PROSTAGLANDINS
INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY TRIYNOIC ACIDS Joan M. Goetz, Howard Sprecher, David G. Cornwell and Rao V. Panganamala
Department of Physiological Chemistry The Ohio State University Columbus, Ohio 43210
ABSTRACT Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from bovine seminal vesicles is inhibited by acetylenic acids. Octadeca-6,9,12-triynoic acid and eicosa-8,11,14-triynoic acid are the most potent inhibitors. These acids hoth contain an m-8 methylene group. Within the 20-carbon acetylenic acid series, inhibition decreases in the sequence eicosa-8,11,14-triynoic acid > eicosa7,10,13-triynoic acid > eicosa-5»8,11-triynoic acid. Furthermore» eicosa-8,11,14-triynoic acid is a more potent inhibitor of arachidonic acid induced platelet aggregation than either eicosa-7,10,13-triynoic acid or eicosa-5,8,11-triynoic acid. The m-8 methylene group is not the only determinent of inhibitory potency since docosa-10,13»16triynoic acid is less potent than its 18 and 20 carbon analogs and all of these acids have an e-8 methylene group.
ACKNOWLEDGMENT This study was supported in part by Research Grant HL-I1897 from the National Institutes of Health and Central Ohio Heart Chapter, Inc., Grant No. 75-39.
INTRODUCTION
Ahern and Downing (1) and Downing e t a l .
(2) showed t h a t e i c o s a -
5,8,11,14-tetraynoic a c i d and o c t a d e c a - 9 , 1 2 - d i y n o i c a c i d i n h i b i t e d both p r o s t a g l a n d i n s y n t h e t a s e o f sheep v e s i c u l a r gland and soybean l i p o x y g e n a s e . They s u g g e s t e d t h a t hydrogen a b s t r a c t i o n from t h e ~-8 methylene group of t h e a c e t y l e n i c a c i d o c c u r r e d in t h e p r e s e n c e o f t h e enzyme system forming an allene which then reacted irreversibly with the enzyme. In order to verify this hypothesis and to establish the structural requirements for acetylenic acid inhibitors of prostaglandin biosynthesis, we studied a series of acetylenic acids, varying in both chain length and acetylenic group positions, by measuring the ability of these acids to inhibit prostaglandin biosynthesis in bovine vesicular glands (BVG) and by measuring the ability of these acids to inhibit platelet aggregation. The results are reported in this paper.
A U G U S T 1976
V O L . 12 NO. 2
187
PROSTAGLANDINS
MATERIALS AND METHODS BVG were purchased from Pel-Freeze Biologicals (Rogers, Arkansas). The microsomal fraction was prepared by the procedure of Takeguchi et al. (3) and lyophilized. [I-14C] eicosa-8,11,14-trienoic acid was-synthesized as described by Budny and Sprecher (4) and was used as the potassium salt. The fatty acid had a radioactive purity of 97% and specific activity of 1.31 x 10 6 dpm/~mole. Eicosa-8,11,14-triynoic acid (20:3, 8a, lla, 14a), eicosa-7,10,13-triynoic acid (20:3, 7a, 10a, 13a), eicosa-5,8,11-triynoic acid (20:3, 5a, 8a, lla), docosa-10,13,16triynoic acid (22:3, 10a, 13a, 16a), octadeca-6,9,12-triynoic acid (18:3, 6a, 9a, 12a), were synthesized by the method described by Sprecher (5). Eicosa-5,8,11,14-tetraynoic acid (20:4, 5a, 8a, lla, 14a) (TYA) was kindly supplied by Hoffman La-Roche (Nutley, N. J.). PGEI, PGFIa and PGD I were kindly supplied by Dr. J. Pike, Upjohn Co. (Kalamazoo, Mich.). All solvents were distilled prior to use and 0.001% 2,6-di -tertbutyl-4 methylphenol was added to ethyl acetate and chloroform. The incubation procedure with potassium [I-14C] eicosa-8,11,14-trienoate (0.05 ~mole), extraction, thin-layer chromatography (TLC) separation of unreacted substrate and prostaglandins, and counting are described elsewhere (6). Inhibitors were dissolved in ethanol and added in 5 to 20 ~i aliquots to the incubation system. Human platelet rich plasma (PRP) was prepared as described elsewhere (7). Arachidonic acid (i mM) was added as the potassium soap to 0.5 ml PRP. Inhibitors were added as ammonium salts to 0.5 ml PRP, 2 minutes prior to the addition of potassium arachidonate. Aggregation was measured as a change in transmitted light with an aggregometer (Chrono-Log, Broomall, Pa.).
