Kernicterus: Report based on a symposium held at the IX International Congress of Paediatrics 9781487583491

Citation preview

KERNICTERUS

KERN ICTER US Report based on a symposium held at the

IX INTERNATIONAL CONGRESS OF PAEDIATRICS Montreal, July 1959

edited by

ANDREW SASS-KORTSAK

UNIVERSITY OF TORONTO PRESS

Copyright, Canada, 1961, by University of Toronto Press Printed in Canada Reprinted in 2018 ISBN 978-1-4875-8217-3 (paper)

PREFACE

THE IXTH INTERNATIONAL CONGRESS OF PAEDIATRICS selected kernicterus as one of the topics for organized discussion in recognition of the significant advances made in our understanding of this problem during the past decade. It was felt that a presentation of these new facts and the ideas emerging from them would be of interest to all paediatricians and, in particular, those handling newborns and prematures. It was felt also that an opportunity for free discussion and the exchange of ideas among those engaged in research work in this field would be of benefit to these people and to their work, as well as to their audience. In these discussions, as well as presenting their achievements and the proven facts, the contributors were asked to bring out the problems that remain to be solved. It was hoped that this approach would stimulate much needed further research in this field. The term "kernicterus" was originally coined to define a macroscopic pathological finding-the yellow staining of the brain nuclei. When it was recognized that this post-mortem finding correlated well with a distinct clinical syndrome, the meaning of the term was widened to include the clinical syndrome as well. Our knowledge concerning the pathogenesis of this clinical and pathological entity has increased a great deal in the past ten years through the work of clinicians as well as of basic scientists. The recognition that the yellow staining of the brain is due to the deposition of indirect reacting, unconjugated bilirubin, the demonstration that the staining of the brain is by no means exclusively localized to the nuclei, and the proof of the actual cytotoxicity of bilirubin led to the suggestion of another name, probably more appropriate, for this condition: "bilirubin encephalopathy." Although this new term defines more adequately our present ideas concerning what was generally meant by "kernicterus," it will require time for the new term to be widely recognized and to be identifiable by all concerned. Hence, rightly or wrongly, the old term was retained as the title of the symposium and of this report. The unexpectedly large audience which attended this symposium at the IXth International Congress of Paediatrics, the many favourable V

vi

PREFACE

comments received and the numerous requests for abstracts of papers suggested that this material should be considered for publication. Permission to make use of the material was obtained from the Organizing Committee of the IXth International Congress of Paediatrics. The entire session had been recorded on tapes by the Congress and these were made available to the Editor by the Congress Committee. Dr. Alan Ross, the General Chairman of the Congress, Dr. Harry J. Ebbs, the Chairman of the Programme Committee, and Dr. Ronald Denton, the General Secretary of the Congress, gave most valuable support not only in connection with the organization of the symposium, but also with the preparation of this report. Special thanks are due to Dr. Audrey K. Brown and Dr. J. F. Lucey for their ideas concerning the subject-matter for the symposium and for their enthusiastic support during the early stages of organization. Miss Laura Scott has given expert secretarial help; her invaluable assistance in connection with the difficult task of the transcription of the recordings is gratefully acknowledged. Of necessity the time allotted for sessions at the Congress was limited, and therefore the discussion had to be restricted to selected topics and comments from the floor had to be kept to a minimum. Participation in the panels was by invitation only and it was not possible to include all of those who had contributed significantly to our understanding of the problems of kemicterus. Their participation in the discussion from the floor was greatly appreciated and showed an excellent spirit of co-operation on their part. The credit for the success of the symposium and for this report is due in large measure to the efforts of the panel chairman and of the contributors. I should like to thank all of the authors for their co-operation in the preparation of these papers for book publication. Finally, your editor wishes to acknowledge the excellent co-operation on the part of the management and staff of the University of Toronto Press. ANDREW SAss--KoRTSAK

CONTENTS

PREFACE

V

CONTRIBUTORS

X

PANEL A . KERNICTERUS OF PREMATURITY: INCIDENCE AND AETIO-

1

LOGICAL FACTORS

Bruce Chown Introduction The Incidence of Kernicterus not due to Haemolytic Disease among Premature Babies V . Mary Crosse Peak Levels of Serum Bilirubin in Normal Premature Infants Ruth C. Harris Experience with Kernicterus in Premature Infants David A. I. Grewar The Neurological Sequelae of Hyperbiliruhinaemia of Prematurity Alfred M. Freedman Jerold F. Lucey Physiological Jaundice Re-examined PANEL

B.

3

4 10

13 20 29

FACTORS INFLUENCING THE LIFE SPAN OF THE RED BLOOD

37

CELL

Introduction Wolf W . Zuelzer Physiological Mechanisms of Red Blood Cell Destruction James H. Jandl The Life Span of Erythrocytes in the Newborn Eugene Kaplan The Relationship between Abnormality of Glutathione Metabolism in Erythrocytes from Newborn Infants and the Occurrence of Drug-induced Haemolytic Anaemia William H. Zinkham Peculiarities of Erythrocytes of the Newborn Audrey K. Brown Metabolism of Glutathione by Erythrocytes from Normal Newborn Infants William H. Zinkham vii

39

41

41

52

54 63

viii

CONTENTS

A Possible Effect of Vitamin K3 on Erythrocyte Metabolism Lars Ernster The Destruction of Red Cells by Antibodies James H . Jandl The Oxygen-Carrying Capacity and Glycolytic Rates of Erythrocytes from Newborn Infants with Erythroblastosis Caused by Rh Incompatibility Abraham Abrahamov PANEL

C.

THE METABOLISM AND EXCRETION OF BILIRUBIN

Introduction Grant H. Lathe The Mechanism of Glucuronide Formation Jeoffrey J. Dutton Studies of Glucuronide Formation Irwin M. Arias Bilirubin Sulfate Rudi Schmid The Effect of Sodium Glucuronate and Glucuronolactone on Bilirubin Conjugation David Yi-Yung Hsia Factors Influencing the Synthesis of Direct Reacting Bilirubin Karel Polacek Familial Defect of Glucuronidation: in vivo Studies James B. Sidbury, Jr. Conjugated Hyperbilirubinaemia: A Defect in Bilirubin Barbara H. Billing Transport Rudi Schmid Bilirubin Metabolism in the Foetus Glucuronide Formation in Pre-Natal and Neonatal Life Geoffrey J. Dutton Foetal and Neonatal Development of the Glucuronide-ConAudrey K. Brown jugating System Conclusion Grant H. Lathe PANEL

D.

THE

PATHOLOGY

OF

KERNICTERUS

AND

THE

68 73 79

83 85

88 94 97 99

102 105 108 112 115

121 128

CYTO-

TOXICITY OF BILIRUBIN

Introduction Rolf Zetterstrom Pathology of Human Kernicterus Albert E . Claireaux Kernicterus in Gunn's Strain of Rats William A. Blanc The in vivo Crystallization of Bilirubin in Hyperbilirubinaemic Infants Teresa J. Vietti Experimental Studies on the Toxicity of Bilirubin Bohdan Rozdilsky Toxicity of Haeme Pigments in Different Test Systems Richard Day Bilirubin Encephalopathy-Cytotoxicity William Waters The Mode of Action of Bilirubin on Mitochondria Lars Ernster

133 135 140 150 153 161 167 170 174

CONTENTS

p ANEL E.

ix

FACTORS INFLUENCING THE DISTRIBUTION OF BILIBUBIN

IN THE BODY

Introduction Richard Day The Effects on Premature Infants of Sulfisoxazole in the NeoWilliam A. Silverman natal Period Factors Influencing the Binding of Bilirubin by Albumin Gerard B. Odell The Effect of Certain Substances on Bilirubin Levels and Occurrence of Kernicterus in Genetically Jaundiced Rats. Lois M. Johnson The Protective Action of Albumin in Bilirubin Toxicity in Newborn Puppies William]. Waters

193 195 196 199

208 219

CONTRIBUTORS Chairman: DR. BRUCE CHOWN Co-chairman: DR. ANDREW SAss-KoRTSAK PANEL CHAIRMEN

Panel A: BRUCE CHOWN, M.D., Professor of Paediatrics, University of Manitoba, and Children's Hospital, Winnipeg 3, Manitoba, Canada Panel B: WOLF W. ZuELZER, M.D., The Children's Hospital of Michigan, Detroit 2, Michigan, U.S.A. Panel C: G. H. LATHE, M.D., c.M., PH.D., Professor of Chemical Pathology, School of Medicine, University of Leeds, Leeds, England Panel D : ROLF ZETTERSTROM, M.D., Professor of Pediatrics, University of Gothenburg, Gothenburg, Sweden Panel E: RICHARD DAY, M.D., Medical Director, Children's Hospital of Pittsburgh, Pittsburgh, Pa., U.S.A. PANEL MEMBERS ABRAHAM ABRAHAMOV, M.D., Chief Physician in Pediatrics, Hadassah University Hospital, and Lecturer, Hebrew Medical School, Jerusalem, Israel IRWIN M. ARIAS, M.D., Department of Medicine, Albert Einstein College of Medicine, New York 61, N.Y., U.S.A. BARBARA H. BILLING, PH.D., Senior Lecturer, Department of Medicine, Royal Free Hospital, Gray's Inn Road, London W.C. 1, England WILLIAM A. BLANC, M.D., Assistant Professor of Pathology, College of Physicians and Surgeons, Columbia University; Assistant Attending Pathologist, Columbia-Presbyterian Medical Center; and Babies Hospital, New York, N.Y., U.S.A. AUDREY K. BROWN, M.D., Assistant Professor of Pediatrics, University of Virginia School of Medicine; Clinical Coordinator and Director of Research, Children's Rehabilitation Center; and Pediatrician, University Hospital, Charlottesville, Virginia, U.S.A. ALBERT E. CLAIREAUX, M.D., F.R.C.P. (EDIN.), Lecturer, Institute of Obstetrics and Gynaecology, University of London, and Queen Charlotte's J\faternity Hospital, Goldhawk Road, London, England V. MARY CROSSE, M.D. (LOND.), D.P.H., M.M.S.A. D. OBST., R.C.D.G., Lecturer, Department of Paediatrics and Child Health, University of Birmingham, and Consultant Paediatrician, Sorrento Maternity Hospital, Birmingham, England GEOFFREY J. DUTTON, PH.D., Department of Biochemistry, Queen's College, University of St. Andrews, Dundee, Scotland X

CONTRIBUTORS

xi

LARS ERNSTER, PH.D., Wenner-Grens Institut, Norrtullsgatan 16, Stockholm, Sweden ALFRED M. FREEDMAN, M.D., Professor of Psychiatry and Chairman of the Department, New York Medical College, New York, N.Y., U.S .A. DAVID A. I. GREWAR, M.C., CH.B., D.C.H., M.R.C.P., Lecturer, Department of Paediatrics, University of Manitoba, and The Children's Hospital, Winnipeg, Manitoba, Canada RUTH C. HARRIS, M.D., Assistant Professor of Pediatrics, College of Physicians and Surgeons, Columbia University, New York 32, N.Y., U.S.A. DAVID Yr-YUNG Hs1A, M.D., Associate Professor of Pediatrics, Northwestern University School of Medicine, and Director of Research, The Children's Memorial Hospital, Chicago, Ill., U.S.A. JAMES H. JANDL, M.D., Thorndike Memorial Laboratory, The Boston City Hospital, Boston 18, Mass., U.S .A. Lms M. JOHNSON, M.D., Department of Pediatrics, State University of New York, Downstate Medical Center, Brooklyn 3, N.Y., U.S.A. EuGENE KAPLAN, M.D., Associate Professor of Pediatrics, Johns Hopkins University, and Assistant Pediatrician-in-Chief, Sinai Hospital of Baltimore, Inc., Greenspring and Belvedere, Baltimore, Maryland, U.S.A. JEROLD F. LUCEY, A.B., M.D., Division of Pediatrics, College of Medicine, University of Vermont, and De Goesbriand Memorial Hospital, Burlington, Vermont, U.S.A. GERARD B. ODELL, B.A. , M.D., Associate Professor of Pediatrics, Johns Hopkins University, and Department of Pediatrics, Johns Hopkins Hospital, Baltimore, Maryland, U.S.A. KAREL POLACEK, M.D., Ustav pro peci o matku a dite, Prague, Czechoslovakia BoHDAN RozmLSKY, M.D., University of Saskatchewan Medical School, Saskatoon, Saskatchewan, Canada Rum SCHMID, M.D., PH.D., Assistant Professor of Medicine, Harvard Medical School, and Thorndike Memorial Laboratory, Boston, Mass., U.S.A. JAMES B. SIDBURY, JR., M.D., Assistant Professor of Pediatrics, Johns Hopkins University, and Johns Hopkins Hospital, Baltimore, Maryland, U.S.A. WILLIAM A. SILVERMAN, M.D., Associate Professor of Clinical Pediatrics, Columbia University Babies Hospital, and Columbia-Presbyterian Medical Center, New York, N.Y., U.S.A. TERESA J. VIETTI, M.D., Foreign Specialist in Research, University of Ankara, and Hacettepe Children's Hospital, Ankara, Turkey WILLIAM J. WATERS, M.D., Department of Pediatrics, State University of New York, Upstate Medical Center, and 1201 State Tower Building, Syracuse, N.Y., U.S.A. WILLIAM H. ZINKHAM, M.D., Assistant Professor of Pediatrics, Johns Hopkins Hospital, Baltimore, Maryland, U.S .A.

