ARACHIDONIC ACID PEROXIDATION, PROSTAGLANDIN SYNTHESIS AND PLATELET FUNCTION GUNDUH. R. RAO,J. M. GERRARD, J. W. EATONand J. G. WHITE Departments of Laboratory Medicine and Pathology, Pediatrics and Medicine, Box 198, Mayo Memorial Building, University of Minnesota, Minneapolis. MN 55455, U.S.A. (Received 6 March 1978; accepted 14 March 1978)
Abstract-The initial oxygenation or peroxidation of arachidonic acid seems to be an essential step for the synthesis of cyclic endoperoxides and prostaglandins. There has been some evidence and considerable interest in the role of superoxide anion, hydroxyl radicals or singlet oxygen as a source of oxygen in the formation of the active species (free radicals). A test capable of detecting active intermediates of lipid peroxidation and useful for studying the role of free radicals has been developed. The test resulted from the discovery that vitamin E markedly enhanced the reduction of nitroblue tetrazolium (NBT) during arachidonic acid peroxidation. Intact platelets, microsomes. sheep vesicular gland enzymes or peroxidases could provide essential enzyme activity. NBT and vitamin E when added to platelet microsornes inhibited the conversion of I4C arachidonic acid to HETE, HHT and thromboxanes. The combination also inhibited aggregation of platelets stimulated by collagen, thrombin, ADP and epinephrine. Prolonged incubation with these agents at the highest concentrations used in the study caused no change in morphology and had no deleterious effect on platelet levels of adenine nucleotides and serotonin. Results of our preliminary studies suggest that NBT and vitamin E can detect intermediates of lipid peroxidation, inhibit the conversion of arachidonic acid, prevent platelet aggregation and the release reaction without damaging the platelets morphologically or biochemically. As both the agents are scavengers of free radicals and in combination exert synergistic effects, the test system may serve as a probe in various free radical mediated events and may offer some degree of protection against free radical mediated pathological processes.
INTRODUCTION
The platelet prostaglandin (PG) endoperoxide synthetase enzyme converts arachidonic acid to PGG2 and PGH2.These labile compounds are then metabolized primarily to the highly active thromboxane A2 or to the hydroxy fatty acid, 12~-hydroxy-5,8,10heptadecatrienoic acid (HHT) and malondialdehyde (MDA) (Hamberg and Samuelsson, 1973, 1974; Hamberg et al., 1974, 1975; Needleman et al., 1976a. b). A large body of literature (Smith and Willis. 1970, 1971; Silver et af., 1972, 1973; Smith et af., 1974; Willis. 1974, Malmsten et al., 1975; Weiss et a/., 1976) now supports the concept that arachidonic acid (AA) transformation plays a major role in platelet physiology. Our interest in the oxidation of AA by platelets was stimulated by the observations of Gerrard et al. (1976) that aminotriazole, an inhibitor of peroxidase, was found to inhibit platelet PG synthesis, secretion and aggregation. These observations suggested that arachidonic acid, released from membrane phospholipids after platelet activation, reacts with the peroxidase and that a product of AA peroxidation is essential for prostaglandin endoperoxide synthesis. Several workers (Barber and Bernheim, 1967; Hamberg et al., 1974; PIaa and Witschi, 1976; Graziano et af., 1976) have suggested that a reactive enzyme, fatty acidoxygen intermediate or an active fatty acid (free radical), is formed during the initial peroxidation process of unsaturated fatty acids. During the course of inves-
tigations to find a method capable of detecting such an interaction in platelet AA peroxidation, we found the nitroblue tetrazolium (NBT) reduction assay to be useful. The ditetrazolium 3’,3’(4,4-di-o-anisyIene)2,2’-di@-nitrophenyl)-bis (5 phenyl) is the most widely employed of the tetrazolium salts, and has been extensively used in studies on normal and abnormal neutrophils to detect the activity of oxidative enzyme systems. When reducing equivalents are available in the medium the yellow dye is reduced to a blue colored product. Recently, superoxide production and reducing activity of platelets have been detected in test systems employing NBT (Marcus et al., 1977). In the present investigation we have found that NBT is a valuable agent for detecting arachidonic acid peroxidation when vitamin E is added t o the system. The findings indicate that peroxidation of AA is an important step for PG synthesis and that prevention of AA peroxidation blocks the platelet PG synthesis, secretion and aggregation. MATERIALS AND METHODS
Platelets for these studies were obtained from healthy adult volunteers after informed consent. Blood drawn from an anticubital vein into siliconized glass or plastic syringes was mixed immediately with 3.8% trisodium citrate or citrate-citric acid, pH6.5, in a ratio of 9 parts blood to 1 part anticoagulant. Platelet rich plasma (C-PRP) was separated by centrifugation at room temperature and sus- . pensions of washed platelets were prepared and resuspended in Caz+, Mg2+ free Hanks’ balanced salt solution
846
GUNDUH. R. RAO et al.
(HBSS) as described in previous publications (Clawson and White, 1971; White, 1968, 1970, 1971). A modification of Kato's (1966) method was used to prepare platelet subcelMar fractions. The washed cells were freeze-thawed and sonicated, then centrifuged at 9.000 x 8 for 15 min at 4°C to remove cellular debris. The supernatant was sedimented in the cold for 30 min at 17,000 x 8 to obtain a mitochondrial fraction and its supernate at 100.OOO x 8 for 1 h to remove microsomes. Supernatants from the last centrifugation served as the soluble fraction. Sheep vesicular gland microsomes for use in these studies were prepared in the same manner. Arachidonic acid and various free fatty acids were obtained from Nu Chek Prep, Elysian, Minnesota. DUtocopherol (Type V) was obtained from Sigma Chemical Co. Unless otherwise noted, all other enzymes and chemicals were obtained from Sigma Chemical Co., St. Louis,
Platelet release reaction. Platelets in C-PRP were labeled with 14C-serotonin (5-hydroxytryptamine, Amersham/ Searle) by a modification (White et a/., 1974) of the method of Jerushalmy and Zucker (1966) for the evaluation of the release reaction. SaMples of prelabeled platelets were exposed to acid soluble collagen or epinephrine and stirred for 3min on an aggregometer at 37°C with and without various inhibitors. After aggregation. the reaction was stopped with the addition of 0.1 M EDTA and the pellet and supernatant separated by centrifugation. Pellet and supernatant fractions containing the labeled serotonin were counted in a scintillation counter.