RESULTS AND DISCUSSION Conversion of [I-14C] eicosa-8,11,14-trienoate acid t o PGE I and PGFIa was measured in the BVG microsomal system. The PGE 1 and PGFI« bands were combined and prostaglandin synthesis was estimated from the radioactivity of the combined fraction. This prostaglandin fraction contained 64.9 ± 2.1% (mean ± S.D. for ii determinations) of the total radioactivity when microsomes were incubated for 30 min. The band corresponding to PGD 1 contained less than 1% of the total radioactivity. Inhibition with triynoic acids, TYA and acetyl salicylic acid (ASA) is expressed as the concentration of inhihitor required to diminish prostaglandin biosynthesis by 50%. Each inhibition experim e n t was compared with its own control. Data are summarized in Table I. Two triynoic acids, 18:3 (6a, 9a, 12a) are very potent inhibitors (Table I) acting well established acetylenic acid TYA (1,2). (Ta, 10a, 13a) and 22:3 (lOa, 13a 16a) show
188
and 20:3 (8a, lla, 14a) in the same fange as the Two triynoic acids, 20:3 intermediate inhibitory
A U G U S T 1976
VOL. 12 NO. 2
PROSTAGLANDINS capacities and one triynoic acid, 20:3 (5a, 8a, lla), has a much lower inhibitory capacity (Table I). All triynoic acids are significantly better inhibitors than ASA (Table I). Within the eicosatriynoic acid series, inhibition decreases in the sequence 20:3 (8a, lla, 14a) > 20:5 (7a, 10a, 13a) > 20:3 (5a, 8a, lla).
TABLE I Inhibition of Prostaglandin Biosynthesis from Eicosa-8,11,14-trienoic Acid by Acetylenic Acids and Acetyl Salicylic Acid in the BVG Microsomal System Inhibitor
Concentration (uM) required to inhibit 50% of total prostaglandin formation
18:3 (6a, 9a, 12a)
2.6
20:3 (8a, lla, ita)
2.6
20:3 (7a, 10a, 13a)
41.0
20:3 (5a, 8a, ii~)
240.0
22:3 (10a, 13a, 16a) 20:4 (5a, 8a, lla, 14a) (TYA) A¢etyl Salicyli¢ Acid (ASA)
66.0 6.6 1000.0
Hamberg et al. (8) showed that the cyclic endoperoxides PGG 2 and PGH 2 and thromboxane A 2 which are all derived from arachidonic acid induced platelet aggregation. Willis et al. (9) found that TYA inhibited arachidonic acid-induced platelet aggregation and Hamberg et al. (I0) later showed that TYA inhibited PGG 2 and PGH 2 formation in platelets. We found that 20:3 (Sa, lla, 14a) was more potent than TYA in the inhibition of arachidonic acid induced platelet aggregation (Fig. i). Furthermore, 20:3 (8a, lla, 14a) was a better inhibitor of arachidonic acid induced aggregation than either 20:3 (7a, 10a, 13a) or 20:3 (5a, 8a, lla). These data are consistent with the results obtained with the BVG microsomal system (Table I).
