following delivery with a decline in progesterone concentrations to normal, the BP declines [145]. The MR mutation denies the ability of spironolactone to bind to the MR and, therefore, spironolactone is ineffective as an antihypertensive drug in Geller syndrome.

Laboratory notes There is no specific biochemical test for Geller syndrome. Geller syndrome is a rare cause of pregnancy-induced hypertension. The diagnosis can be considered when hypokalemic/alkalotic hypertension develops

C=0

HO

O

Cortisol (cpd "f")

CH2-OH

CH2-OH

CH2-OH

HO

OH

HOCH

C=0

O

18-Hydroxycortisol

O=CH C = 0 HO

OH

OH

O

18-Oxocortisol

FIGURE 11.8 Biochemical evidence of apparent mineralocorticoid excess can be seen in the detection of two metabolites of cortisol, 18-hydroxycortisol and 18-oxocortisol, that result from the 18-hydroxylase activity of CYP11B2.

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during pregnancy and other causes have been excluded. Family history may be informative as this is inherited as an autosomal-dominant trait. Possibly genetic analysis of the MR will become commercially available in the future [146].

ENaC gain-of-function mutations (mechanism 1a2) Autosomal-dominant gain-of-function mutations in the β or ϒ chains of ENaC (the amiloride-sensitive epithelial sodium channel) lead to excessive sodium reabsorption by the distal convoluted tube and CD causing Liddle syndrome (familial pseudoaldosteronism type I) [147]. Onset is often in adolescence. Increased sodium reabsorption is followed necessarily by increased water reabsorption, expanded blood volume, and hypertension. The resultant effect is similar to increased levels of mineralocorticoids that produce hypokalemia and alkalosis. Because of hypertension, renin and aldosterone are both suppressed. As noted above, ENaC normally has three types of subunits: α, β, and ϒ. α is the most important subunit. All subunits are structurally similar containing an N-terminal, large extracellular domain, two transmembrane spanning domains, and a C-terminal, short intracellular domain. The ENaC β subunit is encoded by SCNN1B (sodium channel, nonvoltage-gated 1, beta). The ENaC ϒ subunit is encoded by SCNN1G (sodium channel, nonvoltage-gated 1, gamma).

Laboratory notes Hypertension and laboratory evidence of mineralocorticoid excess (i.e., hypokalemia and alkalosis) with suppressed renin and aldosterone without elevations in cortisol or DOC suggest AME, Geller syndrome, or Liddle syndrome. If there is no family history of affected siblings, AME is a consideration because it is inherited as an autosomal recessive disorder. Family history does not help distinguish Geller syndrome from Liddle syndrome, as they are both autosomal-dominant disorders. Hypertension’s appearance or worsening in pregnancy suggests Geller syndrome; however, most hypertension in pregnancy is not caused by monogenic disorders. Practically speaking, if clinical hypermineralocorticoidism (as described earlier) is present early in life, in the absence of pregnancy, AME and Liddle syndrome could be distinguished by family history. Gene sequencing is commercially available for HSD11B2 (the cause of AME), and for SCNN1B and SCNN1G (causes of Liddle syndrome). If urinary metabolite testing were available, the THF/THE ratio would be elevated in AME and normal in Liddle syndrome.

