Reintegro de divisas El reintegro de divisas se realiza a través de los intermediarios cambiarios presentando la declaración de exportación y la

E D- ü 800 H Û . o 8 600 tO _ c X co 400 H 200 H 3 0 0 100 2 0 0 0 Elution Volume (ml)

Fig. 2.11 Elution pattern of 3H alpha tocopherol

in the presence of rat haemoglobin

Rat l i v e r c y t o s o l b i n d i n g s t u d i e s 2 . 1 2 D i s c u s s i o n

The main o b j e c t i v e s o f t h i s s t u d y were t o I s o l a t e t h e a l p h a t o c o p h e r o l b i n d i n g s i t e s p r e s e n t I n r a t l i v e r c y t o s o l and use them t o e s t a b l i s h a CPB assay f o r a l p h a t o c o p h e r o l . A l t h o u g h an agent w i t h a l pha t o c o p h e r o l b i n d i n g a c t i v i t y was I s o l a t e d , I t was p r e s e n t I n l ow c o n c e n t r a t i o n and a t t e m p t s t o I n c r e a s e t h e b i n d i n g were n o t s u c c e s s f u l .

The l i v e r c y t o s o l p r e p a r a t i o n c l e a r l y showed al pha t o c o p h e r o l b i n d i n g a c t i v i t y I n t h e f r a c t i o n s e l u t i n g a t 1 4 0 - 1 60ml , (peak I I I ) , b u t t h e b i n d i n g was l o w e r t h a n t h a t r e p o r t e d by C a t i g n a n i ( 1 9 7 7 ) . T h i s d i s c r e p a n c y c o u l d be due t o t h e use o f d i f f e r e n t r a t s o r t o t h e s h o r t e r d u r a t i o n o f t h e i r v i t a m i n E d e p r i v a t i o n (2 months I n t h e p r e s e n t s t u d y , 3 months I n C a t i g n a n i ' s s t u d y ) . In an a t t e m p t t o I n c r e a s e t h e p e r c e n t a g e b i n d i n g o f al p h a t o c o p h e r o l , two approaches were empl oyed. In t h e f i r s t t h e c y t o s o l p r e p a r a t i o n was d i a l y s e d b e f o r e I s o l a t i n g t h e b i n d i n g s i t e s and I n c u b a t i n g w i t h a l pha t o c o p h e r o l . Si nce a l pha t o c o p h e r o l b i n d i n g was r e p o r t e d t o be r e v e r s i b l e ( C a t i g n a n i , 1977) I t was t h o u g h t t h a t I f some endogenous v i t a m i n E was s t i l l o c c u p y i n g t h e b i n d i n g s i t e s , d i a l y s i s woul d l i b e r a t e t h e a l r e a d y bound a l pha t o c o p h e r o l f r om t h e s e s i t e s . However, upon I n c u b a t i o n w i t h al pha

Rat l i v e r c y t o s o l b i n d i n g s t u d i e s

a l p h a t o c o p h e r o l t o t h e agent p r e s e n t i n r a t l i v e r c y t o s o l was e i t h e r i r r e v e r s i b l e o r t h a t t h e b i n d i n g was so a v i d t h a t adequat e removal o f t h e bound endogenous al pha t o c o p h e r o l by s i mp l e d i a l y s i s was n o t p o s s i b l e .

The second a t t e m p t used i n t h e e f f o r t t o i n c r e a s e t h e b i n d i n g o f a l pha t o c o p h e r o l t o t h e b i n d i n g s i t e s i n v o l v e d a d j u s t i n g t h e pH o f t h e c y t o s o l . Al pha t o c o p h e r o l b i n d i n g has been shown t o be pH d e p e n de n t . In one r e p o r t i t was decr eased t o 50% o f t h e o r i g i n a l l e v e l when t h e pH o f t h e r e a c t i o n m i x t u r e was a d j u s t e d t o pH 6 . 5 ( C a t i g n a n i , 1 9 7 7 ) . In a n o t h e r , t h e l o w e r i n g o f t h e pH o f t h e l i v e r s u p e r n a t a n t f rom 7. 4 t o 5.1 removed some o f t h e s o l u b l e p r o t e i n and most o f t h e l i p i d s o l u b l e phosphat e (Murphy and M a v i s , 19 8 1 ) ; when t h e pH was r e s t o r e d t o 7 . 4 and i n c u b a t e d w i t h ^H al pha t o c o p h e r o l , t h e TTA i n c r e a s e d 1. 3 f o l d . In t h e p r e s e n t s t u d y an a t t e m p t was made t o r e l e a s e t h e bound endogenous a l pha t o c o p h e r o l m o l e c u l e s f r om t h e i r b i n d i n g s i t e s by d e c r e a s i n g t h e pH t o 6 . 5 p r i o r t o d i a l y s i s b u t when such a p r e p a r a t i o n o f b i n d i n g s i t e s was p r o c e s s e d , no s i g n i f i c a n t a l t e r a t i o n i n t h e b i n d i n g was o b s e r v e d . Thus TBA c o u l d n o t be i n c r e a s e d by t h e s e two met hods. I t i s p o s s i b l e t h a t because t h e amount o f b i n d i n g s i t e s p r e s e n t t h e p r e p a r a t i o n was so l ow t h a t any i n c r e m e n t i n t h e b i n d i n g o f ^H a l pha t o c o p h e r o l a f t e r d i a l y s i s o r pH a d j u s t m e n t , may have been so i n s i g n i f i c a n t t h a t i t c o u l d n o t be d e mo n s t r a t e d w i t h t h e t e c h n i q u e s used h e r e . Hence i t