AUGUST 1976
VOL. 12 NO. 2
189
PROSTAGLANDINS
INHIBITOR
ARACHIDONIC AClD
O' ~
%T 50
(So,IIo,14a)
~
mM) TYA
~~,,,~~4m "~"~
M) 20:3 ( "/'a,IOo, 13o }
(2.64mM) 20:3 (5a,8a, Ila)
(ImM) ARACHIDONICACID
IOO TIME (min)
Figure i.
190
Effect of different acetyleni¢ acids on arachidonic acidinduced platelet aggregation.
AUGUST 1976
VOL. 12 NO. 2
PROSTAGLANDINS
Downing e t a l . (2) s t u d i e d s e v e r a l d i y n o i c a c i d s as p r o s t a g l a n d i n i n h i b i t o r s and e s t a b l i s h e d t h a t 18:2 (9a, 12a) had the same i n h i b i t o r y potency as TYA. They proposed t h a t the i n h i b i t o r y e f f e c t was caused by the enzymatic removal of an ~-8 p r o t o n from t h e s e a c e t y l e n i c a c i d s r e s u l t i n g i n the f o r m a t i o n of an a l l e n e . The a11ene then r e a c t e d i r r o v e r s i b l y w±th the enzyme. The enzymatic a b s t r a c t i o n of a p r o t o n from the ~-8 methylene group was o r i g i n a l l y proposed by Hamberg and Samuelsson (11). The importance of the ~-8 methylene group was f i r s t noted by 5 t r u i j k et a l . (12) who showed t h a t 68% of 20:3 (8,11,14) was c o n v e r t e d to p r o s t a g l a n d i n s w h i l e o n l y 18% o f 20:3 (7,10,13) was c o n v e r t e d to p r o s t a g l a n d i n s and no 20:3 ( 5 , 8 , 1 1 ) was c o n v e r t e d t o p r o s t a g l a n d i n s . Out d a t a s u p p o r t t h i s h y p o t h e s i s . The acid with an m-8 methylene group, 20:3 (8a, 11a, 14a), i s a more effective inhibitor than either the acid with an ~-9 methylene group, 20:5 (7a, lOa, 13a), or the acid with an ~-ii methylene group, 20:3 (5a, 8a, lla). The initial studies of Downing et al. (2) suggested that chainlength was not important since 18:2 -~-a~'12a) and 20:4 (Sa, 8a, lla, 14a) had nearly equal inhibitory potencies. Out data confirm their obs¢rvations with 18 carbon and 20 carbon acids. Two m-8 acids, 18:3 (óa, 9a, 12a) and 20:3 (8a, lla, 14a), have the same inhibitory potency. However, a third ~-8 acid, 22:3 (10a, 13a, 16a), is a less effective acetylenic acid inhibitor of prostaglandin biosynthesis. Thus our data show that both chain-length and the acetylenic bond position are important determinants of inhibitory potency.
REFERENCES i.
Ahern, D. G. and D. T. Downing. Inhibition of Prostaglandin Biosynthesis by Eicosa-5,8,11,14-tetraynoi¢ Acid. Biochim. Biophys. Acta 210:456 (1970).
2.
Downing, D. T., J. A. Barve, F. D. Gunstone, F. R. Jacobsberg and M. Lie Ken Jie. Structural Requirements of Acetylenic Fatty Acids for Inhibition of Soybean Lipoxygenase and Prostaglandin Synthetase. Biochim. Biophys. Acta 280:343 (1972).
3.
Takeguchi, C., E. Kohno and C. J. Sih. Mechanism of Prostaglandin Biosynthesis. I. Chara¢terization and Assay of Bovine Prostaglandin Synthetase. Bio¢hemistry 10:2372 (1971).
4.
Budny, J. and H. Sprecher. A Study of Some of the Factors Involved in Regulating the Conversion of Octade¢a-8,11-dienoate to Eicosa-4,7,10,13-tetraenoate in the Rat. Biochim. Biophys. Acta 239:190 (1971).
5.
Sprecher, H. The Synthesis and Metabolism of Hexadeca-4,7,10trienoate, Eicosa-8,11,14-trienoate, Do¢osa-10,13,1ó-trienoate and Docosa-6,9,12,15-tetraenoate in the Rat. Biochim. Biophys. A¢ta 152:519 (1968).
