Nutritional Bioavailability of Calcium

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ACS SYMPOSIUM SERIES 275

Nutritional Bioavailability of Calcium

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

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

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Library of Congress Cataloging in Publication Data 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. Q V 276 N976 1984]

QP535.C2N88 1985 599'.0133 85-3931 ISBN 0-8412-0907-3

American Chemical Society

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PRINTED IN THE UNITED STATES OF AMERICA

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ACS Symposium Series

M . Joan Comstock, Series Editor

Advisory Board

Robert Baker

U.S. Geological Survey

Martin L. Gorbaty

Exxon Research and Engineering Co.

Roland F. Hirsch

U.S. Department of Energy

Herbert D. Kaesz

University of California—Los Angeles

Rudolph J. Marcus

Office of Naval Research

Vincent D. McGinniss

Battelle Columbus Laboratories

Donald E. Moreland

USDA, Agricultural Research Service

W. H. Norton

J. T. Baker Chemical Company

Robert Ory

USDA, Southern Regional Research Center

Geoffrey D. Parfitt

Carnegie-Mellon University

James C. Randall

Phillips Petroleum Company

Charles N. Satterfield

Massachusetts Institute of Technology

W. D. Shults

Oak Ridge National Laboratory

Charles S. Tuesday

General Motors Research Laboratory

Douglas B. Walters

National Institute of Environmental Health

C. Grant Willson

IBM Research Department

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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 pub- lished papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

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PREFACE 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

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

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1 Dietary Calcium Exchangeability and Bioavailability

Evaluation and Potential Uses of an In Vitro Digestion Procedure E. M. WIEN and RUTH SCHWARTZ

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 di- gestion permits study of the chemistry of food calcium under standardized digestion conditions. Investi- gations 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 d i f f e r e n c e 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 r e l a t e d to d i e t a r y 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 i n f l u e n c e the absorption and u t i l i z a t i o n of d i e t a r y calcium under experimental conditions. What now i s l a c k i n g i s a d e t a i l e d knowledge of how these f a c t o r s i n t e r a c t with calcium under normal conditions of i n g e s t i o n i n meals.

We have developed an i n v i t r o d i g e s t i o n procedure, not as a s u b s t i t u t e f o r i n vivo studies, but as a u s e f u l adjunct. Our i n i t i a l o b j e c t i v e was to develop an i n v i t r o procedure f o r measuring exchange- a 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 t r a c e r 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 o b j e c t i v e s 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 p o t e n t i a l

0097-6156/ 85/ 0275-0001 $06.00/0

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

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2 N U T R I T I O N A L B I O A V A I L A B I L I T Y O F C A L C I U M

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 i n t e r a c t i o n s of g a s t r o i n t e s t i n a l secretions with d i e t a r y mineral and other d i e t a r y components which determine mineral s o l u b i  l i t y . A number of i n v i t r o d i g e s t i o n procedures have been developed f o r 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 i r o n 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 i n v e s t i g a t i o n s on i n v i t r o d i g e s t i o n condi

t i o n s and methods f o r f r a c t i o n a t i o n of calcium from d i g e s t s are reported.

In V i t r o Digestion Procedure

The i n v i t r o d i g e s t i o n procedure i s o u t l i n e d 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 d i g e s t i o n was stopped a f t e r peptic d i g e s t i o n . Results of analyses made a f t e r the peptic d i g e s t i o n only w i l l be r e f e r r e d 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 d i g e s t i o n w i l l be r e f e r r e d to as " a f t e r complete d i g e s t i o n " or " a f t e r PPa d i g e s t i o n . "

Exchangeability i s c a l c u l a t e d 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 4 5C 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 i o n i z a b l e 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 p o t e n t i a l 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 d i g e s t i o n . P o t e n t i a l l y a v a i l a b l e 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 w e l l 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)·

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 d i r e c t comparisons of i n v i v o and i n v i t r o estimates of exchangeability and p o t e n t i a l b i o a v a i l a b i l i t y and to t e s t the use of i n v i t r o exchangeability values i n i n v i v o 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 l a b e l e d with 1 + 5C a i n n u t r i e n t s o l u t i o n c u l t u r e . They were used together with ⁴⁷ 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 v i v o 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 a f t e r

