Nutritional Bioavailability of Calcium 9780841209077, 9780841211063, 0-8412-0907-3

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ACS

SYMPOSIUM

SERIES

Nutritional Bioavailability of Calcium Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.fw001

Constance Kies, EDITOR University of Nebraska—Lincoln

Based on a symposium sponsored by the Division of Agricultural and Food Chemistry at the 187th Meeting of the American Chemical Society, St. Louis, Missouri, April 8-13, 1984

American Chemical Society, Washington, D.C. 1985

275

Library of Congress Cataloging in Publication Data

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.fw001

Nutritional bioavailability of calcium. (ACS symposium series, ISSN 0097-6156; 275) "Based on a symposium sponsored by the Division of Agricultural and Food Chemistry at the 187th Meeting of the American Chemical Society, St. Louis, Missouri, April 8-13, 1984." Includes bibliographies and indexes. 1. Calcium—Absorption and adsorption— Congresses. 2. Calcium—Metabolism—Congresses. 3. Intestinal absorption—Congresses. I. Kies, Constance, 1934.II. American Chemical Society. Meeting (187th: 1984: St. Louis, Mo.) III. American Chemical Society. Division of Agricultural and Food Chemistry. IV. Series. [ D N L M : 1. Biological Availability—congresses. 2. Calcium—metabolism—congresses. 3. Nutrition— congresses. QV 276 N976 1984] QP535.C2N88 1985 ISBN 0-8412-0907-3

599'.0133

85-3931

Copyright © 1985 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., 21 Congress Street, Salem, M A 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by A C S of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

ACS Symposium Series M . Joan Comstock, Series Editor

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.fw001

Advisory Board Robert Baker

Robert Ory

U.S. Geological Survey

USDA, Southern Regional Research Center

Martin L. Gorbaty Exxon Research and Engineering Co.

Geoffrey D. Parfitt Carnegie-Mellon University

Roland F. Hirsch U.S. Department of Energy

James C. Randall Phillips Petroleum Company

Herbert D. Kaesz

Charles N. Satterfield

University of California—Los Angeles

Massachusetts Institute of Technology

Rudolph J. Marcus

W. D. Shults

Office of Naval Research

Oak Ridge National Laboratory

Vincent D. McGinniss Battelle Columbus Laboratories

Donald E. Moreland

Charles S. Tuesday General Motors Research Laboratory

Douglas B. Walters

USDA, Agricultural Research Service

National Institute of Environmental Health

W. H. Norton

C. Grant Willson

J. T. Baker Chemical Company

IBM Research Department

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.fw001

FOREWORD The ACS S Y M P O S I U M S E R I E S was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S I N C H E M I S T R Y S E R I E S except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

PREFACE

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.pr001

HIGH INCIDENCE OF OSTEOPOROSIS

among postmenopausal women has created an intensified interest in adequacy of calcium nutritional status among Americans of all ages and sexes. To eat an assumed adequate amount of a nutrient does not necessarily guarantee dietary sufficiency of that nutrient. The degree to which a nutrient is absorbed from the intestinal tract, its efficiency of utilization within the body, and the processes governing its excretion are all contributing factors. The importance of dietary or endogenously synthesized vitamin D has long been recognized as a primary factor influencing the bioavailability of calcium. Some of the most exciting biochemical-nutritional research in recent years has been devoted to determining the mechanisms involved in vitamin D-calcium interactions. This research has been well reviewed in other publications. The objective of the symposium upon which this book is based was to review some of the other lesser-known dietary factors that appear to have an impact on the bioavailability of calcium. For helping plan the symposium and for handling many of the details involved in the preparation of this book, I would like to acknowledge the assistance and work of my secretary, Mrs. Donna Hahn.

CONSTANCE KIES University of Nebraska—Lincoln December 17, 1984

vii

1 Dietary Calcium Exchangeability and Bioavailability Evaluation and Potential Uses of an In Vitro Digestion Procedure E. M. WIEN and RUTH SCHWARTZ

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853 Use of the in vitro digestion procedure for estimating dietary calcium exchange with an extrinsic isotope should facilitate in vivo absorption studies using the "extrinsic tag" technique. Only a peptic digestion stage is required for exchangeability measurement. Attempts to extend use of the the procedure to measure parameters of bioavailability by including a pancreatic digestion stage were partly successful. In vitro digestion permits study of the chemistry of food calcium under standardized digestion conditions. Investigations discussed include the effects of varying pH, bile salts, enzymes and food substrates on calcium solubility; and post-digestion fractionation of calcium complexes. Before bioavailability per se is estimated in vitro, more direct comparisons between in vivo and in vitro measurements are needed.

The concept of b i o a v a i l a b i l i t y was developed to explain the difference between the t o t a l amount of mineral i n a food and the amount which was used by the i n d i v i d u a l consuming the food. Over the past s i x t y years or more, there have been numerous studies related to dietary calcium requirements and b i o a v a i l a b i l i t y (1,2). As a r e s u l t , much i s known about non-calcium food components which influence the absorption and u t i l i z a t i o n of dietary calcium under experimental conditions. What now i s lacking i s a detailed knowledge of how these factors interact with calcium under normal conditions of ingestion i n meals. We have developed an i n v i t r o digestion procedure, not as a substitute for i n vivo studies, but as a useful adjunct. Our i n i t i a l objective was to develop an i n v i t r o procedure f o r measuring exchangea b i l i t y , the f r a c t i o n of the food mineral which exchanges with an e x t r i n s i c isotope tracer added to the food. This was expected to f a c i l i t a t e the measurement of food mineral absorption i n humans by the e x t r i n s i c tag method. Secondary objectives were to determine i f i n v i t r o mineral s o l u b i l i t y could be used to estimate potential

0097-6156/ 85/ 0275-0001 $06.00/0 © 1985 American Chemical Society

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

2

b i o a v a i l a b i l i t y and to explore ways of using the i n v i t r o procedure to study the interactions of g a s t r o i n t e s t i n a l secretions with dietary mineral and other dietary components which determine mineral s o l u b i ­ l i t y . A number of i n v i t r o digestion procedures have been developed for estimating i r o n b i o a v a i l a b i l i t y (e.g. 3-5) and nonheme iron exchangeability (£). The procedure discussed i n t h i s paper evolved from one of them (4) f o r studying other minerals. Most of the work i n our laboratory has been done with calcium. In t h i s report our o r i g i n a l procedure (7) i s described. Then the published i n vivo - i n v i t r o comparison experiments (8) are b r i e f l y summarized and subsequent investigations on i n v i t r o digestion condi­ tions and methods for fractionation of calcium from digests are reported.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

In V i t r o Digestion Procedure The i n v i t r o digestion procedure i s outlined i n Figure 1. The pepsin, pancreatin and two conjugated b i l e s a l t s mixtures - porcine b i l e extract (BE) and bovine "sodium taurocholate" (TC) - were obtained from Sigma Chemical Co. (St. Louis, MO). The commercial preparations were analyzed f o r mineral content and decontaminated i f necessary before use i n the procedure. Usually BE was used i n experiments discussed i n t h i s paper. In some experiments the digestion was stopped a f t e r peptic digestion. Results of analyses made a f t e r the peptic digestion only w i l l be referred to as " a f t e r the Ρ stage"; r e s u l t s of analyses made a f t e r the complete peptic + pancreatic digestion w i l l be referred to as "after complete digestion" or "after PPa digestion." Exchangeability i s calculated as the r a t i o of s p e c i f i c a c t i v i t i e s (dpm C a / u g Ca) of the mixture and supernatant, when the e x t r i n s i c isotope was added i n ionizable form (Figure 1). Exchangeability values are expressed as decimal f r a c t i o n s . The i n v i t r o estimate of potential a v a i l a b i l i t y was defined, somewhat a r b i t r a r i l y , as calcium s o l u b i l i t y (18,000 χ g supernatant) a f t e r complete digestion. P o t e n t i a l l y available calcium was expressed as a percentage of the t o t a l food calcium (Figure 1). With the exception of a low i n v i t r o calcium s o l u b i l i t y value f o r whole milk, our e a r l i e r data compared reasonably well with calcium b i o a v a i l a b i l i t y information i n the l i t e r a t u r e (7)· 45

Comparison of In Vivo and In V i t r o Measurements The i n v i t r o procedure was tested i n " c r i t i c a l " experiments designed to make direct comparisons of i n vivo and i n v i t r o estimates of exchangeability and potential b i o a v a i l a b i l i t y and to test the use of i n v i t r o exchangeability values i n i n vivo experiments. (8). Three foods which were expected to show d i f f e r e n t l e v e l s of calcium s o l u b i ­ l i t y and exchangeability, c o l l a r d s , soybeans and spinach, were i n t r i n s i c a l l y labeled with C a i n nutrient solution culture. They were used together with Ca as an e x t r i n s i c l a b e l i n both i n v i t r o and i n vivo experiments. The r e s u l t s (8>) showed the expected v a r i a t i o n i n exchangeable Ca among the foods; exchangeability was not complete f o r soy or spinach. However, i n vivo and i n v i t r o exchangeability values were nearly i d e n t i c a l f o r each food. The i n v i t r o exchangeability values after 1+5

4 7

tt5

SN

T

= PPa S o l u b i l i t y =(Ca _ /Ca )(100)

45

Analyses ( C a , t o t a l Ca)

l45

Analyses ( Ca, t o t a l Ca)

1 Supernatant (SN-PPa) 1

Centrifuge: 4°C 15 min., 18,000 χ g

Pancreatic Digestion: 37°C, 20 min., shaking

I PPa Mixture (Τ) 1

2

3

N^HC0 to pH 6.8 biie salts to 10 mM pancSeatin to 0.1% (w/v) H 0 to\l5 ml

F i g u r e 1. I n v i t r o d i g e s t i o n procedure. (Reproduced w i t h p e r m i s ­ s i o n from Ref. 7. C o p y r i g h t 1982 J . N u t r . , American I n s t i t u t e of Nutrition.)

P o t e n t i a l b i o a v a i l a b i l i t y (%)

N

45

pPa

Analyses ( Ca, t o t a l Ca)

I Supernatant (SN-P) 1

C e n t r i f i ige: 4°C 15 min. 18,000 χ g

Definitions: Special A c t i v i t y (SA) = dpm Ca/ug Ca Exchangeability= SA^/SAg

1+5

Analyses ( Ca, t o t a l Ca)

< P-Mixture |

f P e p t i c Digest (Ρ) |

+ + + +

Peptic D i g e s t i o n : 37°C, 60 min., continuous shaking

2

+ HC1 to pH 1.5 + peps η to 0.1% (w/v) + H 0 to 9 ml

2

0.5 g dry or freeze-dried food, ground to pass No. 40 sieve (ASMTE-11) + 4.5 g H 0

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

4

PPa digestion were used to correct i n vivo e x t r i n s i c tag absorption i n order to estimate i n t r i n s i c food calcium absorption. The corrected e x t r i n s i c tag absorption agreed well with the i n t r i n s i c tag absorpt i o n i n rats f o r a l l three foods. When the estimates of b i o a v a i l a b i l i t y were compared (8), i n vivo absorption was higher than i n v i t r o s o l u b i l i t y f o r two of the foods: We had expected absorption to be less than s o l u b i l i t y due to physiolog i c a l factors (1,9). Thus, t h i s surprising result led to the reexamination of i n v i t r o digestion conditions which i s reported i n t h i s paper.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

Review of In V i t r o Digestion Conditions Experiments were conducted to determine i f varying the conditions i n the i n v i t r o digestion procedure would affect post-digestion calcium s o l u b i l i t y and i n some cases, exchangeability. This was done with two purposes: to test the use of the i n v i t r o digestion procedure f o r studying factors which might influence calcium b i o a v a i l a b i l i t y and to use the results to modify the standard procedure. Weights and volumes were doubled from the o r i g i n a l procedure (Figure 1) to have more material for analysis. Peptic Digestion Only i n i t i a l pH of the peptic digestion was studied. The pH was set either at 1.5, near the pH optimum f o r pepsin a c t i v i t y (10) or 2.0, to keep the pH closer to the range observed during human gastric digest i o n (11). The r e s u l t s i n Table I show the progressive changes i n calcium "exchangeability" and s o l u b i l i t y from the i n i t i a l s l u r r y through peptic and pancreatic digestion f o r two cow's milk products and four soy products. Varying the peptic pH between 1.5 and 2.0 had l i t t l e effect on exchangeability and s o l u b i l i t y at either the peptic or pancreatic stage. There was l i t t l e relationship between the i n i t i a l slurry and post-digestion values. Exchangeability was determined at the peptic stage, but was incomplete f o r three of the foods. L i t t l e change occurred during pancreatic digestion. S o l u b i l i t y was maximum a f t e r peptic digestion and decreased during pancreatic digest i o n f o r four of the foods. Since the exchangeability did not change during the pancreatic digestion while s o l u b i l i t y decreased, the food calcium and e x t r i n s i c tag must have precipitated from solution at the same rate during pancreatic digestion. Pancreatic Digestion The pancreatic digestion conditions studied included pH, the method of pH control, and b i l e s a l t s mixture and concentration. In addition, experiments were run to determine i f mineral s o l u b i l i t y was affected by enzymatic a c t i v i t y , or only by pH-induced s o l u b i l i t y changes. pH and pH Control. In the o r i g i n a l procedure (Figure 1), the pH was adjusted to pH 6.8 with freshly prepared NaHC0 . The pH generally rose during pancreatic digestion but the magnitude varied with d i f f e r e n t foods and pH adjustment techniques. The data i n Figure 2 are from a number of experiments i n which the pH at the end of pancreatic digestion varied from about 6.2 to 7.2. Although calcium 3

3.46 3.46

4.28 4.28

Defatted Soy flour

Soy Protein Concentrate

G

7.0 7.0

7.1 7.1

6.8 6.8

6. 9 6. 9

6. 7 6.7

I 6.7 6.7

1.5 2.0

1.5 2.0

1.5 2.0

1.5 2.0

1.5 2.0

Po 1.5 2.0

2.2 3.1

1.9 2.8

2.1 2.8

1.8 2.7

1.8 2.4

Ρ 1.8 2.4

PH at !Stage

2

6.3 6.2

6.3 6.3

6.3 6.2

6.4 6.4

6.5 6.5

PPa 6.7 6.6

0.89± .02 0.89± .02

0.46±,.01 0.46± .01

0.51± .01 0.51± .01

0.47± .01 0.47± .01

0.72± .02 0.72± .02

I 0.90± .04 0.90± .04

0.97±. 02 0.98±. 02

0.82±. 02 0.76±. 01

0.81±. 02 0.76±. 01

0.90±. 05 0.86±. 03

1.00±. 03 0.99±. 02

Ρ 1.00±.,02 0.96±. 02

1.02±,.02 1.01±,.00

0.82±,.02 0.76±,.01

0.89± .03 0.79±,.00

0.93± .03 0.89± .02

0.95± .01 0.98± .02

PPa 0.97± .01 1.02± .03

Exchang eab i l i ty,, SA r a t i o 3,4

54.,2±2.6 54.,2±2.6

15.,2±0.3 15.,2±0.3

28.,3±0.7 28.,3±0.7

23.,2±0.4 23..2±0.4

35,,5±1.0 35,,5±1.0

I 64,,7±2.3 64,,7±2.3

94.,3±2.3 96.,5±0.8

80.,8±5.7 68.,3±2.7

71.,5±1.9 65.,1±1.7

81,,1±4.4 83,,9±0.6

98,,4±1.6 96,>4±0.9

Ρ 95,.4±1.8 93,.3±5.0

3

95.• 7±1.9 93,,5±1. 6

52,,2±0. 5 48,,1±1. 0

71,,2±1. 1 66,,6±0. 4

68,,8±2. 2 66,,7±1. 6

48,,0±0. 5 48,,8±0. 8

PPa 93,,7±1. 8 91,,7±1. 7

Solubility, % > 5

In V i t ro Calcium Exchangeab l i l ity and S o l u b i l i t y in Milk and Soy P:roducts as Affected by Stage of Dig estion and I n i t i a 1 Peptic PH

5

3

2

^ g Ca/flask = mg Ca/g dry substrate. I - i n i t i a l slurry; P - i n i t i a l peptic digest, Ρ = at end of peptic digestion and PPa = at end of complete peptic + pancreatic digestion. U n i t s = mean ± S.D. f o r 3 f l a s k s . ^Units of exchangeability: Ratio, s p e c i f i c a c t i v i t y of the mixture: s p e c i f i c a c t i v i t y of the 18,000 χ g supernatant. U n i t s of s o l u b i l i t y (pg Ca i n 18,000 χ g supernatant/yg i n mixture) χ 100.

1.33 1.33

2.23 2.23

Full-fat Soy flour

Soy Protein Isolate

9.72 9.72

1

mg Ca per flask 12.40 12.40

Whole Milk

Skim Milk

Substrate

Table I.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

6

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

lQOh

6.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

pH

F i g u r e 2. R e l a t i o n s h i p between c a l c i u m s o l u b i l i t y and pH a f t e r complete d i g e s t i o n f o r f o u r soy p r o d u c t s . Key: s o l i d l i n e , f u l l f a t soy f l o u r ; long-dashed l i n e , soy p r o t e i n i s o l a t e ; short-dashed l i n e , soy p r o t e i n c o n c e n t r a t e ; and d o t t e d l i n e , d e f a t t e d soy f l o u r

1.