INTRODUCTION Bruce Chown, M.D.

of paediatrics written twenty years ago kernicterus received no more than the most fleeting notice. Up to five years ago interest in the condition was almost confined to its occurrence in babies suffering from haemolytic disease of the newborn. Realization has come slowly that the pathological lesion occurs apart from this disease, but there is still great doubt about its frequency of occurrence and about the conditions under which it develops. This symposium with 33 papers on the panels gives some evidence of the present interest in kernicterus. According to the arrangements which have been made-entirely through the initiative and energy of Dr. Sass-Kortsak-we shall proceed from clinical to pathological and biochemical observations. As I am a clinician, my own panel is clinical. Some of the questions that arise about kernicterus, as about any disease, are: how frequently and under what conditions does it arise; how is it recognized; what, if any, are its after-effects? The papers in this panel will answer some of these questions according to the experience of their authors. The answers will differ, of course, but out of both the similarities and the differences new questions will arise, and in due course the picture will become clear. IN THE STANDARD TEXTBOOKS

s

THE INCIDENCE OF KERNICTERUS (NOT DUE TO HAEMOLYTIC DISEASE) AMONG PREMATURE BABIES 0

V. Mary Crosse, M.D., D.P.H., M.M.S.A. D. OBST., R.C.D.G. t

THE INCIDENCE of kernicterus in the premature baby unit of the Sorrento Maternity Hospital from 1945 to 1954 has been studied. At first very moderate doses of vitamin K were given and the incidence of kernicterus was 1.08 per cent of all admissions. During the next period the dose of vitamin K was increased and was given daily until feeding commenced, but this did not increase the incidence. Later, the number of daily doses of vitamin K to babies showing signs of possible cerebral haemorrhage was increased further, and the incidence of kernicterus immediately went up. Following this, the use of vitamin K was discontinued for a period of six months. The incidence of kernicterus returned to 1.3 per cent, but a few cases of haemorrhagic disease occurred. Consequently, from July 1955, vitamin-K therapy TABLE I INCIDENCE OF KERNICTERUS AMONG PREMATURE BABIES

Dosage of vitamin K A.

Kernicterus ;\lumber of - - - - - - - - - admissions Number of Percentage of cases admissions

BEFORE USE OF REPLACEMENT TRANSFUSIONS (SORRENTO PREMATURE BABY UNIT)

1945-48 1949-52 1953- 54

922 1,027 650

1 mg. at birth 10 mg. daily X 3 10 mg. daily X 5 (or more)

10

11

25

1.08 1.07 3.8

B . WITH USE OF REPLACEMENT TRANSFUSION (THREE PREMATURE BABY UNITS)

Julyl955to June 1957 July 1957 to June 1958

0 . 5mg. atbirth

1,320

5

0 .38

0 . 5 mg. at birth

726

0

0

°From the Sorrento Maternity Hospital and the Department of Paediatrics and Child Health, University of Birmingham. fAddress: 15 Wake Green Rd., Birmingham 13, England. 4

5

V. MARY CROSSE

was reinstituted and 1 mg. was given at birth only; replacement transfusion was also used whenever the level of serum bilirubin rose to 18-20 mg./lO0ml. Table I shows the incidence of kernicterus among the babies in three premature baby units from the commencement of this latter course of treatment. (Just over 700 babies per year are admitted to these three units and the majority weigh less than 2,000 gm. at birth. ) During the first two years there were five cases of kernicterus; all these were preventable, because the level of serum bilirubin was allowed to rise above 22 mg./100 ml. Of these five babies two showed only very slight signs of kernicterus and at the age of two years both were perfectly normal. The following year no further cases of kernicterus occurred. All the babies with serum bilirubin levels over 15 mg. have been followed up to the age of one year or more, to ensure that no case of kernicterus has been missed. Obviously, the cure might be more dangerous than the disease, and the relative risks of transfusion and kernicterus are shown in Table II. TABLE II RELATIVE RISKS FROM KERNICTERI.JS AND FROM EXCHANGE TRANSFUSION

(Three premature baby units)

First 2 years Third year Number of admissions Expected cases of kernicterus (1 . 08% of admissions) Dead Spastic Transfused babies Total number Number to save each kernicterus Results Transfusion deaths Cases of kernicterus (spastic)

1,320

726

1~}14

2f

6\ g

92 6.6

55

1

Nil Nil

5

6.9

During the first two years, 14 cases of kernicterus were expected (judging from our previous incidence of 1.08 per cent), and ten of the afflicted babies would probably have died. In actual fact, there was one death from transfusion and five cases of kernicterus ( all preventable). During the first two years, 92 babies required replacement transfusion, that is, 6.6 babies to save each expected case of kernicterus. During the third year, eight babies were expected to have kernicterus, six of whom would probably die. There were, however, no deaths from transfusion and no cases of kernicterus. There were 55 babies trans-

6

KERNICTERUS OF PREMATURITY

fused, that is, 6.9 babies to save each expected case of kernicterus. All five cases of kernicterus occurred during the first year, and as we have just completed a fourth year without seeing any clinical signs of kernicterus ( these cases have not yet been followed up for one year), we now have a series of more than 2,000 babies over a period of three years without a case of kernicterus. Incidentally, 20 per cent of the transfused babies have required more than one transfusion to keep the serum bilirubin level below 22 mg./lOOml. ( 16 per cent required two, 3 per cent required three, and 1 per cent required four transfusions). The level of serum bilirubin had a very definite influence on the incidence of kernicterus. Table III shows the levels at which the five TABLE III RISK OF KERNICTERUS AT VARIOUS SERUM-BILIRUBIN LEVELS

Maximum serum bilirubin level (mg. per 100 ml.)

Cases of kernicterus Numbers o f - - - - - -·- - - - babies in Percentage of babies group Number in group

Babies less than 2,500 gm. at birth 30 3 Babies less than 2,000 gm. at birth 30 1

0 0

0 0 4 0 1

33 .3

4

33 .3

1

100 .0

19.1

cases of kernicterus occurred: none below 22 mg./1000 ml., four between 22 and 27 mg., and one over 30 mg. All five cases occurred among babies weighing less than 2,000 gm. at birth The incidence of kernicterus increased as the level of the serum bilirubin rose. Other factors also influenced the incidence of kernicterus. Table IV shows the incidence of kernicterus in various factor groups for the three premature baby units during the four years, 1951 to 1954. (This included the two years during which we were giving excessive doses of vitamin K.) The factors which increased the incidence of kemicterus were: low birth-weight, marked prematurity, and birth asphyxia. (The presence of birth asphyxia increased the incidence in each of the three

7

V. MARY CROSSE

TABLE IV INCIDENCE OF KERNICTERUS IN VARIOUS FACTOR SUB-GROUPS (Three premature baby units, 1951-1954; 2,608 babies)

Sub-groups of various factors Birth-weight (gm .) Up to 1,500 1,501-2,000 2,001-2,500 Maturity (completed weeks) Up to 32 33-34 Over 34 Birth asphyxia Present Absent Sex Male Female Single or multiple Single-born Twin or triplet I Twin or triplet II+ Prenatal complications Present Absent Labour complications Present Absent

Kernicterus

Number of babies in sub-group

Cases (60)

Percentage of babies in sub-group

525 1,123 960

21 24 15

4 .0 2 .1 1.6

650 801 1,157

27 22 11

4 .2 2 .7 1.0

511 2,097

17 43

3 .3 2.1

1,185 1,423

35 25

3 .0 1.8

2,031 273 304

45 4 11

2 .2 1.5 3.6

1,490

1,118

22 38

2 .0 2 .6

575 2,033

13 47

2.3 2.3

birth-weight groups.) Males were affected more than females, and single-born babies more than first-born twins or triplets (presumably because in both cases the former were more premature weight for weight). Second-born twins or triplets and third triplets were most likely of all to be affected, probably because they had the highest incidence of birth asphyxia. Generally speaking, complications of pregnancy and labour did not affect the incidence of kernicterus, unless they were associated with birth asphyxia. Since the elimination of kernicterus, we have only been able to show the incidence of babies requiring transfusion for hyperbilirubinaemia ( > 20 mg./100 ml.) and this is shown for 726 babies admitted to the three premature baby units from July 1, 1957, to June 30, 1958, that is, up to one year ago, to allow all babies to be followed up for at least one year. Table V shows the incidence of transfused babies as a percentage of the admissions, and also as a percentage of the babies "at risk," that is, those alive at 48 hrs. (The latter figures are given because a large number of the smallest and

8

KERNICTERUS OF PREMATURITY

TABLE V INCIDENCE OF HYPERBILIRUBINAEMIA IN VARIOUS FACTOR Sue-GROUPS

(Three premature baby units, July 1, 1957-June 30, 1958; 726 babies) Transfused babies (Total Sub-groups of various factors

Birth-weight (gm .) Up to 1,500 1,501-2,000 2,001-2,500 Maturity (completed weeks) Up to 32 33-34 Over 34 Birth asphyxia Present Absent Sex Male Female Single or multiple Single-born Twin or triplet I Twin or triplet I I+ Prenatal complications Present Absent Labour complications Present Absent Early oedema Marked Not marked Rectal temperature on admission Up to 92°F. (33.3°C.) Over 92°F. up to 96°F. Over 96°F. (35°C.)

Percentage of admissions

= 55)

Percentage of those alive at 48 hours ( = babies "at risk")

5.2 9 .7 6 .7

13 . 6

7 .4 9.6 6.6

15.2 10 . 7 7.0

7.4 7.7

11.3 8.6

7.7 7.4

10 . l 8 .6

8 .8 3.2 5.6

10 . 4 3 .8 8 .3

6 .2 8 .5

7.2 10.4

4.1 8.6

5 .9 10.2

14 .3 5. 3

20.3 6 .3

11 .4 14 .0 4.6

29.0 17.4 5.2

11.0

6 .9

least mature infants die before the level of bilirubin has time to rise and this confuses the statistics.) Again, high serum bilirubin levels were associated with low birth-weight, marked prematurity, birth asphyxia, and male sex. This time all multiple-born babies were less affected than first-born babies and this seemed to correlate with the fact that the first-born babies were more often ( and the second-born less often) associated with birth asphyxia in this series than in the first (Table IV). Again complications of pregnancy and labour did not increase the incidence unless they were associated with birth asphyxia. Two new factors appear on this table : early oedema and rectal temperature. The presence of marked early oedema increased the incidence of transfusion, but early oedema was definitely cor-

V. MARY CROSSE

9

related with the degree of prematurity. Between 80 and 85 per cent of the babies were admitted to our hospital after birth and some came from as far as 100 miles away. The rectal temperatures vary tremendously on admission. Babies admitted with a rectal temperature of 92° F . (33.3° C. ), or less, had the highest incidence of transfusion, and those with a temperature over 96°F. ( 35°C.) the lowest. This difference was not due to the degree of prematurity, as the same picture occurred in each of the three birth-weight groups; nor did the low temperature correlate with birth asphyxia. Low body-temperature seemed to be one of the factors which increased the risk of hyperbilirubinaemia. To summarize, I have shown that the incidence of kernicterus increased as the level of serum bilirubin rose; and that the level of serum bilirubin was increased by low birth-weight, marked prematurity, birth asphyxia, low body-temperature, and excessive doses of vitamin K. I have not discussed the part played by sulphonamides and other drugs, such as salicylates, of which I have no experience.

