Nutritional Bioavailability of Zinc - American Chemical Society

10 The Role of Phytate in Zinc Bioavailability and Homeostasis Nutritional Bioavailability of Zinc Downloaded from pubs.acs.org by FUDAN UNIV on 03/19/17. For personal use only.

D. OBERLEAS University of Kentucky, Department of Nutrition and Food Science, Lexington, K Y 40506

Phytate, myo i n o s i t o l hexakis (dihydrogen phosphate) is found i n all plant seeds, many roots and tubers. Its synthesis follows p o l l i n a t i o n and its content increases with maturity. The greatest concentration is found i n legume seeds, bran and germ of cereal grains. Small amounts are found i n many f r u i t s and vegetables except stem and leafy vegetables. Phytate has been shown to be associated with protein, as magnesium salts i n sesame, and i s water soluble i n corn germ. Phytate complexes with divalent elements with varying degrees of tenacity. The s o l u b i l i t y of these complexes varies with pH. Zinc phytate i s least soluble at pH 6 and i s less soluble than calcium or other mineral complexes at this pH. Kinetic synergism of calcium and zinc with phytate causes a complexation less soluble than either separately. Saliva and pancreatic f l u i d secrete large quantities of zinc equivalent to as much as three times the dietary intake which i s also v u l nerable to phytate complexation. The mechanism of phytate action i n the gastrointestinal tract i s related to complexation and subsequent prevention of absorption and reabsorption of zinc. The complexation can be equated to a phytate:zinc molar ratio and the relative hazard may be subsequently estimated from such data. A compound later to be identified as myo-inositol hexakis (dihydrogen phosphate) was f i r s t isolated from plant seeds by Pfeffer i n 1872 (_1) (Figure 1). The relative concentration and widespread distribution i n plant seeds was f i r s t described by Palladin (2) i n 1895 and identity as an i n o s i t o l compound i n 1897 by Winterstein (_3). Its identity as a hexaphosphate ester of i n o s i t o l was confirmed early i n this century (4) and synthesis was accomplished i n 1919 by Posternak 05). The biosynthesis of 0097-615 6/ 83/0210-0145$06.00/0 © 1983 American Chemical Society

Nutritional Bioavailability of Zinc Downloaded from pubs.acs.org by FUDAN UNIV on 03/19/17. For personal use only.

146 NUTRITIONAL

Figure 1. BIOAVAILABILITY OF ZINC

1,2,3,4,5,6-Hexakis(phosphonooxo)cyclohexane [phytate] (39).


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Role of Phytate in Zinc Bioavailability and Homeostasis

phytate has been studied i n corn (6^, 7) and peas ( Zn > Co > Mn > Fe > Ca (36, 37). I t i s i n t e r e s t i n g that calcium, which has u n t i l recent years received the most a t t e n t i o n , i s at the lower end of t h i s s e r i e s . F e r r i c phytate i s known to be l e a s t s o l u b l e i n d i l u t e a c i d . However as a t e r t i a r y component i n the complexation of z i n c (26, 29) and other d i v a l e n t elements ( 3 5 ) , calcium has been shown to promote a synergism t h a t i s unique i n chemical k i n e t i c s . This was f i r s t s t u d i e d i n an i n v i t r o model w h i l e searching f o r a mechanism t o e x p l a i n the experimental observations of calcium and/or phytate as causative agents i n z i n c d e f i c i e n c y (38). Figure 4 i l l u s t r a t e s the q u a n t i t y of p r e c i p i t a t e formed when equimolar concentrations of phytate, z i n c and/or calcium (1:1 or 2:1) are mixed i n an open v e s s e l and pH's adjusted f i r s t to l e s s than 3, than c a r e f u l l y to the appropriate pH. The pH range between 3 and 9 was s e l e c t e d to encompass the p h y s i o l o g i c a l l y important range. The r e s u l t s i n d i c a t e that 1) calcium and phytate, i n equimolar c o n c e n t r a t i o n , are q u i t e s o l u b l e at a l l pH's under these c o n d i t i o n s ; 2) z i n c phytate i s l e s s s o l u b l e than calcium phytate and at pH 6 i s l e s s s o l u b l e than calcium at twice the molar c o n c e n t r a t i o n ; 3) z i n c , calcium and phytate i n a l l combinat i o n s t e s t e d was l e s s s o l u b l e than e i t h e r z i n c or calcium phytate or the sum of these alone at pH 6. The pH 6 i s very important p h y s i o l o g i c a l l y because t h i s i s the approximate pH of the duodenum and upper jejunum, an area of the g a s t r o i n t e s t i n a l t r a c t i n which z i n c must be absorbed. At pH 6 and a 2:1:1 calcium:zinc:phytate molar r a t i o , 98% of the z i n c was i n the p r e c i p i t a t e (28, 38, 39). f

