The Effects of Physicochemical Properties of Food on the Chemical

4 The Effects of Physicochemical Properties of Food on the Chemical Status of Iron F. M. CLYDESDALE

Downloaded by CORNELL UNIV on October 19, 2016 | http://pubs.acs.org Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch004

University of Massachusetts, Department of Food Science and Nutrition, Amherst, MA 01003

Iron bioavailability is affected by valence state, form, solubility, particle size, and complexation which in turn may be affected by the food matrix. Complexation of iron has been found to have either a positive or negative effect on availability, with such compounds as ascorbic acid and fructose increasing availability and oxalates, phytates, phosphates and food fibers perhaps decreasing availability. Availability has also been shown to be directly correlated to acid solubility. We have found that acidity tends to increase ionization as well as favoring the ferrous state which has greater solubility at -1

the pH of the intestine (10 M) than does ferric -18

(10 M). Both reduction potential and dissolved oxygen may also affect ionization and valence state with the former having the most potent effect from results we obtained. These factors, which are inherent in the food system, must be considered i f the problems of iron deficiency are to be combatted successfully.

0097-6156/82/0203-0055$08.50/0 © 1982 American Chemical Society

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

56

NUTRT IO INAL BO IAVAL IABL IT IY OF R ION

The 1977-78 Nationwide Food Consumption Survey (.NFCS) r e c e n t l y p u b l i s h e d by the USDA (-1) o u t l i n e s the f o l l o w i n g f i n d i n g s as r e l a t e d t o i r o n i n t a k e i n the U.S.: 1. Average i r o n intakes o f females 12 t o 50 years were 35% and h0% below the RDA, as i n 1965. 2. The i r o n i n t a k e of i n f a n t s i n 1977 was more than twice the i n t a k e i n 1965. However, the average i n t a k e o f 1 t o 2 year o l d s was much lower, about k5% below the 197^ RDA. These f i n d i n g s were an exception t o the g e n e r a l t r e n d which i n d i c a t e d t h a t food used by households i n 1977 had a higher nut r i e n t d e n s i t y than food used i n 196*5 were i n f a c t r e l a t e d t o s p e c i f i c sex/age groups. Nevertheless, i t i s apparent t h a t problems s t i l l e x i s t w i t h i r o n n u t r i t u r e i n the U.S., c e r t a i n l y i n p a r t due t o the form of i r o n i n the d i e t and what i t i s eaten w i t h , r a t h e r than the t o t a l q u a n t i t y o f i r o n i n the d i e t . From these r e s u l t s i t would seem l o g i c a l t h a t r e s e a r c h must be aimed a t a b e t t e r understanding o f not only the exact mode o f a c t i o n o f i r o n a b s o r p t i o n but a l s o an understanding o f the f a c t o r s which produce and maintain the most b i o a v a i l a b l e forms o f i r o n i n food. T h i s paper w i l l attempt t o address t h i s l a t t e r p o i n t by d i s cussing some o f the physicochemical p r o p e r t i e s o f food which may e f f e c t the chemical status o f i r o n . Obviously not every p r o p e r t y may be considered and t h e r e f o r e t h i s d i s c u s s i o n w i l l be l i m i t e d t o a c o n s i d e r a t i o n of s e l e c t e d complexes, pH, and r e d u c t i o n potenial. I r o n w i t h i n a food matrix provides an extremely r e a c t i v e veh i c l e f o r complexation w i t h a great number o f chemical compounds. In f a c t , i t i s t h i s very r e a c t i v i t y which makes some of the most b i o a v a i l a b l e forms of i r o n so o b j e c t i o n a b l e t o the food processor s i n c e the chemical r e a c t i o n s which occur can d r a s t i c a l l y a f f e c t q u a l i t y . Conversely, the most f u n c t i o n a l l y s u i t a b l e forms o f i r o n are o f t e n not very b i o a v a i l a b l e . T h i s n u t r i t i o n a l / f u n c t i o n a l c o m p a t a b i l i t y , although not a subject o f focus here, should be mentioned s i n c e any f o r t i f i c a t i o n program must c o n s i d e r t h i s comp a t a b i l i t y f a c t o r or i t w i l l be doomed t o f a i l u r e . T h i s problem has been d i s c u s s e d i n some d e t a i l by Lee and Clydesdale'—'and Z o l l e r et a l (2)• Saltman. i n d i s c u s s i n g b i o a v a i l a b i l i t y has s t a t e d t h a t "In essence the r e g u l a t i o n and c o n t r o l o f i r o n metabolism through the i n t e s t i n e as w e l l as across a l l b i o l o g i c a l membranes i s d e t e r mined by the a b i l i t y of the i r o n t o be c h e l a t e d by low molecular weight l i g a n d s " . Whether or not t h i s i s an overstatement remains t o be seen, but nevertheless i t p o i n t s out the p o t e n t i a l r o l e o f ligands- i n i r o n chemistry both i n humans and i n food. The f a c t o r which probably c o n t r i b u t e s most t o the p o t e n t i a l b i o l o g i c a l r o l e o f l i g a n d s i n food i s t h e i r e f f e c t on i r o n s o l u b i l i t y . Ferrous and f e r r i c ions i n s o l u t i o n do not occur i n the f r e e s t a t e , but are hydrated as Fe(H20)g 3 and FeC^Ojg* " i n a c i d and l o s e


