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Changes in lens and erythrocyte glutathione reductase in response to exogenous flavin adenine dinucleotide and liver riboflavin content of rat on riboflavin deficient diet
NUTRITION RESEARCH, Vol. 7, pp. 1203-1208, 1987 0271-5317/87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.
C H A N G E S IN LENS AND E R Y T H R O C Y T E G L U T A T H I O N E R E D U C T A S E IN R E S P O N S E TO E X O G E N O U S FLAVIN A D E N I N E D I N U C L E O T I D E A N D LIVER RIBOFLAVIN C O N T E N T OF RAT ON RIBOFLAVIN D E F I C I E N T DIET K. S e e t h a r a m Bhat 1, ph.D. and P. Vasanth Rao, M.Sc. N a t i o n a l I n s t i t u t e of N u t r i t i o n Indian Council of Medical R e s e a r c h J a m a i Osmania PO, H y d e r a b a d - 500 007 India
ABSTRACT The e f f e c t of e x o g e n o u s flavin adenine d i n u c l e o t i d e (FAD) and h e p a t i c level of r i b o f l a v i n on the a c t i v i t y of l e n t i c u l a r and e r y t h r o c y t e g l u t a t h i o n e r e d u c t a s e (GSH-R) was studied in r a t s fed a diet d e f i c i e n t in riboflavin. By 75 days of feeding, the G S H - R a c t i v i t y , under basal c o n d i t i o n (in the absence of e x o g e n o u s FAD) and the a c t i v a t i o n c o e f f i c i e n t (AC, a c t i v i t y of the e n z y m e in response to e x o g e n o u s F A D to t h a t w i t h o u t it) values w e r e s i g n i f i c a n t l y a l t e r e d in lens as well as e r y t h r o c y t e s of r a t s fed riboflavin d e f i c i e n t diet. Significant r e d u c t i o n in r i b o f l a v i n c o n t e n t of liver was also o b s e r v e d in the e x p e r i m e n t a l animals. T h e s e findings suggest t h a t the A C value for the l e n t i c u l a r G S H - R is as equally a r e l i a b l e b i o c h e m i c a l p a r a m e t e r for assessing r i b o f l a v i n n u t r i t u r e as t h a t of the e r y t h r o c y t e e n z y m e . Key Words:
Lens, e r y t h r o c y t e , liver, g l u t a t h i o n e r e d u c t a s e , riboflavin, a c t i v a t i o n c o e f f i c i e n t , rat. INTRODUCTION
D e t e r m i n a t i o n of flavin adenine d i n u c l e o t i d e (FAD) d e p e n d e n t g l u t a t h i o n e r e d u c t a s e (GSH-R, E.C. 1.6.4.2) in e r y t h r o c y t e s from human s u b j e c t s (1-4) and e x p e r i m e n t a l animals (5-7) has been shown to be a s e n s i t i v e index of r i b o f l a v i n nutriture. In the v i t a m i n d e f i c i e n t s t a t e , the e n z y m e is s t i m u l a t e d following in v i t r o addition of its c o - f a c t o r , FAD and also a f t e r v i t a m i n a d m i n i s t r a t i o n in vivo. In an e a r l i e r study on r a t s from our Institute, it was o b s e r v e d t h a t G S H - R in e r y t h r o c y t e was m o r e s e n s i t i v e to riboflavin d e f i c i e n c y than t h a t of liver e n z y m e , although the v i t a m i n c o n t e n t of the liver d e c r e a s e d at a f a s t e r r a t e than t h a t of e r y t h r o c y t e s (7). F u r t h e r , no response was o b s e r v e d in t h e h e p a t i c e n z y m e a c t i v i t y to in v i t r o addition of FAD. Glutathione reductase is p r e s e n t in lens and its a c t i v i t y d e c r e a s e s during aging and c a t a r a c t f o r m a t i o n (8-11). R e c e n t l y Ono and Hirano (12) have provided e v i d e n c e for t h e e x i s t e n c e of flavin m o n o n u c l e o t i d e (FMN) and F A D synthesising e n z y m e s in lens.
1To whom c o r r e s p o n d e n c e should be addressed.
