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Relative availability of iron to rats from beef, soy protein and a beef-soy protein mixture as determined… Nikolaiczuk, Marcia Jane 1985

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RELATIVE AVAILABILITY OF IRON TO RATS FROM BEEF, SOY PROTEIN AND A BEEF-SOY PROTEIN MIXTURE AS DETERMINED BY IRON REPLETION ASSAY by MARCIA JANE NIKOLAICZUK B.Sc. ( F . S c ) , M c G i l l U n i v e r s i t y , 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1985 ° Marcia Jane N i k o l a i c z u k , 1985 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Food Science The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 Date August 30,1985 DE-6 (3/81) i i ABSTRACT Male weanling Wistar rats were fed a low-iron basal diet for 3 weeks. The iron depleted rats were then divided into 9 groups according to a randomized block design based on body weight. During the repletion period of 2 weeks, one group was fed the low-iron basal diet. The other eight groups received either the basal diet to which was added 5, 10, 15, 20 or 25 mg iron per kg diet as ferrous sulfate or test source diets formulated to provide a t o t a l of 15 mg iron per kg diet from either freeze-dried ground beef, textured defatted soy flour product or a 2.3:1 (w/w) mixture of beef and soy product. A l l diets were i s o c a l o r i c and i s o n i t r o -genous. The r e l a t i v e b i o l o g i c a l value (RBV) of iron in the test source diet was calculated as the ra t i o of the amounts of iron from the reference source (ferrous sulfate) and the test source diet required to give the same response i n hemoglobin or hematocrit. The RBVs ± 95?o confidence l i m i t s , calculated on the basis of f i n a l hemoglobin levels and hematocrit values, were respectively: freeze-dried ground beef, 56 ± 7 % and 62 ± 7 %; f o r t i f i e d textured defatted soy flour product, 81 ± 10 % and 79 ± 10 %; 2.3:1 (w/w) mixture of freeze-dried ground beef and soy flour product, 65 ± 6 % and 68 ± 6 %. The RBVs obtained for the iron in beef and for that i n the soy flour product suggest that the anemic rat might not be a suitable model for normal man when screening such foods for thei r available i r o n . In normal man, the absorption of the iron in beef i s comparable to that of inorganic reference iron, while that in textured soy flour ig_^abnut one t h i r d . i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES vi LIST OF APPENDICES v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 LITERATURE REVIEW 3 A. Availability of iron 3 B. Dietary iron sources 3 C. Chemistry of nonheme iron 6 D. Effect of various protein sources on the absorption of food iron in man 8 1. Animal protein 8 2. Soy protein 10 E. Effect of processing on the availability of meat iron 13 F. Techniques for the determination of iron availability 14 G. The rat as a model system for predicting iron availability in man 21 MATERIALS AND METHODS 24 A. Animal care 24 B. Experimental diets 24 1. General description 24 2. Preparation of test source ingredients 25 (a) Beef 25 (b) Textured defatted soy flour 26 3. Formulation of test source diets 26 C. Biological assay 34 D. Hemoglobin and hematocrit determination 35 E. Calculation of iron bioavailability 36 F. Proximate analysis of diet ingredients and diets 36 G. Sta t i s t i c a l analysis 41 RESULTS AND DISCUSSION 43 A. Composition of the experimental diets 43 B. Feed intake and growth 46 C. Hemoglobin and hematocrit responses 48 D. Regression analysis 51 iv E. Relative biological value of iron in test source diets 53 1. Beef 53 2. Soy protein 55 3. Beef-soy protein mixture 58 F. General discussion 59 CONCLUSIONS 61 REFERENCES CITED 63 APPENDIX 71 LIST QF TABLES Page Table I. Nutrient analysis for TVP® Unflavored-Fortified product 28 Table I I . Composition of basal diet 29 Table I I I . Composition of beef diet 30 Table IV. Composition of beef and soy flour diet 31 Table V. Composition of soy flour diet 32 Table VI. Repletion phase diet treatments 33 Table VII. Proximate composition of test foods and fiber source 44 Table VIII. Proximate composition of basal, beef, beef and soy fl o u r , and soy flour diets 45 Table IX. Feed intake and body weight gain for iron depleted rats fed test sources of iron during the repletion period 47 Table X. Fi n a l hemoglobin levels and hematocrit values for iron depleted rats fed test sources of iron during the repletion period 49 Table XI. Relative b i o l o g i c a l value (RBV) of iron i n iron test source diets (FeS04-7H20= 100.0) 54 v i L I S T OF F I G U R E S Page Figure 1. Processing scheme for TVP® Unflavored-Fortified product 27 Figure 2. Two week hemoglobin response of iron depleted rats to graded levels of ferrous sulfate reference standard 52 v i i LIST OF APPENDICES Page Appendix A. Comparison of experimental and reported values for proximate analysis components 71 Appendix B. Growth and hematological responses for iron depleted rats fed test sources of iron during the repletion period 72 Appendix C. Growth, feed intake and hematological responses for iron depleted rats fed graded levels of iron as ferrous sulfate during the repletion period 73 ACKNOWLEDGEMENTS v i i i I would l i k e to express my appreciation to my Supervisor, Dr. 3. Vanderstoep, for his guidance and support during the course of t h i s research project. I also wish to thank the members of the committee, Dr. S. Nakai, Dr. W. D. Powrie and Dr. R. M. Beames, for their valuable sugges-tions, and the technical s t a f f of the Department of Food Science for their assistance i n the laboratory. I especially wish to thank both Rodger Hadwen and my parents for their encouragement and support throughout the years involved i n t h i s project. F i n a l l y , the fi n a n c i a l assistance provided through the Agriculture Canada Operating Grants Program i s acknowledged. 1 INTRODUCTION Iron i s an essential element for man and other higher forms of l i f e . The major portion of iron in the body i s found within the erythrocytes of the plasma as hemoglobin while smaller quantities are present i n the l i v e r , spleen and bone marrow as f e r r i t i n and hemosiderin, and in muscle tissue as myoglobin. Electron carriers containing iron, such as the cytochromes, are present i n a l l the c e l l s of the body. Together, these iron-containing compounds perform the essential functions of oxygen transport to the tissues and the maintenance of oxidative systems within the tissue c e l l s (Subcommittee on Iron, 1979; Beutler, 1980). The quantity of body iron i s maintained within narrow l i m i t s through the regulation of iron uptake by the i n t e s t i n a l mucosa (Subcommittee on Iron, 1979; Beutler, 1980; Narins, 1980). The amount of dietary iron absorbed by the i n t e s t i n a l mucosa i s influenced by the chemical form of ir o n , the composition of the meal and the iron status of the individual (Narins, 1980; Hallberg, 1981b). In theory, a balanced and varied diet provides a s u f f i c i e n t t o t a l amount of iron to more than adequately meet the needs of the population (Narins, 1980). However, i t i s generally recognized that iron deficiency i s a common n u t r i t i o n a l problem worldwide. Low a v a i l a b i l i t y of dietary iron i s considered to be the most important factor responsible for the development of iron deficiency (Monsen et a l . , 1978). In recent years there has been a growing trend towards a reduction in red meat consumption by the public. This trend has been motivated by concerns for cost and more recently concerns for n u t r i t i o n a l health. 2 Health and Welfare Canada (1977) published Recommendations for Prevention Programs i n Relation to Nutrition and Cardiovascular Disease. N u t r i t i o n i s t s have translated these recommendations into p r a c t i c a l terms, advising the public to reduce both their fat intake and consumption of animal products. The reduction of meat consumption has potentially far-reaching n u t r i -t i o n a l implications. Meat i s a very good source of many nutrients, includ-ing protein, vitamins of the B complex, iron and zinc (Health and Welfare Canada, 1979). A reduction in meat intake necessitates the compensation of nutrients contained in meat with nutrients from other sources, such as cereals, oilseeds and products made from them. The replacement of meat with low-fat meat substitutes, used as extenders or meat simulators, i s a r e a l i s t i c option. In Canada these meat substitutes, derived largely from plant protein sources, must by law be of a nutrient content equivalent to that of the meat replaced or simulated (Health and Welfare Canada, 1981, Section B.14.073). However, the n u t r i t i o n a l a v a i l a b i l i t y of the iron i n the substituted product i s not specified in the regulations, even though iron i s generally recognized as being less available from plant than from meat sources (Sabry, 1976). The objective of this research was to assess the iron a v a i l a b i l i t y in a f o r t i f i e d soy protein product and a mixture of beef and the f o r t i f i e d product to determine whether their a v a i l a b i l i t i e s compared favorably to that of beef. Iron a v a i l a b i l i t y was measured using a bioassay technique, s p e c i f i c a l l y the iron repletion t e s t , using the rat as the assay system. 3 LITERATURE REVIEW k. AVAILABILITY OF IRON The a v a i l a b i l i t y or b i o a v a i l a b i l i t y of iron from a dietary source, be i t a composite meal, single food or iron supplement, describes that portion of the t o t a l iron i n the source which i s absorbed and metabolized to an active species (Mahoney and Hendricks, 1982; O'Dell, 1983). The amount of iron p o t e n t i a l l y available from dietary sources depends on a number of complex and interacting factors. The absorption of iron from dietary sources i s affected by the d i g e s t i b i l i t y of the iron source, the chemical form of the iron as i t occurs i n the source, the l e v e l of iron i n the meal, the iron status of the individual consuming the meal, the presence of other food components in the same meal and the physiological conditions that exist in the digestive t r a c t . The b i o a v a i l a b i l i t y of iron to humans has been extensively reviewed i n the l i t e r a t u r e (Bibeau and Clydesdale, 1976; Bowering et a l . , 1976; Hallberg, 1981b; Morck and Cook, 1981; Cook, 1983; Morris, 1983) and text-books (Subcommittee on Iron, 1979; Beutler, 1980; Narins, 1980; Kies, 1982). The various factors that influence the a v a i l a b i l i t y of iron w i l l be discussed i n subsequent sections. B. DIETARY IRON SOURCES The iron i n a mixed Western diet i s derived from foods of plant and animal o r i g i n , from certain additives and from contamination sources (Morck and Cook, 1981). There are two major chemical forms of iron i n a mixed diet, heme and nonheme, and each i s absorbed by a different mechanism 4 (Monsen et a l . , 1978; Hallberg, 1980; Hallberg, 1981b; Morck and Cook, 1981; Cook, 1983). 1. Heme iron Heme, which contains iron in a porphyrin ring structure, is found in hemoglobin and myoglobin and accounts for about 30-60* of the total iron present in animal tissue (Cook and Monsen, 1976; Schricker et a l . , 1982). Heme iron from muscle foods enters the intestinal mucosal c e l l as the heme moiety with the porphyrin ring intact. Within the mucosal c e l l , the iron-porphyrin complex is catabolized and released ionic iron then enters the same pathways as nonheme iron (Callender et a l . , 1957; Turnbull et a l . , 1962; Conrad et a l . , 1967; Weintraub et a l . , 1968). The proportion of heme iron which humans absorb from a mixed North American diet i s high in comparison to nonheme iron (Bjorn-Rasmussen et a l . , 1974). Because of i t s unique absorptive mechanism and solubility at elevated pH, heme iron i s prevented from interacting with other components of the diet (Morck and Cook, 1981). The only dietary factor thought to influence absorption of heme iron i s the presence of meat (Hallberg et a l . , 1979; Hallberg, 1981b; Morris, 1983). The absorption of purified heme iron by human subjects has been observed to increase by a substantial amount when meat is included in the test meal (Martinez-Torres and Layrisse, 1971; Hallberg et a l . , 1979). Heme iron provides only 5-10% of the total daily iron intake in mixed Western diets. But as the level of heme iron absorption i s high, ranging from 10-25?o depending on whether or not meat is included in the meal (Hall-5 berg et a l . , 1979), i t provides nearly one t h i r d of the iron absorbed each day from mixed diets (Morck and Cook, 1981). The absorption of heme iro n , in comparison to that of nonheme iron, i s less affected by the amount of iron administered or by the iron status of the individual at doses in the usual physiological range (Wheby et a l . , 1970) but at high doses of heme there i s a strong correlation with the subject's iron status (Hallberg et a l . , 1979). 