a n < i

+

1

2


4.

CLYDESDALE

57 Physicochemical Properties of Food

protons as the pH i s r a i s e d t o form the corresponding hydroxides (FE(0H)2 and FE(011)3) i n n e u t r a l and a l k a l i n e s o l u t i o n s v£'• These hydroxides are i n c r e a s i n g l y l e s s s o l u b l e than t h e i r r e s p e c t i v e hydrates and i n the absence of l i g a n d s , at p h y s i o l o g i c a l pH, the s o l u b i l i t y of the two forms i s K T M (+2) and I O " M (+3). These s o l u b i l i t y values however can be a l t e r e d d r a m a t i c a l l y by any number of l i g a n d s i n food such as p r o t e i n s , amino a c i d s , c a r boxy l i e a c i d s , p o l y o l s and phosphates, which i n t u r n might a f f e c t b i o a v a i l a b i l i t y e i t h e r p o s i t i v e l y or n e g a t i v e l y s i n c e a v a r i e t y of s t u d i e s have shown t h a t b i o l o g i c a l systems seem t o u t i l i z e i r o n b e t t e r i f i t i s i n a s o l u b l e form. Therefore, l i g a n d s which form more s o l u b l e complexes might tend t o i n c r e a s e b i o a v a i l a b i l i t y w h i l e those which form more i n s o l u b l e complexes might have the opposite e f f e c t . Saltman et a l c i t e s instances where s o l u b l e polymers such as f e r r i c f r u c t o s e and f e r r i c c i t r a t e have been used t o demonstrate massive d e p o s i t i o n of i r o n i n t i s s u e s of mice, r a t s , guinea p i g s , r a b b i t s , and other animals. F u r t h e r these workers show a t y p i c a l uptake p a t t e r n f o r f e r r i c f r u c t o s e and f e r r o u s s u l f a t e i n guinea p i g s whereby f e r r i c f r u c t o s e i s r e t a i n e d at higher l e v e l s than f e r r o u s s u l f a t e , i n d i c a t i n g greater absorpt i o n (Figure l ) . They e x p l a i n the p a t t e r n shown i n F i g u r e 1 on the s o l u b i l i t y as w e l l as s e p a r a t i n g the k i n e t i c s o f i r o n absorpt i o n i n t o three phases. During the f i r s t phase, there i s very r a p i d e x c r e t i o n of the unabsorbed i r o n v i a the f e c e s , owing i n p a r t t o the formation of unabsorbable p r e c i p i t a t e s of i r o n p o l y mer i n the i n t e s t i n a l lumen. During the second phase, mucosal t i s s u e , which has s t o r e d but not t r a n s p o r t e d i r o n , i s slowly sloughed off and i s excreted. In the t h i r d , i r o n enters the bloodstream and c i r c u l a t e s t o the t i s s u e where i t remains u n t i l i t i s m o b i l i z e d by normal turnover or b i o l o g i c a l demand. However, i t should be recognized t h a t although the e f f i c a c y o f i r o n frtictose complexes has been shown by s e v e r a l workers 2) evidence t o the contrary has been presented by H e i n r i c h ejb a l (£) who s t u d i e d the e f f e c t of l a r g e amounts of f r u c t o s e on t h e r a p e u t i c doses of f e r r i c and f e r r o u s i r o n i n man and found no d i f f e r e n c e s i n absorpt i o n . F u r t h e r , i t should be recognized t h a t r e d u c t i o n of f e r r i c i r o n i n t o the more s o l u b l e f e r r o u s i r o n can occur i n f r u c t o s e solutions which must be taken i n t o account i n any explanation of i n c r e a s e d b i o a v a i l a b i l i t y . However, s i n c e s o r b i t o l , a nonreducing sugar, has been found t o enhance i r o n absorption '12' i t must be concluded t h a t the complexation of i r o n and not s o l e l y valence or s o l u b i l i t y a f f e c t s a b s o r p t i o n . The general phenomenon of carbohydrate-metal complexing i s w e l l known with a review having been w r i t t e n some years ago ' — ' and extensive research having been conducted s i n c e t h a t time. Some s t u d i e s have i n d i c a t e d a s l i g h t improvement i n a b s o r p t i o n from l a c t o s e and sucrose and a decrease due t o s t a r c h (ifL'with glucose having no e f f e c t .


