In Vitro Estimation of Food Iron Bioavailability - ACS Publications

2 In Vitro Estimation of Food Iron Bioavailability DENNIS D. MILLER and BRIAN R. SCHRICKER

Downloaded by UNIV OF NEW SOUTH WALES on August 17, 2015 | http://pubs.acs.org Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

Cornell University, Department of Food Science, Ithaca, NY 14853

A simple, rapid, and inexpensive in vitro method for estimating food iron availability is described. The method involves a simulated gastrointestinal digestion using commercially available enzymes. Soluble, low molecular weight iron is used as an indicator of iron availability. Similar results are obtained when the soluble iron is intrinsic food iron or added extrinsic radioiron. The method is designed to be used with single foods or complex meals. Results obtained with this method compare favorably with published results from human studies using extrinsic radioiron tag methods. The method resembles several published in vitro methods but is unique in two ways: 1. pH adjustment is achieved by dialysis. 2. Low molecular weight soluble iron rather than total soluble iron is used to estimate available iron. I t i s w e l l e s t a b l i s h e d t h a t e v a l u a t i o n of d i e t s f o r i r o n adequacy r e q u i r e s knowledge of both the amount and the a v a i l a b i l i t y of the i r o n present (1). While i n f o r m a t i o n on the i r o n content of foods i s reasonably adequate, knowledge of food i r o n a v a i l a b i l i t y i s incomplete. T h i s gap i n our understanding o f the p o t e n t i a l of d i e t a r y i r o n to meet n u t r i t i o n a l needs e x i s t s because a number o f complex and i n t e r a c t i n g f a c t o r s i n f l u e n c e food i r o n a v a i l a b i l i t y and because i r o n a v a i l a b i l i t y i s d i f f i c u l t to measure. I r o n a b s o r p t i o n from a food i s a f f e c t e d not only by the chemical form of the i r o n i n the food but a l s o by the i r o n s t a t u s of the person consuming the food, the presence of other foods i n the same meal, the amount of a c i d s e c r e t e d by the stomach, the r a t e of passage of the food through the d i g e s t i v e t r a c t , and, most l i k e l y , other f a c t o r s . The experience o f numerous i n v e s t i gators has shown that accurate measurement of i r o n a v a i l a b i l i t y i s a d i f f i c u l t , expensive and time consuming process. The occurrence i n the l i t e r a t u r e of f r e q u e n t l y c o n f l i c t i n g data a t t e s t s to

0097-6156/82/0203-0011$06.00/0 © 1982 American Chemical Society In Nutritional Bioavailability of Iron; Kies, C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

the f a c t that food i r o n a v a i l a b i l i t y measurements are fraught with d i f f i c u l t i e s . The o b j e c t i v e of t h i s paper i s t o d i s c u s s an i n v i t r o method we have developed f o r e s t i m a t i n g food i r o n a v a i l a b i l i t y . The paper w i l l be presented i n three s e c t i o n s : 1. R a t i o n a l e f o r the design and u t i l i t y of an i n v i t r o method. 2. A d e s c r i p t i o n o f the i n v i t r o method. 3. R e s u l t s and e v a l u a t i o n of the method.


