Nutritional Bioavailability of Iron 9780841207462, 9780841209855, 0-8412-0746-1

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
1 Efficiency of Hemoglobin Regeneration as a Method of Assessing Iron Bioavailability in Food Products......Page 8
Literature Cited......Page 16
2 In Vitro Estimation of Food Iron Bioavailability......Page 18
Rationale......Page 19
Description of the Method......Page 25
Results and Evaluation......Page 27
Literature Cited......Page 32
3 Iron Chemistry and Bioavailability in Food Processing......Page 34
Iron Sources......Page 35
Effect of Heat Processing on Iron Bioavailability......Page 37
Effect of Organic Acids and Carbohydrates on Iron Bioavailability......Page 43
Effect of Food Processing on Iron Chemistry......Page 49
Literature Cited......Page 59
4 The Effects of Physicochemical Properties of Food on the Chemical Status of Iron......Page 62
Literature Cited......Page 90
5 Ascorbic Acid: An Enhancing Factor in Iron Absorption......Page 92
The role of ascorbic acid in nonheme iron absorption......Page 93
Estimating bioavailable iron in the diet......Page 96
Dietary intake of iron and ascorbic acid......Page 97
Ascorbic acid:Iron and relationships in abnormal state of metabolism......Page 100
Literature Cited......Page 101
6 Influence of Copper, Zinc, and Protein on Biological Response to Dietary Iron......Page 103
Results......Page 104
Discussion......Page 108
Literature Cited......Page 110
Bioavailability of Iron From Inorganic Compounds Containing Both Phosphorus and Iron......Page 112
Effect of Phosphorus on the Bioavailability of Iron and Zinc in the Total Diet......Page 114
Effect of Phosphorus and Calcium Together on the Bioavailability of Iron and Zinc in the Total Diet......Page 119
Literature Cited......Page 123
8 Phytate, Wheat Bran, and Bioavailability of Dietary Iron......Page 126
Monoferric Phytate-Isolation, Identification and Properties......Page 127
Bioavailability of Ferric Phytates......Page 132
Bioavailability of Iron in Wheat, Wheat Fractions and Wheat Foods......Page 136
Discussion......Page 140
Summary......Page 144
Literature Cited......Page 145
9 Dietary Fiber and the Bioavailability of Iron......Page 147
Affinity of Dietary Fiber Preparations for Iron......Page 149
Effect of pH upon Binding of Iron by Dietary Fiber......Page 150
Stability of Fiber-Bound Iron in Presence of Intestinal segments......Page 151
The Effect of Dietary Fiber upon Absorption of Iron by Animals......Page 152
Dietary Fiber and Iron Absorption by Humans......Page 153
Estimation of the amount of iron complexed by fiber in cereal-rich diets......Page 160
Addenda......Page 161
Literature cited......Page 162
10 Bioavailability of Iron from Bran in Pigs......Page 166
The Iron Situation in Norway......Page 168
Bioavailability of Iron from Bran in Pigs......Page 169
Conclusions Concerning Pig Experiment......Page 171
Conclusion......Page 172
Literature Cited......Page 174
11 Bioavailability of Iron and Other Trace Minerals from Human Milk......Page 175
Lactoferrin's Role in Iron Absorption......Page 176
Trace Mineral Cocentrations in Milk......Page 178
Iron Absorption......Page 179
Literature Cited......Page 181
12 Vegetarianism and the Bioavailability of Iron......Page 184
Factors Affecting Iron Absorption from Vegetarian Diets......Page 185
Iron Content of Vegetarian Diets......Page 187
Comparative Utilization of Iron by Vegetarians and Omnivores......Page 190
Conclusion......Page 196
Literature Cited......Page 198
C......Page 200
H......Page 201
P......Page 202
Z......Page 203

Citation preview

Nutritional Bioavailabilit

f Iro

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Nutritional Bioavailability of Iron Constance Kies, EDITOR University of Nebraska

Sponsored by the Nutrition Subdivision of the Division of Agricultural and Food Chemistry of the American Chemical Society.

ACS

SYMPOSIUM

S E R I E S 203

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1982 In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Library of Congress Cataloging in Publication Data Nutritional bioavailability of iron. (ACS symposium series, ISSN 0097-6156; 203) Papers presented at a symposium held at the American Chemical Society meeting in Atlanta Ga. on Mar. 29-Apr. 3, 1981. Includes bibliographies an 1. Iron—Metabolism—Congresses. 2. Iron in the body—Congresses. I. Kies, Constance, 1933. II. American Chemical Society. Nutrition Subdivision. III. Series. [DNLM: 1. Biological availability—Congresses. 2. Iron—Analysis—Congresses.3.Nutrition—Congresses. 4. Iron—Metabolism—Congresses. QV 183 N976 1981] QP535.F4N8 1982 612'.3924 82-16391 ISBN 0-8412-0746-1 ACSMC8 203 1-205 1982

Copyright © 1982 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article 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. 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 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 thefirstpage 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 ACS 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. PRINTED IN THE UNITED STATES

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In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

ACS Symposium Series M. Joa

Comstock Series Editor

Advisory Board David L. Allara

Marvin Margoshes

Robert Baker

Robert Ory

Donald D. Dollberg

Leon Petrakis

Robert E. Feeney

Theodore Provder

Brian M. Harney

Charles N. Satterfield

W. Jeffrey Howe

Dennis Schuetzle

James D. Idol, Jr.

Davis L. Temple, Jr.

Herbert D. Kaesz

Gunter Zweig

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

FOREWOR SYMPOSU IM SERIES

The ACS 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 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 since symposia may embrace both types of presentation.

IN CHEMSITOY SERIES

ADVANCES

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

PREFACE IF NUTRE INTS FOUND IN FOOD

were digested, absorbed, and made available to the human or animal body at the 100% level, the science and practice of nutrition would be indeed simplified. That nutrients vary in their bioavailability has been well established. The chemical nature of the specific form of the nutrient involved, the chemical and physical characteristics of the foods in which nutrient the nature of the digestiv ents, and the physiological condition of the person consuming the food all may affect bioavailability. However, knowledge of specific individual and interacting factors affecting bioavailability and utilization of nutrients has not yet been fully elucidated and constitutes one of the most active areas of current nutrition research. Iron deficiency anemia is commonly found in both affluent and economically deprived populations. In prevention of this nutritional deficiency disease, both increase in dietary iron and increase in the availability of this dietary iron for population groups at risk should be concurrently addressed. This is a problem for which the solution lies primarily not with the medical community but rather with the providers of food in agriculture and food industry. The chapters were selected to give a broad overview of the topic of bioavailability of iron with special emphasis on topics of concern to food producers. The editor expresses appreciation to all the contributors and to Donna Hahn, who did much of the organizational work involved in preparation of this volume.

CONSTANCE KIES University of Nebraska Lincoln, Nebraska July 1982

ix In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1 Efficiency of Hemoglobin Regeneration as a Method of Assessing Iron Bioavailability in Food Products ARTHUR W. MAHONEY and DELOY G. HENDRICKS Utah State University, Colleges of Family Life and Agriculture, Department of Nutrition and Food Sciences, Logan, UT 84322

The bioavailabilit supplement, food or mea portion of the total iron which is metabolizable. Philosophically, this concept is important because the amount of iron utilized by avian and mammalian species is directly associated with iron need. When assaying iron bioavailability, it is therefore necessary to use an organism whose need will exceed the amount provided. In animal assays of iron bioavailability, iron need is assured by a growth phase and/or creation of iron deficiency through feeding an iron deficient diet and phlebotomy. Because healthy subjects are usually used in human assays of iron bioavailability (Cook et al., 1981; Cook and Monson, 1976; Radhakrishman and Sivaprasad, 1980), i t is inappropriate to compare the data obtained from animal and human assays. In fact i t is questionable i f assays of iron bioavailability yield good information on the quantities of metabolizable iron available when healthy human subjects are used. The Committee on Dietary Allowances, Food and Nutrition Board, National Academy of Sciences (RDA, 1980) has estimated the amount of metabolizable iron (as absorbable iron) from meals consumed by human beings as ranging from 3 to 23 percent depending on the nature of the meal. For adult women of childbearing age, the committee has assumed that 1.5mg iron is lost daily and that 18mg should be consumed to meet this need. They have therefore assumed that approximately 8.3 percent of the dietary iron will be metabolized. For adult men and women over the age of 51 years, they estimate that 1.0mg iron will be lost daily and recommend that 10mg should be consumed to meet this need to offset only approximately 10 percent of the dietary iron being metabolized by these people. It should be noted, however, that what is metabolized from a food under such conditions does not necessarily reflect what is potentially metabolizable. Indeed, the majority of women of childbearing age consume less than the recommended 18mg iron 0097-6156/82/0203-0001$06.0O/0 © 1982 American Chemical Society In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

2

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

and yet are not i r o n d e f i c i e n t (DHEW, 1968-70). Thus, much information i s needed on the m e t a b o l i z a b i l i t y of food i r o n . Two b a s i c methods have been used i n the assay of i r o n b i o a v a i l a b i l i t y (Bing, 1972; Thompson and Raven, 1959). In the absolute method, the change i n t o t a l body i r o n r e l a t i v e to that consumed i s used. T h i s n e c e s s i t a t e s making an estimate of the amount of i r o n present i n the animal body at the i n i t i a t i o n of the experiment and then determining the amount present at the t e r m i n a t i o n . U s u a l l y i n a p p l y i n g t h i s procedure, a r e p r e s e n t a t i v e group of animals i s k i l l e d at the beginning of the experiment to o b t a i n the estimate of t h e i r i n i t i a l body i r o n . Thus, one can o b t a i n an average value f o r body i r o n content r e l a t i v e to weight that can be m u l t i p l i e d with i n i t i a l body weights to estimate i n i t i a l amounts of body i r o n f o r each t e s t animal. Various m o d i f i c a t i o n s of the hemoglobin regeneration procedure have been used(Bing, 1972). In the one described here th t f i r o gained hemoglobin i s estimated of i r o n consumed. An e f f i c i e n c y i r o n i n t o hemoglobin can be computed f o r each t e s t animal knowing i n i t i a l and f i n a l body weights, i n i t i a l and f i n a l hemoglobin c o n c e n t r a t i o n s , the amount of food consumed, and the i r o n content of the food. I t i s c a l c u l a t e d as f o l l o w s : mg Hb Fe

BW x .067 ml b l / g BW X g Hb/100 ml X 3.35mg Fe/g Hb Efficiency = ( ( F i n a l mg Hb Fe - I n i t i a l mg Hb Fe) / mg Fe consumed) X 100