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150

NUTRITIONAL

BIOAVAILABILITY

OF

ZINC

Figure 4. Quantity of precipitate formed at varying molar ratios and pH's. Key: • , Ca.phytate (1:1); Δ , Ca.phytate (2:1); O , Zn:phytate (1:1); X , Ca:Zn:phyta (1:1:1); · , Ca:Zn:phytate (2:1:1). (Reproduced with permission from Ref. 4


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Zinc i s a trace element and both calcium and phytate are present i n many foods i n macro q u a n t i t i e s ; t h e r e f o r e , an i n v i t r o model was developed to study the quantity of z i n c a v a i l a b l e f o r a b s o r p t i o n w i t h these more p h y s i o l o g i c a l r a t i o s . The r e s u l t s , F i g u r e 5, i n d i c a t e that as the r a t i o of calcium:phytate increases there i s a decrease i n the uncomplexed z i n c i n s o l u t i o n which would be a v a i l a b l e f o r absorption (28, 38, 40). EDTA added i n t o t h i s same model increased the s o l u b l e z i n c (38) i n d i c a t i n g that s o l u b l e and absorbable c h e l a t i n g compounds may compete with phytate and make some z i n c a v a i l a b l e f o r absorption or reabsorpt i o n . The i m p l i c a t i o n s of the studies described above are that the i n t e r a c t i o n of phytate w i t h z i n c and calcium i n v o l v e s a chemical rather than a p h y s i o l o g i c a l r e a c t i o n . With the l a r g e d i f f e r e n t i a l between the molecular weight of phytate (660) and the atomic weight of z i n c (65.4), weight or percentage comparisons would be of l i t t l e value. P h y t a t e : z i n c molar r a t i o reduces both of these components to a common denominator which represents a meaningful, chemical comparison. I t i s apparent that some means was needed to express t h i s r e l a t i o n s h i p on a chemical basis that a l s o has p h y s i o l o g i c a l i m p l i c a t i o n s . The use of p h y t a t e : z i n c molar r a t i o was t e s t e d on data from s e v e r a l experiments i n which the d i e t a r y model was i n d e n t i c a l except f o r the concentrations of phytate and z i n c . Since growth r a t e depression i s an e a r l y , s e n s i t i v e and e a s i l y measured parameter, i t was quite s u i t a b l e f o r the dependent v a r i a b l e . The data, Table I , shows the inverse r e l a t i o n s h i p between decreasing p h y t a t e : z i n c molar r a t i o and average growth rate of these r a t s w i t h i n 4 weeks. The s e v e r i t y of c l i n i c a l symptoms are d i r e c t l y Table I .

VARIABILITY OF ZINC DEFICIENCY

Dietary Varient Diet

Phytate

Phytate/Zinc

Gain/Wk +

SD

Zinc

(%)

(mg/kg)

(molar)

(4

Weeks)

1

1.0

2*

495.5

8.8

+ 3.0

(24)

2

1.0

15

66.1

12.5

+ 4.1

(27)

3

0.4

15

26.1

15.3

+ 4.6

(18)

4

0.4

70

5.7

37.5

+ 7.0

(47)

3.2

48.5

+ 4.2

(30)

5

0.4

125

* P r o t e i n EDTA washed. From Oberleas, D.

(39).

151

>

δ > < >

2

>

δ

H

S

H

d

to

65

2 ο

Ν

Figure 5. Uptake of Zn by rat jejunal strips in vitro as affected by calcium and phytate. Data 2 represent activity/mg tissue nitrogen as a percent of the control of each replicate (7 replicates).5 Η (Reproduced with permission from Ref. 40.) ο m


