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An improved method for the extraction of prostaglandins
AN IMPROVEDMETHOD FOR THE EXTRACTION OF PP~STAGLANDINS
G. A. Higgs and J . R. Vane, Department of Pharmacology, I n s t i t u t e of B a s i c Medical S c i e n c e s , Royal C o l l e g e of Surgeons of England, 35/43, L i n c o l n ' s Inn F i e l d s , WC2A 3PN. ABSTRACT
P r e s e n t methods of e x t r a c t i n g p r o s t a g l a n d i n s (laGs) g i v e poor r e c o v e r i e s from s y n o v i a l f l u i d , ~ j ~ r o h a b l y b e c a u s e t h e PGs bind ~Y0 p¥0~ei~ and a r e l o s t in the p r e c i p i t a t i o n s t a g e of e x t r a c t i o n . A d d l t i o n ~ Of the n o n - p o l a r d e t e r g e n t sodium l a u r y l s u l p h a t e p r i o r to e x t r a c t i o n i m ~ o v e s r e c o v e r y of PGs. I t i s s u g g e s t e d t h a t sodium l a u r y l s u l p h a t e competes w i t h PGs f o r the b i n d i n g s i t e s .
Acknowledgements: We wish t o thank Dr. D. H a r t and Dr. J . W o j t e u l e v s k i of t h e W e s t m i n s t e r H o s p i t a l , London, W.C.1. f o r p r o v i d i n g s y n o v i a b f l u i d specimens, and the UpJohn Co., Kalamazoo f o r s u p p l y i n g p r o s t a glandins. The work was supported by a g r a n t from Merck, Sharp and Dohme R e s e a r c h L a b o r a t o r i e s , Rahway, New J e r s e y , U.S.A.
Accepted September5, 1973
PROSTAGLANDINS NOVEMBER
1973
V O L . 4 NO. 5
695
PROSTAGLANDINS
P r e s e n t methods f o r e x t r a c t i n g p r o s t a g l a n d i n s (PGs) from tissue homogenates, seminal fluid or acidified phosphate buffer, into 70-90~ e t h a n o l ( 1 ) , a c e t o n e (2) or e t h y l a c e t a t e (3) g i v e low and i n c o n s i s t e n t r e c o v e r i e s from b l o o d p l a s m a . PGs b i n d w e a k l y t o a l b u m i n ( ~ , 5 ) and a r e p r o b a b l y l o s t in t h e p r o t e i n p r e c i p i t a t i o n s t a g e of the extraction. If precipitation of p r o t e i n i s a v o i d e d by u s i n g ~0-50~ e t h a n o l , t h e PGs can be e x t r a c t e d q u a n t i t a t i v e l y into chloroform ( 6 ) . T h i s method g i v e s h i g h e r r e c o v e r i e s of PGE1, Ez, F l a , F 2 a A 1 and A2 from human b l o o d . We wanted t o m e a s u r e t h e PG a c t i v i t y in s y n o v i a l f l u i d (human) from t h e j o i n t s of p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s . A heavy precipitate formed when t h e f l u i d s were a c i d i f i e d w i t h iN hydrochloric acid prior to extraction with ethyl acetate. Synovial f l u i d has a s i m i l a r t o t a l p r o t e i n c o n c e n t r a t i o n t o t h a t of b l o o d plasma and i n a d d i t i o n c o n t a i n s h i g h c o n c e n t r a t i o n s of h y a l u r o n i e a c i d (7) which makes i t v i s c o u s . Acidification precipitates b o t h of t h e s e components. Acidification f o l l o w e d by e t h y l a c e t a t e e x t r a c t i o n of s y n o v i a l f l u i d c o n t a i n i n g added PGEz ( 2 5 - 1 0 0 n g / m l ) g a v e r e c o v e r i e s of o