In a p p l y i n g t h i s method, weanling male r a t s are given f r e e access to a low-iron d i e t and b l e d to remove about one ml of blood two times 4 days apart. Three days l a t e r , the animals are again b l e d of about 100 m i c r o l i t e r s blood f o r determination of hemoglobin c o n c e n t r a t i o n and are a l l o t t e d to treatments of ten r a t s each such that mean body weights and hemoglobin concentrations are s i m i l a r . The mean hemoglobin concentrations should be between 4 and 6 gm/dl. They are fed the t e s t d i e t s f o r ten days i n amounts that very few o r t s are obtained. Any s p i l l a g e and o r t s are weighed and recorded to account f o r unconsumed d i e t a r y i r o n . The low-iron d i e t should c o n t a i n l e s s than 10 ppm Fe and the t e s t d i e t s should c o n t a i n approximately 35 ppm. T h i s amount of d i e t a r y i r o n has been shown not to exceed the a b i l i t y of t h i s animal p r e p a r a t i o n to u t i l i z e i r o n , s i n c e the regeneration of hemoblobin i r o n i s l i n e a r at l e a s t to 68 ppm d i e t a r y i r o n (Mahoney and Hendricks, 1976). M i l l e r (1977) reported that i r o n gained as hemoglobin was l i n e a r (r=0.94) through i n t a k e s of 5.5mg i r o n as f e r r o u s s u l f a t e i n 11 days. Her r a t s were made anemic by feeding low-iron d i e t f o r 24 days i n p r e p a r a t i o n f o r the hemoglobin regeneration experiment. The f o l l o w i n g c r i t e r i a f o r a good b i o a v a i l a b i l i t y assay are a p p r o p r i a t e , (a) I t must be dose responsive. For an

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1.

MAHONEY AND HENDRC IKS

Assay by Hemoglobin Regeneration

assay to be u s e f u l i n a v a r i e t y of s i t u a t i o n s , i t should not be a f f e c t e d by v a r i a t i o n s i n amounts of i r o n consumed. Therefore, the dose-response r e l a t i o n s h i p should be l i n e a r . (b) I t must d i s c r i m i n a t e w i t h good s e n s i t i v i t y among sources of i r o n and among treatments such as cooking o r p r o c e s s i n g . (c) B i o a v a i l a b i l i t y values obtained should be u n a f f e c t e d by f a c t o r s u n r e l a t e d to the food or i r o n source. Thus, the b i o a v a i l a b i l i t y assay should be i n s e n s i t i v e to v a r i a t i o n s i n c a l o r i c d e n s i t y of the d i e t , a p p e t i t e of the animal, and animal maturity, (d) The procedure should y i e l d r e p r o d u c i b l e r e s u l t s f o r the same i r o n source among experiments and l a b o r a t o r i e s . The e f f i c i e n c y of converting d i e t a r y f e r r o u s s u l f a t e i r o n i n t o hemoglobin by anemic r a t s has been c a l c u l a t e d from the data of many experiments and l a b o r a t o r i e s (Table 1 ) . The 'uncorrected' e f f i c i e n c y v a l u e s represent the values obtained f o r the t o t a l amounts o values represent a mathematica response to only the f e r r o u s s u l f a t e i r o n present i n the d i e t . T h i s estimate was made assuming that the amount of i r o n present i n the low-iron b a s a l d i e t s r e f l e c t s the cumulative i r o n provided by the b a s a l i n g r e d i e n t s of the f e r r o u s s u l f a t e t e s t d i e t s (e. g., c a s e i n , o i l , dextrose, f i b e r , v i t a m i n mixture and m i n e r a l mixture). Thus, knowing the amounts of d i e t consumed by the t e s t animals, one can estimate the c o n t r i b u t i o n of the b a s a l i n g r e d i e n t s to the t o t a l d i e t a r y intake of i r o n of the t e s t animals. T h i s value subtracted from the t o t a l i r o n intake y i e l d s the estimated i r o n intake from f e r r o u s s u l f a t e . S i m i l a r l y , the amount of i r o n gained as hemoglobin by the r a t s f e d the low-iron b a s a l d i e t can be c a l c u l a t e d and subtracted from the t o t a l i r o n gained as hemoglobin by the r a t s f e d the f e r r o u s s u l f a t e t e s t d i e t s , which y i e l d s an estimate of the f e r r o u s s u l f a t e c o n t r i b u t i o n to the i r o n gained as hemoglobin. T h i s v a l u e , r e l a t i v e to the estimated q u a n t i t y of i r o n consumed as f e r r o u s s u l f a t e , was used to compute the ' c o r r e c t e d ' e f f i c i e n c i e s presented i n t a b l e 1. The ' c o r r e c t e d ' values wet^e computed s i m i l a r l y f o r the i r o n sources presented i n t a b l e 2. The v a l i d i t y of t h i s c o r r e c t i o n i s d o u b t f u l when foods are the source of experimental i r o n because the amounts of b a s a l i n g r e d i e n t s are decreased depending on the i r o n content of food t e s t e d , which a f f e c t s the amount of food that must be formulated i n t o the d i e t to provide the d e s i r e d i r o n content. For f e r r o u s s u l f a t e , the average e f f i c i e n c y of c o n v e r t i n g d i e t a r y i r o n i n t o hemoglobin was 52 percent with a c o e f f i c i e n t of v a r i a t i o n of 19 percent (Table 1) . When c o r r e c t e d f o r the b a s a l d i e t a r y i n g r e d i e n t s , the average e f f i c i e n c y was 61 percent, w i t h a c o e f f i c i e n t of v a r i a t i o n of 33 percent. Making the c o r r e c t i o n f o r the b a s a l i n g r e d i e n t s d i d not improve the a n a l y s i s . In two cases, the ' c o r r e c t e d ' e f f i c i e n c y of conversion was g r e a t e r than 100 percent.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

3

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION Table 1.

E f f i c i e n c y o f Converting I r o n i n FeSO^ i n t o Hemoglobin by Anemic Rats

D i e t a r y Fe 33.0 40.4 31.2 27 27

—

27.8 16.2 20.5 29.2 45.0 18.2 25.6 41.8 68.9 11.8 18.9 23.8 23.6 35.2 48.2

13.8 19.8 31.8 12.2 17.2 22.2 27.2 14 20 32 15 22 32 16.5 26.5 46.5 Mean +_ Sd

Efficiency Uncorrected Corrected** 80 111 50 52 38 42 71 45 69 44 54 56 51 70 47 68 48 60 52 61 46 49 44 41 46 41 36 39 57 53 62 60 72 71 54 42 60 57 66 66 49 55 53 59 57 62 49 50 52 85 57 76 48 58 53 148 57 89 47 61 33 39 38 45 43 50 42 46 41 58 60 78 53 60 51 71 67 81 57 64 54 74 57 66 43 47 52 + 10 61 + 20 c

Reference Farmer et a l . (1977) A l l r e d (1976) Mahoney e t a l . (1979) Rahotra e t a l . (1973) Anderson e t a l . (1972) Mahoney et a l . (1974) Blumberg & A r n o l d (1947)

Miller

(1977)

Cardon e t a l . (1980)

Theur e t a l . (1971)

D

Theur e t a l . (1973)°

F r i t z e t a l . (1974)

F r i t z e t a l . (1970)°

Shah e t a l . (1979)

Shah and Belonje (1973a)

Shah and Belonje (1973b)

T-2.67

(P

.02)

Continued on next page.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1.

MAHONEY AND HENDRC IKS

Table

Assay by Hemoglobin Regeneration

1-Continued

Note: Uncorrected e f f i c i e n c i e s of 82, 77, 74, 65, 63, 84, 82, and 65 percent were c a l c u l a t e d using data presented by Cowan et a l . (1967). Because there were i n s u f f i c i e n t data published to c a l c u l a t e the c o r r e c t e d e f f i c i e n c i e s , these data were not included i n Table 1. a The e f f i c i e n c y was c o r r e c t e d by estimating the c o n t r i b u t i o n of jlron i n the basal d i e t to i r o n intake and hematinic response. Supplemental data necessary f o r computations s u p p l i e d by authors. Values greater than 100 percent not included i n the mean. Table 2.

E f f i c i e n c y of Converting Iron From Various Into Hemoglobin By Anemic Rats. D i e t a r y Fe

Source FePO.

Sources

Efficiency

18.8 (1947) 29.2 54.9 118.0 19.8 31.8 55.8 24.2

18 21 14 22 24 23 21

4 18 12 34 32 27 26

32.6 38.2 49.7 Ground Beef 26.6 Beef Shank 31.0 Beef P l a t e 33.0 Bologna 29.0 Beef 26.0 Turkey 23.0 Turkey 30.4 Enriched Flour 24.4 White Bread 10.7 Whole Wheat Flour 28.0 Rice 28.0 48.0 Dried Egg 21.2

17 23 26 34 63 61 46 49 45 43

18 25 30 42 87 79 62 37 74

24 28

33 49

43 30 43 43

54 31 41 41

FePO,

FePO,

u F r i t z e t a l . (1974)°

Mahoney & Hendricks (1976)

c

Mahoney e t al.(1974) Farmer e t al.(1977) Farmer e t a l . (1977) , Mahoney e t a l . (1979) Cardon e t a l . (1980) Mahoney e t a l . (1980) Cardon e t a l . (1980) Mahoney e t a l . (1974) M i l l e r (1977)

C

Mahoney e t al.(1974) Shah e t a l . (1979) Mahoney e t al.(1974)

a

The e f f i c i e n c y was c o r r e c t e d by e s t i m a t i n g the c o n t r i b u t i o n of jlron i n the basal d i e t to i r o n intake and hematinic response. Supplemental data necessary f o r computations s u p p l i e d by authors. Due to c a l c u l a t i o n e r r o r s , the o r i g i n a l value was reported as 45 f o r ground beef and 33 f o r whole wheat f l o u r . c