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OBERLEAS


r e l a t e d to the phytate:zinc molar r a t i o (39). The d i e t s u t i l i z e d i n these experiments contained 1.6 percent calcium which accentuates the experimental e f f e c t of phytate but i s considerably higher than the t y p i c a l human i n t a k e . Three l a b o r a t o r i e s have subsequently and independently confirmed the v a l i d i t y of the phytate:zinc molar r a t i o u t i l i z i n g d i e t a r y models w i t h more reasonable calcium l e v e l s ( 4 ^ , _42, 1*2)· ^1 laborat o r i e s have concluded that w i t h moderate calcium i n t a k e , molar r a t i o s l e s s than 10 are l i k e l y to provide adequate a v a i l a b l e z i n c and molar r a t i o s greater than 10 are a s s o c i a t e d w i t h symptoms of z i n c d e f i c i e n c y ; namely, growth r a t e depression. Though the determination of t h i s c r i t i c a l molar r a t i o was made i n r a t s , i t represents an expression of a chemical r e l a t i o n s h i p and thus a p p l i e s to a l l monogastric species f o r s i m i l a r d i e t a r y phytate:z i n c molar r a t i o s . The molar r a t i o of 10 a l s o compares favorably w i t h the need f o r a molar r a t i o between 3 and 6 at the higher l e v e l s of calcium i n t a k e to provide adequate a v a i l a b l e z i n c .


t n r e e

Homeostatic Fluxes of Zinc Frequently ignored i n many s t u d i e s are the r e l a t i v e l y l a r g e q u a n t i t i e s of z i n c which are r e c y c l e d i n t o the g a s t r o i n t e s t i n a l t r a c t . The most widely studied are the r e l a t i v e l y l a r g e secret i o n s of z i n c i n the p a n c r e a t i c f l u i d (44, 45, 46). The panc r e a t i c c o n t r i b u t i o n to the duodenum may be more than three (3) times the d i e t a r y i n t a k e (44, 45). Some c o n t r i b u t i o n i s a l s o made t h r u the b i l e (44, 46). Zinc i s a l s o secreted i n s i z e a b l e q u a n t i t i e s i n the s a l i v a ( 4 7 ) . With such a l a r g e c o n t r i b u t i o n of z i n c to the t o t a l g a s t r o i n t e s t i n a l p o o l , much of the secreted z i n c must be reabsorbed to prevent the body from experiencing a perpetual zinc d e f i c i t . The r e l a t i v e v u l n e r a b i l i t y of the secreted z i n c to phytate complexation has only r e c e n t l y been demonstrated. The i n j e c t i o n g|i z i n c d e f i c i e n t r a t s i n t r a p e r i t o n e a l l y w i t h a t r a c e r dose of z i n c allows a p o r t i o n of t h i s z i n c to be i n e q u i l i b r i u m w i t h the endogenous metabolic p o o l . This z i n c then i s secreted t h r u the s a l i v a , p a n c r e a t i c f l u i d , and b i l e . Those animals maintained on the phytate c o n t a i n i n g soy p r o t e i n contained 2-4 times the r a d i o a c t i v i t y of the animals fed a c a s e i n p r o t e i n d i e t (Table I I ) . Therefore, not only does phytate a f f e c t the b i o a v a i l a b i l i t y of d i e t a r y z i n c but a l s o the reabsorption of endogenous z i n c and thus has a net e f f e c t on z i n c homeostasis. Since t h i s t o t a l phytate e f f e c t cannot be measured by l a b e l i n g only the d i e t a r y p o o l , the expression of the net e f f e c t as the phytate:zinc molar r a t i o i s the most s e n s i t i v e and accurate method of estimating the r e l a t i v e r i s k of z i n c d e f i c i e n c y i n any i n d i v i d u a l or population.

153

154

NUTRITIONAL

BIOAVAILABILITY

OF

ZINC

TABLE I I . RATIO OF ZINC EXCRETED BY RATS ON SOY PROTEIN DIET VS CASEIN PROTEIN DIET FOLLOWING INTRAPERITONEAL INJECTION

1

3

5

7

9

11

13

%; Ca

1.5

1.4

1.8

3.9

3.3

2.8

2.8

0 . 8 %; Ca

1.3

1.6

1.6

2.3

3.3

2.5

2.5

Days


1.6

Mechanism of A c t i o n In order to formulate a mechanism of a c t i o n of phytate on z i n c homeostasis, c e r t a i n c o n d i t i o n s must be f u l f i l l e d : 1) The process must occur i n the g a s t r o i n t e s t i n a l t r a c t s i n c e phytate i s not absorbed except f o r s m a l l amounts by b i r d s ; 2) calcium must be a t e r t i a r y component i n the t o t a l process but there must be some r e a c t i o n without excessive amounts of calcium; 3) c e r t a i n c h e l a t i n g compounds such as EDTA must be capable of competing w i t h the process and make some z i n c a v a i l a b l e f o r a b s o r p t i o n or r e a b s o r p t i o n and 4) There must be some e x p l a n a t i o n f o r the data which i n d i c a t e s that 40% o r more of the d i e t a r y pool may be a v a i l a b l e f o r absorption (4j3, 4J)). A l l these c o n d i t i o n s are s a t i s f i e d by the f o l l o w i n g formula which i s an expression of the "Law of Mass A c t i o n " :