Rationale Current methodology f o r e s t i m a t i o n of food i r o n a v a i l a b i l i t y u s u a l l y i n v o l v e s one of three approaches: animal bioassays, human bioassays, or i n v i t r o measurements. The most f r e q u e n t l y used animal bioassay i s the r a t hemoglob i n r e p l e t i o n t e s t . F r i t z , et a l . (2), M i l l e r (3), and Rotruck and Luhrsen (4) have d e s c r i b e d the method i n d e t a i l . The method depends on the a b i l i t y of an i r o n source to i n c r e a s e the hemoglob i n c o n c e n t r a t i o n i n anemic r a t s . P r i n c i p a l advantages of t h i s method i n c l u d e : 1. the use o f an i n t a c t b i o l o g i c a l system, 2. the r e l a t i v e s i m p l i c i t y o f the method, and 3. the a v a i l a b i l i t y of the method to a l a r g e number o f researchers. The drawbacks of t h i s method are: 1. the problems a s s o c i a t e d with the e x t r a p o l a t i o n of r e s u l t s from r a t s to humans, 2. the requirement that anemic a n i mals must be used ( r e l a t i v e i r o n a v a i l a b i l i t i e s may d i f f e r between anemic and nonanemic animals), 3. requirement f o r graded l e v e l s of i r o n i n the d i e t s (when whole foods are used t h i s means that the composition of the d i e t s i s u s u a l l y not constant between groups), and 4. the expense a s s o c i a t e d w i t h the method. A second animal bioassay method i s the whole body counting method which has been d e s c r i b e d by Welch and Van Campen (5). In t h i s method, food c o n t a i n i n g a r a d i o a c t i v e t r a c e r i s given t o the animals i n a s i n g l e dose and r e t e n t i o n of the t r a c e r i n the whole animal over time i s measured. Advantages i n c l u d e : 1. the use of an i n t a c t b i o l o g i c a l system, 2. the s i m p l i c i t y o f the method ( f r e quent blood sampling and food consumption measurements are not r e q u i r e d ) , 3. the f l e x i b i l i t y of the method (animals i n d i f f e r e n t treatment groups may or may not r e c e i v e i d e n t i c a l d i e t s throughout the experiment, depending on the study design requirements), and 4. the o p t i o n to use e i t h e r anemic or nonanemic animals. Disadvantages i n c l u d e : 1. questions regarding e x t r a p o l a t i o n of r e s u l t s t o humans, 2, requirements f o r s p e c i a l i z e d equipment (whole body c o u n t e r ) , and 3. questions about extent of exchange of t r a c e r and endogeneous i r o n when an e x t r i n s i c l a b e l i s used. The most f r e q u e n t l y used human bioassay method i s the two pool e x t r i n s i c r a d i o i r o n tag method (6). The method i s based on two assumptions. One, that food i r o n exchanges w i t h two common pools i n the gut (heme and nonheme i r o n pools) and two, that added r a d i o l a b e l e d heme i r o n exchanges completely with food heme iron and added r a d i o l a b e l e d nonheme i r o n exchanges completely with food

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

In Vitro Estimation


2. ML ILER AND SCHRC IKER

13

nonheme i r o n . I n c o r p o r a t i o n o f t h e r a d i o i r o n tag i n t o hemoglobin i s used as the response parameter. T h i s method has proven t o be very s u c c e s s f u l (7). Advantages i n c l u d e : 1. the use of human subjects and, t h e r e f o r e , e l i m i n a t i o n of questions regarding ext r a p o l a t i o n , 2. the s i m p l i c i t y o f the method (a s i n g l e dose cont a i n i n g the r a d i o l a b e l e d food i s administered and a blood sample i s drawn two weeks l a t e r f o r counting). Disadvantages i n c l u d e : 1. the a d m i n i s t r a t i o n of r a d i o i s o t o p e s t o human s u b j e c t s , 2. r e s t r i c t i o n s i n the use o f the method ( r e l a t i v e l y few i n v e s t i g a t o r s are l i c e n s e d t o administer r a d i o i s o t o p e s to human subjects f o r p u r e l y research purposes.), and 3. the wide i n t e r - s u b j e c t v a r i a b i l i t y i n i r o n absorption. (This v a r i a b i l i t y can be l a r g e l y overcome by using each subject as t h e i r own c o n t r o l and by adm i n i s t e r i n g a reference dose). In v i t r o methods have been used t o estimate i r o n a v a i l a b i l i t y f o r a t l e a t 50 years (8). Two approaches w i t h v a r i o u s m o d i f i c a t i o n s have been used. One approach i s t o measure " i o n i z a b l e " o r " i o n i c " i r o n i n foods. T h i s i s done by determining the f r a c t i o n of the t o t a l i r o n i n a food that w i l l r e a c t with a complexing agent such as a , a ' - d i p y r i d y l (8) o r bathophenanthroline (9) t o form a chromagen which can be q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y . A second approach i s t o subject the food t o a simulated g a s t r i c or g a s t r o i n t e s t i n a l d i g e s t i o n using p u r i f i e d p e p t i c and/or panc r e a t i c enzymes with subsequent measurement o f the s o l u b l e i r o n released by the d i g e s t i o n (10-13). Advantages o f i n v i t r o methods i n c l u d e : 1. t h e i r low cost and speed, 2. t h e i r reduced v a r i a b i l i t y compared to i n v i v o methods ( v a r i a b i l i t y caused by d i f f e r e n c e s i n i r o n s t a t u s o f animals and humans i s avoided), and 3. the a b i l i t y t o p r e c i s e l y c o n t r o l c o n d i t i o n s during the determinations. Disadvantages i n c l u d e : 1. u n c e r t a i n t i e s over the use o f an a r t i f i c i a l system, 2. the l e s s than exact d u p l i c a t i o n o f i n v i v o c o n d i t i o n s , 3. the i n a b i l i t y t o account f o r e f f e c t s o f a c t i v e t r a n s p o r t , brush border binding p r o t e i n s , e t c . I t i s c l e a r that each approach has i t s advantages and d i s advantages and that a l l three approaches have provided and w i l l continue t o provide information on food i r o n a v a i l a b i l i t y . It i s a l s o c l e a r that f u r t h e r development and e v a l u a t i o n o f the three approaches w i l l enhance the usefulness o f the data generated by them. Development o f c o n d i t i o n s s u i t a b l e f o r a s u c c e s s f u l i n v i t r o method r e q u i r e s c a r e f u l a t t e n t i o n to c o n d i t i o n s present i n the gut during d i g e s t i o n and t o the behavior of i r o n i n s o l u t i o n . The environment i n the GI t r a c t and the chemical form o f the i r o n both i n the food and i n the d i g e s t a i n t e r a c t t o determine the f r a c t i o n of the food i r o n t h a t i s a v a i l a b l e f o r absorption. The primary determinants o f food i r o n a v a i l a b i l i t y are: 1. the extent o f i r o n r e l e a s e from food and 2. the s o l u b i l i t y , molecular weight, and s t a b i l i t y o f complexes formed from the r e leased i r o n . A l a r g e number o f f a c t o r s i n t e r a c t to i n f l u e n c e these determinants.