WIEN A N D SCHWARTZ

An

In Vitro Digestion

1

Procedure

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

s o l u b i l i t y decreased as pH increased for nearly a l l foods, the rate of change varied among foods, even i n t h i s c l o s e l y - r e l a t e d series ( a l l soy products). Several approaches to c o n t r o l l i n g the pH were t r i e d . These included the use of PIPES, a synthetic buffer with maximum buffering capacity near pH 7 and n e g l i g i b l e binding capacity for minerals (12), and the gradual addition of bicarbonate from a d i a l y s i s tubing "sack" (5.). The method which proved to be most e f f e c t i v e was a procedure i n which CO 2 was bubbled through the digest mixture both during pH adjustment and throughout the pancreatic digestion (13)· With t h i s procedure the pH could be controlled at pH 6.8 ± 0.1. Enzymes. In an e a r l i e r study (14) complete PPa digestion resulted i n calcium s o l u b i l i t y that was at l e a s t 40$ higher than when the peptic digestion step was omitted. I t was not c l e a r whether the enhanced s o l u b i l i t y was due to enzymatic digestion or incubation at acid pH. Therefore, an experiment was run to investigate the r e l a t i v e importance of pH and enzymatic a c t i v i t y . The t e s t substrate was a defatted soy f l o u r . Four minerals, Ca, Mg, Fe and Zn, were measured i n the digests. The r e s u l t s are presented i n Table I I . At the peptic stage, including pepsin increased Fe s o l u b i l i t y , but not Ca s o l u b i l i t y ; Mg Table I I . E f f e c t of Peptic and Pancreatic Enzymes on Mineral S o l u b i l i t y After In V i t r o Digestion Digestion

Final PH 2.1+.0

Ca 82.6±1.7

Peptic

2.6±.0

pH 2, 60 min. + pH 6.8, 30 min. no enzymes

6.7±.l

pH 2, 60 min. no enzymes

% Soluble ±1.2

Fe 2.2±0.7

Zn 101.9±5.3

75.3±0.7

98.0±1.1

17.310.9

93.4±5.4

41.4±3.6

79.0±3.0

19.3±3.5

56.2±4.6

Mg 104

56.2±4.6 Peptic + 44.7±1.2 6.6±.0 48.9±1.2 84.0±1.4 Pancreatic Note: The s u b s t r a t e was a commercial d e f a t t e d soy f l o u r p r o d u c t . Values are mean + S.D. f o r 4 f l a s k s . Each f l a s k c o n t a i n e d 1.0 g substrate. T o t a l m i n e r a l c o n t e n t (\Xglg s u b s t r a t e ) i s Ca, 2778; Mg, 3068; Fe, 87; and Zn, 53. and Zn were completely soluble at pH 2, even without pepsin. A comparison of complete (PPa) digestion with successive non-enzymatic incubations at pH 2 and pH 6.8 indicated that the enzymes greatly increased Fe s o l u b i l i t y and s l i g h t l y increased Ca s o l u b i l i t y . pH and Pancreatic Digestion. The pH range of the i n v i t r o pancreatic digestions (Figure 2) was s i m i l a r to i n vivo conditions, but generally below the pH optima f o r pancreatic enzymes (10,11). To determine i f pH had an i n d i r e c t e f f e c t on calcium s o l u b i l i t y through an e f f e c t on the rate of digestion, protein and carbohydrate digestion and calcium s o l u b i l i t y were measured i n the same PPa digests. The digestions consisted of the standard peptic digestion, followed by pancreatic

8

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

digestion i n which the pH was set at 6.2, 6.5, 6.8 or 7.1. The substrates were complex foods, muffins, which had been prepared f o r another study (15). When 50% of the bran i n the bran muffin was replaced by freeze-dried spinach or lettuce, the t o t a l calcium i n the muffin was increased by 56% or 65?, respectively. The pH had very l i t t l e effect on carbohydrate or protein digestion, but the calcium s o l u b i l i t y dropped by at least k0% i n a l l three muffin formulations over the 0.9 pH unit increase (Table I I I ) . Calcium s o l u b i l i t y was much lower i n the spinach-containing muffins and dropped more sharply with increasing pH than i n the bran and lettuce-containing muffins. The calcium data i n Table I I I confirm that a small change i n digest pH a f f e c t s the r e l a t i v e calcium s o l u b i l i t i e s from d i f f e r e n t foods. B i l e S a l t s . We used crude conjugated b i l e s a l t s mixtures prepared from b i l e of two species, TC (bovine "sodium taurocholate") and BE (porcine b i l e extract) to determine i f the type of concentration of b i l e s a l t s affected PPa calcium s o l u b i l i t y . Each mixture was used at three concentrations (equal weights), approximately 5, 10 and 15 mM, a l l within the range found i n i n t e s t i n a l contents (11). The substrates were three foods with d i f f e r e n t l i p i d compositions: fullfat soy f l o u r , whole milk and whole egg. The "soluble" calcium i s aqueous calcium and does not include the non-emulsified l i p i d layer v i s i b l e i n some of the lower b i l e s a l t concentrations. The r e s u l t s i n Figure 3 are f o r digestions a t pH 6.8. For soy flour and milk, increasing the concentration of either TC or BE s l i g h t l y decreased soluble calcium and there were no consistent differences between the two b i l e s a l t s mixtures. For egg, both mixtures caused a marked decrease i n calcium s o l u b i l i t y . When the experiment was repeated using a pH maintenance procedure which produced a f i n a l pH of pH 6.5, the pattern of r e s u l t s was s i m i l a r but a l l s o l u b i l i t i e s were 5-15Î higher. Fractionation of Calcium from Digests We have t r i e d two non-destructive approaches to fractionation of the mineral complexes i n i n v i t r o digests: u l t r a f i l t r a t i o n and g e l filtration. Only preliminary data from gel f i l t r a t i o n experiments are currently available. An example of the r e s u l t s of u l t r a f i l t r a t i o n i s presented i n Table IV. The fractionation of the digest mixture, which was prepared from a soy protein i s o l a t e , combines the techniques of centrifugation and u l t r a f i l t r a t i o n . Since the digest contained bicarbonate as the main buffer, C 0 was used to apply pressure to the u l t r a f i l t r a t i o n c e l l , rather that N , to avoid forming p r e c i p i t a t e s . Most of the digest calcium was soluble a f t e r centrifugation at 18,000 χ g. Much of i t was bound to large complexes, as judged from the f i l t r a t i o n of less than h a l f the calcium through the 10,000 MWC0 (molecular weight cut­ off) membrane. The nominal MWC0 of the membrane i s not a precise guide to the size of complex which i s f i l t e r e d since one-third of the Ca from CaCl2 was retained by the 1000 MWCO membrane, but the d i s t r i b u t i o n of calcium i n the digest was c l e a r l y d i f f e r e n t from that in the completely ionized solution. For a l l u l t r a f i l t r a t i o n s of the in v i t r o digest, the s t a r t i n g material was the 100,000 χ g supernatant in order to avoid confounding the r e s u l t s with sample deterioration. The r e s u l t s were found to be reproducible and not influenced by the 2

2

+ +

551 562 551 554

319

2

108 106 107 108

14

3

Bran muffin peptide maltose

0.62 0.42 0.37 0.37

(Total= 1.36 mg)

542 540 527 517

322

132 130 134 132

24

0.28 0.19 0.08 0.06

(Total2.12 mg)

573 556 545 531

328

116 113 116 112

17

0.92 0.83 0.63 0.47

(Total* 2.25 mg)

Spinach/bran muffin Lettuce/bran muffin peptide maltose Ca peptide Ca Ca maltose mg/g substrate component i n 18,000 χ g supernatant:

3

2

iFreeze-dried foods were ground to pass a 40-mesh sieve. Freeze-dried spinach or lettuce replace 50% of the bran of the bran muffins i n the spinach/bran and lettuce/bran muffins, respectively. Maltose, the endproduct of amylase a c t i v i t y , was measured by the method of Dahlquist (16). Peptide=supernatant protein which was not p r e c i p i t a t e d when t r i c h l o r o a c e t i c acid (TCA) solution was added to 5% TCA (W/V) (17).

pH 6.2 6.5 6.8 7.1

Initial. (no digestion)

Substrate:! Soluble component:

Table I I I . E f f e c t of In V i t r o Pancreatic Digestion pH on Carbohydrate and Protein Digestion and Calcium S o l u b i l i t y

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

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N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

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Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

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0

5

10

15

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F u l l Fat Soy Flour

Whole Milk

Whole Egg

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F i g u r e 3. Calcium s o l u b i l i t y : dependence on b i l e s a l t p r e p a r a t i o n and c o n c e n t r a t i o n used i n i n v i t r o d i g e s t i o n (pH 6.8-6.9). Key: Δ, crude bovine sodium t a u r o c h o l a t e ; and • , p o r c i n e b i l e e x t r a c t .

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Table IV. F r a c t i o n a t i o n o f Calcium i n an I n V i t r o D i g e s t by C e n t r i f u g a t i o n (CE) and U l t r a f i l t r a t i o n (UF) and Comparison w i t h UF R e s u l t s f o r C a C l 2

Fraction

CaCl

2

Solution

3

1

In Vitro Digest *

F i l t e r a b l e Calcium, 7> Digest Mixture CE:18,000 χ g supernatant CE:100,000 χ g supernatant UF:300,000 MWCO f i l t r a t e UF:10,000 MWCO f i l t r a t e UF:5,000 MWCO f i l t r a t e UF:1,000 MWCO f i l t r a t e

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

1

2

2

2

2

Ca recovery (UF)

1

-

100% 87.4±5.3 67.9±9.1 52.4±6.2 44.9±3.8

95.8% 67.8

30.216.9

98%

93-103%

-

C e n t r i f u g a t i o n at 18,000 χ g, 4°C f o r 15 min. or 100,000 χ g, 4°C for 60 min. U l t r a f i l t r a t i o n was carried out i n an Amicon s t i r r e d c e l l , 65 ml capacity, at 4 C with continuous s t i r r i n g under C0 pressure u n t i l 25 ml f i l t r a t e had been c o l l e c t e d . MWCO = nominal molecular weight cut-off of the membrane. 50 ml of the solution, containing 50 pmoles Ca, was introduced into the UF chamber f o r each determination. Values are f o r duplicate experiments. ^The i n v i t r o digest was prepared by digesting a soy protein i s o l a t e . For each UF determination, 50 ml of the 100,000 g supernatant was introduced into the UF chamber. Values are mean 1 S.D. for four experiments. % f i l t e r a b l e c a l c u l a t i o n : (pg Ca/ml of f i l t r a t e ) / ( y g Ca/ml i n i t i a l solution i n UF chamber) χ 100. The i n v i t r o digest f i l t e r a b l e calcium values were standardized as a % of the o r i g i n a l digest mix­ ture calcium.

2

2

3

5

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N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

order of the series of membranes i n a p a r t i c u l a r experiment. Thus, sample deterioration was not a problem when the sample was stored on ice and CO2 saturation maintained. Recovery of the calcium from the combined f i l t r a t e and "retentate" i n the u l t r a f i l t r a t i o n chamber was 93% or better.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

Discussion Exchangeability. Measurement of food mineral absorption i n humans by the e x t r i n s i c isotope or e x t r i n s i c tag method i s simpler, more f l e x i b l e and would allow more e f f i c i e n t use of stable isotopes than other methods f o r measuring mineral absorption (7.), i f the r e l a t i o n ship between the absorptions of the e x t r i n s i c tag and i n t r i n s i c food mineral i s known. Cook, et a l . (18) and Hallberg, et a l . (19) o r i g i n a l l y demonstrated the v a l i d i t y of the e x t r i n s i c tag f o r i r o n by feeding i t together with foods labeled i n t r i n s i c a l l y with a second isotope. They r e s t r i c t e d use of the technique to meals i n which there was equal absorption of e x t r i n s i c and i n t r i n s i c iron, that i s , when exchangeability was complete. For other minerals, the requirements for i n vivo v a l i d a t i o n of the e x t r i n s i c tag technique, two "safe" isotopes which can be detected i n the presence of one another and the laborious process of i n t r i n s i c a l l y labeling foods, put severe r e s t r i c t i o n s on the use of the e x t r i n s i c tag technique. Also, a lack of information about the degree of exchange between the e x t r i n s i c tag and test meal i r o n has hampered i n t e r p r e t a t i o n of r e s u l t s from some i r o n absorption studies (20). The i n v i t r o digestion procedure should simplify the use of the e x t r i n s i c tag method. Our r e s u l t s indicate that calcium exchangeabil i t y can be determined i n v i t r o since the i n vivo and i n v i t r o calcium exchangeability values were s i m i l a r (8>). No i n t r i n s i c labeling and only one isotope are required. The same procedure provides the information needed to i n t e r p r e t the r e s u l t s of i n vivo e x t r i n s i c tag studies. Also, i t i s not necessary to demonstrate complete exchangea b i l i t y i n order to use the e x t r i n s i c tag technique f o r measuring calcium absorption. I f exchangeability i s known, even i f i t i s not complete i t can be used to calculate i n t r i n s i c food mineral absorption from the e x t r i n s i c tag absorption (8). Hallberg and Bjorn-Rasmussen (6) reached a s i m i l a r conclusion i n t h e i r studies on absorption of "contamination" i r o n . Measurement of calcium exchangeability i s further s i m p l i f i e d i n that only the peptic digestion step of the i n v i t r o procedure i s required since exchangeability does not change a f t e r that (Table I ) . While our data indicate that i n v i t r o digestion provides a simple means to solve some d i f f i c u l t problems of using the e x t r i n s i c tag method f o r measuring calcium absorption, our conclusions are based on a limited number of foods. They would be strengthened i f a wider range of foods i s tested i n direct i n vivo - i n v i t r o comparison studies. The test foods should include foods i n which exchangeability might not be completed during peptic digestion such as foods with " i n d i g e s t i b l e " residues that may be altered and release calcium which may be absorbed i n the lower i n t e s t i n e . Bioavailability. In p r i n c i p l e , the i n v i t r o procedure provides a r e l a t i v e l y fast and inexpensive means to study calcium b i o a v a i l a b i l i t y as a c h a r a c t e r i s t i c of foods. A knowledge of the chemistry of

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Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

the foods themselves i s not s u f f i c i e n t since changes occur during the process of digestion and the t o t a l chemical environment influences the r e l a t i v e binding constants f o r the various complexes which may be formed (21,22). At the same time, the host c h a r a c t e r i s t i c s which r e s t r i c t i n vivo absorption and introduce v a r i a b i l i t y not related to food c h a r a c t e r i s t i c s (1,2,9) are eliminated. Choice of Potential B i o a v a i l a b i l i t y C r i t e r i o n . I t i s usually assumed that calcium must be soluble and probably ionized i n order to be available f o r absorption (£). For the i n v i t r o procedure, as a f i r s t approximation we chose calcium s o l u b i l i t y a f t e r centrifugation at 18,000 χ g as the measure of potential b i o a v a i l a b i l i t y (Figure 1). We assumed that t h i s would probably overestimate the available calcium and l a t e r work based on f r a c t i o n a t i o n might define the bioavailable calcium more p r e c i s e l y . The data i n Table IV i l l u s t r a t e how the choice of c r i t e r i o n f o r " s o l u b i l i t y " could a f f e c t the i n v i t r o estimate of potential a v a i l a b i l i t y , even i f i n v i t r o conditions c l o s e l y resembled i n vivo conditions. Since our i n v i t r o c r i t e r i o n unexpectedly underestimated calcium b i o a v a i l a b i l i t y f o r two of the three foods i n the direct i n vivo - i n v i t r o comparison (8), i t was necessary to determine the i n v i t r o digestion conditions which might be l i m i t i n g s o l u b i l i t y before addressing the choice of appropriate criterion. Digestion Conditions. Peptic conditions were not emphasized since calcium s o l u b i l i t y i s high at the peptic stage (Table I) and chyme release to the duodenum i s more dependent on p a r t i c l e size than completeness of digestion (23,24). Analyses of the pancreatic digestion conditions indicate that the pH of the pancreatic digest was more important for determining calcium s o l u b i l i t y than enzymatic a c t i v i t y (Figure 2, Tables I I and III) or b i l e s a l t s (Figure 3) for most foods tested. In e a r l i e r studies (7,8) the pH increased 0.2-0.7 pH u n i t s during the pancreatic digestion, presumably due to a combination of bicarbonate decomposition, digestion endproduct release and the variable buffering capacity of the foods. Even though these pH's are i n the range found i n the small i n t e s t i n e (11), the observed pH d r i f t occured i n a range which i s c r i t i c a l for calcium s o l u b i l i t y (£). The f i n a l pH was apparently too high to result i n the net effect seen i n vivo (8). "Standard" Digestion Conditions. As a result of the analyses of digestion conditions we have modified our i n v i t r o digestion to s t a r t the peptic digestion at pH 2.0 instead of 1.5, and to control the pH at 6.8 i n the pancreatic stage by continuous aeration with CO . We also substituted b i l e extract f o r taurocholate since, although calcium s o l u b i l i t y was similar, other minerals were more soluble i n the b i l e extract-containing digests (13)· The term "standard" i s not meant to denote a digestion procedure which should be routinely used to determine p o t e n t i a l l y available calcium. Since the number of foods tested so f a r i s l i m i t e d , i t w i l l be more useful to think of the "standard" procedure as a set of conditions to be used to see how well we understand food chemistry and calcium s o l u b i l i t y i n the g a s t r o i n t e s t i n a l environment. I t should be used f o r measuring the r e l a t i v e s o l u b i l i t y of calcium from foods and meals, but mostly i n the context of comparisons with i n vivo r e s u l t s to define factors which require further study.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