PEAK LEVELS OF SERUM BILIRUBIN IN NORMAL PREMATURE INFANTS 0

Ruth C. Harris, M.D. t+

IN 1955 AND 1956, at the Babies Hospital in New York City, it was decided that the natural course of bilirubinaemia should be studied in our premature population during the first week of life. The babies included in this group all weighed less than 2,000 gm. at birth, and a large proportion of them were less than 1,500 gm. Venous blood samples were obtained daily from each infant and analysed for bilirubin content by the Hsia modification of the method of Evelyn and Malloy. In evaluating the normal ranges of bilirubin, all babies with infection or with blood group and particularly Rh patterns which might have caused the formation of maternal antibodies were eliminated. Of 206 nursery admissions, 114 babies were completely studied until the peak of bilirubinaemia had been passed. Of these, we found 60 surviving infants who were clinically normal. These infants all received either oxytetracycline or tetracycline during the first 120 hrs. of life in dosage of either 5 or 10 mg./kg. 24 hrs. All infants received less than 10 mg. of a vitamin K analogue. Table I shows that 73 per cent of the infants had peak plasma bilirubin levels below 15 mg./100 ml. and that the largest number reached this peak on the fourth day of life. Only 10 per cent of the infants had levels over 20 mg./100 ml., and these peaks occurred at a later time. The highest value attained was 25.1 mg./100 ml. Of the total group, 88 per cent reached peak concentration of bilirubin by the fifth day of life. °From the Babies Hospital and the Department of Pediatrics, College of Physicians and Surgeons, Columbia University. Supported by a grant from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, Public Health Service ( A-1173). tAddress : 3975 Broadway, New York 32, N.Y., U.S.A. tCo-authors : Garrison Rapmund, M.D., and John M. Bowman, M.D. 10

11

RUTH C. HARRIS

TABLE I* PEAK PLASMA BILIRUBIN CONCENTRATIONS OF 60 SURVIVING COMPATIBLE NoN-ERYTHROBLASTOTIC PREMATURE INFANTS CORRELATED WITH DAY OF PEAK CONCENTRATION

Peak day

2 3 4 5 6 7

TOTALS Percentage

Peak bilirubin concentration (mg. per 100 ml.)

21

0 0 1 3 0 0 4

0 0 1 2 1 0 4

0 0 0

0 0 0 0 0 0 0

0 0 0 1 1 1 3

0 0 0 0 1 0 1 10

0 0 1 1 0 0 2

2

17

0 0 2

Total cases

9 12 16 16 6 1 60 100

*Reproduced with the permission of the A.M.A. Journal of Diseases of Children.

Table II indicates the peak plasma bilirubin levels in relation to birth-weight, colour, and sex. Infants under 1,250 gm. had the highest average peak levels and, conversely, those of highest birth-weight had the lowest peak bilirubin levels. White infants under 1,500 gm. birth-weight had much higher levels than did Negro babies of the same weight. The abnormally high average of the Negro group weighing between 1,501 and 1,750 gm. TABLE II* MEAN PEAK PLASMA BILIRUBIN CONCENTRATIONS SUBDIVIDED ACCORDING TO BIRTH-WEIGHT, RACE, AND SEX OF 60 COMPATIBLE PREMATURE INFANTS Mean peak plasma bilirubin according to weight group Group

No. of infants

All

60

White

41

Negro

1\)

Male

28

Female

32

1,251-1,500

1,501-1,750

1,751-2,000

(14) 12 .2

(21) 11.6

(17) 11.2

(5) 15.5 (3) 10.3

(6) 15.3 (8) 11 . 5

(16) 10.6 (5) 14 . 9

(14) 11.6 (3) 9.3

(2) 12 . 3 (6) 13 .9

(7) 12.8 (7) 13.4

(9) 14.4 (12) 9.5

(10) 13 . 4 (7) 8.1

1,250 gm.

(8)t 13 5

gm.

gm.

gm.

*Reproduced with the permission of the A.M.A. Journal of Diseases of Children. Number of infants per weight group given in parentheses.

t

12

SERUM BILIRUBIN IN PREMATURES

was weighted by the inclusion of the highest bilirubin value ( 25.l) obtained in this series. Male infants weighing more than 1,500 gm. had slightly higher bilirubin levels than females. While the total number of patients studied is too small to provide conclusive statistical information, this study has been helpful to us in appraising our premature population and the problems associated with hyperbilirubinaemia.

EXPERIENCES WITH KERNICTERUS IN PREMATURE INFANTS 0 David A. I. Grewar, M.B., Ch.B., D.C.H., M.R.C.P. t

of the occurrence of kernicterus in non-erythroblastotic premature infants ( 1-4), and the recommendation now being made more or less enthusiastically for exchange transfusion in such infants as a means of preventing kernicterus ( 5-11), occasioned the following retrospective survey of live-born premature infants over the period from 1953 through 1958. The newborn infants who died in the nursery during this period have been reviewed for autopsy evidence of kernicterus ( autopsy rate, 96 per cent) . It will be seen from Figure 1 that during 1953 through

AN APPRECIATION

mil EBF 0 NO EBF /J)

::,

3-

a::

w

I~

z a::

w ~

NEONATAL DEATHS LIVE BORN PREMATURES

YEAR INCIDENCE

OF

2I-

~

[J

tJ

43

38

38

33

204

220

240

224

221

202

1953

1954

1955

1956

1957

1958

~

KERNICTERUS

OF'

PREMATURITY -

I 46

25

223

1311 TOTALS

0.2%.

FIGURE 1. Deaths with kernicterus-premature infants (Women's Pavilion, Winnipeg General Hospital) . Autopsy rate 96%. °From the Women's Pavilion, Winnipeg General Hospital, and the Department of Paediatrics, University of Manitoba. fAddress: Children's Hospital, Winnipeg 3, Man., Canada.

13

14

KERNICTERUS IN PREMATURE INFANTS

1958 there were 1,311 live-born premature infants of whom 223 died in the neonatal period. Kernicterus was found in six of those who died. Excluding three children in whom kernicterus was associated with immune haemolytic disease, there remain three examples of "nonerythroblastotic kernicterus of prematurity," representing an incidence of 0.2 per cent for all live-born premature babies and of 1.3 per cent for neonatal deaths of premature babies. These are low incidences compared with those of some other published series ( 1, 12-14). A study of the three cases of non-erythroblastotic kernicterus of prematurity ( Fig. 2) reveals that all occurred following an abnormal pregnancy of one kind or another. All occurred in babies who were in no better than fair condition at the time of birth and who all had some form of respiratory distress during the nursery period preceding the onset of jaundice. In two of the three, bilirubin levels were measured. The cord blood levels were 2 mg./100 ml. and 1.5 mg./100 ml., rising to peaks of 14 mg. and 17 mg. respectively. In both cases, the peak levels were measured on the day of death. It is significant that at autopsy, besides kernicterus, all three infants showed other relevant -

- CASE

S

-

- -- -- - -· -- ··· - --·-··GEST MATERNAL BIRTH

BW

3•

NUflSERY I

32

TOXAEMIA

EASY

BLEEDING

FAIR

2:

)

4

PATHOLOGY 6

~

-•D!-o

, t - .IOCE --,

(13601

t--NSTUI..U•y

7

INTRA. PUL. HAEM . BRONCHIAL EPITHEL SLOUGHED

S. AUlllUS

.-... ,. -----..

PNEUMONIA KERNICTERUS

z

3'

34

(1400)

TOXAEMIA BLEEDING (2nd TAU

A.R.M. BREECH FAIR

--•os- - ,

TENTORIAL TEAR SUBOURAL HAEM. COARCT. AORTA KERNICTERUS

+--JOCE-----#

+--P.S.~

+ T

II 1609)

26

BLEEDING I NTERMIT

BREECH POOR

.,_.,._.----+ ~.1oct---,

.

'-CU ➔

SUBARACH . HAEM. INTRA PUL HAEM . ATELECTASIS PNEUMONIA KERNICTERUS

2. Kernicterus of prematurity without erythroblastosis (Women's Pavilion, Winnipeg General Hospital) . FIGURE

15

DAVID A . J. GREWAR

pathological conditions : two had pneumonia and one had a large tentorial tear with subdural haemorrhage. It is unlikely that exchange transfusion would have altered the outcome in these three cases. Interest centres next on the live-born prematures discharged alive from the nursery and whether they show any indication of a disability in the neuropsychiatric sense, which might be attributable to non-erythroblastotic kernicterus of prematurity ( 11). Of 393 premature infants discharged alive from the nursery during the period 1955 and 1956, it has been possible to follow 307 ( 78 per cent). Twelve infants died following discharge from the nursery, all of whom were autopsied and none showed evidence of kemicterus. It is of interest, however, that one infant who died at the age of five weeks from gastroenteritis and pneumonia showed some focal gliosis in the lentiform nuclei. This infant's neonatal period featured severe respiratory distress and a peak bilirubin of 10.5 mg. One could speculate that had this infant died during the nursery period we might have found kernicterus with staining evident in the region of the lentiform nuclei, when hypoxia rather than hyperbilirubinaemia would have to assume the blame. In Figure 3 the incidence of neuropsychiatric handicap is shown with a somewhat arbitrary division into cerebral palsy, mental defect, epilepsy, retardation, and behaviour problem. The peak bilirubin level, NO.

FATE DEATH SPASTIC C.P.

MAX. DEGREE

10

15 2

- -

4

-

4

-

3

I

I

MENTA' DEFC-CT

4

3

EPILEPSY

2

I

RETAROATIONI'

5

2

BEHAVIOR PROB"

3

I

NUCLEI -

**

20+

2

9

-

ATTRIBUTABLE

JAUNDICE 20

12

ATHETOIO C. P.

* I DEATH

-

-

-

I

I

-

- -

SEVERE

-

KERNICTERUS o* 0 0 0

0 ? 2**

0

AT 5/52 GE ANO PNEUMONIA , AUTOPSY ALSO SHOWED FOCAL GLIOSIS LENTIFORM Bl LI. PEAK 10 .5 MG "lo.

I CASE OF EBF (RH) EXTRANS X3 BILI PEAK I9MG ~ SPEECH OELAY

I CASE "NON EBF"

BILI PEAK 22MG"lo WITH 1082 CF TWIN 10 120 (BILI. PEAK 13)

FIGURE 3. Neuropsychiatric handicap in liveborn premature infants (Women's Pavilion, Winnipeg General Hospital, 1955 and 1956). Discharged alive from nursery, 307 babies. Follow-up period, l½ years or more.