n l y 12-20~ (mean = 17~, 10 e x p e r i m e n t s ) when a s s a y e d on t h e r a t fundus (8). The r e c o v e r y was i n c r e a s e d t o 2~-59~ (mean = 39~, l 0 e x p e r i m e n t s ) by u s i n g t h e method which g i v e s good r e c o v e r y from b l o o d p l a s m a , i . e . a d d i t i o n of ~0-50~ e t h a n o l , a c i d i f y i n g w i t h f o r m i c a c i d and e x t r a c t i n g i n t o c h l o r o f o r m ( 6 ) . Albumin has a g r e a t e r a f f i n i t y f o r l e s s p o l a r u n s a t u r a t e d f a t t y a c i d s such as o l e a t e t h a n f o r PGs ( ~ , 9 ) and Dr. F. Dea s u g g e s t e d t o us t h a t a n o n - p o l a r h y d r o p h o b i c compound such a s t h e d e t e r g e n t sodium l a u r y l s u l p h a t e would compete w i t h PGs f o r p r o t e i n b i n d i n g
sites. The e t h a n o l / c h l o r o f o r m e x t r a c t i o n p r o c e d u r e (6) was u s e d t o e x t r a c t PGs from samples of s y n o v i a l f l u i d . To e s t i m a t e t h e r e c o v e r y of added PGs, each sample was d i v i d e d i n t o two and PGE2, E. o r F2~ ( 2 5 - 1 0 0 n g / m l ) was added t o one h a l f ; t h e endogenous 1 c o n c e n t r a t i o n was measured in t h e o t h e r . For joint fluid (values c o r r e c t e d f o r r e c o v e r y ) t h i s v a r i e d from 0-62 n g / m l (mean = 8 . 7 ,
28 fluids). T a b l e 1 shows t h e e f f e c t s of d i f f e r e n t c o n c e n t r a t i o n s sodium l a u r y l s u l p h a t e (Sigma London, C h e m i c a l Co. L t d . ) on t h e e f f i c i e n c y of e x t r a c t i o n of t h e added PGs.
696
NOVEMBER 1973
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VOL. 4 NO. 5
PROSTAGLANDINS
Table I Recovery of added PGs from synovial fluid with different concentrations of sodium lauryl sulphate.
C o n c e n t r a t i o n of sodium l a u r y l sulphate (mg/ml)
P e r c e n t a g e r e c o v e r y of PGs Range (number of e x p e r i m e n t s ) Mean ± s.e.m.
2~-59% (10 e x p e r i m e n t s ) 39~ ± 2.90 1.0
39-100~ ( l l e x p e r i m e n t s ) 5 ~ ± 5.22
5.0
~ - 1 0 0 ~ (13 e x p e r i m e n t s ) 76% ~ ~.15
I0.0
31-~9% (4 experiments)
,u
,
39?; PG a c t i v i t y was assayed (10) on the r a t fundus, c h i c k rectum and r a t c o l o n , s u p e r f u s e d a t 5 ml/min with K r e b s ' b i c a r b o n a t e s o l u t i o n a t 37°C bubbled w i t h 5~ carbon d i o x i d e in oxygen and c o n t a i n i n g a m i x t u r e of a n t a g o n i s t s as p r e v i o u s l y d e s c r i b e d 411). A d d i t i o n of sodium t a u r y l s u l p h a t e improved t h e r e c o v e r y of added PGs. The o p t i m a l c o n c e n t r a t i o n of d e t e r g e n t was 5 mg/ml and h i g h e r c o n c e n t r a t i o n s d e c r e a s e d r e c o v e r y . A d d i t i o n of sodium l a u r y l s u l p h a t e to t h e samples had no e f f e c t on t h e t h r e e a s s a y t i s s u e s . T r i t i a t e d PGE1 (5-6 3H-I~E1), R a d i o c h e m i c a l s , Amersbam) was added to s y n o v i a l f l u i d ~nd the e x t r a 6 t o b t a i n e d w i t h the improved method ( u s i n g 5 mg/ml sodium l a u r y l s u l p h a t e ) was s u b j e c t e d to t h i n l a y e r chromatography. Eastman s i l i c a g e l chromatogram s h e e t s were used with t h e upper phase of e t h y l a c e t a t e : w a t e r : i s o - o c t a n e : a c e t i c a c i d , 411 : 10 : 5 : 2) 412) as a s o l v e n t system. 