MO.
Addition of arachidonic acid t o frozen-thawed, sonicated platelets in the presence of N B T caused the slow formation of a weak blue color indicating only a small amount or reduction of NBT (Table 1). Vitamin E was added t o the reaction mixtures as an antioxidant and was expected t o inhibit formation of reduced NBT. Instead, vitamin E markedly potentiated the NBT reduction and development of a deep blue color in the reaction mixture (Fig. 1). The results could not be explained by a direct effect of vitamin E on NBT. since the mixture of vitamin E and N B T alone caused no reduction of the tetrazolium dye. Furthermore, both arachidonic acid and some constituent of the frozen-thawed platelets were essential to the reduction of NBT. The results indicated that vitamin E was contributing significantly to the reduction of N B T by arachidonic acid and platelet homogenate.
Nitroblue retrazolium assay. The test system used in our studies to detect free radical formation was reduction of NBT which yields a purple-blue formazan composed of reduced. insoluble NBT. D-a-tocopherol was dissolved in ethanol and used as an alcohol preparation in all the experiments. Samples without added vitamin E had similar volumes of ethanol added to the vials. For quantitation. the reduced NBT was extracted into ethyl acetate and read at 540nm. Typical reaction mixtures had 0 S m t total volume and consisted of the sodium salt of various free fatty acids, enzymes or platelet suspensions, inhibitors when studied. NBT and vitamin E. The exact concentrations of each of the compounds in this assay are presented as footnotes to the Tables. Conversion of "C-AA. Metabolities formed during the oxidation of AA by the two main pathways (lipoxygenase and cyclo-oxygenase), leading to the formation of 12~-hydroxy-5.8,10,14-eicosatetraenoicacid (HETE). and the two nonprostanoate compounds C17 hydroxytrienoic acid (HHT) and a hemiacetal derivative (PHD). were followed by the method of Hamberg and Samuelsson (1974). using ' 4C-arachidonic acid (Applied Science) as the substrate. Labeled AA was diluted with cold AA (Nu Chek Prep) to a specific activity of 25-28mCi/mM, made up as a sodium salt and diluted to 0.1 M Tris buffer pH 7.0. One m/ aliquots of each reaction mixture were incubated for 3 min at 37°C with identical amounts of labeled AA (about I pg). At the end of the incubation period, 10 m t of ethanol was added to stop the reaction. The procedure used to separate, identify and quantitate the metabolites has been described previously (Hamberg and Samuelsson, 1974; Gerrard er a/., 1976).
RESULTS
Enhancement of N B T reduction by vitamin E
Injluence of oxidative enzymes on A A peroxidation Studies were cotrducted t o evaluate the effect of various oxidative enzymes on arachidonic acid peroxidation. Several platelet fractions, including a microsoma1 fraction and a soluble fraction, contained the same enzyme activity found in frozen-thawed, sonicated platelets essential for the reduction of NBT (Table 2). Sheep vesicular gland fractions, purified
Table I. The influence of vitamin E and platelet enzymes on NBT reduction stimulated by arachidonic acid Platelet Arachidonic homogenate (100pg,hn/ VitaminE acid 50pg/m/ Protein) 1 mg/m/
+ + + -
+ + +
+ ++
NBT 100pcg/m/
Blue color
+ + + +
+ -
O.D. at 540 nm* 0.100 f 0.008$ 0.030 f 0.006
0.028 f 0.006 0.022 f 0.006
@Meanf SD (n = 3). 8 Significantly different from controls P < 0.01. Components of the reaction mixture (+) were added together and stirred at 37°C for 15 min. Absent components (-) were replaced by the appropriate buffer control. Reduced NBT was extracted into ethyl acetate and measured spectrophotometrically. Markedly enhanced reduction of NBT occurred when arachidonic acid, platelet homogenate and vitamin E were all present.
Arachidonic acid peroxidation Time - dependent Reduction of Nitroblue Tetrazolium (NET) with Platelet Homogenates ( P H I and Arachidonic Acid (An) in the Presence of Vitomin E
a47
in the presence of molecular oxygen and form reactive intermediates capable of mediating the reduction of NBT in the presence of vitamin E.
The role of the activated fatty acid in transformation of arachidonic acid by platelets
050 0
040 E
0
2
4
8
16
Time (min)
To determine whether activated AA is important in PG synthesis by platelets, NBT and vitamin E separately and in combination were added to samples of frozen-thawed, sonicated platelets and then stirred with I4C-AA for 3 min. NBT alone partially inhibited platelet production of HETE and H H T (Fig. 2). Vitamin E in combination with NBT resulted in marked inhibition of HETE and H H T formation as well as a significant decrease in the production of thromboxane B2. Inhibition of the formation of HETE, H H T and thromboxane B, suggests that initial peroxidation of the fatty acid is important for subsequent transformation by platelet lipoxygenase and cyclooxygenase.
Figure 1. Components of the reaction mixture (AA 50pg/m/, pH 100pg protein/m/. Vitamin E 1 mg/m/. NBT 100pg/m/) were added together and stirred at 37°C for various time periods. Components deleted in the reaction mixture were replaced by the appropriate buffer controls. Reduced NBT was extracted into ethyl acetate and measured spectrophotometrically. Marked enhanced reduction of NBT occurred when arachidonic acid, platelet homogenate and vitamin E were all present.