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

5

6

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION In ten cases, the 'corrected* values were l e s s than the uncorrected ones. Because of t h i s i n c o n s i s t e n c y and because c o r r e c t i o n does not reduce v a r i a b i l i t y w i t h i n nor among experiments, attempting to c o r r e c t f o r the i r o n c o n t r i b u t i o n of the b a s a l i n g r e d i e n t s to the hematinic response does not seem to improve t h i s assay of i r o n b i o a v a i l a b i l i t y . D i e t a r y i r o n l e v e l does not seem to a f f e c t the e f f i c i e n c y with which d i e t a r y i r o n i s converted i n t o hemoglobin when f e r r o u s s u l f a t e (Table 1) or when f e r r i c orthophosphate (Table 2) i s the primary source of d i e t a r y i r o n . T h i s i s a l s o true f o r white bread (Table 2); however, the source of the i r o n i n the enriched f l o u r used i n the bread i s unknown. That the e f f i c i e n c y of converting food i r o n i n t o hemoglobin i s not a f f e c t e d by d i e t a r y i r o n c o n c e n t r a t i o n i s important to b i o a v a i l a b i l i t y t e s t i n g because i t i s o f t e n d i f f i c u l t to formulate d i e t s w i t h p r e c i s e amounts of i r o n , e s p e c i a l l y when foods are the source The e f f e c t s of carbohydrat with which d i e t a r y i r o n i s converted i n t o hemoglobin have been s t u d i e d . M i l l e r and Landes (1976) used s t a r c h , sucrose or glucose as the carbohydrate source and f e r r o u s s u l f a t e as the i r o n source. The r e s p e c t i v e e f f i c i e n c i e s of converting d i e t a r y i r o n i n t o hemoglobin were 72, 65, and 46 percent. Amine and Hegsted (1971) obtained s i m i l a r carbohydrate e f f e c t s studying i r o n a b s o r p t i o n . Glucose i s the most commonly used source of d i e t a r y carbohydrate i n s e m i p u r i f i e d d i e t s . Pennell et a l . (1976) reported that b e t a - l a c t o s e i n p l a c e of sucrose reduced the r e l a t i v e b i o l o g i c a l value of i r o n as sodium i r o n pyrophosphate when fed to r a t s . However, a l p h a - l a c t o s e or glucose i n p l a c e of the sucrose d i d not a f f e c t the b i o a v a i l a b i l i t y of t h i s i r o n source. S i m i l a r l y , the source of f a t can a f f e c t the b i o a v a i l a b i l i t y of d i e t a r y i r o n ; but, the l e v e l of d i e t a r y f a t has no e f f e c t (Mahoney et a l . , 1980). The c a s e i n c o n c e n t r a t i o n of d i e t s fed r a t s does not a f f e c t i r o n a b s o r p t i o n (Amine and Hegsted, 1971, Carmichael et a l . , 1975); however, e f f e c t of p r o t e i n source was not s t u d i e d by these authors. Thus, the sources of carbohydrate and f a t can markedly a f f e c t the u t i l i z a t i o n of d i e t a r y i r o n and should be considered as important v a r i a b l e s i n b i o a v a i l a b i l i t y experiments. The amount of p r o t e i n , however, does not seem as critical. Among experiments, the v a r i a b i l i t y of the e f f i c i e n c y of converting i r o n from f e r r o u s s u l f a t e i n t o hemoglobin (Table 1) was much g r e a t e r than when f e r r i c orthophosphate (Table 2) was the i r o n source. T h i s v a r i a b i l i t y i s d i s t u r b i n g s i n c e f e r r o u s s u l f a t e i s commonly used as a reference source of i r o n f o r b i o a v a i l a b i l i t y experiments, as w e l l as an i r o n supplement clinically. T y p i c a l l y , t h i s v a r i a b i l i t y i s d e a l t w i t h by expressing the hematinic responses of the unknowns r e l a t i v e to f e r r o u s s u l f a t e (Shah et a l . , 1979; C o c c o d r i l l i et a l . , 1976; Amine et a l . , 1972).

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1.

MAHONEY AND HENDRC IKS

Assay by Hemoglobin Regeneration

Using the e f f i c i e n c y of c o n v e r t i n g d i e t a r y i r o n i n t o hemoglobin, e f f e c t s of food p r o c e s s i n g procedures on the b i o a v a i l a b i l i t y of i r o n i n meat have been s t u d i e d . Farmer e t a l . (1977) showed that the b i o a v a i l a b i l i t y o f i r o n from mechanically deboned meat was l e s s than that from hand deboned meat; but, more metabolizable i r o n was a v a i l a b l e i n the mechanically deboned product because of i t s g r e a t e r i r o n content. There was no d i f f e r e n c e , however, between the i r o n b i o a v a i l a b i l i t y from mechanically deboned and hand deboned turkey frame meat ( A l l r e d , 1976). The d i f f e r e n c e i n i r o n b i o a v a i l a b i l i t y between the mechanically deboned turkey and the mechanically deboned beef might be a t t r i b u t e d to d i f f e r e n c e s i n abrasiveness o f the meat and bone mixture on the machinery, which would modify the amount and form o f i r o n i n the two products (Farmer e t a l . , 1977). The b i o a v a i l a b i l i t y of meat i r o n i s decreased due t o c u r i n g . T h i s decrease i s dose dependen n i t r i t e begins t o accumulat n i t r i t e was a s s o c i a t e d w i t h an apparent i n c r e a s e i n i r o n b i o a v a i l a b i l i t y , which was explained on the b a s i s o f some n i t r i c oxide b i n d i n g t o hemoglobin, rendering a f r a c t i o n o f i t unable t o c a r r y oxygen and thus s t i m u l a t i n g hematopoiesis. Severe atmospheric o x i d a t i o n o f beef r e s u l t s i n depressed i r o n b i o a v a i l a b i l i t y and growth i n r a t s while s i m i l a r o x i d a t i o n of turkey meat d i d not (Cardon et a l . , 1980). Based on the l i m i t e d data a v a i l a b l e , the r e l a t i v e b i o l o g i c a l v a l u e s o f i r o n sources are s i m i l a r whether determined by the s l o p e - r a t i o assay o r by e f f i c i e n c y o f conversion o f d i e t a r y i r o n i n t o hemoglobin (Table 3 ) . The most descrepancies are observed when the r e l a t i v e b i o l o g i c a l value i s estimated by method " c " i n Table 3. Much a d d i t i o n a l research i s r e q u i r e d t o determine the u t i l i t y of the simpler method of e v a l u a t i n g i r o n b i o a v a i l a b i l i t y by e f f i c i e n c y of converting d i e t a r y i r o n i n t o hemoglobin. I t does, however, take l e s s time than the s l o p e - r a t i o n method, apply t o food s t u f f s of r e l a t i v e l y low i r o n c o n c e n t r a t i o n ( I f o n , 1981), provide f o r d i r e c t measurements o f i r o n u t i l i z a t i o n , and apply to human subjects such as blood donors, anemic s u b j e c t s (Norby and S o l w e l l , 1977) and i n f a n t s (Garry, e t a l . , 1981). I t , t h e r e f o r e , has many p o t e n t i a l advantages as means o f evaluating iron b i o a v a i l a b i l i t y .

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

1

8

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

Table 3.

Comparison of B i o l o g i c a l Values o f D i f f e r e n t Sources R e l a t i v e to Ferrous S u l f a t e

Iron Source FePO.

R e l . B i o l . Value 51 d

FePO, FePO? FePO,

56 44.5 + 4.8°

FePO,

46

FePO FePO? FePO, i n Breakfast C e r e a l FePO, i n Breakfast C e r e a l

75 c

b

v

2 3

Enriched F l o u r Enriched F l o u r White Bread Turkey, raw Turkey, raw Whole egg, d r i e d Egg y o l k Beef, raw Beef, cooked

4.7 (18 * 0)

Solubility (mg/100 ml) 0.67 (13°C) 70.0 (16°C) 5.1 (18°C) 22.0

Ferric oxalate

9.4

very soluble

Zinc oxalate

4.9

0.79 (18°C)

Manganous oxalate (2H 0)

3.9

3.1 (25°C)

Cobalt oxalate

4.7

insoluble

Copper oxalate

6.3

2.5 (25°C)

2

Reprinted, with permission, from Ref. 35. Sp r i nge r-Ver1ag.

Copyright

1978,

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Physicochemical Properties of Food

4. CLYDESDALE

71

able from Fe-oxalate and FeCL^. A l s o the a d d i t i o n o f 0.75$ o x a l i c a c i d t o the d i e t d i d not depress i r o n absorption and i f anything appeared t o enhance i r o n u t i l i z a t i o n by r a t s . This study seemed t o be q u i t e c l e a r i n showing t h a t the oxa l a t e s do not seem t o i n h i b i t i r o n absorption. Therefore, other parameters w i l l have t o be evaluated t o e x p l a i n poor i r o n b i o a v a i l a b i l i t y from foods which might happen t o contain o x a l a t e s . No d i s c u s s i o n of complexation of i r o n would be complete without mentioning a s c o r b i c a c i d . Studies too numerous t o l i s t have c l e a r l y d e f i n e d the p o s i t i v e e f f e c t s of a s c o r b i c a c i d on i n c r e a s i n g the b i o a v a i l a b i l i t y of non-heme i r o n t o both animals and humans. The i n t e r a c t i o n s of Vitamin C and i r o n have r e c e n t l y been reviewed by Lynch and Cook (3Z)» However, upon examination i t would seem t h a t the e f f e c t s of a s c o r b i c are due t o f a c t o r s which i n c l u d e , but are not l i m i t e d t o , complex formation. I f one was w i l l i n t t s i m p l i f i c a t i o n th t important f a c t o r s i n v o l v e i r o n b i o a v a i l a b i l i t y migh 1. pH 2. complexation 3. o x i d a t i o n - r e d u c t i o n p o t e n t i a l Since the e f f e c t s o f pH and r e d u c t i o n p o t e n t i a l on i r o n b i o a v a i l a b i l i t y are t o be discussed next i t would seem l o g i c a l t o i n clude a d i s c u s s i o n of a s c o r b i c a c i d i n t h i s s e c t i o n . I f one examines the f o l l o w i n g r e a c t i o n , F e 3 (aq) + e" — > Fe (aq) (water s o l u t i o n , 25°C) i t i s found t h a t the standard r e d u c t i o n p o t e n t i a l i s +770mv i n d i c a t i n g a tendency t o occur spontaneously i n foods, since most foods have a standard r e d u c t i o n p o t e n t i a l o f UOOmv or s l i g h t l y l e s s . However, i f we examine the r e d u c t i o n h a l f - r e a c t i o n i n b a s i c solution, F e ( 0 H ) (s) + e~ * Fe(.0H) (s) + (OH)" i t i s found t h a t the standard r e d u c t i o n p o t e n t i a l i s -560mv i n d i c a t i n g non-spontaneity i n foods. I t i s c l e a r t h a t pH plays a r o l e i n maintaining i r o n ( I I ) i n s o l u t i o n . S o l u b i l i t y o f i r o n ( I I ) at low pH i s obviously a most important f a c t o r but the p o s s i b l e e f f e c t of the standard r e d u c t i o n p o t e n t i a l of the F e 3 redox couple at d i f f e r e n t pH values should not be overlooked. T h i s may e x p l a i n the r e s u l t s of L e i c h t e r and J o s l y n (3&)» Lee and Clydesdale (32) and others who found t h a t r e g a r d l e s s of the source the i r o n found i n bread and non-yeast leavened baked goods (high pH foods) r e s p e c t i v e l y , was mainly i n the i r o n ( I I I ) s t a t e and/or i n s o l u b l e . Such chemical changes w i l l obviously e f f e c t b i o a v a i l a b i l i t y and perhaps e x p l a i n some o f the r e s u l t s r e p o r t e d . For i n s t a n c e , B r i s e and H a l l b e r g (ko) determined t h a t 200-500 mg a s c o r b i c a c i d more than t r i p l e d the b i o a v a i l a b i l i t y of 30 mg o f i r o n administered as ferrous s u l f a t e while 100 mg or l e s s had l i t t l e e f f e c t . Simil a r l y , Cook and Monsen (kl) determined that the i n c r e a s e i n i r o n absorption from a semisynthetic meal was d i r e c t l y p r o p o r t i o n a l t o +