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NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

Lipid-containing Foods. The decreased calcium s o l u b i l i t y i n the presence of b i l e s a l t s (Figure 3) suggests factors which require further study. B i l e s a l t s have been shown to enhance calcium absorp­ t i o n from sparingly soluble s a l t s (25) but also enhance lipase a c t i v i t y (26). Since no absorption occurs i n the i n v i t r o system, the l i b e r a t e d f a t t y acids could form insoluble calcium soaps (27). In contrast, i n vivo calcium absorption i s not i n h i b i t e d by even large amounts of f a t i n the d i e t unless there i s a pre-existing malabsorp­ t i o n problem (1,2,9). Presumably, l i p i d hydrolysis products are absorbed from the i n t e s t i n e f a s t enough to prevent insoluble calcium soap formation. The complex l i p i d s of egg yolk (28), and t h e i r possible e f f e c t s on l i p a s e a c t i v i t y (29) may explain the marked decrease i n egg calcium s o l u b i l i t y with increasing b i l e s a l t s concen­ t r a t i o n . More information i s needed to determine i f t h i s i s a problem i n vivo. In any case, these questions should not prevent the use of the i n v i t r o procedure f o r measuring exchangeability i n l i p i d containing foods, since exchangeability i s determined at the peptic stage, as shown f o r whole milk and f u l l - f a t soy f l o u r i n Table I. Fractionation of Digest Calcium. The i n v i t r o digestion procedure provides a means of producing the s t a r t i n g material for a more detailed study of the calcium complexes i n i n t e s t i n a l digests. This may be desirable i n a number of s i t u a t i o n s : 1 . To determine how the degree of exchange between an e x t r i n s i c isotope and the i n t r i n s i c calcium i n the food or meal i s affected by the method of incorporating the isotope, the calcium source i t s e l f or foods fed with i t . 2. To determine how controlled manipulation of digestion conditions influences the d i s t r i b u t i o n of calcium among possible ligands from a food or meal. 3 . To describe the calcium complexes formed during a standardized digestion for a number of foods and food mixtures, f o r comparisons, or to test hypotheses r e l a t i n g food components to i n t e s t i n a l calcium complexes. Fractionation Methods. U l t r a f i l t r a t i o n and gel f i l t r a t i o n are non­ destructive methods which, based on l i m i t e d experience, can be used for fractionation of mineral complexes from digests. In e a r l i e r studies mineral absorption on the g e l material was a problem. Lonnerdal ( 3 0 ) introduced a method of treating dextran gels with sodium borohydride i n order to eliminate the mineral-binding s i t e s on the g e l . In preliminary studies we have recovered more than 90% of Ca, Mg, Fe, Zn and Ρ i n samples applied to a borohydride-treated gel column (Sephadex G-50, Pharmacia Fine Chemicals, Piscataway, NJ). Recovery of Ca (Table IV) and Mg, Fe and Zn from u l t r a f i l t r a t i o n was also good. In our experience with u l t r a f i l t r a t i o n , use of C 0 pressure to force the f i l t e r a b l e material through the membrane i n the u l t r a f i l ­ t r a t i o n procedure introduced a possible source of error. The high pressure caused more C 0 to be dissolved i n the digest supernatant and the pH i n the chamber decreased to about 6.2. This may have caused a s h i f t of the mineral among ligands ( 3 1 ) . I t should be possible to formulate a mixture of C02 and N2 to maintain the pH i n the chamber, but we have not pursued t h i s . 2

2

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In Vitro Digestion

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15

Gel f i l t r a t i o n may be best used to analyze fractions already separated from a digest supernatant by u l t r a f i l t r a t i o n , as used i n a recent study by Sandstrom, et a l . (32). A more precise separation of complexes can be obtained with gel f i l t r a t i o n , but the size of sample which can be applied i s l i m i t e d . Thus, i n many situations, the sample must be concentrated before being applied to the gel column. E i t h e r p r e - p u r i f i c a t i o n or sample concentration could introduce possible s h i f t s i n mineral binding which should be understood f o r proper interpretation of the r e s u l t s (33).

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch001

Summary Use of an i n v i t r o simulated g a s t r o i n t e s t i n a l digestion procedure i n calcium b i o a v a i l a b i l i t y research has been discussed. Two d i s t i n c t types of uses were described: (1) measurement of exchangeability to f a c i l i t a t e dietary calcium absorption studies, and (2) study of the fate of food calcium i n the g a s t r o i n t e s t i n a l environment with regard to i t s potential a v a i l a b i l i t y for absorption. Ideas have been incorporated from many sources and only limited testing has been possible so f a r . We have t r i e d to indicate the advantages of such a procedure as well as where more testing of the v a l i d i t y of the ideas for calcium b i o a v a i l a b i l i t y research i s required.

Acknowledgments The research was supported i n part by grants from NIH Grant 18569 and USDA Cooperative Agreement 58-320A4-9-91.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Allen, L.H. Am. J. Clin. Nutr. 1982, 35, 783-808. Irwin, M.I.; Kienholz, E.W. J. Nutr. 1973, 103, 1019-95. Jacobs, Α.; Greenman, D.A. Brit. Med. J. 1969, 1, 673-6. Narasinga Rao, B.S.; Prabhavathi, T. Am. J. Clin. Nutr. 1978, 31, 169-75. Miller, D.D.; Schricker, B.R.; Rasmussen, R.R.; Van Campen, D. Am. J. Clin. Nutr. 1981, 34, 2248-56. Hallberg, L.; Bjorn-Rasmussen, E. Am. J. Clin. Nutr. 1981, 34, 2808-15. Schwartz, R.; Belko, A.Z.; Wien, E.M. J. Nutr. 1982, 112, 497504. Wien, E.M.; Schwartz, R. J. Nutr. 1983, 113, 388-93. Wilkinson, R. In "Calcium, Phosphate and Magnesium Metabolism"; Nordin, B.E.C., Ed.; Churchill-Livingston: Edinburgh, 1976; pp. 36-112. Harper, H.A. "Review of Physiological Chemistry"; Lange: Los Altos, 1975, 15th ed.; p. 230. Fordtran, J.S.; Locklear, T.W. Am. J. Digest. Dis. 1966, 11, 503-21. Good, N.E.; Winget, G.D.; Winter, W.; Connolly, T.N.; Izawa, S.; Singh, R.M.M. Biochemistry 1966, 5, 467-77.

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13. Schwartz, R. 1984, In preparation. 14. Belko, A.Z. M.S. Thesis, Cornell University, Ithaca, 1980. 15. Schwartz, R.; Spencer, H.; Welsh, J.E. Am. J. Clin. Nutr. 1984, 39, 571-6. 16. Dahlquist. A. Scand. J. Clin. Lab. Invest. 1962, 14, 145-51. 17. Layne, E. In "Methods in Enzymology"; Colowick, S. P.; Kaplan, N.O. Eds.; Academic: New York, 1956, Vol. III., pp. 448-50. 18. Cook, J.D.; Layrisse, M.; Martinez-Torres, C.; Walker, R.; Monsen, E.; Finch, C.A. J. Clin. Invest. 1972, 51, 805-15. 19. Hallberg, L.; Bjorn-Rasmussen, E. Scand. J. Haematol. 1972, 9, 193-7. 20. Consaul, H.R.; Lee, K. J. Agr. Food Chem. 1983, 31, 684-9. 21. Leigh, M.J.; Miller, D.D. Am. J. Clin. Nutr. 1983, 38, 202-13. 22. Schubert, J. In "Iron Metabolism"; Gross, F., Ed.; SpringerVerlag: Berlin, 1964; pp. 466-94. 23. Davenport, H.W. "Physiology of the Digestive Tract"; Yearbook Medical Publ.: Chicago, 1982, 5th ed. 24. Arnold, J.G.; Dubois, A. Digest. Dis. Sci. 1983, 28, 737-41. 25. Webling, D. D'A.; Holdworth, E.S. Biochem. J. 1966, 100, 65260. 26. Rathelot, J.; Julien, R.; Canioni, P.; Coeroli, C.; Sarda, L. Biochemie 1975, 57, 1117-22. 27. Patton, J.S.; Carey, M.C. Science 1979, 204, 145-8. 28. Parkinson, T.L. J. Sci. Fd. Agric. 1966, 17, 101-11. 29. Patton, J.S.; Carey, M.C. Am. J. Physiol. 1981, 241, G328-36. 30. Lonnerdal, B. In "Trace Element Analytical Chemistry in Medicine and Biology"; Bratter, P.; Schramel, P. Eds.; WalterDeGruyter: Berlin, 1980; pp. 439-46. 31. Danielson, B.G.; Pallin, E.; Sohtell, M. Uppsala J. Med. Sci. 1982, 87, 43-53. 32. Sandstrom, B.; Keen, C.L.; Lonnderdal, B. Am. J. Clin. Nutr. 1983, 38, 420-8. 33. Chaberek, S.; Martell, A.E. "Organic Sequestering Agents"; John Wiley: New York, 1959; p. 101. RECEIVED October 15, 1984

2 Assaying Calcium Bioavailability in Foods Applicability of the Rat as a Model A R T H U R W. M A H O N E Y and DELOY G. HENDRICKS

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

Department of Nutrition and Food Sciences, Utah State University, Logan, U T 84322

All middle aged adults lose bone which becomes debilitating when sufficient mineral is lost and fractures occur whether as chronic compression fractures of the vertebrae or as acute fractures of the femoral neck. Evidence is accumulating that adult bone loss is the result of insufficient consumption of bioavailable calcium. Several strategies for assaying calcium bioavailability are discussed. Information is presented supporting the rat as a model for predicting human calcium u t i l i z a t i o n . This cannot be fully evaluated, however, because animal data have been obtained using growing rats fed controlled amounts of calcium and human data have been obtained from adult subjects who have received liberal amounts of calcium. Calcium absorption data are needed from animal and human subjects having similar nutritional and physiological characteristics and which have consumed identical calcium sources. Adult bone loss i s one of the most d e b i l i t a t i n g health problems in modern western society f o r e l d e r l y people. Although bone i s l o s t by both men and women as they age ( 7 3 , 9 5 ) , women suffer from osteoporosis more frequently and severely than do men. Bone loss i s detected by radiodensity and photon absorption techniques. Because 20 to 50 percent of bone mineral may be l o s t before the loss i s detected by radiodensity techniques ( 1 , 2 ) , i t i s probable that bone mineral i s being l o s t much e a r l i e r than age 40 to 45 in women and age 60 in men as i s commonly thought ( 3 , 4 ) . Photon absorptiometry has a precision of 2 to 4 percent r e l a t i v e to bone mineral content of the same bone. Measurements on the r a d i i and ulnae are highly correlated (r = 0 . 8 5 ) with bone mineral content of the femoral neck {2). Using photon absorptiometry, Mazess et a l . (5) reported that bone mineral declines beginning approximately at age 50 f o r both men and women. It i s estimated that the average rate of t h i s bone loss amounts to approximately 10 mg calcium d a i l y f o r men and 20 mg calcium f o r women before menopause. After menopause t h i s loss i s 0097-6156/ 85/ 0275-0017506.00/ 0 © 1985 American Chemical Society

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

18

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

approximately 4 0 to 1 2 0 mg calcium d a i l y { § ) . Calculating from the data reported by Mazess et a l . ( 5 J , approximately 5 0 mg of bone mineral i s l o s t d a i l y by women over age 5 0 . It i s generally believed that the larger the bone mass before age-onset bone loss occurs the less l i k e l y the development of d e b i l i t a t i n g bone loss a f t e r age 6 5 ( 3 , 6 , 7 ) . Bone strength however declines much e a r l i e r in l i f e beginning approximately age 2 0 f o r both men and women (8). In animals bone strength i s d i r e c t l y related with i t s mineral content ( 9 - 1 2 ) . These are related to the amounts of dietary calcium and phosphorus ( 1 1 , 1 2 ) or other factors a f f e c t i n g mineral metabolism ( 9 , 1 0 ) . In people, however, the ash, calcium and phosphorus concentrations of bones do not seem to change with age ( 1 3 ) even though human bone strength ( 8 ) and bone mass ( 3 , 1 4 ) clearTy do decline with aging. Havvi et a l . ( 1 3 ) also found descrepancies between bone densities and r a d i o l o g i c i T l y detected osteoporosis vs. bone mineral contents. In spite of these descrepancies, bone loss i s a major d i f f i c u l t y facing aging people. Evidence i s accumulating for dietary calcium deficiency being an e n t i t y in human n u t r i t i o n ( 6 , 7 , 1 5 , 1 6 ) . In a study of 1 3 0 normal perimenopausal women, Heany et a l . ( 1 5 J found that t h e i r calcium balance averaged - 2 5 to - 3 0 milligrams d a i l y . By regression analysis they determined that these women required an intake of 1241 mg (with a 95% confidence interval of 1 1 6 6 to 1 3 1 6 mg Ca) calcium d a i l y to maintain calcium balance. From other data, i t i s estimated that 3 5 mmol ( 1 4 0 0 mg) calcium d a i l y i s needed to maintain calcium balance in women aged 3 5 to 5 0 and postmenopausal women need 4 7 . 5 mmol ( 1 9 0 0 mg) calcium d a i l y {6). A l l of these values are well above the current Recommended Dietary Allowance of 8 0 0 mg calcium d a i l y f o r adult women. The average d a i l y calcium intakes of American women above age 2 3 vary from 5 1 5 to 6 0 4 milligrams for d i f f e r e n t age groups ( 1 7 ) . An average d a i l y calcium intake of 9 4 4 (Sd = 3 4 3 ) milligrams was found for 1 0 0 premenopausal Canadian women ( 1 8 ) . Calcium i s the only nutrient that i s associated with incidence of bone fracture ( 6 ) . Calcium intake i s highly correlated with the mineral content of the bones of experimental animals ( 1 1 ). Thus, calcium could be considered the most frequently d e f i c i e n t nutrient in the U.S.A. Anything that could r e s u l t in greater intakes of calcium and/or improved calcium b i o a v a i l a b i l i t y would be p o t e n t i a l l y important in preventing or delaying d e b i l i t a t i n g bone loss in the e l d e r l y . Approximately, 4 6 percent of a l l calcium consumed by Americans is from dairy products ( 1 7 ) . Scythes et a l . ( 1 8 ) found that dairy products contributed 66.7~percent of the calcium consumed by Canadian pre-menopausal women. Others suggest that dairy products contribute approximately 7 5 percent of the calcium consumed ( 1 9 ) . Neither data set includes calcium taken as supplements. About nine percent of the population consume calcium supplements ( 2 0 ) . Dairy products, however, contribute only 1 3 . 8 to 1 8 . 2 percent of the energy consumed ( 1 7 , 1 8 ) . C l e a r l y , dairy products are a r i c h source of dietary calcium (approximately 1 3 7 0 mg per 1 0 0 0 kcal) and can contribute major quantities of calcium to the diets of those who consume them.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

2.

MAHONEY A N D HENDRICKS

A Model for

Assaying

Ca

Bioavailability

19

Calcium retention i s dependent on two f a c t o r s , absorption and excretion. Normal subjects have been observed to have apparent calcium absorptions of 2 3 (sd = 1 2 ) to 2 7 (sd = 1 7 ) percent of the calcium from normal diets ( 2 1 , 2 2 ) . For 2 0 women aged 5 5 to 6 5 consuming 6 2 9 (se = 9 2 ) milligrams dietary calcium d a i l y , the apparent absorption was 3 2 . 1 (se = 1 . 9 ) percent ( 2 3 ) . An apparent calcium absorption of 2 9 . 5 percent (n = 1 3 0 ) may be calculated from data published by Heaney et a l . ( 1 5 ) . Apparent absorption values from 2 9 to 4 2 percent may be calcûTated from data published by Linkswiler ( 2 4 , 2 5 ) . However, much lower apparent absorption values of 6 to 1 5 percent may also be calculated from data published from the same laboratory ( 2 6 ) . Although there i s considerable v a r i a b i l i t y in the apparent absorption values determined from many s t u d i e s , a conservative value of 2 5 percent seems r e a l i s t i c for normal people consuming typical d i e t s . Calcium retention i s also affected by variations in urinary excretion. Dietary factors a f f e c t i n g calcium b i o a v a i l a b i l i t y have been recently reviewed ( 1 9 ) . Linkswiler and her students have shown that dietary protein i s a major f a c t o r contributing to urinary calcium excretion ( 2 4 , 2 5 , 2 7 , 2 8 ) . Renal acid excretion increases with protein intake" Lutz ( 2 9 ) has found that sodium bicarbonate ingestion w i l l a l k a l i n i z e the urine and reverse the renal excretion of calcium by people treated with a high protein d i e t . Renal acid secretion and c a l c u r i a occur during short-term starvation ( 3 0 ) . Ingestion of 5 grams of calcium lactate ( 6 5 0 mg Ca) corrects the acidosis of short-term starvation and improves the calcium balance; however, sodium bicarbonate alone markedly reduces the starvation acidosis but does not improve the calcium balance ( 3 0 ) as i t did above ( 2 9 ) for people treated with high protein d i e t . Thus correction of acidosis does not seem to be the primary factor in c o n t r o l l i n g urinary calcium excretion. Dietary phosphorus also affects calcium metabolism. Polyphosphate decreases calcium absorption in young men while orthophosphate supplement does not ( 2 6 J . However, in the rat a l l forms of phosphate decrease calcium absorption about equally ( 3 1 ) . However, widely divergent dietary calcium:phosphorus ratios do not seem to a f f e c t calcium u t i l i z a t i o n by people as long as there i s adequate phosphorus intake ( 3 2 ) . In general phosphorus stimulates calcium retention in man ( 3 2 J 7 Many other dietary factors have been reported to a f f e c t calcium b i o a v a i l a b i l i t y . Phytate, f i b e r , c e l l u l o s e , uronic a c i d s , sodium a l g i n a t e , oxalate, fat (only in the presence of steatorrhea), and alcohol have been reported to decrease calcium b i o a v a i l a b i l i t y ( 1 5 ) . Lactose and medium chain t r i g l y c e r i d e increase i t ( 1 5 ) . FTïïoride also affects calcium retention primarily by stimulating bone formation thereby decreasing calcium excretion ( 3 3 - 3 8 ) . The effects of f l u o r i d e on calcium u t i l i z a t i o n have been variable (34,38,39).