16

KERNICTERUS IN PREMATURE INFANTS

where measured, is indicated and where no bilirubin estimation was made the degree of jaundice, if reported clinically severe, is denoted. In only one area, that of "retardation" ( which here implies unspecified delay in motor, language, or intellectual development), do we see a hint that kernicterus might have contributed to our experience of neuropsychiatric handicap. In this group there were two cases where jaundice was notable. One of these we can exclude, since it was an example of severe Rh erythroblastosis foetalis treated by multiple exchange transfusions; the bilirubin level reached a peak of 19 mg. and at the age of three years there was some delay in speech development. The other child, the second born of premature twins (birth-weights: 3 lb., 3 oz., and 3 lb., 6 oz., respectively), had a peak bilirubin of 22 mg. This child compared unfavourably in the field of intelligence with her twin sister, whose peak bilirubin was 12 mg. The I.Q of these twins was 82 and 120 respectively. It must also be noted, however, that the twins were descendants of a family with a neuropathic background and, furthermore, the twin with the high bilirubin was in severe respiratory distress during the nursery period, whereas her twin sister had an uneventful nursery period. In this group of prematures, therefore, there is no very convincing evidence of neuropsychiatric handicap attributable to kernicterus. The validity of the above statement is questionable since bilirubin estimations were not universal. In the period 1955 through 1956, however, 116 consecutively born premature infants did have bilirubin estimations which were continued whenever possible until the bilirubin showed a decline. There were: 48 newborns with a peak bilirubin of less than 10 mg., 52 with a peak between 10 and 15 mg., 13 with a peak between 15 and 20 mg., 3 with a peak of over 20 mg., None with a peak over 25 mg. This group was carefully studied and follow-up examinations included 1.Q. testing and EEG recordings wherever the bilirubin had exceeded 15 mg. The incidence of neuropsychiatric handicap is graphically shown in Figure 4. There does not appear to be any relation between the degree of jaundice as measured by the bilirubin level and the occurrence of disability. It is appreciated that the bilirubin levels in this study are lower than in many of the published accounts of hyperbilirubinaemia of prematurity ( 5-7, 15, 16) and we have, therefore, attempted to define

17

DAVID A. I. GREWAR WEIGHT

TO 1000

RANGE

1000 TO 1501 TO

1500

2000

2001 TO

2500

40-

35303520-

1510-

5-

OVERIO ,: UNDER IO,no"I. 510-

1520-

253035-

0 CEREBRAL PALSY SPASTIC 0 CERE8RAL PALSY ATHETOI D

.Q) BEHAVIOR PROBLEM

'v EPILEPSY

◊ "RETARDATION'" 0 MENTAL DEFECT I VE

40

Figure 4. Incidence of neuropsychiatric handicap in 116 premature infants with birth-weight and peak bilirubin values.

and study some of the factors which might influence the degree of hyperbilirubinaemia. Serial bilirubin determinations were carried out in 64 consecutively born full-term newborns and these were added to the groups of 116 prematures discussed above. A number of variables, of which we had reasonably accurate measurement, were then correlated with the peak bilirubin value by statistical method in the total number of 280 newborn babies. 0 The variables were: birth-weight, cord bilirubin, gestational period, infant's condition at birth as determined by Apgar rating, infant's sex. It was found that only the birth-weight and the cord bilirubin level had any real influence on the peak bilirubin values. These two variables, however, were regarded as only going about one-third of the way to explain the total influence of factors on the peak bilirubin reached. The remaining measure of 0

Details of the methods and the complete results will be published later.

18

KERNICTERUS IN PREMATURE INFANTS

influence which could explain the variation in incidence of hyperbilirubinaemia reported from different centres we can only speculate upon, since we have not devised any suitable method of measurement for analysis ( 17-21). In summary, the incidence of non-erythroblastotic kemicterus in premature infants in our experience has been very low. Where it has been found at autopsy, it has not been associated with strikingly high bilirubin values, but rather with additional pathological conditions, especially in the lungs or brain. A follow-up of live-born premature infants gave no indication of non-erythroblastotic kemicterus as a contributory factor in neuropsychiatric handicap nor was it possible in our experience to relate the incidence of neuropsychiatric handicap to bilirubin levels in the premature. It is appreciated that in this series bilirubin levels seldom exceeded 20 mg./100 ml. The influence of birth-weight, cord bilirubin value, gestational period, sex, and condition at birth as determined by Apgar rating were studied with reference to peak bilirubin values. It was only possible to relate the birth-weight and the cord bilirubin to the degree of hyperbilirubinaemia. There must be other factors which we cannot measure or do not understand, which influence the degree of hyperbilirubinaemia and possibly the incidence of kemicterus. It has not been our practice to employ exchange transfusion in the non-erythroblastotic premature with hyperbilirubinaemia and in our experience there is no convincing evidence that it would have altered the incidence of death or disablement in this group. REFERENCES

I. AmEN, R., CORNER, E., and TovEY, G. Kernicterus and prematurity. Lancet

1950, 1, 1153. 2. ZUELZER, W.W., and MUDGETr, R. T. Kernicterus : Etiologic study based on an analysis of 55 cases. Pediatrics 1950, 6, 452. 3. GovAN, A. D., and ScoTr, J. M. Kernicterus and prematurity. Lancet 1953, 1,611. 4. BLACK-SCHAFFER, B., KAMBES, S., FURUTA, N., and MOLONEY, W. C. Neonatal jaundice and kernicterus, Am. J. Dis. Child. 1954, 87, 737. 5. CROSSE, V. M., WALLIS, P. G., and WALSH, A. M. Replacement transfusion as a means of preventing kernicterus of prematurity. Arch. Dis. Childh. 1958, 33,403. 6. MEYER, T. C. A study of serum bilirubin levels in relation to kemicterus and prematurity. Arch. Dis. Childh. 1956, 31, 75. 7. CORNER, B. Hyperbilurinaemia in premature infants treated by exchange blood transfusion. Proc. Roy. Soc. Med. 1958, 51, 1019. 8. NEWNS, T. H., and NORTON, Hyperbilirubinaemia in prematurity. Lancet 1958, 2, 1138.

DAVID A . I. GREWAR

19

9. DUNDON, S. The prevention of kernicterus in the premature: Hyperbilurinaemia unassociated with blood groups incompatibility. J. Irish Med. Assoc. 1956, 99, 226. 10. BROWN, A. K., and ZuELZER, W.W. Studies in hyperbilirubinemia: I, Hyperbilirubinemia of the newborn unrelated to isoimmunization. Am. J. Dis. Child. 1957, 93, 263. 11. KocH, T. A., JoNES, D. V., DINE, M. S., and WAGNER, E. Hyperbilurinemia in premature infants: A follow up study. J. Pediat. 1959, 55, 23. 12. CROSSE, V. M., MEYER, T. C., and GERRARD, J. W. Kernicterus and prematurity. Arch. Dis. Childh. 1955, 30, 501. 13. CLAIREAUX, A. E., CoLE, P. G., and LATHE, G. H. Icterus of the brain of the newborn. Lancet 1953, 2, 1226. 14. GERRARD, J. W. Kernicterus in prematurity. Canad. M.A.J. 1957, 77, 661. 15. HoLMAN, G. Studies on Physiologic hyperbilirubinemia of negro and white premature infants. Pediatrics 1958, 22, ll5. 16. FoRFAR, J. O.; KEAY, A. J.; ELLIOTI, W. D.; and CuMMING, R. A. Exchange transfusion in neonatal hyperbilurinaemia. Lancet 1958, 2, ll31. 17. BouND, J. P., and TELFER, T. P. Effect of vitamin K dosage on plasma bilirubin levels in premature infants. Lancet 1956, 1, 720. 18. CREMER, R. J., PERRYMAN, P. W., and RICHARDS, D. H. Influence of light on hyperbi1urinemia of infants. Lancet 1958, 1, 1094. 19. BILLrNG, B., COLE, A. G., and LATilE, G. H. Increased plasma bilirubin in newborn infants in relation to birth weight. Brit. Med. J. 1954, 2, 1263. 20. BROWN, A. K., and ZuELZER, W. W. Studies on the neonatal development of the glucuronide conjugating system. J. clin. Invest. 1958, 37, 332. 21. MILLER, C. A., and REED, H. R. The relation of serum concentrations of bilirubin to respiratory function of premature infants. Pediatrics 1958, 21, 362.

THE NEUROLOGICAL SEQUELAE OF HYPERBILIRUBINAEMIA OF PREMATURITY" Alfred M. Freedman, M.D. tt

FoR the past three years a research team at the State University of New York College of Medicine has been engaged in an anterospective study to test the hypothesis that increasing amounts of circulating bilirubin in the blood stream of non-erythroblastotic premature infants are associated with increasing incidence of the manifestations of cerebral injury. A total of 600 infants, with birth-weights not exceeding 2,100 gm., have been admitted to this study. The bulk of this population is Negro. Serum bilirubin has been determined daily during the neonatal period to establish the peak value. Physical, neurological, and psychological examinations were carried out at frequent intervals. The present report will focus upon the findings of the neurological and psychological examination of the infants at a chronological age of four months. The neurological examination is based upon the methods of McGraw ( 1), Gesell ( 2), and Andre-Thomas ( 3). Indices of neurological development have been qualified by means of a numerical scoring system that has been validated. Thus, statistical analysis is facilitated. Two indices were quantified in this way: ( i) development in the gross motor area based upon six examination procedures; (ii) the strength of grasp reflex. Two other items were utilized in the analysis of the premature at four months of age: (iii) the visual fixation and eye following items of the Cattell Infant Scale; (iv) amplitude and normality of the Moro reflex. The gross motor grasp reflex and visual examinations are scored in ~From the State University of New York, Downstate Medical Center, Brooklyn. Supported by United State Public Health Grant B11-04 ( Cl Sl) and United Cerebral Palsy Foundation. fAddress : New York Medical College, Fifth Avenue at 106th Street, New York, N.Y., U.S.A. tCo-authors: Martin D. S. Braine, Ph.D., Caryl B. Heimer, M.D., Muriel Kowlessar, M.D., Walter J. O'Connor, M.D., and Helen Wort.is, M.S.

20

21

ALFRED M. FREEDMAN

reference to a "corrected chronological age" ( CCA). The CCA is obtained by subtracting from the chronological age in weeks the degree of prematurity in weeks. The latter was obtained from the regression curve of the gestational period on birth-weight determined for the Kings County Hospital population by Drs. Schuyler Kohl and Martin Braine of the State University of New York. Figure 1 portrays the development in the gross motor area of the 212 infants examined at four months of age, plotted separately for sex

12. \lJ

'0"' 11

u

rJ')

....,.,., u

f-c

.... C

u

E

0.. ,...

...., u ;.. u

0

10

9

8

/1(' ......

..... ....4

1

........,,.......,'"' 6 ,...

::E :,i :,i

,... ....,

'"' ~

Fe male.

5 4 3

2-4

Male

Negro

~

A-.l

White

o--.o

IJ,---A

8-10 11-13 14-16 Corrected Age in Weeks

5-1

FIGURE 1. The relationship between gross motor score at a chronological age of four months and corrected chronological age in weeks'.

22

NEUROLOGICAL SEQUELAE OF HYPERBILIRUBINAEMIA

and colour. There is an insufficient number of subjects in some of the groups and age categories and the curves, therefore, appear incomplete in the figure. The consistent relationship between increasing gross motor development and advancing corrected chronological age is reassuring evidence of the validity of this test. Similar relationships in general are found between the development of visual fixation, decline of the grasp reflex, and loss of the Moro reflex and the corrected chronological age. Table I shows the number of high and low bilirubin subjects in the four groups. It should be noted that a child is classified as "high bilirubin" if the maximum bilirubin level in the neonatal period rose TABLE I NUMBER OF INFANTS WITH HIGH AND

High (over 15.0 mg.) Group Negro Negro White White

male female male female

Low

Low (0.0-12.9 mg.)

BILIRUBl:-1

High and low

Examined

Total

Examined

Total

Examined

Total

19

34 38 9 18

4-5 56 10

!l9 18

72

64 80 16 22

106 137 27 37

24 6 11

11

HJ

above 15.0 mg./100 ml. In all, 18 per cent of the infants had peak bilirubin between 15.0 mg. and 19.9 mg. while 6 per cent had peak values above 20 mg. This is a total of 24 per cent above 15 mg., in the "high" category. The "low bilirubin" group comprised infants whose maximum bilirubin level never rose above 12.9 mg./100 ml. The number of subjects given a developmental examination at four months is also shown in Table I. Since there were so few subjects in the white groups, no statistical analysis of the results has been made for the white infants. In the gross motor development area, a total of 133 children were examined. Of these, 39 were in the high bilirubin category and 94 were in the low bilirubin category. The jaundiced infants obtained a mean score of 7.79 and the non-jaundiced 9.75. The difference is significant ( t 2.93 with 132 df, p .01). Figure 2 shows the performance of the high and low bilirubin infants in the area of gross motor performance as a function of the CCA separately for the male and female groups. An analysis of covariance was performed to compare the motor development of the high and low bilirubin babies, partialling out CCA which covaries

=

=

23

ALFRED M. FREEDMAN

13

12

11

Males Low Bilirubin~ ,,'

10 ~ 0 u

'J°l

9

....