1203
1204
K.S. BHAT and P.V. RAO
To i n v e s t i g a t e the relationship b e t w e e n l e n t i c u l a r and e r y t h r o c y t e G S H - R a c t i v i t y and liver riboflavin c o n t e n t , the a c t i v i t y of GSH-R in lens as well as e r y t h r o c y t e and riboflavin c o n t e n t of the liver w e r e d e t e r m i n e d . In addition, t h e e f f e c t of in v i t r o addition of F A D on the a c t i v i t y of GSH-R was also studied in lens and e r y t h r o c y t e from t h e s e rats, MATERIALS A N D METHODS Ten m a l e NIN-Wistar a riboflavin d e f i c i e n t diet riboflavin s u p p l e m e n t e d (8 rats was fed ad libitum, to the food c o n s u m p t i o n (Pair-fed). The rats w e r e tained at 22-25~
strain rats weighing 35-54 g w e r e fed ad libitum on (7). Two groups of rats, 10 in each group, r e c e i v i n g mg/kg) diet s e r v e d as controls. One group of c o n t r o l while the o t h e r group was fed with a q u a n t i t y equal of the riboflavin d e f i c i e n t group in the previous day c a g e d individually in s c r e e n - b o t t o m e d c a g e s and m a i n -
A t the end of 75 days of feeding, rats w e r e s a c r i f i c e d a f t e r o v e r n i g h t fasting under e t h e r anaesthesia. Blood was drawn from the orbital plexus into h e p a r i n i z e d tube kept cold. Blood was c e n t r i f u g e d at 2000 x g for 15 min in the cold. The plasma and the b u l l y c o a t w e r e r e m o v e d with p a s t e u r e p i p e t t e . The residual plasma and buffy c o a t w e r e r e m o v e d by resuspending the e r y t h r o c y t e s in 5 v o l u m e s of ice cold 0.9% NaC1 solution and c e n t r i f u g i n g at 2000 x g for 10 min. The e r y t h r o c y t e s w e r e washed t h r i c e in the s a m e medium. A l i q u o t e s of the packed e r y t h r o c y t e s ( h a e m a t o c r i t , 96%) w e r e dispensed into a m b e r c o l o u r e d s c r e w capped glass tubes, f r o z e n and s t o r e d at - 2 0 ~ till f u r t h e r analysis. The processing of e r y t h r o c y t e s was c a r r i e d out under dim light. Lens was c a r e f u l l y e x c i s e d through p o s t e r i o r approach. Each lens was h o m o g e n i z e d (10%) in 0.2 M T r i s - E D T A buffer, pH 8.2 in a P o t t e r - E l v e n j e m glass h o m o g e n i z e r and the h o m o g e n a t e was c e n t r i f u g e d at 20000 X g in a Sorvall RC 513 superspeed c e n t r i f u g e at 4 ~ for 30 rain. The liver was rapidly r e m o v e d and s t o r e d at - 2 0 ~ G l u t a t h i o n e r e d u c t a s e a c t i v i t y in both lens and e r y t h r o c y t e was assayed a c c o r d i n g to Bayoumi and Rosalki (13). The c o n t e n t s of the r e a c t i o n m i x t u r e are shown in Table 1. The r e a c t i o n was followed k i n e t i c a l l y for the first 5 min TABLE 1 C o n t e n t of the G l u t a t h i o n e R e d u c t a s e E n z y m e Assay M i x t u r e Lens
Erythrocyte
Potassiumphosphate b u f f e r
78
Enzyme preparation
80 pl (2000 g s u p e r n a t a n t )
EDTA
1.6 jJ m o l e
100 }H (1:15 h a e m o lyzate} 4 ~ mole
GSSG
2 p mole
5 p mole
FAD*
jJ mole (pH 7.4)
10 n mole Incubated at 3 7 ~
0.2 m mole (pH 7.4)
25 n mole for 25 min.
NADPH
0.16 y m o l e
0.4 F mole
Total volume
1.0 ml
2.45 ml
* When p r e s e n t
GSH-REDUCTASE AND RIBOFLAVIN DEFICIENCY
1205
( m a x i m u m l i n e a r r a t e ) by t h e d e c r e a s e in o p t i c a l d e n s i t y a t 340 nm in a G i l f o r d s p e c t r o p h o t o m e t e r (Model 250) e q u i p p e d with a r e c o r d e r (Model 6050). T h e d e c r e a s e in o p t i c a l d e n s i t y was l i n e a r with t i m e and e n z y m e c o n c e n t r a t i o n . Protein concent r a t i o n of t h e lens was d e t e r m i n e d by t h e m e t h o d of L o w r y e t al (14) w i t h c r y s t a lline b o v i n e s e r u m a l b u m i n as a p r o t e i n s t a n d a r d . A c t i v a t i o n c o e f f i c i e n t (AC) v a l u e for GSH-R, t h e s t i m u l a t o r y e f f e c t of e x o g e n o u s FAD, on t h e e n z y m e was c a l c u l a t e d by dividing t h e a c t i v i t y of t h e e n z y m e in t h e p r e s e n c e of a d d e d c o - f a c t o r by t h a t w i t h o u t it to p r o v i d e e v i d e n c e of r i b o f l a v i n d e f i c i e n c y (15,16). R i b o f l a v i n c o n t e n t of t h e l i v e r was a s s a y e d m i c r o b i o l o g i c a l l y using L. c a s e i as t h e t e s t o r g a n i s m (17). S i g n i f i c a n c e of d i f f e r e n c e s b e t w e e n groups was e v a l u a t e d by t h e s t u d e n t ' s ' t ' t e s t . RESULTS A N D DISCUSSION A t t h e e n d of 75 days of f e e d i n g w i t h t h e r i b o f l a v i n s u p p l e m e n t e d d i e t , t h e r a t s in ad l i b i t u m (274 + 3.9g) and p a i r - f e d (108 + 3.3g) groups w e r e s i g n i f i c a n t l y h e a v i e r (P/-. 