2. Nonheme iron Nonheme iro n , the predominant form of iron i n the mixed Western diet, i s present i n foods such as cereals, vegetables, f r u i t s and eggs and accounts for the remaining portion of iron i n animal tissues. Other sources of nonheme iron include compounds added to f o r t i f y foods and con-taminating iron introduced during the harvesting, processing, cooking or storing of food (Monsen et a l . , 1978; Hallberg, 1980; Hallberg, 1981a; Morck and Cook, 1981). The absorption of nonheme iron occurs from a common pool formed by foods ingested in the same meal. The implication of the pool concept i s that the absorption of nonheme iron from a meal depends not only on the t o t a l amount of dietary nonheme iron but also on the effects of various intraluminal factors, which either enhance or i n h i b i t the a v a i l a b i l i t y of iron (Hallberg, 1980; Hallberg, 1981b). The absorption of nonheme iron i s markedly influenced by the iron status of the i n d i v i d u a l . As body iron stores become depleted more of the nonheme iron from the diet i s absorbed; once the iron stores become replenished the l e v e l of nonheme iron absorption declines (Monsen et a l . , 6 1978). Absorption of nonheme iron by an individual with adequate iron stores w i l l be about 2% from a meal containing high levels of food com-ponents which i n h i b i t the a v a i l a b i l i t y of iron. At the other extreme, an iron deficient subject may absorb as much as 20% of nonheme iron from a meal containing an abundance of absorption enhancers (Monsen et a l . , 1978). The t o t a l amount of iron i n the diet has an effect on the proportion of iron that i s absorbed; t h i s effect being more pronounced on the assimi-l a t i o n of nonheme iron than that of heme iron. Generally as the level of iron i n the diet increases, the proportion of the iron that i s absorbed decreases, but the absolute amount increases (Narins, 1980). As previously mentioned, a small proportion of the nonheme iron of the diets of developed countries i s introduced through contamination from the environment (Hallberg, 1980; Hallberg, 1981a; Morck and Cook, 1981). The a v a i l a b i l i t y of contaminating iron occurring as insoluble oxides and hydroxides remains to be determined as i t does not exchange with soluble radioiron s a l t s used to measure food iron absorption (Hallberg, 1980). C. CHEMISTRY OF NONHEME IRON The chemical characteristics of iron such as valence, s o l u b i l i t y and chelation have been recognized as being i n f l u e n t i a l i n the b i o a v a i l a b i l i t y of an iron source (Lee and Clydesdale, 1979a). Iron has several oxidation states, ranging from Fe +^ to F e - 2 , depending on the chemical environment. The f e r r i c form (Fe + 3) and the ferrous form (Fe +2) are the only states occurring naturally i n foods. The iron present i n f o r t i f i c a t i o n sources may occur i n one of three valence states, F e + 3 , F e + 2 or Fe° (Lee and Clydesdale, 1979a). 7 Ferrous and ferric ions in solution do not occur in the free state, but are hydrated as Fe(H2n06+-' and F e i ^ Q ) ^ 2 in acid and lose protons as the pH i s raised to form the corresponding hydroxides in neutral and alkaline solutions. These hydroxides become more insoluble with increas-ing pH. At neutral pH, in the absence of ligands, ferrous hydroxide has a solubility of about 10~^ M while that in the ferric form is much less soluble, having a solubility of 10~^M (Lee and Clydesdale, 1979a). Iron in the ferrous state is absorbed more efficiently by man than iron in the ferric state (Brise and Hallberg, 1962). It remains to be determined whether this phenomenon is due to the higher degree of solubility of ferrous iron or to a selective absorptive mechanism (Lee and Clydesdale, 1979a; Nojeim and Clydesdale, 1981). Many common food components are effective chelating agents or ligands and can prevent the precipitation of iron in the neutral to mildly alkaline environment of the small intestine (Lee and Clydesdale, 1979a), the site at which the majority of dietary iron is absorbed (Wheby et a l . , 1964). The absorption of chelated iron, however, may be enhanced or inhibited depending on the nature of the specific iron complex formed, which includes i t s solubility, molecular weight and st a b i l i t y (Monsen and Page, 1978; Lee and Clydesdale, 1979a; Clydesdale, 1982). Enhanced iron absorption has been observed when such organic acids as ascorbic acid, sugars such as fructose, and amino acids like cysteine are present in the meal. It is postulated that these compounds form soluble low molecular weight, relatively weak chelates with iron. These chelates keep the iron in solution during transit through the neutral to alkaline 8 environment of the upper small intestine (Monsen et a l . , 1978; Lee and Clydesdale, 1979a; Morck and Cook, 1981). Ascorbic acid and cysteine, acting as reducing agents, have been reported to increase iron absorption by reducing f e r r i c iron to the more soluble ferrous form (Lee and Clydesdale, 1979a; Morck and Cook, 1981). Food components reported to have a negative effect on iron absorption include tannins, EDTA, oxalates, calcium and phosphate s a l t s , phospho-proteins, phytate and carbonates. Various mechanisms have been proposed as the reason these iron-complexing compounds decrease the a v a i l a b i l i t y of dietary i r o n ; among the suggestions are the formation of insoluble precipitates or strongly bound soluble complexes with f e r r i c iron at neutral pH (Bibeau and Clydesdale, 1976; Monsen and Page, 1978; Morck and Cook, 1981). D. EFFECT OF VARIOUS PROTEIN SOURCES ON THE ABSORPTION OF FOOD IRON IN MAN 1. Animal protein A "meat factor" present in meat, poultry and f i s h has been shown i n humans to raise the rate of nonheme iron absorption from a meal by as much as four times (Cook and Monsen, 1976). As the quantities of these substances i n a complex or composite meat increase, iron absorption increases. Neither protein nor animal protein per se enhance iron absorption. While beef, lamb, pork, chicken, l i v e r and fis h increase nonheme iron absorption, milk, cheese, and eggs do not increase and may decrease iron absorption (Cook and Monsen, 1976). I t was shown years ago by Layrisse et a l . (1969) that the absorption of iron from different vegetable foods was markedly increased when they were served together with 9 meat and f i s h . I n i t i a l l y , t h i s effect was considered to be due to an effect of amino acids formed during the digestion of meat (Martinez-Torres and Layrisse, 1970). The results of lat e r studies, however, and especially the observation that egg albumen does not enhance dietary iron absorption, i n spite of the fact that i t has about the same amino acid composition as meat and f i s h , are not consistent with the i n i t i a l interpretation (Cook and Monsen, 1976). Bjorn-Rasmussen and Hallberg (1979) performed a series of studies with humans in an attempt to gain some insight into the mechanism of action of meat on the absorption of nonheme iron. The effect of beef on absorption of food iron was studied i n patients with proved gastric achlorhydria to see i f the effect was related to the stimulus of the secretion of HC1; the effect of various foods (beef, chicken, f i s h , thymus and egg albumen) was compared; the effect of cysteine or a mixture of amino acids i n amounts and proportions corresponding to the contents of beef was studied; minced beef was boiled with water, and the effects of broth and meat residue were measured separately; the effect of beef meat on the absorption of inorganic ferrous and f e r r i c s a l t s (given alone or together with sodium phytate) was studied. The test meal consisted of a maize porridge that had been e x t r i n s i c a l l y labelled with ^Fe as ferrous sulfate. Measurements of iron retention showed the r e l a t i v e enhancement of iron absorption was the same for both normal and achlorhydric subjects. The meat products (beef, f i s h , chicken and c a l f thymus) a l l increased iron absorption to the same extent. Neither egg albumen, cysteine or a water extract of beef did, however, affect the absorption of food iron. Beef 10 increased the absorption of a solution of inorganic iron given without food only when the iron s a l t was t r i v a l e n t or when sodium phytate was added to the solution. I t was concluded that meat acts by counteracting luminal factors that i n h i b i t iron absorption; the most probable mechanism for t h i s action being the formation of a luminal c a r r i e r which transports the iron to the mucosal c e l l membrane. Hallberg et a l . (1979) conducted studies on a mixed group of human subjects, which included both normal adults and blood donors,to determine the effect of meat on hemoglobin iron absorption. Testmeal A (meat-free) consisted of two wheat/rye r o l l s made from u n f o r t i f i e d flour (radiolabelled with Hb-^^Fe), served with margarine, cheese, marmalade, cereal with milk, and coffee. The meat-containing test meal (B) consisted of meal (A) plus two beef patties. Both meals contained 5 mg radiolabelled heme iron. I t was found that the addition of meat to the meal increased the percent Hb-^Fe absorption from 10.2% i n the meat-freemeal to 16.0% when meat was included. The mean ratios of Hb- : reference iron (FeSO^) absorption were 0.20 and 0.31 for the meat-free meal and meat-containing meal, respectively. The authors postulated that absorption-enhancing effect of meat on both heme and nonheme iron might be due to stimulation of the digestion of food (by pancreatic or b i l i a r y secretions) so that iron i n either form i s more e f f i c i e n t l y released and made available for absorption. 2. Soy protein Human studies have demonstrated that the addition of soy protein-derived food products to a meal can either i n h i b i t or enhance the absorp-t i o n of nonheme iron already present in the meal. The effect soy protein 11 has on dietary nonheme iron absorption largely depends upon the composition of the meal to which i t i s added, that i s , to what extent the meal contains or lacks enhancing or inh i b i t o r y food components. Cook et a l . (1981), using an e x t r i n s i c tag method with human subjects, demonstrated that soy, as a protein source, depressed dietary nonheme iron absorption. These researchers substituted, i n protein-equivalent amounts, a wide range of soy products, including f u l l fat soy flour , textured soy flour and soy i s o l a t e , into a semisynthetic egg albumen-based meal. A l l diets were adjusted with FeCl3 to provide the same amount of iron. The mean nonheme iron absorption values were observed to f a l l from 5.5% i n the unsubstituted meal to 1.0, 1.9 and 0.4% respectively. Hallberg and Rossander (1984), in a human study using e x t r i n s i c tag methodology, investigated the effect of adding soy protein to a simple Latin American cereal-based meal to see whether the absorption of nonheme iron could be improved. The basal diet, consisting of maize chapattis, black beans and r i c e , was high i n phytic acid and lacked enhancing factors. There was only a marginal improvement in percent absorption of dietary nonheme iron with the addition of soy; the percent iron absorption from the basal diet alone being 3.5% while the addition of defatted soy flour increased the iron absorption to 4.0%. However, th i s addition of soy t r i p l e d the quantity of nonheme iron absorbed from 0.17 to 0.51mg as a result of the high iron content of the soy product. Studies examining the effect of replacing a portion of meat in a hamburger meal with soy protein product have shown that under such circumstances the absorption of nonheme iron i s inhibited (Cook et a l . , 12 1981; Hallberg and Rossander, 1982). The overall resultant reduction in absorption is due to a combination of the inhibitory action of soy and the reduction in the enhancing action of the meat (Morris, 1983). Cook et a l . (1981), in a human study applying the extrinsic labelling technique, found that the percent nonheme iron absorption decreased from 3.2% in the beef protein-based meal to 1.24 and 1.51% when 3:1 and 2:1 ratios (w/w) of meat to unhydrated textured defatted soy flour, respec-tively, were fed. Hallberg and Rossander (1982), who extrinsically labelled both the heme and nonheme iron of a hamburger meal, demonstrated that when half of the beef of a hamburger was substituted by a protein-equivalent amount of textured soy flour or defatted soy flour the percent nonheme iron absorp-tion in normal human subjects decreased from 8.4% to 7.2 and 5.2%, respec-tively. This observed decrease in nonheme iron absorption was attributed to the reduction of beef, an animal protein demonstrated to stimulate iron absorption (Cook and Monsen, 1976; Hallberg et a l . , 1979). However, the amount of nonheme iron absorbed from the soy containing meals compared favorably to that from the beef-based meal; the high iron content of the soy products being the reason for this similarity. The inhibition observed in this particular study was not alleviated when dephytinized soy was included in the meal. Upon examining the effect of partial substitution of soy for beef protein (1:1) on the amounts of heme and nonheme iron absorbed, i t was found that the total amount of iron (heme and nonheme) absorbed from the beef and beef-substituted meals did not differ. 