95¢ of the l a b e l corresponded t o the a u t h e n t i c PGE s p o t ; the r e m a i n i n g a c t i v i t y appeared in a peak which corresponded to PGAI and PGB1. D e h y d r a t i o n of PGEs to form PGAs o c c u r s a f t e r a c i d i f i c a t i o n but b e f o r e adding o r g a n i c s o l v e n t (13) and may a l s o r e s u l t from the p r e s e n c e of a c i d in the organic s o l v e n t during e x t r a c t i o n . The c o n v e r s i o n can be minimised by e x t r a c t i n g r a p i d l y a f t e r a c i d i f i c a t i o n and by washing the o r g a n i c s o l v e n t w i t h s m a l l volumes of w a t e r m~til t h e pH of t h e washings is neutral.
NOVEMBER
1973
V O L . 4 NO. 5
697
PROSTAGLANDINS
Thus t h e a d d i t i o n of sodium l a u r y l s u l p h a t e t o s y n o v i a l f l u i d i m m e d i a t e l y p r i o r to e x t r a c t i o n improves t h e r e c o v e r y of added PGs. This p r o c e d u r e does not i m p a i r t h e p h a r m a c o l o g i c a l a c t i v i t y or t h e c h r o m a t o g r a p h i c m o b i l i t y of t e s t e d PGs. The improvement in r e c o v e r y of the more p o t a r PGF2~ i s t h e same as t h a t f o r PGE1 and E2. We have a l s o used c i t r a t e d blood plasma and found t h e g r e a t e s t improvement in r e c o v e r y of PGs with a c o n c e n t r a t i o n of sodium l a u r y l s u l p h a t e of 0.5 mg/ml ( r e c o v e r y , 81~ ± 5.39 s . e . m . ; r a n g e , 56-100; 8 e x p e r i m e n t s ) .
698
NOVEMBER
1973
V O L . 4 NO. 5
PROSTAGLANDINS
REFER~CES i.
Samuelsson, B. Identification of smooth m u s c l e - s t i m u l a t i n g f a c t o r in bovine b r a i n . P r o s t a g l a n d i n s and r e l a t e d f a c t o r s 25, Biochim. Biophys. A c t a . 8~:218, 196q.
2.
Ambache, N. Irin and a h y d r o x y - a c i d from b r a i n , Biochem. Pharmac. 12: 421, 1963.
3.
Anggard, E. The isolation and determination of prostaglandins in lungs of sheep, guinea pig, monkey and man, Biochim. Pharmac. 14: 1507, 1965.
.
O
Unger, W. G. B i n d i n g of p r o s t a g l a n d i n to human serum albumin, J . Pharm. Pharmac. 2&: ~07, 1972. 5.
Raz, A. Comparative b i n d i n g of p r o s t a g l a n d i n s A2, F 2 a a n d E2 %0 human plasma p r o t e i n s , Biochem. J. 130: 631, 1972.
6.
Unger, W. G., I . F. S t a m f o r d , and A. B e n n e t t . E x t r a c t i o n of p r o s t a g l a n d i n s from human b l o o d , N a t u r e , Lond. 255: 336, 1971.
7.
B e l l , G. H., J . N. Davidson, and H. S c a r b o r o u g h . Textbook of P h y s i o l o g y and B i o c h e m i s t r y , L i v i n g s t o n e , 1965.
8.
Vane, J . R. A s e n s i t i v e method f o r t h e a s s a y of 5 - h y d r o x y t r y p famine, B r i t . J . Pharmacol. 12: 3aa, 1957.