0.001) t h a n t h e a n i m a l s fed r i b o f l a v i n d e f i c i e n t d i e t (58 + 3.3g). T h e m e a n food i n t a k e by t h e r a t s a t t h e end of t h e e x p e r i m e n t a l perTod was 14.8 + 0.75 g / d a y in ad l i b i t u m g r o u p as a g a i n s t 4.9 + 0.33 g / d a y o f t h e r i b o f l a v i n d e f i c i e n t group. S i m i l a r t r e n d was also r e p o r t e d from this i n s t i t u t e e a r l i e r (7). S i g n i f i c a n t c h a n g e s w e r e also s e e n in lens w e t w e i g h t ( P < 0 . 0 0 1 ) and t o t a l p r o t e i n c o n t e n t ( P ~ 0.025) of t h e lens b e t w e e n r i b o f l a v i n d e f i c i e n t (33.2 +_ o.39 m g and 17.1 + 0.37 r a g / l e n s r e s p e c t i v e l y ) and c o n t r o l s (37.1 + 0.37 m g and 19.4 + 0.56 m g / l e n s r e s p e c t i v e l y ) . H o w e v e r , t h e c o n t e n t of lens pr-oteins, e x p r e s s e d p e r g w e t w e i g h t was not s i g n i f i c a n t l y d i f f e r e n t in t h e e x p e r i m e n t a l (514 + 9.3) and c o n t r o l groups (501 + 16.1 and 523 + 15.0). Riboflavin deficiency h a s b e e n s h o w n to r e s u l t in d e c r e a ~ d r i b o f l a v i n c o ' t e n t and a c t i v i t i e s of f l a v i n n u c l e o t i d e d e p e n d e n t e n z y m e s in t i s s u e s (18-20). In t h e p r e s e n t study, s i g n i f i c a n t r e d u c t i o n in r i b o f l a v i n c o n t e n t of t h e l i v e r was o b s e r v e d in r i b o f l a v i n d e f i c i e n t r a t s {17.3 + 0.72 )ag/g liver) a g a i n s t b o t h ad l i b i t u m and p a i r fed c o n t r o l a n i m a l s (44.7 + 1.9-2 a n d 45.1 + 1.48 jag/g l i v e r r e s p e c t i v e l y ) w h i c h was s i m i l a r to t h a t r e p o r t e d by B a m j i a n d S h a r a d a (7). T h e a c t i v i t y of G S H - R b o t h in lens a n d e r y t h r o c y t e s , in t h e a b s e n c e of e x o g e n o u s F A D was s i g n i f i c a n t l y { P < 0 . 0 0 1 ) l o w e r e d in r a t s on r i b o f l a v i n d e f i c i e n t r a t i o n (50%) c o m p a r e d to r i b o f l a v i n s u p p l e m e n t e d r a t s (Table 2). On t h e o t h e r hand, G S H - R a c t i v i t y in t h e p r e s e n c e of t h e c o f a c t o r did not s i g n i f i c a n t l y d i f f e r in t h e v i t a m i n d e f i c i e n t and s u p p l e m e n t e d r a t s . A d d i t i o n of F A D c a u s e d a s i g n i f i c a n t i n c r e a s e ( P < 0.001) in G S H - R a c t i v i t y in r i b o f l a v i n d e f i c i e n t t i s s u e s c o m p a r e d to c o n t r o l s , w i t h t h e r e s u l t , t h e A C v a l u e increased significantly (P<0.001) in lens as well as e r y t h r o c y t e s in r i b o f l a v i n deficient rats. S i m i l a r r e s u l t s w e r e also o b t a i n e d w h e n t h e e n z y m e a c t i v i t y was e x p r e s s e d p e r g p r o t e i n ( r e s u l t s not given). E r y t h r o c y t e G S H - R a c t i v i t y c o e f f i c i e n t v a l u e s h a s b e e n p r o v e d to b e a useful tool in p r o v i d i n g b i o c h e m i c a l e v i d e n c e for t h e d e f i c i e n c y of r i b o f l a v i n {15,16}. F u r t h e r , t h e A C v a l u e for e r y t h r o c y t e G S H - R was s h o w n to be a b e t t e r i n d e x of r i b o f l a v i n d e f i c i e n c y s t a t e t h a n t h a t of liver e n z y m e (7). A p r e v i o u s s t u d y (21) r e p o r t e d 40% d e c r e a s e in e r y t h r o c y t e G S H - R a c t i v i t y of r i b o f l a v i n d e f i c i e n t r a t s c o m p a r e d to c o n t r o l s a f t e r 21 days on t h e vitamin deficient diet. In t h e s e r a t s t h e a c t i v i t y of t h e e n z y m e was also f o u n d to b e l o w e r e d in lenses. S i m i l a r l y , a b o u t 70% d e c r e a s e was o b s e r v e d in e r y t h r o c y t e e n z y m e a c t i v i t y of r a t s f e d a r i b o f l a v i n d e f i c i e n t d i e t for 35 days (5). An association between r i b o f l a v i n d e f i c i e n c y a n d lens o p a c i t y h a s b e e n r e p o r t e d (22-25). S i n c e lens m e t a b o l i s m a p p e a r s to be s i m i l a r to t h a t of e r y t h r o c y t e s (26), it is o f i n t e r e s t to s e e w h e t h e r t h e r e s p o n s e o f e x o g e n o u s F A D on G S H - R a c t i v i t y in lens is s i m i l a r to t h a t o b s e r v e d in e r y t h r o c y t e s . The response o b s e r v e d to t h e in v i t r o a d d i t i o n of F A D on G S H - R a c t i v i t y in lens is v e r y s i m i l a r t o t h a t o b s e r v e d in e r y t h r o c y t e s ( T a b l e 2). This f u r t h e r s u g g e s t s t h a t t h e t h e s y n t h e t i c r a t e of t h e a p o e n z y m e was not a f f e c t e d d u r i n g r i b o f l a v i n d e f i c i e n c y