A U G U S T 1976
VOL. 12 NO. 2
191
PROSTAGLANDINS
6.
Panganamala, R. V., N. R. Brownlee, H. Sprecher and D. G. Cornwell. Evaluation of Superoxide Anion and Singlet Oxygen in the Biosynthesis of Prostaglandins from Eicosa-8,11,14-trienoic Acid. Prostaglandins 7:21 (1974).
7.
Panganamala, R. V., H. R. Sharma, H. Sprecher, J. C. Geer and D. G. Cornwell. A Suggested Role for Hydrogen Peroxide in the Biosynthesis of Prostaglandins. Prostaglandins 8:5 (1974).
8.
Hamberg, M, J. Svensson and B. Samuelsson. Thromboxanes: A New Group of Biologically Active Compounds Derived from Prostaglandin Endoperoxides. Proc. Nat. Aca d. Sci. USA 72:2994 (1975).
9.
Willis, A. L., D. C. Kuhn and H. J. Weiss. Acetylenic Analog of Arachidonate that Acts like Aspirin on Platelets. Science 183:
327 (1974). i0.
Hamberg, M. and B. Samuelsson. Prostaglandin Endoperoxides. Novel Transformation of Arachidonic Acid in Human Platelets. Proc. Nat. Acad. Sci. USA 71:3400 (1974).
ii.
Hamberg, M. and B. Samuelsson. On the Mechanism of the Biosynthesis of Prostaglandins E l and FIa. J. Biol. Chem. 242:5336
(1967). 12.
192
Struijk, C. B., R. K. Beerthuis, H. J. J. Pabon and D. A. van Dorp. Specificity in the Enzymic Conversion of Polyunsaturated Fatty Acids into Prostaglandins. Rec. Trav. Chim. 85:1235 (1966).
AUGUST 1976
VOL. 12 NO. 2
INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY TRIYNOIC ACIDS Joan M. Goetz, Howard Sprecher, David G. Cornwell and Rao V. Panganamala
Department of Physiological Chemistry The Ohio State University Columbus, Ohio 43210
ABSTRACT Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from bovine seminal vesicles is inhibited by acetylenic acids. Octadeca-6,9,12-triynoic acid and eicosa-8,11,14-triynoic acid are the most potent inhibitors. These acids hoth contain an m-8 methylene group. Within the 20-carbon acetylenic acid series, inhibition decreases in the sequence eicosa-8,11,14-triynoic acid > eicosa7,10,13-triynoic acid > eicosa-5»8,11-triynoic acid. Furthermore» eicosa-8,11,14-triynoic acid is a more potent inhibitor of arachidonic acid induced platelet aggregation than either eicosa-7,10,13-triynoic acid or eicosa-5,8,11-triynoic acid. The m-8 methylene group is not the only determinent of inhibitory potency since docosa-10,13»16triynoic acid is less potent than its 18 and 20 carbon analogs and all of these acids have an e-8 methylene group.
ACKNOWLEDGMENT This study was supported in part by Research Grant HL-I1897 from the National Institutes of Health and Central Ohio Heart Chapter, Inc., Grant No. 75-39.
INTRODUCTION
Ahern and Downing (1) and Downing e t a l .
(2) showed t h a t e i c o s a -
5,8,11,14-tetraynoic a c i d and o c t a d e c a - 9 , 1 2 - d i y n o i c a c i d i n h i b i t e d both p r o s t a g l a n d i n s y n t h e t a s e o f sheep v e s i c u l a r gland and soybean l i p o x y g e n a s e . They s u g g e s t e d t h a t hydrogen a b s t r a c t i o n from t h e ~-8 methylene group of t h e a c e t y l e n i c a c i d o c c u r r e d in t h e p r e s e n c e o f t h e enzyme system forming an allene which then reacted irreversibly with the enzyme. In order to verify this hypothesis and to establish the structural requirements for acetylenic acid inhibitors of prostaglandin biosynthesis, we studied a series of acetylenic acids, varying in both chain length and acetylenic group positions, by measuring the ability of these acids to inhibit prostaglandin biosynthesis in bovine vesicular glands (BVG) and by measuring the ability of these acids to inhibit platelet aggregation. The results are reported in this paper.