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0.5 g dry or freeze-dried food, ground to pass No. 40 sieve (ASMTE-11) + 4.5 g H20 + HC1 to pH 1.5 + peps + H20 to 9 ml

η to 0.1% (w/v) fPeptic Digest (Ρ) |

<

P-Mixture |

Peptic Digestion: 37°C, 60 min., continuous shaking + N^HC03 to pH 6.8 + biie salts to 10 mM + pancSeatin to 0.1% (w/v) + H20 to\l5 ml Analyses (¹⁺⁵Ca, total Ca)

Centrifi 15 min. ige: 4°C 18,000 χ g I Supernatant (SN-P) 1

Pancreatic Digestion: 37°C, 20 min., shaking Analyses (^tt5Ca, total Ca) I PPa Mixture (Τ) 1 Definitions: Special Activity (SA) = dpm⁴⁵Ca/ug Ca Exchangeability= SA^/SAgN Potential bioavailability (%) = PPa Solubility =(CaSN_pPa/CaT)(100)

Analyses (⁴⁵Ca, total Ca)

Centrifuge: 4°C 15 min., 18,000 χ g 1 Supernatant (SN-PPa) 1 Analyses (^l45Ca, total Ca) Figure 1. In vitro digestion procedure. (Reproduced with permis sion from Ref. 7. Copyright 1982 J. Nutr., American Institute of Nutrition.)

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PPa d i g e s t i o n were used to c o r r e c t 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 w e l l with the i n t r i n s i c tag absorp- t i o n i n r a t s 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 v i v o 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 l e s s than s o l u b i l i t y due to physiolo- g i c a l f a c t o r s (1,9). Thus, t h i s s u r p r i s i n g r e s u l t l e d to the reexamination of i n v i t r o d i g e s t i o n conditions which i s reported i n t h i s paper.

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 d i g e s t i o n procedure would a f f e c t 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 t e s t the use of the i n v i t r o d i g e s t i o n procedure f o r studying f a c t o r s which might i n f l u e n c e calcium b i o a v a i l a b i l i t y and to use the r e s u l t s 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 f o r a n a l y s i s .

Peptic Digestion

Only i n i t i a l pH of the peptic d i g e s t i o n was studied. The pH was set e i t h e r 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 c l o s e r to the range observed during human g a s t r i c diges- t 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 d i g e s t i o n 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 e f f e c t on exchangeability and s o l u b i l i t y at e i t h e r the peptic or pancreatic stage. There was l i t t l e r e l a t i o n s h i p between the i n i t i a l s l u r r y and post-digestion values. Exchangeability was deter- mined at the peptic stage, but was incomplete f o r three of the foods.

L i t t l e change occurred during pancreatic d i g e s t i o n . S o l u b i l i t y was maximum a f t e r peptic d i g e s t i o n and decreased during pancreatic diges- t i o n f o r four of the foods. Since the exchangeability did not change during the pancreatic d i g e s t i o n 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 p r e c i p i t a t e d from s o l u t i o n at the same r a t e during pancreatic d i g e s t i o n .

Pancreatic Digestion

The pancreatic d i g e s t i o n conditions studied included pH, the method of pH c o n t r o l , and b i l e s a l t s mixture and concentration. In a d d i t i o n , experiments were run to determine i f mineral s o l u b i l i t y was a f f e c t e d 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 C o n t r o l . In the o r i g i n a l procedure (Figure 1), the pH was adjusted to pH 6.8 with f r e s h l y prepared NaHC03 . The pH generally rose during pancreatic d i g e s t i o n but the magnitude v a r i e d 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 d i g e s t i o n v a r i e d from about 6.2 to 7.2. Although calcium

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Table I. In Vit ro Calcium Exchangeab lil ity and Solubilit y in Milk and Soy P: roducts as Affected by Stage of Dig estion and Initia 1 Peptic PH Substrate mg Ca per PH at ! Stage

2 Exchang eab ili ty, , SA ratio 3,4 Solubility, %

3> 5 flask

1 I Po Ρ PPa I Ρ PPa I Ρ PPa Skim 12.40 6. 7 1.5 1.8 6.7 0.90± .04 1.00±. ,02 0.97± .01 64, ,7±2.3 95, .4±1.8 93, ,7±1. 8 Milk 12.40 6. 7 2.0 2.4 6.6 0.90± .04 0.96±. 02 1.02± .03 64, ,7±2.3 93, .3±5.0 91, ,7±1. 7 Whole 9.72 6. 7 1.5 1.8 6.5 0.72± .02 1.00±. 03 0.95± .01 35, ,5±1.0 98, ,4±1.6 48, ,0±0. 5 Milk 9.72 6. 7 2.0 2.4 6.5 0.72± .02 0.99±. 02 0.98± .02 35 , ,5±1.