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Insulin resistance (mechanism 1b): obesity, type 2 diabetes, the metabolic syndrome, and acromegaly: multiple mechanisms of hypertension Obesity, type 2 diabetes, the metabolic syndrome, and acromegaly cause hypertension, at least in part, via the development of an insulin-resistant state [148]. Certainly Cushing syndrome also causes insulin resistance; therefore hypertension from Cushing syndrome can result from multiple mechanisms. The discussion of obesity [including obesity related to atypical antipsychotics (e.g., clozapine and olanzapine) [149]] is beyond the scope of this chapter. Acromegaly is a disease of excess growth hormone (GH) secretion with a subsequent elevation in insulin-like growth factor-I (IGF-I). Acromegaly is suggested by large hands and feet, facial soft tissue and mandibular growth, skin tags, intestinal polyposis, diabetes mellitus, myopathy, and organomegaly (e.g., enlarged heart and liver). As many as half of all acromegalic patients display hypertension [150]. Similar to the metabolic syndrome, obesity-associated hypertension, and type 2 diabetes, in response to the insulin resistance of GH excess, hyperinsulinism develops in acromegaly. Hyperinsulinism, in turn, can cause sodium retention, produce vascular hypertrophy, increase Na1/K1 ATPase pump activity, and stimulate increased sympathetic activity [151,152]. By causing sodium retention with increased fluid retention, renin suppression is observed in acromegaly. Although both diabetes mellitus and obesity are associated with hypertension [153], other secondary causes of hypertension in such situations should be excluded. In stage III diabetic nephropathy (recognized by the presence of persistent microalbuminuria without dipstick proteinuria), hypertension may be observed [154]. In stage IV and V diabetic nephropathy, hypertension is essentially universal and accompanied by dipstick-positive proteinuria that can progress to nephrotic syndrome and/or end-stage renal disease. Diabetic nephropathy can result from any type of diabetes based upon the severity of the hyperglycemia and with sufficient duration of diabetes. Therefore diabetic nephropathy can affect individuals with type 1 or type 2 diabetes [155,156]. Insulin resistance is not a feature of type 1 diabetes other than during diabetic ketoacidosis (DKA). During DKA because of insulin deficiency there is an approximate 20% decline in the cellular content of insulin-responsive facilitative glucose transporters (GLUT4; SLC2A4, solute carrier family 2 member 4; chromosome 17p13.1). As well, with acute insulin deficiency, reduced quantities of GLUT4 are located on the plasma membranes prohibiting normal glucose uptake by muscle and fat cells. With insulin replacement during treatment for DKA, GLUT4 deficiency is resolved and GLUT4 will be properly localized on plasma membranes at normal concentrations.

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Laboratory notes Diabetes mellitus is discussed in the hyperglycemia Chapter ___. Proper evaluation for acromegaly includes measurement of the IGF-I concentration and measurement of GH following a 75-g oral glucose load. IGF-I concentrations need to be compared with age- and sex-matched reference intervals [157]. Using current generation assays for GH, after a 75-g oral glucose load, GH is normally ,1 ng/mL. In acromegaly, GH is not suppressed 2 h after the oral glucose load and IGF-I is greatly elevated. Pheochromocytoma: direct effects on vascular smooth muscle and myocardium (mechanism 2a) Catecholamines can cause hypertension by stimulating the heart via β1adrenergic receptors increasing contractility and heart rate. In addition, the direct action of catecholamines on α1-receptors in vascular smooth muscle raises PVR. Pheochromocytoma is most often a benign catecholaminesecreting tumor of the adrenal medulla [158]. Because catecholamines can stimulate renin secretion via β2-adrenergic receptors, RAAS activation can also be present in patients with pheochromocytoma [159].

Laboratory testing Because the frequency of pheochromocytoma is very low, measuring catecholamines and metabolites in urine or blood is not cost-effective unless the patient has (1) a history of paroxysmal or severe hypertension or (2) there is suspicion of a genetic syndrome with increased risk for pheochromocytoma (e.g., multiple endocrine neoplasia type 2, type 1 neurofibromatosis, von HippelLindau syndrome, SturgeWeber syndrome, Carney triad, or CarneyStratakis dyad) [160]. Twenty-four-hour urinary total metanephrines is the most specific test for the detection of pheochromocytoma. On the other hand, the plasma-free metanephrine measurement is the most sensitive test for the detection of pheochromocytoma [161163]. Hyperthyroidism (mechanism 2a) and hypothyroidism A normal action of thyroid hormone is to increase the sensitivity of tissues to β-adrenergic stimulation that: (1) increases heart rate and increases stroke volume (via increased contractility) that raise cardiac output, (2) decreases PVR, and (3) decreases diastolic pressure [164]. Thus hyperthyroidism most often causes systolic hypertension with a widened pulse pressure. Indeed, B30% of Graves’ disease patients demonstrate systolic hypertension. Hyperthyroidism can also produce hypercalcemia from increased bone turnover, and hypercalcemia can cause BP elevations.

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Approximately 15%30% of adults with hypothyroidism display elevated BP [165]. The mechanism of hypertension in hypothyroidism is unclear. Some experts have proposed that hypothyroidism raises PVR and increases arterial wall stiffness [166].