Zn*"*" + Phytate

-——

Zn(Ca)Phytate EDTA

One must recognize that the t o t a l z i n c pool a v a i l a b l e f o r s a t i s f y i n g the c o n d i t i o n s of t h i s r e a c t i o n must i n c l u d e the endogenous secreted z i n c which may represent 3-4 times the d i e t a r y i n t a k e even when an adequate l e v e l of z i n c i s consumed. Whereas phytate i s a p o l y f u n c t i o n a l compound and the s o l u b i l i t y product of such a mixed s a l t i s d i f f i c u l t to c a l c u l a t e , the concept of expressing the phytate and z i n c as molar r a t i o s i s simple t o c a l c u l a t e and u s e f u l i n the determination of r e l a t i v e z i n c s t a t u s ( 5 0 ) . P o t e n t i a l P o p u l a t i o n Hazards The e a r l i e s t t e s t s of the p h y t a t e : z i n c molar r a t i o concept i n humans was provided by data s u p p l i e d by Reinhold ( 5 1 , 5 2 ) . He analyzed a l a r g e number of samples of Middle E a s t e r n bread f o r phytate and z i n c . These breads were of two types; "Bazari and Sangak" which are leavened breads s o l d i n the urban areas and "Tanok" which i s an unleavened bread consumed i n the v i l l a g e s of the Middle East (Table I I I ) . For many of the v i l l a g e r s , "Tanok",


10.

OBERLEAS


c o n s t i t u t e s over 90% of t h e i r d i e t . I t i s from these v i l l a g e populations that c l i n i c a l z i n c d e f i c i e n c y was f i r s t described (31) and i s e a s i l y diagnosed (53). At a z i n c c o n c e n t r a t i o n of 30 mg/kg (Reinhold, personal communication) the unleavened "Tanok" has a phytate:zinc molar r a t i o of about 23 whereas the leavened bread had molar r a t i o s of about 10-12. The l a t t e r would imply an element of r i s k but not t o the extent of e x h i b i t i n g c l i n i c a l symptoms. Since the "tanok" bread c o n s t i t u t e s the major p o r t i o n of the v i l l a g e d i e t s , i f an estimate of consumption of bread were 1 kg/day, c l i n i c a l z i n c d e f i c i e n c y was prevalent a t a molar r a t i o of 23 i n s p i t e of a z i n c i n t a k e of 30 mg/day. The l a t t e r would be twice the current Recommended D i e t a r y Allowance (RDA) i n the United S t a t e s . TABLE I I I . PHYTATE CONTENT OF MIDDLE EASTERN BREADS Bazari

Sangak

Tanok

Samples (N)

56

50

126

Phytate (mg/100 g)

326

388

684

4.94

5.88

10.36

Phytate (m moles) Zinc

Estimated at 30 mg/kg f o r a l l breads (0.46 m moles)

Phytate/Zinc (molar) From:

10.8

12.8

22.6

Oberleas (39).

A current study of d i e t a r y r e c a l l s from EFNEP s u b j e c t s on s e l f - s e l e c t e d d i e t s from three areas of Kentucky i n d i c a t e that from a s y s t e m a t i c a l l y s e l e c t e d sample of 1294 subjects a p p r o x i mately 800 had p h y t a t e : z i n c molar r a t i o s greater than 10 and about 600 had r a t i o s greater than 15. A s m a l l group of subjects had molar r a t i o s greater than 15 and z i n c i n t a k e s greater than 15 mg/day. Beyond t h i s , one has but to look a t t y p i c a l d i e t s consumed throughout the world and imagine that z i n c d e f i c i e n c y , although not always expressing i t s e l f w i t h overt c l i n i c a l symptoms, may a f f e c t a very l a r g e segment of the world's p o p u l a t i o n . P a r t i c u l a r l y v u l n e r a b l e are the poorer segments of the p o p u l a t i o n which must depend on c e r e a l g r a i n s and legume seeds as the major source of p r o t e i n and c a l o r i e s . W i t h i n the United S t a t e s , the most " a t r i s k " p o p u l a t i o n may be represented by adolescents who f r e q u e n t l y i n c l u d e c e r e a l s , peanut b u t t e r and pasta as major d i e t a r y components, "vegans", o r those s u b s t i t u t i n g , soybean based s y n t h e t i c meats f o r animal p r o t e i n . As a n a l y t i c a l techniques f o r e s t i m a t i n g phytate become a v a i l a b l e , computer data bases can be updated t o c o n t a i n accurate estimates of the phytate and z i n c concentrations of foods. When