Role of arachidonic acid peroxidation in platelet function
horseradish peroxidase, lactoperoxidase and soybean lipoxidase also duplicated the activity found in platelet homogenate. When these reactions were carried out under N, atmosphere, there was marked inhibition. These results indicated that several oxidative enzymes have the capacity to interact with fatty acids
Since NBT and vitamin E together inhibited formation of products from AA, we studied the role of AA peroxidation in platelet function. Vitamin E or NBT alone partially inhibited the second wave of epinephrine induced platelet aggregation. When combined, the two agents were more effective in blocking the platelet response to epinephrine than either one alone
Table 2. The influence of various oxidative enzymes on NBT reduction Arachidonic acid
Oxidative enzyme source (protein concentration
O.D. at
100 pg/m4
Vitamin E 1 mg/m/
NBT
50 aim/
100 pg/m/
540 nm*
+
-
+
+
0.024 f 0.009
+
Platelet homogena te
+
+
0.096
+
Platelet microsomes
+
+
0.104 f 0.01 24
+
Sheep vesicular gland microsomes
+
+
0.1 12 f 0.01 I #
+
Horseradish peroxidase
+
+
0.108 f 0.0168
+
Soybean lipoxygenase
+
+
0.100
0.01 18
*
0.012g
* Mean & SE ( n = 4). 8 Statistically different from controls (P< 0.01). The influence of various oxidative enzymes on the reduction of NBT in the presence of vitamin E was measured 15 min after addition of arachidonic acid. With no oxidative enzyme present ( - ) tittle NBT reduction occurred. Isolated platelet microsornes, sheep vesicular gland microsomes, horseradish peroxidase or soybean lipoxygenase could all initiate substantial NBT reduction when arachidonic acid and vitamin E were both present. Omission of arachidonic acid, or vitamin E separately with each enzyme, as done with the platelet homogenate in Table 1, gave values in the same range as the control sample with no oxidative enzyme present (data not shown).
GUNDUH. R. RAO et
848
a/.
Distance from Origin (cm) Figure 2. Transformation of arachidonic acid by platelets studied by thin-layer radiochromatography. AA was stirred in buffer for 3 min. extracted, converted to methyl ester derivatives and resolved by thin-layer chromatography. The chromatogram shown on the upper left corner reveals a single peak (Me-AA). When the AA was stirred with a platelet homogenate for 3 min. most of the AA was converted to HETE. HHT or thromboxane B, (Upper right). Addition of NBT alone (lower left) or NBT and vitamin E (Lower right) significantly reduced the conversion of AA.
(Fig. 3). Similar inhibitory effects of NBT and vitamin E were observed when platelets were stimulated with other agents such as collagen, ADP. thrombin and arachidonic acid. In addition, half the concentrations of NBT and vitamin E which separately caused little inhibition also blocked the platelet response to the same stimuli (Fig. 4).
Effects of NET (100pg/mll and Vitamin E ( 1 U/ml) on Epinephrine (5.5 x 10-6) Induced Aggregation
AT*
-
-
NBT and vitamin E also affected the secretory responses of the pladelets. Concentrations of NBT or vitamin E which sdparately had moderate effects on the release of serotonin almost completely blocked the release reaction in response to epinephrine, ADP, collagen, thrombin and arachidonic acid when the two agents were used in combination (Table 3). Effects of NBT ( l O O p q / m l ~ond Vilomin E ( 0 7 5 U / m l ) on Thrombln (0.1 W m l ) Induced Aggregotion
NBT + Vit E
Im m l e
*AT -change in lqht lrmsmission
Figure 3. Tracings obtained from samples of C-PRP exposed to the same concentration of epinephrine after incubation with NBT, vitamin E,both agents or buffer alone. NBT and vitamin E delay the onset of the second wave of aggregation when used separately. Together, NBT and vitamin E at concentrations which delay aggregation separately completely block epinephrine induced aggregation. '
*AT-chonqe --
In Iiqhl
transmlsslm
Figure 4. Tracings obtained from samples of C-PRP exposed to the same concentrations of thrombin after incubation with NBT. vitamin E, both agents or buffer alone. NBT delayed the onset of second wave of aggregation. Whereas vitamin E , partially inhibited the second wave However, in combi ation even at half the concentration of these agentq a $nplete block of thrombin induced aggregation was obtained.
Arachidonic acid peroxidation
849
Table 3. Effect of NBT (100 pg), Vitamin E ( I I.U.) and their combination on the release of ‘‘C-serotonin from platelets stimulated by various aggregating agents Per cent release. Aggregating agent
No agg. agent
NBT
Vit. E
2.8 f 0.27 6.3 0.17 6.1 & 0.39 3.7 f 0.41 4.2 k 0.26
9.7f 0.76 8.4 f 0.28 38.5 3.0 18.7 & 0.57 12.6f 0.42
30.2 k 0.82 9.8 f 0.66 25.6 k 4.2 7.1 f 0.55 14.8 k 0.12
Epinephrine 5.5pM ADP 4 p M Collagen 50 pg/mt Thrombin 0.2U/m/ Arachidonic acid 250 pg
NBT
+ vit. E
3.4 f 0.26 3.1 k 0.12 7.3 f 0.62 2.6 _+ 0.25 4.3 f 0.22
No inhibitors
48.4 k 0.72 27.8 f 3.3 63.5 k 2.3 57.4 f 0.57 50.2 f 0.26
* Mean and
the standard error ( n = 3). Influence of NBT, vitamin E or NBT-vitamin E on the secretion of labeled serotonin from prelabeled platelets. Serotonin prelabeled platelets were aggregated with various agents for 3 min. Serotonin released varied considerably when NBT or vitamin E was tested separately. Combination of both agents totally blocked the release reaction.