Strategies for determining calcium b i o a v a i l a b i l i t y The term b i o a v a i l a b i l i t y implies that f r a c t i o n of a n u t r i e n t , drug or toxicant that i s u t i l i z e d r e l a t i v e to the amount consumed. Calcium i s fed to the test subject in amounts below what the subject w i l l u t i l i z e . This ensures that a l l of the calcium provided can be absorbed and metabolized. Then, that f r a c t i o n

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

20

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

which i s u t i l i z e d r e l a t i v e to that given i s considered to be the amount of calcium in the source that i s metabolizable. There are two primary approaches to determining b i o availability: (a) Direct measurement of uptake into the body can be done using pharmacokinetic methods, quantitating the accumulation of radioactive n u t r i e n t s , or by quantitating the accumulation of a unique mineral or compound above an expected background l e v e l . The uptake into the body can be estimated i n d i r e c t l y by t r a d i t i o n a l metabolic balance methods (31,40). One can also use changes in blood concentrations of minerals, compounds or physiological markers in conjunction with body weight data to calculate an estimate of mineral or compound uptake (41-45). (b) The second approach i s to determine the uptake of a test mineral or compound r e l a t i v e to the uptake of a stable reference source of that mineral or compound (11,46). Calcium carbonate has frequently been used as a reference source in animal studies of calcium b i o a v a i l a b i l i t y . Nearly a l l of the calcium in the body i s located in bone. Bone i s very s e n s i t i v e to dietary factors such as the amount of calcium present in the d i e t and the a v a i l a b i l i t y of that calcium when a l l other nutrients are present in adequate amounts (46, 47). This i s e s p e c i a l l y true of the growing animal which i s u t i I i z e d in most b i o a v a i l a b i l i t y studies. Adult animals, however, may also be used. Krook et al (48) caused osteoporosis in adult dogs in 42 weeks by feeding a low-calcium high-phosphorus d i e t . The bones were r a d i o l o g i c a l l y normal a f t e r 28 weeks of calcium repletion (48). The ash contents of the vertebral bones of these dogs were much more responsive to dietary calcium and phosphorus manipulation than were the humeri and femora (48). The rat appears to be a good animal model that might be developed f o r predicting calcium b i o a v a i l a b i l i t y for human beings. Various dietary and physiological factors a f f e c t human and rat calcium absorption s i m i l a r l y (Table I ) . The greatest discrepancy among studies seems to be human and rat responses to changes in dietary phosphorus; increases in dietary phosphorus consistently decrease calcium absorption by the rat but does not consistently decrease i t in man. However, the calcium absorption response was s i m i l a r for rats.and humans for 8 of 9 dietary and physiological factors reviewed (Table I ) . This i s good evidence that the rat may be a p r a c t i c a l model for estimating human dietary calcium utilization. An attempt was made to c o l l a t e data on human and rat apparent calcium absorption values f o r several calcium sources. Absorption values were so variable within species and calcium sources that a c o r r e l a t i o n could not be j u s t i f i e d . Much of t h i s v a r i a b i l i t y may be due to methodological differences between the design of the rat and the human experiments. Most of the animal experiments were conducted using rapidly growing rats which were fed modest amounts of calcium but which have high calcium requirements. On the other hand, most of the human experiments were conducted using adult subjects consuming l i b e r a l amounts of calcium. Some degree of standardization of methodologies for rats and human experimentation must be done before a reasonable comparison can be made on the c o r r e l a t i o n between the calcium absorption responses of these two species.

2.

MAHONEY A N D HENDRICKS

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

Table I.

A Model for Assaying

Ca

Comparison of Various Dietary and Physiological Factors on Apparent Calcium Absorption by Rats and Humans

Factor

Human Beings

Ca absorption decreases with age

True True (over age 60) True (over age 30)

Gastric a c i d i t y necessary for absorption of poorly soluble Ca source Ortho phosphate

True True in B i l l r o t h II patient False (Total gastrectomy) No change S l i g h t decrease Decrease

Rat

True False

True

Decrease Polyphosphate

Decrease

Increased dietary protein

Increase

Intestine adapts True to Low CA intake by increasing absorption Lactose Increase No change Pregnancy

Increase

Lactation

Increase?

a

Ca

-47

21

Bioavailability

uptake in serum.

Decrease

Reference 59,60 61 16 62,63 64 65-67 68 69 9,10,31 26,70-72 28 27,73 31,74,75 26 31 25,27,76 & 77

Increased absorptive cap 78 No change in absorptive cap 79 No change 81 Increase 80,82 16,83 True 84,85,99 · 68 ,86 87 Increase 88 No change 89-91 92-93 Increase 94,95 Increase

94,96,97

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

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N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

Although present data cannot be used to determine the c o r r e l a t i o n between human and rat calcium absorption responses, Bressani (49) found a high c o r r e l a t i o n between nitrogen balance index in children and nitrogen growth index in r a t s . Using published data, a high correlation (r = 0.94) f o r percent iron u t i l i z a t i o n between rats and human beings was found (Mahoney & Hendricks, unpublished data). A l s o , iron absorption by rats and humans responded s i m i l a r l y to 18 of 19 dietary and physiological factors reviewed which are known to a f f e c t iron u t i l i z a t i o n (Mahoney & Hendricks, unpublished data). Again, phosphorus seemed to be the d i f f e r i n g factor for these two species. We believe that t h i s evidence along with that presented in Table I i s i n d i c a t i v e of the potential u t i l i t y of the rat for quantitative preduction human u t i l i z a t i o n of many nutrients including calcium. This w i l l require much concerted research to determine. Metabolic Balance Methods. T h e o r e t i c a l l y , the amount of mineral retained in the body should be determinable by balance methods. Heroux and Peter (50) attempted to do t h i s f o r calcium and magnesium in rats fed three d i e t s . For rats fed t h e i r stock d i e t , they predicted from balance data that the carcasses would contain 23.8 g calcium and 605 mg magnesium. By a n a l y s i s , the carcasses contained 4.45 g calcium and 152 mg magnesium. However, the relationship between calcium balance data (X) and carcass data (Y) were c l o s e l y related (Y = 1.05X - . 0 3 , r = 0.99 f o r group mean data) in the rat data of Whittemore et a l . (51). The metabolic balance technique has received much c r i t i c i s m (40,50,52-54). The intake and c o l l e c t i o n errors are usually not random, the intake usually being s l i g h t l y overestimated and the output being s l i g h t l y underestimated, seldom the reverse (53). As a r e s u l t a s l i g h t positive error in the balance of nutrients is usually encountered (50). Thus, alternate methods f o r determining the b i o a v a i l a b i l i t y of nutrients are sought. The most meaningful methods, however, w i l l be those that present the b i o a v a i l a b i l i t y of test sources in terms of the amount of the nutrient u t i l i z e d r e l a t i v e to that consumed. C e r t a i n l y , meticulously executed balance studies w i l l continue to be very valuable for evaluating nutrient u t i l i z a t i o n . Isotope Methods. The isotopes of calcium have r e l a t i v e l y short h a l f - l i v e s and are r e a d i l y counted using l i q u i d s c i n t i l l a t i o n or gamma counters as appropriate to the nuclide. Calcium isotopes may be quantitated in the excreta, blood, tissues or in the whole body. This has made them useful f o r many n u t r i t i o n a l metabolic studies. However, because of safety concerns, radioactive isotopes are cumbersome to work with and many researchers are unwilling to administer them to human beings. This has limited the use of isotopes to those studies in which alternate methods are not a v a i l a b l e or are imprecise. Methodologies f o r stable isotopes of calcium, which may be safely used in human being, are becoming available for use in metabolism s t u d i e s . These w i l l be p r a c t i c a l alternatives to radioactive isotopes in the future. Isotopic methods f o r estimating calcium absorption have been evaluated by several researchers (49,55-58). From the human data of Harrison et a l . (55), the relationship between percent calcium absorption determined by isotope d i l u t i o n (Y) and excreta counting

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

2.

MAHONEY A N D HENDRICKS

A Model for Assaying

Ca

Bioavailability

23

(X) may be expressed as: Y = 0.83 X + 2 . 4 , r = 0.77. Also from t h e i r data, the relationship between percent calcium absorption by isotope d i l u t i o n (Y) and absorption determined by whole body counting plus urine was: Y = 0.90 X + 3 . 9 , r = 0.96. From the human data of Agnew et a l . (56), the relationship between calcium retention determined by whole body counting (Y) or by excreta counting (X) was: Y = 0.87 X - 2 . 0 , r = 0.93. From human data (58), the relationship between percent calcium absorption determined by isotope d i l u t i o n (Y) and absorption determined by fecal counting (X) was e x c e l l e n t : Y = 0.99 X + 1.04, r = 0.99. S i m i l a r l y , the relationship between percent calcium absorption determined by isotope d i l u t i o n (Y) and calcium balance (X) was e x c e l l e n t : Y = 0.92 X - . 0 2 , r = 0.97 calculated from rat data (51). It is c l e a r from the above that calcium retention determined by balance data consistently are greater than when determined by carcass analysis or whole body counting; however, there i s a good c o r r e l a t i o n between the two methods. This i s e s p e c i a l l y true when the calcium i s quantitated by isotope counting in the excreta in the balance method. Relati ye Cal c i urn Bi oavai1abi1i t y . Determination of the b i o a v a i l a b i l i t y of calcium in low-calcium food sources i s d i f f i c u l t using metabolic balance techniques. It can be done, however, using changes in bone composition in growing animals fed test sources compared with animals fed a reference source such as calcium carbonate. A dose-response curve for the reference calcium source is created by feeding diets containing d i f f e r e n t amounts of calcium; and, the bone response i s determined. The bone response of the animals fed the test calcium source may then be compared with the response expected f o r an equal dose of calcium from the reference source. This i s done by determining the equation f o r the l i n e a r portion of the dose-response curve f o r the reference substance by the method of least squares. Using t h i s equation, one can then calculate the response anticipated for the reference substance at the actual dose of the test substance. Relative b i o a v a i l a b i l i t y (RB) may then be calculated as f o l l o w s : d o _ Actual Response of test d i e t Response estimated from CaC0

y 3

i A

n

n

, u u

If t h i s model i s s e l e c t e d , one must then decide what variables to use for the ordinate and the abscissa. The parameters must be d o s e - s e n s i t i v e , free of confounding v a r i a b l e s , e a s i l y determined and preferably l i n e a r . We have evaluated t h i s approach for estimating the b i o a v a i l a b i l i t y of calcium in mechanically deboned meat products (11 ). T y p i c a l l y , correlations between various bone parameters and dietary calcium are very high (r = 0.943 to 0.999). This i s consistent with what others have found for s i m i l a r parameters (46,47). These correlations are also s i m i l a r to the those (r = 0.947 to 0.982) between the amount of calcium consumed and calcium retained (11) a good index procedure. A very important advantage of the r e l a t i v e b i o a v a i l a b i l i t y assay i s that experimental parameters may be selected which are e a s i l y quantitated. Thus, Tso et a l . (11) determined the r e l a t i o n s h i p between dietary calcium concentration (X, g/kg) and

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

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NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

bone weight (Y, mg): Y = 8.88 X + 6 6 . 7 , r = 0.99. Relative b i o a v a i l a b i l i t i e s determined with t h i s relationship were s i m i l a r to those determined by calcium retained compared with the calcium consumed or by percent bone ash r e l a t i v e to dietary calcium concentration. They found that bone breaking strength was also a good parameter f o r determining r e l a t i v e b i o a v a i l a b i l i t y . Similar results were found on r e c a l c u l a t i n g the rat data reported by Wong and LaCroix (57). Using t h e i r data, r e l a t i v e calcium b i o a v a i l a b i l i t i e s determined by using bone weight (Y, mg) vs. dietary calcium (X, g/kg) were s i m i l a r in two experiments to those computed by s l o p e - r a t i o . There was a good c o r r e l a t i o n (r = 0.92) between r e l a t i v e b i o a v a i l a b i l i t i e s determined by bone weight (Y, mg) or bone calcium (Y, mg) vs. dietary calcium (X, g/kg). Thus, r e l a t i v e b i o a v a i l a b i l i t i e s are readily determined using e a s i l y quantitated parameters of calcium metabolism. Furthermore, r e l a t i v e b i o a v a i l a b i l i t i e s determined as described above may be used to evaluate calcium b i o a v a i l a b i l i t y in sources having low calcium concentrations. From the foregoing, i t i s clear that r e l a t i v e b i o a v a i l a b i l i t i e s f o r various sources may be determined using e a s i l y analyzed parameters of calcium metabolism. Determining the amount of calcium consumed (X, mg) and the dry weight of some e a s i l y dissected bone (Y, mg) seem l o g i c a l parameters for evaluating r e l a t i v e calcium b i o a v i l a b i l i t y . By doing t h i s on an individual animal b a s i s , one accounts for variations in food intake and weight gain which i s not done when calcium dose i s expressed as concentration in diet or bone. This i s p a r t i c u l a r l y important when evaluating calcium b i o a v a i l a b i l i t i e s of food sources which can cause marked variations in animal acceptance of test d i e t s . The data may then be analyzed with appropriate s t a t i s t i c a l models f o r determining r e l a t i v e b i o a v a i l a b i l i t i e s . Relative b i o a v a i l a b i l i t y determinations are l i m i t e d by what i s known about the quantitative absorption of the reference substance. One cannot confidently predict absorption of the test substances based on r e l a t i v e b i o a v a i l a b i l i t y data. Relative b i o a v a i l a b i l i t y data, however, can be used to rank the test sources and to provide a basis for comparison among experiments. Acknowledgments Paper 2975 of the Utah State University Experiment S t a t i o n .

Agricultural

Literature Cited 1. Lutwak, L. J . Am. Dietet. Assoc. 1964, 44, 173-175. 2. Mazess, R.B. In: Barzel, U.S., Ed., "Osteoporosis II", Grune & Stratton, Inc., New York, NY, 1979. 3. Garn, S.M. "The Earlier Gain and the Later Loss of Cortical Bone in Nutritional Perspective", Charles C. Thomas, Publisher, Springfield, IL 1970. 4. P a r f i t t , A.M. Medical Times 1981, November. 5. Mazess, R.B.; Peppler, W.W.; Chesney, R.W.; Lange, Τ . Α . ; Lindgren, U.; Smith, E., Jr. Calcif Tissue Int. 1984, 36, 8-13. 6. P a r f i t t , A.M. Lancet 1983, 2, 1181-1184. 7. A v i o l i , L.V. Fed. Proc. 1981, 40, 2418-2422. 8. Lindhal, O. Acta Orthop. Scand. 1976, 47, 11-19.

2.

MAHONEY A N D HENDRICKS

9. 10. 11. 12. 13. 14.