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The nonheme i r o n i n food i s most l i k e l y bound to food components, probably p r o t e i n . The r e l e a s e of i r o n from these components i s a complex process. I t i s reasonable to assume that the b i n d i n g a f f i n i t y of the food components f o r i r o n , the d i g e s t i b i l i t y of the food components, the presence of reductants and accepting l i g a n d s , the pH i n the GI t r a c t are a l l f a c t o r s which i n f l u e n c e r e l e a s e of the i r o n from the food. Binding a f f i n i t y and d i g e s t i b i l i t y are c h a r a c t e r i s t i c s of the food and may vary s i g n i f i c a n t l y between foods. Reductants and accepting l i g a n d s are a l s o l a r g e l y c o n t r i b u t e d by the food. D i g e s t i o n products may a c t as reductants and/or accepting l i g a n d s . Food components such as ascorbate, c i t r a t e , and simple sugars may c o n t r i b u t e subs t a n t i a l l y to the r e l e a s e of the i r o n from food. Since f a c t o r s which are c h a r a c t e r i s t i c s of the food would not d i f f e r between i n v i v o and i n v i t r o s i t u a t i o n s , the major v a r i a b l e s t h a t must be c o n t r o l l e d i n an i n v i t r o s i m u l a t i o n are pH, enzyme concentrations and a c t i v i t i e s , and d i g e s t i o n times. The observation that i r o n absorption i s reduced when g a s t r i c a c i d s e c r e t i o n i s compromised (14) suggests that g a s t r o i n t e s t i n a l pH does i n f l u e n c e food i r o n a v a i l a b i l i t y . The explanation f o r t h i s appears to be r e l a t e d to the importance of a c i d f o r the r e lease of i r o n from food. Bezwoda et a l . (15) measured the capac i t y to s o l u b i l i z e i r o n i n bread of g a s t r i c j u i c e from normal and i r o n d e f i c i e n t s u b j e c t s . G a s t r i c j u i c e s with pH values above 2 had l i m i t e d c a p a c i t y to s o l u b i l i z e bread i r o n w h i l e below pH 2, s o l u b i l i z a t i o n of i r o n increased l i n e a r l y with decreasing pH. Even though the s t a b i l i t i e s of metal complexes i n foods are unknown, i t i s to be expected that low pHs w i l l r e s u l t i n g r e a t e r r e l e a s e of food i r o n s i n c e metal c h e l a t e s t a b i l i t i e s decrease with decreasing pH (14). I t i s apparent, t h e r e f o r e , that pH must be c a r e f u l l y c o n t r o l l e d i n any i n v i t r o method designed to estimate food iron availability. S e l e c t i o n of an appropriate pH f o r use i n i n v i t r o p e p t i c d i g e s t i o n s i s d i f f i c u l t s i n c e the pH i n the i n v i v o s i t u a t i o n i s q u i t e v a r i a b l e . However, i t i s g e n e r a l l y accepted that i n g e s t i o n o f food stimulates g a s t r i c a c i d s e c r e t i o n and that g a s t r i c a c i d i t y i s one f a c t o r i n v o l v e d i n the r e g u l a t i o n of g a s t r i c a c i d s e c r e t i o n . Malagelada et a l . (16) showed t h a t i n g e s t i o n of a meal by human s u b j e c t s increased g a s t r i c pH from about 2 to about 5. The peak pH of a 5 was reached r a p i d l y . I t then g r a d u a l l y f e l l with time and s t a b i l i z e d at pH 1.5 to 2 by the end of the second hour. Walsh et a l . (17) found t h a t the r a t e of g a s t r i c a c i d s e c r e t i o n i n normal human subjects i n response to a meal i s suppressed at a g a s t r i c pH of 2.5 compared to a g a s t r i c pH of 5.5. T h i s suggests that the stomach " t i t r a t e s " i t s contents to an a c i d pH and that stomach pHs f o l l o w i n g d i f f e r e n t meals should be s i m i l a r . I t seems reasonable, t h e r e f o r e , to assume that adjustment of the pH to 2 p r i o r to i n v i t r o pepsin d i g e s t i o n would approximate the i n v i v o situation. Ferrous and f e r r i c ions are present i n the hydrated form i n