A U G U S T 1976
V O L . 12 NO. 2
187
PROSTAGLANDINS
MATERIALS AND METHODS BVG were purchased from Pel-Freeze Biologicals (Rogers, Arkansas). The microsomal fraction was prepared by the procedure of Takeguchi et al. (3) and lyophilized. [I-14C] eicosa-8,11,14-trienoic acid was-synthesized as described by Budny and Sprecher (4) and was used as the potassium salt. The fatty acid had a radioactive purity of 97% and specific activity of 1.31 x 10 6 dpm/~mole. Eicosa-8,11,14-triynoic acid (20:3, 8a, lla, 14a), eicosa-7,10,13-triynoic acid (20:3, 7a, 10a, 13a), eicosa-5,8,11-triynoic acid (20:3, 5a, 8a, lla), docosa-10,13,16triynoic acid (22:3, 10a, 13a, 16a), octadeca-6,9,12-triynoic acid (18:3, 6a, 9a, 12a), were synthesized by the method described by Sprecher (5). Eicosa-5,8,11,14-tetraynoic acid (20:4, 5a, 8a, lla, 14a) (TYA) was kindly supplied by Hoffman La-Roche (Nutley, N. J.). PGEI, PGFIa and PGD I were kindly supplied by Dr. J. Pike, Upjohn Co. (Kalamazoo, Mich.). All solvents were distilled prior to use and 0.001% 2,6-di -tertbutyl-4 methylphenol was added to ethyl acetate and chloroform. The incubation procedure with potassium [I-14C] eicosa-8,11,14-trienoate (0.05 ~mole), extraction, thin-layer chromatography (TLC) separation of unreacted substrate and prostaglandins, and counting are described elsewhere (6). Inhibitors were dissolved in ethanol and added in 5 to 20 ~i aliquots to the incubation system. Human platelet rich plasma (PRP) was prepared as described elsewhere (7). Arachidonic acid (i mM) was added as the potassium soap to 0.5 ml PRP. Inhibitors were added as ammonium salts to 0.5 ml PRP, 2 minutes prior to the addition of potassium arachidonate. Aggregation was measured as a change in transmitted light with an aggregometer (Chrono-Log, Broomall, Pa.).
RESULTS AND DISCUSSION Conversion of [I-14C] eicosa-8,11,14-trienoate acid t o PGE I and PGFIa was measured in the BVG microsomal system. The PGE 1 and PGFI« bands were combined and prostaglandin synthesis was estimated from the radioactivity of the combined fraction. This prostaglandin fraction contained 64.9 ± 2.1% (mean ± S.D. for ii determinations) of the total radioactivity when microsomes were incubated for 30 min. The band corresponding to PGD 1 contained less than 1% of the total radioactivity. Inhibition with triynoic acids, TYA and acetyl salicylic acid (ASA) is expressed as the concentration of inhihitor required to diminish prostaglandin biosynthesis by 50%. Each inhibition experim e n t was compared with its own control. Data are summarized in Table I. Two triynoic acids, 18:3 (6a, 9a, 12a) are very potent inhibitors (Table I) acting well established acetylenic acid TYA (1,2). (Ta, 10a, 13a) and 22:3 (lOa, 13a 16a) show
188
and 20:3 (8a, lla, 14a) in the same fange as the Two triynoic acids, 20:3 intermediate inhibitory
A U G U S T 1976
VOL. 12 NO. 2
PROSTAGLANDINS capacities and one triynoic acid, 20:3 (5a, 8a, lla), has a much lower inhibitory capacity (Table I). All triynoic acids are significantly better inhibitors than ASA (Table I). Within the eicosatriynoic acid series, inhibition decreases in the sequence 20:3 (8a, lla, 14a) > 20:5 (7a, 10a, 13a) > 20:3 (5a, 8a, lla).