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15. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

A Model for Assaying

Ca

Bioavailability

25

Mahoney, A.W.; Hendricks, D.G. Nutr. Metabol. 1974, 16, 375-382. Mahoney, A.W.; Holbrook, R.S.; Hendricks, D.G. Nutr. Metabol. 1975, 18, 310-317. Tso, N . ; McLaughlin, K . ; Mahoney, A.W.; Hendricks, D.G. J . Nutr. 1984, 114, 946-952. Crenshaw, T.D.; Peo, E.R., Jr.; Moser, B.D. J. Animal S c i . 1981, 53, 827-835. Havvi, E.; Reshef, Α.; Schwartz, Α.; Guggenehim, K . ; Bernstein, D.S.; Hegsted, D.M.; Stare, F . J . Israel J . Med. Sci. 1971, 7, 1055-1062. Newton-John, H.F.; Morgan, D.B. Clin. Orthop. Rel. Res. 1970, 71, 229-252. Heaney, R.P.; Recker, R.R.; Saville, P.D. Am. J. Clin. Nutr. 1977, 30, 1603-1611. Heaney, R.P.; Gallagher, J . C . ; Johnston, C.C.; Neer, R.; Parfitt, A.M.; Chir, M.B.; Whedon, G.D. Am. J. Clin. Nutr. 1982, 36, 986-1013. Nationwide Food Consumption Survey 1977-78. Preliminary Report No. 2. "Food and Nutrient Intakes of Individuals in 1 day in the United States, Spring 1977", SEA/USDA, Washington, D.C., 1980. Scythes, C.A.; Gibson, R.S.; Draper, H.H. Nutr. Res. 1982, 2, 385-396. Allen, L.H. Am. J. C l i n . Nutr. 1982, 35, 783-808. Schutz, H.G.; Read, M . ; Bendel, R.; Bhalla, V . ; Harrill, I . ; Sheehan, E.T.; Standal, B.R. Am. J. C l i n . Nutr. 1982, 36, 897901. Kocian, J.; Skala, I.; Bakos, K. Digestion 1973, 9, 317-324. Wilz, D.R.; Gray, R.W.; Dominguez, J . H . ; Lemann, J., Jr. Am. J . Clin. Nutr. 1979, 32, 2052-2060. Recker, R.R.; Saville, P.D.; Heaney, R.P. Ann. Int. Med. 1977, 6, 649-655. Anand, C.R.; Linkswiler, H.M. J. Nutr. 1974, 104, 695-700. Walker, R.M.; Linkswiler, H.M. J . Nutr. 1972, 102, 1297-1302. Zemel, M.B.; Linkswiler, H.M. J . Nutr. 1981, 111, 315-324. Hegsted, M . ; Schuette, S.A.; Zemel, M.B.; Linkswiler, H.M. J . Nutr. 1981, 111, 553-562. Schuette, S.A.; Linkswiler, H.M. J. Nutr. 1982, 112, 338-349. Lutz, J . Am. J. Clin. Nutr. 1984, 39, 281-288. Kocian, J.; Brodan, V. Nutr. Metab. 1979, 23, 391-398. Mahoney, A.W.; Hendricks, D.G. J. Food S c i . 1978, 43, 14731476. Spencer, H . ; Kramer, L.; Osis, D. Am. J. Clin. 1982, 36, 776-787. Jowsey, J.; Riggs, B . L . ; Kelly, P . J . ; Hoffman, D.L. Am. J. Med. 1972, 53, 43-49. Cohn, S.H.; Donbrowski, C.S.; Hauser, W.; Atkins, H.L. Am. J . Clin. Nutr. 1971, 24, 20-28. Spencer, H . ; Lewin, I . ; Osis, D.; Samachson, J. Am. J. Med. 1970, 49, 814-822. Chang, Y . O . ; Pan, M . ; Varnell, T. Nutr. Rpts. I n t l . 1977, 16, 539-547. Ericsson, Y. Calc. Tiss. Res. 1972, 9, 39-53.

26

38. 39. 40. 41. 42. 43. 44. 45. Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67.

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

Rich, C.; Ensinck, J.; Ivanovich, P. J . Clin. Invest. 1964, 43, 545-556. Riggs, B.L.; Hodgson, S.F.; Hoffman, D.L.; Kelly, P.J.; Johnson, K.A.; Taves, D. JAMA 1980, 243, 446-449. Lentner, C.; Lauffenburger, T.; Guncago, J.; Dambachner, M.; Haas, H.G. Metabolism 1975, 24, 461-471. Mahoney, A.W.; Van Orden, C.C.; Hendricks, D.G. Nutr. Metab. 1974, 17, 223-230. Reizenstein, P. Br. J. Haematol. 1975, 31, 265-268. Saarinen, U.M.; Siimes, M.A. Pediatric Res. 1979, 13, 143-147. Adolph, W.H.; Wang, C.H.; Smith, A.H. J . Nutr. 1938, 16, 291297. Pennell, M.D.; Davies, M.I.; Rasper, J.; Motzok, I. J . Nutr. 1976, 106, 265-274. Ranhotra, G.S.; Gelroth, J.Α.; Torrence, F.A.; Bock, M.A.; Winterringer, G.L. J. Nutr. 1981, 111, 2081-2086. Forbes, R.M.; Weingartner, K.E.; Parker, H.M.; Bell, R.R.; Erdman, J.W., Jr. J . Nutr. 1979, 109, 1652-1650. Krook, L . ; Lutwak, L . ; Henrikson, P.; Kallfelz, F.; Hirsch, C.; Romanus, B.; BeLanger, F.; Marier, J.R.; Sheffy, B.E. J. Nutr. 1971, 101, 233-246. Bressani, R. In: Bodwell, C.E., ed., "Evaluation of Proteins for Humans", AVI, Inc., Westport, CN 1977, pp. 81-118. Heroux, O.; Peter, D. J . Nutr. 1975, 105, 1157-1167. Whittemore, C.T.; Thompson, Α.; Atherton, D. Br. J. Nutr. 1973, 30, 425-436. Hegsted, D.M. J. Nutr. 1976, 106, 307-311. Forbes, G.B. Nutr. Rev. 1973, 31, 297-300. Ferretti, J . L . ; Bazan, J . L . ; Puche, R.C. Medicina (Buenos Aires) 1976, 36, 83-92. Harrison, J.E.; McNeill, K.G.; Wilson, D.R.; Oreopoulos, D.G.; Krondl, Α.; Finlay, J.M. Clin. Biochem. 1973, 6, 237-235. Agnew, J.E.; Kehayoglou, A.K.; Holsworth, C.D. Gut 1969, 10, 590-597. Wong, N.P.; LaCroix, D.E. Nutr. Rpts. Intl. 1980, 21, 673-680. DeGrazia, J.Α.; Ivanovich, P.; Fellows, H.; Rich, C. J. Lab. Clin. Med. 1965, 66, 822-829. Ireland, P.; Fordtran, J.S. J . Clin. Invest. 1973, 52, 26722681. Alevizaki, C.C.; Ikkos, D.G.; Singhelakis, P. J . Nuclear Med. 1973, 14, 760-762. Bullamore, J.R.; Gallagher, J.C.; Wilkinson, R.; Nordin, B.E.C.; Marshall, D.H. Lancet 1970, 2, 535-537. Armbrecht, H.J.; Zenser, T.V.; Bruns, M.Ε.H.; Davis, B.B. Am. J . Physiol. 1979, 236, E769-E774. Armbrecht, H.J.; Gross, C . J . ; Zenser, T.V. Arch. Biochem. Biophys. 1981, 210, 179-185. Hironaka, R.; Draer, H.H.; Kastelic, J . J . Nutr. 1960, 71, 356-360. Hunt, J.N.; Johnson, C. Digest. Dis. & Sci. 1983, 28, 417-421. Ivanovich, P.; Fellows, H.; Rich, C. Ann. Intern. Med. 1967, 66, 917-923. Nicolaysen, R.; Ragard, R. Scand. J. Clin. Lab. Invest. 1955, 7, 298-299.

2.

MAHONEY A N D HENDRICKS

68. 69. 70.

71. 72. 73.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch002

74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99.

A Model for Assaying

Ca

Bioavailability

27

Kocian, J.; Brodan, V. Digestion 1975, 12, 193-200. Bradley, E . L . , III,; Isaacs, J.; Hersh, T.; Dvidson, E.D.; Millikan, W. Ann. Surgery 1975, 182, 415-429. Kim, Y. "Effect of Level of Protein, Calcium and Phosphorus Intake on Calcium, Phosphorus and Magnesium Metabolism in the Young Adult Male", Ph.D. Dissertation, University of Wisconsin, Madison, 1977. Spencer, H . ; Kramer, L.; Osis, D.; Norris, C. J. Nutr. 1978, 108, 447-457. Malm, O.J. Scand. J . Clin. Lab. Invest. 1953, 5, 75-84. Leichsenring, J . M . ; Norris, L.M.; Lamison, S.A.; Wilson, E.D.; Patton, M.B. J. Nutr. 1951, 45, 407-418. Tanaka, Y . ; Frank, H . ; DeLuca, H. Science 1973, 181, 564-566. Shah, B.G.; Meranger, J.C. Can. J. Physiol. Pharm. 1970, 48, 675-680. Schwartz, R.; Woodcock, N.A.; Blakely, J . D . ; MacKellar, I. Am. J. C l i n . Nutr. 1973, 26, 519-523. McCance, R.A.; Widdowson, E.M.; Lehmann, H. Biochem. J. 1942, 36, 686-691. Moyer, G . L . ; Wilson, H.D.; Schedl, H.P. Am. J. Digest. Dis. 1978, 23, 545-549. Shenolikar, I.S. Nutr. Metabol. 1974, 16, 10-14. Bottom, J.S. "The Effect of High Protein, Low Pyridoxine Diet on Calcium Retention in Rats", M.S. Thesis, Utah State University, Logan, Utah, 1978. Howe, J . C . ; Beecher, G.R. Nutr. Rpt. I n t l . 1981, 24, 919-929. Engstrom, G.W.; DeLuca, H.F. J. Nutr. 1963, 81, 218-222. Irving, J.T. "Calcium and Phosphorus Metabolism", Academic Press, NY, 1973, p. 40. Kimberg, D.V.; Schachter, D.; Schenker, H. Am. J. Physiol. 1961, 200, 1256-1262. Petith, M.M.; Schedl, H.P. Am. J. Physiol. 1976, 231, 865-871. Kabayashi, Α.; Kawai, S.; Ohbe, Y . ; Nagashima, Y. Am. J. Clin. Nutr. 1975, 28, 681-683. M i l l s , R.; Breiter, H . ; Kempster, E . ; McKey, B . ; Pickens, M . ; Outhouse, J . J . Nutr. 1940, 20, 467-476. Forbes, R.M. J. Nutr. 1964, 83, 225-233. Ali, R.; Evans, J . L . J. Nutr. 1967, 93, 273-279. Evans, J.L.; Ali, R. J. Nutr. 1967, 92, 417-424. Urban, E . ; Pena, M. Digestion 1977, 15, 18-27. Shenolikar, I.S. Am. J. Clin. Nutr. 1970, 23, 63-67. Pitkin, R.M. Am. J. Obstet. Gynecol. 1975, 121, 724-737. Halloran, B . P . ; DeLuca, H.F. Am. J. Physiol. 1980, 239, E64E68. Graves, K . L . ; Wolinsky, I. J . Nutr. 1980, 110, 2420-2432. Boass, Α.; Toverud, S.U.; Pike, J.W.; Haussler, M.R. Endocrinology 1981, 109, 900-907. Kostial, K.; Gruden, N . ; Durakovic, A. Calc. Tiss. Res. 1969, 4, 13-19. Hansard, S . L . ; Crowder, H.M. J. Nutr. 1957, 62, 325-339. Krawitt, E . L . ; Stubbert, P.Α.; Ennis, P.H. Am. J. Physiol. 1973, 224, 548-551.

RECEIVED January 19, 1985

3 Phosphates and Calcium Utilization in Humans MICHAEL B. ZEMEL

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202

High phosphate diets cause decreased Ca absorption, secondary hyperparathyroidism, accelerated bone resorption and soft tissue calcification in some animals, but not in normal humans. Although phos­ phates may decrease Ca absorption in man at very high (> 2000 mg/day) Ca intakes, they do not do so at more moderate Ca levels and enhance Ca absorption at very low levels (< 500 mg/day). Phosphates increase renal tubular reabsorption and net retention of Ca. At low Ca intakes, phosphates stimulate parathyroid hormone (PTH) secretion without causing net bone resorption. Increasing both dietary Ca and Ρ causes a decrease in PTH-mediated bone resorption; polyphosphates and phos­ phorus in food cause greater reductions than does in­ organic orthophosphate, as these sources are slowly released in digestion. The effects of phosphates on the absorption and u t i l i z a t i o n of calcium have been the topic of continuing controversy. Phosphates have been widely believed to reduce calcium absorption as a result of the formation of insoluble calcium phosphate s a l t s i n the gut. How­ ever, data from several human studies do not support t h i s conclusion. Although phosphate supplementation may cause decreases i n calcium absorption i n man at very high l e v e l s of calcium intake, i . e . 2000 mg per day or more, i t does not appear to do so when calcium intake i s 1500 mg per day or less (1,2). Furthermore, Fox and Care (3) found inorganic phosphate to be without effect on calcium absorption from Thiry-Vella jejunal loops i n pigs. In addition, phosphates do not consistently cause p r e c i p i t a t i o n of calcium from foods during i n v i t r o digestions (4). Table I shows the e f f e c t s of 1% ortho-, t r i poly-,or hexametaphosphate added to ground beef or soy protein concentrate on calcium s o l u b i l i t y following simulated g a s t r i c and complete g a s t r o i n t e s t i n a l digestions. Gastric digestions were accomplished by incubating samples i n a pepsin-HCl mixture for two hours at 37°C, and "complete" g a s t r o i n t e s t i n a l digestions were accomplished by r a i s i n g the pH of the samples to 7.0 with sodium

0097-6156/85/0275-Ό029$06.00/0 © 1985 American Chemical Society

30

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

Table I . E f f e c t s o f 1% Orthophosphate, T r i p o l y p h o s p h a t e , and Hexametaphosphate on C a l c i u m S o l u b i l i t y from Ground Beef or Soy P r o t e i n C o n c e n t r a t e S u b j e c t e d t o I n V i t r o G a s t r i c and G a s t r o i n t e s t i n a l D i g e s t i o n s Soluble Calcium (% of Control) Calcium Source Gastric Digestion Control +o rthophosphat e +tr ipolypho sphat e +h ex ame t aph ο spha t e

Soy

Concentration 100.of 73.4* 73.2^ 72.9 b

Ground Beef 100.0,a 41. οί; 43.6^ 38.5 b

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Gastrointestinal Digestion Control +orthophosphate +tripolyphosphate +hexametaphosphate

100.0ia 85.0 97.4 127.8° D

a

loo. o:a 145.5 427.7 400.0

D

C

C

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t (p < 0.05) d i f f e r e n c e s f o r each t r e a t m e n t .

bicarbonate and incubating for two additional hours with pancreatic and b i l e extracts. Although a l l three phosphates tested caused substantial decreases i n calcium s o l u b i l i t y following the g a s t r i c digestion, t h i s trend was reversed upon completion of the second phase of the digestion. Only orthophosphate caused a decrease i n calcium s o l u b i l i t y from the soy following the complete digestion, while a l l three phosphates enhanced calcium s o l u b i l i t y from the meat. Thus, phosphates are u n l i k e l y to i n t e r f e r e with calcium absorption due to the formation of insoluble s a l t s i n the gut. In contrast,recent human studies indicate that increasing dietary phosphorus, i n the form of orthophosphate, may cause an increase i n the apparent absorption of calcium (5,6). Table I I shows the e f f e c t s of 1 g supplements of phosphorus on calcium absorption during two experiments with 18 young adult males maintained at low (< 400 mg/day) l e v e l s of calcium intake; i n both experiments, the phosphorus supplement caused a s i g n i f i c a n t reduction i n f e c a l c a l ­ cium. However,in a third study,which employed a s l i g h t l y higher l e v e l (500 mg/day) of calcium intake, orthophosphate supplementation was without effect on calcium absorption (7) Thus, available evidence indicates that orthophosphates may enhance calcium absorption i n man at very low l e v e l s of calcium intake (< 400 mg/day), reduce i t at very high l e v e l s of intake (> 2000 mg/day), but exert no effect on calcium absorption throughout a broad range of moderate calcium intakes (500-1500 mg/day). Consequently, i f phosphates are to affect calcium u t i l i z a t i o n , they must do so post-absorptively.

3.

ZEMEL

Phosphates

Table I I . Calcium Intake

and

Ca Utilization

in Humans

31

E f f e c t s of Orthophosphate on C a l c i u m A b s o r p t i o n Phosphorus Intake

Fecal Calcium

Apparent Absorption Of Calcium

mg/day Study 1 399 399

a

a

835 1835

a

a

401 374

b

- 2 +25

b

Study 2

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

356 356

a

a

866 1866

a

b

329 284

a

+27 +72

b

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t d i f f e r e n c e s (p < 0 . 0 5 ) f o r each study.