8

QI

7

C

8

g-.

, ~ 5 0 s 4

3 ~ "'"' 2 ~

Females

10

.. e8-1

'Hi3h Bi/,·rubin

6

12 11

,

1s Cl

,,

,/

13

,o ,,

3

2 "' 2

C

1 0

2.-6

7-U

12-16

Corrected Age in Weeks

0

IZ-16 Corrected Age in Weeks 2.-6

7-11

FIGURE 2. The performance of the high and low bilirubin infants at the age of four months in the area of gross motor performance as a function of corrected chronological age separately for the male and female groups.

with motor development. It was found that among the boys, the high bilirubin infants obtained significantly lower motor development scores than the low bilirubin infants. ( F = 7.34, 1 and 54 df, significant at .01 level) ( 4). In the girls, high bilirubin had no effect on motor development ( F is less than 1) ( 4). On the test of visual fixation and following, there were no differences between the high and the low bilirubin groups of either sex ( F less than 1 in each sex). Figure 3 shows the performance of the high and low bilirubin children of each sex as a function of CCA. Figure 4 compares the strength of the grasp reflex of the high and low bilirubin infants of each sex. Complete measures were obtained on 125 children. Of these, 42 were high bilirubin babies ( 19 boys and 23 girls), and 83 low bilirubin (30 boys and 53 girls). Since in this premature population there are sex differences both in average strength of the reflex and in the rate of dec!ine with age, the sexes are always analysed separately. The average strength of grasp in

24

NEUROLOGICAL SEQUELAE OF HYPERBILIRUBINAEMIA

...0

7

...

6

!-o

s

2 2-6

7-11

12-16

2-6

1-11

12-16

Corrected Age in Weeks

Corrected Age in Weeks

FIGURE 3. The similarity in the performance in test of visual fixation of the "high" and "low bilirubin" children of each sex at four months as a function of the corrected chronological age.

7

7

Males ~

' ',,

Low Bilirubin

'"' '"o-· -----~

..c

~3 C

.... :.;

cJ)

2 1 __________

2-6

7-11

12-16

Corrected Age in Weeks

1 __________

2-6

7-11

12-16

Corrected Age in Weeks

FIGURE 4. The strength of the grasp reflex of the "high" and "low bilimbin" infants of each sex at four months as a function of corrected chronological age.

25

ALFRED M. FREEDMAN

the 19 jaundiced males was 2.32 and in the 30 non-jaundiced males 4.74. The difference was significant ( t = 3.16 with 57 df, p less than .01). Analysis of covariance of grasp strength and CCA in the males revealed a significant difference between the high and low bilirubin groups. The high bilirubin infants having the weaker grasp (F 10.07, 1 and 55 df, significant at the .01 level) ( 4) . Again, in the girls, jaundice appeared to have no effect on grasp ( F is less than 1) ( 4). The frequency of the abnormality of the Moro reflex was also compared in the high and low bilirubin infants, as indicated in Table II.

=

TABLE II NEONATAL JAUNDICE AND DEVIANT MORO RESPONSE*

Bilirubin

Normal Moro Abnormal Moro TOTAL

High

Low

31 6

90 3 93

37

Chi square = 5.06 (significant at 5% level)

*4 infants with no reaction are omitted.

Since the infants vary in their maturity, a full Moro (bilateral extension of arms, digits, and legs), a partial Moro, or a "startle" response ( that is, a blink or body jerk without any bilateral extension of extremities), was considered "normal." A symmetry of response was considered "abnormal." A complete absence of any reaction to the stimulus was called "absent response" and also considered "abnormal." The stimulus was administered to a total of 130 infants of whom 37 were high and 93 low bilirubin infants; nine responses were classified "abnormal" and four "absent." The few abnormal responses seemed about equally frequent in the girls and boys, and the sexes were not analysed separately. When "abnormal" responses alone were considered and "absent" responses omitted from consideration, it was found that six of 37 high and three of 93 low bilirubin infants responded abnormally to the stimulus: chi square ( with Yates Correction) = 5.06 ( significant at .05 level) ( 4). If those with no response are included, it is significant at the .02 level ( 4). This is indicated in Table III. In Table IV the differences between the high and the low bilirubin babies are summarized and the direction of the significant differences stated. It can be seen that at four months of age the male premature

26

NEUROLOGICAL SEQUELAE OF HYPERBILIRUBINAEMIA

TABLE III :\EONATAL JAUNDICE AND DEVIANT MORO RESPONSE*

Bilirubin

Normal Moro Abnormal Moro TOTAL

High

Low

31

90

39

95

8

Chi square = 5 . 70 (significant at 2% level)

5

*4 infants with no reaction classed as "abnormal Moro."

TABLE IV DIFFERENCES BETWEEN HIGH AND

Group

Low

Test

Significance

Motor Visual Grasp

.01 NS* .01

Negro females

Motor Visual Grasp

NS NS NS

Both sexes

Moro

.02

:\'egro males

BILIRUBIN INFANTS

Direction Low better Low stronger

Low more normal

*NS = no significant difference.

is more susceptible and the female more resistant to the effects of hyperbiliru binaemia. A preliminary study has been done of the neurological examination at one year. It appears at the present time that the difference in the males between those with high bilirubin and those with low bilirubin is still maintained at the one-year examination in the gross motor area. SUMMARY AND CONCLUSIONS

1. Four-month-old prematurely born infants with a history of high

serum bilirubin level during the early neonatal period, but no erythroblastosis, are significantly slower in gross motor development than those with a history of low bilirubin. 2. The difference is demonstrable only among the males. 3. This gross motor difference seems to hold at the one-year level also.

ALFRED M. FREEDMAN

27

4. It may be that with further accumulation of cases to permit analysis of females with bilirubin above 20 mg./100 ml., an impairment of gross motor development may be demonstrated in both sexes. 5. One cannot state unequivocally that the slow motor development is due to injury to the central nervous system. This may be a peripheral phenomenon. 6. Visual fixation and following is the same in infants with low and high bilirubin. 7. Male infants with a history of high bilirubin have a weaker grasp reflex at the four-months examination than the low bilirubin males. 8. Rather than more rapid maturation, it is felt that these results reflect impaired motor development or weaker muscular strength. 9. The vulnerability of the male in this study is similar to the findings of others. Allen and Diamond ( 5) showed greater mortality of male infants with erythroblastosis foetalis compared to females. In the Gunn strain rats studied by Dr. Lois Johnson (6), significantly more males developed kernicterus than females. 10. Exchange transfusions have been advocated in premature infants whose serum bilirubin appears destined to exceed the 18 mg./ 100 ml. level. Before such a hazardous procedure can be recommended, one would certainly wish a clear-cut demonstration of the damage wrought by excessive bilirubin. This can only be done by an anterospective study such as is described here, but no unequivocal statement on this point can be made as yet. It is apparent that there is some effect in those children who had a high neonatal bilirubin. We are not certain if any difference in motor or intellectual performance will be found when the children are three or four years old. Furthermore, the difference seems to be confined to males. 11. It would certainly appear wiser at present to delay definite commitment for or against exchange transfusions in premature infants and to avoid dispute or polemic concerning one course of action. When the results of such studies as this one are available, an appropriate programme of management can be elucidated. 12. Of the children being followed at present ( more than 500), three definite cases of cerebral palsy have been identified. One is in the low bilirubin group, one in the middle, and one barely in the high. 13. No cases of kernicterus have been found in babies who have been included in this study and who have succumbed and have come to autopsy. Only one of the survivors showed clinical signs somewhat suggestive of kernicterus. The disparity between these results and those of other series may be accounted for by the large proportion

28

NEUROLOGICAL SEQUELAE OF HYPERBILIRUBINAEMIA

of Negroes in our population. However, one must strongly consider differences in medical management or the existence of as yet unrecognized deleterious influences of medical origin. REFERENCES 1. McGRAW, M. B.

2. 3. 4. 5. 6.

The neuromuscular maturation of the human infant. New York, Columbia University Press, 1943. GESELL, A., and AMATRUDA, C. S. Developmental diagnosis. New York, Paul B. Roeber, Inc., 1951. ANDRE-THOMAS, et SAINTE-ANNE-DARGASSIES, S. Etudes neurologiques sur le nouveau-ne et le jeune nourrisson. Paris, Masson, 1952. McNEMAR, Q. Psychological statistics, 2nd ed. New York, John Wiley & Sons, 1955. ALLEN, F. H., Jr., and DIAMOND, L. K. Prevention of kernicterus: Management of erythroblastosis fetalis according to current knowledge. J.A.M.A. 1954, 155, 1209. JoHNSON, Lms. Personal communication.

PHYSIOLOGICAL JAUNDICE RE-EXAMINED 0

Jerold F. Lucey, A.B., M.D. tt

IN A'ITEMPTING to gather comparable data on the frequency and degree of hyperbilirubinaemia and the incidence of kernicterus among premature infants, I have been impressed by the wide differences which occur between various premature nurseries. I believe that the differences encountered are real, despite the many pitfalls involved in evaluating these data. The first question which I attempted to answer was, "What is the normal serum bilirubin concentration of a premature newborn infant?" Figure 1 and Table I are summaries of reports of the mean concentrations of serum bilirubin of over 800 premature infants (1-14) . In Figure 1 I should like to call attention to the considerable difference between the infants observed by Dine in Cincinnati ( 4) and Hottinger in Basel ( 1) and those in the other reports. In Table I, the same type of data is expressed differently to answer the question, "What percentage of premature infants develop high concentrations of serum bilirubin?" If all these reports are averaged, it will appear that 12 per cent of premature infants develop concentrations of serum bilirubin of over 20 mg./100 ml. and that 30 per cent develop concentrations of serum bilirubin of over 30 mg. Hyperbilirubinaemia is of common occurrence. Note, however, the wide differences between the results of Holman ( 8), Dine ( 4), and Hottinger ( 1) and those of the other authors. Comparable figures as to the autopsy incidence of non-erythroblastotic kernicterus in premature infants are difficult to collect. They must be selected from premature nurseries where exchange transfusions have not been done. Stillbirths should be excluded. Table II °From the Department of Pediatrics, University of Vermont College of Medicine. Supported by the Playtex Park Research Foundation Grant 76.C and a grant-inaid from Hoffmann-La Roche Fund. fAddress: Department of Pediatrics, University of Vermont, College of Medicine, Burlington, Vermont, U.S.A. jCo-author: Timothy J. Driscoll.

29

30

PHYSIOLOGICAL JAUNDICE

"

24

[

A N

22

8 I

REPORTED BILIRUBIN MEAN CONCENTRATIONS IN

20

l I R

18

B

16

u

INFANTS

PREMATURE

14

C 0 N C

12

COOE

[

10

N T R A T

•lZ!ll

8 6

') ~

4 (>IG.'tJ

2

5

AGE

IN

6

7

8

WEIGHT

RANGE (GM .)

NO. OF INFANTS

MEYER

(2500

46

n

MILLER

L750-2!500

57

nr nr

OBRINSKY

(2000

JELIU

1350-2500

:,,:

MILLER

IOCXH750

12

lZI

CROSSE

(2500

47

1ZIO:.

TAYLOR

(2500

Z7

27 106

lZ!ll. HSIA

9B0-2500

12

Ir.

RAPMUND

20 K >5 >10

Selection excludes

Author ref. Weight (gm.)

Rapmund ,t al. Hsia et al. Hsia et al.

Days No. Incompatiof sampied cases Sepsis bles Sulfa

139

Waahed MICROSOMES

20

,:.:1d~~fl ATP

UTP

GA

GA ATP

GA UTP

GA UTP ATP

58

.i . . d . ,_, ,d ATP

'f

UTP ATP UTP "GA-1-P" G+P +Ml+SF

UOPGA

+Ml+SF

FIGURE 1. Formation of 4-methyl-umbelliferone-glucuronide by various substrates and subcellular fractions of homogenates of rat liver. Abbreviations: 4-MUMB: 4-methyl-umbelliferone, KRP: Krebs-Ringer-phosphate solution, GA: glucuronic acid, GA-I-P: glucuronic acid-I-phosphate, G-I-P : glucose-I-phosphate, UDPGA: uridine diphosphate glucuronic acid, Mi: microsomal fraction, SF: soluble fraction.