0 96, >4±0.9 48, ,8±0. 8 Full-fat 2.23 6. 9 1.5 1.8 6.4 0.47± .01 0.90±. 05 0.93± .03 23. ,2±0.4 81, ,1±4.4 68, ,8±2. 2 Soy flour 2.23 6. 9 2.0 2.7 6.4 0.47± .01 0.86±. 03 0.89± .02 23. .2±0.4 83, ,9±0.6 66, ,7±1. 6 Defatted 3.46 6. 8 1.5 2.1 6.3 0.51± .01 0.81±. 02 0.89± .03 28. ,3±0.7 71. ,5±1.9 71, ,2±1. 1 Soy flour 3.46 6. 8 2.0 2.8 6.2 0.51± .01 0.76±. 01 0.79±, .00 28. ,3±0.7 65. ,1±1.7 66, ,6±0. 4 Soy Protein 4.28 7. 1 1.5 1.9 6.3 0.46±, .01 0.82±. 02 0.82±, .02 15. ,2±0.3 80. ,8±5.7 52, ,2±0. 5 Concentrate 4.28 7. 1 2.0 2.8 6.3 0.46± .01 0.76±. 01 0.76±, .01 15. ,2±0.3 68. ,3±2.7 48, ,1±1. 0 Soy Protein 1.33 7. 0 1.5 2.2 6.3 0.89± .02 0.97±. 02 1.02±, .02 54. ,2±2.6 94. ,3±2.3 95. • 7±1. 9 Isolate 1.33 7. 0 2.0 3.1 6.2 0.89± .02 0.98±. 02 1.01±, .00 54. ,2±2.6 96. ,5±0.8 93, ,5±1. 6 ^g Ca/flask = mg Ca/g dry substrate. 2 I - initial slurry; PG - initial peptic digest, Ρ = at end of peptic digestion and PPa = at end of complete peptic + pancreatic digestion. 3Units = mean ± S.D. for 3 flasks. ^Units of exchangeability: Ratio, specific activity of the mixture: specific activity of the 18,000 χ g supernatant. 5 Units of solubility (pg Ca in 18,000 χ g supernatant/yg in mixture) χ 100.

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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 c o m p l e t e 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 ; l o n g - d a s h e d l i n e , soy p r o t e i n i s o l a t e ; s h o r t - d a s h e d 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

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1. W I E N A N D S C H W A R T Z An In Vitro Digestion Procedure 1

s o l u b i l i t y decreased as pH increased f o r nearly a l l foods, the rate of change v a r i e d among foods, even i n t h i s c l o s e l y - r e l a t e d s e r i e s ( 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 b u f f e r with maximum b u f f e r i n g capacity near pH 7 and n e g l i g i b l e binding capacity f o r minerals (12), and the gradual a d d i t i o n 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 d i g e s t i o n (13)· With t h i s procedure the pH could be c o n t r o l l e d at pH 6.8 ± 0.1.

Enzymes. In an e a r l i e r study (14) complete PPa d i g e s t i o n r e s u l t e d 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 d i g e s t i o n 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 d i g e s t i o n or incubation at a c i d pH.

Therefore, an experiment was run to i n v e s t i g a t e the r e l a t i v e impor- tance 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 d i g e s t s . The r e s u l t s are presented i n Table I I . At the peptic stage, i n c l u d i n g 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 A f t e r In V i t r o Digestion

Digestion F i n a l % Soluble

PH Ca Mg Fe Zn

pH 2, 60 min.

no enzymes

2.1+.0 82.6±1.7 104 ±1.2 2.2±0.7 101.9±5.3

Peptic 2.6±.0 75.3±0.7 98.0±1.1 17.310.9 93.4±5.4 pH 2, 60 min. +

pH 6.8, 30 min.