1

1 6


58



100

Days 59

Figure 1. Kinetic pattern of Fe retention by a guinea pig given a single initial dose of isotope per os. Key: • , ferric fructose; O, ferrous sulfate. (Reproduced, with permission, from Ref. 6. Copyright 1976, Institute for Clinical Nutrition.)



4.

CLYDESDALE


The d i f f e r e n c e s i n b i o a v a i l a b i l i t y found with c e r t a i n complexes from study t o study are f r u s t r a t i n g at b e s t . I t seems d i f f i c u l t t o i s o l a t e the exact a f f e c t of a g i v e n l i g a n d and the part i c u l a r complex i t might form. For i n s t a n c e , Derman ejb a l '±2/ r e c e n t l y found t h a t i r o n a b s o r p t i o n from maize and sorghum beer was more than t w e l v e - f o l d g r e a t e r than from a g r u e l made from the c o n s t i t u e n t s used t o prepare the beer. The authors p o s t u l a t e d t h a t at l e a s t three f a c t o r s are r e s p o n s i b l e f o r t h i s : l ) the r e moval of s o l i d s during fermentation, 2) the presence of e t h a n o l , and 3) the presence of l a c t i c a c i d . However, to suggest a part i c u l a r l i g a n d t o e x p l a i n t h i s e f f e c t would be almost impossible. The d i f f i c u l t y i s a l s o probably due i n p a r t t o the chemistry i n v o l v e d . Bachran and Bernhard (2-L'reported the formation of a s o l u b l e l a c t o s e . PeClg complex along w i t h a p r e c i p i t a t e of a l a c t o s e - i r o n g e l , an i n s o l u b l e l a c t o s e . Fe(0H)2 adduct and i n s o l uble Fe(OH)p. The amount of each of these forms was found t o vary i n t h i s work which was based on c a r e f u l l y c o n t r o l l e d model systems, t h e r e f o r e , i t would not be very s u r p r i s i n g t o f i n d tremendous v a r i a t i o n i n food m a t e r i a l s where c o n d i t i o n s are subject t o the vaga r i e s o f nature. Such v a r i a t i o n s would d i r e c t l y a f f e c t the amount of s o l u b l e and i n s o l u b l e complexes, and perhaps the s t r e n g t h o f the c h e l a t i n g bonds, both of which would a f f e c t the b i o a v a i l a b i l ity. Other carbohydrates which act as l i g a n d s are i n c l u d e d i n t h a t d i f f u s e c l a s s i f i c a t i o n o f m a t e r i a l s known as d i e t a r y f i b e r . A l though a l l d i e t a r y f i b e r s are not carbohydrates, i t seems advantageous t o d i s c u s s them as a group, s i n c e they are g e n e r a l l y t r e a t e d i n t h a t manner. The l i t e r a t u r e i s l i t e r a l l y b u l g i n g with r e p o r t s on the b i o a v a i l a b i l i t y of i r o n from p l a n t m a t e r i a l s . In g e n e r a l , i t has been found t h a t both i n t r i n s i c and added i r o n i n vegetables i s l e s s a v a i l a b l e than i n other sources as summarized by L a y r i s s e and Martinez-Torres '25.'(Figure 2 ) . However, other r e s e a r c h seems t o provide c o n f l i c t i n g data and a p a u c i t y of cons i s t e n t explanations f o r e i t h e r i n c r e a s e d or decreased b i o a v a i l a bility. T h i s i s not a r e f l e c t i o n o f the r e s e a r c h , but more a r e f l e c t i o n of the complexity o f the human/food/nutrient i n t e r a c t i o n c h a i n . Although many chemical c o n s t i t u e n t s are i n v o l v e d , such as phosphorous '22/, p r o t e i n s 'Ur£2.', phytates, which have been e x t e n s i v e l y s t u d i e d by M o r r i s and E l l i s o f the USDA and r e c e n t l y by Cheryan (21), and c a r b o x y l i c a c i d s , i t seems t h a t a great d e a l of the b i n d i n g may be due t o the d i e t a r y f i b e r s . One component of d i e t a r y f i b e r , p e c t i n , has long been known t o form c h e l a t e s with a number of d i v a l e n t metals. Haug and Smidsrod ^^±J r e p o r t e d on the s e l e c t i v i t y of some a n i o n i c polymers, i n c l u d i n g p e c t a t e s , f o r d i v a l e n t metal ions and Schweiger et a l (23) have presented data i n support of the formation of both i n t e r molecular and i n t r a m o l e c u l a r chelates of p e c t i n with d i v a l e n t metals. Recently Camire and Clydesdale (2U) r e p o r t e d s i g n i f i c a n t b i n d i n g o f i r o n , calcium, magnesium, and z i n c by both p e c t i n and


0

0

8


1-

1

Lettuce

73

• •

42

2-4 mg

Wheat

• •

13

2-4 mg l - l 7 m g

•• •

137

3-4 mg

Com

17

38

1 B

3 mg

3-4 mg

Soybeon

E

II

3 mg

Veoi liver

§

39

H

warn

3-4 mg

34

Hemoglobin

1-2 mg

Fish muscle

Food of animal origin

96

3-4 mg 520

Veoi Totol muscle

Figure 2. Iron absorption by adults from a range of foods. The bars represent the mean absorptions and standard errors, calculated from the logarithms of the percentage absorptions. (Reproduced, with permission, from Ref. 15. Copyright 1971, Grune and Stratton, Inc.)