155


156

n u t r i t i o n a l bioavailability of zinc

good data bases become e s t a b l i s h e d a simple and noninvasive t e c h nique of c a l c u l a t i n g p h y t a t e : z i n c molar r a t i o from d i e t a r y r e c a l l s w i l l provide a s e n s i t i v e and accurate estimate of the r e l a t i v e r i s k of z i n c d e f i c i e n c y f o r any p o p u l a t i o n . Since t h i s represents a p h y s i c a l constant the molar r a t i o of 10 or l e s s represents and adequate standard provided some minimal i n t a k e of approximately 5 mg of zinc/day i s consumed. L i k e w i s e , the RDA f o r z i n c , c u r r e n t l y at a somewhat a r b i t r a r y 15 mg/day needs t o be modified to r e f l e c t the e f f e c t of phytate on z i n c homeostasis as expressed by the p h y t a t e : z i n c molar r a t i o . Z i n c from almost any m i n e r a l source i s r e a d i l y a v a i l a b l e f o r a b s o r p t i o n (54). There f o r e , i n the absence of phytate, z i n c d e f i c i e n c y represents l i t t l e more than an academic c u r i o s i t y ; the e f f e c t e x h i b i t e d by phytate makes z i n c d e f i c i e n c y a r e a l and prevalent problem. Acknowledgment This was supported i n part by the Science and Education A d m i n i s t r a t i o n of the U.S. Department of A g r i c u l t u r e under Grant No. 5901-0410-8-0076-0 from the Competive Research Grants O f f i c e .

Literature Cited 1. Pfeffer, E. Pringsheims Jb. Wiss. Bot. 1872, 8, 429,475. 2. Palladin, W. Z. Biol. 1895, 31, 199. 3. Winterstein, E. Ber. dtsch. chem. Ges. 1897, II, 2299. 4. Suzuki, U . ; Yoshimura, K.; Takaishi, M. Coll. Agric. B u l l . , Tokyo Imp. Univ. 1907, 7, 503. 5. Posternak, S. Compt. Rend. 1919, 169, 138-40. 6. De Turk, E.E.; Holbert, J.R.; Hawk, B.W. J. Agric. Res. 1933, 46, 121-41. 7. Earley, E.B.; De Turk, E.E. J. Am. Soc. Agron. 1944, 36, 803-14. 8. Fowler, H.D. J. Sci. Food Agric. 1957, 8, 333-41. 9. O'Dell, B . L . ; deBoland, A.R.; Koirtyohann, S.H. J. Agric. Food Chem. 1972, 20, 718-21. 10. Fontaine, T.D.; Pons, W.A., J r . ; Irving, G.W., Jr. 1946, 164, 487-507. 11. Smith, A.K.; Rackis, J.J. J. Am. Chem. Soc. 1957, 79, 633-7. 12. deBoland, A.R.; Garner, G.B.; O'Dell, B.L. J. Agric. Food Chem. 1975, 23, 1186-9. 13. Morris, E.R.; Ellis, R. J. Nutr. 1976, 106, 753-60. 14. Raulin, J. Ann. Sci. Nat. Bot. Biol. Vegetale 1869, 11, 93. 15. Bertrand, G.; Benzon Β. Compt. Rend. 1922, 175, 289-92. 16. Todd, W.R.; Elvehjem, C.A.; Hart E.B. Am. J. Physiol. 1934, 107, 146-56. 17. Stirn, F.E.; Elvehjem, C.A.; Hart E.B. J. Biol. Chem. 1935, 109, 347-59. 18. Tucker, H.F.; Salmon, W.D. Proc. Soc. Exp. Biol. Med. 1955, 88, 613-6.


10.

OBERLEAS


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October 13,1982

Nutritional Bioavailability of Zinc - American Chemical Society

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