Phosphates and Bone Mineralization High phosphate diets low i n calcium have been shown to cause bone démineraiization and soft tissue c a l c i f i c a t i o n i n several lower animal species (8-13), and Draper and co-workers (14-16) found similar e f f e c t s i n rats and mice fed diets containing adequate or high l e v e l s of calcium. Increasing the l e v e l of phosphorus intake from 0.6 to 1.2% resulted i n an accelerated loss of ^ C a f deeply labelled bone and a corresponding decrease i n the calcium and phosphorus content of the skeleton; these e f f e c t s were eliminated by parathyroidectomy. The authors concluded that excess dietary phosphorus stimulates parathyroid hormone (PTH) mediated bone resorption. However, i n tissue culture, inorganic phosphate i n h i b i t s PTH-induced bone resorption (17) and instead stimulates increased bone collagen synthesis (18) and mineralization (19). In addition, Anderson et a l . (20) reported that monkeys fed high phosphate diets low i n calcium (0.3% Ca) or adequate i n calcium (0.95% Ca) developed no s i g n i f i c a n t c l i n i c a l , radiographic, or h i s t o l o g i c a l evidence of bone disease during a 7-year observation period. In attempting to reconcile these findings, i t should be pointed out that rats may not be appropriate models for the study of calcium metabolism i n humans. U n l i k e humans, the r a t does not undergo e p i p h y s e a l p l a t e c l o s u r e and does not have a s i g n i f i c a n t h a v e r s i a n remodeli n g sequence (21). Furthermore, r a t s e x c r e t e o n l y 1 - 2 % of t h e i r c a l cium i n t a k e i n t h e i r u r i n e whereas humans e x c r e t e a p p r o x i m a t e l y 2 0 307o or more. T h i s f a c t i s e s p e c i a l l y s i g n i f i c a n t , s i n c e most of the known e f f e c t s of phosphates on calcium retention i n humans are effected by a l t e r a t i o n s i n urinary calcium. Increasing the l e v e l of phosphorus intake has long been known to exert a hypocalciuretic effect (22,23). B e l l et a l . (24) suggested that t h i s effect may be secondary to an increase i n PTH secretion, but phosphorus supplementation has been reported to decrease urinary calcium even i n parathyroidectomized rats (14). Goldsmith and coworkers (25) found phosphate supplements to cause an increase i n PTH r

o

m

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

32

secretion i n women with osteoporosis; however, i l e a c crest biopsies from the women showed increases i n both bone formating and bone sorbing surfaces. I t was suggested that the phosphate i n i t i a l l y caused retention of calcium by the skeleton and a subsequent reduc­ t i o n i n serum calcium l e v e l s ; t h i s reduction i n serum calcium was presumed to be responsible for the increase i n PTH secretion. I t was further suggested that providing a calcium supplement along with the phosphorus may prevent the phosphate-induced decrease i n serum c a l ­ cium and the resultant Increase i n PTH-mediated bone resorption; thus net bone mineral accretion would be permitted.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Orthophosphates We have conducted two human metabolic studies (5,6) to compare the e f f e c t s of increasing phosphorus intake on calcium u t i l i z a t i o n In healthy young adults maintained at low (ca. 400 mg/day) and high (ca. 1200 mg/day) l e v e l s of calcium intake. Increasing dietary phos­ phorus, as orthophosphate, caused a s l i g h t reduction i n f e c a l calcium and a substantial reduction i n urinary calcium losses (Table I I I ) .

Table I I I . A b s o r p t i o n , E x c r e t i o n , and R e t e n t i o n o f C a l c i u m as A f f e c t e d by C a l c i u m and Orthophosphates

Diet

Intake

Apparent Absorption

Urinary Excretion

Calcium Balance

mg/day Low Ca Low Ρ

399

a

Low Ca High P(ortho)

399

a

High Ca High P(ortho)

1194

-

2

a

, + 25° +177

c

196

a

-198

a

115

b

- 90

b

+

C

176°

1

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t d i f f e r e n c e s (P < 0.05).

Consequently, calcium balance improved, although negative calcium balance was s t i l l evident i n a l l subjects. Simultaneously i n ­ creasing calcium and phosphorus intake, however, caused an increase in the apparent absorption of calcium and a decrease i n urinary c a l ­ cium; these changes were s u f f i c i e n t to a t t a i n calcium balance. An i n d i c a t i o n of the mechanism of the hypocalciuretic effect of phos­ phorus i s presented i n Table IV. The phosphate supplement caused an increase i n f r a c t i o n a l renal tubular reabsorption of calcium without affecting glomerular f i l t r a t i o n rate; even the combined supplements of calcium and phosphate caused an increase i n calcium reabsorption, a l b e i t a smaller one than that oberseved with the low calcium-high phosphorus d i e t . Diet was without s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on plasma u l t r a f i l t r a b l e calcium l e v e l s . However, the phosphate supplement did cause a decrease i n mean u l t r a f i l t r a b l e calcium l e v e l s

3.

ZEMEL

Phosphates

and

Ca Utilization

in

33

Humans

at the low l e v e l of calcium intake, while no such decrease was ob­ served when calcium and phosphate supplements were given simultane­ ously. Furthermore, the phosphate supplement caused an increase i n urinary c y c l i c AMP excretion, indicating an increase i n PTH secretion, while simultaneously supplementing the diet with calcium and phos­ phate caused reductions i n the urinary excretion of both c y c l i c AMP and hydroxyproline, indicating a decrease i n PTH-mediated bone resorption. Thus, i t appears that a high calcium-high phosphorus diet may provide a mechanism to increase net skeletal calcium reten­ t i o n without stimulating PTH release.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Table IV.

Fractional Renal Ca Reabsorption (%)

Diet Low

Plasma U l t r a Urinary Urinary f i l t r a b l e Ca Cyclic AMP Hydroxyproline (mg/100 ml) (ymoles/day) (mg/day)

Ca

Low Ρ Low

Renal H a n d l i n g of Calcium as A f f e c t e d by Calcium and Orthophosphates

97.9

a

89.7

b

98.I

e

5.53

a

Ca

High P(ortho) High Ca High P(ortho)

2.14

3.22 5.13

27.12

a

b

24.50

a

16.76

b

a

1.64° 5.52

a

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t (p < 0.05) d i f f e r e n c e s . It i s of interest to note that changes i n calcium balance were p a r a l l e l e d by changes i n phosphorus balance (Table V ) . Despite gen­ erous phosphorus intakes ( i n excess of the RDA) and approximately 60-70% absorption, subjects were i n negative phosphorus balance when calcium intake was low. Phosphorus balance became p o s i t i v e only when calcium balance was positive, and there was a s i g n i f i c a n t p o s i t i v e correlation between the two. These data, along with the aforemention­ ed changes i n c y c l i c AMP and h y d r o x y p r o l i n e e x c r e t i o n (Table IV) serve to r e i n f o r c e the concept t h a t d i e t a r y c a l c i u m and phosphorus exert mutually b e n e f i c i a l e f f e c t s , as the two elements are retained toget­ her i n the skeleton. This concept i s also supported by survey data. In a cross-sectional study of bone status and fracture rates i n two regions of Yugoslavia, Matkovic et a l . (26) reported that metacarpal c o r t i c a l width, c o r t i c a l area and c o r t i c a l density were a l l lower and the fracture rate was higher i n the region with the lower c a l ­ cium intake than i n the region with the higher calcium intake. The differences i n density tended to diminish past age 55 i n the women, but was s t i l l evident even at age 75. Although the authors a t t r i ­ buted these e f f e c t s to differences i n calcium intake between the two regions, inspection of t h e i r data indicates a two-fold increase i n phosphorus intake as w e l l .

34

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

Table V . A b s o r p t i o n , E x c r e t i o n , and R e t e n t i o n of Phosphorus as A f f e c t e d by C a l c i u m and Orthophosphates

Diet

Intake

Apparent Absorption

Urinary Excretion

Retention

jng/day__

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Low Ca Low Ρ

835°

Low Ca High P(ortho)

1835

High Ca High P(ortho)

1835

u

486° 1307 1239

u

679*

-193°

1416"

-109°

1197°

+ 42^

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t (p < 0.05) d i f f e r e n c e s .

Polyphosphates Condensed (poly) phosphates may exert d i f f e r e n t e f f e c t s on calcium u t i l i z a t i o n than the aforementioned e f f e c t s of simple (ortho-) phos­ phates. Polyphosphates have a much greater a f f i n i t y f o r calcium than do orthophosphates, and soluble calcium-polyphosphate complexes are r e a d i l y formed i n the g a s t r i c and i n t e s t i n a l environments. In addi­ tion, polyphosphates must be hydrolyzed by an i n t e s t i n a l alkaline phosphatase (27) p r i o r to absorption. We have found polyphosphates to be incompletely (80.5%) hydrolyzed to orthophosphate during the digestive process i n young adult males when calcium intake was low; only 56% of a 1 g phosphorus supplement was absorbed from a polyphos­ phate sources as compared to 71% from an orthophosphate source (5). It i s possible that, by v i r t u e of their incomplete hydrolysis i n the g a s t r o i n t e s t i n a l t r a c t , polyphosphates may represent a "slow-re­ lease" form of phosphorus that does not e l i c i t as great a PTH r e ­ sponse as orthophosphate. A 75-day repeated measures human meta­ b o l i c study was recently conducted to compare the e f f e c t s of 1 g phosphorus supplements, given either an orthophosphate or as hexa metaphosphate, on calcium and phosphorus u t i l i z a t i o n i n ten young adult males consuming a low (356 mg/day) or a h i g h (1166 mg/day) l e v ­ e l o f c a l c i u m ( 6 ) . Polyphosphate h y d r o l y s i s ( T a b l e V I ) was found t o be reduced when the l e v e l of calcium intake increased. Hexametaphosphate and a l l of i t s detectable hydrolysis products, except f o r pyrophosphate,were s t a b i l i z e d against further hydrolysis to orthophosphate. Consequently, the f r a c t i o n of f e c a l phosphorus i n the form of polyphosphate increased from 62.5% on the low calcium, high polyphosphate diet to 74.3% on the high calcium, high polyphosphate d i e t . As a r e s u l t of t h i s increase i n residual f e c a l polyphosphate, the hexametaphosphate caused a greater increase i n f e c a l calcium l o s s e s when c a l c i u m i n t a k e was h i g h than when i t was low (Table V I I ) . In c o n t r a s t t o orthophosphate, hexametaphosphate caused a decrease i n c a l c i u m a b s o r p t i o n , as i n d i c a t e d by an i n c r e a s e i n f e c a l c a l c i u m , a t both l e v e l s o f c a l c i u m i n t a k e . However, the polyphosphate supplement

3.

ZEMEL

Phosphates

Table V I .

and Ca Utilization

in

35

Humans

D i s t r i b u t i o n o f F e c a l Phosphates as A f f e c t e d by L e v e l of C a l c i u m I n t a k e Diet

Phosphate Species

Low Calcium High Polyphosphate

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Expressed

*

37.6 26.5 9.1 2.5 3.9 20.5 62.5

ortho pyro tripoly trimeta tetra hexameta t o t a l poly Note:

High Calcium High Polyphosphate

25.7 ·6* 13.2 4.0* 6.3* 23.2* 74.3* 2 7

as a percentage o f t o t a l f e c a l phosphorus,

vindicates a s i g n i f i c a n t

difference

(p< 0.01) i n each row.

Table V I I . Calcium E x c r e t i o n and R e t e n t i o n as A f f e c t e d by Orthophosphate, Hexametaphosphate ( P o l y p h o s p a t e ) , and C a l c i u m

Diet

Calcium Intake

Low CaLow Ρ

356

Low CaHigh Ρ(ortho)

356

Low CaHigh Ρ(poly)

356

Fecal Excretion

a

329

a

284

a

338

b

832

b

959

High CaHigh Ρ (ortho)

1166

High CaHigh Ρ(poly)

1166

Urinary Excretion

a

13 7

b

ioi

c

116

d

158

e

169

a

b

Balance

-110

a

- 29

b

C

- 98

a

d

+176

e

+ 38

c

d

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t (p < 0.05) d i f f e r e n c e s .

caused a 54 mg/day i n c r e a s e i n f e c a l c a l c i u m when compared t o o r t h o phosphate a t t h e lower l e v e l o f c a l c i u m i n t a k e , w h i l e a t the h i g h e r l e v e l of c a l c i u m i n t a k e a much l a r g e r i n c r e a s e o f 127 mg/day was observed. A l l s u b j e c t s were i n n e g a t i v e c a l c i u m balance when consuming the b a s a l low c a l c i u m , low phosphorus d i e t (Table V I I ) , the mean c a l c i u m l o s s b e i n g 110 mg/day. The orthophosphate supplement s i g n i f i c a n t l y reduced t h i s l o s s t o 29 mg/day, due t o decreases i n both u r i n a r y and f e c a l c a l c i u m l o s s e s . The polyphosphate supplement, however, caused

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

36

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

an increase i n f e c a l calcium and was not as hypocalciuretic as the orthophosphate; as a consequence i t did not improve calcium balance. Simultaneously increasing the levels of calcium and orthophosphate intake resulted i n net retention of calcium f o r a l l subjects. The improvement i n calcium balance compared to that seen when orthophos­ phate was given without a calcium supplement was due to a substantial increase i n the apparent absorption of calcium which was accompanied by only a s l i g h t increase i n urinary calcium losses. The high c a l ­ cium, high polyphosphate diet caused s i g n i f i c a n t l y greater urinary and f e c a l losses of calcium than did the high calcium, high orthophosphate diet; nonetheless, calcium balance was attained on both high calcium d i e t s . As with the f i r s t study (5), calcium and phosphorus balances varied together, and there was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between the two. Phosphorus balance became p o s i t i v e only when calcium balance was p o s i t i v e , during the two high calcium treatment periods ( T a b l e V I I I ) . The c a l c i u m supplement caused a s i g n i f i c a n t i n c r e a s e i n f e c a l phosphorus losses, but t h i s was more than compensated for by the substantial decrease i n urinary phosphorus caused by the calcium supplements. Calcium has been shown to affect the renal handling of phosphorus through both PTH-dependent and PTH independent mechanisms (28-30). Calcium causes a direct increase i n renal tubular reabsorp­ tion of phosphorus (29) and i n h i b i t s the PTH-dependent renal adenyl­ ate cyclase (30). However, the decreases i n urinary c y c l i c AMP (Table 9) which resulted from calcium supplementation i n the present study suggest a PTH-dependent mechanism. I t i s noteworthy t h a t a l t h o u g h phosphorus a b s o r p t i o n was lower on the h i g h c a l c i u m , h i g h polyphosphate d i e t than on the h i g h c a l c i u m , h i g h orthophosphate d i e t , t h e r e was no s i g n i f i c a n t d i f f e r e n c e between

Table V I I I . Phosphorus Excretion and Retention as Affected by Orthophosphat e, Hexametaphosphate (Polyphosphate), and Calcium

Diet

Phosphorus Intake

Fecal Excretion

Urinary Excretion

Balance

mg/day Low CaLow Ρ

-221

866

a

376

a

Low CaHigh Ρ(ortho)

1866

b

437

a

1450

Low CaHigh Ρ(poly)

1866

b

634

b

1343°

High CaHigh Ρ(ortho)

1866

b

522

c

1289

High CaHigh Ρ(poly)

1866

b

799

d

1037°

711

a

b

d

-

a

21

-lll

b

a

+

55

C

+

30

C

Note: Nonmatching s u p e r s c r i p t s i n each column denote s i g n i f i c a n t (p< 0.05) differences.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

3.

ZEMEL

Phosphates

and Ca Utilization

in

37

Humans

the e f f e c t s of the two diets on phosphorus retention; t h i s was due to the substantial decrease i n urinary phosphorus on the high calcium, high polyphosphate diet from that observed on the high calcium-high orthophosphate d i e t . Thus, phosphorus absorbed from the calcium-polyphosphate supplement was u t i l i z e d more e f f i c i e n t l y than that absorbed from the calcium-orthophosphate supplement. S i m i l a r l y , Schuette and Linkswiler (31) reported lower u t i l i z a t i o n of phosphorus when an orthophosphate supplement was the source than when foods were the phosphorus source; the lower u t i l i z a t i o n was due to an increase i n urinary phosphorus excretion. The authors noted that, since phos­ phorus i s absorbed as inorganic phosphate, a phosphorus supplement ( i . e . orthophosphate) w i l l be rapidly absorbed and cause an immediate r i s e i n c i r c u l a t i n g phosphorus l e v e l s and subsequent s p i l l a g e into urine. I t therefore appears that those phosphorus sources which are slowly absorbed, such as polyphosphates, may be u t i l i z e d more e f f i ­ c i e n t l y . This concept i s supported by observed changes i n urinary c y c l i c AMP i n response to varying dietary orthophosphate, polyphos­ phate and c a l c i u m (Table I X ) . C y c l i c AMP e x c r e t i o n i n c r e a s e d when t h e phosphate supplements were given without calcium and the greater i n ­ crease was associated with the orthophosphate supplement. In con­ t r a s t , simultaneously increasing the l e v e l s of calcium and phosphorus intakes caused a decrease i n urinary c y c l i c AMP, and the largest de­ crease resulted from the combined calcium-polyphosphate supplement. Furthermore, the calcium-polyphosphate supplement also caused a de­ crease i n bone resorption, as indicated by a s i g n i f i c a n t decrease i n hydroxyproline excretion. Thus these data indicate that, i n the presence of adequate calcium, polyphosphates act as a "slow-release"

T a b l e IX. U r i n a r y C y c l i c AMP and H y d r o x y p r o l i n e as A f f e c t e d by Orthophosphate, Hexametaphosphate, and C a l c i u m 1

Diet

Urinary C y c l i c AMP (ymoles/day)

Low Ca Low Ρ

4.32

Low CaHigh Ρ(ortho)

4.71

Low CaHigh Ρ(poly)

a

b

Urinary Hydroxyproline^ (mg/day)

27 . i o

a

25 .82

a

4.53

a

28 .14

a

High CaHigh Ρ(ortho)

4.22

a

31 .03

a

High CaHigh Ρ(poly)

4.10

c

24 .80°

b

No s i g n i f i c a n t d i f f e r e n c e s were observed a t t t = 0.05; nonmatching s u p e r s c r i p t s i n t h i s column denote d i f f e r e n c e s a t Oi = 0.06. 2 Nonmatching s u p e r s c r i p t s i n t h i s column denote s i g n i f i c a n t (p < 0.05) d i f f e r e n c e s .