13 E. EFFECT OF PROCESSING ON THE AVAILABILITY OF MEAT IRON The results of early studies on the effect of heat processing on the availability of heme iron to humans have been inconsistent. Boiling of i n t r i n s i c a l l y labelled rabbit hemoglobin for 3 minutes decreased the intestinal absorption of hemoglobin iron from 11 to 7% and from 22 to 12% in normal and iron deficient subjects, respectively (Callender et a l . , 1957). On the other hand, Turnbull et a l . (1962) reported that cooking hemoglobin in a boiling water bath for 15 minutes did not alter the absorption of the hemoglobin iron in iron deficient subjects. More recently, Schricker et a l . (1982) demonstrated that dry heat processing (100UC for 20 min) of fresh ground beef resulted in an increase of 110% in the nonheme iron content of the sample (9.9 yyg/g in the raw sample compared to 20.9^g/g in the cooked). This increase in nonheme iron concentration apparently results from release of iron from the heme complex, possibly through a mechanism involving the oxidative cleavage of the porphyrin ring (Schricker et a l . , 1982). It seems possible that different cooking methods could result in different proportions of heme and nonheme iron in meat. Since the availabilities of heme and nonheme iron in humans are substantially different, more information on the effects of cooking on heme degradation is needed (Schricker et a l . , 1982). The results of rat feeding studies employing hemoglobin repletion methodology have shown that the effect of heat treatment on meat iron availability i s variable. Oldham (1941) found that the hemoglobin gain in rats fed with oven-dried meat was higher than for those fed with unheated, vacuum dried meat. A recent study by Jansuittivechakul et a l . (1985) investigated the effect of autoclaving, boiling and baking on the 14 a v a i l a b i l i t y of iron i n beef i n anemic r a t s . I t was found that cooking meat did not s i g n i f i c a n t l y a f f e c t iron b i o a v a i l a b i l i t y compared with uncooked product. Furthermore, the decrease i n heme iron i n meat due to cooking was not associated with any decrease i n b i o a v a i l a b i l i t y of meat ir o n as fed to r a t s . F . TECHNIQUES FOR THE DETERMINATION OF IRON AVAILABILITY 1. Chemical Balance The e a r l i e s t attempts to measure the quantity of dietary iron a v a i l -able for absorption were made with balance techniques whereby estimates of iron absorption were based on the differences between o r a l intake and f e c a l l o s s of iron (Moore, 1968). The chemical balance technique i s the only method that d i r e c t l y measures iron absorption from the whole diet ( H a l l -berg, 1981b). This method, however, i s i n s e n s i t i v e , imprecise, and time consuming, and i t gives no information about iron absorption from d i f f e r e n t meals (Hallberg, 1981b). The technical errors of the method include the inherent lack of precision i n measuring the small difference between o r a l intake and f e c a l loss and the i m p o s s i b i l i t y of d i f f e r e n t i a t i n g between endogenous loss and unabsorbed i r o n (Moore, 1968). 2. Radioisotopic l a b e l l i n g Radioisotopic l a b e l l i n g techniques allow the most precise measure-ments of dietary iron absorption i n humans and animals (Narins, 1980). Estimates of iron absorption from a given source are based on the measure-ment of r a d i o a c t i v i t y remaining 10 to 20 days following the consumption of food containing a known amount of radioiron (Narins, 1980). Radioiron 15 absorption may be determined by either measuring erythrocyte incorporation or whole-body retention of radioiron (Narins, 1980). The whole-body reten-t i o n of radioiron i s regarded as being not only r e l i a b l e and sensitive but indeed the only completely satisfactory quantitative method but i s not widely used i n human studies due to the expense of the whole-body counter (Narins, 1980). Though less accurate, the measurement of blood radio-a c t i v i t y i s the method most extensively used in human studies of iron absorption (Narins, 1980). The errors of t h i s method include the v a r i -a b i l i t y of the red blood c e l l s i n incorporating the radioiron and i n the use of estimates of t o t a l blood volumes from height and weight figures (Narins, 1980). Biosynthetic l a b e l l i n g ( i n t r i n s i c l a b e l l i n g ) was f i r s t introduced by Moore and Dubach (1951) and i s considered by some to be the most val i d approach to measuring absorption of food iron (Narins, 1980). This l a b e l -l i n g technique involves the b i o l o g i c a l incorporation of radioiron into single food items. Vegetable foods are labelled by growing them i n hydro-ponic medium containing radioiron while animal foods or f i s h are s i m i l a r l y tagged by i n j e c t i n g or feeding radioiron in the months prior to s a c r i f i c e (Narins, 1980). Studies with i n t r i n s i c a l l y labelled foods demonstrated that iron absorption from ind i v i d u a l foods differed markedly (Moore and Dubach, 1951). Also, when two iron-containing foods were fed together, the amount of iron absorbed from each food differed from that when fed separately, thereby demonstrating that a food interaction had taken place (Layrisse et a l . , 1968). Thus, knowing the iron absorption from single foods does not 16 provide a v a l i d estimate of the absorption of iron from a whole meal (Hall-berg, 1981b). Disadvantages of the i n t r i n s i c l a b e l l i n g technique include the time, expense and d i f f i c u l t y involved i n the preparation of biosyn-t h e t i c a l l y labelled foods (Narins, 1980). The application of th i s l a b e l -l i n g technique to the estimation of iron absorption from complete meals i s limited by the a v a i l a b i l i t y of only two radioiron isotopes (Narins, 1980). This type of l a b e l l i n g , however, remains the only one available to study insoluble forms of i n t r i n s i c food iron (Narins, 1980). The development of the e x t r i n s i c radioiron tag technique, whereby a radioactive tracer i s added to either a complete meal or one of the components of the meal prior to i t s consumption, has made i t possible to obtain an estimate of the absorption of the i n t r i n s i c iron from selected single foods and complex meals (Hallberg, 1980; Narins, 1980; Hallberg, 1981b; Consaul and Lee, 1983; Van Campen, 1983). The v a l i d i t y of the method i s based on the assumptions that (1) heme iron and nonheme iron from the diet form two separate pools of iron i n the gastrointestinal t r a c t ; and (2) these dietary iron pools can be independently and simultaneously labelled through the use of two different radioiron isotopes that exchange completely with their respective iron pools. The heme iron pool i s labelled with biosynthetically radioiron-labelled hemoglobin and the nonheme pool with an ex t r i n s i c inorganic radioiron tracer (Hallberg, 1980; Hallberg, 1981b; Van Campen, 1983). The ex t r i n s i c tag technique i s not applicable to a l l food items, nor can i t be used in determining the absorption of insoluble forms of iron (Hallberg, 1980; Hallberg, 1981b; Consaul and Lee, 1983; Van Campen, 1983). While the ex t r i n s i c l a b e l l i n g 17 technique offers a convenient alternative to the more specialized, time-consuming techniques of preparing i n t r i n s i c a l l y labelled foods, i t may be associated with an error of 20% when estimating b i o a v a i l a b i l i t y of iron (Weaver et a l . , 1984). 3. I_n v i t r o Various iri v i t r o techniques have been developed to estimate the amount of dietary iron that i s potentially available for absorption ( M i l l e r and Schricker, 1982). Early methods interpreted available iron to be the measurable ionizable iron i n foods. Ionizable iron has been determined as that fraction of the t o t a l iron in a food that w i l l react with a complexing agent, such as a, a ' - d i p y r i d y l , t r i p y r i d y l t r i a z i n e or bathophenanthroline, to form a chromagen which can be quantitated spectrophotometrically (Narins, 1980; M i l l e r and Schricker, 1982). Determinations of ionizable iron i n foods are of l i t t l e physiological significance, however, since the conditions of the gastrointestinal tract which influence iron a v a i l a b i l i t y are not considered (Narasinga-Rao and Prabhavathi, 1978). Recent ir± v i t r o methods have concentrated on simulating gastro-i n t e s t i n a l digestion using p u r i f i e d peptic and/or pancreatic enzymes with subsequent measurement of either the soluble or dialyzable iron ( M i l l e r and Schricker, 1982; Van Campen, 1983). M i l l e r et a l . (1981) has refined the procedure used by previous researchers to better simulate _in vivo digestion conditions. Schricker et a l . (1981) compared the JLn v i t r o method of M i l l e r et a l . (1981) with rat and human e x t r i n s i c radioiron tag techniques. Complex meals were used i n the comparison. The a b i l i t y of this i n v i t r o method to 18 show s t a t i s t i c a l l y s i g n i f i c a n t differences between iron a v a i l a b i l i t y in the various meals substantially agreed with that of the human jm vivo method whereas there was less agreement between the in_ v i t r o and rat _in vivo methods. Advantages of jln v i t r o methods include their rapidity and low cost, the i r reduced v a r i a b i l i t y compared to jLn vivo methods and the a b i l i t y to precisely control conditions during the determinations. j[ri v i t r o methods are viewed as being useful i n preliminary studies of iron a v a i l a b i l i t y (Schricker et a l . , 1981). Limitations of the method include the inexact duplications of in_ vivo conditions and the i n a b i l i t y to account for the effects of factors involved i n the absorptive process such as active trans-port and brush border binding proteins ( M i l l e r and Schricker, 1982). Given these l i m i t a t i o n s , _in v i t r o measurement of iron a v a i l a b i l i t y must be regarded as a r e l a t i v e rather than an absolute indication of a v a i l a b i l i t y ( M i l l e r et a l . , 1981 ). 