21 Effect of a Glucose-Lysine Reaction Mixture on Protein and Carbohydrate Digestion and Absorption
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RICKARD ÖSTE, INGER BJÖRCK, ARNE DAHLQVIST, MARGARETHA JÄGERSTAD, and PER SJÖDIN University of Lund, Department of Applied Nutrition, Chemical Center, P.O. Box 740, S-220 07 Lund, Sweden HANS SJÖSTROM The Panum Institute, Department of Biochemistry C, Copenhagen, Denmark A low-molecular-weight fraction from a glucose -lysine reaction mixture reduced the plasma level of lysine originating from dietary protein, when given to rats (1.5% w/w in diet). This fraction inhibited in vitro trypsin, carboxypeptidase A, and carboxypeptidase Β as well as aminopeptidase Ν of the brush border. A high-molecular-weight frac tion from the reaction mixture strongly inhibited, competitively, invertase and lactase. When intu bated into rats, however, i t did not affect the absorption of sucrose (ratio sucrose/reaction fraction 100:1 w/w). The low-molecular-weight fraction had only a slight effect on the carbo hydrate -hydrolyzing enzymes. When p r o t e i n s a r e h e a t e d t o g e t h e r w i t h c a r b o h y d r a t e s a d e crease i n t h e n u t r i t i v e v a l u e i s f r e q u e n t l y o b s e r v e d . The d e g r e e o f t h i s d e c r e a s e i s d e p e n d e n t on a number o f f a c t o r s , i n c l u d i n g w a t e r a c t i v i t y , t y p e and amount o f r e d u c i n g s u g a r , t y p e o f p r o t e i n , as w e l l as t h e e x t e n t o f h e a t t r e a t m e n t (J_). The l o s s o f p r o t e i n q u a l i t y may be e x p r e s s e d a s r e d u c e d b i o l o g i c a l v a l u e (BV) and d i g e s t i b i l i t y ( T D ) , as a p p a r e n t i n a c o n v e n t i o n a l n e t p r o t e i n u t i l i z a t i o n (NPU) a s s a y w i t h r a t s . When t h e h e a t t r e a t meant i s m i l d , a l o s s i n t h e BV o f t e n c o r r e s p o n d s t o t h e l o s s o f l y s i n e c a u s e d by t h e M a i l l a r d r e a c t i o n , p r o v i d e d l y s i n e i s t h e l i m i t i n g amino a c i d i n t h e p r o t e i n . When t h e h e a t i n g i s more p r o n o u n c e d , t h e r e d u c t i o n i n BV i s o f t e n f o u n d t o be g r e a t e r i n t h e p r o t e i n and may a l s o c a u s e a r e d u c t i o n o f TD. Reviews o f t h e e f f e c t o f t h e M a i l l a r d r e a c t i o n i n p r o t e i n n u t r i t i o n have r e c e n t l y been p u b l i s h e d by Mauron {2} and Dworschak ( 3 ) . T h e r e a r e some r e p o r t s on p o s s i b l e mechanisms b e h i n d t h e s e e f f e c t s o f s e v e r e h e a t t r e a t m e n t . A d r i a n 0 ) has shown t h a t w a t e r s o l u b l e premelanoidins from a g l u c o s e - g l y c m e r e a c t i o n mixture r e d u c e t h e p r o t e i n d i g e s t i b i l i t y and a f f e c t t h e u t i l i z a t i o n o f 0097-6156/83/0215-0405$06.00/0 © 1983 American Chemical Society Waller and Feather; The Maillard Reaction in Foods and Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
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a b s o r b e d amino a c i d s . V a l l e - R i e s t r a (4) s u g g e s t s t h a t t h e r e duced u p t a k e o f a s e v e r e l y h e a t e d g l u c o s e - e g g albumen m i x t u r e i s the r e s u l t of a lowered p a n c r e a t i c s e c r e t i o n of d i g e s t i v e enzymes. The o b s e r v e d d e c r e a s e i n b i o l o g i c a l v a l u e ( t h e r e s u l t o f enhanced u r i n a r y n i t r o g e n e x c r e t i o n ) may p a r t l y be e x p l a i n e d by an u p t a k e o f i n d i g e s t i b l e l o w - m o l e c u l a r r e s i d u e s f r o m t h e g u t (4, 5, 6). H " h e p r e s e n t s t u d y was p e r f o r m e d i n o r d e r t o examine w h e t h e r t h e compounds i n a g l u c o s e - l y s i n e r e a c t i o n m i x t u r e p e r se may i n f l u e n c e t h e d i g e s t i o n and u p t a k e o f p r o t e i n s . S i n c e s u c h an e f f e c t was n o t e d , t h e s t u d y was e x t e n d e d t o i n c l u d e t h e d i g e s t i o n and u p t a k e o f c a r b o h y d r a t e s as w e l l . Glucose-lysine