et a!. However, the earlier workers did not use vita-
DISCUSSION
min E together with the NBT. The hypothesis that peroxidation of arachidonic acid has a significant role in PGG2 synthesis was further supported by our studies, on the influence of NBT-vitamin E on platelet aggregation and secretion (White et al., 1977). Steiner and others have shown previously that vitamin E alone is a partial inhibitor of platelet function (Steiner and Anastasi, 1976; Fong, 1976), and our studies have confirmed that observation. However, the vitamin E-NBT combination was much more NBT 22 2H’ + NBTH2. effective in inhibiting epinephrine, collagen, ADP, Yellow Blue thrombin and arachidonic acid induced aggregation NBT was used in this study to detect the peroxida- than either agent alone. In addition, this combination tion of arachidonic acid during platelet prostaglandin effectively blocked the release reaction as evidenced biosynthesis. The discovery that vitamin E poten- by the inhibition of 14C serotonin release from pretiated NBT reduction in the presence of platelets labeled platelets. The results suggest that the prevenstimulated by AA facilitated the study of this reaction. tion of AA peroxidation blocks platelet PGGz synSeveral different oxidative enzymes were found which thesis, aggregation and the release reaction. The “acticould substitute for the platelet enzymes, including vated species” detected by vitamin E and NBT is not horse-radish peroxidase, lactoperoxidase, and soy- superoxide nor a breakdown product of H 2 0 , since bean lipoxygenase. These results suggest a primary neither superoxide dismutase nor catalase inhibit the interaction between certain oxidative enzymes and reaction. The specific nature of the species, perhaps long chain fatty acids resulting in the generation of a free radical. is as yet uncertain and further investigaan active species, very probably the initiation of fatty tion of its nature is in progress. In conclusion, the NBT-vitamin E test system deacid free radical formation. Such a free radical initiation reaction has been postulated to occur as the first veloped in this investigation is a sensitive technique step in lipid peroxidation reactions. In addition, free for detecting peroxidation of fatty acids (probably radical generating systems can activate platelets and fatty acid free radicals). Application to the initial steps this activation process is not inhibited by superoxide of prostaglandin synthesis suggests that conversion of AA to an active intermediate is required for its inserdismutase (Handin et al., 1977). The vitamin E-NBT combination not only detects tion into either the lipoxygenase or cyclo-oxygenase “activated species” but by its very nature removes and pathways. The inhibitory effect of NBT-vitamin E on thus prevents the participation of such an inter- platelet aggregation and secretion supports this hymediate in the synthetic process. Our studies showed pothesis. On the basis of this study, it is reasonable that conversion of AA to HHT and thromboxane B, to suggest that the formation of an active inter(PHD), products of platelet FGGz synthesis, was sig- mediate of endogenous arachidonic acid may be a nificantly suppressed by the combination of NBT and fundamental step regulating the response of platelets vitamin E. The result differs from findings of Marnett to most, if not all, aggregating agents.
The present investigation has resulted in the development of a novel test system for detecting peroxidation of fatty acids by oxidative enzymes. Essential components of the test system include (1) an oxidative enzyme, (2) one of a variety of fatty acid, (3) molecular oxygen, (4) vitamin E and ( 5 ) nitroblue tetrazolium. NBT is a yellow dye which is converted to its reduced “blue” form when reducing equivalents are arailable.
+ +
REFERENCES
Barber, A. A. and F. Bernheim (1967) Adv. Gerontol. Res. 2, 355-403. Clawson, C.C. and J. G. White (1971) Am. J . Path. 65, 367-380. P.A.P. 2 8 4 1 5 - 0 6
850
GUNDUH. R. RAO et a/. Fong, J. S. C. (1976) Experientia 32. 639-641. Gerrard, J. M., J. G. White, G. H. R. Rao and D. Townsend (1976) Am. J . Path. 83, 283294. Graziano, J. H.. D. R. Miller, R. W. Grady and A. Cerami (1976) Br. J. Haematol. 32, 251-356. Handin, R. 1.. R. Karabin and G. I. Boxer (1977) J. Clin. Invest. 59, 959-965. Hamberg, M. and B. Samuelsson (1973) Proc. Natl. Acad. Sci. U.S.71. 3824-3828. Hamberg. M. and B. Samuelsson (1974) Proc. Narl. Acad. Sci. U.S.71, 34W3404. Hamberg. M.. J. Svensson. T. Wakabayashi and B. Samuelsson (1974) Proc. Natl. Acad. Sci. U.S. 71. 345-349. Hamberg, M.. J. Svensson and B. Samuelsson (1975) Proc. Natl. Acad. Sci. U.S. 72, 29942998. Jerushalmy, Z. and M. B. Zucker (1966) Thromb. Diath. Haemorrh. 15, 413419. Kato. R. (1966) J . Biochem. 59. 574-583. Malmsten, C.. M. Hamberg. J. Svensson and B. Samuelsson (1975) Proc. Natl. Acad. Sci. U.S. 72. 1446 1450. Marcus, A. J., S. T. Silk. L. B. Safier and H. L. Ullman (1977) J . Clin. Invest. 59. 149-158. Marnett, L. J.. P. Wlodawaer and B. Samuelsson (1975) J. Biol. Chem. 250, 8510-8517. Needelman. P., S. Moncada. S. Bunting, J. R. Vane, M. Hamberg and B. Samuelsson (1976a) Nature 261, 558-560. Needleman. P., M. Minkes and A. Raz (1976b) Science 193, 163-165. Plaa, G. L. and H. Witschi (1976) Ann. Rev. Pharmac. Toxicol. 16, 125-129. Smith. J. B. and A. L. Willis (1970) Br. J . Pharmac. 40, 545-546. Smith, J. B. and A. L. Willis (1971) Nature (New Biol.) 231, 235-237. Smith. J. B., C. Ingerman. J. J. Kocsis and M. J. Silver (1974) J. Clin. Invest. 53, 1468-1472. Silver, M. J.. J. B. Smith, C. Ingerman and J. J. Kocsis (1972) Prostaglandins 1, 429-436. Silver, M. J., J. B. Smith, C. Ingerman and J. J. Kocsis (1973) Prostaglandins 4, 863-875. Steiner. M. and J. Anastasi (1976) J . Clin. Invest. 57, 732-737. Weiss, H. J.. A. L. Willis, D. C. Kuhn and H. Brand (1976) Br. J . Haematol. 32, 257-271. Willis. A. L.. F. M.Vane, D. C. Kuhn. C. G. Scott and M. Petrin (1974) Prostaglandins 8, 453463. White, J. G. (1968) Blood 31, 606-622. White, J. G . (1970) Am. J . Path. 58. 19-28. White J . G. (1971) In The Circulating Platelet (Edited by S. A. Johnson), Academic Press. New York. White, J. G.. G. H. R. Rao and R. D. Estensen (1974) Am. J. Path. 75, 301-312. White, J. G., G. H.R. Rao and J. M. Gerrard (1977) Am. J. Path. (in press).