Laboratory testing Testing for thyroid disease is straightforward: TSH and FT4 should be measured. If hyperthyroidism is a consideration, T3 should also be measured. Hypercalcemia: Direct effects on vascular smooth muscle (mechanism 2b) Hypercalcemia can cause hypertension [167]. In dogs, hypercalcemia causes hypertension via a direct effect of calcium on vascular smooth muscle causing vasoconstriction and via the effect of calcium raising catecholamine secretion [168,169]. Therefore elevated calcium levels can have a direct vasoconstrictive effects. In epidemiologic studies, low dietary calcium or magnesium intake is associated with hypertension [170]. Approximately 50%70% of patients with primary hyperparathyroidism are hypertensive, whereas B35% of patients with hypercalcemia from other etiologies are hypertensive [171]. Hypertension can be observed in pseudohypoparathyroidism [172]. This may result from a decline in vasodilatory response to parathyroid hormone (PTH) (e.g., PTH resistance).

Laboratory testing If total calcium is elevated, it is important to ensure that this is not due to hemoconcentration. Therefore confirmation of hypercalcemia by finding an elevated ionized calcium concentration is recommended. Whereas total calcium can be “corrected” for elevated or depressed albumin concentrations (e.g., for every change in albumin of 1 g/dL compared with 4 g/dL, the total calcium changes by 0.8 mg/dL), ionized calcium is a direct measurement and is preferred. When hypercalcemia is detected, the assessment should include a measurement of PTH, phosphate, and alkaline phosphatase. Elevated PTH in hypercalcemic patients with normal renal function is most commonly observed in primary hyperparathyroidism. If the PTH is suppressed or inappropriately normal, considerations as causes of the hypercalcemia include hypercalcemia of malignancy (e.g., elevated PTH-related peptide), local osteolytic hypercalcemia, immobilization, vitamin D or A toxicity, Addison disease, hypo- or hyperthyroidism, Paget disease, granulomatous disease (e.g., sarcoidosis), thiazides, lithium, milk-alkali syndrome, and benign familial hypercalcemia. In hyperparathyroidism, PTH is elevated, phosphate is typically depressed (because of hyperphosphaturia), and hypercalciuria is

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present (potentially leading to nephrolithiasis or nephrocacinosis). Renin is low to normal in cases of hypercalcemic hypertension. The evaluation of hypercalcemia is further described in Chapter ___.

SIADH: vasopressin excess with direct effects on vascular smooth muscle (mechanism 2c) The rare case of the syndrome of inappropriate secretion of ADH (also known as vasopressin; SIADH) with hypertension may result from the direct vasoconstrictor effect of high concentrations of ADH [173].

Laboratory testing Hyponatremia together with a reduced volume of urine output and urine sodium of B 5 . 40 meq/L (certainly a UNa1 . 20 mEq/L) in an appropriate clinical setting (e.g., head trauma, pulmonary disease, etc.) are consistent with SIADH. In SIADH urine osmolality should be greater than serum osmolality. Vasopressin need not be measured to establish the diagnosis of SIADH. Hypertension in pregnancy—abnormal angiogenesis (mechanism 2c) Toxemia of pregnancy is a broad term for a group of disorders characterized by material hypertension and proteinuria and/or end-organ damage [174]. In the absence of maternal seizures, such disorders are termed “preeclampsia.” The development of maternal seizures advances this disorder to “eclampsia.” When coexistent with hemolysis, thrombocytopenia, and liver dysfunction, the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome is diagnosed [175]. Whereas the pathogenesis of toxemia is controversial, one cause appears to involve abnormalities of placental angiogenesis [176]. Toxemia of pregnancy is believed to result from inadequate implantation of the placenta into the uterine wall [177]. Failure of normal placental implantation leads to the secretion of fms-like tyrosine kinase 1 (sFlt-1; FLT1 gene; chromosome 13q12.3), which antagonizes vascular endothelial growth factor (VEGF). VEGF is necessary for normal placental angiogenesis. Other factors involved in preeclampsia may include increased concentrations of soluble endoglin (sEng), and lower concentrations of placental growth factor (PlGF) and vascular endothelial growth factor A (VEGF-A) [178180]. The ratio of sFlt-1 to PlGF, a proangiogenic protein, is increased prior to the development of preeclampsia.

Laboratory testing There is no specific endocrine testing for preeclampsia or eclampsia.