4. Animal bioassay E f f o r t s to develop an appropriate b i o l o g i c a l assay to assess iron a v a i l a b i l i t y were begun years ago when interest arose regarding the f o r t i -f i c a t i o n of foods with iron (Elvehjem et a l . , 1933; Smith and Otis, 1937; Freeman and B u r r i l l , 1945). Pla and F r i t z (1970) revived efforts in t h i s area by introducing an assay method involving young anemic rats (or chicks) which u t i l i z e d changes i n hemoglobin concentration (or hematocrit level) as the c r i t e r i o n for assessing the a v a i l a b i l i t y of the iron source. Following collaborative study ( F r i t z et a l . , 1970; Pla and F r i t z , 1971; Pla et a l . , 1973; F r i t z et a l . , 1974) the method, as f i r s t conceived 19 by Pla and F r i t z (1970), was refined and proposed to the Association of O f f i c i a l A nalytical Chemists. The rat hemoglobin repletion test was subsequently adopted as the standard method of analysis for the b i o a v a i l -a b i l i t y of iron by the Association of O f f i c i a l A n a l y t i c a l Chemists (AOAC, 1975, Sections 43.188-43.190). Since that time, changes recommended as a result of further collaborative study ( F r i t z et a l . , 1975; 1978) have been incorporated into the standard method (AOAC, 1980, Sections 43.217-43.219). This procedure involves the feeding of a fast growing s t r a i n of male weanling rats a basal diet low i n iron for a period of about 4 weeks. The rats are determined to be s u f f i c i e n t l y anemic when hemoglobin levels are less than 6 g/dL. Once s a t i s f a c t o r i l y depleted the rats are divided into groups of >_ 8 animals of similar mean body weight or hemoglobin l e v e l . Groups of test animals are placed on 0, 6, 12, and 24 mg Fe/kg diet, sup-p l i e d by the reference standard FeS04'7rl20. Other groups of animals are placed on diets that contain levels of test samples that provide similar concentrations of iron. The rats are i n d i v i d u a l l y housed in stainless s t e e l cages and have free access to deionized d i s t i l l e d water throughout the repletion period. The diets are fed jad libitum for two weeks. At the end of the repletion period, t a i l blood i s collected from each rat and hemoglobin levels are determined. Fin a l hemoglobin values achieved by the iron test source are compared to those of the reference standard by the p a r a l l e l lines technique of analysis ( B l i s s and White, 1967). The results are expressed as the r e l a t i v e b i o l o g i c a l value (RBV) of the test source r e l a t i v e to ferrous sulfate which i s assigned a value of 100. 20 This technique has several advantages. The method employs an intact b i o l o g i c a l system, the method i t s e l f i s r e l a t i v e l y simple and available to a large number of researchers ( M i l l e r and Schricker, 1982). The experi-ments are of a r e l a t i v e l y short duration (about one month) and a large number of iron sources can be screened f a i r l y rapidly (Van Campen, 1983). The experimental animals are a r e l a t i v e l y homogeneous population, and repr o d u c i b i l i t y among laboratories i s generally good i f i d e n t i c a l or nearly i d e n t i c a l conditions are used (Van Campen, 1983). F i n a l l y , t h i s technique does take both the absorption and u t i l i z a t i o n of iron into account, although the r e l a t i v e contribution of the two processes cannot be separated (Van Campen, 1983). There are also several disadvantages to the method. Formulation of the diet becomes a problem when t h i s hemoglobin repletion technique i s used to evaluate the i n t r i n s i c iron of whole foods ( F r i t z and Pla, 1972). The requirement for graded levels of iron i n the diets means that when whole foods are used the composition of diets i s usually not constant between groups ( M i l l e r and Schricker, 1982; Van Campen, 1983). However, the addi-tion of whole foods to the basal diet i s done in a manner to maintain protein and energy levels as nearly constant as possible between diets so that the diets have a similar effect on the growth of the animals ( F r i t z and P l a , 1972). There i s a difference of opinion among researchers as to the proper data analysis technique for the calculation of r e l a t i v e b i o l o g i c a l values of iron sources (Van Campen, 1983). F r i t z et a l . (1975) compared several s t a t i s t i c a l treatments ( p a r a l l e l l i n e s , slope-ratio and graphic) for the 21 calculation of r e l a t i v e b i o l o g i c a l values of collaborative iron samples. It was observed that the different methods yielded similar conclusions regarding r e l a t i v e b i o l o g i c a l values of the iron sources. F r i t z and co-workers postulated that i f there were large s i g n i f i c a n t differences i n iron u t i l i z a t i o n between iron sources, these differences would be obvious regardless of the s t a t i s t i c s applied to the data. The hemoglobin repletion test probably overestimates the RBV of iron sources. The feeding of suboptimal levels of iron to depleted rats during a rapid stage of growth provides conditions whereby the animals can be expected to make the most e f f i c i e n t use of any iron given (Thompson and Raven, 1959). However, i t i s necessary to use depleted animals i n order to measure a response to dietary iron ( M i l l e r and Schricker, 1982). The results obtained by the hemoglobin repletion test are r e l a t i v e , rather than absolute, estimates of iron absorption. This l i m i t s the technique to ranking or screening a variety of iron sources (Van Campen, 1983). F i n a l l y , there are many problems associated with the extrapolation of results from rats to humans (M i l l e r and Schricker, 1982; Van Campen, 1983). G. THE RAT AS A MODEL SYSTEM FOR PREDICTING IRON AVAILABILITY IN MAN The rat i s often used as an experimental model for predicting iron a v a i l a b i l i t y for humans. More often than not, physiological differences between the two species account for the different iron a v a i l a b i l i t y values obtained i n the two species for the same iron sources. 22 Numerous studies have shown that ferrous iron i s absorbed and u t i l i z e d more e f f i c i e n t l y by man than f e r r i c iron (Narins, 1980), whereas rats absorb the ferrous and f e r r i c forms equally well (Elwood, 1965). The rat does appear to be a v a l i d model for screening inorganic supplementary iron souces for man. The a v a i l a b i l i t i e s of selected inorganic iron sources to rats and man, as determined by animal repletion studies and human r e l a t i v e plasma iron responses following test sources of iron, were ranked in the same order by both species (ferrous sulfate > reduced iron > f e r r i c orthophosphate and sodium iron pyrophosphate) (Pla and F r i t z , 1971). The rat i s not a suitable model for evaluating the enhancing effect of ascorbic acid on nonheme dietary iron absorption. F r i t z and Pla (1972) have postulated that species such as rats, which do not need a dietary source of ascorbic acid, produce enough ascorbic acid in their bodies to minimize any effect from dietary sources. Among 21 food sources of iron tested in a collaborative study by the rat repletion technique ( F r i t z et a l . , 1970) no clear d i s t i n c t i o n i n a v a i l -a b i l i t y was observed between animal and vegetable foods. An extensive collaborative human study using the i n t r i n s i c radioiron l a b e l l i n g tech-nique, determined that the iron of animal origin was more available to humans than that from vegetable sources (Martinez-Torres and Layrisse, 1974). The absorption of hemoglobin iron by the rat, regardless of i t s iron status, i s much lower than that demonstrated i n normal (11%) and iron deficient human subjects (22%) (Callender et a l . , 1957; Turnbull et a l . , 23 1962). In studies using radioiron l a b e l l i n g techniques, Weintraub et a l . (1965) Found that i n the normal adult rat, the absorption of a test dose of porphyrin iron (2%) was s i g n i f i c a n t l y less than that of a comparable dose of iron i n the form of ferrous sulfate (11%). The comparably low level of absorption of hemin iron (1%) by the normal rat demonstrated that the poor absorption of hemoglobin iron was not due to the i n a b i l i t y of the rat to s p l i t off the heme from the globin. I t was observed that iron deficiency did not improve the absorption of hemoglobin iron (3%) while i t had an effect on the absorption of ferrous sulfate iron (30%). That t h i s i n a b i l i t y to absorb heme iron was of physiological significance was demonstrated by producing a state of iron deficiency in animals raised on a diet with hemoglobin as the only source of iron. The results of their research led them to suggest that possibly the rat lacks the absorptive pathway for heme iron found in man and that perhaps there i s a difference i n the two species regarding the e p i t h e l i a l c e l l s ' a b i l i t y to sequester iron as the salt or i n the heme ring. 24 MATERIALS AND METHODS A. ANIMAL CARE Ninety male weanling Wistar rats, 35.7 ± 4.7 g in weight, were obtained from Woodlyn Laboratories Ltd., Guelph, Ont. The rats, prior to the i r a r r i v a l , had received only maternal milk; the shipping cages con-tained potato pieces but not stock diet. Upon a r r i v a l , they were housed singly in screen-bottomed stainless steel cages in f a c i l i t i e s of the Animal Science Department, U.B.C. The temperature of the animal room was main-tained at 23-25uC; the l i g h t i n g was regulated automatically to provide alternate 12-hour periods of l i g h t and darkness ( l i g h t on from 8:00 a.m. to 8:00 p.m.). Food and deionized d i s t i l l e d water were given _ad libitum throughout the experimental period. B. EXPERIMENTAL DIETS 1. General description Nine experimental diets were used: a semipurified low-iron basal diet; the same basal diet to which was added 5, 10, 15, 20 or 25 mg iron per kg diet, supplied by the FeSO^^r^O reference standard (food grade FeS04*7H20, Mallinckrodt, St. Louis, M0); and three test source diets which were formulated to provide a t o t a l of 15 mg iron per kg diet from either freeze-dried raw lean ground beef, a textured defatted soy flour meat extender or a 2.3:1 (w/w) combination of the freeze-dried lean ground beef and the textured defatted soy flour. Each of the nine diet treatments was assigned an alphabetic code as summarized in Table VI. The composition of the low-iron basal diet and that of the mineral and vitamin mixes are 25 shown i n Table II while those of the diets containing test foods are outlined i n Tables I I I , IV and V. A l l diets were formulated to supply the nutrient requirements of the growing rat (NRC, 1978), with the exception of ir o n ; the reference sources for ingredient nutrient contents being Agriculture Handbook 8 (Watt and M e r r i l l , 1963), s c i e n t i f i c l i t e r a t u r e , supplier information and data obtained by chemical analysis. The diet ingredients were mixed by hand using a stainless steel bucket and culinary whisk. Efforts to preserve the n u t r i t i o n a l and sensory properties of these diets included their being stored i n the dark in sealed p l a s t i c bags at -22UC between experiments and 4 UC during the feeding t r i a l s . 2. Preparation of test source ingredients a. Beef The t o t a l quantity of raw lean ground beef required for the prepara-tion of beef-containing test diets was purchased at one time from a l o c a l r e t a i l outlet. The mean fat content of the beef, as determined by the Goldfisch ether extraction method on duplicate samples, was calculated to be 15.63% on a wet basis (63.33% moisture). The beef was pooled and mixed, weighed into 400 g l o t s , wrapped i n pre-weighed sheets of aluminum f o i l and frozen overnight. The beef was freeze-dried (Thermovac Freeze Dryer, Model FDC-10, Thermovac Industries Corp., Marshalltown, IA), the average f i n a l moisture content achieved being 2.12%. The freeze-dried beef was then chopped i n a Waring blender (Waring Products Division, Dynamics Corp. of America, New Hartford, CT) followed by hand grinding i n porcelain mortar with a pestle. The resulting fibrous meal was stored i n the dark at -22UC i n sealed p l a s t i c bags u n t i l being incorporated into the test diets. 