.
Goodman, D. S. The i n t e r a c t i o n of human serum albumin with long chain f a t t y a c i d a n i o n s , J . Am. Chem. Soc. 8U: 3892, 1958.
10.
F e r r e i r a , S. H., and J . R. Vane. P r o s t a g l a n d i n s : t h e i r d i s a p ~ p e a r a n c e from and r e l e a s e i n t o t h e c i r c u l a t i o n , Nature, Lond. 216: 868, 1967.
11.
Gilmore, N . , J . R. Vane, and J . H. W i l e y . P r o s t a g l a n d i n s r e l e a s e d by the s p l e e n , N a t u r e , Land. 218: 1135, 1968.
12.
F l o w e r , R. J . , H. S. Cbeung, and D. W. Cushman. q u a n t i t a t i v e d e t e r m i n a t i o n of p r o s t a g l a n d i n s and m a l o n d i a l d e h y d e formed by t h e a r a c h i d o n i c oxygenase ( p r o s t a g l a n d i n s y n t b e t a s e ) system of b o v i n e s e m i n a l v e s i c l e , Prostaglandins. Submitted f o r p u b l i c a t i o n .
13.
Shaw, J a n e , E . , and P. W. Ramwell. S e p a r a t i o n , i d e n t i f i c a t i o n and e s t i m a t i o n of p r o s t a g l a n d i n s , Methods of B i o c h e m i c a l A n a l y s i s . 17: 325, 1969.
NOVEMBER
1973
V O L . 4 NO. 5
699
G. A. Higgs and J . R. Vane, Department of Pharmacology, I n s t i t u t e of B a s i c Medical S c i e n c e s , Royal C o l l e g e of Surgeons of England, 35/43, L i n c o l n ' s Inn F i e l d s , WC2A 3PN. ABSTRACT
P r e s e n t methods of e x t r a c t i n g p r o s t a g l a n d i n s (laGs) g i v e poor r e c o v e r i e s from s y n o v i a l f l u i d , ~ j ~ r o h a b l y b e c a u s e t h e PGs bind ~Y0 p¥0~ei~ and a r e l o s t in the p r e c i p i t a t i o n s t a g e of e x t r a c t i o n . A d d l t i o n ~ Of the n o n - p o l a r d e t e r g e n t sodium l a u r y l s u l p h a t e p r i o r to e x t r a c t i o n i m ~ o v e s r e c o v e r y of PGs. I t i s s u g g e s t e d t h a t sodium l a u r y l s u l p h a t e competes w i t h PGs f o r the b i n d i n g s i t e s .
Acknowledgements: We wish t o thank Dr. D. H a r t and Dr. J . W o j t e u l e v s k i of t h e W e s t m i n s t e r H o s p i t a l , London, W.C.1. f o r p r o v i d i n g s y n o v i a b f l u i d specimens, and the UpJohn Co., Kalamazoo f o r s u p p l y i n g p r o s t a glandins. The work was supported by a g r a n t from Merck, Sharp and Dohme R e s e a r c h L a b o r a t o r i e s , Rahway, New J e r s e y , U.S.A.