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Approach to the patient If primary hyperaldosteronism accounts for up to B10% of all cases of adult hypertension, endocrine causes of hypertension are actually relatively common and deserve routine diagnostic consideration during the evaluation of the patient. Secondary causes of hypertension are even more common in children than in adults. Flynn [181] reported that 99% of cases of hypertension in infants are secondary, 70%85% of cases of hypertension in children are secondary, whereas, the frequency of secondary hypertension in adolescents is similar to that observed in adults (5%15%). The initial laboratory studies recommended by the 2017 AAC/AHA guideline for all adults presenting with hypertension can suggest three important causes of secondary hypertension: (1) renal disease (abnormal urinalysis, creatinine, eGFR), (2) diabetes mellitus [hyperglycemia (fasting plasma glucose)], or (3) hypermineralocorticoidism (hypokalemia). The CBC provides information about anemia and general health. The lipid profile, in part, assesses risk for atherosclerotic CVD. Sodium is monitored during antihypertensive therapy (as is potassium and creatinine). Because hypercalcemia can be a clinically silent cause of hypertension, the measurement of calcium is part of the battery of screening tests. TSH is also included as a screening test for hyper- and hypothyroidism. Of the many endocrine causes of hypertension, for most disorders, while hypertension may be present, it is usually other features of the endocrine disease that lead to its diagnosis (Table 11.8). In only two endocrine disorders is

TABLE 11.8 Endocrine causes of hypertension, their usual clinical presentation, and their evaluation. Endocrinopathy causing hypertension

Usual clinical presentationa of the disorder

Evaluation

Estrogen-induced hypertension

History of oral contraceptive use or current pregnancy

hCG (pregnancy test)

Hypermineralocorticoidism

Hypertension, 1 / 2 hypokalemia, 1 / 2 alkalosis

Renin, aldosterone

Cushing syndrome

Centripetal obesity, moon facies, buffalo hump, diabetes, osteopenia/fractures, stria, psychosis

24-UFC, MN salivary cortisol, AM cortisol postPM dexamethasone; lowdose-high-dose dexamethasone suppression test or IPSS testing (Continued )

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TABLE 11.8 (Continued) Endocrinopathy causing hypertension

Usual clinical presentationa of the disorder

Evaluation

Pheochromocytoma

Hypertension, 1 / 2 episodic, anxiety, tachycardia, sweating with episodes

Urinary metanephrines, plasma-free metanephrines

Acromegaly

Soft tissue growth, facial coarsening, acral growth, organomegaly, diabetes

IGF-I, GH postglucose

Hypercalcemia

Malaise, gastrointestinal upset, renal lithiasis

Plasma calcium

Hyperthyroidism

Tachycardia, weight loss, diarrhea, smooth skin and hair, oligomenorrhea

TSH, FT4, T3

Hypothyroidism

Bradycardia, weight gain, constipation, dry skin and hair, reduced mental acuity, oligomenorrhea

TSH, FT4

Vasopressin excess from SIADH

Hyponatremia, concentrated urine, underlying illness (CNS, respiratory, etc.)

Hyponatremia with concentrated urine specific gravity or osmolality

Diabetes mellitus

Hyperglycemia

FBG, PG 2-g OGTT, HbA1c

Obesity



Diagnosed by physical examination

Toxemia of pregnancy

History of current pregnancy

Clinical diagnosis

24-UFC, 24-hour urinary free cortisol; FBG, fasting blood glucose; hCG, human chorionic gonadotropin; IPSS, inferior petrosal venous sinus sampling; OGTT, oral glucose tolerance test; PG, plasma glucose; SIADH, Syndrome of inappropriate ADH (antidiuretic hormone). a This is not an exhaustive list of the features of the endocrinopathies.

hypertension a major presenting problem: hypermineralocorticoidism and pheochromocytoma. Because hypokalemia has been reported in pheochromocytoma, hypokalemia cannot be used to distinguish pheochromocytoma and hypermineralocorticoidism. Furthermore, because hypokalemia is not a universal finding in

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Incr. aldosterone

Incr. cortisol

Yes

No

Yes

1

Aldosterone PRA ratio

No

Yes

Evaluate for hypercortisolism (including cortisol resistance)

Primary hyperaldosteronism

No

6

5

3

≥20–25

Continued Figure 10

Incr. DOC 4

2

End-organ disorder (Figure 11)

Rule out: DOComa 11β-Hydroxylase def. 17α-Hydroxylase def.