1206
K.S. BHAT and P.V. RAO
b o t h in lens a n d e r y t h r o c y t e s . R e c e n t s t u d y on r a t s h a s d e m o n s t r a t e d d e c r e a s e d c a p a c i t y of t h e lens t o s y n t h e s i z e flavin n u c l e o t i d e s d u r i n g r i b o f l a v i n d e f i c i e n c y (27). W h e t h e r t h i s d e c r e a s e d c a p a c i t y of t h e lens to s y n t h e s i z e f l a v i n n u c l e o t i d e s d u r i n g r i b o f l a v i n d e f i c i e n c y is due to low r i b o f l a v i n c o n t e n t of t h e t i s s u e r e m a i n s to be investigated, While t h e p r e s e n t work w a s in p r o g r e s s Ono a n d H i r a n o (28) f r o m J a p a n r e p o r t e d F A D s t i m u l a t e d r e s p o n s e of t h e G S H - R in lens o f r a t s m a i n t a i n e d on r i b o f l a v i n d e f i c i e n t d i e t . It is s u g g e s t e d t h a t t h e l i m i t i n g f a c t o r for G S H - R a c t i v i t y d u r i n g r i b o f l a v i n d e f i c i e n c y is c o - f a c t o r s a t u r a t i o n c a p a c i t y of t h e t i s s u e (28). TABLE 2 G l u t a t h i o n e R e d u c t a s e A c t i v i t y in Lens a n d E r y t h r o c y t e Ad libitum fed c o n t r o l (10)
Pair-fed c o n t r o l (10)
from Rats Riboflavin d e f i c i e n t (10)
Lens G l u t a t h i o n e R e d u c t a s e ( p m o l e N A D P H o x i d i z e d -h p e r 100 m g p r o t e i n ) -
FAD
2.63 + 0.201
2.44 + 0.252
1.35 + 0 . 0 8 1 "
+
FAD
3.07 + 0.205
2.93 + 0.199
2.58 + 0:076
1,17 + 0.O35
1.23 + 0.049
1.97 + 0 . 1 1 8 #
Activation Coe f f i c i e n t Value
Erythrocyte
Glutathione Reductase
( p m o l e N A D P H o x i d i z e d -h p e r ml e r y t h r o c y t e ) -
FAD
34.2 + 2.39
31.9 + 2.49
19.4 + 1.55#
+
FAD
39.2 + 2.50
37.6 + 3.29
35.6 + 3.86
1.15 + 0.003
1.17 + 0.017
1.82 + 0 . 0 8 6 #
Activation Coe f f i c i e n t Value
D a t a r e p r e s e n t m e a n and s t a n d a r d e r r o r . F i g u r e s in p a r e n t h e s i s i n d i c a t e n u m b e r of a n i m a l s . * P < 0.005, s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l r a t s . # P<0.001,
significantly different from control rats.
The results of the present study taken together with those reported earlier (4,6,7,28) s u g g e s t t h a t t h e A C v a l u e in r e s p o n s e to e x o g e n o u s F A D for t h e lens a n d e r y t h r o c y t e G S H - R is a s e n s i t i v e b i o c h e m i c a l i n d e x for t h e a s s e s s m e n t o f r i b o f l a v i n s t a t u s in r a t s . E x p e r i m e n t s a r e in p r o g r e s s t o s e e t h e v a l i d i t y of t h i s t e s t in h u m a n s i t u a t i o n . ACKNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e Dr. B.S. N a r a s i n g a Rao, D i r e c t o r , N a t i o n a l I n s t i t u t e of N u t r i t i o n , H y d e r a b a d , for his k e e n i n t e r e s t in t h e s t u d y . Mrs. P. L a x m i Rajkumar provided technical assistance. P.V.R. is a r e c i p i e n t of s e n i o r f e l l o w s h i p f r o m C o u n c i l of S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h , India.
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Lowry OH, Rosebrough N J, Farr AL, Randall R J. Protein m e a s u r e m e n t with the Follin phenol reagent. J. Biol. Chem. 1951; 193:265-75.
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Sauberlich HE, Dowdy RP, Skala JH. Laboratory tests for the of nutritional status. CRC Crit. Rev. Lab. Sci. 1973; 4:215-340.
16.
Bamji MS. Laboratory tests for the assessment of vitamin nutritional status. In: Briggs, MH, ed. Vitamins in Human Biology and Medicine. Florida: CRC Press, Inc., 1981: 1-27.
assessment
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I?.
Clegg KM, Kodicek E, Mistry SP. A modified medium for Lactobacillus casei for the assay of B vitamins. Biochem. J. 1952; 50:326-331,
18,
Bhat KS, Belavady B, Riboflavin nutritional status of rats essential fat t y acid deficiency. J. Vitaminol, 1970; 16:149-53,
19,
Goldsmith GA. Riboflavin deficiency. York: Plenum Press, 1975: 221-44.
20.
Lane M, Smith FE, Alfrey Jr CP, Experimental dietary and antagonist-induced human ariboflavin deficiency. In: Rivilin, RS, ed. Riboflavin, New York: Plenum Press, 1975: 245-77.
21.
Srivastava SK, Beutler E. Galactose Biochem, Med, 1972; 6:372-79,
22.
