5 Dietary Phytate and Calcium Bioavailability ERNST GRAF1 and J O H N W. EATON2 1
2
The Pillsbury Company, Minneapolis, M N 55414 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, M N 55455
Phytic acid (myo-inositol hexaphosphoric acid) comprises 1 to 5% by weight of legumes, cereals, oil seeds and nuts and t h u s is p r e s e n t in numerous f o o d commodities. This compound has been indicted as a major cause of several mineral deficiency syndromes, through the formation of insoluble metallophytate complexes. Our recent results demonstrate, however, that polyvalent cations form soluble complexes with phytate at high phytate to metal ratios and that phytate does not impair the absorption of45Ca2+administered to mice by gavage. Thus, many of the supposedly adverse effects of dietary phytate and the resulting mineral deficiencies may merely reflect g e n e r a l d i e t a r y inadequacy. Furthermore, some d i e t a r y phytate may actually be beneficial duetoitsabilityto suppress iron-mediated oxidative processes. Indeed, phytic acid may reduce the incidence of colonic cancer and function as a natural food preservative. C a l c i u m i s t h e most abundant m i n e r a l i n t h e human body. About 99% i s d e p o s i t e d as phosphate m a t r i c e s i n bones and teeth. I n addition to t h i s s t r u c t u r a l r o l e , C a i s of c e n t r a l i m p o r t a n c e t o most a s p e c t s o f c e l l p h y s i o l o g y , b o t h a s a c o f a c t o r f o r e x t r a c e l l u l a r enzymes and a s an i n t r a c e l l u l a r regulator. I n t r a c e l l u l a r C a has profound e f f e c t s on a v a r i e t y o f c e l l u l a r and enzyme f u n c t i o n s , i n c l u d i n g e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n a l l forms o f muscle, e x c i t a t i o n - s e c r e t i o n c o u p l i n g a t nerve endings and i n both e x o c r i n e and e n d o c r i n e g l a n d s , e x o c y t o s i s , membrane t r a n s p o r t , c e l l morphology, gene e x p r e s s i o n , and vision. R e c o g n i z i n g t h e importance o f adequate C a intake, 2 +
2 +
2 +
0097-6156/85/0275-0051$06.00/0 © 1985 American Chemical Society
NUTRITIONAL BIOAVAILABILITY O F C A L C I U M
52
the United S t a t e s n a t i o n a l Research C o u n c i l recommends an i n t a k e of 800 mg C a (recommended d a i l y allowance or RDA) and 1200 mg d u r i n g pregnancy and l a c t a t i o n . I n o r d e r t o meet t h e s e g u i d e l i n e s , c e r t a i n f o o d s t a p l e s i n t h e U.S. are f o r t i f i e d w i t h Ca . Despite t h i s widespread e f f o r t , Ca d e f i c i e n c y i s s t i l l v e r y common as e v i d e n c e d by t h e high i n c i d e n c e of o s t e o p o r o s i s . This h e a l t h problem p a r t i a l l y a r i s e s from the f a c t that maintenance of C a homeostasis r e q u i r e s not only adequate d i e t a r y i n t a k e , but i n t e s t i n a l a b s o r p t i o n and subsequent u t i l i z a t i o n o f Ca . B i o a v a i l a b i l i t y of C a i s a f f e c t e d by numerous p h y s i o l o g i c a l c o n d i t i o n s , i n c l u d i n g age, s e x , g e n e t i c make-up, s t r e s s , h o r m o n a l s t a t u s , h e a l t h s t a t u s , and n u t r i t i o n a l habits. In a d d i t i o n to these i n t r i n s i c f a c t o r s , c e r t a i n d i e t a r y components, such as f i b e r and o x a l a t e , form i n s o l u b l e complexes w i t h C a and i n t e r f e r e w i t h i t s absorption. Another p u t a t i v e c u l p r i t i n t h i s category i s p h y t i c a c i d . P h y t i c a c i d (Figure 1) c o n s t i t u t e s 1 t o 5% by weight of most n u t r i t i o n a l l y important p l a n t seeds (Table I ) and 2 +
2 +
2 +
2 +
2 +
Table I . P h y t i c A c i d Content of S e l e c t e d Seeds
Millet Barley Sorghum Corn Rye Dehydrated peas Oat Wheat Soybeans Sunflower seeds Peanuts Wild r i c e Lima beans Sesame seeds
Phytic Acid (% w/w) 0.7 0.7 0.9 0.9 0.9 0.9 1.0 1.1 1.4 1.9 1.9 2.2 2.5 5.3
Reference 2 2 2 2 2 3 2 2 4 5 3 5 5 3
t y p i c a l l y accounts f o r 60 t o 90$ of the t o t a l phosphorus. I t u s u a l l y o c c u r s i n d i s c r e t e r e g i o n s o f the s e e d s , such as t h e a l e u r o n e l a y e r o f wheat and r i c e ( 6 ) , where i t i s b e l i e v e d t o s e r v e as an a n t i o x i d a n t and p r o t e c t a g a i n s t o x i d a t i v e damage d u r i n g storage (7). Due t o the a b i l i t y o f p h y t i c a c i d t o p r e c i p i t a t e p o l y c a t i o n i c n u t r i l i t e s (8,9), i t s p r e s e n c e i n f o o d has c o n c e r n e d n u t r i t i o n i s t s f o r s e v e r a l decades. S e v e r a l r e c e n t r e v i e w s e x t e n s i v e l y d i s c u s s t h e c h e m i s t r y and n u t r i t i o n a l r a m i f i c a t i o n s o f d i e t a r y phytate (4,10-15). The e f f e c t o f p h y t i c a c i d on C a bioavailability i s s t i l l i n d i s p u t e . Some e a r l y n u t r i t i o n i s t s reported r a c h i t o g e n i c p r o p e r t i e s o f d i e t a r y p h y t a t e based on f e e d i n g s t u d i e s u s i n g puppies (16-18) and e p i d e m i o l o g i c a l s t u d i e s on B r i t i s h - b o r n c h i l d r e n o f A s i a n i m m i g r a n t s 2 +
5.