%

3

ICH

•

9

II

20
Fe (aq) (water s o l u t i o n , 25°C) i t i s found t h a t the standard r e d u c t i o n p o t e n t i a l i s +770mv i n d i c a t i n g a tendency t o occur spontaneously i n foods, since most foods have a standard r e d u c t i o n p o t e n t i a l o f UOOmv or s l i g h t l y l e s s . However, i f we examine the r e d u c t i o n h a l f - r e a c t i o n i n b a s i c solution, F e ( 0 H ) (s) + e~ * Fe(.0H) (s) + (OH)" i t i s found t h a t the standard r e d u c t i o n p o t e n t i a l i s -560mv i n d i c a t i n g non-spontaneity i n foods. I t i s c l e a r t h a t pH plays a r o l e i n maintaining i r o n ( I I ) i n s o l u t i o n . S o l u b i l i t y o f i r o n ( I I ) at low pH i s obviously a most important f a c t o r but the p o s s i b l e e f f e c t of the standard r e d u c t i o n p o t e n t i a l of the F e 3 redox couple at d i f f e r e n t pH values should not be overlooked. T h i s may e x p l a i n the r e s u l t s of L e i c h t e r and J o s l y n (3&)» Lee and Clydesdale (32) and others who found t h a t r e g a r d l e s s of the source the i r o n found i n bread and non-yeast leavened baked goods (high pH foods) r e s p e c t i v e l y , was mainly i n the i r o n ( I I I ) s t a t e and/or i n s o l u b l e . Such chemical changes w i l l obviously e f f e c t b i o a v a i l a b i l i t y and perhaps e x p l a i n some o f the r e s u l t s r e p o r t e d . For i n s t a n c e , B r i s e and H a l l b e r g (ko) determined t h a t 200-500 mg a s c o r b i c a c i d more than t r i p l e d the b i o a v a i l a b i l i t y of 30 mg o f i r o n administered as ferrous s u l f a t e while 100 mg or l e s s had l i t t l e e f f e c t . Simil a r l y , Cook and Monsen (kl) determined that the i n c r e a s e i n i r o n absorption from a semisynthetic meal was d i r e c t l y p r o p o r t i o n a l t o +