TABLE I Inhibition of Prostaglandin Biosynthesis from Eicosa-8,11,14-trienoic Acid by Acetylenic Acids and Acetyl Salicylic Acid in the BVG Microsomal System Inhibitor
Concentration (uM) required to inhibit 50% of total prostaglandin formation
18:3 (6a, 9a, 12a)
2.6
20:3 (8a, lla, ita)
2.6
20:3 (7a, 10a, 13a)
41.0
20:3 (5a, 8a, ii~)
240.0
22:3 (10a, 13a, 16a) 20:4 (5a, 8a, lla, 14a) (TYA) A¢etyl Salicyli¢ Acid (ASA)
66.0 6.6 1000.0
Hamberg et al. (8) showed that the cyclic endoperoxides PGG 2 and PGH 2 and thromboxane A 2 which are all derived from arachidonic acid induced platelet aggregation. Willis et al. (9) found that TYA inhibited arachidonic acid-induced platelet aggregation and Hamberg et al. (I0) later showed that TYA inhibited PGG 2 and PGH 2 formation in platelets. We found that 20:3 (Sa, lla, 14a) was more potent than TYA in the inhibition of arachidonic acid induced platelet aggregation (Fig. i). Furthermore, 20:3 (8a, lla, 14a) was a better inhibitor of arachidonic acid induced aggregation than either 20:3 (7a, 10a, 13a) or 20:3 (5a, 8a, lla). These data are consistent with the results obtained with the BVG microsomal system (Table I).
AUGUST 1976
VOL. 12 NO. 2
189
PROSTAGLANDINS
INHIBITOR
ARACHIDONIC AClD
O' ~
%T 50
(So,IIo,14a)
~
mM) TYA
~~,,,~~4m "~"~
M) 20:3 ( "/'a,IOo, 13o }
(2.64mM) 20:3 (5a,8a, Ila)
(ImM) ARACHIDONICACID
IOO TIME (min)
Figure i.
190
Effect of different acetyleni¢ acids on arachidonic acidinduced platelet aggregation.
AUGUST 1976
VOL. 12 NO. 2
PROSTAGLANDINS
Downing e t a l . (2) s t u d i e d s e v e r a l d i y n o i c a c i d s as p r o s t a g l a n d i n i n h i b i t o r s and e s t a b l i s h e d t h a t 18:2 (9a, 12a) had the same i n h i b i t o r y potency as TYA. They proposed t h a t the i n h i b i t o r y e f f e c t was caused by the enzymatic removal of an ~-8 p r o t o n from t h e s e a c e t y l e n i c a c i d s r e s u l t i n g i n the f o r m a t i o n of an a l l e n e . The a11ene then r e a c t e d i r r o v e r s i b l y w±th the enzyme. The enzymatic a b s t r a c t i o n of a p r o t o n from the ~-8 methylene group was o r i g i n a l l y proposed by Hamberg and Samuelsson (11). The importance of the ~-8 methylene group was f i r s t noted by 5 t r u i j k et a l . (12) who showed t h a t 68% of 20:3 (8,11,14) was c o n v e r t e d to p r o s t a g l a n d i n s w h i l e o n l y 18% o f 20:3 (7,10,13) was c o n v e r t e d to p r o s t a g l a n d i n s and no 20:3 ( 5 , 8 , 1 1 ) was c o n v e r t e d t o p r o s t a g l a n d i n s . Out d a t a s u p p o r t t h i s h y p o t h e s i s . The acid with an m-8 methylene group, 20:3 (8a, 11a, 14a), i s a more effective inhibitor than either the acid with an ~-9 methylene group, 20:5 (7a, lOa, 13a), or the acid with an ~-ii methylene group, 20:3 (5a, 8a, lla). The initial studies of Downing et al. (2) suggested that chainlength was not important since 18:2 -~-a~'12a) and 20:4 (Sa, 8a, lla, 14a) had nearly equal inhibitory potencies. Out data confirm their obs¢rvations with 18 carbon and 20 carbon acids. Two m-8 acids, 18:3 (óa, 9a, 12a) and 20:3 (8a, lla, 14a), have the same inhibitory potency. However, a third ~-8 acid, 22:3 (10a, 13a, 16a), is a less effective acetylenic acid inhibitor of prostaglandin biosynthesis. Thus our data show that both chain-length and the acetylenic bond position are important determinants of inhibitory potency.