no enzymes

6.7±.l 41.4±3.6 79.0±3.0 19.3±3.5 56.2±4.6

Peptic + Pancreatic

6.6±.0 48.9±1.2 84.0±1.4 44.7±1.2 56.2±4.6 N o t e : The s u b s t r a t e was a c o m m e r c i a l d e f a t t e d soy f l o u r p r o d u c t . V a l u e s a r e 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 sub- s t r a t e . 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) d i g e s t i o n with successive non-enzymatic incubations at pH 2 and pH 6.8 i n d i c a t e d that the enzymes g r e a t l y 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 r a t e of d i g e s t i o n , protein and carbohydrate d i g e s t i o n and calcium s o l u b i l i t y were measured i n the same PPa d i g e s t s . The digestions consisted of the standard peptic d i g e s t i o n , followed by pancreatic

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d i g e s t i o n i n which the pH was s e t a t 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 f r e e z e - d r i e d spinach or l e t t u c e , the t o t a l calcium i n the muffin was increased by 56% or 65?, r e s p e c t i v e l y . The pH had very l i t t l e e f f e c t on carbohydrate or p r o t e i n d i g e s t i o n , but the calcium s o l u b i l i t y dropped by a t l e a s t k0% i n a l l three muffin formulations over the 0.9 pH u n i t 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 i n c r e a s i n g pH than i n the bran and l e t t u c e - c o n t a i n i n g 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) t o determine i f the type of concentration of b i l e s a l t s a f f e c t e d PPa calcium s o l u b i l i t y . Each mixture was used a t 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: f u l l - f a t soy f l o u r , whole milk and whole egg. The " s o l u b l e " calcium i s aqueous calcium and does not include the non-emulsified l i p i d l a y e r 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 f l o u r and milk, increasing the concentration of e i t h e r TC or BE s l i g h t l y decreased soluble calcium and there were no consistent d i f f e r e n c e s 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.

F r a c t i o n a t i o n of Calcium from Digests

We have t r i e d two non-destructive approaches t o f r a c t i o n a t i o n of the mineral complexes i n i n v i t r o d i g e s t s : u l t r a f i l t r a t i o n and g e l f i l t r a t i o n . Only preliminary data from g e l f i l t r a t i o n experiments are c u r r e n t l y a v a i l a b l e .

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 f r a c t i o n a t i o n of the digest mixture, which was prepared from a soy protein i s o l a t e , combines the techniques of c e n t r i f u g a t i o n 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 02 was used t o apply pressure t o the u l t r a f i l t r a t i o n c e l l , rather that N², t o 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 c e n t r i f u g a t i o n at 18,000 χ g. Much of i t was bound t o large complexes, as judged from the f i l t r a t i o n of l e s s than h a l f the calcium through the 10,000 MWC0 (molecular weight cut

o f f ) membrane. The nominal MWC0 of the membrane i s not a p r e c i s e guide t o the s i z e of complex which i s f i l t e r e d since one-third of the C a+ + 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 i n the completely i o n i z e d s o l u t i o n . For a l l u l t r a f i l t r a t i o n s of the i n v i t r o digest, the s t a r t i n g material was the 100,000 χ g supernatant i n order t o avoid confounding the r e s u l t s with sample d e t e r i o r a t i o n . The r e s u l t s were found to be reproducible and not influenced by the

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Table III. Effect of In Vitro Pancreatic Digestion pH on Carbohydrate and Protein Digestion and Calcium Solubility Substrate:! Bran muffin Spinach/bran muffin Lettuce/bran muffin Soluble component: maltose

2 peptide

3 Ca maltose peptide Ca maltose peptide Ca component in 18,000 χ g supernatant: mg/g substrate Initial. 319 14 (Total= 322 24 (Total-328 17 (Total* (no digestion) 1.36 mg) 2.12 mg) 2.25 mg) pH 6.2 551 108 0.62 542 132 0.28 573 116 0.92 6.5 562 106 0.42 540 130 0.19 556 113 0.83 6.8 551 107 0.37 527 134 0.08 545 116 0.63 7.1 554 108 0.37 517 132 0.06 531 112 0.47 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 in the spinach/bran and lettuce/bran muffins, respectively. 2Maltose, the endproduct of amylase activity, was measured by the method of Dahlquist (16). 3Peptide=supernatant protein which was not precipitated when trichloroacetic acid (TCA) solution was added to 5% TCA (W/V) (17).