+ 2

3

2

+

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

72

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

the amount of a s c o r b i c a c i d added over a range of 25 t o 1000 mg. The apparent dependency o f the e f f i c a c y of a s c o r b i c a c i d on conc e n t r a t i o n seems t o i n d i c a t e t h a t the ascorbate e i t h e r formed a complex and/or c o n t r i b u t e d t o the s o l u b i l i t y , and/or maintained the i r o n i n the f e r r o u s s t a t e , s i n c e the standard r e d u c t i o n potent i a l f o r ascorbate i n water i s +400MV. S i m i l a r conclusions might a l s o be drawn from Hodson (h2) who found t h a t a f t e r 2 t o 5 months storage o f a l i q u i d weight c o n t r o l d i e t a r y w i t h an excess of asc o r b i c a c i d , the i r o n added as f e r r o u s s u l f a t e remained i n the f e r r o u s valence whereas the i r o n added as f e r r i c ortho phosphate has been s o l u b i l i z e d , i o n i z e d and reduced t o the b i v a l e n t form. These r e s u l t s i n d i c a t e t h a t perhaps ascorbate may e f f e c t b i o a v a i l a b i l i t y more than another complexing and reducing agent such as f r u c t o s e because i t i s a l s o an a c i d . I n an attempt t o c l a r i f y the chemical e f f e c t s o f pH and a s c o r b i c a c i d on i r o n valence model systems as w e l l a t o food m a t e r i a l s . They used a phthalate/HGl/NaOH b u f f e r system, s i n c e i t covered a s u i t a b l e pH range and d i d not r e a c t with added i r o n . Four i r o n sources; hydrogen reduced elemental ( E l ) , f e r r o u s s u l f a t e monohydrate (FS), f e r r i c ortho phosphate (FOP), and sodium f e r r i c EDTA t r i h y d r a t e (SFEDTA) were added t o a s e r i e s of b u f f e r s ranging from pH 2.2 t o 6.2. As w e l l , these same sources were evaluated at d i f f e r e n t molar l e v e l s of ascorbate, s i m i l a r t o those used by B r i s e and H a l l b e r g (kQ). I t was found t h a t pH was indeed a f a c t o r i n the i o n i z a t i o n and valence of the four i r o n compounds evaluated. EI and FS were completely converted t o f e r rous i o n w i t h i n k& hours at pH k.2 and below. I o n i z a t i o n o f FOP and SFEDTA was slower, incomplete, and r e s u l t e d i n l e s s f e r r o u s and more f e r r i c i r o n . At pH 2.7> most o f the i o n i z e d ( s o l u b l e ) i r o n remained i n or was converted t o the f e r r o u s form over a one month storage p e r i o d . The more r e a c t i v e ( l e s s o x i d i z e d ) i r o n compounds, EI and FS, remained 100$ i o n i c ( s o l u b l e ) at pH U.2 but showed some gradual o x i d a t i o n t o the t r i v a l e n t s t a t e . These r e s u l t s are c o n s i s t e n t w i t h the d i s c u s s i o n on redox p o t e n t i a l s mentioned previously. A s c o r b i c a c i d showed a r a t h e r c o n t r a d i c t o r y r o l e , at l e a s t at f i r s t glance. I t seemed t o promote the r e d u c t i o n of i r o n at low pH and the o x i d a t i o n of i r o n at higher pH v a l u e s . For i n s t a n c e , i n the s t u d i e s w i t h FS and EI at pH 2.7 i n the presence o f a s c o r b a t e , n e a r l y 100$ of the EI and FS added was i o n i z e d and i n the f e r r o u s valence, where i t remained f o r the d u r a t i o n o f the study (one month). However, at pH 6.2, the presence of ascorbate g r e a t l y i n c r e a s e d the i o n i z a t i o n ( s o l u b i l i z a t i o n ) o f both EI and FS but the newly i o n i z e d i r o n remained i n the f e r r o u s valence form f o r only k8 hours before f u r t h e r o x i d a t i o n began t o occur. In the case of EI about 50$ o f the i o n i c i r o n was i n the f e r r o u s valence, and 50$ i n the f e r r i c s t a t e a f t e r k weeks. While w i t h FS a t pH 6.2, f e r r i c hydroxide p r e c i p i t a t e s occurred without aseorbate uttzhln one week. The presence o f ascorbate i n h i b i t e d

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

4.

CLYDESDALE

Physicochemical Properties of Food

73

the formation of these hydroxides keeping most of the i r o n i n the i o n i c f e r r i c form as measured "by the method of Lee and Clydesdale ( 3 9 ) which u t i l i z e d "bathophenanthroline. I t i s p o s s i b l e that the ascorbate i n h i b i t e d the formation of the hydroxides by forming a complex with the f e r r i c i r o n i n the same manner as described by Conrad and Schade (kk). I f t h i s was the case, the iron-ascorbate complex must have been d i s r u p t e d by the iron-bathophenanthroline a n a l y s i s , s i n c e the a n a l y s i s i n d i c a t e d the presence of i o n i c f e r r i c i r o n . These observations by Nojeim and Clydesdale (V3) as w e l l as other observations i n the l i t e r a t u r e on the mode of a c t i o n of a s c o r b i c a c i d cannot be explained simply. For example, i f the e f f i c a c y of a s c o r b i c a c i d i n i r o n n u t r i t u r e was due s o l e l y t o i t s r o l e as c h e l a t i n g agent then i t should not enhance b i o a v a i l a b i l i t y i n almost every i n s t a n c e . Further confusion a r i s e s when i t i s noted that low pH a s c o r b i c a c i d promotes the r e d u c t i o n of i r o n (h5), but i t s presence at high pH seems to promote the o x i d a t i o n of i r o n (k3). Smith an have pro-oxidant p r o p e r t i e l i p i d p e r o x i d a t i o n i n milk. They considered t h a t these were a t t r i b u t a b l e to two p r o p e r t i e s of a s c o r b i c a c i d : the a b i l i t y t o reduce c u p r i c copper t o the cuprous form, and a s p e c i f i c a s s o c i a t i o n between a s c o r b i c a c i d and copper that i n some unexplained manner increases prooxidant a c t i v i t y . These studies support the observed prooxidant e f f e c t of asc o r b i c a c i d at high pH (milk) i n the presence of copper but not i n the presence of i r o n . In order t o e x p l a i n the e f f e c t s observed by Nojeim and Clydesdale (U3), I would l i k e to propose a mechanism f o r the act i o n of a s c o r b i c a c i d i n the presence of i r o n which might e x p l a i n how i t could act as both a reducing agent and a prooxidant as w e l l as perhaps shedding some more l i g h t on i t s r o l e i n the chemi s t r y and thus the b i o a v a i l a b i l i t y of i r o n . This proposed mechanism i s based on the i n t e r r e l a t i o n s h i p between s o l u b i l i t y , pH, r e d u c t i o n p o t e n t i a l , and c h e l a t i o n i n a s o l u t i o n of i r o n and a s c o r b i c a c i d . At low pH, i t w i l l be remembered, F e 3 and F e are s o l u b l e and probably e x i s t as t h e i r r e s p e c t i v e hydrates with the standard r e d u c t i o n p o t e n t i a l of F e 3 (aq) +e~ -*Fe 2 (aq) being +770mv. In the presence of ascorbate which has a standard r e d u c t i o n potent i a l of +UU0mv the formation of F e w i l l take p l a c e spontaneously h8) and r e a c t i o n 1 (Figure 6) w i l l go to the r i g h t . However, at the same time, at a low pH. both a F e ^ -ascorbate and a F e 3 -ascorbate complex may form (M+, h8), represented by r e a c t i o n s 2 and k (Figure 6 ) . Upon a d d i t i o n of f e r r i c i r o n t o an a s c o r b i c a c i d s o l u t i o n r e a c t i o n 2 w i l l probably take p l a c e more r a p i d l y than r e a c t i o n 1, but i n time r e a c t i o n 1 w i l l predominant i f the pH i s maintained at a low l e v e l , as observed by Nojeim and Clydesdale (1*3), with the o v e r a l l e f f e c t being r e d u c t i o n . Thus ascorbate, due t o i t s r e d u c t i o n p o t e n t i a l r e l a t i v e t o i r o n at a low pH, and +

+

+ 2

+

+ 2

+

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

+

74

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

Fe

3+

+ Ligand + e"

4-

Ligand + e~ 4 Figure 6.

-+

Fe

2+

+ Ligand

2

Fe * — Ligand

Interrelationship between iron (II), iron (III), and their respective ligand complexes in solution.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

4.