15

In Vitro Estimation

2. ML ILER AND SCHRC IKER

a c i d i c aqueous s o l u t i o n s (18). As the pH of a s o l u t i o n of these aquated ions i s i n c r e a s e d , deprotonation of the complexed water molecules occurs and hydroxo- and/or oxo-aquo s p e c i e s are formed (19). T h i s process i s c a l l e d h y d r o l y s i s and may be represented as (19) x[Fe(H 0) ]


2

6

3 +

+ y H0 2

^ [Fe (0H) (H 0)J x

y

2

(

3

x

~

y

)

+

+ y H 0

+

3

where n i s the degree of h y d r a t i o n of the h y d r o l y s i s product. As the above equation suggests, p o l y n u c l e a t e d s p e c i e s may be p r o duced i n the r e a c t i o n . Polymers with a molecular weight of 150,000 have been observed (19). C h e l a t i n g agents such as c i t r a t e may prevent p o l y m e r i z a t i o n i f added i n s u f f i c i e n t excess (19) . Many of the c h e l a t e s are, however, q u i t e unstable and add i t i o n of concentrated base can cause p r e c i p i t a t i o n even when c h e l a t i n g l i g a n d s are present. Upward pH adjustment with NaHCO^ r a t h e r than NaOH appears to be l e s s l i k e l y to cause p r e c i p i t a t i o n (20) . As the i r o n i s r e l e a s e d from food i n the a c i d i c environment of the stomach, l i g a n d s r e l e a s e d i n the d i g e s t i o n process combine with the i r o n to form c h e l a t e s . These c h e l a t e s should i n h i b i t p o l y m e r i z a t i o n and p r e c i p i t a t i o n of i r o n as the stomach contents are n e u t r a l i z e d i n the duodenum provided that l o c a l i z e d regions of h i g h pH caused by too r a p i d a d d i t i o n o f concentrated base are avoided. T h i s suggests that pH adjustment i n an i n v i t r o simulat i o n i s a c r i t i c a l step. When the products of g a s t r i c d i g e s t i o n reach the duodenum, bicarbonate s e c r e t e d by the pancreas begins to n e u t r a l i z e the stomach a c i d . The c o n c e n t r a t i o n of bicarbonate i n p a n c r e a t i c j u i c e ranges from about 70 to about 150 meq/£ (21). In a study i n v o l v i n g human s u b j e c t s , Murthy e t a l . (22) found the pH o f duodenal a s p i r a t e s to range from 4.7 to 7.2 f o l l o w i n g a d m i n i s t r a t i o n of a Lundh Test meal. S e l e c t i o n of a p p r o p r i a t e concentrations f o r d i g e s t i v e enzymes i s a d i f f i c u l t undertaking. A l a r g e range of concentrat i o n s used i n i n v i t r o s t u d i e s have been reported. Akeson and Stahmann (23) used 15 mg pepsin and 40 mg p a n c r e a t i n per gram of p r o t e i n . Narasinga Rao and Prabhavathi (11) used pepsin concent r a t i o n s ranging from 12 to 60 mg per g of food. They reported no d i f f e r e n c e i n i r o n r e l e a s e when t h i s range of p e p s i n concent r a t i o n s was used. Lease (24) used 20 mg of pepsin per gram of food. H a z e l l et a l . (12) used 10 mg pepsin and 10 mg p a n c r e a t i n per gram of p r o t e i n . While d i f f e r e n t concentrations of enzymes w i l l produce d i f f e r e n t r a t e s o f d i g e s t i o n , the a c t u a l enzyme conc e n t r a t i o n s are not c r i t i c a l provided they are p r e c i s e l y d u p l i c a ted when comparisons between foods or meals are being made. Appropriate d i g e s t i o n times f o r an i n v i t r o system l i k e w i s e cannot be determined with p r e c i s i o n . Rates o f passage of d i g e s t a are determined by s e v e r a l i n t e r a c t i n g f a c t o r s i n c l u d i n g the osmol a r i t y of the meal, the r e l a t i v e amounts of l i q u i d and s o l i d i n