Abstract-The initial oxygenation or peroxidation of arachidonic acid seems to be an essential step for the synthesis of cyclic endoperoxides and prostaglandins. There has been some evidence and considerable interest in the role of superoxide anion, hydroxyl radicals or singlet oxygen as a source of oxygen in the formation of the active species (free radicals). A test capable of detecting active intermediates of lipid peroxidation and useful for studying the role of free radicals has been developed. The test resulted from the discovery that vitamin E markedly enhanced the reduction of nitroblue tetrazolium (NBT) during arachidonic acid peroxidation. Intact platelets, microsomes. sheep vesicular gland enzymes or peroxidases could provide essential enzyme activity. NBT and vitamin E when added to platelet microsornes inhibited the conversion of I4C arachidonic acid to HETE, HHT and thromboxanes. The combination also inhibited aggregation of platelets stimulated by collagen, thrombin, ADP and epinephrine. Prolonged incubation with these agents at the highest concentrations used in the study caused no change in morphology and had no deleterious effect on platelet levels of adenine nucleotides and serotonin. Results of our preliminary studies suggest that NBT and vitamin E can detect intermediates of lipid peroxidation, inhibit the conversion of arachidonic acid, prevent platelet aggregation and the release reaction without damaging the platelets morphologically or biochemically. As both the agents are scavengers of free radicals and in combination exert synergistic effects, the test system may serve as a probe in various free radical mediated events and may offer some degree of protection against free radical mediated pathological processes.
INTRODUCTION
The platelet prostaglandin (PG) endoperoxide synthetase enzyme converts arachidonic acid to PGG2 and PGH2.These labile compounds are then metabolized primarily to the highly active thromboxane A2 or to the hydroxy fatty acid, 12~-hydroxy-5,8,10heptadecatrienoic acid (HHT) and malondialdehyde (MDA) (Hamberg and Samuelsson, 1973, 1974; Hamberg et al., 1974, 1975; Needleman et al., 1976a. b). A large body of literature (Smith and Willis. 1970, 1971; Silver et af., 1972, 1973; Smith et af., 1974; Willis. 1974, Malmsten et al., 1975; Weiss et a/., 1976) now supports the concept that arachidonic acid (AA) transformation plays a major role in platelet physiology. Our interest in the oxidation of AA by platelets was stimulated by the observations of Gerrard et al. (1976) that aminotriazole, an inhibitor of peroxidase, was found to inhibit platelet PG synthesis, secretion and aggregation. These observations suggested that arachidonic acid, released from membrane phospholipids after platelet activation, reacts with the peroxidase and that a product of AA peroxidation is essential for prostaglandin endoperoxide synthesis. Several workers (Barber and Bernheim, 1967; Hamberg et al., 1974; PIaa and Witschi, 1976; Graziano et af., 1976) have suggested that a reactive enzyme, fatty acidoxygen intermediate or an active fatty acid (free radical), is formed during the initial peroxidation process of unsaturated fatty acids. During the course of inves-
tigations to find a method capable of detecting such an interaction in platelet AA peroxidation, we found the nitroblue tetrazolium (NBT) reduction assay to be useful. The ditetrazolium 3’,3’(4,4-di-o-anisyIene)2,2’-di@-nitrophenyl)-bis (5 phenyl) is the most widely employed of the tetrazolium salts, and has been extensively used in studies on normal and abnormal neutrophils to detect the activity of oxidative enzyme systems. When reducing equivalents are available in the medium the yellow dye is reduced to a blue colored product. Recently, superoxide production and reducing activity of platelets have been detected in test systems employing NBT (Marcus et al., 1977). In the present investigation we have found that NBT is a valuable agent for detecting arachidonic acid peroxidation when vitamin E is added t o the system. The findings indicate that peroxidation of AA is an important step for PG synthesis and that prevention of AA peroxidation blocks the platelet PG synthesis, secretion and aggregation. MATERIALS AND METHODS
Platelets for these studies were obtained from healthy adult volunteers after informed consent. Blood drawn from an anticubital vein into siliconized glass or plastic syringes was mixed immediately with 3.8% trisodium citrate or citrate-citric acid, pH6.5, in a ratio of 9 parts blood to 1 part anticoagulant. Platelet rich plasma (C-PRP) was separated by centrifugation at room temperature and sus- . pensions of washed platelets were prepared and resuspended in Caz+, Mg2+ free Hanks’ balanced salt solution
846
GUNDUH. R. RAO et al.
(HBSS) as described in previous publications (Clawson and White, 1971; White, 1968, 1970, 1971). A modification of Kato's (1966) method was used to prepare platelet subcelMar fractions. The washed cells were freeze-thawed and sonicated, then centrifuged at 9.000 x 8 for 15 min at 4°C to remove cellular debris. The supernatant was sedimented in the cold for 30 min at 17,000 x 8 to obtain a mitochondrial fraction and its supernate at 100.OOO x 8 for 1 h to remove microsomes. Supernatants from the last centrifugation served as the soluble fraction. Sheep vesicular gland microsomes for use in these studies were prepared in the same manner. Arachidonic acid and various free fatty acids were obtained from Nu Chek Prep, Elysian, Minnesota. DUtocopherol (Type V) was obtained from Sigma Chemical Co. Unless otherwise noted, all other enzymes and chemicals were obtained from Sigma Chemical Co., St. Louis,
Platelet release reaction. Platelets in C-PRP were labeled with 14C-serotonin (5-hydroxytryptamine, Amersham/ Searle) by a modification (White et a/., 1974) of the method of Jerushalmy and Zucker (1966) for the evaluation of the release reaction. SaMples of prelabeled platelets were exposed to acid soluble collagen or epinephrine and stirred for 3min on an aggregometer at 37°C with and without various inhibitors. After aggregation. the reaction was stopped with the addition of 0.1 M EDTA and the pellet and supernatant separated by centrifugation. Pellet and supernatant fractions containing the labeled serotonin were counted in a scintillation counter.