Day PL, Darby WJ, Cosgrove KW. The arrest of the use of riboflavin. J, Nutr. (suppl) 1938; 15:83-90,
23.
Bhat KS, Gopalao C. Human c a t a r a c t and galactose metabolism. Nutr. Metab. 1974; 17:1-8.
24.
Prchal JT, Conrad ME, Skalka HW. Association of presenile c a t a r a c t s with heterozygosity for g a l a c t o s a e m i c states and with riboflavin deficiency, Lancet 1978; i: 12-13.
25,
Dunce GE. Nutrition and C a t a r a c t , In: Tobin, RB, Mehlman Advances in Modern Human Nutrition, Vol. 1. Illinois: Pathotox Inc., 1980: 1-31,
26,
Srivastava SK. Red Blood North Holland, Inc., 1980.
27.
Ono S, Hirano H, Hamajima S, Horiuchi S, E f f e c t of riboflavin deficiency on the synthesis of ester forms of riboflavin in rat lens. J. Nutr. Sci. Vitam. 1981; 27:599-604,
28.
Ono S, Hirano H. FAD-induced in vitro a c t i v i t y of glutathione in the lens of B 2 deficient rats. Curr. Eye Res. 1984; 3:663-65.
Cell
and
Accepted for publication July 6, 1987.
In:
Rivlin,
cataract
Lens
RS,
in
ed,
riboflavin
suffering
from
Riboflavin,
New
deficient
nutritional
Metabolism.
New
cataract
rats.
by
MA, eds. Publishers,
York:
Elsevier
r ed u ct ase
C H A N G E S IN LENS AND E R Y T H R O C Y T E G L U T A T H I O N E R E D U C T A S E IN R E S P O N S E TO E X O G E N O U S FLAVIN A D E N I N E D I N U C L E O T I D E A N D LIVER RIBOFLAVIN C O N T E N T OF RAT ON RIBOFLAVIN D E F I C I E N T DIET K. S e e t h a r a m Bhat 1, ph.D. and P. Vasanth Rao, M.Sc. N a t i o n a l I n s t i t u t e of N u t r i t i o n Indian Council of Medical R e s e a r c h J a m a i Osmania PO, H y d e r a b a d - 500 007 India
ABSTRACT The e f f e c t of e x o g e n o u s flavin adenine d i n u c l e o t i d e (FAD) and h e p a t i c level of r i b o f l a v i n on the a c t i v i t y of l e n t i c u l a r and e r y t h r o c y t e g l u t a t h i o n e r e d u c t a s e (GSH-R) was studied in r a t s fed a diet d e f i c i e n t in riboflavin. By 75 days of feeding, the G S H - R a c t i v i t y , under basal c o n d i t i o n (in the absence of e x o g e n o u s FAD) and the a c t i v a t i o n c o e f f i c i e n t (AC, a c t i v i t y of the e n z y m e in response to e x o g e n o u s F A D to t h a t w i t h o u t it) values w e r e s i g n i f i c a n t l y a l t e r e d in lens as well as e r y t h r o c y t e s of r a t s fed riboflavin d e f i c i e n t diet. Significant r e d u c t i o n in r i b o f l a v i n c o n t e n t of liver was also o b s e r v e d in the e x p e r i m e n t a l animals. T h e s e findings suggest t h a t the A C value for the l e n t i c u l a r G S H - R is as equally a r e l i a b l e b i o c h e m i c a l p a r a m e t e r for assessing r i b o f l a v i n n u t r i t u r e as t h a t of the e r y t h r o c y t e e n z y m e . Key Words:
Lens, e r y t h r o c y t e , liver, g l u t a t h i o n e r e d u c t a s e , riboflavin, a c t i v a t i o n c o e f f i c i e n t , rat. INTRODUCTION
D e t e r m i n a t i o n of flavin adenine d i n u c l e o t i d e (FAD) d e p e n d e n t g l u t a t h i o n e r e d u c t a s e (GSH-R, E.C. 1.6.4.2) in e r y t h r o c y t e s from human s u b j e c t s (1-4) and e x p e r i m e n t a l animals (5-7) has been shown to be a s e n s i t i v e index of r i b o f l a v i n nutriture. In the v i t a m i n d e f i c i e n t s t a t e , the e n z y m e is s t i m u l a t e d following in v i t r o addition of its c o - f a c t o r , FAD and also a f t e r v i t a m i n a d m i n i s t r a t i o n in vivo. In an e a r l i e r study on r a t s from our Institute, it was o b s e r v e d t h a t G S H - R in e r y t h r o c y t e was m o r e s e n s i t i v e to riboflavin d e f i c i e n c y than t h a t of liver e n z y m e , although the v i t a m i n c o n t e n t of the liver d e c r e a s e d at a f a s t e r r a t e than t h a t of e r y t h r o c y t e s (7). F u r t h e r , no response was o b s e r v e d in t h e h e p a t i c e n z y m e a c t i v i t y to in v i t r o addition of FAD. Glutathione reductase is p r e s e n t in lens and its a c t i v i t y d e c r e a s e s during aging and c a t a r a c t f o r m a t i o n (8-11). R e c e n t l y Ono and Hirano (12) have provided e v i d e n c e for t h e e x i s t e n c e of flavin m o n o n u c l e o t i d e (FMN) and F A D synthesising e n z y m e s in lens.
1To whom c o r r e s p o n d e n c e should be addressed.