CLYDESDALE

75 Physicochemical Properties of Food

i t s a b i l i t y t o de-complex w i l l act as a reducing agent w i t h time. T h i s i m p l i e s t h a t with time i n an a c i d s o l u t i o n c o n t a i n i n g F e 3 and ascorbate t h a t the F e 2 form w i l l predominate and complexes of F e 3 -ascorbate w i l l tend t o d e s t a b i l i z e . As the pH i s r a i s e d s e v e r a l chemical events occur which tend to r e d i r e c t the flow i n F i g u r e 6 which was j u s t o u t l i n e d . As p o i n t e d out p r e v i o u s l y , the hydrates o f F e 3 and F e begin t o l o s e protons as the pH i s r a i s e d , thus forming t h e i r r e s p e c t i v e hydroxides which are i n c r e a s i n g l y l e s s s o l u b l e , with the standard r e d u c t i o n p o t e n t i a l of Fe(0H)3 (s) + e — > Fe (0H)2 (s) = (OH)" (aq) being -560mv. T h i s means t h a t a t high pH values the standard r e d u c t i o n p o t e n t i a l s o f the two h a l f r e a c t i o n s f a v o r the formation of Fe(0H)3 and r e a c t i o n 1 (Figure 6) w i l l go t o the l e f t , a cond i t i o n opposite t o t h a t which occurs at low pH v a l u e s . Since most foods have a standard r e d u c t i o n p o t e n t i a l o f +U00 mv or l e s s , the formatio f Fe(0H)3 with without ascorbate. Therefore, i s r a i s e d would seem t by forming a reasonably s t a b l e F e 3 -ascorbate complex, thus f a v o r i n g the downward d i r e c t i o n o f r e a c t i o n 2 (Figure 6) and thereby promoting more o x i d a t i o n t o F e 3 by i n d i r e c t l y f a v o r i n g the l e f t ward flow i n equation 1 ( F i g u r e 6 ) . Such s t a b i l i t y was found by Conrad and Schade (kk) at pH values from k t o 9Thus, at h i g h pH v a l u e s , the o v e r a l l e f f e c t which would be noted i n a s o l u t i o n of i r o n and ascorbate would be o x i d a t i o n . T h i s p o s t u l a t i o n f o r a r e a c t i o n pathway t o e x p l a i n the seemi n g l y c o n t r a d i c t o r y data which i m p l i c a t e s a s c o r b i c a c i d as both a reductant and oxidant i s not intended t o be a f i n a l e x p l a n a t i o n . However, i t does seem t o f i t many o f the observations i n the l i t e r a t u r e which under other explanations seem t o be almost imposs i b l e from a chemical standpoint or simply t o be c o n t r a d i c t o r y . Conrad and Schade (kk) c o u l d not form a s o l u b l e F e 3 - a s c o r bate complex s t a r t i n g a t an a l k a l i n e pH, but c o u l d at an a c i d pH and found i t t o be s t a b l e even under a l k a l i n e c o n d i t i o n s . T h i s suggests t h a t from a p r a c t i c a l viewpoint, f o r t i f i c a t i o n might be best accomplished with an a c i d s o l u t i o n c o n t a i n i n g a s c o r b i c a c i d and i r o n I I I or some p u r i f i e d e x t r a c t of the f e r r i c - a s c o r b a t e complex analagous t o Saltman*s suggestion f o r the use of a f e r r i c f r u c t o s e complex. However, Sayers et a l (Jf£) suggests t h a t even i f a f e a s i b l e method were found f o r supplementing foods with asc o r b i c a c i d and i n o r g a n i c i r o n , n u t r i t i o n a l b e n e f i t s would only be a n t i c i p a t e d with uncooked or b o i l e d foods s i n c e they found t h a t a s c o r b i c a c i d e f f i c a c y was l o s t due t o o x i d a t i v e d e s t r u c t i o n at the high temperatures r e q u i r e d f o r baking. In order t o more f u l l y understand the mode o f a c t i o n of a s c o r b i c a c i d and s u b s t a n t i a t e the foregoing hypothesis, we are c u r r e n t l y i n v e s t i g a t i n g the s t a b i l i t y constants of the complexes i n much more d e t a i l . The thermodynamic s t a b i l i t y constants between F e -ascorbate and F e 3 - ascorbate are important s i n c e t h e i r r e l a t i v e values w i l l +

+

+

+

+ 2

:

+

+

+

+ 2

+

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

76

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

not only a f f e c t the s t a b i l i t y and amount of Fe or Fe ° i n sol u t i o n but a l s o w i l l a f f e c t the r e a c t i o n flow as shown i n F i g u r e 6, and determine to some extent the exchange of i r o n with other l i g a n d s i n food as w e l l as with the b i o l o g i c a l t r a n s f e r of i r o n to t r a n s f e r r i n . However, F o r t h and Rummel (j>0) argue t h a t the thermodynamic s t a b i l i t y constants of i r o n are of l i m i t e d value i n d e f i n i n g the s t a b i l i t y of complexes and chelates i n b i o l o g i c a l media. They base t h i s view on the o b s e r v a t i o n t h a t the thermodynamic s t a b i l i t y constant i s an e q u i b i l i b r i u m constant d e t e r mined i n a d e f i n i t e medium and as such provides no i n f o r m a t i o n regarding the v e l o c i t y of a s s o c i a t i o n and d i s s o c i a t i o n of comp l e x e s , e s p e c i a l l y when the complex formation takes p l a c e i n the presence of competing l i g a n d s or metals and i n a s s o c i a t i o n with o x i d o r e d u c t i o n processes. In other words, they s t a t e , a high thermodynamic s t a b i l i t y constant does not i n d i c a t e t h a t a complex i s i n e r t . Therefore, they propose t h a t when a s s e s s i n g the s t a b i l i t y of i r o n complexe i n the k i n e t i c r a t h e r tha suggest t h a t the h a l f - t i m e of the i r o n exchange of complexes can be used as a measure of the k i n e t i c s t a b i l i t y based i n p a r t on the extensive s t u d i e s of Aaso et a l (51.), Bates ejt a l (5£, 53.), and B i l l u p s et a l (5k). I t can be seen i n Table IV that d e s p i t e the small d i f f e r e n c e s i n the thermodynamic s t a b i l i t y constants, these i r o n chelates have very d i f f e r e n t k i n e t i c s t a b i l i t i e s as measured by the exchange of i r o n with t r a n s f e r r i n , a b i o l o g i c a l acceptor f o r i r o n . This argument i s l o g i c a l and s c i e n t i f i c a l l y accurate with respect to the t r a n s f e r of i r o n i n the body. However, i t does not address the p o t e n t i a l importance of the thermodynamic s t a b i l i t y constants of the two common valence forms of i r o n ( I I and III) with l i g a n d s i n food. When i r o n i s added to a food the environment i s going t o a f f e c t the valence s t a t e as has been discussed p r e v i o u s l y . One of the parameters which might maintain a p a r t i c u l a r valence s t a t e i n the face of adverse environmental c o n d i t i o n s , such as pH or redox p o t e n t i a l , i s the s t a b i l i t y of i t s complex. Theref o r e , e i t h e r the F e or F e 3 form might be maintained i f one formed a complex with a g r e a t e r thermodynamic s t a b i l i t y than the other as discussed p r e v i o u s l y . Therefore, i t would seem t h a t the importance of the thermodynamic s t a b i l i t y constant should not be discounted because i t could have a great deal of relevance with respect t o s o l u b i l i t y and maintenance of a s p e c i f i c i r o n valence w i t h i n a given food system. Nojeim and Clydesdale (U3) and Nojeim et a l (.55) a l s o attempted t o u t i l i z e the r e s u l t s obtained i n t h e i r model systems as a p r e d i c t o r of the e f f e c t s of both pH and r e d u c t i o n p o t e n t i a l of a c t u a l foods on the chemical status of i r o n . I t would be most h e l p f u l t o be able to p r e d i c t the chemical f a t e of added i r o n simply by measuring the pH or r e d u c t i o n p o t e n t i a l of the food. + 2

+

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

4. CLYDESDALE Table IV.

Fe

Physicochemical Properties of Food

11

K i n e t i c s t a b i l i t y o f some Fe ^ chelates compared w i t h t h e i r thermodynamic s t a b i l i t y constants

Chelates o f :

Nitrilotriacetic

Thermodynamic Half-Time o f Iron Exchange S t a b i l i t y Constant with Iron-Free T r a n s f e r r i n (Kinetic S t a b i l i t y )

acid

23

3 sec.

C i t r i c acid

25

8 hours

Ethylenediaminetetraacetic acid

25

k days

Reprinted, with permission, from Ref. 50. Copyright 1973, American P h y s i o l o g i c a l S o c i e t y .

In order t o evaluate pH as a p r e d i c t o r o f i r o n s t a t u s i n food, four i r o n sources, E I , FS, FOP and SFEDTA were added t o three foods o f d i f f e r e n t pH v a l u e s ; cranberry j u i c e , tomato j u i c e and a c h e m i c a l l y leavened b i s c u i t dough. The percent i o n i z a t i o n and conversion t o f e r r o u s i r o n were measured and i t was found t h a t t h e chemical changes i n the added i r o n f o l l o w e d the same trends observed i n the p h t h a l a t e b u f f e r s . F i g u r e s 7 and 8 show the r e s u l t s obtained with both the b u f f e r s and the foods i n terms of the percentage i o n i z a t i o n o f the i r o n and the percentage o f t h a t i o n i z e d i n the f e r r o u s s t a t e , r e s p e c t i v e l y . From these r e s u l t s , i t may be seen t h a t pH, though not q u a n t i t a t i v e l y , i s an important parameter t o c o n s i d e r when p r e d i c t i n g the general trends of chemical changes which i r o n might undergo when added t o a food. As p o i n t e d out p r e v i o u s l y the redox p o t e n t i a l o f t h e reduct i o n o f f e r r i c t o f e r r o u s i o n i s +770 mv r e l a t i v e t o the standard hydrogen e l e c t r o d e (SHE). T h i s means t h a t t h i s r e a c t i o n w i l l be d r i v e n i n the forward d i r e c t i o n whenever f e r r i c i o n i s present i n a system whose o v e r a l l redox p o t e n t i a l i s lower than +770 mv. How much lower the system i s may be r e l a t e d t o t h e l e v e l o f comp l e t i o n t o which the r e a c t i o n i s c a r r i e d . S u r p r i s i n g l y , as p o i n t e d out by Nojeim ($6), t h e study o f r e d u c t i o n p o t e n t i a l s as p o s s i b l e p r e d i c t o r s o f the chemical f a t e o f i r o n i n food has l a r g e l y been neglected. I n f a c t , l i t e r a t u r e d i s c u s s i n g any e f f e c t s o f redox p o t e n t i a l on food chemistry i s sparse. Most o f

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

TOMATO JUICE pH 4.3

B I S C U I T DOUGH pH 6.5

SFEDTA 18/4

Figure 7. Percentage ionization of iron additives predicted by buffers and actually found in foods. EI, elemental iron; FS, ferrous sulfate; FOP, ferric orthophosphate; SFEDTA, sodium ferric EDTA trihydrate. (Reproduced, with permission, from Ref. 43. Copyright 1981, Institute of Food Technologists.)