96

STUDIES OF GLUCURONIDE FORMATION

While the mechanism of glucuronide formation was being studied, it was observed in our laboratory that the glucuronides of 4-methylumbelliferone ( 4-methyl-7-hydroxy-coumarin) and o-aminophenol are formed on incubation of the appropriate receptor, glucuronic acid, ATP, UTP, a dialysed soluble fraction and a washed microsomal fraction of rat liver homogenates. When glucuronic acid-1-phosphate was substituted for glucuronic acid and ATP in this system, comparable glucuronide formation occurred. Figure I illustrates the formation of 4-methyl-umbelliferone glucuronide under these experimental conditions. The mechanism responsible for these reactions may be as follows: Glucuronic acid-I-phosphate+ UTP~ UDP glucuronic acid pyrophosphate (pyrophosphorylase in soluble fraction) 4-methyl umbelliferone ( or o-aminophenol) + UDP glucuronic acid ~ UDP + appropriate glucuronide (glucuronyl transferase activity in microsomal fraction)

This pathway has been demonstrated in the mung bean seedling by Hassid and associates. Glucuronide formation has not been demonstrated when glucuronic acid-1-phosphate and UTP were appropriately incubated with a glucuronide receptor and rat liver slices. This observation may represent the inability of some of the substrates to enter the hepatic cell. To date, we have been unable to synthesize bilirubin glucuronide by this pathway. The role of this pathway for UDPglucuronic acid formation in the mammalian organism requires further study. REFERENCES 1. DurroN, G. J., and STOREY, I. D. E. Uridine compounds in glucuronic acid metabolism. Biochem. J. 1954. 57, 275. 2. LELoIR, L. F. Enzymatic transformation of UDPG into galactose derivatives. Arch. Biochem. & Biophys. 1951, 33, 186. 3. CAPUTIO, R., LELOIR, L. F., CARUINI, C. E., and PALAVINI, A. C. Isolation of the coenzyme of the galactose phosphate-glucose phosphate transformation. J. biol. Chem. 1950, 184, 333. 4. SMITH, E. E. B., and MILLS, G. T. Uridine nucleotide compounds of liver. Biochem. & Biophys. Acta 1954, 13, 386. 5. REm, E. Distribution of UDPG pyrophosphorylase in rat liver. Biochem. & Biophys. Acta 1959, 32, 251. 6. KALCKAR, H. M. and MAXWELL, E. S. Biosynthesis and metabolic function of UDPG in mammalian organisms and its relevance to certain in born errors. Physiol. Rev. 1958, 38, 77. 7. STROMINGER, J. L., MAXWELL, E. S., AXELROD, J., and KALCKAR, H. M. Enzymatic formation of UDPGA. J. biol. Chem. 1957, 224, 79.

BILIRUBIN SULFATE,:,

Rudi Schmid, M.D., Ph.D. t

As DR. LATHE has pointed out in his introductory remarks, most compounds which are excreted in conjugated form have several pathways by which conjugation can occur. Steroids, for instance, can form glucuronides, sulfates, and possibly other water-soluble conjugates. Salicylate, can form salicyluric acid ( by conjugation with glycine), glucuronides, gentisic acid, and perhaps other metabolic products whch are more water-soluble, less toxic, and better suited for excretion. In most instances, one particular pathway of conjugation is predominant. For salicylate, this is probably formation of salicyluric acid, that is, conjugation with glycine, which accounts for 70 to 80 per cent of the excreted salicylate metabolites. For bilirubin, the predominant pathway is undoubtedly formation of glucuronides, but several investigators have noted that in human bile, the conjugated water-soluble bilirubin fraction contains a pigment conjugate which obviously is not a glucuronide. Dr. Isselbacher has clearly demonstrated that this fraction is a sulfate ( 1). Sulfates are formed by a soluble enzyme system which is present in the liver and which transfers "active" sulfate to the receptor which in this instance is bilirubin. The attachment of the sulfate occurs probably at the phenolic group of bilirubin in contrast to the glucuronic acid which is attached to the propionic acid radical. This renders the sulfate stable to alkali hydrolysis, in contrast to the glucuronide which is hydrolysed in alkali at room temperature. It is therefore possible to obtain a rough estimate of the magnitude of the sulfate fraction by determining the percentage of conjugated bilirubin which is stable to hydrolysis at alkaline pH. From Dr. Isselbacher's data ( 1), it is apparent that, in human bile, approximately 15 per cent of the conjugated bilirubin is excreted as a 0 Dr. K. J. Isselbacher was unable to participate in this symposium. In his absence Dr. Rudi Schmid kindly consented to deliver this paper which is based on the studies of Dr. Isselbacher. fAddress : Thorndike Memorial Laboratory, Boston City Hospital, Boston, Mass., U.S.A.

97

98

BILIRUBIN SULFA TE

sulfate. In addition he has obtained evidence to suggest that human bile contains small amounts of conjugated pigments other than glucuronides and sulfates, but the nature of these water-soluble bilirubin fractions is not known. REFERENCE

J., and McCARTHY, E. A. Studies on bilirubin sulfate and other nonglucuronide conjugates of bilirubin. J. din. Invest. 1959. 38, 645.

1. IssELBACHER, K.

THE EFFECT OF SODIUM GLUCURONATE AND GLUCURONOLACTONE ON BILIRUBIN CONJUGATION°

David Yi-Yung Hsia, M.D.t

THE PRESENT STUDY was undertaken to evaluate the relative physiological roles of glucose, glucuronolactone, and free glucuronic acid as precursors of the glucuronide moiety of conjugates. Fasting adult mongrel dogs were anaesthetized with intravenous Nembutal, the cystic duct was ligated to exclude the gall bladder from the system, and cannulae were placed in the common bile duct, the urinary bladder, and both femoral veins. Following collection of control specimens of hepatic bile, venous blood, and urine, 220 mg./kg. body weight of sodium glucuronide, glucuronolactone, and glucose, uniformly labelled with C14, were given intravenously over a 1 hr. period. An intravenous infusion of an alkaline solution of bilirubin, 15 mg./kg. body weight, was started simultaneously and given in 20 min. Bile and urine were collected for 3 hr. periods from the start of the infusions and serial blood specimens were obtained during this time. Each of the experiments was repeated on a second dog. The concentrations of bilirubin were determined in each specimen by the Van den Bergh reaction. Aliquots of each specimen of urine and bile were assayed for radioactivity. Aliquots of each bile specimen were also coupled with excessive diazotized sulphanilic acid. The azo-pigments were extracted with butanol and separated by ascending chromatography according to the method described by Schmid. The chromatograms were dried and scanned for radioactivity and contact radioautograms of the paper chromatograms were prepared on X-ray film . At the same time, 30 ml. of aliquots of urine from dogs 2, 4, and 6 were acidified with methaphosphoric acid and extracted with ether and the extracts assayed for radioactivity. °From the Genetic Clinic of the Children's Memorial Hospital, and the Departments of Pathology, Pediatrics, and Medicine, Northwestern University School of Medicine, Chicago. Aided by grants from the Elizabeth McCormick Memorial Fund and the Association for the Aid of Crippled Children. ¼Co-authors: Shiiley G. Driscoll and Robert M. Dowben. 99

100

GLUCURONATE AND BILIRUBIN CONJUGATION

10

~ 8

0

01 E

zs

iii

::>

a: :::;

ai 4 :::E ::>

a:

IJ.l2 (/)

0

3 2 HOURS FIGURE 1. Serum bilirubin levels during experiments. Dog # 1 (zeroes), Dog #2 (triangles), Dog #3 (squares). Reproduced with the kind permis~ion of the American Journal of Physiology.

0

The levels of hyperbilirubinaemia induced in three animals given sodium glucuronide ( the ones with the open circle), glucuronolactone ( the ones with the triangles) and glucose ( the ones given in the squares) are shown in Figure 1. In each instance only a small fraction of the bilirubin infused was excreted in the urine. Recovery of the injected isotope from the 3 hr. urine samples was next studied. When sodium gluruconide was injected, 28.5 and 56.3 per cent of the C 14 appeared in the urine compared with 36.7 and 16.0 per cent following the administration of glucuronolactone and 14.1 and 3.4 with glucose. An ether extract of acidified urine contained most of the conjugated glucuronides but almost no free glucuronic acid. The fraction of administered label in the ether extracts of urine from three dogs, was 4.42 X 10- 4 when sodium glucuronate was the substrate, 3.28 X 10--4 when glucuronolactone was administered, and 11.9 X IQ--4 following glucose injection or less than 1 per cent of the injected label in each case. We then studied the fraction of administered radiocarbon recovered per milligram of bilirubin in the bile in each experiment. In the dogs given sodium glucuronide, 4.16 X 10-il and 0.833 X 10--6 of injected label were recovered per milligram of bilirubin in bile. For the dogs

101

DAVID YI-YUNG HSIA

Solvent Front

Start

Sodium Glucuronate

Glucuronolactone

Glucose

F1cuRE 2. Radioautogram of paper chromatogram of biles. The locations of the colored spots corresponding to bilirubin glucuronide are marked in ink. Reproduced with the kind permission of the American Journal of Physiology.

given glucuronolactone the comparable values were 49.8 X 10-6 and 79.4 X 10-6 and, for the dogs treated with glucose, 3.39 X 10-6 and 4.91 X 10-6 injected isotope was so recovered. Upon separation of the azo-pigments from bile by paper chromatography, which is shown in Figure 2, no label was found in the spot of bilirubin glucuronide when labelled sodium g)ucuronide was given as a precursor and a very faint suggestion of activity in the spot, which does not show on the figure, when the lactone was used, but definite radioactivity was found in the site when glucose was the substrate. Under the conditions of these experiments glucose was best utilized in the formation of bilirubin glucuronide. Glucuronolactone was incorporated to small extent, serving as a poor substrate compared with glucose and sodium glucuronate was an even less effective precursor. These data suggest that the reported reductions in neonatal hyperbilirubinaemia by infusions of large doses of sodium glucuronate are not accomplished by the conjugation of the sodium glucuronate with bilirubin.

FACTORS INFLUENCING T.HE SYNTHESIS OF DIRECT REACTING BILIRUBIN °

Karel Polacek, M.D. t

THE AIM OF THE WORK was to study means of influencing the removal of bilirubin as a form of treatment of clinical haemolytic disease of the newborn. The conjugation of bilirubin has been studied in vitro in rat liver slices in the presence of several factors which might theoretically be expected to facilitate or inhibit this reaction. Fresh liver slices were produced from adult male rats, placed in buffered Ringer-bicarbonate medium with the addition of mixed human plasma and bilirubin. The final concentration of bilirubin was 20 mg./100 ml. at pH 7.4. Further experimental substances were added to this medium. Incubation was carried out in a Warburg shaker at 37° C. for 1 hr. The medium was then centrifuged and direct reacting bilirubin was estimated at pH 5.0, at which point the difference between direct and indirect bilirubin is the greatest. This determination was carried out by a modification of the method of Malloy and Evelyn ( 2). The results were calculated as gamma bilirubin conjugated per gram of wet tissue and each value presented is the average of five values from a single experiment. Normal rat liver on the average conjugated 37 gamma per gram of wet tissue. The inhibitors tested were borneol, potassium cyanide, carbon monoxide, and anoxia. The last two factors were tested by applying them to the living animal until it died or was sacrificed. The results are shown in Table I. Inhibition is expressed in terms of percentage of control activity, each value being the average of one experimental series. There was marked variability of results - not sursprising since each applied factor involves only one link of a complex cycle, the total result de°From the Research Hospital for Mothers and Children, Prague. fAddress: Ustav pro peci o matku a dite, Prague, Czechoslovakia. !Co-authors : Vera Jirsova, M.D., Milan Jirsa, M.D. , and Milos Janovsky, M.D. 102

103

KAREL POLACEK

TABLE I INFLUENCE OF INHIBITORS OF CONJUGATION Agent tested (amounts in 5 ml. of solution) Borneo! 40-80 gamma KCN 0.001 M CO 5-15 min. CO up to death .'\noxia: transitory Anoxia: Up to death

I nhibitio11

(%)

-39, -50, -39, -80, -46, -87,

-66, -55, -57 -96, -52, -92,

-69, -71 -61 -100 -55, -95, -100

pending on the concentration of the remaining links, the level of the enzymes, etc. It can be seen from the Table I that all the factors listed above produced a marked degree of inhibition, which in some cases was complete. The mechanism of this varies. Borneol competes with bilirubin, that is, it is also conjugated by glucuronic acid, while KCN, CO, and anoxia probably block enzymes, especially those concerned with ATP production. TABLE II INFLUENCE OF PRESUMED STIMULATING AGENTS OF CONJUGATION* Agent tested (amounts in 5 ml. of solution)

Stimulation or inhibition (%)

Glucose (0.2 gm.) -5, -7, +6 Glucose (0. 12 gm.) + ATP (1 mg.) -26 -43 -60 + insulin (1 unit) 0, -2, -1'1 ATP (1 mg.) UDP (1 mg.) 0, + 10, +23, +28 DPNH (0.5 mg.) 0, +5, +10, +12 Glucuronolactone (1 mg.) +23, +30, +35, +52, +61 Glucuronic acid (3 mg.) +3, +4, +8, +19, +23, +25 Thiocaprylic acid (0.5-1 mg.) -57, -60, -73, -76, -79, -80 *ATP: adenosine triphosphate; UDP : uridine diphosphate; DPNH : reduced diphosphopyridine nucleotide.