38

NUTRITIONAL BIOAVAILABILITY OF CALCIUM

source of phosphorus which allows for net deposition of bone mineral without depressing c i r c u l a t i n g calcium l e v e l s and, consequently, without stimulating PTH secretion.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

Conclusions Data presented herein indicate that although orthophosphates cause a s l i g h t increase i n calcium absorption when calcium intake i s low and polyphosphates cause a s l i g h t decrease, phosphates exert l i t t l e s i g n i f i c a n t effect on calcium absorption throughout a broad range (500 - 1500 mg/day) of normal calcium intakes. Phosphates do exert a s i g n i f i c a n t hypocalciuretic effect and thereby cause an improvement i n calcium retention. However, at very low l e v e l s of calcium intake phosphates may reduce c i r c u l a t i n g calcium l e v e l s and , as a consequence, e l i c i t an increase i n parathyroid hormone-mediated bone resorption which p a r t i a l l y o f f s e t s the improvement i n calcium retention. On the other hand, high l e v e l s of calcium and phosphorus intakes are associated with substantial retention of both minerals and a decrease i n parathyroid hormone-mediated bone resorption. These r e s u l t s also indicate that the e f f e c t s of dietary phosphates on c a l cium and phosphorus u t i l i z a t i o n i n man i s i n part dependent upon the phosphate source. Those phosphates which are slowly digested and absorbed, such as food-bound phosphates or a polyphosphate, appear to be more e f f i c i e n t l y u t i l i z e d then inorganic orthophosphate.

Literature Cited 1. Spencer, H.; Kramer, L; Osis, D.; Norris, C. J. Nutr. 1978, 108, 447-457. 2. Kim, Y.; Linkswiler, H.M. Fed. Proc. 1980, 39, 895. 3. Fox, J.; Care, A.D. Br. J. Nutr. 1978, 39, 431-439. 4. Zemel, M.B. Unpublished Data. 5. Zemel, M.B.; Linkswiler, H.M. J. Nutr. 1981, 111, 315-324. 6. Zemel, M.B.; Soullier, B.A.; Steinhardt, N.J. Fed. Proc. 1983, 42, 397. 7. Hegsted, M.; Schuette, S.A.; Zemel, M.B.; Linkswiler, H.M. J. Nutr. 1981, 111, 553-562, 8. Krook, L.; Barret, R.B.; Usui, K.; Wolke, R.E. Cornell Vet. 1963, 224-240. 9. Rowland, R.N.; Capen, C.C.; Nagode, L.A. Path. Vet. 1968, 5, 504-519. 10. Hammond, R.H.; Storey, E. Calc. Tiss. Res. 1970, 4, 291-304. 11. Morris, M.L. Jr.; Teeter, S.M.; Collins, D.R. J. Am. Vet. Med. Assoc. 1971, 477-488. 12. Laflamme, G.H.; Jowsey, J. J. Clin. Invest. 1972, 51, 2834-2840. 13. Jowsey, J.; Reiss, E.; Canterbury, J.M. Acta Orthop. Scand. 1974, 45, 801-808. 14. Anderson, G.H.; Draper, H.H. J. Nutr. 1972, 102, 1123-1132. 15. Draper, H.H.; Sie, T.L.; Bergen, J.G. J. Nutr. 1972, 102, 11331142. 16. Krishnarao, G.V.G.; Draper, H.H. J. Nutr. 1972, 102, 1143-1146. 17. Raisz, L.G.; Neimann, I. Endocrinology 1969, 85, 446-452. 18. Raisz, L.G. Fed. Proc. 1970, 29, 1176-1178. 19. Flanagan, B.; Nichols, G. Jr. J. Clin. Invest. 1969, 48, 607612.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch003

3.

ZEMEL

Phosphates

and Ca Utilization

in

Humans

39

20. Anderson, M.P.; Hunt, R.D.; Griffiths, H.J.; McIntyre, K.W., Zimmerman, R.E. J. Nutr. 1977, 107, 834-839. 21. Parfitt, A.M. Metabolism 1976, 25, 809-844. 22. Malm, O.J. Scand. J. Lab. Invest. 1952, 5, 75-84. 23. Farquharson, R.F.; Salter, W.T.; Tibbets, D.M.; Aub. J. C. J. Clin. Invest.1931, 10, 221-249. 24. Bell, R.R.; Draper, H.H.; Tzeng, D.Y.M. J. Nutr. 1977, 107, 42-50. 25. Goldsmith, R.S.; Jowsey, J.; Dube, W.J.; Riggs, B.L.; Arnaud, C.D.; Kelly, P.J. J. Clin. Endocrinol. Metab. 1976, 43, 523532. 26. Matkovic, V.; Kostial, K.; Simonovic, I.; Buzina, R.; Broderec, Α., Nordin, B.E.C. Am. J. Clin. Nutr. 1979, 32, 540-549. 27. Ivey, F.J.; Shaver, K. J. Agric. Food Chem. 1977, 25, 125-130. 28. Howard, J.E.; Hopkins; T.R.; Connor, T.B. J. Clin. Endocrinol. 1953, 13, 1-19. 29. Lavender, A.R.; Pullman, T.N. Am. J. Physiol. 1963, 205, 10251032. 30. Amiel, C.; Kuntziger, H.; Covette, S.; Coureau, C.; Bergounioux, N. J. Clin. Invest. 1976, 57, 256-263. 31. Schuette, S.A.; Linkswiler, H.M. J. Nutr. 1982, 112, 338-349. RECEIVED

October

15, 1984

4 Calcium and Phosphate Needs of Preterm Infants Requiring Prolonged Intravenous Feeding P. J. KNIGHT1

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

Departments of Surgery and Pediatrics, University of Kansas School of Medicine, Wichita, KS 67214

Premature infants who develop a gastrointestinal disease that precludes adequate oral nutrition may develop fractures and rickets when their growth is sustained by prolonged intravenous feeding. These fractures and rickets are caused by a deficiency of bone mineral substrate (calcium and/or phosphate). The goals of this chapter are: to review the previously established data regarding the amounts of calcium and phosphate normally accreted by fetuses in utero and by infants following birth, to look at the incidence of fractures and rickets with the administration of varying amounts of calcium and phosphate, to examine the physicochemical limitations of calcium and phosphate solubility in a single intravenous solution, to determine the appropriate ratio of calcium to phosphate in these solutions to maximize retention of calcium and phosphate, and to look at the clinical application of these findings. Normal Calcium and Phosphate Accretion P r i o r analyses (1-2) of the mineral content of ashed fetuses, s t i l l borns and infant cadavers showed that the rate o f calcium and phosphate accretion i s highest at the end of pregnancy and that approximately h a l f of the calcium and phosphate i n the i n f a n t ' s body at term b i r t h i s accumulated i n the l a s t eight weeks of pregnancy Figure 1. Consequently infants who are born prematurely s t a r t l i f e with much lower stores of calcium and phosphate than i f they had remained i n the uterus f o r the f u l l 40 weeks of gestation. The enteral absorption of calcium and phosphate after b i r t h depends on a number of f a c t o r s , including the amounts ingested, the types of foods eaten and the level o f vitamin D. Under optimal circumstances, the amounts of calcium and phosphate incorporated into the body per kilogram per day following b i r t h are only about onet h i r d of those accumulated i n utero. Since the growth of the baby's body mass i s faster than the absorption of calcium and phosphate, the infant must normally borrow from and r e d i s t r i b u t e his e x i s t i n g bone 1Current address: 818 N. Emporia, Suite 200, Wichita, KS 67214

0097-6156/85/0275-0041$06.00/0 © 1985 American Chemical Society

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

42

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

0

4

8

12

16

20

24

28

32

36

40

Weeks Gestation Figure 1. Total amounts and rate of accretion of calcium and phosphorus in the body of the human fetus with gestation. (Reproduced with permission from Réf. 1. Copyright 1965 W. B. Saunders Company.) mineral stores following b i r t h . Stated another way, the r a t i o of bone mineral measured as calcium r e l a t i v e to bone protein matrix measured as nitrogen must f a l l (3)-Figure 2. Since the structural strength of bone depends i n part on i t s mineral content, eventually the mineral to osteoid r a t i o w i l l f a l l to the point where bones break with minor trauma. Bones a c t i v e l y growing in the presence of a deficiency of calcium or phosphate develop excessive accumulations of noncalcified c a r t i l a g e at t h e i r growth centers (epiphyseal p l a t e s ) , which are recognized on both physical examination and x-ray f i l m as the deformity c a l l e d r i c k e t s . Since preterm infants s t a r t extrauterine l i f e with low bone mineral s t o r e s , these babies are p a r t i c u l a r l y susceptible to developing fractures and r i c k e t s as they grow. Incidence of Fractures and Rickets To define the incidence of fractures and r i c k e t s that we were encountering in infants who required prolonged parenteral feeding, we reviewed the roentgenograms of a series of preterm infants who developed necrotizing e n t e r o c o l i t i s and who required at least four weeks of t o t a l parenteral n u t r i t i o n a l support (4). These data are recorded i n Table I.

4.

KNIGHT

Ca and Phosphate

Needs of Preterm

43

Infants

4-5r

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

2-5k

Ο 14 2 0

28

36

J

2




j 11-12 Years

Figure 2. Calcium-nitrogen r a t i o of the nonepiphyseal parts of the human femur during development. (Reproduced with permission from Réf. 1. Copyright 1965 W. B. Saunders Company.)

Table I.

Incidence of Demineralization, Fractures, and Rickets at Low and Moderate Calcium Intake Low Calcium Intake 1974-78 Ca = 2.5 mM P04 = 9.7 mM

Roentgenograph!cally normal bones Demineralization only Pathologic fractures (both r i c k e t s and f r a c tures) Total patients Percent with fractures Percent with r i c k e t s

1 5 6

Moderate Calcium Intake 1979-80 Ca = 10 mM P04 = 5 mM 0 4 1 (0)

(4) 12 50% 33%

5 20% 0%

Infants treated before 1979 received protein hydrolysates cont a i n i n g high concentrations of phosphate which limited the concent r a t i o n of calcium that could be used without causing p r e c i p i t a t i o n . Beginning i n 1979 c r y s t a l l i n e amino acid solutions which contained less obligatory phosphate became a v a i l a b l e ; these allowed greater l a t i t u d e i n the concentrations of calcium and phosphate that could be achieved. The data i n Table I suggest that the severity of demineralization and the incidence of fractures and r i c k e t s w i l l decrease i f more calcium i s added to the parenteral alimentation solution.

44

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

S o l u b i l i t y Limitations for Ca and Ρ In 1978, on the basis of a few measurements of urine calcium and phosphate excretion as well as an awareness of the previously mentioned work regarding the amounts of calcium and phosphate normally accreted in utero and p o s t n a t a l l y , i t became apparent that the demineralization, fractures and r i c k e t s we were seeing i n our infants were caused by calcium d e f i c i e n c y . Consequently we i n ­ creased the amount of calcium added to the parenteral alimentation s o l u t i o n s . If more than 12.5 mM of the calcium were added to a l i t e r of hyperalimentation s o l u t i o n , gross p r e c i p i t a t i o n would occur i n the feeding s o l u t i o n . If 10 mM of calcium were added per l i t e r , c r y s t a l l i n e precipitated began to build up on the inside of our ban urn-impregnated s i l i c o n e rubber central venous catheters. This c r y s t a l l i n e p r e c i p i t a t e resulted i n gradual occlusion and functional loss of these l i n e s . After several f a l s e s t a r t s and s i x l o s t catheters, chemical and c r y s t a l analysis showed that the p r e c i p i t a t e inside these catheters was CaHPOy,. consultation Knowledge of some with the Handbook of Physics and Chemistry {5) resolved the problem of the p r e c i p i t a t e s . Barium s u l f a t e i s incorporated in the s i l i c o n e rubber catheters to make them radioopaque; the Handbook shows that BaHPO* i s only s l i g h t l y soluble. The barium ions i n the catheter were apparently a t t r a c t i n g the HPO* ions from the hyperalimentation s o l u t i o n . The concentration of phosphate at the solution-catheter interface became high enough that CaHPO* began to p r e c i p i t a t e out. Propogation of the CaHPO* c r y s t a l l a t t i c e from the hyperalimentation s o l u t i o n then continued u n t i l the catheter became plugged and use­ l e s s . At s l i g h t l y higher concentrations of calcium, CaHPO^ would d i r e c t l y p r e c i p i t a t e out i n the bags of feeding s o l u t i o n . The s o l u b i l i t y of calcium and phosphate i s pH dependent as shown in Figure 3 . The pH of these feeding solutions ranges from 5.2 to 6.5 depending on the amounts of acetate, amino acids and the other buffers in the system. Because of the l i m i t a t i o n i n the amount of calcium that could be added to these high phosphate protein hydrolysates available in 1978, we began to alternate one s o l u t i o n high i n calcium with one s o l u t i o n high in phosphate every 12 hours (6). Because of the immense surface area of the bone c r y s t a l l a t t i c e , i t was possible by alternating solutions to get good retention of both calcium and phosphate as determined by the measured excretion of these ions i n the urine. When the c r y s t a l l i n e amino acid alimentation solutions low in obligatory phosphate became available in 1979, we thought we should determine how much calcium and phosphate could be simul­ taneously added to a single s o l u t i o n . By s e r i a l l y a l t e r i n g the con­ centrations of calcium and of phosphate in racks of glass t e s t tubes containing hyperalimentation s o l u t i o n , the maximal amounts that would remain i n solution were determined. These data are shown graphically i n Figure 4. The best f i t curve i s a hyperbola, x-y = k; the chemical explanation for t h i s curve i s the s o l u b i l i t y product for calcium dibasic phosphate, [Ca ][HP04=] = K ++

$p

Optimal Ca to Ρ Ratio Since the amounts of calcium and phosphate that can be added

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

4. K N I G H T

Ca and Phosphate

Needs of Preterm

45

Infants

Figure 3 . Interaction among pH, calcium, and phosphate i n hyperalimentation s o l u t i o n s . The pH, calcium values to the right of phosphate isoconcentration curves are associated with p r e c i p i t a t i o n of dibasic calcium phosphate, while pH, calcium values to the l e f t of phosphate isoconcentration curves are associated with s o l u b i l i t y of calcium and phosphates i n hyperalimentation s o l u t i o n . (Reproduced with permission from Ref. 6 Copyright 1980 American Medical Association.) V

CaHP0 PRECIPITATION CURVE IN 10% DEXTROSE, 1% TRAVASOL AT T*7 C, pH=6 4

e

I

5

10

15

20

25

30

mM Ca Figure 4. P r e c i p i t a t i o n became v i s i b l e at the points marked by the dots. The shape of the curve (hyperbola) i s described by the equation CaxP = mM^. Solutions with CaxP products above the curve show p r e c i p i t a t i o n , while solutions with CaxP products below the curve remain free of p r e c i p i t a t e . (Reproduced with permission from Ref. 4. Copyright 1983 American Society f o r Parenteral and Enteral N u t r i t i o n . )

N U T R I T I O N A L BIOAVAILABILITY O F C A L C I U M

46

simultaneously to any parenteral alimentation s o l u t i o n are l i m i t e d , we wanted to determine the r a t i o of calcium to phosphate that would maximize retention of these elements by the body. We had a ballpark estimate for t h i s optimal Ca/P r a t i o from several pieces of data. In a d u l t s , bone i s the repository for 98% of the body's calcium and 85% of the body's phosphate; i n i n f a n t s , the skeleton contains about 96% of the body's calcium and 70% of i t s phosphate. The Ca/P r a t i o of bone mineral (hydroxyapatite or 3[Ca2(P0 )]-Ca(0H) can be expressed i n various units y i e l d i n g d i f f e r e n t values: 4

Moles

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

Ca

!» •

Equivalents 1.67

20 18

1.11

2

Weight 10 χ 40 6 χ 31

2.15

The remaining non-osseous body calcium i s l a r g e l y dissolved i n body f l u i d s ; the ionized portion of t h i s soluble calcium mediates muscle contraction including l i f e - s u s t a i n i n g cardiac muscle and respiratory muscle contraction. Acute serum calcium d e f i c i e n c i e s r a r e l y develop because the body's homeostatic mechanisms can mobilize calcium from bone to maintain the serum calcium level u n t i l none i s l e f t i n bone. The remaining non-osseous phosphate i s : s t r u c t u r a l l y incorporated i n the phospholipids of c e l l membranes and i n the nucleic a c i d s , i s the p r i n c i p l e i n t r a c e l l u l a r buffer of the body, and i s the primary mediator of i n t r a c e l l u l a r energy t r a n s f e r . Acute phosphate d e f i ­ ciencies do occur; they can present as cardiac (7) or respiratory (8) or peripheral muscle f a i l u r e (9) or as infections due to the i n ­ a b i l i t y of polymorphonuclear leukocytes to phagocytize (10). Since the effects of phosphate deficiency can occur acutely ancTare l i f e threatening as opposed to the fractures and r i c k e t s of chronic body calcium d e p l e t i o n , one should e r r on the side of giving more phosphate than calcium. Any excess phosphate i s excreted i n the urine. Our simple approach to determining the optimal Ca/P r a t i o for intravenous feeding solutions was to simply a l t e r the r a t i o of calcium to phosphate i n these solutions and measure the only external loss of calcium and phosphate which was i n the urine. We i n i t i a l l y assumed that the difference between the intake and urinary loss of calcium and phosphate would measure the retention of these elements. The results of seven balance studies at varying Ca/P r a t i o s are shown in Figure 5. The observed calcium/phosphate r a t i o of 4.5 at the intercept of the calcium and phosphate retention curves that should minimize the sum of the urine calcium plus urine phosphate losses was d i f f i c u l t to believe i n view of both the known Ca/P r a t i o of bone and the amounts we were adding to these s o l u t i o n s . This d i s p a r i t y between the optimal r a t i o determined experimentally and what we had assumed t h i s r a t i o should be on the basis of known body composition i s p a r t i a l l y reconciled by the experiment of Sutton and Barltrop. They fed preterm infants stable C a ^ and observed that up to 20% of the isotope absorbed was subsequently excreted i n the s t o o l . Our infants also were undoubtedly having unmeasured calcium losses from the b i l e , pancreatic j u i c e and succus entericus secreted into t h e i r i n t e s t i n e 4

4.