CRANBERRY JU CE pH 2.7

FOP 1/0

I

I

H

2 C

00

-J

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982. 2.7

JUICE pH

T O M A T O

J U I C E 4.3

pH

6.S

B I S C U I T

DOUGH

Figure 8. Percentage of ionized iron in the ferrous form predicted from buffers and actually found in foods. EI, elemental iron; FS, ferrous sulfate; FOP, ferric orthophosphate; SFEDTA, sodium ferric EDTA trihydrate. (Reproduced, with permission, from Ref. 43. Copyright 1981, Institute of Food Technologists.)

pH

CRANBERRY

80

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

the work i n t h i s area has been on how redox p o t e n t i a l a f f e c t s m i c r o b i a l growth. The f i r s t evidence of a r e l a t i o n s h i p between redox p o t e n t i a l and i r o n valence was observed by K i r c h et a l (57) • A v a r i e t y o f i r o n f o r t i f i e d foods were subjected t o an a r t i f i c i a l g a s t r i c d i g e s t i o n w i t h pepsin and/or HC1. A f t e r these treatments redox pot e n t i a l and pH measurements were recorded. Reduction of f e r r i c t o f e r r o u s i o n was a l s o analyzed. I t was found t h a t pH and redox p o t e n t i a l values were s i m i l a r between each of the foods. This could have been a r e s u l t of measurement a f t e r the acid/enzyme d i g e s t i o n s . Redox p o t e n t i a l s were a l l w i t h i n 75 mv e i t h e r way of +U25 mv, SHE. A c c o r d i n g l y , r e d u c t i o n t o f e r r o u s i o n was expected and observed. Where the degree o f r e d u c t i o n was low, the degree o f complex formation was h i g h . In f u r t h e r work Bergeim and K i r c h (58) s t u d i e d the r e d u c t i o n of i r o n i n a c t u a l g a s t r i c d i g e s t i o n . Samples were taken f r o n the stomachs o f subjects s h o r t l y a f t e r i n g e s t i o n o f the same i r o R e s u l t s were comparabl o f the i r o n i n each food was observed i n v i v o . Even though no l i n e a r r e l a t i o n s h i p was seen they concluded t h a t the degree o f r e d u c t i o n depends on the redox p o t e n t i a l of the e n t i r e food system r a t h e r than on the c o n c e n t r a t i o n of one potent reducing compound present i n a l i m i t e d amount. In other words a s c o r b i c a c i d added t o an i r o n f o r t i f i e d food would not promote the r e d u c t i o n of f e r r i c t o f e r r o u s i o n unless the o v e r a l l redox p o t e n t i a l were f a v o r a b l e . T h i s b e l i e f was supported through the r e s e a r c h o f Unnikrishnan et a l (59) which i n v o l v e d the study of the e f f e c t of copper and m i c r o b i a l metabolism on o x i d a t i o n - r e d u c t i o n r e a c t i o n s occurring i n milk. These authors observed decreases i n the redox p o t e n t i a l of the system upon a d d i t i o n o f reducing agents. The p o t e n t i a l i n c r e a s e d as a s c o r b i c a c i d became o x i d i z e d t o dehydroascorbic a c i d . Reducing agents are o f t e n added t o foods f o r t h e i r a n t i oxidant p r o p e r t i e s . But even i n foods devoid o f these a d d i t i v e s , there e x i s t many innate redox couples. Endogenous electrochemic a l l y a c t i v e compounds i n c l u d i n g vitamins C and E, organic a c i d s , unsaturated f a t s , reducing sugars, quinones, oxygen and p o l y v a l e n t metal ions a l l c o n t r i b u t e t o a food's o v e r a l l redox p o t e n t i a l . I t should a l s o be noted t h a t these compounds and thus a food's redox p o t e n t i a l are subject t o changes during p r o c e s s i n g . I t seems apparent then, t h a t the f i n a l form o f i r o n i n a food system should be d i r e c t l y i n f l u e n c e d by the r e d u c t i o n p o t e n t i a l o f t h a t system and anything which a f f e c t s the r e d u c t i o n p o t e n t i a l might a f f e c t the b i o a v a i l a b i l i t y of i r o n i n the system. The enhancement of b i o a v a i l a b i l i t y o f i r o n by the reducing compounds a s c o r b i c a c i d and f r u c t o s e i s w e l l known and has been d i s c u s s e d . However, i t should be reemphasized t h a t the a d d i t i o n of these, and other reducing agents, may i n p a r t i n c r e a s e i r o n b i o a v a i l a b i l i t y by t h e i r e f f e c t on redox p o t e n t i a l as w e l l as by t h e i r a b i l i t y

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

4.

CLYDESDALE

81 Physicochemical Properties of Food

t o form absorption enhancing chelates as suggested by Conrad and Schade (kk) and Saltman (k). Nojeim e t a l (j>£) u t i l i z e d an e l e c t r o l y t i c c e l l model system, f r e e o f oxygen, b u f f e r e d w i t h phthalate t o pH k.2 and designed t o provide a redox p o t e n t i a l between +300 and +650 mv t o evaluate the e f f e c t o f redox p o t e n t i a l on the i o n i z a t i o n and valence o f four i r o n compounds; E I , FS, FOP, and SFEDTA, d e s c r i b e d p r e v i o u s l y (ij3). Data obtained were used t o p r e d i c t i o n i z a t i o n and valence trends i n a c t u a l food systems o f d i f f e r e n t redox p o t e n t i a l s . Redox p o t e n t i a l was found t o have no s i g n i f i c a n t e f f e c t on the i o n i z a t i o n o f any o f the f o u r compounds evaluated. However, i n the c a s e o f E I and FS lower p o t e n t i a l s i n the environment favored the reduced form o f i r o n . T h i s i s t o be expected s i n c e a g r e a t e r d i f ference between the +770 mv p o t e n t i a l o f the f e r r i c t o f e r r o u s couple and the p o t e n t i a l o f i t s chemical environment would cause the r e d u c t i o n t o be more spontaneous In the case o f SFEDT l i t t l e e f f e c t was seen f a c t t h a t i n these two cases s o l u b i l i t y was low (10$) producing a t o t a l o f only 2.5 ppm i n s o l u t i o n b r i n g i n g i n t o question the v a l i d i t y o f the a n a l y t i c a l technique used t o d i f f e r e n t i a t e the b i v a l e n t from the t r i v a l e n t form. In u t i l i z i n g these r e s u l t s t o p r e d i c t i o n i z a t i o n and valence i n food m a t e r i a l s ; tomato j u i c e (Eh=2l*0), b i s c u i t dough (Eh=3^0), cranberry j u i c e (Eh=i*00) one would expect t h a t the percentage i r o n i o n i z e d would be the same i n each food s i n c e redox p o t e n t i a l was found t o have no e f f e c t i n the model system on i o n i z a t i o n . However research p r e v i o u s l y c i t e d (k3) showed t h a t t h i s was not the case as was seen i n F i g u r e 7. Obviously, t h i s means t h a t reduct i o n p o t e n t i a l o f the food m a t e r i a l i s not a major f a c t o r i n determining i o n i z a t i o n o f added i r o n . Model system r e s u l t s were more c o n s i s t e n t with a c t u a l foods, however, i n the p r e d i c t i o n o f the amount o f b i v a l e n t i r o n formed (Figure 9 ) . The percentage f e r r o u s i r o n was r e l a t i v e l y high f o r a l l f o u r i r o n compounds i n tomato j u i c e and cranberry j u i c e where i o n i z a t i o n was a l s o high but not i n b i s c u i t dough where i o n i z a t i o n was low. This study shows a r e l a t i o n s h i p , although not h i g h l y c o r r e l a t e d , between redox and chemical behavior o f i r o n , f u r t h e r emphasizing the need t o evaluate r e d u c t i o n p o t e n t i a l s p r i o r t o fortification. The need t o understand the chemical behavior o f i r o n i n foods i s e s s e n t i a l t o a c l e a r understanding o f subsequent b i o l o g i c a l behavior. There are many f a c t o r s which impinge upon the chemic a l status o f i r o n i n food with the physicochemical being o n l y one. However, i t i s hoped t h a t the i n t e r r e l a t i o n s h i p and importance o f some o f these f a c t o r s might be considered more f u l l y . I n t h i s way, a s p i r i t o f s c i e n t i f i c cooperation might very w e l l prov i d e answers t o the problems which seem t o i n h i b i t the existence of a p o p u l a t i o n r e p l e t e i n terms o f i r o n n u t r i t u r e .

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

JUICE Eh

BISCUIT

Eb

D O U G H CRANBERRY 340mv

JUICE 400mv

Figure 9. Percentage of ionized iron in the ferrous state predicted from model systems with a known Eh and found in foods of varying Eh values. EI, elemental iron; FS, ferrous sulfate; FOP, ferric orthophosphate; SFEDTA, sodium ferric EDTA trihydrate. (Reproduced, with permission, from Ref. 55. Copyright 1981, Institute of Food Technologists.)

Eh 240mv

TOMATO

4. CLYDESDALE

Physicochemical Properties of Food

83

Acknowledgments This chapter i s Paper No. 2050, Massachusetts A g r i c u l t u r a l Experiment S t a t i o n , U n i v e r s i t y of Massachusetts at Amherst. This work was supported i n part from Experiment S t a t i o n Project No. NE-116 and a grant from the General M i l l s Foundation.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

