Chapter 19
Speciation Chemistry of Foods Problems and Prospects D. J . McWeeny, H. M . Crews, and R. C. Massey
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Food Science Laboratory, Food Science Directorate, Ministry of Agriculture, Fisheries, and Food, Colney Lane, Norwich NR4 7UQ, England
A 2-stage i n v i t r o enzyme system has been used to simulate g a s t r i c and i n t e s t i n a l digestion of food. Trace element species i n the digest can then be separated chromatographically using ICP-MS as an o n - l i n e s p e c i f i c detector. This has been used i n examination of the cadmium species i n kidney, arsenic species i n turkey and aluminium species i n tea infusions. An a l t e r n a t i v e approach developed i n c o l l a b o r a t i o n with the U n i v e r s i t y of Wales at C a r d i f f involves computer modelling to predict the major chemical forms of the method. The merits and l i m i t a t i o n s of the two techniques are discussed.
Not a l l c h e m i c a l forms of the i n o r g a n i c elements a r e e q u a l l y b i o l o g i c a l l y a c t i v e . Thus methyl mercury i s more t o x i c than i n o r g a n i c mercury s a l t s , a r s e n o - b e t a i n e i s much l e s s t o x i c than i n o r g a n i c a r s e n i c , the b i o l o g i c a l v a l u e of i r o n i n the form of haem as compared t o i n o r g a n i c s a l t s i s w e l l known and c o b a l t a s cyanocobalamin i n l i v e r has i m p o r t a n t v i t a m i n a c t i v i t y . On the o t h e r hand, i n the c o n v e n t i o n a l approaches t o t r a c e i n o r g a n i c a n a l y s i s i n food i t i s u s u a l t o take p a r t i c u l a r c a r e t o c o n v e r t a l l c h e m i c a l compounds c o n t a i n i n g the a n a l y t e element i n t o a s i n g l e c o n v e n i e n t s p e c i e s - and t o measure i t i n t h i s form. I n so d o i n g a l l i n f o r m a t i o n about the o r i g i n a l c h e m i c a l form of the a n a l y t e i s d e l i b e r a t e l y destroyed. Two f a i r l y r e c e n t developments o f f e r the p r o s p e c t o f b e i n g a b l e t o i n v e s t i g a t e the m e t a l s i n food i n t h e i r o r i g i n a l c h e m i c a l form on a g e n e r a l b a s i s . One i s based on an enzymolysis/chromatography/ i n s t r u m e n t a l approach (_1_), the o t h e r on a thermodynamic/computer m o d e l l i n g approach (2^). The p r e s e n t paper aims t o o u t l i n e both of t h e s e , and t o attempt t o a s s e s s them i n terms of t h e i r p o t e n t i a l and l i m i t a t i o n s i n two p a r t i c u l a r r e s p e c t s i e t h e i r a b i l i t y t o : - work s u c c e s s f u l l y a t normal a n a l y t e c o n c e n t r a t i o n s - a v o i d a l t e r i n g the s p e c i a t i o n s i g n i f i c a n t l y . 0097-6156/91/0-0248$06.00/0 Published 1991 American Chemical Society
In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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E n z y m o l y s i s / C h r o m a t o g r a p h i c / i n s t r u m e n t a l Approach T h i s approach i s based upon the view t h a t i n terms of b i o l o g i c a l a c t i o n the i m p o r t a n t i n f o r m a t i o n r e l a t e s t o the c h e m i c a l s p e c i e s which e x i s t i n the s m a l l i n t e s t i n e - the major p o i n t a t which they w i l l be absorbed ( i f a t a l l ) by human b e i n g s . From the p o i n t of view of the e n v i r o n m e n t a l s c i e n t i s t t h i s may not be t r u e - and e n z y m o l y s i s , i n which the f o o d i s s u b j e c t e d t o the d i g e s t i v e enzymes n o r m a l l y encountered i n the g a s t r o i n t e s t i n a l t r a c t - may be i r r e l e v a n t . The procedure i n v o l v e s a s t e p w i s e t r e a t m e n t w i t h p e p s i n a t a c i d pH f o l l o w e d by t r y p s i n a t n e u t r a l pH. T h i s t e c h n i q u e was used i n i t i a l l y t o e s t a b l i s h whether or not t h e r e a r e s i g n i f i c a n t d i f f e r e n c e s between foods i n terms of t h e i r t r a c e element s p e c i a t i o n . T h i s was done s i m p l y i n terms of the e x t e n t t o which the m e t a l s a r e p r e s e n t i n a w a t e r - s o l u b l e form a f t e r t r e a t m e n t w i t h d i g e s t i v e enzymes ( 1_). Without g o i n g t o any s o p h i s t i c a t e d s e p a r a t i o n t e c h n i q u e s , t h i s s i m p l e t r e a t m e n t g i v e s some i n d i c a t i o n of the e f f e c t s which can be encountered. Thus, the major d i f f e r e n c e s a r e r e a d i l y demonstrable i n the s o l u b i l i t i e s of Cu, Fe, Zn, Cd, Pb i n the s u p e r n a t a n t from the enzymic d i g e s t s of a range of f o o d s Q , 3 ) . I n most c a s e s , o n l y p a r t of the t r a c e element i s s o l u b l e , but i n o t h e r s the m a j o r i t y i s s t i l l bound t o the u n d i g e s t e d r e s i d u e - the r e l a t i v e amounts v a r y from element t o element, and from f o o d t o f o o d . Some f u r t h e r i n d i c a t i o n of the c o m p l e x i t y of the s i t u a t i o n i n v o l v e d comes from a s i m p l e a d d i t i o n a l experiment i n which the d i g e s t i s s p i k e d w i t h a s m a l l amount of the element of i n t e r e s t , i n the form of an i n o r g a n i c s a l t . I n the case of some f o o d / m e t a l c o m b i n a t i o n s the added s a l t and the n a t u r a l l y p r e s e n t element behave q u i t e differently. T h i s i n d i c a t e s t h a t i n the d i g e s t the l a t t e r i s not p r e s e n t as a s i m p l e s a l t - nor i n a form which i s i n r a p i d e q u i l i b r i u m w i t h such s a l t s . T h i s l i n e o f e n q u i r y has been t a k e n a s t a g e f u r t h e r i n comparisons o f the s i t u a t i o n s i n i n d i v i d u a l f o o d s w i t h those i n v o l v i n g m i x t u r e s of f o o d s ( 4 ) - the l a t t e r b e i n g what we n o r m a l l y eat. T h i s work demonstrated c l e a r l y t h a t t h e r e a r e d i f f e r e n c e s when food i t e m s a r e c o n s i d e r e d i n c o m b i n a t i o n eg i n terms o f the s o l u b i l i t y of Zn and Cd i n bread and from c r a b when d i g e s t e d s e p a r a t e l y or i n c o m b i n a t i o n ; t h e r e were a l s o s i g n i f i c a n t d i f f e r e n c e s between w h i t e and wholemeal bread i n terms of t h e i r e f f e c t upon the z i n c from c r a b . On the b a s i s of these r e l a t i v e l y s i m p l e e x p e r i m e n t s i t has been concluded t h a t t h e r e a r e r e a l major d i f f e r e n c e s between f o o d s i n the s p e c i a t i o n of the t r a c e elements they c o n t a i n . Subsequent e x p e r i m e n t s have aimed t o e l u c i d a t e the c h e m i s t r y of these d i f f e r e n c e s and t o s e p a r a t e and i d e n t i f y the substances i n v o l v e d . Where e q u i l i b r i a between s p e c i e s e x i s t i t i s a r g u a b l e t h a t i t i s not n e c e s s a r y t o d i s t i n g u i s h between s p e c i e s which e q u i l i b r a t e i n the t i m e s c a l e of a chromatographic s e p a r a t i o n - t h i s b e i n g no g r e a t e r than the d u r a t i o n over which s p e c i e s a r e a v a i l a b l e f o r a b s o r p t i o n from the gut. Work based on t h i s premise i s d e s c r i b e d below.
In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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BIOLOGICAL TRACE ELEMENT RESEARCH
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Chromatographic s e p a r a t i o n of s o l u b l e m e t a l l i c s p e c i e s i s , i n p r i n c i p l e , s t r a i g h t f o r w a r d - one can use g e l permeation, a b s o r p t i o n o r ion-exchange systems f o r t h e purpose - but u n t i l r e c e n t l y d e t e c t i o n systems were i n s u f f i c i e n t l y s e n s i t i v e f o r on l i n e measurements on chromatographic e l u a t e s d e r i v e d from f o o d s c o n t a i n i n g normal l e v e l s o f t r a c e elements, a l t h o u g h i t was p o s s i b l e t o measure a l i q u o t s from e l u a t e s by O f f - l i n e ' g r a p h i t e f u r n a c e atomic a b s o r p t i o n s p e c t r o m e t r y ^ ) . The advent o f ICP-MS i n s t r u m e n t s has changed t h a t ; t h e s e ' i n s t r u m e n t s have an adequate sensitivity f o r many t r a c e elements i n food - and moreover , can be used as an ' o n - l i n e ' chromatography d e t e c t o r ( 6 ) . The p o t e n t i a l o f t h i s approach t o m e t a l s p e c i a t i o n i n food d i g e s t s