26 b. Textured defatted soy flour A single sample of Textured Vegetable Protein (Unflavored, F o r t i f i e d TVP®, Archer Daniels Midland Company, Decatur, IL), an extruded defatted soy flour product marketed i n North America as a meat extender, was ob-tained through B r i t i s h Canadian Importers, Vancouver, B.C. The processing steps involved i n the preparation of t h i s soy product are outlined i n Figure 1; the nutrient content of the r e t a i l e d product i s reported i n Table I. The soy product was physically converted to a form suitable for mixing into test diets by f i r s t chopping i t i n a Waring blender followed by hand grinding with a porcelain mortar and pestle. The product, in i t s f i n a l form, was a powder which passed through mesh No. 18 (1.00 mm diam). The ground soy product was stored at -22"C i n sealed p l a s t i c bags u n t i l incorporation into test diets. 3. Formulation of test source diets The three test diets containing food sources of iron were designed to be quantitatively similar with respect to iron contribution of the test sources as well as be ali k e i n protein, fat and c a l o r i c content. Cellulose powder was added to adjust the diets accordingly. Overall, t h i s group of diets was formulated to contain protein and energy levels comparable to that supplied by the basal diet. The r a t i o of freeze-dried ground beef to unhydrated soy product (2.3:1, w/w) i n the beef and soy flour diet was selected to approximate the formula recommended by the supplier for preparing a basic extended ground beef loaf: 10 lb extended beef = 7 lb raw ground beef + 1 lb meat extender 27 Figure 1. Processing scheme for TVP* Unflavored-Fortified product.^»2,3 Whole Raw Soybeans Heat 'rack, d e i u l l , flake Hypocotyl (3%) ("Germ") Cotyledons (8958) ( F u l l Fat Flakes) Solvent extract Defatted Soy Flakes Grind ani c l a s s i f y Hulls ( 8 » ) ("Bran") Defatted Soy Flour Blend to a s t i f f ' s l u r r y with water Extlude Dry and^classify I Toast F o r t i f y wilh nutrients (Amino acids, vitamins, minerals) Nutrient F o r t i f i e d Textured Defatted Soy Flour Product ''Archer Daniels Midland Company, Decatur, IL. ^Processing information obtained through B r i t i s h Canadian Importers, Vancouver, B.C. -'The percentage figures for the morphological components of the soybean seed are expressed on a wet weight basis (Circle and Smith, 1978). 28 Table I. Nutrient analysis for T V P W Unflavored-Fortified product.^ per 100 g per 100 g Water (58) 6.0 Total carbohydrate (g) 31.5 Fiber (g) 3.0 Total protein (g) 52.0 Amino acids Tryptophan (mg) 343 Threonine (mg) 2170 Isoleucine (mg) 2464 Leucine (mg) 4075 Lysine (mg) 3164 Methionine (mg) 596 Cystine (mg) 409 Phenylalanine (mg) 2603 Tyrosine (mg) 1619 Valine (mg) 2617 Arginine (mg) 3777 Histidine (mg) 1347 Total fat (g) 1.0 Calories 280.0 Vitamins A D E C Folacin (mg) Thiamin (mg) Riboflavin (mg)-' Niacin (mg)3 B 6 (mg)3 B 1 2 (/<g)3 B i o t i n ty<g) Pantothenate (mg)3 Minerals (g) Calcium (mg) Phosphorus (mg) Iron (mg)^. Iodine (/<g) Chlorine (mg) Sodium (mg) Potassium (mg) Magnesium (mg) Manganese (mg) Zinc (mg) Copper (mg) 0 0 0 0 .35-.36 .60-.64 .60 16.0 1.4 5.7 73.0 2.0 6.0 220 570 10 500 19 700 2200 260 2.6 5.5 1.3 ^Archer Daniels Midland Company, Decatur, IL. ^The endogenous iron content of the soy flour (8 mg/100 g, wb) i s sup-plemented with ferrous sulfate to bring the t o t a l iron content of the product to 10 mg/100 g, wb. 3Nutrients added i n excess of levels reported for TVP® Unflavored, Non-fortified product. 29 Table I I . Composition of basal diet. Ingredient Skim milk powder2 53.00 Glucose 35.96 Corn o i l 3 5.00 Cellulose powder^ 2.70 Potassium chloride 2.05 Vitamin mix^ 1.00 Choline chloride 0.20 Potassium phosphate monobasic 0.07 Mineral mix^ 0.02 '4.2 ppm total iron (db). ^Lucerne brand (Canada Safeway Ltd., Winnipeg, Man.). 3Mazola brand (Canada Starch Co., Inc., Montreal, P.Q.). ^Alphacel (ICN Nutritional Biochemicals, Cleveland, OH). ^AIN vitamin mixture 76 (American Institute of Nutrition, 1977) (10663 -U.S. Biochemical Corp., Cleveland, OH) supplying per kg diet: 6.0 mg thiamin HC1, 6.0 mg riboflavin, 30.0 mg nicotinic acid, 7.0 mg pyridoxine HC1, 2.0 mg f o l i c acid, 10.0/<g cyanocobalamin, 16.0 mg D-calcium panto-thenate, 0.2 mg D-biotin, 4,000 I.U. vitamin A, 1,000 I.U. vitamin D3, 50 I.U. vitamin E and 50^g vitamin K. ^Mineral mix supplying per kg diet: 0.85 mg l^C^Oy, 20.0 mg CuS04'5H20, 2.25 mg NaF, 0.20 mg KI, 154.0 mg MnS04«H20, 0.22 mg Na2Se03 and 15.0 mg ZnO. Table I I I . Composition of beef diet. Ingredient Skim milk powder 29.50 Cellulose powder 23.00 Glucose 21.99 Freeze-dried raw ground beef^ 20.01 Corn o i l 3.00 Potassium chloride 0.92 Vitamin mix2 1.00 Choline chloride 0.20 Calcium carbonate 0.28 Potassium phosphate monobasic 0.07 Mineral mix2 0.02 Sodium chloride 0.01 'Moisture content of 2.12%. 2As for basal diet (Table II). 31 Table IV. Composition of beef and soy flour diet. Ingredient 0' ra Skim milk powder 30.78 Glucose 21.29 Cellulose powder 19.79 Freeze-dried raw ground beef 12.59 Corn o i l 7.30 Textured defatted soy flour^ 5.55 Potassium chloride 1.18 Vitamin mix3 1.00 Choline chloride 0.20 Calcium carbonate 0.25 Potassium phosphate monobasic 0.05 Mineral mix3 0.02 ''Moisture content of 2.12%. ^Ground, Unflavored, Fortified TVP® (Archer Daniels Midland Company, Decatur, IL). 3As for basal diet (Table II). 32 Table V. Composition of soy flour diet. Ingredient Skim milk powder 32.97 Glucose 19.98 Textured defatted soy flour 1 14.99 Corn o i l 14.59 Cellulose powder 13.99 Potassium chloride 2.11 Vitamin mix2 1.00 Choline chloride 0.20 Calcium carbonate 0.15 Mineral mix2 0.02 1 Ground, Unflavored, Fortified . TVP® (Archer Daniels Midland Company, Decatur, IL). 2As for basal diet (Table II). 33 Table VI. Repletion phase diet treatments. Treatment code Diet treatment A Basal d i e t 1 B Basal diet + 5 mg iron (as FeS04*7H20)/kg diet C Basal diet + 10 mg iron (as FeSO^-Tr^OO/kg diet D Basal diet + 15 mg iron (as FeS04,7H20)/kg diet E Basal diet + 20 mg iron (as FeSO^Th^OVkg diet F Basal diet + 25 mg iron (as FeSO^'Vr^OVkg diet G Freeze-dried ground beef H Freeze-dried ground beef + textured defatted soy flour (2.3:1, w/w) I Textured defatted soy flour Diet also used i n depletion phase to make rats anemic. In repletion phase the diet served as a negative control. 34 + 2 lb water, while at the same time maintaining the t o t a l iron contribu-tion by these ingredients at 15 ppm. C . BIOLOGICAL ASSAY The a v a i l a b i l i t y of the iron contained in the test diets was deter-mined using the hemoglobin repletion procedure described by Pla and F r i t z (1970) and late r adopted by the AOAC as o f f i c i a l f i r s t action (AOAC, 1980, Sections 43.217-43.219). The rats were fed the low-iron basal diet u n t i l they were s a t i s f a c t o r i l y depleted of their iron stores i . e . hemoglobin < 6 g/dL, hematocrit < 29% (Pla and F r i t z , 1970). Two weeks after the rats were put on the basal diet their hematological indices were measured. Blood samples were taken by t a i l c u t t i n g after anesthetizing the rats with anhydrous ether (for anesthesia) (J. T. Baker Chemical Co., P h i l l i p s b u r g , NJ). Blood for hemoglobin determinations was di r e c t l y drawn from the t a i l vein into oxalate coated calibrated sampling pipets (Corning Glass Works, Corning, NY). S i m i l a r l y , blood samples for hematocrit measurement were drawn into even bore heparinized micro-hematocrit c a p i l l a r y tubes (Fisher S c i e n t i f i c Co., Fair Lawn, N3) to the 3/4 mark; the tubes were sealed at one end with clay. Only single blood samples were taken for each test per sampling session due to the rapid onset of blood c l o t t i n g within the t a i l vein. After two weeks i t was found that the rats were not s a t i s f a c t o r i l y depleted so they were continued on the basal diet for an additional week. At the end of the t h i r d week measurements of the blood parameters were repeated and accepted as being s u f f i c i e n t l y low to terminate the depletion phase of the experiment. At the end of 21 days of depletion, the mean 35 values (± standard deviation) for hemoglobin and hematocrit were 3.47 ± 0.73 g/dL and 14.69 ± 2.25%, respectively (Appendix B and C). During the depletion phase individual rat weights and food consumptions were recorded weekly. The rats were started on the repletion phase the same day they were assessed to be s u f f i c i e n t l y iron deplete. The rats were grouped according to s i m i l a r i t y in weight (Appendix B and C); i n turn, these groups were randomly assigned to one of the nine experimental diets described e a r l i e r (Table VI). Food and deionized d i s t i l l e d water were provided ad libitum throughout the repletion phase. As only 30 rats were ordered at a time, the depletion/repletion procedure was conducted on three occasions so that for the nine diets tested there were 10 observations for each type of response measured. After two weeks on the experimental diets, individual blood samples were taken for hemoglobin and hematocrit determinations. Individual rat weights and food consumptions were recorded weekly. D. HEMOGLOBIN AND HEMATOCRIT DETERMINATION Hemoglobin was determined by the cyanmethaemoglobin method of Crosby et a l . (1954). 0.02 mL of blood was diluted with 5 mL of Drabkin's reagent and the absorbance of the solution was read at 540 nm using a Beckman double beam spectrophotometer (Beckman Industries, Fullerton, CA) which had been set at zero absorbance with Drabkin's reagent. The concentration of cyanmethemoglobin pigment was calculated from a standard curve prepared with cyanmethemoglobin standards (Laboratory Centre for Disease Control, Department of National Health and Welfare, Ottawa, Ont.) made up i n Drabkin's reagent. The results were expressed as g hemoglobin/dL. 36 Hematocrit values were determined according to the method of Cohen (1967). B l o o d - f i l l e d hematocrit tubes were centrifuged (International Micro-capillary Centrifuge, Model MB, International Equipment Co., Needham Heights, MA) at 12,000 rpm for 6 min. Hematocrit {% packed red c e l l volume) was calculated by measuring with a 150 mm ruler the height of the packed red c e l l s and dividing t h i s value by the t o t a l height of red c e l l s and plasma. E. CALCULATION OF IRON BIOAVAILABILITY The hematological responses of rats fed the basal diet containing graded levels of ferrous sulfate reference standard were plotted as a standard curve and the equation of the l i n e determined by linear regression analysis using an Apple I I personal computer (Apple Computer, Inc., Cuper-t i n o , CA) and curve f i t t i n g program package (Warme, 1980). The responses of rats fed test sources of iron were related to the standard curve by comparing the iron levels required to achieve the same hematological response. The r e l a t i v e b i o l o g i c a l value (RBV) of the test source was calculated according to the following equation mgFe/kg from FeS04*7H20 RBV test source = 100 x ; giving the same mgFe/kg from test source response i n hemoglobin (or hematocrit) (Pla and F r i t z , 1971). F . PROXIMATE ANALYSIS OF DIET INGREDIENTS AND DIETS Proximate analysis was performed on the iron test source ingredients, the low-iron basal diet and the three test source diets. The defatted soy protein product and diets containing the soy product were analyzed for 37 phytic acid content. Basal diet containing graded levels of ferrous sulfate was analyzed for moisture and total iron content only. Chemical analysis of the cellulose powder included quantitative determination of moisture, neutral detergent fiber, total iron and magnetically extractable iron. 1. Moisture The AOAC vacuum oven method for wheat flour (AOAC, 1980, Section 14.002), with some modification, was used to determine the moisture content of the diets and individual ingredients. Triplicate samples (2 g) were accurately weighed into pre-dried, desiccator-cooled, weighed aluminum dishes (50.8 mm diam x 22.4 mm depth) with tight f i t t i n g slip-over covers (Canlab, Richmond, B.C.). Samples were dried to a constant weight in a vacuum oven (National Appliances Co., Portland, OR) under 91.5 kPA (27 inches Hg) at 100UC for defatted soy flour and cellulose powder samples and 80"C for diet and freeze-dried beef samples. The loss in sample weight was recorded as moisture. 