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ο

\

^^^^

— ι 1 ι %js

0 5 10 15 0 5 10 15 0 5 10 15 F u l l Fat Soy Flour Whole M i l k Whole Egg

« o m m F i g u r e 3. C a l c i u m 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, c r u d e b o v i n e 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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1. W I E N A N D S C H W A R T Z An in Vitro Digestion Procedure

T a b l e I V . F r a c t i o n a t i o n o f C a l c i u m 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 l2

F r a c t i o n C a C l2 S o l u t i o n3 In V i t r o Digest1* F i l t e r a b l e Calcium, 7>

Digest Mixture - ^100%

CE:18,000 χ g supernatant1 - ^87.4±5.3

CE:100,000 χ g supernatant1 - ^67.9±9.1

UF:300,000 MWCO f i l t r a t e2 - ^52.4±6.2

UF:10,000 MWCO f i l t r a t e2 - ^44.9±3.8

UF:5,000 MWCO f i l t r a t e2 95.8% -

UF:1,000 MWCO f i l t r a t e2 67.8 30.216.9

Ca recovery (UF) 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.

2U l t r a f i l t r a t i o n was c a r r i e d 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 C02 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 c u t - o f f of the membrane.

350 ml of the s o l u t i o n , 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 d i g e s t i n g a soy p r o t e i n 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. f o r four experiments.

5% 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 s o l u t i o n 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.

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order of the s e r i e s of membranes i n a p a r t i c u l a r experiment. Thus, sample d e t e r i o r a t i o n was not a problem when the sample was stored on i c e 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.

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 s t a b l e 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 l a b e l e d 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 i r o n , that i s , when exchangeability was complete. For other minerals, the requirements f o r i n v i v o 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 l a b e l i n g 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 t e s t 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 d i g e s t i o n procedure should s i m p l i f y the use of the e x t r i n s i c tag method. Our r e s u l t s i n d i c a t e that calcium exchangeabi- l 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 l a b e l i n g and only one isotope are required. The same procedure provides the information needed t o i n t e r p r e t the r e s u l t s of i n v i v o e x t r i n s i c tag s t u d i e s . Also, i t i s not necessary t o demonstrate complete exchange- a 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 t o c a l c u l a t e 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 f u r t h e r s i m p l i f i e d i n that only the peptic d i g e s t i o n 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 i n d i c a t e that i n v i t r o d i g e s t i o n 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 l i m i t e d number of foods. They would be strengthened i f a wider range of foods i s tested i n d i r e c t i n v i v o - i n v i t r o comparison s t u d i e s . The t e s t foods should include foods i n which exchangeability might not be completed during peptic d i g e s t i o n such as foods with

" i n d i g e s t i b l e " residues that may be a l t e r e d and release calcium which may be absorbed i n the lower i n t e s t i n e .

B i o a v a i l a b i l i t y . 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 f a s t 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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1. W I E N A N D S C H W A R T Z An In Vitro Digestion Procedure 13 the foods themselves i s not s u f f i c i e n t since changes occur during the process of d i g e s t i o n 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 v i v o absorption and introduce v a r i a b i l i t y not r e l a t e d to food c h a r a c t e r i s t i c s (1,2,9) are eliminated.

Choice of P o t e n t i a l 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 u s u a l l y assumed that calcium must be soluble and probably i o n i z e d i n order to be a v a i l a b l e 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 c e n t r i f u g a t i o n at 18,000 χ g as the measure of p o t e n t i a l 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 a v a i l a b l e 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 b i o a v a i l a b l e 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 p o t e n t i a l 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 c o n d i t i o n s . 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 d i r e c t i n v i v o - i n v i t r o comparison (8), i t was necessary to determine the i n v i t r o d i g e s t i o n 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 c r i t e r i o n .

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 s i z e than completeness of d i g e s t i o n (23,24).

Analyses of the pancreatic d i g e s t i o n conditions i n d i c a t e that the pH of the pancreatic digest was more important f o r 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 I I I ) or b i l e s a l t s (Figure 3) f o r most foods t e s t e d . 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 d i g e s t i o n , presumably due to a combination of bicarbonate decomposition, d i g e s t i o n endproduct release and the v a r i a b l e b u f f e r i n g 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 f o r calcium s o l u b i l i t y (£). The f i n a l pH was apparently too high t o r e s u l t i n the net e f f e c t seen i n v i v o ( 8 ) .