The stimulating agents tested were mainly substances which enter into the conjugation cycle. It can be seen from Table II that glucose and glucose plus ATP plus insulin either did not affect conjugation or produced some degree of inhibition. UDP and DPN increased the formation of direct bilirubin, but this effect was both small and variable. Glucuronolactone, which theoretically would not be expected to affect conjugation, showed a marked positive action, amounting to a 35 per cent increase on the average. On the other hand, glucuronic

104

FACTORS INFLUENCING BILIRUBIN CONJUGATION

acid per se affected conjugation to a rather small and variable extent. Thiocapryllic acid, which clinically has a marked hepato-protective effect, clearly depressed the formation of direct bilirubin. These results are interesting from both theoretical and practical viewpoints: 1. The marked inhibition produced by anoxia shows that foetal and neonatal anoxia may play an important role in lowering the excretion or removal of bilirubin. This is in agreement with the results of Miller and Reed ( 3) who found that newborn infants with respiratory difficulties, clinically manifest by a high respiratory rate, have a significantly higher level of bilirubin than normal newborn babies. We have also observed ( 4) that newborn babies with haemolytic disease have higher levels of bilirubin in the cord blood when they are anaemic, the height of bilirubinaemia being related to the degree of anaemia, even though one might presume, according to the law of mass action, that haemolysis would decrease with a falling red cells count. It would appear that hepatic anoxia may play a role here. 2. The finding that glucuronolactone produces a rise in the rate of bilirubin conjugation is interesting in view of the disagreement on the clinical effect of glucuronic acid administration in haemolytic disease of the newborn. Danoff et al. ( 1) observed a fall in bilirubin after glucuronic acid administration, but their results have not been generally confirmed. The present results suggest that, in addition to the classical cycle, the liver can produce UDPglucuronic acid by other pathways, such as those described by Arias in this volume. At present we are studying the clinical effect of glucuronolactone administration, but our results are still incomplete. REFERENCES 1. DANOFF, S., GRANT, CH., BOYER, A., HOLT, L. E., Jr.

Reduction of indirect bilirubinemia in vivo. Lancet 1958, l, 316. 2. JrnsovA, V., JIRSA, M., JANOVSKY, M. lmPortance of the quantitative determination of direct and indirect bilirubin in hemolytic disease of the newborn. Acta Paediat. Sci. 1958, 47, 179. 3. MILLER, C. A., and REED, H. R. The relation of serum concentrations of bilirubin to respiratory function of premature infants. Pediatrics 1958, 21, 362. 4. POLACEK, K. The clinical assessment of haemolytic disease of the newborn. Arch. Dis. Childh. 1955, 30, 217.

FAMILIAL DEFECT OF GLUCURONIDATION:

IN VIVO STUDIES 0 James 8. Sidbury, Jr., M.D. tt

FAMILIAL NON-HAEMOLYTIC JAUNDICE ( Crigler-Najjar Syndrome) ( 1) is characterized by the appearance of marked jaundice in the early neonatal period which persists throughout life. Many affected infants die with kernicterus. The bilirubin in the serum is almost entirely the indirect reacting type by the Van den Bergh Test. Early studies had indicated that this syndrome was familial. When it became apparent that direct and indirect reacting bilirubin differed in that the former is conjugated with glucuronic acid ( 2, 3) , it was reasonable to suspect that the defect in these patients was in their glucuronidating mechanism. It seemed possible too that other members of the family may also show such a defect, albeit to a lesser degree than those clinically affected. We designed experiments to test the glucuronidating mechanism of such patients and the family members in vivo. Trichlorethanol, which represented an aliphatic alcohol, was given by mouth to an affected individual and his parents, and the results compared with three normal adults. The patient excreted about 10 per cent as much trichlorethanol glucuronide in 24 hrs. as the normal controls, whereas one parent excreted 60 per cent and the other 25 per cent. When 4-C 14 cortisol was administered intravenously to an affected individual, the half-life of the free cortisol in the plasma was prolonged while the glucuronide fraction was decreased. Urine collected over the succeeding 48 hrs. contained 60 per cent less radio-metabolites of cortisol conjugated with glucuronic acid than those of normal adults,

°From the Department of Pediatrics, Johns Hopkins University School of Medicine, and the Harriet Lane Home, Johns Hopkins Hospital, Baltimore. Aided by grants from the National Institutes of Health, Department of Health, Education and Welfare, U. S. Public Health Service. tAddress : Johns Hopkins Hospital, Baltimore, Maryland, U.S.A. fCo-authors: Barton Childs, M. D., and C. J. Migeon, M. D . 105

106

FAMILIAL DEFECT OF GLUCURONIDATION

0

NORMAL

[I HETEROZYGOTE ■

D

~ D

l l l l

NT

NT NT

D JDH

HOMOZYGOTE

MEH NT

ffi J l l l ~ ~

NT NT

FIGURE 1. The pedigree of two families, having memb~rs with familial nonhaemolytic jaundice, designating those individuals who had an abnormally low salicylate glucuronide in their urine following a test dose of sodium salicylate. Solid shading indicates an individual with the disease ( homozygote), and the half shading denotes a clinically normal individual with an abnormal urinary salicylate glucuronide value ( heterozygote). The distribution of normals, carriers of the abnormal gene, and affected individuals agrees well with that expected from Mendelian transmission of the abnormal gene. Reproduced by permission of the editors of Pediatrics.

while radioactivity remaining in the urine after the various extraction procedures was 50 per cent greater. This decrease in the urinary glucuronide was, in fact, due to a defect in the glucuronidating mechanism. This was conclusively demonstrated by intravenous administration of tetrahydrocortisone (THE), which undergoes no further chemical change in the body except conjugation and excretion, and again an elevated level of unconjugated THE and a decreased level of THE glucuronide was found in the plasma. These studies tested glucuronide conjugation of a hydroxyl group on a saturated ring compound. Sodium salicylate was administered to two affected individuals and the members of two families in which affected patients had occurred, and the subsequent 24 hr. urine was analysed for the free salicylate, the glycine, and glucuronide conjugates of salicylate. The response

JAMES B. SIDBURY, JR.

107

was tested for different dosages of salicylate in all individuals when possible ( .5, LO, 2.0, and 3.0 gm.). The percentage of salicylate glucuronide present as the acyl glucuronide was also determined in most specimens. Normal controls were obtained for different age groups and different doses. It was found that the affected individuals have a markedly decreased salicylate glucuronide conjugation, and the parents and some members of the families had values intermediate between the affected and the normal subjects. The salicylate molecule is an aromatic ring compound; however, glucuronidation can occur through either the hydroxyl or the carboxyl radical. The data are interpreted to indicate that familial non-haemolytic jaundice is a recessive condition involving a defect of glucuronidation. The abnormal gene involved shows incomplete dominance with reference to glucuronide conjugation but recessive with respect to jaundice. By inference, depression of the glucuronidating activity within the liver below a certain undetermined level results in jaundice characterized by high levels of indirect reacting bilirubin. REFERENCES

J. F., and NAJJAR, V. A. Familial non-hemolytic jaundice with Kernicterus. Pediatrics 1952, 10, 169. 2. BILLING, B. H., CoLE, P. G., and LATHE, G. H. The excretion of bilirubin as a diglucuronide giving the direct Van den Bergh reaction. Biochem. J. 1957, 65,774. 3. ScHMID, R. The id-mtification of "direct-reacting" bilirubin as bilirubin glucuronide, J. biol. Chem. 1957, 229, 841. 1. CRIGLER,

CONJUGATED HYPERBILIRUBINAEMIA: A DEFECT IN BILIRUBIN TRANSPORT°

Barbara H. Billing, Ph.D. t

DURING THE LAST FEW YEARS we have come to accept the hypothesis that if bilirubin can be conjugated, then its excretion from the liver cell into the bile will automatically follow. Recently, however, our attention has been drawn to two types of non-haemolytic familial jaundice in which the conjugated pigments as well as bilirubin accumulate in the plasma, in the absence of extra hepatic or intrahepatic obstruction. In the group of patients originally described by Sprintz and Nelson (1) and Dubin and Johnson (2), there is a characteristic pigmentation of the parenchymal cells, but in the two families described by Rotor et al. ( 3) no abnormal liver histology was found. In the newborn infant raised plasma-conjugated bilirubin levels may sometimes be found at a time when the non-conjugated pigment level is falling. In all these patients there appears to be a defect in the mechanism whereby conjugated bilirubin is excreted, and in an attempt to throw some light on this problem I should like to present the results of an investigation made with Dr. Schiff and Dr. Oikawa of Cincinnati on a patient with the Rotor syndrome ( 4). The patient was a young married woman of 29 who was asymptomatic but had been icteric since the age of 16. Her sister was also jaundiced. Liver biopsies performed at a two-year interval indicated a normal liver histology. An oral cholecystogram was normal, but her excretion of Cholegraffin® was defective. In contrast to patients with Gilbert's disease, both conjugated and free bilirubin were found in the serum and as a result bile pigments were detected in the urine. Liver function tests, including alkaline phosphatase and serum transaminases, were within normal limits and suggested that there was no mechanical obstruction to account for the jaundice. The only ~From the Department of Surgery, Postgraduate Medical School of London. fAddress: Department of Medicine, Royal Free Hospital, Gray's Inn Road, London, W.C.l., England. 108

109

BARBARA H . BILLING

abnormality that was noted, in addition to the presence of bile pigments, was a very high bromsulphalein ( BSP) retention; the retained dye was all in the unconjugated form. The total serum bile pigment concentrations varied between 4.0 and 7.6 mg./ 100 ml. of which 45 per cent was in the unconjugated form. Of the conjugated pigments 58 per cent were in the form of Pigment I, which according to Bollman and his collaborators, at the Mayo Clinic, would support a diagnosis of hepatocellular disease rather than obstructive jaundice. Dr. Isselbacher showed that 60 per cent of the conjugated pigments were alkali-stable and were not hydrolysed by ,8-glucuronidase, which suggests that an unusually high proportion of the bilirubin may be conjugated as a sulphate. The bile pigments isolated from duodenal drainage were, however, mainly in the form of glucuronides so that it seems likely that there is a greater difficulty in excreting the sulphate than the glucuronide conjugates. Similar observations have been made in a French Algerian subject who had a plasma bile pigment concentration of over 20 mg./100 ml., with a normal liver. The effect of an intravenous injection of 2 mg. bilirubin/ kg. body weight on serum bile pigment concentration was studied ( Fig. 1) . In the normal subject only a very slight rise in conjugated pigment was observed and the rise in bilirubin level was temporary. In the patient

"' l 0

Q

'i;-;

NORM-4L

8

ROroR SYND!i'OME ln-

II»

.., 80

.