reaction mixture
E q u i m o l a r amounts o f g l u c o s e ( 4 . 5 0 g) and L - l y s i n e h y d r o c h l o r i d e ( 4 . 5 5 g) were d i s s o l v e d i n 100 ml o f d i s t i l l e d w a t e r and t h e s o l u t i o n was b o i l e d u n d e r r e f l u x f o r 24 h. The pH was k e p t c o n s t a n t d u r i n g t h e r e a c t i o n by a p H - s t a t - c o n t r o l l e d a d d i t i o n o f 5N NaOH. The p H - s t a t s e t t i n g was 6 . 5 . A f t e r t h e r e a c t i o n t h e pH o f t h e m i x t u r e a t room t e m p e r a t u r e was a b o u t 5 . 3 . The m i x t u r e was d i a l y z e d a g a i n s t 3x2 1 d i s t i l l e d w a t e r i n a S p e c t rapor s a c k , which a c c o r d i n g t o the manufacturer (Spectrum M e d i c a l I n d u s t r i e s , I n c . , Los A n g e l e s , U . S . A . ) had an e x c l u s i o n l i m i t o f 6 0 0 0 - 8 0 0 0 d a l t o n s . The d i a l y s a t e was e v a p o r a t e d , t h e r e s i due d i s s o l v e d i n 0.1 M ammonium-acetate b u f f e r , and p l a c e d on a C o n A - S e p h a r o s e column t o s e p a r a t e u n r e a c t e d g l u c o s e . The e l u a t e was c o n c e n t r a t e d and c o n s t i t u t e d t h e l o w - m o l e c u l a r - w e i g h t (LMW) f r a c t i o n used i n t h i s s t u d y . The y i e l d s f r o m r e p e a t e d p r e p a r a t i o n s were 7 . 4 - 7 . 7 g . The r e t e n t a t e f r o m t h e d i a l y s i s was c e n t r i f u g a t e d t o remove i n s o l u b l e m a t e r i a l , and t h e n c o n c e n t r a t e d by e v a p o r a t i o n . The y i e l d was 0 . 9 - 1 . 2 g and c o n s t i t u t e d t h e h i g h m o l e c u l a r - w e i g h t (HMW) f r a c t i o n . E f f e c t s on p r o t e i n u t i l i z a t i o n A n i m a l a s s a y s . When a s s e s s i n g t h e n a t u r e o f a p r o t e i n q u a l i t y r e d u c t i o n , t h e c o n v e n t i o n a l methods o f p r o t e i n q u a l i t y meas u r e m e n t have c e r t a i n l i m i t a t i o n s . F o r e x a m p l e , a r e d u c e d TD i n an NPU a s s a y i s n o t n e c e s s a r i l y t h e r e s u l t o f a r e d u c e d p r o t e i n d i g e s t i o n . O b v i o u s l y t h e same r e s u l t w i l l be o b t a i n e d i f t h e i n c r e a s e o f f e c a l n i t r o g e n i s c a u s e d by an enhanced e x c r e t i o n o f endogenous p r o t e i n o r i f t h e r e i s a f i x a t i o n o f m e t a b o l i c n i t r o g e n by t h e c o l o n i c m i c r o - f l o r a . To be a b l e t o s p e c i f i c a l l y s t u d y t h e d i g e s t i o n o f an e x o genous p r o t e i n i n r a t s , a method was e l a b o r a t e d as f o l l o w s : A s o l u t i o n o f [ U - C l - l y s i n e was i n j e c t e d i n t h e w i n g - v e i n o f a l a y i n g hen a t t h e t i m e o f maximal e g g - a l b u m e n s y n t h e s i s . The eggs were r e c o v e r e d , t h e e g g - w h i t e s e p a r a t e d and d i a l y z e d t o r e move g l u c o s e and t h e n l y o p h i l i z e d . An a c i d h y d r o l y s a t e was s e p a l i f
Waller and Feather; The Maillard Reaction in Foods and Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
21.