16


the meal, the s i z e of the meal and the carbohydrate, p r o t e i n , and f a t content of the meal (25). However, choice of an appropriate pepsin d i g e s t i o n time may be based on estimates of stomach emptying times and r a t e s of d i g e s t i o n . Grimes and Goddard (26) showed t h a t , f o l l o w i n g a bread and water meal, about 70% of the s o l i d phase (bread) and from 1 to 30% of the l i q u i d phase (water) r e mained i n the stomach one hour a f t e r the meal was consumed by human s u b j e c t s . Malagelada et a l . (16) showed w i t h human subj e c t s that g a s t r i c volume rose q u i c k l y to the volume of the meal and remained at that volume f o r about one hour. The g a s t r i c v o l ume then f e l l g r a d u a l l y u n t i l i t reached b a s a l l e v e l s about 3 hours from the time the meal was consumed. Narasingao Rao and Prabhavathi showed i n an i n v i t r o system that i r o n r e l e a s e d from food was the same when pepsin i n c u b a t i o n times were v a r i e d from 50 to 180 minutes. Selecton of an i n v i t r o p a n c r e a t i n d i g e s t i o n time i s even more d i f f i c u l t s i n c e d i g e s t a i s c o n s t a n t l y e n t e r i n g and l e a v i n g the duodenum and jejunum. I t i s reasonable to s e l e c t a d i g e s t i o n time that produces s i g n i f i c a n t d i g e s t i o n . Care must be taken to use i d e n t i c a l d i g e s t i o n times when comparisons are being made between foods or meals. Once the c o n d i t i o n s f o r an i n v i t r o method f o r e s t i m a t i o n of i r o n a v a i l a b i l i t y have been e s t a b l i s h e d , s e l e c t i o n of an approp r i a t e response parameter must be made. T o t a l s o l u b l e or " i o n i z a b l e " i r o n present i n the f i l t r a t e o r supernate obtained from an i n v i t r o d i g e s t i o n has been the most f r e q u e n t l y used response parameter (10-13). The foregoing d i s c u s s i o n provides a b a s i s f o r s e l e c t i o n of a r e l i a b l e indicator of i r o n a v a i l a b i l i t y . F i g u r e 1 summarizes i n schematic form the changes that occur as food moves through the d i g e s t i v e t r a c t . The assumption i s made that absorbable i r o n i s present i n the duodenum as a low molecular weight s o l u b l e c h e l a t e . I t i s f u r t h e r assumed t h a t the other forms of i r o n that may be present do not c o n t r i b u t e s i g n i f i c a n t l y to absorbable i r o n . These assumptions seem j u s t i f i e d f o r the f o l l o w i n g reasons: 1. Iron may be absorbed as the i n t a c t c h e l a t e or the chelate may t r a n s f e r i t s i r o n t o an acceptor on the mucosal c e l l s u r f a c e . Absorption and exchange would be much more r a p i d with s o l u b l e forms of i r o n s i n c e i n s o l u b l e forms would have l i m i t e d contact with the mucosal c e l l s u r f a c e . 2. Iron bound to l a r g e molecular weight l i g a n d s may be a v a i l able but absorption would be l i m i t e d to an i r o n t r a n s f e r mechanism s i n c e l a r g e molecules are g e n e r a l l y not absorbed i n t a c t . Large molecular weight s o l u b l e c h e l a t e s of a v a i l a b l e i r o n would most l i k e l y i n v o l v e p r o t e i n s as the l i g a n d and d i g e s t i o n would q u i c k l y transform them i n t o low molec u l a r weight c h e l a t e s . 3. Polymerized i r o n , even when s o l u b l e , i s probably not r e a d i l y a v a i l a b l e . Bates et a l . (20), i n s t u d i e s designed to measure i r o n exchange r a t e s between chelates and t r a n s f e r r i n , showed that polymerized i r o n was t r a n s f e r r e d to t r a n s f e r r i n