+ 2

3

2

+



72


the amount of a s c o r b i c a c i d added over a range of 25 t o 1000 mg. The apparent dependency o f the e f f i c a c y of a s c o r b i c a c i d on conc e n t r a t i o n seems t o i n d i c a t e t h a t the ascorbate e i t h e r formed a complex and/or c o n t r i b u t e d t o the s o l u b i l i t y , and/or maintained the i r o n i n the f e r r o u s s t a t e , s i n c e the standard r e d u c t i o n potent i a l f o r ascorbate i n water i s +400MV. S i m i l a r conclusions might a l s o be drawn from Hodson (h2) who found t h a t a f t e r 2 t o 5 months storage o f a l i q u i d weight c o n t r o l d i e t a r y w i t h an excess of asc o r b i c a c i d , the i r o n added as f e r r o u s s u l f a t e remained i n the f e r r o u s valence whereas the i r o n added as f e r r i c ortho phosphate has been s o l u b i l i z e d , i o n i z e d and reduced t o the b i v a l e n t form. These r e s u l t s i n d i c a t e t h a t perhaps ascorbate may e f f e c t b i o a v a i l a b i l i t y more than another complexing and reducing agent such as f r u c t o s e because i t i s a l s o an a c i d . I n an attempt t o c l a r i f y the chemical e f f e c t s o f pH and a s c o r b i c a c i d on i r o n valence, Nojeim and Clydesdale (k3) investigated model systems as w e l l as attempting t o e x t r a p o l a t e t h e i r f i n d i n g s t o food m a t e r i a l s . They used a phthalate/HGl/NaOH b u f f e r system, s i n c e i t covered a s u i t a b l e pH range and d i d not r e a c t with added i r o n . Four i r o n sources; hydrogen reduced elemental ( E l ) , f e r r o u s s u l f a t e monohydrate (FS), f e r r i c ortho phosphate (FOP), and sodium f e r r i c EDTA t r i h y d r a t e (SFEDTA) were added t o a s e r i e s of b u f f e r s ranging from pH 2.2 t o 6.2. As w e l l , these same sources were evaluated at d i f f e r e n t molar l e v e l s of ascorbate, s i m i l a r t o those used by B r i s e and H a l l b e r g (kQ). I t was found t h a t pH was indeed a f a c t o r i n the i o n i z a t i o n and valence of the four i r o n compounds evaluated. EI and FS were completely converted t o f e r rous i o n w i t h i n k& hours at pH k.2 and below. I o n i z a t i o n o f FOP and SFEDTA was slower, incomplete, and r e s u l t e d i n l e s s f e r r o u s and more f e r r i c i r o n . At pH 2.7> most o f the i o n i z e d ( s o l u b l e ) i r o n remained i n or was converted t o the f e r r o u s form over a one month storage p e r i o d . The more r e a c t i v e ( l e s s o x i d i z e d ) i r o n compounds, EI and FS, remained 100$ i o n i c ( s o l u b l e ) at pH U.2 but showed some gradual o x i d a t i o n t o the t r i v a l e n t s t a t e . These r e s u l t s are c o n s i s t e n t w i t h the d i s c u s s i o n on redox p o t e n t i a l s mentioned previously. A s c o r b i c a c i d showed a r a t h e r c o n t r a d i c t o r y r o l e , at l e a s t at f i r s t glance. I t seemed t o promote the r e d u c t i o n of i r o n at low pH and the o x i d a t i o n of i r o n at higher pH v a l u e s . For i n s t a n c e , i n the s t u d i e s w i t h FS and EI at pH 2.7 i n the presence o f a s c o r b a t e , n e a r l y 100$ of the EI and FS added was i o n i z e d and i n the f e r r o u s valence, where i t remained f o r the d u r a t i o n o f the study (one month). However, at pH 6.2, the presence of ascorbate g r e a t l y i n c r e a s e d the i o n i z a t i o n ( s o l u b i l i z a t i o n ) o f both EI and FS but the newly i o n i z e d i r o n remained i n the f e r r o u s valence form f o r only k8 hours before f u r t h e r o x i d a t i o n began t o occur. In the case of EI about 50$ o f the i o n i c i r o n was i n the f e r r o u s valence, and 50$ i n the f e r r i c s t a t e a f t e r k weeks. While w i t h FS a t pH 6.2, f e r r i c hydroxide p r e c i p i t a t e s occurred without aseorbate uttzhln one week. The presence o f ascorbate i n h i b i t e d



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Physicochemical Properties of Food