ÔSTE ET A L .
Protein and Carbohydrate
Digestion
407
and Absorption
r a t e d by t h i n l a y e r c h r o m a t o g r a p h y and s u b j e c t e d t o a u t o r a d i o g r a p h y . I t was t h e r e b y shown t h a t t h e r a d i o a c t i v i t y p r e s e n t i n t h e e g g - w h i t e was d e r i v e d a l m o s t e x c l u s i v e l y f r o m p r o t e i n - b o u n d l y s i n e (45000 dpm/mg p r o t e i n ) . O n l y t r a c e s o f r a d i o a c t i v i t y c o u l d be f o u n d i n n o n - l y s i n e r e s i d u e s . A c o n t r o l d i e t was p r e p a r e d a s f o l l o w s : Tha l a b e l l e d e g g w h i t e p r o t e i n was m i x e d w i t h a b a s a l d i e t i n t h e p r o p o r t i o n 2 : 9 8 . The b a s a l d i e t c o n t a i n e d 10% c a s e i n , 10% s u c r o s e , a b o u t 70% s t a r c h and 5% m a i z e o i l , a s w e l l a s v i t a m i n s and m i n e r a l s . A s m a l l amount o f [ H ] - l y s i n e was added t o t h i s m i x t u r e , g i v i n g a r a t i o o f t r i t i u m t o ( C ) dpm o f a p p r o x i m a t e l y 1 0 : 1 . The e x p e r i m e n t a l d i e t was o b t a i n e d by a d d i n g a s m a l l amount o t t h e g l u c o s e l y s i n e reaction mixture to the control d i e t . These d i e t s were t h e n f e d t o m a l e S p r a g u e - D a w l e y r a t s , w e i g h i n g 100-120 g e a c h . The r a t s were p l a c e d i n i n d i v i d u a l c a g e s and were f a s t e d o v e r n i g h t . E a r l y i n t h e m o r n i n g e a c h r a t was g i v e n 1.5 g o f t h e c o n t r o l o r t h e e x p e r i m e n t a l d i e t t o be consumed ac[ l i b i tum. We have o b s e r v e d t h a t o v e r n i g h t f a s t e d r a t s i n t h e m o r n i n g i m m e d i a t e l y w i l l consume a t l e a s t 2 g o f f o o d , p r o v i d e d t h e a n i mal room i s k e p t d a r k and t h e f o o d i s n o t t o o u n t a s t y . In t h e s e e x p e r i m e n t s a l l t h e r a t s c o m p l e t e l y consumed t h e p o r t i o n w i t h i n 15 m i n . T h i s p r o c e d u r e was a c c u r a t e enough t o o b v i a t e i n t u b a t i o n as a means f o r a d m i n i s t e r i n g t h e d i e t s . E x a c t l y two h o u r s a f t e r f i n i s h i n g t h e meal each r a t was a n e s t h e s i z e d w i t h e t h e r , b l o o d was sampled by h e a r t p u n c t u r e , and t h e ( C ) / ( H ) - r a t i o i n t h e plasma was m e a s u r e d . In t h e f i r s t e x p e r i m e n t t h e e x p e r i m e n t a l d i e t c o n t a i n e d 1.5% o f t h e LMW f r a c t i o n . T a b l e I shows t h e ( * C ) / ( H ) - r a t i o s f o u n d i n t h e p l a s m a . The r a t i o o b t a i n e d i n t h e c o n t r o l d i e t was a b o u t t h e same a s t h a t i n t h e d i e t ( 0 . 1 2 0 ) . In t h e g r o u p f e d w i t h t h e e x p e r i m e n t a l d i e t a s t a t i s t i c a l l y s i g n i f i c a n t 15% d e c r e a s e was o b s e r v e d . In t h e s e c o n d e x p e r i m e n t t h e e x p e r i m e n t a l d i e t c o n t a i n e d 1.7% o f t h e HMW f r a c t i o n . W i t h t h i s f r a c t i o n a somewhat u n e x p e c t e d e f f e c t was o b s e r v e d . T h e r e was a s m a l l , b u t h i g h l y s i g n i f i c a n t i n c r e a s e i n t h e ( C/ H)-ratio ( T a b l e I ) . T h i s c o u l d be e x p l a i n e d by a decrease i n t h e uptake o f f r e e l y s i n e from t h e small i n t e s t i n e , since a considerable part of the o r i g i n a l l y protein-bound ( C ) l a b e l l e d l y s i n e m i g h t be a b s o r b e d i n t h e f o r m o f s m a l l p e p t i d e s ( 8 ) . T a b l e I a l s o shows t h e a b s o l u t e v a l u e s o f ( H ) and ( C ) i n tïïe plasma o f t h e r a t s f e d t h e HMW f r a c t i o n . The s t a n d a r d d e v i a t i o n s o f t h e s e d a t a a r e h i g h e r t h a n t h e one c a l c u l a t e d f r o m t h e ( C)/( H ) - r a t i o and t h e i n c r e a s e d a v e r a g e o f ( C ) i n t h e e x p e r i m e n t a l group i s n o t s t a t i s t i c a l l y s i g n i f i c a n t . The l e v e l s o f ( H ) a r e a b o u t t h e same i n b o t h g r o u p s . These r e s u l t d i d n o t i n d i c a t e a s p e c i f i c a f f e c t on t h e l y s i n e u p t a k e . Enzyme a s s a y s As shown p r e v i o u s l y t h e LMW f r a c t i o n had a r e p r e s s i n g e f f e c t on t h e p r o t e i n d i g e s t i o n i n t h e i n v i v o e x p e r i ment. A c c o r d i n g l y , i t was o f i n t e r e s t t o s t u d y i n v i t r o t h e e f f e c t o f t h i s f r a c t i o n on t h e k i n e t i c s o f r e a c t i o n s c a t a l y z e d by p r o t e a s e s and p e p t i d a s e s p r e s e n t i n t h e g a s t r o - i n t e s t i n a l t r a c t . S
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l l f
1 4
ll
lk
3
3
3
l l f
3
lk
3
l l f
l l f
3
Waller and Feather; The Maillard Reaction in Foods and Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
408
MAILLARD
In T a b l e II a r e shown t h e r e s u l t s f r o m k i n e t i c s t u d i e s w i t h c o m m e r c i a l l y a v a i l a b l e g a s t r i c and p a n c r e a t i c enzymes. T r y p s i n was s t r o n g l y i n h i b i t e d , a t l e a s t a t a l o w c o n c e n t r a t i o n o f c a s e i n as s u b s t r a t e . The h y d r o l y s i s o f b e n z o y l a r g i n i n e ethyl ester (BAEE) by t r y p s i n was n o n - c o m p e t i t i v e l y i n h i b i t e d , g i v i n g a 30% r e d u c t i o n o f V x a t 0.5 mg/ml o f t h e LMW f r a c t i o n . C a r b o x y p e p t i d a s e A, and t o a l e s s e r e x t e n t c a r b o x y p e p t i d a s e B, were n o n - c o m p e t i t i v e l y i n h i b i t e d as w e l l . P e p s i n and c h y m o t r y p s i n were n o t a f f e c t e d by t h e c o n d i t i o n s u s e d i n t h e s e a s s a y s . The a c t i o n o f t h e g a s t r i c and p a n c r e a t i c enzymes c a u s e s t h e r e l e a s e o f s m a l l p e p t i d e s as w e l l as f r e e amino a c i d s , t h e p e p t i d e f r a c t i o n b e i n g t h e q u a n t i t a t i v e l y d o m i n a n t one ( 9 ) . Thus f u r t h e r h y d r o l y s i s i s c r u c i a l , i f t h e d i e t a r y p r o t e i n i s t o be c o m p l e t e l y u t i l i z e d by t h e o r g a n i s m . The f i n a l s t a g e s o f h y d r o l y s i s i s a s s o c i a t e d w i t h t h e i n t e s t i n a l mucosal c e l l s . L a r g e r p e p t i d e s a r e p r o b a b l y h y d r o l y z e d by enzymes a t t h e b r u s h b o r d e r membrane. D i - and t r i p e p t i d e s may be a b s o r b e d as s u c h and h y d r o l y z e d i n t r a c e l l u l a r ^ ( 9 , J_0). There are a t l e a s t t h r e e p e p t i d a s e s i n the brush border o f t h e s m a l l i n t e s t i n e : A m i n o p e p t i d a s e A , w h i c h has an a f f i n i t y f o r p e p t i d e - b o u n d a c i d amino a c i d s O j _ ) , a m i n o p e p t i d a s e N, w h i c h has a b r o a d s p e c i f i c i t