In Vitro Estimation


ML ILER AND SCHRC IKER

Mucosal Cell

Duodenum

Stomach

[-•Fe-H 0 4 i

Fe-lig

2

s

HCO3 +

Enzymes

L+Fe-LIGj ±

Figure 1. Proposed model for changes that occur in nonheme iron as food moves through GI tract. Abbreviations: lig , low molecular weight soluble ligand; LIG , large molecular weight soluble ligand; LIG , large molecular weight insoluble ligand; Poly Fe , soluble polynuclear (polymerized) iron; Poly Fe insoluble polynuclear (polymerized) iron. s

a

t

s

it



18


at a very slow r a t e while i r o n i n the low molecular weight n i t r i l o t r i a c e t a t e c h e l a t e was almost instantaneously t r a n s f e r r e d t o the t r a n s f e r r i n . Polymerized i r o n may be depolymerized by l i g a n d s and/or reducing agents (27) and thereby enter the low molecular weight i r o n c h e l a t e p o o l . C o n s i d e r a t i o n of the f a c t o r s d i s c u s s e d above provides a r e a sonably good r a t i o n a l e f o r the design of an i n v i t r o method to estimate food i r o n a v a i l a b i l i t y . B r i e f l y , i t seems apparent that an i n v i t r o method should: 1. Simulate i n v i v o d i g e s t i o n c o n d i t i o n s . 2. Permit pH c o n t r o l . 3. Provide f o r gradual pH adjustment with a m i l d base. 4. D i s t i n g u i s h between low and h i g h molecular weight s o l u b l e iron. 5. Accommodate food mixtures (meals). D e s c r i p t i o n of the Method D e t a i l s of the method have been d e s c r i b e d elsewhere (28,29). A flow diagram of the method i s shown i n F i g u r e 2. B r i e f l y , the method i n v o l v e s the f o l l o w i n g steps: 1. Water i s added to mixtures of foods (meals) so that the water content of d i f f e r e n t meals i s approximately the same. 2. The meals are blended i n a food blender to a creamy c o n s i s tency . 3. The pH of the blended meals i s adjusted to 2 with 6 N HC1 and the samples are spiked with ^ F e . 4. Pepsin i s added and the meals are incubated i n a shaking water bath a t 37°C f o r 2 hours. 5. A l i q u o t s of the pepsin d i g e s t are analyzed f o r a) t i t r a t a b l e a c i d i t y (the number of e q u i v a l e n t s of KOH r e q u i r e d t o t i t r a t e a 20 g a l i q u o t c o n t a i n i n g 5 ml of the p a n c r e a t i n b i l e mixture to pH 7.5), b) nonheme i r o n c o n c e n t r a t i o n , and c) ^ F e a c t i v i t y . 6. A d i a l y s i s bag c o n t a i n i n g an amount of NaHC03 i n 25 ml of water e q u i v a l e n t to the p r e v i o u s l y determined t i t r a t a b l e a c i d i t y i s added to a 20 g a l i q u o t of the pepsin d i g e s t . The sample i s incubated f o r 30 minutes at 37°C i n a shaking water bath. During t h i s time, the pH i n c r e a s e s to about 5. 7. F i v e ml of a p a n c r e a t i n - b i l e a c i d mixture i s added to each d i g e s t i o n v e s s e l ( i t i s added to the contents o u t s i d e the d i a l y s i s bag). Incubation i s continued f o r 2 hours. 8. The d i a l y s i s bag i s removed, r i n s e d i n d i s t i l l e d water, and emptied of i t s contents (the d i a l y s a t e ) . 9. The d i a l y s a t e i s weighed and analyzed f o r ^ F e a c t i v i t y and bathophenanthroline r e a c t i v e i r o n . 10. R e s u l t s are expressed as percent of t o t a l nonheme and r a d i o i r o n i n the o r i g i n a l a l i q u o t t h a t i s present i n the d i a l y s i s bag at the end of the d i g e s t i o n . In order to evaluate the method, meals were prepared that 5