MO.
Addition of arachidonic acid t o frozen-thawed, sonicated platelets in the presence of N B T caused the slow formation of a weak blue color indicating only a small amount or reduction of NBT (Table 1). Vitamin E was added t o the reaction mixtures as an antioxidant and was expected t o inhibit formation of reduced NBT. Instead, vitamin E markedly potentiated the NBT reduction and development of a deep blue color in the reaction mixture (Fig. 1). The results could not be explained by a direct effect of vitamin E on NBT. since the mixture of vitamin E and N B T alone caused no reduction of the tetrazolium dye. Furthermore, both arachidonic acid and some constituent of the frozen-thawed platelets were essential to the reduction of NBT. The results indicated that vitamin E was contributing significantly to the reduction of N B T by arachidonic acid and platelet homogenate.
Nitroblue retrazolium assay. The test system used in our studies to detect free radical formation was reduction of NBT which yields a purple-blue formazan composed of reduced. insoluble NBT. D-a-tocopherol was dissolved in ethanol and used as an alcohol preparation in all the experiments. Samples without added vitamin E had similar volumes of ethanol added to the vials. For quantitation. the reduced NBT was extracted into ethyl acetate and read at 540nm. Typical reaction mixtures had 0 S m t total volume and consisted of the sodium salt of various free fatty acids, enzymes or platelet suspensions, inhibitors when studied. NBT and vitamin E. The exact concentrations of each of the compounds in this assay are presented as footnotes to the Tables. Conversion of "C-AA. Metabolities formed during the oxidation of AA by the two main pathways (lipoxygenase and cyclo-oxygenase), leading to the formation of 12~-hydroxy-5.8,10,14-eicosatetraenoicacid (HETE). and the two nonprostanoate compounds C17 hydroxytrienoic acid (HHT) and a hemiacetal derivative (PHD). were followed by the method of Hamberg and Samuelsson (1974). using ' 4C-arachidonic acid (Applied Science) as the substrate. Labeled AA was diluted with cold AA (Nu Chek Prep) to a specific activity of 25-28mCi/mM, made up as a sodium salt and diluted to 0.1 M Tris buffer pH 7.0. One m/ aliquots of each reaction mixture were incubated for 3 min at 37°C with identical amounts of labeled AA (about I pg). At the end of the incubation period, 10 m t of ethanol was added to stop the reaction. The procedure used to separate, identify and quantitate the metabolites has been described previously (Hamberg and Samuelsson, 1974; Gerrard er a/., 1976).
RESULTS
Enhancement of N B T reduction by vitamin E
Injluence of oxidative enzymes on A A peroxidation Studies were cotrducted t o evaluate the effect of various oxidative enzymes on arachidonic acid peroxidation. Several platelet fractions, including a microsoma1 fraction and a soluble fraction, contained the same enzyme activity found in frozen-thawed, sonicated platelets essential for the reduction of NBT (Table 2). Sheep vesicular gland fractions, purified
Table I. The influence of vitamin E and platelet enzymes on NBT reduction stimulated by arachidonic acid Platelet Arachidonic homogenate (100pg,hn/ VitaminE acid 50pg/m/ Protein) 1 mg/m/
+ + + -
+ + +
+ ++
NBT 100pcg/m/
Blue color
+ + + +
+ -
O.D. at 540 nm* 0.100 f 0.008$ 0.030 f 0.006
0.028 f 0.006 0.022 f 0.006
@Meanf SD (n = 3). 8 Significantly different from controls P < 0.01. Components of the reaction mixture (+) were added together and stirred at 37°C for 15 min. Absent components (-) were replaced by the appropriate buffer control. Reduced NBT was extracted into ethyl acetate and measured spectrophotometrically. Markedly enhanced reduction of NBT occurred when arachidonic acid, platelet homogenate and vitamin E were all present.
Arachidonic acid peroxidation Time - dependent Reduction of Nitroblue Tetrazolium (NET) with Platelet Homogenates ( P H I and Arachidonic Acid (An) in the Presence of Vitomin E
a47
in the presence of molecular oxygen and form reactive intermediates capable of mediating the reduction of NBT in the presence of vitamin E.
The role of the activated fatty acid in transformation of arachidonic acid by platelets
050 0
040 E
0
2
4
8
16
Time (min)
To determine whether activated AA is important in PG synthesis by platelets, NBT and vitamin E separately and in combination were added to samples of frozen-thawed, sonicated platelets and then stirred with I4C-AA for 3 min. NBT alone partially inhibited platelet production of HETE and H H T (Fig. 2). Vitamin E in combination with NBT resulted in marked inhibition of HETE and H H T formation as well as a significant decrease in the production of thromboxane B2. Inhibition of the formation of HETE, H H T and thromboxane B, suggests that initial peroxidation of the fatty acid is important for subsequent transformation by platelet lipoxygenase and cyclooxygenase.
Figure 1. Components of the reaction mixture (AA 50pg/m/, pH 100pg protein/m/. Vitamin E 1 mg/m/. NBT 100pg/m/) were added together and stirred at 37°C for various time periods. Components deleted in the reaction mixture were replaced by the appropriate buffer controls. Reduced NBT was extracted into ethyl acetate and measured spectrophotometrically. Marked enhanced reduction of NBT occurred when arachidonic acid, platelet homogenate and vitamin E were all present.