Rat l i v e r c y t o s o l b i n d i n g s t u d i e s

was c o n c l u d e d t h a t , a l t h o u g h an a g e n t c a p a b l e o f b i n d i n g al p h a t o c o p h e r o l was i s o l a t e d i n t h e r a t l i v e r c y t o s o l p r e p a r a t i o n s , i t was n o t p r e s e n t i n s u f f i c i e n t q u a n t i t y f o r use i n t h e devel opment o f a CPB as s a y .

Si nce t h e p o o l e d f r a c t i o n s a t 1 4 0 - 1 60ml (peak I I I p o s s e s s i n g TBA) were r e d d i s h i n c o l o u r , an a t t e m p t was made t o i d e n t i f y t h e pi gment u s i n g s p e c t r o p h o t o m e t r y . Thi s r e v e a l e d absor bance peaks s i m i l a r t o t h e a b s o r p t i o n s pec t r um g i v e n by a haemogl obi n p r e p a r a t i o n , i n c l u d i n g t h e pr esence o f t h e c h a r a c t e r i s t i c S o r e t band between 400- 420nm, (Wei nkove and McDowel l , 1 9 8 8 ) . A b s o r p t i o n bands a t 540nm and 578nm a l s o i n d i c a t e d t h e pr esence o f o x y haemogl obi n ( M i a l e , 1982) i n bot h p r e p a r a t i o n s . Th i s suggest ed t h a t an agent p r e s e n t i n r e d b l o o d c e l l s may have been p a r t l y r e s p o n s i b l e f o r t h e b i n d i n g o f a l p h a t o c o p h e r o l seen i n t h e l i v e r homogenat es. T h i s o b s e r v a t i o n was f u r t h e r s u b s t a n t i a t e d when a m i x t u r e o f t h e commer ci al p r e p a r a t i o n o f r a t haemogl obi n and a l p h a t o c o p h e r o l produced a peak o f r a d i o a c t i v i t y i n t h e 1 4 0 - 1 60ml c h r o m a t o g r a p h i c f r a c t i o n s ( F i g . 2 . 1 1 ) . I t appeared t h e r e f o r e t h a t a component o f r e d b l o o d c e l l s f r om whi ch t h e haemogl obi n was p r e p a r e d was c a p a b l e o f b i n d i n g al pha t o c o p h e r o l . The pr esence o f a l pha t o c o p h e r o l b i n d i n g s i t e s

Rat l i v e r c y t o s o l b i n d i n g s t u d i e s

t o possess TBA i n some r e p o r t s . For exampl e, Mowri e t a l . , ( 1981) f ound TBA t o be p r e s e n t i n h e a r t , s p l e e n and l u n g as w e l l as i n l i v e r c y t o s o l a l t h o u g h C a t i g n a n i ( 1977) obser ved i t o n l y i n t h e l i v e r . C o n t a m i n a t i o n o f t h e v a r i o u s t i s s u e c y t o s o l s w i t h components o f r e d b l o o d c e l l s may e x p l a i n t h e s e f i n d i n g s .

The e l u t i o n p a t t e r n o f n eat a l p h a t o c o p h e r o l on gel f i l t r a t i o n ( F i g . 2 . 9 B) c o u l d be a n o t h e r p o s s i b l e e x p l a n a t i o n f o r t h e f i n d i n g by Murphy and Mavi s ( 1981) o f t h e pr esence o f TBA i n s e v e r a l d i f f e r e n t t i s s u e s . Al pha t o c o p h e r o l was shown t o e l u t e i n two peaks, one a t t h e v o i d