REFERENCES i.
Ahern, D. G. and D. T. Downing. Inhibition of Prostaglandin Biosynthesis by Eicosa-5,8,11,14-tetraynoi¢ Acid. Biochim. Biophys. Acta 210:456 (1970).
2.
Downing, D. T., J. A. Barve, F. D. Gunstone, F. R. Jacobsberg and M. Lie Ken Jie. Structural Requirements of Acetylenic Fatty Acids for Inhibition of Soybean Lipoxygenase and Prostaglandin Synthetase. Biochim. Biophys. Acta 280:343 (1972).
3.
Takeguchi, C., E. Kohno and C. J. Sih. Mechanism of Prostaglandin Biosynthesis. I. Chara¢terization and Assay of Bovine Prostaglandin Synthetase. Bio¢hemistry 10:2372 (1971).
4.
Budny, J. and H. Sprecher. A Study of Some of the Factors Involved in Regulating the Conversion of Octade¢a-8,11-dienoate to Eicosa-4,7,10,13-tetraenoate in the Rat. Biochim. Biophys. Acta 239:190 (1971).
5.
Sprecher, H. The Synthesis and Metabolism of Hexadeca-4,7,10trienoate, Eicosa-8,11,14-trienoate, Do¢osa-10,13,1ó-trienoate and Docosa-6,9,12,15-tetraenoate in the Rat. Biochim. Biophys. A¢ta 152:519 (1968).
A U G U S T 1976
VOL. 12 NO. 2
191
PROSTAGLANDINS
6.
Panganamala, R. V., N. R. Brownlee, H. Sprecher and D. G. Cornwell. Evaluation of Superoxide Anion and Singlet Oxygen in the Biosynthesis of Prostaglandins from Eicosa-8,11,14-trienoic Acid. Prostaglandins 7:21 (1974).
7.
Panganamala, R. V., H. R. Sharma, H. Sprecher, J. C. Geer and D. G. Cornwell. A Suggested Role for Hydrogen Peroxide in the Biosynthesis of Prostaglandins. Prostaglandins 8:5 (1974).
8.
Hamberg, M, J. Svensson and B. Samuelsson. Thromboxanes: A New Group of Biologically Active Compounds Derived from Prostaglandin Endoperoxides. Proc. Nat. Aca d. Sci. USA 72:2994 (1975).
9.
Willis, A. L., D. C. Kuhn and H. J. Weiss. Acetylenic Analog of Arachidonate that Acts like Aspirin on Platelets. Science 183:
327 (1974). i0.
Hamberg, M. and B. Samuelsson. Prostaglandin Endoperoxides. Novel Transformation of Arachidonic Acid in Human Platelets. Proc. Nat. Acad. Sci. USA 71:3400 (1974).
ii.
Hamberg, M. and B. Samuelsson. On the Mechanism of the Biosynthesis of Prostaglandins E l and FIa. J. Biol. Chem. 242:5336
(1967). 12.
192
Struijk, C. B., R. K. Beerthuis, H. J. J. Pabon and D. A. van Dorp. Specificity in the Enzymic Conversion of Polyunsaturated Fatty Acids into Prostaglandins. Rec. Trav. Chim. 85:1235 (1966).
AUGUST 1976
VOL. 12 NO. 2