y ( 1 2 ) , and d i p e p t i d y l p e p t i d a s e I V , w h i c h r e l e a s e s d i p e p t i d e s f r o m t h e N - t e r m i n a l end o f p e p t i d e s w i t h a p r e f e r e n c e f o r X-PRO t e r m i n a l s (j_3). In T a b l e I I I a r e shown t h e e f f e c t o f a l o w c o n c e n t r a t i o n o f t h e LMW f r a c t i o n on t h e a c t i v i t y o f t h e s e enzymes i n e x t r a c t s o f hog i n t e s t i n e . A m i n o p e p t i d a s e Ν was f o u n d t o be s t r o n g l y i n h i b i t e d by 0.25 mg/ml o f t h e f r a c t i o n . A m i n o p e p t i d a s e A and d i p e p t i d y l p e p t i d a s e IV were n o t i n h i bited. Around 40% o f t h e LMW f r a c t i o n i s absorbed from the small i n t e s t i n e i n t h e r a t ( 1 4 ) . A p a r t o f t h e f r a c t i o n may t h u s be present i n the e p i t h e l i a l c e l l s at the time of i n t r a c e l l u l a r p e p t i d e h y d r o l y s i s . The e f f e c t o f t h e LMW f r a c t i o n on t h e a c t i v i t y o f two c y t o s o l e n z y m e s , p r e s e n t i n a p r e p a r a t i o n f r o m hog intestine, i s shown i n T a b l e I I I . G l y c y l l e u c i n e d i p e p t i d a s e , w h i c h has a b r o a d s p e c i f i c i t y ( 1 5 ) , was i n h i b i t e d a t 0.7 mg/ml of the f r a c t i o n , w h i l e p r o l i n e "oTpeptidase, which c a t a l y z e s the h y d r o l y s i s o f X-PRO ( J 6 ) , was n o t . S i n c e the g l u c o s e - l y s i n e r e a c t i o n m i x t u r e used i n t h i s study c o n s i s t e d o f a number o f d i f f e r e n t s u b s t a n c e s , i t was o f i n t e r e s t t o study whether the observed i n h i b i t o r y e f f e c t i n v i t r o c o u l d be a t t r i b u t e d t o some s p e c i f i c c o m p o u n d ( s ) . In o r d e r t o o b t a i n a s e p a r a t i o n , an a l i q u o t o f t h e LMW f r a c t i o n , r a d i o l a b e l e d by [U- Χ] g l u c o s e added t o t h e r e a c t a n t s , was a p p l i e d on a Sephadex G-50 column and e l u t e d w i t h w a t e r . The UV a b s o r b a n c e was r e c o r d e d and t h e e l u a t e was c o l l e c t e d i n f r a c t i o n s . The d e g r e e o f i n h i b i t i o n e f f e c t e d by s m a l l samples o f e q u a l volume f r o m e a c h f r a c t i o n and e x e r t e d on c a r b o x y p e p t i d a s e A and p u r i f i e d a m i n o p e p t i d a s e Ν was d e t e r m i n e d as w e l l as t h e r a d i o a c t i v i t y m a
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REACTIONS
τ
Waller and Feather; The Maillard Reaction in Foods and Nutrition ACS Symposium Series; American Chemical Society: Washington, DC, 1983.
21.
ÔSTE E T A L .
Protein and Carbohydrate Digestion and Absorption
Measurements
TABLE I o f ( H ) and (
Experiment
3
(
l l f
C ) i n r a t plasma
(i*c)***
( H)
0.100 ± 0 ,.0027
1969 ± 2 3 5
18251 ± 1 3 8 2
0.106 ± 0 .. 0 0 3 6 * *
1817 ± 1 6 4
18568 ± 1 8 4 6
1 ! ,
409
0/( Η) 3
3
LMW f r a c t i o n Control (6) Experimental
0.117 ± 0 ..014 (7)
0.102 ± 0 ..010*
Downloaded by FUDAN UNIV on November 27, 2016 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch021
HMW f r a c t i o n C o n t r o l (10) Experimental
(10)
Α Π v a l u e s a r e mean ± s t a n d a r d d e v i a t i o n , A new c o n t r o l d i e t was p r e p a r e d b e f o r e each e x p e r i m e n t . W i t h i n p a r e n t h e s i s number o f r a t s . From O s t e e t a l . ( 7 ) * ** ***
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