1203
1204
K.S. BHAT and P.V. RAO
To i n v e s t i g a t e the relationship b e t w e e n l e n t i c u l a r and e r y t h r o c y t e G S H - R a c t i v i t y and liver riboflavin c o n t e n t , the a c t i v i t y of GSH-R in lens as well as e r y t h r o c y t e and riboflavin c o n t e n t of the liver w e r e d e t e r m i n e d . In addition, t h e e f f e c t of in v i t r o addition of F A D on the a c t i v i t y of GSH-R was also studied in lens and e r y t h r o c y t e from t h e s e rats, MATERIALS A N D METHODS Ten m a l e NIN-Wistar a riboflavin d e f i c i e n t diet riboflavin s u p p l e m e n t e d (8 rats was fed ad libitum, to the food c o n s u m p t i o n (Pair-fed). The rats w e r e tained at 22-25~
strain rats weighing 35-54 g w e r e fed ad libitum on (7). Two groups of rats, 10 in each group, r e c e i v i n g mg/kg) diet s e r v e d as controls. One group of c o n t r o l while the o t h e r group was fed with a q u a n t i t y equal of the riboflavin d e f i c i e n t group in the previous day c a g e d individually in s c r e e n - b o t t o m e d c a g e s and m a i n -
A t the end of 75 days of feeding, rats w e r e s a c r i f i c e d a f t e r o v e r n i g h t fasting under e t h e r anaesthesia. Blood was drawn from the orbital plexus into h e p a r i n i z e d tube kept cold. Blood was c e n t r i f u g e d at 2000 x g for 15 min in the cold. The plasma and the b u l l y c o a t w e r e r e m o v e d with p a s t e u r e p i p e t t e . The residual plasma and buffy c o a t w e r e r e m o v e d by resuspending the e r y t h r o c y t e s in 5 v o l u m e s of ice cold 0.9% NaC1 solution and c e n t r i f u g i n g at 2000 x g for 10 min. The e r y t h r o c y t e s w e r e washed t h r i c e in the s a m e medium. A l i q u o t e s of the packed e r y t h r o c y t e s ( h a e m a t o c r i t , 96%) w e r e dispensed into a m b e r c o l o u r e d s c r e w capped glass tubes, f r o z e n and s t o r e d at - 2 0 ~ till f u r t h e r analysis. The processing of e r y t h r o c y t e s was c a r r i e d out under dim light. Lens was c a r e f u l l y e x c i s e d through p o s t e r i o r approach. Each lens was h o m o g e n i z e d (10%) in 0.2 M T r i s - E D T A buffer, pH 8.2 in a P o t t e r - E l v e n j e m glass h o m o g e n i z e r and the h o m o g e n a t e was c e n t r i f u g e d at 20000 X g in a Sorvall RC 513 superspeed c e n t r i f u g e at 4 ~ for 30 rain. The liver was rapidly r e m o v e d and s t o r e d at - 2 0 ~ G l u t a t h i o n e r e d u c t a s e a c t i v i t y in both lens and e r y t h r o c y t e was assayed a c c o r d i n g to Bayoumi and Rosalki (13). The c o n t e n t s of the r e a c t i o n m i x t u r e are shown in Table 1. The r e a c t i o n was followed k i n e t i c a l l y for the first 5 min TABLE 1 C o n t e n t of the G l u t a t h i o n e R e d u c t a s e E n z y m e Assay M i x t u r e Lens
Erythrocyte
Potassiumphosphate b u f f e r
78
Enzyme preparation
80 pl (2000 g s u p e r n a t a n t )
EDTA
1.6 jJ m o l e
100 }H (1:15 h a e m o lyzate} 4 ~ mole
GSSG
2 p mole
5 p mole
FAD*
jJ mole (pH 7.4)
10 n mole Incubated at 3 7 ~
0.2 m mole (pH 7.4)
25 n mole for 25 min.