2. Crude protein Protein content was determined by the macro-Kjeldahl technique of the AOAC (AOAC, 1980, Section 2.057) with the following modifications. Trip-licate 1 g samples were digested for 1 h in 35 mL H2SO4 (cone) plus added catalyst (10.4 g of a K2SO4-CUSO4 - pumice mixture, 10:.3:1 w/w/w). Dis-t i l l a t e s were titrated with HC1 (0.1091 N) previously standardized with THAM (tri s hydroxy methylamino methane) (Fisher Scientific Co., Fair Lawn, N3); the number of mLs HC1 per titration being recorded. 38 % N (db) of each sample was calculated by the following equation N HC1 x 14.007 x mL HC1 used x 100 g dry weight of sample x 1000 Values for crude protein content (%, db) were obtained by multiplying the % N (db) by the following nitrogen to protein conversion factors: 6.38 for the milk-based basal diet and 6.25 for the beef or beef and soy-based diets. 3. Ether extract Crude fat determinations were performed i n t r i p l i c a t e using a Gold-fis c h fat extraction unit (Labconco Corp., Kansas City, M0). Prior to ex-t r a c t i o n , a l l samples were vacuum oven dried to a constant weight and held in a desiccator containing s i l i c a gel. S i m i l a r l y , the beakers used i n the extraction procedure were dried, weighed and desiccated u n t i l the time of extraction. 1 g dried samples were transferred to Whatman cellulose ex-t r a c t i o n thimbles (22 mm diam x 80 mm ht) (VWR S c i e n t i f i c Inc., Brisbane, CA) containing a small amount of glass wool packing at the t i p . The sam-ples were extracted for 4 h on the low heat setting of the unit with 35 mL petroleum ether. The extraction beakers were removed from the unit, placed i n a fume hood to v o l a t i l i z e the remaining ether (6 h), oven dried (85 UC) overnight (8 h), desiccator cooled (1 h) and weighed. The crude fat contents of the beakers were calculated and reported as % ether extract (db). 4. Neutral detergent fiber The neutral detergent fiber content of samples was analyzed i n t r i p -39 l i c a t e by the micro-digestion procedure developed by Waldern (1971) employ-ing the neutral-detergent solution of Van Soest and Wine (1967). Sample preparation included extraction for 6 h with petroleum ether using Soxhlet extraction apparatus (Fisher S c i e n t i f i c Co., Fair Lawn, N3) followed by overnight oven drying at 50"C to v o l a t i l i z e the solvent. Particulate matter remaining after the f i l t r a t i o n of digested sample was vacuum oven dried overnight (12 h) at 100"C under 91.5 kPa, cooled i n a desiccator over s i l i c a gel and weighed. Sample fiber contents were calculated from the recorded dry weights employing a correction for crude fat content and reported as % neutral detergent fiber (db). 5. Ash The ash content of samples was determined in t r i p l i c a t e using the AOAC dry ashing technique for wheat flour (AOAC, 1980, Section 14.006) with the following modification. The samples were carbonized on a hot plate prior to their being ignited in the muffle furnace. Sample ash contents were calculated from recorded ash weights and reported as % ash (db). 6. Iron a. Total iron Total iron concentration of samples was determined in t r i p l i c a t e on wet ashed samples by atomic absorption spectrophotometry. A l l glassware used during the a n a l y t i c a l procedure was washed, soaked overnight in I N HC1 and rinsed with deionized d i s t i l l e d water. A l l reagent solutions used i n t o t a l iron determination were prepared with deionized d i s t i l l e d water. 40 The wet ashing procedure involved the digesting of 1.2 g samples with 15 mL of a mixture of 25% hydrochloric and 65% n i t r i c acids (9:1, v/v) and 10 mL of water for 1/2 h over a hot water bath (80-85UC) as described by Maurer (1977). The digested sample was cooled, made up to volume (50 mL) with water and f i l t e r e d through ashless f i l t e r paper (Whatman 541, VWR S c i e n t i f i c Inc., Brisbane, CA). The iron concentration of the c l a r i f i e d solution was measured at 248.3 nm by a double beam atomic absorption spectophotometer (Perkin Elmer Corp., Norwalk, CT, Model 306) using an i r o n - s p e c i f i c hollow cathode lamp, 5 cm single-slot burner head and a i r -acetylene flame. The instrument was calibrated with iron standards pre-pared from C e r t i f i e d Iron Reference Solution (Fisher S c i e n t i f i c Co., Fair Lawn, NO) diluted with HCI-HNO3 acid mixture and deionized d i s t i l l e d water. A reagent blank was carried through the entire ashing procedure for every f i v e samples analyzed. Sample readings obtained by atomic absorption spectrophotometry were corrected for their respective reagent blank readings before calculating the iron concentration of samples on a dry weight basis. b. Magnetically extractable iron Total iron analysis of the cellulose powder indicated t h i s diet ingredient to contain a high l e v e l of iron. Further analysis was performed i n order to determine the derivation of t h i s iron. The magnetically extractable iron content was determined on duplicate samples of cellulose powder according to the method described by Lee and Clydesdale (1979b). The teflon coated magnets bearing the iron extracted from the sample slurry (5 g sample blended with 70 mL deionized d i s t i l l e d 41 water) were soaked i n concentrated HC1 u n t i l the iron had dissolved from the magnets. The HCl-iron solution was made up to volume with deionized d i s t i l l e d water and the iron concentration measured by atomic absorption spectrophotometry as described i n Section 6a. The instrument was c a l i -brated with iron standards prepared from C e r t i f i e d Iron Reference Solution (Fisher S c i e n t i f i c Co., Fair Lawn, NJ") diluted with 20% HC1. The concen-t r a t i o n of iron in the sample slurry was calculated on a dry weight basis. 7. Phytic acid The method of Latta and Eskin (1980) was used to determine the phytic acid contents of the defatted soy flour product and diet samples containing the soy product. Aliquots of sample extract were eluted through chloride anion-exchange resin. Inorganic phosphorus was eluted with 0.1 M NaCl followed by elution of phytate with 0.7 M NaCl. The absorbance of a solu-tion of phytate extract and Wade reagent (3:1, v/v) was read at 500 nm using a Cary 210 spectrophotometer (Varian Associates Inc., Palo A l t o , CA) which had been set at zero absorbance with deionized d i s t i l l e d water. The concentration of phytic acid was calculated from a standard curve prepared with sodium phytate standards ( i n o s i t o l hexaphosphoric acid, corn type V, 97% pure, sodium sal t ) (Sigma Chemical Co., St. Louis, M0) made up in deionized d i s t i l l e d water and reacted with Wade reagent. The procedure for phytic acid determination was performed in t r i p l i c a t e for each sample and the calculated sample concentrations reported on a dry weight basis. 42 G . S T A T I S T I C A L A N A L Y S I S The raw data collected for the bi o l o g i c a l assay was analyzed in a one-way analysis of variance, using the UBC MFAV program package (Le, 1978) available for use on the UBC Amdahl 470 V/8 computer. Duncan's multiple range test (Zar, 1974) was used to perform multiple comparisons among means. 43 RESULTS AND DISCUSSION A. COMPOSITION OF THE EXPERIMENTAL DIETS The composition of the test foods and fiber source used in form-ulating the diets and that of the 9 experimental diets are presented i n Tables VII and VI I I , respectively. Values experimentally determined are compared to reported values i n Appendix A. The results of analysis indicate the overall design objective for the 9 experimental diets, that being maintaining the protein and energy levels constant between diets, was achieved. I t i s well established that these n u t r i t i o n a l factors influence growth. At suboptimal levels of dietary iron the degree of anemia i s correlated with the growth of the young animal ( F r i t z and P l a , 1972). The group of diets containing iron from test sources were designed to supply on a per kg basis similar levels of protein, f a t , calories and iron (15 ppm). The l e v e l of protein and calories were kept constant for growth purposes; the levels of fat in each diet was held constant i n an effort to maintain the same sa t i a t i n g effect between diets. It has been hypothesized that the emptying of food into the intestine i s necessary for feeding to stop and for the satiety sequence to appear (Liebling et a l . , 1974). It i s well established that the presence of fat in the diet delays gastric emptying. Therefore, i t would appear reasonable to assume that a constant l e v e l of fat between diets would be expected to reduce differences i n feeding behavior. The decision to select 15 ppm as the iron test dose was based on the results of rat repletion assays performed on graded levels of ferrous sulfate (10, 20 and 40 ppm iron) by Amine et a l . (1972). In t h i s Table VIII. Proximate composition of basal, beef, beef and soy flour, and soy flour diets. Neutral Diet Dry Crude Ether Detergent Total Phytic Treatment Matter Energy 3 Protein Extract Fiber Ash Iron Acid (») (kcal/g) {%) {%) {%) (ppm) (?o) Basal 95.891»2 3.85 18.50 4.82 2.54 6.71 4.204 (0.23) (0.22) (0.21) (0.09) (0.09) (0.45) Beef 96.66 3.56 19.25 11.29 21.80 4.45 27.80 _ (0.34) (0.53) (0.09) (0.41) (0.05) (1.49) Beef + soy flour 96.77 3.64 19.25 12.52 19.95 5.07 28.55 U.11 (2.3:1,w/w) (0.19) (0.13) (0.05) (0.13) (0.01 ) (0.02) (0.02) Soy flour 96.92 3.78 19.72 13.65 12.92 6.27 30.63 0.33 (0.14) (0.58) (0.31) (0.62) (0.05) (2.18) (0.01) 1Mean ± (standard deviation) of 3 determinations. ^Expressed on dry weight basis. ^Calculated on basis of data values in Watt and M e r r i l l (1963). ^The t o t a l iron content of the basal diet to which was added 5, 10, 15, 20 or 25 mg iron FeS04-7H20)/kg was 9.3, 14.1, 18.4 , 23.7 and 28.4 ppm (db), respectively. Table VIII. Proximate composition of basal, beef, beef and soy flour, and soy flour diets. Neutral Diet Dry -f Crude Ether Detergent Total Phytic Treatment Matter Energy-5 Protein Extract Fiber Ash Iron Acid (?o) (kcal/g) {%) {%) (ppm) (S) Basal 95.891>2 3.85 18.50 4.82 2.54 6.71 4.204 (0.23) (0.22) (0.21) (0.09) (0.09) (0.45) Beef 96.66 3.56 19.25 11.29 21.80 4.45 27.80 — (0.34) (0.53) (0.09) (0.41) (0.05) (1.49) Beef + soy flour 96.77 3.64 19.25 12.52 19.95 5.07 28.55 0.11 (2.3:1,w/w) (0.19) (0.13) (0.05) (0.13) (0.01) (0.02) (0.02) Soy flour 96.92 3.78 19.72 13.65 12.92 6.27 30.63 0.33 (0.14) (0.58) (0.31) (0.62) (0.05) (2.18) (0.01) Wean ± (standard deviation) of 3 determinations. ^Expressed on dry weight basis. ^Calculated on basis of data values in Watt and M e r r i l l (1963). *The t o t a l iron content of the basal diet to which was added 5, 10, 15, 20 or 25 mg iron (as FeS04»7H20)/kg was 9.3, 14.1, 18.4, 23.7 and 28.4 ppm (db), respectively. 46 study i t was found that the dose-response l i n e remained linear up to a l e v e l of 20 ppm iron but departed from l i n e a r i t y thereafter. As well, the hemoglobin concentrations of animals repleted with ferrous sulfate up to the l e v e l of 20 ppm were below that considered normal for the rat, while those of rats supplied with 40 ppm iron had reached normal levels by the end of the t h i r d week of repletion. An iron test dose of 15 ppm was therefore selected on the assumption that the ferrous sulfate reference dose-response curve would be linear in t h i s region of the curve and the rats would s t i l l be engaged in the process of hemoglobin regeneration. The t o t a l iron content of these diets, while similar in l e v e l , ranging from 27.8 to 30.6 ppm (db), was about twice that o r i g i n a l l y intended. This was due to the iron contributed by the fiber source. As Table VII shows, the t o t a l iron content of the cellulose powder was approx-imately 77 ppm (db) of which 18 ppm (db) was magnetically extractable and assumed to originate from the metal surface of the m i l l used to reduce the cellulose to a fine powder. The addition of a non-nutritive fiber source, at levels ranging from 150 to 230 g per kg of diet formulation, was neces-sary i n order to maintain protein, fat and c a l o r i c levels constant between the diets. B . F E E D I N T A K E A N D GROWTH The feed intake and growth for rats fed diets containing food test sources are reported i n Table IX while those for rats repleted by graded levels of ferrous sulfate appear i n Appendix C. The mean feed intake of rats fed either of the beef-containing diets was greater than that of animals fed the ferrous sulfate supplemented basal 47 Table IX. Feed intake and body weight gain for iron depleted rats fed test sources of iron during the repletion period. Diet treatment Feed intake Weight gain ( g ) 3 (g) Basal + 25 mg iron 181.8ab1»2 77.6a (as FeS04-7H20)/kg (30.5) (16.3) Beef 216.1c /1.4a (16.4) (12.7) Beef + soy flour 199.6bc 74.8a (2.3:1, w/w) (20.4) (16.2) Soy flour 176.1a 77.0a (18.3) (15.9) 'Mean of 10 animals ± (standard deviation). 