73

the formation of these hydroxides keeping most of the i r o n i n the i o n i c f e r r i c form as measured "by the method of Lee and Clydesdale ( 3 9 ) which u t i l i z e d "bathophenanthroline. I t i s p o s s i b l e that the ascorbate i n h i b i t e d the formation of the hydroxides by forming a complex with the f e r r i c i r o n i n the same manner as described by Conrad and Schade (kk). I f t h i s was the case, the iron-ascorbate complex must have been d i s r u p t e d by the iron-bathophenanthroline a n a l y s i s , s i n c e the a n a l y s i s i n d i c a t e d the presence of i o n i c f e r r i c i r o n . These observations by Nojeim and Clydesdale (V3) as w e l l as other observations i n the l i t e r a t u r e on the mode of a c t i o n of a s c o r b i c a c i d cannot be explained simply. For example, i f the e f f i c a c y of a s c o r b i c a c i d i n i r o n n u t r i t u r e was due s o l e l y t o i t s r o l e as c h e l a t i n g agent then i t should not enhance b i o a v a i l a b i l i t y i n almost every i n s t a n c e . Further confusion a r i s e s when i t i s noted that low pH a s c o r b i c a c i d promotes the r e d u c t i o n of i r o n (h5), but i t s presence at high pH seems to promote the o x i d a t i o n of i r o n (k3). Smith and Dunkley (_U6, kj) found a s c o r b i c a c i d to have pro-oxidant p r o p e r t i e s i n r e l a t i o n t o o x i d i z e d f l a v o r and l i p i d p e r o x i d a t i o n i n milk. They considered t h a t these were a t t r i b u t a b l e to two p r o p e r t i e s of a s c o r b i c a c i d : the a b i l i t y t o reduce c u p r i c copper t o the cuprous form, and a s p e c i f i c a s s o c i a t i o n between a s c o r b i c a c i d and copper that i n some unexplained manner increases prooxidant a c t i v i t y . These studies support the observed prooxidant e f f e c t of asc o r b i c a c i d at high pH (milk) i n the presence of copper but not i n the presence of i r o n . In order t o e x p l a i n the e f f e c t s observed by Nojeim and Clydesdale (U3), I would l i k e to propose a mechanism f o r the act i o n of a s c o r b i c a c i d i n the presence of i r o n which might e x p l a i n how i t could act as both a reducing agent and a prooxidant as w e l l as perhaps shedding some more l i g h t on i t s r o l e i n the chemi s t r y and thus the b i o a v a i l a b i l i t y of i r o n . This proposed mechanism i s based on the i n t e r r e l a t i o n s h i p between s o l u b i l i t y , pH, r e d u c t i o n p o t e n t i a l , and c h e l a t i o n i n a s o l u t i o n of i r o n and a s c o r b i c a c i d . At low pH, i t w i l l be remembered, F e 3 and F e are s o l u b l e and probably e