"Standard" Digestion Conditions. As a r e s u l t of the analyses of d i g e s t i o n conditions we have modified our i n v i t r o d i g e s t i o n to s t a r t the peptic d i g e s t i o n a t pH 2.0 instead of 1.5, and to c o n t r o l the pH at 6.8 i n the pancreatic stage by continuous aeration with CO . We a l s o substituted b i l e extract f o r taurocholate since, although calcium s o l u b i l i t y was s i m i l a r , other minerals were more soluble i n the b i l e extract-containing d i g e s t s (13)·

The term "standard" i s not meant to denote a d i g e s t i o n procedure which should be r o u t i n e l y used to determine p o t e n t i a l l y a v a i l a b l e 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 u s e f u l to think of the "standard" procedure as a s e t of conditions t o be used t o see how w e l l 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 f a c t o r s which require f u r t h e r study.

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L i p i d - c o n t a i n i n g 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 f a c t o r s which require f u r t h e r study. B i l e s a l t s have been shown to enhance calcium absorp

t i o n from s p a r i n g l y s o l u b l e s a l t s (25) but a l s o enhance l i p a s e 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 i n s o l u b l e calcium soaps (27). In contrast, i n v i v o 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 p r e - e x i s t i n g 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 i n s o l u b l e calcium soap formation. The complex l i p i d s of egg yolk (28), and t h e i r p o s s i b l e 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 i n c r e a s i n g 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 v i v o . 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.

F r a c t i o n a t i o n of Digest Calcium. The i n v i t r o d i g e s t i o n procedure provides a means of producing the s t a r t i n g m a t e r i a l f o r a more d e t a i l e d study of the calcium complexes i n i n t e s t i n a l d i g e s t s . T h i s may be d e s i r a b l e 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 extrinsic isotope and the i n t r i n s i c calcium i n the food or meal i s a f f e c t e d by the method of i n c o r p o r a t i n g the isotope, the calcium source i t s e l f or foods fed with i t .

2. To determine how c o n t r o l l e d manipulation of d i g e s t i o n c o n d i t i o n s i n f l u e n c e s the d i s t r i b u t i o n of calcium among p o s s i b l e ligands from a food or meal.

3 . To describe the calcium complexes formed during a standardized d i g e s t i o n f o r a number of foods and food mixtures, f o r comparisons, or to t e s t hypotheses r e l a t i n g food components to i n t e s t i n a l calcium complexes.

F r a c t i o n a t i o n Methods. U l t r a f i l t r a t i o n and g e l f i l t r a t i o n are non

d e s t r u c t i v e methods which, based on l i m i t e d experience, can be used f o r f r a c t i o n a t i o n of mineral complexes from d i g e s t s . 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 g e l 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 a l s o good.

In our experience with u l t r a f i l t r a t i o n , use of C 02 pressure to f o r c e 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 p o s s i b l e source of e r r o r . The high pressure caused more C 02 to be d i s s o l v e d i n the d i g e s t 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 ) . It 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 .

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1. W I E N A N D S C H W A R T Z An In Vitro Digestion Procedure 15 Gel f i l t r a t i o n may be best used to analyze f r a c t i o n s 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 p r e c i s e separation of complexes can be obtained with gel f i l t r a t i o n , but the s i z e of sample which can be applied i s l i m i t e d . Thus, i n many s i t u a t i o n s , the sample must be concentrated before being a p p l i e d to the g e l 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 p o s s i b l e s h i f t s i n mineral binding which should be understood f o r proper i n t e r p r e t a t i o n of the r e s u l t s (33).

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 d i g e s t i o n 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 d i e t a r y calcium absorption s t u d i e s , and (2) study of the f a t e 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 p o t e n t i a l a v a i l a b i l i t y f o r absorption. Ideas have been incorporated from many sources and only l i m i t e d t e s t i n g has been possible so f a r . We have t r i e d to i n d i c a t e the advantages of such a procedure as well as where more t e s t i n g of the v a l i d i t y of the ideas f o r 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.

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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, 652- 60.