U.S. Dept. of Agriculture, "Family Economics Review", Science & Education Administration, USDA, Beltsville, Md., Spring 1980, 22 pp. Lee, K.; Clydesdale, F.M. CRC Critical Reviews in Food Science and Nutrition 1979, 11, 117. Zoller, J.M.; Wolinsky, I.; Paden, C.A.; Hoskin, J.C.; Lewis, K.C.; Lineback, D.R.; McCarthy, R.D. Food Technol. 1980, 34, 38. Saltman, P., J. Chem Spiro, T.G.; and Saltman, R. "Iron in Biochemistry and Medicine", Jacobs, A. and Wormwood, M. Eds. Academic Press, NY 1974. Saltman, P.; Hegenauer, J.; Christopher, J . Ann. Clin. Lab Sci. 1976, 6, 167. Pollack, S.; Kaufman, R.N.; Crosby, W.H. Blood 1964, 24. 577. Bates, G.W.; Hegenauer, J.C.; Renner, J.; Saltman, P.; Spiro, G. Bioinorg. Chem 1973, 2, 311. Heinrich, H.C.; Gabbe, E.E.; Bruggemann, J.; Oppitz, K.H. Nutr. Metab. 1974, 17, 236. Loria, A.; Medal, L.S.; Elizando, J. Am. J . Clin. Nutr. 1962, 10, 124. Rendleman, J.A. Jr. "Advances in Carbohydrate Chemistry"; Ed. Woltram, M. Academic Press, NY, 1966; 21, 209. Amine, E.K.; Hegsted, D.M. J . Agric. Food Chem. 1975, 22, 740. Derman, D.P.; Bothwell, T.H.; Torrance, J.D.; Bezwoda, W.R.; MacPhail, A.P.; Kew, M.C.; Sayers, M.H.; Disler, P.B.; Charlton, R.W. Br. J . Nutr. 43, 1980, 271. Bachran, K.; Bernhard, R.A. J . Agric. Food Chem. 1980, 28, 536. Layrisee, M.; Martinez-Torres, C. "Progress in Hematology", Vol. 7. Brown, E.B. and Moore, C.V. Eds. Grune and Stratton, NY, 1971; p 137. Mahoney, A.W.; Hendricks, D.G. J . Food Sci. 1978, 43, 1473. Van Campen, D. J . Nutr. 1973, 103, 139. Welch, R.M.; Van Campen, D.R. J . Nutr. 1975, 105, 253. Nelson, K.J.; Potter, N.N. J . Food Sci. 1979, 44, 104. Nelson, K.J.; Potter, N.N. J . Food Sci. 1980, 45, 52. Cheryan, M. CRC Critical Rev. Food Sci. Nutr. 1980, 13, 297. Haug, A.; Smidsrod, O. Acta Chem. Schan. 1970, 24, 843. Schweiger, R.G.; Kolloid-Z., Z. X. Polymere. 1966, 208, 28. Camire, A.L.; Clydesdale, F.M. J . Food Sci. 1981, 46, 548. Barry, J.A.; Halsey, G.D. Jr. J . Phys. Chem. 1963, 67, 1698.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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26. Angyal, S.J. Pure Applied Chem. 1973, 35, 131. 27. Grant, G.T.; Morris, E.R.; Rees, D.A.; Smith, P.J.C.; Thorr,D. FEBS Letters 1973, 32, 195. 28. Furda, I. "Dietary Fibers, Chemistry and Nutrition", Ed. Inglett, G.E. and Falkehag, S.I. Academic Press: NY, 1979;p31. 29. Nagyvary, J.; Bradbury, E.L. Biochem and Biophysical Res. Comm. 1977, 77, 592. 30. Ranhotra, G.S.; Lee, C.; Gelroth, J.A.; Nutr. Rept. Intern. 1979, 19, 851. 31. Ranhotra, G.C.; Lee, C.; Gelroth, J.A.; Cereal Chem. 1979, 56, 156. 32. Lee, K.; Clydesdale, F.M. J. Food Sci. 1980, 45, 1500. 33. Anderson, N.E.; Clydesdale, F.M. J. Food Sci. 1980, 45, 1533. 34. Anderson, N.E.; Clydesdale, F.M. J. Food Sci. 1980, 45, 336. 35. Hodgkinson, A.; Zarembski, P.M. Calc. Tiss. Res. 1968, 2, 115. 36. Van Campen, D.R.; Welch R.M J Nutr 110 1618 37. Lynch, S.R., Cook 38. Leichter, J.; Joslyn 39. Lee, K.; Clydesdale, F.M. J. Food Sci. 1979, 44, 549. 40. Brise, H.; Hallberg, L. Acta. Med. Scand. Supp. 1962, (Stockholm) 171, (Supp. 376) 51. 41. Cook, J.D.; Monsen, E.R. Amer. J. Clin. Nutr. 1977, 30, 235. 42. Hodson, A.Z. J. Agric. Food Chem. 1980, 18, 946. 43. Nojeim, S.J.; Clydesdale, F.M.; J. Food Sci. 1981, 46, 606. 44. Conrad, M.E.; Schade, S.G. Gastroenterology. 1968, 55, 35. 45. Erdey, L.; Svehla, G. "Ascorbinometric Titrations". Akademiai Kiado, Budapest 1973; 183 pp. 46. Smith, G.J.; Dunkley, W.L. J. Dairy Sci. 1962, 45, 170. 47. Smith, G.J.; Dunkley, W.L. J. Food Sci. 1962, 27, 127. 48. Gorman, J.E.; Clydesdale, F.M. J. Food Sci. 1982, In press. 49. Sayers, M.H.; Lynch, S.R.; Jacobs, P.; Charlton, R.W.; Bothwell, T.W.; Walker, R.B.; Mayet, F. British J. Haem. 1973, 24, 209. 50. Forth, W.; Rummel, W. Physiol. Rev. 1973, 53, 724. 51. Aaso. R.; Malmström, B.G.; Saltman, R.; Vanngard, T. Biochem. Biophys. Acta. 1963, 75, 203. 52. Bates, G.W.; Billups, C.; Saltman, P. J. Biol. Chem. 1967, 242, 2810. 53. Bates, G.W.; Billups, C.; Saltman, P.; J. Biol. Chem. 1967, 242, 2816. 54. Billups, C.; Pape, L.; Saltman, P. J. Biol. Chem. 1967, 242, 4284. 55. Nojeim, S.J.; Clydesdale, F.M.; Zajicek, O.T. J. Food Sci. 1981, 46, 606. 56. Nojeim, S.J. "M.S. Thesis", Univ. of Mass., Amherst, MA 1981; 96 pp. 57. Kirch, E.R.; Bergeim, O.; Kleinberg, J.; James, J. J. Biol. Chem. 1947, 171, 687. 58. Bergeim, O.; Kirch, E.R. J. Biol. Chem. 1948, 172, 591. 59. Unnikrishnan, V.; Rao, D.S.; Rao, M.B. J. Milk and Food Tech. 1976, 39, 397. RECEIVED June 2, 1982.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

5 Ascorbic Acid: An Enhancing Factor in Iron Absorption ELAINE R. MONSEN University of Washington, Division of Human Nutrition, Dietetics, and Foods and Department of Medicine, Seattle, WA 98195

Ascorbic acid concomitantl d enhance heme iron absorptio the model estimating bioavailable dietary iron. The 500 mgs iron store Reference Individual is assumed to absorb 23% of ingested heme iron (estimated at 40% of meat, fish or poultry iron) and 3-8% of ingested nonheme iron (plant iron plus remaining meat, fish, poultry iron). The quantity of enhancing factors consumed at a specific meal, i.e. mgs ascorbic acid plus gms cooked meat/fish/poultry, determines % absorption of nonheme iron: 1.

ΣEF < 75: % = 3 + 8.93 log

2.

ΣEF

-

+ ≥ 75:

n

%= 8

HANES 2 (1976 - 80) reports low intake of iron and ascorbic acid for large population segments, especially females below poverty line, e.g. at 10th percentile 4 mg iron and 7 mg ascorbic acid per day. Prevention of iron deficiency in populations not sustaining chronic blood loss is possible by judicious selection of diets which enhance the bioavailability of dietary iron. The recent decades have produced significant research on the availability of iron asitisaffected by various dietary components, those which enhance as well as those which inhibit iron absorption. This has allowed for the first time the quantification of dietary effects on a trace metal and the development of a model whereby the quantity of bioavailable iron in a diet may be estimated. This paper will discuss four related areas: the major 0097-6156/82/0203-0085$06.00/0 © 1982 American Chemical Society In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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s e c t i o n w i l l be focused on the r o l e o f a s c o r b i c a c i d i n nonheme i r o n absorption; the second s e c t i o n w i l l d i s c u s s b r i e f l y the model f o r e s t i m a t i n g the q u a n t i t y o f a v a i l a b l e i r o n ; the t h i r d s e c t i o n w i l l d e a l w i t h c u r r e n t estimates o f d i e t a r y i n t a k e o f i r o n and a s c o r b i c a c i d i n the United S t a t e s ; and the l a s t s e c t i o n w i l l d i s c u s s b r i e f l y the i n t e r r e l a t i o n s h i p s of a s c o r b i c a c i d and i r o n i n abnormal s t a t e s of metabolism. The r o l e of a s c o r b i c a c i d i n nonheme i r o n a b s o r p t i o n Radioisotope techniques have allowed p r e c i s e measurement o f dietary i r o n absorption. I n i t i a l s t u d i e s u t i l i z e d t e s t meals o f i n d i v i d u a l food which had been i n t r i n s i c a l l y l a b e l e d with radio-a c t i v e i r o n p r i o r to h a r v e s t i n g (1,2), U t i l i z a t i o n o f these s i n g l e food meals allowed a rank order to be e s t a b l i s h e d among the t e s t e d foods. Subjects could serve as t h e i r own c o n t r o l s when an i d e n t i c a l r e f e r e n c T h i s reference dose of at a higher r a t e i n i r o n d e f i c i e n t s u b j e c t s than i n i r o n r e p l e t e s u b j e c t s ; a r a t i o o f the a b s o r p t i o n o f the t e s t meal to the r e f e r e n c e dose allowed comparisons to be made between i n d i v i d u a l s u b j e c t s . As these e a r l y s t u d i e s were l i m i t e d to study o f s i n g l e food items an e f f o r t was made to extend the technique by developing designs u t i l i z i n g e x t r i n s i c a l l y tagged t e s t meals (3,4). U t i l i z a t i o n o f these techniques has given evidence that d i e t a r y i r o n forms two separate pools i n the gut, one a p o o l o f heme i r o n and the other a p o o l of nonheme i r o n . The predominate source o f i r o n i n human d i e t s i s i n the form o f nonheme i r o n ( 5 ) , A s c o r b i c a c i d as an enhancing f a c t o r i n nonheme i r o n absorpt i o n has been f r e q u e n t l y observed, C a l l e n d e r , e t a l . , f e d i n t r i n s i c a l l y l a b e l e d hen eggs i n a b r e a k f a s t meal composed o f bread, b u t t e r , jam, eggs, and t e a o r c o f f e e to 26 s u b j e c t s ; when an a d d i t i o n o f 100 mis o f orange j u i c e was added t o t h i s meal a b s o r p t i o n i n c r e a s e d to 280% o f the c o n t r o l meal (6), Cook, e t a l . , provided a meal o f i n t r i n s i c a l l y l a b e l e d maize to sub^ j e c t s and observed that when 500 mgs o f a s c o r b i c a c i d was added that a b s o r p t i o n o f both i n t r i n s i c and e x t r i n s i c a l l y l a b e l e d i r o n was 6 times higher (3), Sayers, e t a l , , u t i l i z e d both i n t r i n s i c a l l y and e x t r i n s i c a l l y l a b e l e d maize, wheat and soybean, p r o v i d i n g a d d i t i o n a l evidence o f the a p p l i c a b i l i t y o f the extrints i c a l l y l a b e l e d model (_7), To the s u b j e c t s whom they gave maize and 50 mgs o f a s c o r b i c a c i d , a b s o r p t i o n was i n c r e a s e d from 8,.8% to 14.9%; to those subjects given maize p l u s 100 mg o f a s c o r b i c a c i d a b s o r p t i o n was increased to 22,6%, The importance o f the p r o c e s s i n g procedure was i n d i c a t e d i n that the t e s t meals to which a s c o r b i c a c i d was added p r i o r to high temperature baking, no enhancement o f i r o n a b s o r p t i o n was observed, suggesting that the h i g h temperatures i n a c t i v a t e d the a s c o r b i c a c i d , Bjorn-Rasmussen gave meals o f maize p l u s 4.5 mgs o f i r o n ; to these meals were added 0-200 mgs o f a s c o r b i c a c i d (8). I n -

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

5.