NADPH
0.16 y m o l e
0.4 F mole
Total volume
1.0 ml
2.45 ml
* When p r e s e n t
GSH-REDUCTASE AND RIBOFLAVIN DEFICIENCY
1205
( m a x i m u m l i n e a r r a t e ) by t h e d e c r e a s e in o p t i c a l d e n s i t y a t 340 nm in a G i l f o r d s p e c t r o p h o t o m e t e r (Model 250) e q u i p p e d with a r e c o r d e r (Model 6050). T h e d e c r e a s e in o p t i c a l d e n s i t y was l i n e a r with t i m e and e n z y m e c o n c e n t r a t i o n . Protein concent r a t i o n of t h e lens was d e t e r m i n e d by t h e m e t h o d of L o w r y e t al (14) w i t h c r y s t a lline b o v i n e s e r u m a l b u m i n as a p r o t e i n s t a n d a r d . A c t i v a t i o n c o e f f i c i e n t (AC) v a l u e for GSH-R, t h e s t i m u l a t o r y e f f e c t of e x o g e n o u s FAD, on t h e e n z y m e was c a l c u l a t e d by dividing t h e a c t i v i t y of t h e e n z y m e in t h e p r e s e n c e of a d d e d c o - f a c t o r by t h a t w i t h o u t it to p r o v i d e e v i d e n c e of r i b o f l a v i n d e f i c i e n c y (15,16). R i b o f l a v i n c o n t e n t of t h e l i v e r was a s s a y e d m i c r o b i o l o g i c a l l y using L. c a s e i as t h e t e s t o r g a n i s m (17). S i g n i f i c a n c e of d i f f e r e n c e s b e t w e e n groups was e v a l u a t e d by t h e s t u d e n t ' s ' t ' t e s t . RESULTS A N D DISCUSSION A t t h e e n d of 75 days of f e e d i n g w i t h t h e r i b o f l a v i n s u p p l e m e n t e d d i e t , t h e r a t s in ad l i b i t u m (274 + 3.9g) and p a i r - f e d (108 + 3.3g) groups w e r e s i g n i f i c a n t l y h e a v i e r (P/-. 0.001) t h a n t h e a n i m a l s fed r i b o f l a v i n d e f i c i e n t d i e t (58 + 3.3g). T h e m e a n food i n t a k e by t h e r a t s a t t h e end of t h e e x p e r i m e n t a l perTod was 14.8 + 0.75 g / d a y in ad l i b i t u m g r o u p as a g a i n s t 4.9 + 0.33 g / d a y o f t h e r i b o f l a v i n d e f i c i e n t group. S i m i l a r t r e n d was also r e p o r t e d from this i n s t i t u t e e a r l i e r (7). S i g n i f i c a n t c h a n g e s w e r e also s e e n in lens w e t w e i g h t ( P < 0 . 0 0 1 ) and t o t a l p r o t e i n c o n t e n t ( P ~ 0.025) of t h e lens b e t w e e n r i b o f l a v i n d e f i c i e n t (33.2 +_ o.39 m g and 17.1 + 0.37 r a g / l e n s r e s p e c t i v e l y ) and c o n t r o l s (37.1 + 0.37 m g and 19.4 + 0.56 m g / l e n s r e s p e c t i v e l y ) . H o w e v e r , t h e c o n t e n t of lens pr-oteins, e x p r e s s e d p e r g w e t w e i g h t was not s i g n i f i c a n t l y d i f f e r e n t in t h e e x p e r i m e n t a l (514 + 9.3) and c o n t r o l groups (501 + 16.1 and 523 + 15.0). Riboflavin deficiency h a s b e e n s h o w n to r e s u l t in d e c r e a ~ d r i b o f l a v i n c o ' t e n t and a c t i v i t i e s of f l a v i n n u c l e o t i d e d e p e n d e n t e n z y m e s in t i s s u e s (18-20). In t h e p r e s e n t study, s i g n i f i c a n t r e d u c t i o n in r i b o f l a v i n c o n t e n t of t h e l i v e r was o b s e r v e d in r i b o f l a v i n d e f i c i e n t r a t s {17.3 + 0.72 )ag/g liver) a g a i n s t b o t h ad l i b i t u m and p a i r fed c o n t r o l a n i m a l s (44.7 + 1.9-2 a n d 45.1 + 1.48 jag/g l i v e r r e s p e c t i v e l y ) w h i c h was s i m i l a r to t h a t r e p o r t e d by B a m j i a n d S h a r a d a (7). T h e a c t i v i t y of G S H - R b o t h in lens a n d e r y t h r o c y t e s , in t h e a b s e n c e of e x o g e n o u s F A D was s i g n i f i c a n t l y { P < 0 . 0 0 1 ) l o w e r e d in r a t s on r i b o f l a v i n d e f i c i e n t r a t i o n (50%) c o m p a r e d to r i b o f l a v i n s u p p l e m e n t e d r a t s (Table 2). On t h e o t h e r hand, G S H - R a c t i v i t y in t h e p r e s e n c e of t h e c o f a c t o r did not s i g n i f i c a n t l y d i f f e r in t h e v i t a m i n d e f i c i e n t and s u p p l e m e n t e d r a t s . A d d i t i o n of F A D c a u s e d a s i g n i f i c a n t i n c r e a s e ( P < 0.001) in G S H - R a c t i v i t y in r i b o f l a v i n d e f i c i e n t t i s s u e s c o m p a r e d to c o n t r o l s , w i t h t h e r e s u l t , t h e A C v a l u e increased significantly (P<0.001) in lens as well as e r y t h r o c y t e s in r i b o f l a v i n deficient rats. S i m i l a r r e s u l t s w e r e also o b t a i n e d w h e n t h e e n z y m e a c t i v i t y was e x p r e s s e d p e r g p r o t e i n ( r e s u l t s not given). E r y t h r o c y t e G S H - R a c t i v i t y c o e f f i c i e n t v a l u e s h a s b e e n p r o v e d to b e a useful tool in p r o v i d i n g b i o c h e m i c a l e v i d e n c e for t h e d e f i c i e n c y of r i b o f l a v i n {15,16}. F u r t h e r , t h e A C v a l u e for e r y t