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2 .3 .4 &NIIOXIDANIS IN.. HDMAN, ELQgP. PLASMA
2 .3 .4 .1 BIPASSAI STUDIES. - TOTAL ANTIQXIDANT ACTIVITY ( k O k )
T o ta l AGA was m easured u sin g an a d a p ta tio n o f th e m ethod o f S to ck s £jb. jaL (1 9 7 4 ).
2 . 3 .4 . 1 .1 AHIMAL, SOURCE
F re sh o x - b ra in was o b ta in e d from S t.A ndrew s s la u g h te r house from f r e s h l y s la u g h te re d an im als and was tr a n s p o r te d to th e la b o r a to r y packed in i c e .
2 .3 .4 .1 .2 PREPARATION CF STANDARD HOMQGENATE
The ox b r a in was s tr ip p e d o f i t s m eninges and a l l b lo o d c l o t s w ere washed o f f in ic e - c o ld 0 .1 5M sodium c h lo r i d e ; The t i s s u e was th e n chopped i n t o sm a ll p ie c e s and hom ogenized f o r 2 min u sin g a P o t te r E lvehjem ty p e t i s s u e g r in d e r , in f o u r tim es i t s w e ig h t o f ic e - c o ld PBS (40mM p o ta ssiu m d ih y d ro g e n p h o s p h a te /d ip o ta s s iu m hydrogen p h o sp h a te , pH 7 .4 i n 0 .1 42M sodium c h l o r i d e ) . The hom ogenate was c e n tr if u g e d f o r 15min a t 3500xg. The s u p e r n a ta n t f l u i d was t r a n s f e r r e d to 20ml d is p o s a b le c o n ta in e r s . T hese w ere s to r e d a t -20°C f o r up to 8 w eeks.
2 . 3 . 4 . 1 . 3 AUTOXIDATION
A sam ple of th e sto c k b r a in hom ogenate was thaw ed a t room te m p e ra tu re and im m ed ia te ly d i lu t e d w ith th r e e tim e s i t s volume o f PBS. 5.0m l p o r tio n s o f th e d i l u t e hom ogenate w ere t r a n s f e r r e d i n t o a s e r i e s o f c o n ta in e r s . Sam ples ( 5pUl) o f plasm a o r PBS ( c o n tr o l) w ere ad d ed . A liq u o ts f o r z e ro -tim e MDA e s tim a tio n s
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w ere removed im m e d ia te ly . The c o n ta in e r s w ere th e n t r a n s f e r r e d to a 37^C w a te r b a th and in c u b a tio n was c o n tin u e d f o r e x a c tly
1h o u r•
2 . 3 . 4 . 1 . 4 MDA-ESTIMATION
MDA c o n c e n tr a tio n in th e z e ro -tim e and one h our a l i q u o t s w ere m easured by th e t h i o b a r b i t u r i c a c id r e a c ti o n (S in n h u b e r and Yu, 1958; S to c k s and Dormandy, 1971; S to ck s e t a l . ,1 9 7 4 ). 2.0m l TCA ( 2 8 0 g /l) was added to 4.0m l o f hom ogenate and th e p r e c i p i t a t e d p r o te in rem oved by c e n tr if u g a ti o n . Then 4.0m l - s u p e rn a ta n t was h e a te d w ith 1,0m l o f 1 0 g /l (w /v) t h i o b a r b i t u r i c
o
a c id f o r 15min a t 100 C. A bsorbance was m easured a t 532nra.
R e s u lts w ere c a lc u la te d as nmol MDA p e r ml p lasm a.
2 .3 .4 .1 .5 CALCULATION OF ANTIOXIDANT ACTIVITY
i The a n tio x id a n t a c t i v i t y o f plasm a was e x p re s s e d i n term s I o f p e rc e n ta g e i n h i b i t i o n o f sp o n ta n e o u s a u to x id a tio n as m easured i in th e c o n tr o l hom ogenate. The c a lc u la t io n was based on th e
i
‘ fo llo w in g e q u a tio n :
nmol o f MDA/ml (T ^)-nm ol o f MDA/ml (Tq)
nmol o f MDA/ml (C ^)-nm ol o f MDA/ml (0 ^) Cq = C o n tro l a t 0 tim e
0^ = C o n tro l a t 1 hour Tq = T e s t a t 0 tim e T^ = T e st a t 1 h o u r
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2 .3 .4 .2 VITAMIN E ES.JIMAIIQM
2 .3 .4 .2 .1 STANDARD ALPHA-TQCQPHERQL
A s to c k s o lu t i o n o f Im g/m l was d is s o lv e d in m eth an o l and s to r e d i n dark b o t t l e s a t -2 0 °C . The w orking s ta n d a rd was p re p a re d by d i l u t i n g s to c k s o lu t i o n (1 :5 0 v /v ) in m eth an o l ( f i n a l c o n c e n tr a tio n 2 0 m g /l), s to r e d i n d ark b o t t l e s a t -20^C and n e v e r exposed to n a tu r a l i ll u m i n a ti o n . W orking s ta n d a rd was p re p a re d f r e s h e v e ry 2 w eeks.