Accepted September5, 1973
PROSTAGLANDINS NOVEMBER
1973
V O L . 4 NO. 5
695
PROSTAGLANDINS
P r e s e n t methods f o r e x t r a c t i n g p r o s t a g l a n d i n s (PGs) from tissue homogenates, seminal fluid or acidified phosphate buffer, into 70-90~ e t h a n o l ( 1 ) , a c e t o n e (2) or e t h y l a c e t a t e (3) g i v e low and i n c o n s i s t e n t r e c o v e r i e s from b l o o d p l a s m a . PGs b i n d w e a k l y t o a l b u m i n ( ~ , 5 ) and a r e p r o b a b l y l o s t in t h e p r o t e i n p r e c i p i t a t i o n s t a g e of the extraction. If precipitation of p r o t e i n i s a v o i d e d by u s i n g ~0-50~ e t h a n o l , t h e PGs can be e x t r a c t e d q u a n t i t a t i v e l y into chloroform ( 6 ) . T h i s method g i v e s h i g h e r r e c o v e r i e s of PGE1, Ez, F l a , F 2 a A 1 and A2 from human b l o o d . We wanted t o m e a s u r e t h e PG a c t i v i t y in s y n o v i a l f l u i d (human) from t h e j o i n t s of p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s . A heavy precipitate formed when t h e f l u i d s were a c i d i f i e d w i t h iN hydrochloric acid prior to extraction with ethyl acetate. Synovial f l u i d has a s i m i l a r t o t a l p r o t e i n c o n c e n t r a t i o n t o t h a t of b l o o d plasma and i n a d d i t i o n c o n t a i n s h i g h c o n c e n t r a t i o n s of h y a l u r o n i e a c i d (7) which makes i t v i s c o u s . Acidification precipitates b o t h of t h e s e components. Acidification f o l l o w e d by e t h y l a c e t a t e e x t r a c t i o n of s y n o v i a l f l u i d c o n t a i n i n g added PGEz ( 2 5 - 1 0 0 n g / m l ) g a v e r e c o v e r i e s of o n l y 12-20~ (mean = 17~, 10 e x p e r i m e n t s ) when a s s a y e d on t h e r a t fundus (8). The r e c o v e r y was i n c r e a s e d t o 2~-59~ (mean = 39~, l 0 e x p e r i m e n t s ) by u s i n g t h e method which g i v e s good r e c o v e r y from b l o o d p l a s m a , i . e . a d d i t i o n of ~0-50~ e t h a n o l , a c i d i f y i n g w i t h f o r m i c a c i d and e x t r a c t i n g i n t o c h l o r o f o r m ( 6 ) . Albumin has a g r e a t e r a f f i n i t y f o r l e s s p o l a r u n s a t u r a t e d f a t t y a c i d s such as o l e a t e t h a n f o r PGs ( ~ , 9 ) and Dr. F. Dea s u g g e s t e d t o us t h a t a n o n - p o l a r h y d r o p h o b i c compound such a s t h e d e t e r g e n t sodium l a u r y l s u l p h a t e would compete w i t h PGs f o r p r o t e i n b i n d i n g
sites. The e t h a n o l / c h l o r o f o r m e x t r a c t i o n p r o c e d u r e (6) was u s e d t o e x t r a c t PGs from samples of s y n o v i a l f l u i d . To e s t i m a t e t h e r e c o v e r y of added PGs, each sample was d i v i d e d i n t o two and PGE2, E. o r F2~ ( 2 5 - 1 0 0 n g / m l ) was added t o one h a l f ; t h e endogenous 1 c o n c e n t r a t i o n was measured in t h e o t h e r . For joint fluid (values c o r r e c t e d f o r r e c o v e r y ) t h i s v a r i e d from 0-62 n g / m l (mean = 8 . 7 ,
28 fluids). T a b l e 1 shows t h e e f f e c t s of d i f f e r e n t c o n c e n t r a t i o n s sodium l a u r y l s u l p h a t e (Sigma London, C h e m i c a l Co. L t d . ) on t h e e f f i c i e n c y of e x t r a c t i o n of t h e added PGs.
696
NOVEMBER 1973
of
VOL. 4 NO. 5
PROSTAGLANDINS
Table I Recovery of added PGs from synovial fluid with different concentrations of sodium lauryl sulphate.
C o n c e n t r a t i o n of sodium l a u r y l sulphate (mg/ml)
P e r c e n t a g e r e c o v e r y of PGs Range (number of e x p e r i m e n t s ) Mean ± s.e.m.