h r o c y t e G S H - R was s h o w n to be a b e t t e r i n d e x of r i b o f l a v i n d e f i c i e n c y s t a t e t h a n t h a t of liver e n z y m e (7). A p r e v i o u s s t u d y (21) r e p o r t e d 40% d e c r e a s e in e r y t h r o c y t e G S H - R a c t i v i t y of r i b o f l a v i n d e f i c i e n t r a t s c o m p a r e d to c o n t r o l s a f t e r 21 days on t h e vitamin deficient diet. In t h e s e r a t s t h e a c t i v i t y of t h e e n z y m e was also f o u n d to b e l o w e r e d in lenses. S i m i l a r l y , a b o u t 70% d e c r e a s e was o b s e r v e d in e r y t h r o c y t e e n z y m e a c t i v i t y of r a t s f e d a r i b o f l a v i n d e f i c i e n t d i e t for 35 days (5). An association between r i b o f l a v i n d e f i c i e n c y a n d lens o p a c i t y h a s b e e n r e p o r t e d (22-25). S i n c e lens m e t a b o l i s m a p p e a r s to be s i m i l a r to t h a t of e r y t h r o c y t e s (26), it is o f i n t e r e s t to s e e w h e t h e r t h e r e s p o n s e o f e x o g e n o u s F A D on G S H - R a c t i v i t y in lens is s i m i l a r to t h a t o b s e r v e d in e r y t h r o c y t e s . The response o b s e r v e d to t h e in v i t r o a d d i t i o n of F A D on G S H - R a c t i v i t y in lens is v e r y s i m i l a r t o t h a t o b s e r v e d in e r y t h r o c y t e s ( T a b l e 2). This f u r t h e r s u g g e s t s t h a t t h e t h e s y n t h e t i c r a t e of t h e a p o e n z y m e was not a f f e c t e d d u r i n g r i b o f l a v i n d e f i c i e n c y
1206
K.S. BHAT and P.V. RAO
b o t h in lens a n d e r y t h r o c y t e s . R e c e n t s t u d y on r a t s h a s d e m o n s t r a t e d d e c r e a s e d c a p a c i t y of t h e lens t o s y n t h e s i z e flavin n u c l e o t i d e s d u r i n g r i b o f l a v i n d e f i c i e n c y (27). W h e t h e r t h i s d e c r e a s e d c a p a c i t y of t h e lens to s y n t h e s i z e f l a v i n n u c l e o t i d e s d u r i n g r i b o f l a v i n d e f i c i e n c y is due to low r i b o f l a v i n c o n t e n t of t h e t i s s u e r e m a i n s to be investigated, While t h e p r e s e n t work w a s in p r o g r e s s Ono a n d H i r a n o (28) f r o m J a p a n r e p o r t e d F A D s t i m u l a t e d r e s p o n s e of t h e G S H - R in lens o f r a t s m a i n t a i n e d on r i b o f l a v i n d e f i c i e n t d i e t . It is s u g g e s t e d t h a t t h e l i m i t i n g f a c t o r for G S H - R a c t i v i t y d u r i n g r i b o f l a v i n d e f i c i e n c y is c o - f a c t o r s a t u r a t i o n c a p a c i t y of t h e t i s s u e (28). TABLE 2 G l u t a t h i o n e R e d u c t a s e A c t i v i t y in Lens a n d E r y t h r o c y t e Ad libitum fed c o n t r o l (10)
Pair-fed c o n t r o l (10)
from Rats Riboflavin d e f i c i e n t (10)
Lens G l u t a t h i o n e R e d u c t a s e ( p m o l e N A D P H o x i d i z e d -h p e r 100 m g p r o t e i n ) -
FAD
2.63 + 0.201
2.44 + 0.252
1.35 + 0 . 0 8 1 "
+
FAD
3.07 + 0.205
2.93 + 0.199
2.58 + 0:076
1,17 + 0.O35
1.23 + 0.049
1.97 + 0 . 1 1 8 #
Activation Coe f f i c i e n t Value
Erythrocyte
Glutathione Reductase
( p m o l e N A D P H o x i d i z e d -h p e r ml e r y t h r o c y t e ) -
FAD
34.2 + 2.39
31.9 + 2.49
19.4 + 1.55#
+
FAD
39.2 + 2.50
37.6 + 3.29
35.6 + 3.86
1.15 + 0.003
1.17 + 0.017
1.82 + 0 . 0 8 6 #
Activation Coe f f i c i e n t Value
D a t a r e p r e s e n t m e a n and s t a n d a r d e r r o r . F i g u r e s in p a r e n t h e s i s i n d i c a t e n u m b e r of a n i m a l s . * P < 0.005, s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l r a t s . # P<0.001,
significantly different from control rats.
The results of the present study taken together with those reported earlier (4,6,7,28) s u g g e s t t h a t t h e A C v a l u e in r e s p o n s e to e x o g e n o u s F A D for t h e lens a n d e r y t h r o c y t e G S H - R is a s e n s i t i v e b i o c h e m i c a l i n d e x for t h e a s s e s s m e n t o f r i b o f l a v i n s t a t u s in r a t s . E x p e r i m e n t s a r e in p r o g r e s s t o s e e t h e v a l i d i t y of t h i s t e s t in h u m a n s i t u a t i o n . ACKNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e Dr. B.S. N a r a s i n g a Rao, D i r e c t o r , N a t i o n a l I n s t i t u t e of N u t r i t i o n , H y d e r a b a d , for his k e e n i n t e r e s t in t h e s t u d y . Mrs. P. L a x m i Rajkumar provided technical assistance. P.V.R. is a r e c i p i e n t of s e n i o r f e l l o w s h i p f r o m C o u n c i l of S c i e n t i f i c a n d I n d u s t r i a l R e s e a r c h , India.
GSH-REDUCTASE AND RIBOFLAVIN DEFICIENCY
1207
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Cell
and
Accepted for publication July 6, 1987.
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