can be i l l u s t r a t e d by some r e c e n t s t u d i e s . Cadmium S p e c i a t i o n i n P i g Kidney The e n z y m o l y s i s procedure was employed t o examine t h e s t a t e o f cadmium i n cooked p i g k i d n e y d u r i n g d i g e s t i o n . Preliminary e x p e r i m e n t s on s i m p l y b u f f e r e x t r a c t s o f t h e uncooked p i g k i d n e y r e v e a l e d the presence o f t h r e e cadmium b i n d i n g s p e c i e s whose SEC r e t e n t i o n t i m e s corresponded t o m o l e c u l a r w e i g h t s o f 1.2 χ 1 0 , 7.0 χ 10^ and 6-9 χ 1 0 d a l t o n s . On c o o k i n g , t h e two h i g h e r m o l e c u l a r weight s p e c i e s were not observed i n d i c a t i n g t h a t they had been heat d e n a t u r e d . However, the lower m o l e c u l a r weight component was s t i l l p r e s e n t and i t s m o l e c u l a r s i z e and t h e r m a l s t a b i l i t y suggested t h a t cadmium was a s s o c i a t e d w i t h a m e t a l l o t h i o n e i n (MT). On s i m u l a t e d g a s t r i c d i g e s t i o n o f t h e cooked p i g k i d n e y a l l o f t h e cadmium p r e s e n t i n s o l u t i o n e l u t e d as a s i n g l e peak whose r e t e n t i o n time corresponded t o t h a t o f i o n i c cadmium. T h i s s t r o n g l y s u g g e s t s t h a t t h e m e t a l had d i s s o c i a t e d from t h e b i n d i n g s i t e s on MT under t h e a c i d i c c o n d i t i o n s . On subsequent s i m u l a t e d i n t e s t i n a l d i g e s t i o n o f t h e g a s t r i c d i g e s t a s i n g l e peak was a g a i n observed but i n t h i s case i t s r e t e n t i o n time corresponded t o t h a t o f MT. These f i n d i n g s i n d i c a t e t h a t t h e MT p r o t e i n i s r e s i s t a n t t o g a s t r o i n t e s t i n a l e n z y m o l y s i s and i s a b l e t o r e b i n d cadmium a t t h e n e u t r a l pH a t which t h e i n t e s t i n a l d i g e s t was performed. T h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h t h e r e s u l t s of a n i m a l f e e d i n g e x p e r i m e n t s i n which o r a l l y a d m i n i s t e r e d Cd-MT and i o n i c cadmium a r e t r a n s p o r t e d d i f f e r e n t l y f o l l o w i n g a b s o r p t i o n , the former being d e p o s i t e d i n t h e k i d n e y ( t h e t a r g e t organ f o r cadmium t o x i c i t y ) and t h e l a t t e r i n t h e l i v e r . 6
3
I n these s t u d i e s cadmium c o n c e n t r a t i o n s i n t h e e x t r a c t s / d i g e s t s were i n some c a s e s around 10~9g/ml but p r o v i d i n g t h a t s t r i n g e n t p r e c a u t i o n s a r e t a k e n w i t h a p p a r a t u s , r e a g e n t s and procedures t o avoid contamination during the s p e c i a t i o n i n v e s t i g a t i o n s , t h e i n s t r u m e n t a l s e n s i t i v i t y i s adequate f o r s t u d i e s a t t h e l e v e l s encountered i n 'normal' t i s s u e e x t r a c t s . A r s e n i c i n Turkey M u s c l e A r s e n i c a l drugs a r e w i d e l y employed i n i n t e n s i v e p o u l t r y p r o d u c t i o n and t h i s r a i s e s t h e q u e s t i o n o f the i n c i d e n c e o f drug r e s i d u e s i n e d i b l e t i s s u e from b i r d s f e d medicated r a t i o n s - and t h e e f f e c t o f w i t h d r a w a l p e r i o d s p r i o r t o s l a u g h t e r on t h e s e l e v e l s . 3-nitro A-hydrophenyl a r s e n i c a c i d i s w i d e l y used i n t h i s way and an
In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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a n a l y t i c a l procedure b r o a d l y s i m i l a r t o t h a t d e s c r i b e d above has been used i n the study of the a r s e n i c s p e c i e s i n muscle from b i r d s r a i s e d on feed c o n t a i n i n g t h i s substance (Massey, R.C; B a x t e r , M.; B u r r e l l , J.A.; Crews, H.M.; Ebdon, L.; Dean, J.R. u n p u b l i s h e d d a t a ) . I n t h i s case a t r y p s i n d i g e s t was used and the chromatog r a p h i c s e p a r a t i o n was by r e v e r s e phase chromatography w i t h ICP-MS d e t e c t i o n of a r s e n i c a t m/e 75. I t was p o s s i b l e t o demonstrate t h a t o n l y very low drug r e s i d u e l e v e l s e x i s t e d i n these b i r d s i e . up t o 15 ng/g (as As) i n muscle compared w i t h l e v e l s