2~-59% (10 e x p e r i m e n t s ) 39~ ± 2.90 1.0
39-100~ ( l l e x p e r i m e n t s ) 5 ~ ± 5.22
5.0
~ - 1 0 0 ~ (13 e x p e r i m e n t s ) 76% ~ ~.15
I0.0
31-~9% (4 experiments)
,u
,
39?; PG a c t i v i t y was assayed (10) on the r a t fundus, c h i c k rectum and r a t c o l o n , s u p e r f u s e d a t 5 ml/min with K r e b s ' b i c a r b o n a t e s o l u t i o n a t 37°C bubbled w i t h 5~ carbon d i o x i d e in oxygen and c o n t a i n i n g a m i x t u r e of a n t a g o n i s t s as p r e v i o u s l y d e s c r i b e d 411). A d d i t i o n of sodium t a u r y l s u l p h a t e improved t h e r e c o v e r y of added PGs. The o p t i m a l c o n c e n t r a t i o n of d e t e r g e n t was 5 mg/ml and h i g h e r c o n c e n t r a t i o n s d e c r e a s e d r e c o v e r y . A d d i t i o n of sodium l a u r y l s u l p h a t e to t h e samples had no e f f e c t on t h e t h r e e a s s a y t i s s u e s . T r i t i a t e d PGE1 (5-6 3H-I~E1), R a d i o c h e m i c a l s , Amersbam) was added to s y n o v i a l f l u i d ~nd the e x t r a 6 t o b t a i n e d w i t h the improved method ( u s i n g 5 mg/ml sodium l a u r y l s u l p h a t e ) was s u b j e c t e d to t h i n l a y e r chromatography. Eastman s i l i c a g e l chromatogram s h e e t s were used with t h e upper phase of e t h y l a c e t a t e : w a t e r : i s o - o c t a n e : a c e t i c a c i d , 411 : 10 : 5 : 2) 412) as a s o l v e n t system. 95¢ of the l a b e l corresponded t o the a u t h e n t i c PGE s p o t ; the r e m a i n i n g a c t i v i t y appeared in a peak which corresponded to PGAI and PGB1. D e h y d r a t i o n of PGEs to form PGAs o c c u r s a f t e r a c i d i f i c a t i o n but b e f o r e adding o r g a n i c s o l v e n t (13) and may a l s o r e s u l t from the p r e s e n c e of a c i d in the organic s o l v e n t during e x t r a c t i o n . The c o n v e r s i o n can be minimised by e x t r a c t i n g r a p i d l y a f t e r a c i d i f i c a t i o n and by washing the o r g a n i c s o l v e n t w i t h s m a l l volumes of w a t e r m~til t h e pH of t h e washings is neutral.
NOVEMBER
1973
V O L . 4 NO. 5
697
PROSTAGLANDINS
Thus t h e a d d i t i o n of sodium l a u r y l s u l p h a t e t o s y n o v i a l f l u i d i m m e d i a t e l y p r i o r to e x t r a c t i o n improves t h e r e c o v e r y of added PGs. This p r o c e d u r e does not i m p a i r t h e p h a r m a c o l o g i c a l a c t i v i t y or t h e c h r o m a t o g r a p h i c m o b i l i t y of t e s t e d PGs. The improvement in r e c o v e r y of the more p o t a r PGF2~ i s t h e same as t h a t f o r PGE1 and E2. We have a l s o used c i t r a t e d blood plasma and found t h e g r e a t e s t improvement in r e c o v e r y of PGs with a c o n c e n t r a t i o n of sodium l a u r y l s u l p h a t e of 0.5 mg/ml ( r e c o v e r y , 81~ ± 5.39 s . e . m . ; r a n g e , 56-100; 8 e x p e r i m e n t s ) .
698
NOVEMBER
1973
V O L . 4 NO. 5
PROSTAGLANDINS
REFER~CES i.
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NOVEMBER
1973
V O L . 4 NO. 5
699