.,,:, C

i 60 E

0

A

;, 40

..,

t

Q,.

: I

C..,

I 1

I

'"' ::,

D 6~~

20 0

t,0>

I

I

C

~

*-

Q: I O ::, ~ I I.,-;;

5

10

15 20 Age (days)

25

30

FIGURE 2. o-Aminophenyl glucuronide synthesis by mouse liver preparations at various ages, expressed as percentage of mean adult values. Synthesis by slices, triangle; by homogenates, closed circle ( from small litter), open circle ( from large litter). ( Reproduced by permission of the Biochemical Journal.)

This work was continued with foetal guinea pig, to study the prenatal phase. The guinea pig foetus, besides being larger, is born in a much more advanced state than that of the mouse. Figure 3 sums up the results. The earliest foetal livers, up to seven weeks, had only negligible amounts of this enzyme activity in homogenates, and the slice syntheses were of course low as well. All the way through ( for both mouse and guinea pig) the synthesis in liver homogenates ( where the UDPglucuronic acid is added) was higher than in liver slices ( where the UDPglucuronic acid has to be manufactured on the spot); so that it looked as if there were two effects: first, shortage of UDPtransglucuronylase, and secondly, a shortage of UPPglucuronic acid,

118

GLUCURONIDE FORMATION IN NEONATES

100 V :::, ~

> .... 80 :::,

"O ~

C

E60 ~

0

4)

0,()

~ ...,

C

4)

40

V

I..

4)

0...

20

KLHU KLHU KLHU KLHU;KLHU 5-7 7-8 8-9 9-birth i 1-2 Foetal age-group (weeks)

I

: Age (days}

FIGURE 3. o-Aminophenyl glucuronide synthesis and UDPglucuronic acid levels in guinea pig tissue at various ages, expressed as percentage of mean adult values. K, kidney slices; L, liver slices; H, liver homogenate; U, UDPglucuronic acid in liver.

We estimated as best we could the amount of formed UDPglucuronic acid in the livers and found that it, too, was low at first and increased gradually. We have found that increasing activity of the enzyme UDPglucose dehydrogenase, catalysing the oxidation of UDPglucose to UDPglucuronic acid, is probably responsible for this. Dr. Brown, in a subsequent paper, will cover the subject in more detail. This effect was confirmed with other aglycons including those which form "ester" glucuronides, and in other animals including man. Human foetuses, 2-4 months of age, had virtually no UDP-transglucuronylase

GEOFFREY

J.

DUTTON

119

activity in kidney or liver slices or in liver homogenates, and scarcely any UDPglucuronic acid in the latter organ. This was not due to inevitable slight delays in working with human tissues, for adult mouse preparations put through exactly the same procedure showed almost their usual activity. This low glucuronide conjugation is probably a common foetal characteristic which may continue into neonatal life temporarily, as in prematurity, or permanently if, for example, by a genetic defect UDP-transglucuronylase never increases satisfactorily at all. We found virtually no activity also in placenta preparations of various ages, and the foetus would therefore appear to rely entirely on the maternal tissues for any "detoxication" by conjugation with glucuronic acid. Yet one fact makes us think the foetus might not be quite so dependent after all. When we were investigating other foetal tissues we examined the gastrointestinal tract which has a fair activity in the adult, though not so great, weight for weight, as the liver. We found that stomach homogenates from foetal guinea pigs, even at six or seven weeks, could form about twice as much "ether" or "ester" glucuronides as could similar preparations from adult gastric mucosa. We also observed UDPglucose dehydrogenase activity, so that foetal stomach, like adult ( 5), could make UDPglucuronic acid readily from UDPglucose. If we used slices of foetal stomach, addition of glucose increased synthesis considerably, presumably by increasing respiration ( and so ATP) and by supplying the glucose units. Glucuronate was about a third as effective as glucose, and glucurone virtually ineffective, under our conditions. Conjugation of bilirubin in foetal liver develops similarly to that of these other aglycons. This will be discussed in more detail in the next paper. Before I finish, however, I would mention that preliminary results suggest that in the stomach indirect Van den Bergh reacting bilirubin can be converted to the direct reacting pigment, but that is by no means to say that even a conjugate is formed: we may have merely altered the solubility of bilirubin, and until we have isolated a conjugate chromatographically, destroyed it by ,8-glucuronidase, and inhibited that destruction by saccharolactone we cannot begin to speak about a glucuronide. SUMMARY

Evidence is presented for the absence of glucuronide synthesis from liver and kidney of early foetal animals, and for its gradual development with age; this is correlated with the levels of UDP-transglu-

120

GLUCURONIDE FORMATION IN NEONATES

curonylase and UDPglucuronic acid. The significance of the resulting dependence on maternal conjugation is discussed in relation to prematurity and genetic defects. Presence of this glucuronide-synthesizing mechanism in foetal guinea pig stomach is reported and its importance suggested.

REFERENCES

J., and GREIG, C. G. Observations on the distribution of glu· curonide synthesis in tissues. Biochem. J. 1957, 66, 52p. DUTTON, G. J. Glucuronide synthesis in foetal liver and other tissues. Biochem. J. 1959, 71, 141. KARUNAIRATNAM, M. C., KERR, L. M. H., and L5llfl, G. A. The glucuronide-synthesizing system in the mouse and its relationship to beta-glucuronidase. Biochem. J. 1949, 45, 496. HARTIALA, K. J. V., and PULKKINEN, M. Studies on detoxication mechanisms. IV. Glucuronide synthesis in foetal rabbit. Ann. med. exper. et biol. Fenniae, Helsinki, 1955, 33, 246. DurroN, G. J., and STEVENSON, I. H. Synthesis of glucuronides and of uridine diphosphate glucuronic acid in kidney cortex and gastric mucosa. Biochem. Biophys. Acta. 1959, 31, 568.

1. DurroN, G.

2. 3. 4. 5.

FOETAL AND NEONATAL DEVELOPMENT OF THE GLUCURONIDE-CONJUGATING SYSTEM" Audrey K. Brown, M.D. t

IT HAS LONG BEEN RECOGNIZED that newborn infants, particularly premature infants become jaundiced in the first few days of life. This jaundice is due to an accumulation of indirect reacting or free bilirubin. Recently, it has been shown that indirect bilirubin must be converted to the direct reacting type which is chiefly bilirubin diglucuronide, before it is excreted by the liver (1--3). Formation of bilirubin diglucuronide seems to involve the same series of enzymatic steps as are necessary for the detoxification of many substances such as phenols and alcohols ( 4-7). In the preceding papers the authors have described the enzymatic sequence leading to the formation of the active glucuronic acid donor and terminating in its transfer to suitable acceptors such as bilirubin. We investigated the possibility that neonatal jaundice was fundamentally related to the inability of liver of the newborn to perform adequately the enzymatic processes needed for the conversion of bilirubin to bilirubin diglucuronide and undertook a sequential exploration of the steps involved ( 8-9). We investigated first the terminal step in the series which effects transfer of an active glucuronic acid moiety from the donor, uridine disphosphoglucuronic acid. This involves the enzyme glucuronyl transferase:

+

Bilirubin 2UDPglucuronic acid - Bilirubin diglucuronide 2UDP ( uridine diphosphate)

+

Diminished activity at this step would lead to accumulation of free or ~From the Child Research Center of Michigan and the Children's Hospital of \1ichigan and supported by a grant from the Public Health Service, RG 5105, and Lambda Tau Delta Cerebral Palsy Fund. tAddress: University of Virginia, Charlotteville, Virginia, U.S.A. 121

122

DEVELOPMENT OF TH£ CONJlJCA'tlNG SYSTEM

indirect reacting bilirubin. Glucuronyl transferase is located in microsomes which can be isolated from liver cells by ultracentrifugation. This liver fraction was prepared from foetal, newborn and adult guinea pigs and rats. To pinpoint the specific defect, pure UDPglucuronic acid was added to the system so that only the glucuronyl transferase activity would have to be supplied by the liver. Glucuronide acceptors such as bilirubin, ortho-aminophenol, and phenolphthalein were tested in the system. We first demonstrated that, when indirect bilirubin was the acceptor, only adult liver microsomes formed bilirubin glucuronide. When 1 - -· "O

•

E .035 0

"O ·c

0

.030

•

~

::,

u

i,025

•

0

C

Q)

-g_.020 0 C

E -.. additions of the buffer were made. To one of experimental cuvettes, 20 >-.. volumes of 400 mg./100 ml. sodium salicylate solution were added and to the third cuvette 20 >-.. volumes of a caffeine sodium benzoate solution ( 64 mg. / ml.) . After each addition, the optical density of the sample was measured at 465 m,u, and the difference in optical density between the experimental cuvettes and control cuvette was expressed as the percentage change of optical density (% ~ O.D. 465) . The results are plotted in the two curves shown in the insert ( Fig. 2). The optical density at 465 m,u under these conditions is a direct measure of the amount of protein-bound bilirubin. The percentage change in optical density at 465 m,u is then a measure of the amount of bilirubin dissociated from the protein owing to the addition of the salicylate or caffeine sodium benzoate, because the dissociated bilirubin has its maximal absorption at 420-440 mµ. After the final addition to the three cuvettes, the spectral absorption of the samples was measured again between 410 and 470 mµ. The three resultant curves are also plotted in Figure 2. The shape of the control curve has not changed and the loss in absorption is due to dilution. However, the curve obtained following the addition of salicylate shows less absorption at 465 m,u, owing to dissociation of protein-bound bilirubin and a greater absorption between 420-430 m,u, owing to an increase in the amount of free bilirubin. The sample, to which caffeine sodium benzoate was added, shows a qualitatively similar pattern, but the change is of greater magnitude. It should be noted that the total amount of bilirubin in the three samples was identical. Figure 3 illustrates a similar experiment where we have compared three drugs, salicylate, sulfisoxazole, and sulfam3thoxypyradazine, with regard to their ability to dissociate protein-bound bilirubin. ( For

202

BINDING OF BILIRUBIN BY ALBUMIN

. 500..-------------------------SPECTRAL EFFECT OF AN IONS ON

SERUM

Bl LI RUBIN

.400

.300 ci 0

.200 E20 LO

(.!)

v

ci 0

.100

-. of anion addition

400

20

40

60

80

500

mµ FIGURE 3. Changes in the spectral absorption of icteric serum produced by organic anions. The serum was from an erythroblastotic infant with a concentration of 0.8 mg./100 ml. direct reacting and 21.2 mg./100 ml. total bilirubin. The serum was prepared as described for Figure 2. The anion solutions used were 400 mg./ 100 ml. aqueous solutions of sodium salicylate, sulfisoxazole, and sulfamethoxypyradazine. The insert below is a plot of the per cent of proteinbound bilirubin dissociated ( % d O D 465 mµ) against the volume of anion solution added to the cuvette containing the samples of diluted serum. The spectral curves above the insert ar~ the samples before and after 300A of buffer or anion solution had been added. Closed circle: Control and experimental samples before additions. Closed circle: Control sample after addition of 300A buffer. Open circle: Sample after 300A sulfisoxazole solution was added. d: Sample after 300A of sulfamethoxypyradazine solution was added. X: Sample after 300A of sodium salicylate solution was added. ( Reproduced by permission of the Editors of the Journal of Pediatrics. )

o:

204

BINDING OF BILIRUBIN BY ALBUMIN

details of this experiment, see the legends of Figure 3.) It is apparent from this study that the long-acting sulfonamide might have a greater ability, in vivo, than sulfisoxazole to dissociate bilirubin from plasma proteins. We studied this reaction in a simpler test system also. Model solutions were made by mixing a solution of crystalline bilirubin in 0.05 N deaerated sodium hydroxide, with a solution of crystalline bovine albumin in isotonic phosphate buffer. The mixtures were adjusted to pH 7.4 and contained different molar ratios of bilirubin to Effect of anion cone. on

protein bound bi lirubin .

(sal;cylate).

12

:::l.

X

10

I. 2

X

X

~

0

t-'-I" 0

8

~x .6

6

0