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.; Walter De Gruyter:

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

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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 D E L O Y G. H E N D R I C K S

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 utilization. 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 elderly 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 = 0.85) 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 this bone loss amounts to approximately 10 mg calcium d a i l y f o r men and 20 mg calcium for women before menopause. After menopause t h i s loss i s

0097-6156/ 85/ 0275-0017506.00/ 0

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approximately 4 0 to 1 2 0 mg calcium daily { § ) . Calculating from the data reported by Mazess et a l . ( 5 J , approximately 5 0 mg of bone mineral is l o s t daily by women over age 5 0 . It is 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 after 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 for both men and women (8). In animals bone strength is 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 affecting 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 radiologiciTly detected osteoporosis vs. bone mineral contents.

In spite of these descrepancies, bone loss is 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 entity in human nutrition ( 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 1 2 4 1 mg (with a 95% confidence interva l of 1 1 6 6 to 1 3 1 6 mg Ca) calcium daily to maintain calcium balance. From other data, i t i s estimated that 3 5 mmol ( 1 4 0 0 mg) calcium daily is 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 dail y fo r adult women. The average dail y calcium intakes of American women above age 2 3 vary from 5 1 5 to 6 0 4

milligrams for different age groups ( 1 7 ) . An average daily calcium intake of 9 4 4 (Sd = 3 4 3 ) milligrams was found for 1 0 0 premenopausal Canadian women ( 1 8 ) . Calcium is the only nutrient that i s

associated with incidence of bone fracture ( 6 ) . Calcium intake is highly correlated with the mineral content of the bones of

experimental animals ( 1 1 ). Thus, calcium could be considered the most frequently deficient nutrient in the U.S.A. Anything that could result 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 potentially 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 rich 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.

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2. M A H O N E Y A N D H E N D R I C K S 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 is considerable

v a r i a b i l i t y in the apparent absorption values determined from many studies, 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 is also affected by variations in urinary excretion. Dietary factors affecting 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 is a major factor 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 calcuria 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 controlling urinary calcium excretion.

Dietary phosphorus also affects calcium metabolism. Poly- phosphate 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 affect 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 affect 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 alginate, 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 triglycerid 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 fluoride on calcium u t i l i z a t i o n have been variable

( 3 4 , 3 8 , 3 9 ) .

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 fraction of a nutrient, 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 is 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 fraction

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which is u t i l i z e d r e l a t i v e to that given is considered to be the amount of calcium in the source that is metabolizable.

There are two primary approaches to determining b i o -

a v a i l a b i l i t y : (a) Direct measurement of uptake into the body can be done using pharmacokinetic methods, quantitating the accumulation of radioactive nutrients, 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 traditional 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 is to determine the uptake of a test mineral or compound relative 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 is located in bone.

Bone i s very sensitive to dietary factors such as the amount of calcium present in the diet 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 is especially 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 after 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 for 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 affect 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 similar for rats.and humans for 8 of 9 dietary and physiological factors reviewed (Table I). This is good evidence that the rat may be a practical model for estimating human dietary calcium

u t i l i z a t i o n .

An attempt was made to collate data on human and rat apparent calcium absorption values for several calcium sources. Absorption values were so variable within species and calcium sources that a correlation could not be j u s t i f i e d . Much of this 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 correlation between the calcium absorption responses of these two species.

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2. M A H O N E Y A N D H E N D R I C K S A Model for Assaying Ca Bioavailability 21

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

Factor Human Beings Rat Reference

Ca absorption True 59,60 decreases with age True (over age 60) 61

True (over age 30) 16 True 62,63 False 64

Gastric acidity True 65-67 necessary for True in B i l l r o t h II 68

absorption of poorly patient 69 soluble Ca source False (Total True 9,10,31

gastrectomy)

Ortho phosphate No change 26,70-72 Slight decrease 28

Decrease 27,73 Decrease 31,74,75

Polyphosphate Decrease 26 Decrease 31

Increased dietary Increase 25,27,76 &

protein 77 Increased

absorptive

cap 78 No change in

absorptive

cap 79 No change 81

Increase 80,82

Intestine adapts True 16,83 to Low CA intake by True 84,85,99

increasing absorption · Lactose Increase 68 ,86

No change 87 Increase 88

No change 89-91

Pregnancy Increase 92-93 Increase 94,95

Lactation Increase?

Increase 94,96,97 a -47 Ca uptake in serum.