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Enhancement by Ascorbic Acid

87

cremental i n c r e a s e s i n i r o n a b s o r p t i o n were observed as a s c o r b i c a c i d was i n c r e a s e d . I n another study, Cook and Monsen gave 63 male subjects 700 k i l o c a l o r i e meals composed o f s e m i s y n t h e t i c i n g r e d i e n t s o f dextrimaltose, corn o i l and ovalbumin; to these meals were added a s c o r b i c a c i d ranging from 0 to 1000 mgs (9)» With each i n c r e a s e i n a s c o r b i c a c i d an i n c r e a s e i n i r o n absorpt i o n occurred. The r a t e o f a b s o r p t i o n appeared to be l o g a r i t h m i c a l l y r e l a t e d to the a s c o r b i c a c i d content. I t was f u r t h e r shown i n t h i s experiment that a s c o r b i c a c i d must be present i n the stomach a t the same time that the nonheme i r o n i s present, Nonheme i r o n was absorbed a t a high r a t e from meals i n t o which the a s c o r b i c a c i d was i n c o r p o r a t e d . However i f the a s c o r b i c a c i d was given 4 o r 8 hours before the t e s t meal there was no enhancing e f f e c t observed. S e v e r a l i n v e s t i g a t o r s have looked a t the e f f e c t o f mixtures of foods on nonheme i r o a b s o r p t i o n L a y r i s s t e s t e d meal that are reminiscent o f thos Venezuela: nonheme i r o meals that i n c l u d e d a s c o r b i c a c i d c o n t a i n i n g foods, the highest r a t e r e s u l t i n g from meals c o n t a i n i n g 66 mgs o f ascorbate cont r i b u t e d by 150 gms o f papaya (10). H a l l b e r g , e t a l . , t e s t e d 37 subjects with a d i e t c o n s i s t i n g o f a mixture o f r i c e , cabbage, c o l l a r d s , s t r i n g beans, c h i l i paste, f i s h sauce and coconut cream (11), When a f r u i t mixture c o n t a i n i n g banana, papaya and oranges was added t o t h i s v e g e t a l mixture, a b s o r p t i o n was reported to increase three f o l d . A s c o r b i c a c i d content was c a l c u l a t e d from food t a b l e s as being 35 mgs. Further a d d i t i o n of 80 gms o f l e a n beef to the v e g e t a l and f r u i t meal had an a d d i t i v e e f f e c t and enhanced the a b s o r p t i o n o f the b a s a l d i e t six-fold. Rossander, e t a l , , t e s t e d the impact o f adding 150 mis o f orange j u i c e t o a b r e a k f a s t c o n s i s t i n g o f bread, b u t t e r , marmalade, cheese and c o f f e e ; with the a d d i t i o n o f the orange j u i c e , a b s o r p t i o n increased from 3,7% t o 8% (12), When the b r e a k f a s t meal i n c l u d e d tea i n p l a c e o f c o f f e e , the enhancing e f f e c t o f the orange j u i c e was l e s s pronounced, A recent study compared v e g e t a r i a n meals (13), The f i r s t composed o f beans, r i c e , cornbread and apples contained 7 mg a s c o r b i c a c i d ; nonheme i r o n a b s o r p t i o n was 2.2%; the second meal, composed o f c a u l i f l o w e r , red kidney beans, white bread, cottage cheese and pineapple, contained 74 mg a s c o r b i c a c i d : from t h i s meal 16,9% of the nonheme i r o n was absorbed. Reviewing the s t u d i e s i n which nonheme i r o n a b s o r p t i o n has been assessed a t v a r i o u s l e v e l s o f a s c o r b i c a c i d i n t e s t meals composed o f e i t h e r s i n g l e food items o r food mixtures i t appears t h a t , w i t h i n any i n d i v i d u a l study, a d d i t i o n a l increments o f a s c o r b i c a c i d c o n s i s t e n t l y increased a b s o r p t i o n o f nonheme i r o n (Table 1), Considering the wide d i f f e r e n c e s i n experimental c o n d i t i o n s , the v a r i o u s s t u d i e s i n c o r p o r a t i n g 12.5 to 1000 mg a s c o r b i c a c i d i n d i c a t e c l e a r l y the enhancing e f f e c t that a s c o r b i c a c i d has on nonheme i r o n a b s o r p t i o n .

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Ascorbic Acid mg. 12.5 25 25 35 50 50 50 50 66 70 70 74 100 100 100 200 250 500 500 1000

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

Table 1 The E f f e c t o f A s c o r b i c A c i d on Absorption of Nonheme Iron i n Humans Iron Absorption Ratio Test Meal + Ascorbic Acid Maize 700 K c a l SS* Maize V e g e t a l meal + f r u i t s Maize 700 K c a l SS Maize Bread, egg, + orange j u i c e Maize + 150 g papaya Bread, cheese, t e a + orange j u i c e Bread, cheese, c o f f e e + orange j u i c e 600-700 K c a l v e g e t a r i a n Maize 700 K c a l SS Maize Maize 700 K c a l SS Maize 700 K c a l SS 700 K c a l SS

1.7 3.0 2.7 1.7 2.5 2.6 2,8 5.0 1.2 2,2 7,7 2,6 4.1 4,6 6.1 4,7 6.0 6.2 9.6

*Semi-synthetic meal composed o f d e x t r i m a l t o s e , corn o i l and ovalbumin.

In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

Ref.

9 8 11 7 9 8 6 10 12 12 13 7 9 8 8 9 3 9 9

89 Enhancement by Ascorbic Acid S e v e r a l d i e t a r y f a c t o r s have been i d e n t i f i e d which i n h i b i t nonheme i r o n a b s o r p t i o n . Tea and tannates appear p a r t i c u l a r l y i n h i b i t i n g (14). Rossander, e t a l . , reported only a s l i g h t i n crease i n a b s o r p t i o n when orange j u i c e was added to a t e a c o n t a i n i n g b r e a k f a s t meal (12). Other food a d d i t i v e s such as EDTA may have an i n h i b i t i n g e f f e c t on nonheme i r o n a b s o r p t i o n i f the EDTA i s i n h i g h molar concentrations r e l a t i v e to nonheme i r o n (JL5). A s c o r b i c a c i d has a l s o been shown to i n t e r a c t with t h e r a p e u t i c i r o n . Derman, e t a l . , have reported that a s c o r b i c a c i d i n c r e a s e s a b s o r p t i o n of v a r i o u s i r o n f o r t i f i c a t i o n compounds i n i n f a n t formulas i n c e r e a l s ; t h i s t h r e e - f o l d i n c r e a s e i n i r o n a b s o r p t i o n induced by a s c o r b i c a c i d was observed i n multiparous women (16). El-Hawary, e t a l . , s t u d i e d 97 i n f a n t s and young c h i l d r e n and observed that a s c o r b i c a c i d increased a b s o r p t i o n from a f o u r mg i r o n supplement as f e r r o u s s u l f a t e (17). McPhail, e t a l . , have a l s i r o n from e i t h e r f e r r o u of three to s i x molar concentrations of a s c o r b i c a c i d (18), When these mixtures of a s c o r b i c a c i d and i r o n f o r t i f i c a t i o n compounds were added to foods such as maize p o r r i d g e before the foods were cooked a b s o r p t i o n was not enhanced.

5.

MONSEN

E s t i m a t i n g b i o a v a i l a b l e i r o n i n the d i e t Extensive research on the a b s o r p t i o n of i r o n from v a r i o u s types of meals has allowed g u i d e l i n e s to be developed by which the amount of d i e t a r y i r o n a v a i l a b l e f o r a b s o r p t i o n may be estimated. I r o n i s the f i r s t t r a c e mineral to be thus t r e a t e d and thus serves as a model f o r other n u t r i e n t s (19), The model f o r e s t i m a t i n g b i o a v a i l a b l e i r o n i s based on the concept that i r o n forms a) a p o o l of heme i r o n which i s r e a d i l y a v a i l a b l e to humans and i s u n e f f e c t e d by other d i e t a r y components and b) a p o o l of nonheme i r o n which i s of low b i o a v a i l a b i l i t y unless enhancing f a c t o r s are present concommitantly (20). Recognition i s made of the importance of the extent of an i n d i v i d u a l ' s i r o n s t o r e s as i t w i l l i n f l u e n c e the amount of nonheme i r o n and heme i r o n that would be absorbed. The conservat i v e model suggests u t i l i z a t i o n of an i n d i v i d u a l with 500 mgs of i r o n s t o r e s as the r e f e r e n c e i n d i v i d u a l f o r whom b i o a v a i l a b l e i r o n may be c a l c u l a t e d . As the purpose of such a model i s to compare one d i e t with another, i t i s immaterial what l e v e l of i r o n s t o r e s an i n d i v i d u a l has i n that the comparison between d i e t s w i l l not be a f f e c t e d . The c a l c u l a t e d values w i l l , of course, be lower f o r the amount of t o t a l a v a i l a b l e i r o n with an i n d i v i d u a l with moderate i r o n s t o r e s of 500 mgs than they would be f o r an i n d i v i d u a l w i t h zero i r o n s t o r e s . The enhancing f a c t o r s considered f o r the a b s o r p t i o n of nonheme i r o n are a s c o r b i c a c i d and meat, f i s h and p o u l t r y . The

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e a r l i e r model c l a s s i f i e d meals as low, moderate o r h i g h a v a i l ability; t h i s produced a r t i f i c i a l s t a i r step increments. Thus, the model has been f u r t h e r r e f i n e d t o show that the enhancing f a c t o r s a r e a d d i t i v e i n t h e i r e f f e c t and smoothly i n c r e a s e d i e t a r y nonheme i r o n a b s o r p t i o n i n a l o g a r i t h m i c f a s h i o n (21). Thus the amount o f nonheme i r o n a v a i l a b l e f o r a b s o r p t i o n from each meal o r snack can be estimated from the summation o f enhancing f a c t o r s (milligrams o f a s c o r b i c a c i d p l u s grams o f cooked m e a t / f i s h / p o u l t r y ) . Absorption o f nonheme i r o n f o r the r e f e r e n c e i n d i v i d u a l with 500 mg i r o n s t o r e s i s assumed to change from 3% f o r a meal w i t h no enhancing f a c t o r s to 8% from a meal o f 75 enhancing f a c t o r s . The formulas f o r c a l c u l a t i n g the r a t e o f a b s o r p t i o n o f nonheme i r o n f o r the r e f e r e n c e i n d i vidual are: For EF