In Vitro Estimation

FOODS + H 0 Downloaded by UNIV OF NEW SOUTH WALES on August 17, 2015 | http://pubs.acs.org Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch002

2

^

Blend

^HCI+

5 9

Fe

pH 2 MEAL HOMOGENATE Pepsin, 2 hr, 37° PEPSIN DIGEST

Dialysis bag 30 min, 37° Pancreatin-bile Titratable 2 hr, 37° Remove bag [Nonheme Fe] Rinse, empty, weigh |-V ^ a c , d , t y

g

DIALYSATE, pH 7

i

Count Batho assay

% Dialyzable F e and nonheme Fe 59

Figure 2.

Schematic of the in vitro method. See text for details.


20


would permit zable i r o n . meal) and two (2,30). The

food s u b s t i t u t i o n s that might a l t e r l e v e l s of d i a l y One meal was of our own f o r m u l a t i o n (the standard were r e p l i c a t e s o f meals used by Cook and Monsen composition of these meals i s shown i n Table I .

Table I:


Standard

Composition of Test Heals Used i n Iron A v a i l a b i l i t y T r i a l s * STD,

Cook & Monsen

SS, Cook & Monsen

Beef; l e a n ground

Beef; ground

Albumin; egg

Bread; white, enriched

Potatoes;

Dextrose

Cornmeal

Corn o i l

Beans; snap, f r o z e n

Bread; white, enriched

CaHP0

4

M i l k ; f l u i d , whole

Margarine

K HP0

4

Water

Peaches

Fe s o l u t i o n

Ice m i l k

Water

dry

2

Water The STD and SS meals were o r i g i n a l l y formulated by Cook and Monsen (7,30). Results and

Evaluation

The 2 hour p e p s i n i n c u b a t i o n time was based on work reported by Narasinga Rao (11) and on l i t e r a t u r e r e p o r t s on g a s t r i c emptyi n g time (16,26). The s l i g h t l y longer p a n c r e a t i n i n c u b a t i o n time of 2 1/2 hours was chosen f o r two reasons. F i r s t , i t was necessary to delay a d d i t i o n of the p a n c r e a t i n - b i l e mixture f o r 30 minutes a f t e r the d i a l y s i s bag was added and the i n c u b a t i o n begun. The delay allowed the pH to r i s e to about 5 and thereby prevented i n a c t i v a t i o n of the p a n c r e a t i c enzymes which does occur at lower pHs (22). Secondly, t h i s time p e r i o d produced s u f f i c i e n t d i a l y s ate i r o n concentrations f o r accurate bathophenanthroline measurements. F i g u r e 3 shows a time course f o r d i a l y z a b l e i r o n changes during the second i n c u b a t i o n . D i a l y z a b l e i r o n r i s e s r a p i d l y to e q u i l i b r i u m l e v e l s when the semisynthetic meal i s used. T h i s suggests that p a n c r e a t i n d i g e s t i o n has l i t t l e e f f e c t on a v a i l a b l e i r o n i n a meal composed of p u r i f i e d i n g r e d i e n t s . For the s t a n dard meal, on the other hand, d i a l y z a b l e i r o n i n c r e a s e s s t e a d i l y over the e n t i r e i n c u b a t i o n p e r i o d . T h i s suggests that d i g e s t i o n by p a n c r e a t i c enzymes does play a r o l e i n food i r o n a v a i l a b i l i t y . Data from Malagelada et a l . (16) provided the; b a s i s f o r choosing a pH of 2 f o r the pepsin d i g e s t i o n . A f i n a l d i a l y s a t e pH of 7 was chosen f o r the p a n c r e a t i n d i g e s t i o n on the b a s i s of the data of Murthy e t a l . (22). Attempts were made to adjust the