KNIGHT

Ca and Phosphate

Needs of Preterm

Infants

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

100 Λ A

Figure 5. The areas above the measured calcium and measured phosphate retention curves represent the percent of the i n t r a ­ venously administered calcium and phosphate that was l o s t i n the urine. The combined percent losses (Ca + P) are minimized at the intercept of the curves. The dotted l i n e represents an assumed endogenous fecal loss of 20% of the infused calcium added to the measured urinary calcium losses. The Ca/P r a t i o that minimizes the percent calcium and phosphate losses i s then approximately 3 . 0 . (Reproduced with permission from Ref. 4. Copyright 1983 American Society f o r Parenteral and Enteral N u t r i t i o n . )

47

48

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

and t h i s calcium was not reabsorbed. If one assumes that 20% of absorbed calcium i s subsequently excreted into the bowel, the calcium retention curve would move downward, r e s u l t i n g in an i n ­ tercept with the phosphate retention curve at a Ca/P r a t i o of about 3.0-Figure 5. Although t h i s Ca/P r a t i o s t i l l seems a b i t high, Ca/P ratios between 2.0 and 3.0 can be maintained for prolonged periods without calcium or phosphate depletion occurring and with the urine excretion both of these elements being low but measurable.

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

C l i n i c a l Application Using the i n v i t r o determined maximal Ca χ Ρ product (mM ) of 75 for barium-impregnated s i l i c o n e rubber catheters and of 100 for s t a i n l e s s steel needles and polyethylene catheters and the Ca/P r a t i o deter­ mined by the interception of the retention curves at a value of 2.0 to 3 . 0 , one has two equations with two unknowns. Solving these equations simultaneously gives the average optimal concentrations for calcium and phosphate in the alimentation s o l u t i o n s . These are concentrations that w i l l both stay i n solution and w i l l maximize retention for the average p a t i e n t . Although these average concen­ t r a t i o n s are useful s t a r t i n g p o i n t s , one would l i k e to be able to fine-tune these concentrations to each individual p a t i e n t ' s metab­ olism. This metabolism i s determined by the p a t i e n t ' s parathormone and c a l c i t o n i n l e v e l s , the active vitamin D l e v e l , the body's a c i d base status and other poorly understood f a c t o r s . These factors acting on the renal tubular c e l l s determine the percent of the calcium and phosphate f i l t e r e d by the glomeruli that i s reabsorbed. In the absence of renal tubular defects, measuring the concentrations of calcium and phosphate excreted in any individual i n f a n t ' s urine indicated in which d i r e c t i o n the Ca/P r a t i o should be changed to achieve t h i s f i n e - t u n i n g . When the Ca/P r a t i o i s appropriate, both calcium and phosphate should be measurable; absence of measurable calcium or phosphate i n the urine means the r a t i o must be changed. Our data from premature infants indicate that i f both the urine calcium and the urine phosphate are between 2 and 10 mg/dl, the r a t i o of these elements i n the alimentation s o l u t i o n i s appropriate. A Lesson from the Body The product of the Ca χ Ρ concentrations maintained in human serum has a t e n - f o l d safety factor over the amounts that cause precip­ i t a t i o n . Some of t h i s safety factor i s because only s l i g h t l y more than half of the serum calcium i s a c t i v e l y available (ionized calcium), while the remainder i s e i t h e r bound to protein or chelated by c i r t a t e i o n . I f c i t r a t e buffer i s added to the alimentation solutions to a concentration of 5 mM, the Ca χ Ρ product can excède 1800 without p r e c i p i t a t i o n of CaHPO. occurring. This effect of c i t r a t e i s not s o l e l y due to chelation since 5 mM of c i t r a t e can only bind 7.5 mM of calcium. The c i t r a t e ions appear to prevent CaHPOc r y s t a l s from propogating and coming out of s o l u t i o n as shown by Patterson (_12). Since recognizing the Ca χ Ρ products and the Ca/P r a t i o which are appropriate for preterm infants on prolonged i n t r a ­ venous alimentation, we have had no further cases of fractures or r i c k e t s in our preterm infants on prolonged intravenous n u t r i t i o n .

4.

KNIGHT

Ca and Phosphate Needs of Preterm

Infants

Literature Cited

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch004

1.

Widdowson, E. M.; McNance, R. A. Pediatr. Clin. of N. Amer. 1965, 12, 595-614. 2. Shaw, J. C. L. Pediatr. Clin. of N. Amer. 1973, 20, 333-58. 3. Dickerson, J. W. T. Biochem. J. 1962, 82, 56-61. 4. Knight, P. J . ; Heer, D.; Abdenour, G. J. Ε. P. N. 1983, 7, 110-14. 5. Weast, R. "Handbook of Physics and Chemistry"; Cleveland, C. R. C. Press, 1974; p. Β 71 and Β 78. 6. Knight, P. J . ; Buchanan, S.; Clatworthy, H. W. J. A. M. A. 1980, 243, 1244-46. 7. O'Connor, L. R.; Wheeler, W. S.; Bethune, J. E. New Engl. J. Med. 1977, 297, 901-03. 8. Newman, J. H.; Neff, Τ. Α . ; Ziporin, P. New Engl. J. Med. 1977, 296, 1101-03. 9. Weintraub, M. I. J. A. M. A. 1976, 235, 1040-41. 10. Craddock, P. R.; Yawata, Y.; VanSaten, L.; et.al. New Engl. J. Med. 1974, 290, 1403-07. 11. Sutton, Α . ; Barltrop, D. Nature 1973, 242, 265. 12. Patterson, D. Nature 1954, 173, 75-76. RECEIVED December 26, 1984

5 Dietary Phytate and Calcium Bioavailability ERNST GRAF1 and J O H N W. EATON2 1

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch005

2

The Pillsbury Company, Minneapolis, M N 55414 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, M N 55455

Phytic acid (myo-inositol hexaphosphoric acid) comprises 1 to 5% by weight of legumes, cereals, oil seeds and nuts and t h u s is p r e s e n t in numerous f o o d commodities. This compound has been indicted as a major cause of several mineral deficiency syndromes, through the formation of insoluble metallophytate complexes. Our recent results demonstrate, however, that polyvalent cations form soluble complexes with phytate at high phytate to metal ratios and that phytate does not impair the absorption of45Ca2+administered to mice by gavage. Thus, many of the supposedly adverse effects of dietary phytate and the resulting mineral deficiencies may merely reflect g e n e r a l d i e t a r y inadequacy. Furthermore, some d i e t a r y phytate may actually be beneficial duetoitsabilityto suppress iron-mediated oxidative processes. Indeed, phytic acid may reduce the incidence of colonic cancer and function as a natural food preservative. C a l c i u m i s t h e most abundant m i n e r a l i n t h e human body. About 99% i s d e p o s i t e d as phosphate m a t r i c e s i n bones and teeth. I n addition to t h i s s t r u c t u r a l r o l e , C a i s of c e n t r a l i m p o r t a n c e t o most a s p e c t s o f c e l l p h y s i o l o g y , b o t h a s a c o f a c t o r f o r e x t r a c e l l u l a r enzymes and a s an i n t r a c e l l u l a r regulator. I n t r a c e l l u l a r C a has profound e f f e c t s on a v a r i e t y o f c e l l u l a r and enzyme f u n c t i o n s , i n c l u d i n g e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n a l l forms o f muscle, e x c i t a t i o n - s e c r e t i o n c o u p l i n g a t nerve endings and i n both e x o c r i n e and e n d o c r i n e g l a n d s , e x o c y t o s i s , membrane t r a n s p o r t , c e l l morphology, gene e x p r e s s i o n , and vision. R e c o g n i z i n g t h e importance o f adequate C a intake, 2 +

2 +

2 +

0097-6156/85/0275-0051$06.00/0 © 1985 American Chemical Society

NUTRITIONAL BIOAVAILABILITY O F C A L C I U M

52

the United S t a t e s n a t i o n a l Research C o u n c i l recommends an i n t a k e of 800 mg C a (recommended d a i l y allowance or RDA) and 1200 mg d u r i n g pregnancy and l a c t a t i o n . I n o r d e r t o meet t h e s e g u i d e l i n e s , c e r t a i n f o o d s t a p l e s i n t h e U.S. are f o r t i f i e d w i t h Ca . Despite t h i s widespread e f f o r t , Ca d e f i c i e n c y i s s t i l l v e r y common as e v i d e n c e d by t h e high i n c i d e n c e of o s t e o p o r o s i s . This h e a l t h problem p a r t i a l l y a r i s e s from the f a c t that maintenance of C a homeostasis r e q u i r e s not only adequate d i e t a r y i n t a k e , but i n t e s t i n a l a b s o r p t i o n and subsequent u t i l i z a t i o n o f Ca . B i o a v a i l a b i l i t y of C a i s a f f e c t e d by numerous p h y s i o l o g i c a l c o n d i t i o n s , i n c l u d i n g age, s e x , g e n e t i c make-up, s t r e s s , h o r m o n a l s t a t u s , h e a l t h s t a t u s , and n u t r i t i o n a l habits. In a d d i t i o n to these i n t r i n s i c f a c t o r s , c e r t a i n d i e t a r y components, such as f i b e r and o x a l a t e , form i n s o l u b l e complexes w i t h C a and i n t e r f e r e w i t h i t s absorption. Another p u t a t i v e c u l p r i t i n t h i s category i s p h y t i c a c i d . P h y t i c a c i d (Figure 1) c o n s t i t u t e s 1 t o 5% by weight of most n u t r i t i o n a l l y important p l a n t seeds (Table I ) and 2 +

2 +

2 +

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch005

2 +

2 +

Table I . P h y t i c A c i d Content of S e l e c t e d Seeds

Millet Barley Sorghum Corn Rye Dehydrated peas Oat Wheat Soybeans Sunflower seeds Peanuts Wild r i c e Lima beans Sesame seeds

Phytic Acid (% w/w) 0.7 0.7 0.9 0.9 0.9 0.9 1.0 1.1 1.4 1.9 1.9 2.2 2.5 5.3

Reference 2 2 2 2 2 3 2 2 4 5 3 5 5 3

t y p i c a l l y accounts f o r 60 t o 90$ of the t o t a l phosphorus. I t u s u a l l y o c c u r s i n d i s c r e t e r e g i o n s o f the s e e d s , such as t h e a l e u r o n e l a y e r o f wheat and r i c e ( 6 ) , where i t i s b e l i e v e d t o s e r v e as an a n t i o x i d a n t and p r o t e c t a g a i n s t o x i d a t i v e damage d u r i n g storage (7). Due t o the a b i l i t y o f p h y t i c a c i d t o p r e c i p i t a t e p o l y c a t i o n i c n u t r i l i t e s (8,9), i t s p r e s e n c e i n f o o d has c o n c e r n e d n u t r i t i o n i s t s f o r s e v e r a l decades. S e v e r a l r e c e n t r e v i e w s e x t e n s i v e l y d i s c u s s t h e c h e m i s t r y and n u t r i t i o n a l r a m i f i c a t i o n s o f d i e t a r y phytate (4,10-15). The e f f e c t o f p h y t i c a c i d on C a bioavailability i s s t i l l i n d i s p u t e . Some e a r l y n u t r i t i o n i s t s reported r a c h i t o g e n i c p r o p e r t i e s o f d i e t a r y p h y t a t e based on f e e d i n g s t u d i e s u s i n g puppies (16-18) and e p i d e m i o l o g i c a l s t u d i e s on B r i t i s h - b o r n c h i l d r e n o f A s i a n i m m i g r a n t s 2 +

5.

G R A F A N D EATON

2+

Dietary

Phytate and Ca

53

Bioavailability

(19,20). These c o n c l u s i o n s were s e r i o u s l y questioned (212ÏÏ7 and recent r e s u l t s i n d i c a t e t h a t t h e b i o a v a i l a b i l i t y of C a i s t h e same f r o m a c a s e i n d i e t as f r o m a h i g h p h y t a t e s o y c o n c e n t r a t e (25). O b e r l e a s c o n t e n d s t h a t , i n the p r e s e n c e o f adequate amounts o f C a and v i t a m i n D, d i e t a r y p h y t a t e i s n o t r a c h i t o g e n i c , even though i t may b i n d s u b s t a n t i a l amounts o f Ca' " ' (15). T h i s c o n t r o v e r s y , the r e l a t i v e p a u c i t y o f a v a i l a b l e i n f o r m a t i o n , and t h e growing incidence of C a d e f i c i e n c y prompted us t o i n v e s t i g a t e f u r t h e r the chemical i n t e r a c t i o n s between C a and phytate and t o a s s e s s i t s e f f e c t on t h e b i o a v a i l a b i l i t y of C a a d m i n i s t e r e d t o mice by gavage. 2 +

2 +

2

1

2 +

2 +

2 +

Publication Date: April 2, 1985 | doi: 10.1021/bk-1985-0275.ch005

Methods P h y t i c a c i d s o l u t i o n s were p r e p a r e d by t i t r a t i n g sodium phytate ( S i g m a C h e m i c a l Company) w i t h H C l ; t h e c o n c e n t r a t i o n was d e t e r m i n e d by a n a l y z i n g f o r i n o r g a n i c p h o s p h a t e a f t e r wet a s h i n g w i t h H 2 S O J . - H N O 3 (3:2) f o r 45 m i n u t e s . The c o n c e n t r a t i o n o f CaCl2 s t o c k s o l u t i o n s was measured by atomic a b s o r p t i o n spectrometry. Ca s o l u b i l i t y was determined by i n c u b a t i n g C a and p h y t a t e i n 100 mM HEPES ρ Η 7 . 1 a t 25°C f o r 2 h o u r s , c e n t r i f u g i n g and m e a s u r i n g ^->Ca i t h e s u p e r n a t a n t by l i q u i d s c i n t i l l a t i o n aounting (26). The b i n d i n g o f C a t o p h y t i c a c i d was a s c e r t a i n e d by measuring C a p o t e n t i o m e t r i c a l l y w i t h a Radiometer C a s e l e c t i v e electrode (2£). For the q u a n t i t a t i o n o f jln v i v o a b s o r p t i o n o f ^->Ca , 200 μ ΐ o f r a d i o a c t i v e s o l u t i o n s were a d m i n i s t e r e d by g a s t r i c gavage t o male mice kept on a d e i o n i z e d water d i e t d u r i n g t h e p r e v i o u s 18 h o u r s . A f t e r 4 h o u r s b l o o d was o b t a i n e d by a x i l l a r y i n c i s i o n and ^ C a extracted w i t h TCA and determined by l i q u i d s c i n t i l l a t i o n counting (26). I r o n - m e d i a t e d g e n e r a t i o n o f h y d r o x y l r a d i c a l (*0H) was monitored by t h e hypoxanthine-xanthine oxidase method as p r e v i o u s l y d e s c r i b e d (28). F o r m a l d e h y d e produced by reaction o f ·0Η w i t h DMS0 was determined s p e c t r o p h o t o m e t r i c a l l y by the Hantzsch r e a c t i o n (29). L i p i d p e r o x i d a t i o n was measured by d e t e r m i n i n g m a l o n d i a l d e h y d e (MDA) s p e c t r o p h o t o m e t r i c a l l y by t h e t h i o b a r b i t u r i c a c i d method (30). 2 +

2 +

ς

2+

n

2 +

2 +

2 +

ac

2+

2 +

w

a

s

R e s u l t s and D i s c u s s i o n T r a d i t i o n a l l y , p o l y v a l e n t c a t i o n - p h y t a t e complexes have been c o n s i d e r e d u n i f o r m l y i n s o l u b l e , and t h i s dogma has been t h e b a s i s o f most methods f o r t h e d e t e r m i n a t i o n o f phytate (31). I n 1976, however, s o l u b l e monoferric phytate was i s o l a t e d (32), and most r e c e n t l y , Ca