Role of arachidonic acid peroxidation in platelet function
horseradish peroxidase, lactoperoxidase and soybean lipoxidase also duplicated the activity found in platelet homogenate. When these reactions were carried out under N, atmosphere, there was marked inhibition. These results indicated that several oxidative enzymes have the capacity to interact with fatty acids
Since NBT and vitamin E together inhibited formation of products from AA, we studied the role of AA peroxidation in platelet function. Vitamin E or NBT alone partially inhibited the second wave of epinephrine induced platelet aggregation. When combined, the two agents were more effective in blocking the platelet response to epinephrine than either one alone
Table 2. The influence of various oxidative enzymes on NBT reduction Arachidonic acid
Oxidative enzyme source (protein concentration
O.D. at
100 pg/m4
Vitamin E 1 mg/m/
NBT
50 aim/
100 pg/m/
540 nm*
+
-
+
+
0.024 f 0.009
+
Platelet homogena te
+
+
0.096
+
Platelet microsomes
+
+
0.104 f 0.01 24
+
Sheep vesicular gland microsomes
+
+
0.1 12 f 0.01 I #
+
Horseradish peroxidase
+
+
0.108 f 0.0168
+
Soybean lipoxygenase
+
+
0.100
0.01 18
*
0.012g
* Mean & SE ( n = 4). 8 Statistically different from controls (P< 0.01). The influence of various oxidative enzymes on the reduction of NBT in the presence of vitamin E was measured 15 min after addition of arachidonic acid. With no oxidative enzyme present ( - ) tittle NBT reduction occurred. Isolated platelet microsornes, sheep vesicular gland microsomes, horseradish peroxidase or soybean lipoxygenase could all initiate substantial NBT reduction when arachidonic acid and vitamin E were both present. Omission of arachidonic acid, or vitamin E separately with each enzyme, as done with the platelet homogenate in Table 1, gave values in the same range as the control sample with no oxidative enzyme present (data not shown).
GUNDUH. R. RAO et
848
a/.
Distance from Origin (cm) Figure 2. Transformation of arachidonic acid by platelets studied by thin-layer radiochromatography. AA was stirred in buffer for 3 min. extracted, converted to methyl ester derivatives and resolved by thin-layer chromatography. The chromatogram shown on the upper left corner reveals a single peak (Me-AA). When the AA was stirred with a platelet homogenate for 3 min. most of the AA was converted to HETE. HHT or thromboxane B, (Upper right). Addition of NBT alone (lower left) or NBT and vitamin E (Lower right) significantly reduced the conversion of AA.
(Fig. 3). Similar inhibitory effects of NBT and vitamin E were observed when platelets were stimulated with other agents such as collagen, ADP. thrombin and arachidonic acid. In addition, half the concentrations of NBT and vitamin E which separately caused little inhibition also blocked the platelet response to the same stimuli (Fig. 4).
Effects of NET (100pg/mll and Vitamin E ( 1 U/ml) on Epinephrine (5.5 x 10-6) Induced Aggregation
AT*
-
-
NBT and vitamin E also affected the secretory responses of the pladelets. Concentrations of NBT or vitamin E which sdparately had moderate effects on the release of serotonin almost completely blocked the release reaction in response to epinephrine, ADP, collagen, thrombin and arachidonic acid when the two agents were used in combination (Table 3). Effects of NBT ( l O O p q / m l ~ond Vilomin E ( 0 7 5 U / m l ) on Thrombln (0.1 W m l ) Induced Aggregotion
NBT + Vit E
Im m l e
*AT -change in lqht lrmsmission
Figure 3. Tracings obtained from samples of C-PRP exposed to the same concentration of epinephrine after incubation with NBT, vitamin E,both agents or buffer alone. NBT and vitamin E delay the onset of the second wave of aggregation when used separately. Together, NBT and vitamin E at concentrations which delay aggregation separately completely block epinephrine induced aggregation. '
*AT-chonqe --
In Iiqhl
transmlsslm
Figure 4. Tracings obtained from samples of C-PRP exposed to the same concentrations of thrombin after incubation with NBT. vitamin E, both agents or buffer alone. NBT delayed the onset of second wave of aggregation. Whereas vitamin E , partially inhibited the second wave However, in combi ation even at half the concentration of these agentq a $nplete block of thrombin induced aggregation was obtained.
Arachidonic acid peroxidation
849
Table 3. Effect of NBT (100 pg), Vitamin E ( I I.U.) and their combination on the release of ‘‘C-serotonin from platelets stimulated by various aggregating agents Per cent release. Aggregating agent
No agg. agent
NBT
Vit. E
2.8 f 0.27 6.3 0.17 6.1 & 0.39 3.7 f 0.41 4.2 k 0.26
9.7f 0.76 8.4 f 0.28 38.5 3.0 18.7 & 0.57 12.6f 0.42
30.2 k 0.82 9.8 f 0.66 25.6 k 4.2 7.1 f 0.55 14.8 k 0.12
Epinephrine 5.5pM ADP 4 p M Collagen 50 pg/mt Thrombin 0.2U/m/ Arachidonic acid 250 pg
NBT
+ vit. E
3.4 f 0.26 3.1 k 0.12 7.3 f 0.62 2.6 _+ 0.25 4.3 f 0.22
No inhibitors
48.4 k 0.72 27.8 f 3.3 63.5 k 2.3 57.4 f 0.57 50.2 f 0.26
* Mean and
the standard error ( n = 3). Influence of NBT, vitamin E or NBT-vitamin E on the secretion of labeled serotonin from prelabeled platelets. Serotonin prelabeled platelets were aggregated with various agents for 3 min. Serotonin released varied considerably when NBT or vitamin E was tested separately. Combination of both agents totally blocked the release reaction.