In Vitro Estimation



Figure 3. Time course for changes in dialyzable iron during the second digestion step. See text for a description of meals and digestion conditions. Key: A * standard, colorimetric; A , standard, radioactive; | , SS, colorimetric; Q SS radioactive. t



22


pH of the pepsin d i g e s t by dropwise a d d i t i o n of d i l u t e NaHC03 or NaOH but t h i s approach produced v a r i a b l e and p o o r l y r e p r o d u c i b l e values f o r d i a l y z a b l e i r o n . Adjustment of pH by d i a l y s i s produced the l e a s t v a r i a b l e and most r e p r o d u c i b l e d i a l y z a b l e i r o n v a l u e s . In a d d i t i o n , t h i s method of pH adjustment permits d i a l y s i s of iron to occur during the n e u t r a l i z a t i o n process, a s i t u a t i o n which more c l o s e l y resembles the i n v i v o process. Even with t h i s method of pH adjustment, however, the f i n a l d i a l y s a t e pHs d i d vary. In order to determine the e f f e c t of v a r i a t i o n i n f i n a l pH on d i a l y z a b l e i r o n , an experiment was run using d i f f e r e n t amounts of bicarbonate i n the d i a l y s i s bags. The amounts used were based on t i t r a t a b l e a c i d i t y measurements made with d i f f e r e n t end p o i n t s . A l i q u o t s were t i t r a t e d to f i n a l pHs ranging from 6.5 to 8.5. Add i t i o n of NaHC03 based on these t i t r a t i o n s produced f i n a l d i a l y s ate pHs ranging from about 6 to about 7.5. F i g u r e 4 shows t h a t i n the range of pH v a r i a b i l i t y observed when d i f f e r e n t meals are run i n the i n v i t r o procedure ( f i n a l pHs i n the a c i d i t y t i t r a t i o n of 7.0 to 8.0), the e f f e c t of pH on d i a l y z a b l e i r o n i s s m a l l . I t i s i n t e r e s t i n g to note that the d i r e c t i o n of pH e f f e c t s are d i f f e r e n t f o r the semisynthetic and standard meals. The decrease observed w i t h i n c r e a s i n g pH i n the semisynthetic meal would be expected i f d i g e s t i o n were not a f a c t o r s i n c e h i g h e r pHs would cause i n c r e a s e d formation of polymerized i r o n . The i n c r e a s e observed i n the standard meal can be explained by an i n c r e a s e i n p a n c r e a t i c enzyme a c t i v i t y a t higher pHs. Table I I shows e f f e c t s on d i a l y z a b l e i r o n caused by v a r i o u s s u b s t i t u t i o n i n t o the standard meal. These data have been r e ported elsewhere ( M i l l e r et a l . 28, S c h r i c k e r et a l . 29). Table I I :

* t Percent D i a l y z a b l e Iron ' , E f f e c t s of S e l e c t e d Foods

Substitution

Colorimetric

None, Standard meal

4.08

+

0.31

Ham

5.05

+

0.80

Whole wheat f o r white bread

2.06

+

Spinach f o r green beans

5.73

+

4.59

+

Tea f o r m i l k

2.80

+

0.31

Cola f o r m i l k

6.14

+

0.61°

24.96

+

f o r beef

Water f o r m i l k

Orange j u i c e f o r m i l k

Values represent means + S.E.

Radioactive 3.80

+

0.31

a

5.14

+

0.23

0.42

b

1.49

+

0.10

0.33

a

5.04

+

0.19

a

4.16

+

0.14

a

1.52

+

0.21

b

7.82

+

0.34°

25.83

+

0.86

a

0.32

ac

b

0.83

d

a

b

f o r three o b s e r v a t i o n s .

t In each column, means followed by d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (P

In Vitro Estimation of Food Iron Bioavailability - ACS Publications

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