x i s t as t h e i r r e s p e c t i v e hydrates with the standard r e d u c t i o n p o t e n t i a l of F e 3 (aq) +e~ -*Fe 2 (aq) being +770mv. In the presence of ascorbate which has a standard r e d u c t i o n potent i a l of +UU0mv the formation of F e w i l l take p l a c e spontaneously h8) and r e a c t i o n 1 (Figure 6) w i l l go to the r i g h t . However, at the same time, at a low pH. both a F e ^ -ascorbate and a F e 3 -ascorbate complex may form (M+, h8), represented by r e a c t i o n s 2 and k (Figure 6 ) . Upon a d d i t i o n of f e r r i c i r o n t o an a s c o r b i c a c i d s o l u t i o n r e a c t i o n 2 w i l l probably take p l a c e more r a p i d l y than r e a c t i o n 1, but i n time r e a c t i o n 1 w i l l predominant i f the pH i s maintained at a low l e v e l , as observed by Nojeim and Clydesdale (1*3), with the o v e r a l l e f f e c t being r e d u c t i o n . Thus ascorbate, due t o i t s r e d u c t i o n p o t e n t i a l r e l a t i v e t o i r o n at a low pH, and +

+

+ 2

+

+ 2

+


+


74


Fe

3+

+ Ligand + e"

4-

Ligand + e~ 4 Figure 6.

-+

Fe

2+

+ Ligand

2

Fe * — Ligand

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


4.

CLYDESDALE


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

+

+


+

+ 2

:

+

+

+

+

+ 2

+



76


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

+


4. CLYDESDALE Table IV.

Fe

11

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

Chelates o f :

Nitrilotriacetic Downloaded by CORNELL UNIV on October 19, 2016 | http://pubs.acs.org Publication Date: November 1, 1982 | doi: 10.1021/bk-1982-0203.ch004


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

acid

23

3 sec.

C i t r i c acid

25

8 hours

Ethylenediaminetetraacetic acid

25

k days

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

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



TOMATO JUICE pH 4.3

SFEDTA 18/4

B I S C U I T DOUGH pH 6.5

FOP 1/0

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

CRANBERRY JU CE pH 2.7


I

I

H

2 C

00

-J

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

JUICE pH

T O M A T O

J U I C E 4.3

pH

6.S

B I S C U I T

DOUGH

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

pH

CRANBERRY



80


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



4.

CLYDESDALE


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



JUICE Eh

BISCUIT

Eb

D O U G H CRANBERRY 340mv

JUICE 400mv

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

Eh 240mv

TOMATO


4. CLYDESDALE


83

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

Literature Cited 1.


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

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