et a!. However, the earlier workers did not use vita-
DISCUSSION
min E together with the NBT. The hypothesis that peroxidation of arachidonic acid has a significant role in PGG2 synthesis was further supported by our studies, on the influence of NBT-vitamin E on platelet aggregation and secretion (White et al., 1977). Steiner and others have shown previously that vitamin E alone is a partial inhibitor of platelet function (Steiner and Anastasi, 1976; Fong, 1976), and our studies have confirmed that observation. However, the vitamin E-NBT combination was much more NBT 22 2H’ + NBTH2. effective in inhibiting epinephrine, collagen, ADP, Yellow Blue thrombin and arachidonic acid induced aggregation NBT was used in this study to detect the peroxida- than either agent alone. In addition, this combination tion of arachidonic acid during platelet prostaglandin effectively blocked the release reaction as evidenced biosynthesis. The discovery that vitamin E poten- by the inhibition of 14C serotonin release from pretiated NBT reduction in the presence of platelets labeled platelets. The results suggest that the prevenstimulated by AA facilitated the study of this reaction. tion of AA peroxidation blocks platelet PGGz synSeveral different oxidative enzymes were found which thesis, aggregation and the release reaction. The “acticould substitute for the platelet enzymes, including vated species” detected by vitamin E and NBT is not horse-radish peroxidase, lactoperoxidase, and soy- superoxide nor a breakdown product of H 2 0 , since bean lipoxygenase. These results suggest a primary neither superoxide dismutase nor catalase inhibit the interaction between certain oxidative enzymes and reaction. The specific nature of the species, perhaps long chain fatty acids resulting in the generation of a free radical. is as yet uncertain and further investigaan active species, very probably the initiation of fatty tion of its nature is in progress. In conclusion, the NBT-vitamin E test system deacid free radical formation. Such a free radical initiation reaction has been postulated to occur as the first veloped in this investigation is a sensitive technique step in lipid peroxidation reactions. In addition, free for detecting peroxidation of fatty acids (probably radical generating systems can activate platelets and fatty acid free radicals). Application to the initial steps this activation process is not inhibited by superoxide of prostaglandin synthesis suggests that conversion of AA to an active intermediate is required for its inserdismutase (Handin et al., 1977). The vitamin E-NBT combination not only detects tion into either the lipoxygenase or cyclo-oxygenase “activated species” but by its very nature removes and pathways. The inhibitory effect of NBT-vitamin E on thus prevents the participation of such an inter- platelet aggregation and secretion supports this hymediate in the synthetic process. Our studies showed pothesis. On the basis of this study, it is reasonable that conversion of AA to HHT and thromboxane B, to suggest that the formation of an active inter(PHD), products of platelet FGGz synthesis, was sig- mediate of endogenous arachidonic acid may be a nificantly suppressed by the combination of NBT and fundamental step regulating the response of platelets vitamin E. The result differs from findings of Marnett to most, if not all, aggregating agents.
The present investigation has resulted in the development of a novel test system for detecting peroxidation of fatty acids by oxidative enzymes. Essential components of the test system include (1) an oxidative enzyme, (2) one of a variety of fatty acid, (3) molecular oxygen, (4) vitamin E and ( 5 ) nitroblue tetrazolium. NBT is a yellow dye which is converted to its reduced “blue” form when reducing equivalents are arailable.
+ +
REFERENCES
Barber, A. A. and F. Bernheim (1967) Adv. Gerontol. Res. 2, 355-403. Clawson, C.C. and J. G. White (1971) Am. J . Path. 65, 367-380. P.A.P. 2 8 4 1 5 - 0 6
850
GUNDUH. R. RAO et a/. Fong, J. S. C. (1976) Experientia 32. 639-641. Gerrard, J. M., J. G. White, G. H. R. Rao and D. Townsend (1976) Am. J . Path. 83, 283294. Graziano, J. H.. D. R. Miller, R. W. Grady and A. Cerami (1976) Br. J. Haematol. 32, 251-356. Handin, R. 1.. R. Karabin and G. I. Boxer (1977) J. Clin. Invest. 59, 959-965. Hamberg, M. and B. Samuelsson (1973) Proc. Natl. Acad. Sci. U.S.71. 3824-3828. Hamberg. M. and B. Samuelsson (1974) Proc. Narl. Acad. Sci. U.S.71, 34W3404. Hamberg. M.. J. Svensson. T. Wakabayashi and B. Samuelsson (1974) Proc. Natl. Acad. Sci. U.S. 71. 345-349. Hamberg, M.. J. Svensson and B. Samuelsson (1975) Proc. Natl. Acad. Sci. U.S. 72, 29942998. Jerushalmy, Z. and M. B. Zucker (1966) Thromb. Diath. Haemorrh. 15, 413419. Kato. R. (1966) J . Biochem. 59. 574-583. Malmsten, C.. M. Hamberg. J. Svensson and B. Samuelsson (1975) Proc. Natl. Acad. Sci. U.S. 72. 1446 1450. Marcus, A. J., S. T. Silk. L. B. Safier and H. L. Ullman (1977) J . Clin. Invest. 59. 149-158. Marnett, L. J.. P. Wlodawaer and B. Samuelsson (1975) J. Biol. Chem. 250, 8510-8517. Needelman. P., S. Moncada. S. Bunting, J. R. Vane, M. Hamberg and B. Samuelsson (1976a) Nature 261, 558-560. Needleman. P., M. Minkes and A. Raz (1976b) Science 193, 163-165. Plaa, G. L. and H. Witschi (1976) Ann. Rev. Pharmac. Toxicol. 16, 125-129. Smith. J. B. and A. L. Willis (1970) Br. J . Pharmac. 40, 545-546. Smith, J. B. and A. L. Willis (1971) Nature (New Biol.) 231, 235-237. Smith. J. B., C. Ingerman. J. J. Kocsis and M. J. Silver (1974) J. Clin. Invest. 53, 1468-1472. Silver, M. J.. J. B. Smith, C. Ingerman and J. J. Kocsis (1972) Prostaglandins 1, 429-436. Silver, M. J., J. B. Smith, C. Ingerman and J. J. Kocsis (1973) Prostaglandins 4, 863-875. Steiner. M. and J. Anastasi (1976) J . Clin. Invest. 57, 732-737. Weiss, H. J.. A. L. Willis, D. C. Kuhn and H. Brand (1976) Br. J . Haematol. 32, 257-271. Willis. A. L.. F. M.Vane, D. C. Kuhn. C. G. Scott and M. Petrin (1974) Prostaglandins 8, 453463. White, J. G. (1968) Blood 31, 606-622. White, J. G . (1970) Am. J . Path. 58. 19-28. White J . G. (1971) In The Circulating Platelet (Edited by S. A. Johnson), Academic Press. New York. White, J. G.. G. H. R. Rao and R. D. Estensen (1974) Am. J. Path. 75, 301-312. White, J. G., G. H.R. Rao and J. M. Gerrard (1977) Am. J. Path. (in press).