2 .3 .4 .2 .2 INTERNAL STANDARD (TOCOL)
T ocol was a g i f t o f Roche P h a rm a c e u tic a ls (Welwyn G arden C ity , H e r tf o r d s h ir e ALT SAY, E ngland) and was o b ta in e d as a s to c k s o lu t i o n i n m ethanol (a p p ro x im a te ly 2 m g /l). The s o lu t i o n was s to r e d i n a d ark c o n ta in e r a t. -20°C . The w orking i n t e r n a l s ta n d a r d was p re p a re d by d i l u t i n g s to c k i n t e r n a l s ta n d a r d (1 :5 0 v /v ) i n m eth a n o l. The w orking i n t e r n a l s ta n d a rd was s to r e d in daric b o t t l e s a t -20^C and u n d er th e s e c o n d itio n s , i t was s t a b l e i n d e f i n i t e l y . The c a l i b r a t i o n s o lu t i o n (1 :1 0 v /v ) was p re p a rin g by d i l u t i n g th e w orking i n t e r n a l s ta n d a rd (1 0 0 :9 0 0 v /v ) in m e th a n o l. The c a l i b r a t i o n s o lu t i o n was p re p a re d f r e s h f o r each e s tim a tio n .
2 .3 .4 .2 .3 UALIBBATIDN. STANDARD.
E qual volum es o f a lp h a - to c o p h e ro l (2 0 m g /l) and to c o l (1 :1 0 v /v ) s to r e d a t -20°C i n dark c o n ta in e r . They w ere p re p a re d f r e s h e v e ry c o u p le o f d a y s.
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2 .3 .4 .2 .4 EXTRACTION PROCEDURE
D u p lic a te 50(^1 a li q u o ts o f plasm a w ere p ip e tte d i n t o p re-w ash ed (m eth an o l th e n w ith hexane) g l a s s e x tr a c t i o n tu b e s . To each tu b e 2 ^ 1 w orking to c o l s ta n d a rd (1 :5 0 v / v ) , was added w ith a a m icro s y rin g e and mixed th o ro u g h ly w ith a v o r te x m ix e r. W hile th e tu b e c o n te n ts w ere mixed on a v o rte x m ix e r, 5 0 ^ 1 e th a n o l w ere added slo w ly to d e p r o te in iz e th e sam p le. Then 2.0m l hexane (HPLC g ra d e ) w ere added to e x tr a c t th e sam ple fo llo w e d by m ixing w ith a v o r te x m ix er f o r 1 m in u te c o n tin u o u s ly . The tu b e c o n te n ts w ere c e n tr if u g e d f o r 5 m in u tes a t 1 2 ,0 0 0 x g . A f te r c e n tr if u g a ti o n , th e s u p e r n a ta n t was tr a n s f e r r e d to a c o n ic a l 10x100mm g l a s s tu b e and th e aqueous la y e r r e - e x tr a c t e d w ith 2.0m l hexane f o r 1 m in u te as b e f o r e . The combined o rg a n ic e x t r a c t s w ere e v a p o ra te d a t 40°C u n d er a stre a m o f n itr o g e n on a D ri-B lo c k . The r e s id u e was r e - d is s o lv e d in 500ul m ethanol and c e n tr if u g e d f o r 5 m in u tes a t 12,0 0 0 x g . Then th e m eth an o l e x t r a c t was s to r e d in a d a rk c o n ta in e r a t -20°C u n t i l a n a ly s is by HPLC.
2 .3 .4 .2 .5 HPLC CONDITIONS AND ANALYSIS 2 .3 .4 .2 .5 .1 INSTRUMENTATION
H P L C - .R Q D B L
A GILSON l iq u i d chrom atograph equipped w ith a Model 702 G ra d ie n t M anager, Model 802 M onom etric M odule, Model 303 l i q u i d d e liv e r y m odule, in c o n ju n c tio n w ith a Model HM/HPLC Holochrom e U.V-VIS d e te c to r w ere used f o r t h i s s tu d y .
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g m a m o G B A P H i
colum nS t a i n l e s s s t e e l colum n packed w ith r e v e rs e d -p h a s e ZORBAX ODS (C^g) ( p a r t i c l e s iz e 5~6um) 4.6mm i . d x 2.5cm
s u p p lie d by DuPont in s tr u m e n ts , W e llin g to n , D elaw are.
MOBILE PHASE. FLOW RATE AND PRESSURE
E lu tio n was perform ed w ith m ethanol a t a flo w r a t e o f 2ml p e r m in u te ( p r e s s u r e = 2000 P SI),
DETECTOR WAVELENGTH
The column e f f l u e n t was m onitored a t 292nm a t a s e n s i t i v i t y s e t t i n g o f 0 .0 2 .
GUARD COLUMN
A D u-Pont s t a i n l e s s s t e e l column
(4.6mm i . d x 5cm) packed w ith ODS was u se d .
BE.ÇPBDER
Shim adzu D ata P ro c e s s o r Chrom atopac C-RIB was em ployed (Shim adzu C o o p e ra tio n ,
A n a ly tic a l In s tru m e n ts P la n ts , K yoto, J a p a n ) .