2Me ans i n a column followed by the same l e t t e r are not s i g n i f i c a n t l y different (P > 0.05) as determined by Duncan's multiple range test. 3Expressed on dry weight basis. 48 diet or the diet containing soy flou r . However, th i s higher level of consumption of diets containing beef was not reflected i n the weight gain of animals fed these diets. The lower c a l o r i c density of the beef-containing diets (Table VIII) did not account for the increased consumption. The differences in mean feed intake for the three test source diets appear to correlate with the crude fat content of these diets (Table V I I I ) , however, no definite conclusion can be made regarding the effect of fat l e v e l on animal feeding behavior. A comparison of the mean weight gains for a l l 9 diets showed there to be no si g n i f i c a n t differences (P > 0.05) in body weight gain by rats fed the basal diet supplemented with 10, 15, 20 and 25 ppm iron as ferrous sulfate or the three test source diets. Furthermore, the weight gains for these diets were s i g n i f i c a n t l y greater (P < 0.05) than those for rats fed either the basal diet without ferrous sulfate or the basal diet supple-mented with 5 ppm iron as ferrous sulfate. The greater gain i n weight observed in the animals fed higher levels of iron could be interpreted as an improved health status resulting from a faster rate of iron repletion (Mahoney and Hendricks, 1976). The weight gain by rats supplemented with ferrous sulfate compared favorably to experimental values obtained in studies using similar methodology (Rotruck and Luhrsen, 1979; Shah et a l . , 1983). C. HEMOGLOBIN AND HEMATOCRIT RESPONSES The f i n a l hemoglobin levels and hematocrit values for rats fed iron as food test sources or as ferrous sulfate are reported i n Table X and Appendix C, respectively. 49 Table X. Final hemoglobin levels and hematocrit values for iron depleted rats fed test sources of iron during the repletion period. Diet treatment Fin a l hemoglobin F i n a l hematocrit (g/dO {%) Basal + 25 mg iron 10.1b 1, 2 35.5bc (as FeS0V7H 20)/kg (1.8) (4.6) Beef 7.7a 3U.6a (0.9) (2.5) Beef + soy flour 8.7a 32.7ab (2.3:1, w/w) (0.7) (2.5) Soy flour 10.8b 37.8c (1,3) (4.4) Mean of 10 animals ± (standard deviation). Means i n a column followed by the same l e t t e r are not s i g n i f i c a n t l y different (P > 0.05) as determined by Duncan's multiple range test. 50 The hemoglobin levels and hematocrit values for rats fed the basal diet supplemented with graded levels of ferrous sulfate reference standard increased progressively as the iron dose became higher, the mean hemato-l o g i c a l response at each iron l e v e l being s i g n i f i c a n t l y higher (P < 0.05) than the response at the dose below. The dose-response relationship observed in th i s study f u l f i l l s what i s considered to be an appropriate c r i t e r i o n of a good b i o a v a i l a b i l i t y assay (Mahoney and Hendricks 1982). The hematological responses of rats fed the highest level of iron as added ferrous sulfate (25 ppm, db) were below the reported normal range of values for the rat (12-17.5 g hemoglobin/dL blood, 39-53% hematocrit) (Spector, 1956), thus indicating that at t h i s level of iron the rats were s t i l l a ctively involved i n the regeneration of hemoglobin and erythrocytes. This suboptimal hematological response by the rats i s also regarded as a c r i t e r i o n of a good assay. At the end of two weeks of repletion the mean hemoglobin concentra-tion of rats fed the soy flour diet was not s i g n i f i c a n t l y different (P > 0.05) from that of rats fed the basal diet supplemented with 25 ppm iron as ferrous sulfat e ; the f i n a l hemoglobin concentrations of rats consuming these two diets were s i g n i f i c a n t l y higher (P < 0.05) than those of rats fed either the beef diet or beef and soy flour diet. There was considerable overlap of mean response values for rats repleted with the test source diets and the ferrous sulfate supplemented basal diet when the f i n a l hematocrit value was used as the c r i t e r i o n of response to the iron source. The hematocrit values for the soy flour diet was s i g n i f i c a n t l y greater (P < 0.05) than those for the beef diet and beef 51 and soy flour diet; the response by rats fed the supplemented basal diet was s i g n i f i c a n t l y greater (P < 0.05) than that by rats repleted with the beef diet. D. REGRESSION ANALYSIS Standard hematological response curves for the iron reference salt (ferrous sulfate) were derived from the hemoglobin concentrations and hematocrit values determined for blood samples taken from rats repleted for two weeks with the low-iron basal diet and basal diet supplemented with graded levels of ferrous sulfate. The response data were plotted against the iron concentrations of the 6 reference diets, corrected for the iron contributed by the basal diet ingredients (Figure 2), using the linear least squares curve f i t t i n g technique. Visual inspection of the data points in re l a t i o n to the regression l i n e obtained suggested that the curve was indeed linear up to 15 ppm added iron, but beyond th i s iron l e v e l there was a downward trend i n the f i t of data points to the curve. The slopes of in d i v i d u a l l y calculated dose-response regression lines ( f i n a l hemoglobin) i n the range of 0 to 15, 0 to 20 and 0 to 25 ppm added iron were respectively 0.391, 0.341 and 0.324, thus confirming curvature to be present. However, despite the curvature, responses to even the highest l e v e l of iron had to be included in the curve in order to accommodate the calculation of RBVs of iron i n the test source diets. Thus, the curves from which the RBVs were calculated were found to be well described by the following regression equations: F i n a l hemoglobin Y = 2.673 + 0.324X r 2 = 0.8328 s y . x = 1.215 F i n a l hematocrit Y = 12.775 + 1.034X r 2 = 0.8292 s y . x = 3.862 Figure 2. Two Meek hemoglobin response of iron depleted rats to graded levels of ferrous sulfate reference standard. IRON DOSE (PPM), AS ADDED FERROUS SULFATE 53 The standard response curve for hemoglobin i s presented i n Figure 2. The d i s t r i b u t i o n of data points about the regression l i n e i s representative of that obtained for the hematocrit response curve as well. Alternatively, the hemoglobin response data for a l l levels of iron were plotted by the polynomial least squares curve f i t t i n g technique. The r 2 value obtained by t h i s p l o t t i n g technique was 0.8467. E. RELATIVE BIOLOGICAL VALUE OF IRON IN TEST SOURCE DIETS The r e l a t i v e b i o a v a i l a b i l i t y of the iron in the beef, beef and soy flo u r , and soy flour diets are reported i n Table XI. The RBVs were calculated using the t o t a l iron values for the test source diets as determined by chemical analysis (Table VIII). Had the t o t a l iron values for the test source diets been corrected for iron contributed by the cellulose powder, then the RBVs calculated on the basis of either hematolo-g i c a l response for a l l food test sources would have greatly exceeded that demonstrated for ferrous sulfate, which would have been in contradiction to the l i t e r a t u r e reported for the rat. As the majority of animal researchers, i n evaluating the r e l a t i v e iron a v a i l a b i l i t y in beef, soy and beef-soy mixtures, employed hemoglobin repletion methodology, a l l RBVs referred to from the present study are those calculated from f i n a l hemoglobin responses. 1. Beef The RBV of iron i n freeze-dried raw ground beef (56%) compared favorably to the value of 53% reported by others (Shah et a l . , 1983) for Table XI. Relative biological value (RBV) of iron in iron test source diets (FeS04*7H20= 100.0). RBV3 RBV3 Diet treatment Final hemoglobin 95% confidence l i m i t s Final hematocrit 95% confidence l i m i t s Beef 55.7a1 48.6 - 62.8 61.9a 55.3 - 68.5 Beef + soy flour (2.3:1, w/w) 65.3b 59.7 - 71.0 67.6a 61.2 - 74.0 Soy flour 81.3c 71.7 - 90.9 79.0b 68.5 - 89.5 'Mean of 10 animals. 2Means in a column followed by the same l e t t e r are not s i g n i f i c a n t l y different (P > 0.05) as determined by Duncan's multiple range test. 3 R e l a t i v e biological value = 100 x (mgFe/kg from FeSO^^r^OVtmg Fe/kg from test source) giving the same hematological response. 55 anemic rats. The somewhat lower RBV of 41% reported by Rotruck and Luhrsen (1979) may possibly have been due to cooking of beef before feeding. The low a v a i l a b i l i t y of beef iron to rats in the present study was not unexpected. Weintraub et a l . (1965) demonstrated that the rat, regardless of i t s iron status, does not absorb porphyrin iron as well as i t does iron i n the inorganic form as ferrous sulfate. In human studies the absorption of i n t r i n s i c a l l y labelled iron of veal muscle was not s i g n i f i c a n t l y different from that of a tracer dose of iron as f e r r i c chloride or iron ascorbate (Layrisse and Martinez-Torres, 1972). Thus, the r e l a t i v e a v a i l a b i l i t y of iron in veal muscle, which i s simil a r to that i n ground beef, was 100% of the standard, almost twice that observed i n the present study with rats. 2. Soy protein The RBV of iron i n the ferrous sulfate f o r t i f i e d textured defatted soy product (81%) f a l l s within the broad range of values reported in the l i t e r a t u r e for a variety of commercially available soy protein products. Rat feeding studies employing hemoglobin repletion methodology have shown the mean re l a t i v e iron a v a i l a b i l i t y from f u l l fat soy flour (SF) to be 81% (Picciano et a l . , 1984); from soy protein isolate (SI) to be 61% (Steinke and Hopkins, 1978) and 82-100% (Rotruck and Luhrsen, 1979); and from soy protein concentrate (SC) to be 62% (Shah et a l . , 1983) and 92% (Picciano et a l . , 1984). Rat feeding studies employing radiolabelled test meals (Schricker et a l . , 1983), reported the re l a t i v e iron a v a i l a b i l i t y from SF, SC and SI to be 70 to 90% that of ferrous sulfate added to casein-based diets. I t would appear from the above mentioned l i t e r a t u r e values that 56 there is a general lack of ability on the part of the rat to discriminate between the availability of iron from different soy protein products, but that on the whole, the iron of soy protein is highly available. 