G R A F A N D EATON
2+
Dietary
Phytate and Ca
53
Bioavailability
(19,20). These c o n c l u s i o n s were s e r i o u s l y questioned (212ÏÏ7 and recent r e s u l t s i n d i c a t e t h a t t h e b i o a v a i l a b i l i t y of C a i s t h e same f r o m a c a s e i n d i e t as f r o m a h i g h p h y t a t e s o y c o n c e n t r a t e (25). O b e r l e a s c o n t e n d s t h a t , i n the p r e s e n c e o f adequate amounts o f C a and v i t a m i n D, d i e t a r y p h y t a t e i s n o t r a c h i t o g e n i c , even though i t may b i n d s u b s t a n t i a l amounts o f Ca' " ' (15). T h i s c o n t r o v e r s y , the r e l a t i v e p a u c i t y o f a v a i l a b l e i n f o r m a t i o n , and t h e growing incidence of C a d e f i c i e n c y prompted us t o i n v e s t i g a t e f u r t h e r the chemical i n t e r a c t i o n s between C a and phytate and t o a s s e s s i t s e f f e c t on t h e b i o a v a i l a b i l i t y of C a a d m i n i s t e r e d t o mice by gavage. 2 +
2 +
2
1
2 +
2 +
2 +
Methods P h y t i c a c i d s o l u t i o n s were p r e p a r e d by t i t r a t i n g sodium phytate ( S i g m a C h e m i c a l Company) w i t h H C l ; t h e c o n c e n t r a t i o n was d e t e r m i n e d by a n a l y z i n g f o r i n o r g a n i c p h o s p h a t e a f t e r wet a s h i n g w i t h H 2 S O J . - H N O 3 (3:2) f o r 45 m i n u t e s . The c o n c e n t r a t i o n o f CaCl2 s t o c k s o l u t i o n s was measured by atomic a b s o r p t i o n spectrometry. Ca s o l u b i l i t y was determined by i n c u b a t i n g C a and p h y t a t e i n 100 mM HEPES ρ Η 7 . 1 a t 25°C f o r 2 h o u r s , c e n t r i f u g i n g and m e a s u r i n g ^->Ca i t h e s u p e r n a t a n t by l i q u i d s c i n t i l l a t i o n aounting (26). The b i n d i n g o f C a t o p h y t i c a c i d was a s c e r t a i n e d by measuring C a p o t e n t i o m e t r i c a l l y w i t h a Radiometer C a s e l e c t i v e electrode (2£). For the q u a n t i t a t i o n o f jln v i v o a b s o r p t i o n o f ^->Ca , 200 μ ΐ o f r a d i o a c t i v e s o l u t i o n s were a d m i n i s t e r e d by g a s t r i c gavage t o male mice kept on a d e i o n i z e d water d i e t d u r i n g t h e p r e v i o u s 18 h o u r s . A f t e r 4 h o u r s b l o o d was o b t a i n e d by a x i l l a r y i n c i s i o n and ^ C a extracted w i t h TCA and determined by l i q u i d s c i n t i l l a t i o n counting (26). I r o n - m e d i a t e d g e n e r a t i o n o f h y d r o x y l r a d i c a l (*0H) was monitored by t h e hypoxanthine-xanthine oxidase method as p r e v i o u s l y d e s c r i b e d (28). F o r m a l d e h y d e produced by reaction o f ·0Η w i t h DMS0 was determined s p e c t r o p h o t o m e t r i c a l l y by the Hantzsch r e a c t i o n (29). L i p i d p e r o x i d a t i o n was measured by d e t e r m i n i n g m a l o n d i a l d e h y d e (MDA) s p e c t r o p h o t o m e t r i c a l l y by t h e t h i o b a r b i t u r i c a c i d method (30). 2 +
2 +
ς
2+
n
2 +
2 +
2 +
ac
2+
2 +
w
a
s
R e s u l t s and D i s c u s s i o n T r a d i t i o n a l l y , p o l y v a l e n t c a t i o n - p h y t a t e complexes have been c o n s i d e r e d u n i f o r m l y i n s o l u b l e , and t h i s dogma has been t h e b a s i s o f most methods f o r t h e d e t e r m i n a t i o n o f phytate (31). I n 1976, however, s o l u b l e monoferric phytate was i s o l a t e d (32), and most r e c e n t l y , Ca