20% of the iron in the soy product used in the present study was derived from ferrous sulfate, an inorganic iron source which is recognized as being highly available to the rat (Pla and F r i t z , 1970). The effect of soy protein on the availability of this added iron was not investigated in the present study. However, as the RBV of the iron in the soy diet was high, i t would appear that the effect of this particular soy product on the availability of the supplemental iron was minimal. At the commencement of this study, the inhibitory effect of phytic acid on iron absorption was a highly controversial issue. Phytic acid, the hexaphosphate of myoinositol, is a strong chelating agent capable of binding d i - and trivalent metal ions. This is particularly true of calcium, copper, zinc, magnesium and iron _in vitro at physiological pH. However, mineral interactions in vivo are not clear and are complicated by protein-mineral phytate interactions (Erdman, 1979). The origin of the postulate that phytic acid inhibits nonheme iron absorption is unclear, but i t would appear to be largely based on the inhibitory effect of non physiological doses of purified sodium phytate on iron absorption in rats and in man (Rotruck and Luhrsen, 1979). It is currently f e l t that the inhibitory effect of purified phytic acid i s dependent on the chemical form and concentration (Morris, 1983). Recently, i t has been reported that the utilization of iron by iron deficient rats was unaffected when sodium phytate was added to the diet at levels up to 4% 57 (Hunter, 1981). The present understanding in regard to the action of phytic acid on food iron a v a i l a b i l i t y i s that the pu r i f i e d form and that of endogenous food phytate may d i f f e r considerably from each other (Rotruck and Luhrsen, 1979; Morris, 1983). In view of the current research and the excellent a v a i l a b i l i t y of iron i n the soy product used i n the present study, i t i s unlikely that the phytic acid content of the diet, present at the level of 0.33% (db), had an inhibit o r y effect on dietary iron absorption. Shah et a l . (1983) found there was no difference in the b i o a v a i l a b i l i t y to rats of iron i n either soy or rapeseed protein concentrate despite a three-fold difference i n phytic acid concentration. Human studies employing radiolabelled iron-containing meals have indicated that the iron of soybeans and various processed soy products i s not highly available to t h i s species. Reported mean ratios for soy iron: reference iron absorption include 0.17 for boiled soybeans and 0.33 for baked soybeans fed to infants (Ashworth et a l . , 1973); 0.15 for baked soybeans fed to adults (Cook et a l . , 1972); 0.10 for boiled soybeans fed to adults (Lynch et a l . , 1984); 0.18 for f u l l fat soy f l o u r , 0.35 for textured soy flour and 0.08 for soy protein i s o l a t e , a l l fed to adults (Cook et a l . , 1981). The results of the present study as well as other rat data appearing i n the l i t e r a t u r e indicate that the a v a i l a b i l i t y of iron of soy to rats i s several-fold higher than that obtained i n studies with humans. I t would therefore appear that the anemic rat i s not a suitable model for man i n predicting the absolute a v a i l a b i l i t y of iron in such foods. 58 3. Beef-soy protein mixture The RBV of iron in the meat-soy mixture (65%) was s i g n i f i c a n t l y better (P < 0.05) than that of the meat iron (56%), but i t was s i g n i f i c a n t l y lower (P < 0.05) than that of the iron i n the soy product (81%). Shah et a l . (1983) repleted rats with a 2:1 (w/w) rat i o of freeze-dried ground beef to soy protein concentrate product. These researchers also observed that the RBV of the iron in the beef-soy combination (62%) was s i g n i f i c a n t l y greater than the RBV of iron in beef (53%). The absorption of nonheme iron in normal man was depressed by 61 and 53%, respectively, when a soy-extended beef patty, containing either a 3:1 or 2:1 (w/w) r a t i o of raw ground beef to unhydrated textured soy flo u r , was included i n the meal (Cook et a l . , 1981). This observed decrease in nonheme iron absorption was attributed to a combination of the in h i b i t o r y action of soy and the reduction i n the enhancing action of the meat. In the present study, despite the reduction i n the proportion of beef protein i n the beef-soy mixture over that in the diet containing only ground beef, the RBV of the iron i n the beef-soy mixture was 17% higher than that of the iron i n beef. Thus the promotion of iron absorption by the "meat factor", which i s operative in man (Cook and Monsen, 1976; Hallberg et a l . , 1979), was not evident i n the present study with rats. S i m i l a r l y , the inhibitory effect by soy protein on nonheme iron absorption demonstrated i n man (Cook et a l . , 1981; Hallberg and Rossander, 1982) was not observed i n the present study. Furthermore, in man i t has been shown that the addition of 100g ground beef to a soy isola t e meal increased absorption nearly four-fold, 59 from 0.36 to 1.44% (Morck et a l . , 1982). In contrast, i n the present study the RBV of the iron in soy was 25% higher than that of the beef-soy mixture. Based on the two examples given for man and the results of the present study, i t appears that the effects of soy and beef protein, when present together on iron u t i l i z a t i o n i n the anemic rat, are not the same as those observed in normal man. F. General discussion No definite statement can be made with regards to the effect cellulose powder might have had on the absorption of iron from the test source diets. Admittedly, the l e v e l at which t h i s fiber source was added, ranging from 14 to 20%, mostly certainly exceeded the 5% recommended for most rat formulations (American Institute of N u t r i t i o n , 1977). A recent review by Reinhold (1982) of the effect of dietary fiber on iron b i o a v a i l a b i l i t y in anemic rats suggests that, at the l e v e l provided i n the present study, some i n h i b i t i o n of hemoglobin regeneration would be expected. I f the fiber did i n h i b i t the absorption of iron, i t presumably did so through one of two mechanisms. The fiber may have physically entrapped iron i n the gut, thereby preventing potentially available iron from coming i n contact with iron receptor sit e s in the i n t e s t i n e . Alternatively, iron that might have been absorbed in the large intestine could have been metabolized by the microbial f l o r a of the cecum before reaching the colon (McCall et a l . , 1962; Reinhold, 1982). A f i n a l comment i s i n order with regard to the methodology employed i n the present study and the manner in which the RBVs of the iron in the test sources were calculated. The assay design involved repleting anemic 6U rats with a single l e v e l of iron provided by the test source and determining the potency of the iron source r e l a t i v e to that of the ferrous sulfate reference through the use of a graphic technique. However, the AOAC method (1980) stipulates that three levels of iron provided by both the test source and reference s a l t be assayed and that the responses for each be analyzed by a p a r a l l e l lines technique. Amine et a l . (1972) has commented that when RBVs are determined from a single level of test source ir o n , one i s unable to determine the v a l i d i t y of the assay or the precision of the estimated potency. However, owing to the differences in the nutrient content of the test sources used in this study and the formulary objectives imposed, i t would have been d i f f i c u l t indeed to design the diets to provide three levels of iron. There i s l i t t l e information i n the l i t e r a t u r e about the forms in which the i n t r i n s i c iron of soy occurs. E l l i s and Morris (1981) have reported that a portion of the iron i n soy may be present as monoferric phytate. In l i g h t of the differences observed i n the a v a i l a b i l i t y of soy iron i n rats and man, i t would be useful i f the chemical forms of iron could be determined for the various soy protein products available. Perhaps such information would assist in explaining the different iron a v a i l a b i l i t i e s observed between man and rat for soy protein as an iron source. 61 CONCLUSIONS The objective of this research project was to assess whether the iron a v a i l a b i l i t y i n a f o r t i f i e d textured defatted soy protein product or a mixture of beef and the f o r t i f i e d product was comparable to that of beef. The iron a v a i l a b i l i t y i n these food sources was estimated by measuring the hemoglobin or hematocrit response of anemic rats following two weeks of repletion on diets supplying an equal le v e l of iron from these sources. A r e l a t i v e b i o l o g i c a l value was assigned on the basis of the amount of iron furnished by reference ferrous sulfate that produced equal curvative responses. The RBVs of the iron in the three diets, calculated on the basis of f i n a l hemoglobin and hematocrit values, were respectively: freeze-dried ground beef, 56% and 62%; f o r t i f i e d soy protein product, 81% and 79%; 2.3:1 (w/w) combination of beef and soy product, 65% and 68%. The r e l a t i v e a v a i l a b i l i t y of iron i n the three diets compared favorably to the values reported from rat studies employing similar methodology. Human studies, investigating the re l a t i v e a v a i l a b i l i t y of iron i n meat and soy protein products have demonstrated an opposite trend. In humans, the absorption of the iron in beef i s comparable to or better than that of the reference source, while that of the iron in soy i s at best one t h i r d of the reference standard. Human studies, using e x t r i n s i c radioiron techniques, have clearly demonstrated the inhibitory effect of soy protein on the absorption of other nonheme iron present i n the same meal as well as the enhancing effect of beef protein on iron absorption. 62 Under the conditions of the present study with rats, neither of these dietary factors appeared to be operative. In the present study, judging from the high RBV obtained for the iron in the f o r t i f i e d soy protein pro-duct, i t would appear that the effect of the soy protein on the a v a i l a b i -l i t y of the iron i n the soy product was minimal. The cellulose powder, present i n high levels i n a l l three diets, was more l i k e l y the si g n i f i c a n t dietary component i n h i b i t i n g iron absorption; i t s mode of action presumably being one of physical entrapment. While the o r i g i n a l intent of th i s study was f u l f i l l e d , the l i m i t a -tions of the information obtained must be recognized. 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Prentice-Hall, Inc., Englewood C l i f f s , N3. 71 Appendix A. Comparison of experimental and reported values for proximate analysis components. Analysis Control sample Experimental Reported or Value 1 Expected value Nitrogen (%, db) Ether extract (58, db) Neutral detergent fiber {%, db) Total iron (ppm, db) Magnetically extractable iron (ppm, db) Phytic acid {%, db) Skim milk powder 84 g mixture of skim milk powder, cellulose, glucose + 16 g corn o i l cellulose powder (Alphacel) raw ground beef 36.69 (0.17) 16.34 (0.62) Fe^, hydrogen reduced, 10 mg Rapeseed meal 97.92 (0.20) 62.15 (0.30) 1,000,000 (28284) 4.64 (0.10) 37.292 16.43 99.OO3 67.844 1,000,000 4.795 1Mean ± (standard derivation) of 2 or 3 determinations. 2Watt and Merrill (1963); item 369. 3Polizzoto et a l . (1983). 4Watt and Merrill (1963); item 1328. ^Experimental value reported by laboratory of Department of Foods and Nutrition, University of Manitoba, Winnipeg, Manitoba, through which sample was obtained. Appendix B. Growth and hematological responses for iron depleted rats fed test sources of iron during the repletion period. Basal Beef + + 25 ppm iron Beef Soy flour Soy flour Diet treatment (as FeSO^ 7H20)/Kg (2.3:1, w/w) BW i n i t i a l (g) 85.2 ± 22.6a 1, 2 127.0 ± 5.5c 116.2 ± 4.2c 100.8 ± 6.6b BW f i n a l (g) 162.8 ± 29.7a 198.3 ± 12.6c 191.0 ± 17.9bc 177.7 ±12.9ab Hb i n i t i a l (g/dL) 3.5 ± 0.8a 3.1 ± 0.5a 3.1 ± 0.6a 3.1 ± 0.9a Hb gain (g/dL) 6.6 ± 1.7b 4.6 ± 0.9a 5.7 ± 0.6b 7.7 ± 1.0c Hematocrit i n i t i a l (55) 13.2 ± 2.7a 15.3 ± 1.2b 15.4 1.7b 15.5 ± 2.4b Hematocrit gain {%) 22.4 ± 4.5b 15.3 ± 1.9a 17.3 ± 2.6a 22.3 ± 3.5b Mean of 10 animals (± standard deviation). •Means in a row followed by the same l e t t e r are not s i g n i f i c a n t l y different (P > 0.05) as determined by Duncan's multiple range test. Appendix C. Growth, feed intake and hematological responses for iron depleted rats fed graded levels of iron as ferrous sulfate during the repletion period. Basal Basal Basal Basal Basal Basal + 5 ppm iron + 10 ppm iron + 15 ppm iron + 20 ppm iron + 25 ppm iron Diet treatment BW i n i t i a l (g) 111.1 ± 20.0b 1, 2 101.2 20.2b 104.5 BW gain (g) 23.7 ± 9.1a 55.3 ± 10.6b 73.2 Feed intake ( g ) 3 131.4 ± 18.8a 157.3 ± 25.3b 188.3 Hb i n i t i a l (g/dL) 3.8 ± 0.06a 3.6 0.06a 3.6 Hb gain (g/dL) -1.4 ± 0.6a 0.6 ± 0.7b 2.3 Hematocrit i n i t i a l (58) 14.4 ± 2.3a 14.7 ± 2.6a 14.3 Hematocrit -2.6 ± 1.6a 3.2 ± 3.6b 9.0 12.8b 98.5 ± 13.6b 101.3 ± 21.4b 85.2 ± 22.6a ± 10.8c 70.2 ± 18.4c 80.2 ± 14.3c 77.6 ± 16.3c ± 23.5c 184.4 ± 17.9c 195.8 ± 22.0c 181.8 ± 30.5c ± 0.7a 4.1 ± 0.9a 3.5 ± 0.5a 3.5 ± 0.8a ± 0.9c 4.1 ± 0.9d 5.5 ± 1.1e 6.6 ± 1.7f ± 2.4a 15.4 ± 2.4a 14.0 ± 1.9a 13.2 ± 2.7a ± 3.7c 15.4 ± 2.6d 19.4 ± 3.1e 22.4 ± 4.5f 1Mean of 10 animals (± standard deviation). 2Means in a row followed by the same l e t t e r are not s i g n i f i c a n t l y different (P > 0.05) as determined by Duncan's multiple range test. ^Expressed on dry weight basis. 

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