Biochemistry of the Eicosanoids: Cyclooxygenase and Lipoxygenase Products of Polyunsaturated Fatty Acids

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1 Lipids in Modern Nutrition, edited by M. Horisberger and U. Bracco. Nestle Nutrition, Vevey/Raven Press, New York Biochemistry of the Eicosanoids: Cyclooxygenase and Lipoxygenase Products of Polyunsaturated Fatty Acids Elisabeth Granstrom Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden Certain polyunsaturated fatty acids can be metabolized by oxygenation into a large family of biologically active substances, the so-called prostanoids or eicosanoids. This family includes the prostaglandins, thromboxanes, prostacyclins, leukotrienes, lipoxins, and also a number of related mono-, di-, and trihydroxy compounds as well as epoxygenated fatty acids. Most of these substances are potent compounds and display a wide variety of effects in many biological systems. Among these are effects on blood pressure, smooth muscle contractility, kidney function, cyclic nucleotide levels, glandular excretion, hormone secretion, platelet aggregation, vessel tone, temperature regulation, lipolysis, chemotaxis, immune response, cell growth and differentiation, electrolyte and water balance, etc. In combination with the fact that the eicosanoids are almost ubiquitous in the mammalian body, it is not surprising that these substances are supposed to be involved in many physiological as well as pathological conditions in the organism. Thus, altered eicosanoid levels are sometimes found to coincide with the appearance of certain symptoms, and normalization occurs upon successful treatment of the condition with drugs known to interfere with eicosanoid biosynthesis or metabolism. The major classes of eicosanoids will be described below, and some of their possible biological roles will be discussed. PRECURSOR FATTY ACIDS The probably most important precursor fatty acid in this field is arachidonic acid, 5,8,11,14-all-cw-eicosatetraenoic acid (20:4to6), which gives rise to prostaglandins (PGs) and thromboxanes (TXs) of the 2-series and leukotrienes (LTs) of the 4-series (the figure indicates the number of double bonds in the product, see below). Other polyunsaturated fatty acids are however also of interest, namely 8,11,14-eicosatrienoic acid (dihomo-7-linolenic acid, 20:3«6), precursor of the 1-series of PGs and TXs and one 3-series of LTs; 5,8,11-eicosatrienoic acid, pre- 59

2 60 BIOCHEMISTRY OF EICOSANOIDS cursor of the "normal" 3-series of LTs; and 5,8,11,14,17-eicosapentaenoic acid (timnodonic acid, 20:5w3), which gives rise to the PGs and TXs of the 3-series as well as LTs of the 5-series. Recently, however, C 22 fatty acids also have come into focus as precursors of correspondingly elongated PGs and TXs and possibly other products as well. For example, adrenic acid (22:4<D6) can be metabolized into biologically active dihomo-pgs. The oxygenation of docosapenta- and docosahexaenoic acids (22:5w6 and 22:6o)3) has also attracted attention recently. In general, the precursor fatty acid does not occur free in the unstimulated cell but is esterified to glycerophospholipids almost exclusively in the 2-acyl position. The release of the precursor is considered to be the rate-limiting step in the biosynthesis of PGs and related substances. This hydrolysis can be achieved by a phospholipase A 2, or by phospholipase C and then by other lipases, depending on cell type, phospholipid category, and possibly other factors. After release, the parent fatty acid is metabolized via three different types of pathways. The best known of these is catalyzed by the enzyme fatty acid cyclooxygenase and leads to the formation of prostaglandin endoperoxides, which in turn are the immediate precursors of prostaglandins, thromboxanes, and prostacyclins. Other metabolic oxygenations are catalyzed by various lipoxygenases. Several such enzymes with different positional specificity for the introduction of oxygen have been identified. The products of the lipoxygenase pathways include hydroperoxy fatty acids, mono-, di-, and trihydroxy fatty acids, the leukotrienes and the recently discovered lipoxins. Finally, the fatty acid can also be metabolized by the action of various monooxygenases into wl or a>2 hydroxy acids or by epoxidation at the double bonds. Comparatively little is as yet known about the products of these latter pathways. For reviews on the release of the polyunsaturated fatty acids from their phospholipid precursors, see, e.g., refs PROSTAGLANDINS As indicated above, oxygenation can take place at many different positions of the fatty acid. In, for example, arachidonic acid, enzymatic oxygenation can occur at carbon-5, carbon-8, carbon-9, carbon-11, carbon-12, and carbon-15. In the best known metabolic pathway in this field, namely the cyclooxygenase pathway, the enzyme introduces oxygen at carbon-11 in a dioxygenase type reaction, and the resulting ll-peroxy compound then cyclizes to form a 9,11-endoperoxide structure (Fig. 1). Simultaneously, ring closure occurs between carbon-8 and carbon-12, the A 14 cis double bond isomerizes to A 13 trans, and another oxygen radical attacks at carbon-15. The resulting compound is the first product with the prostanoic acid skeleton (Fig. 2), and its systemic name is, when originating in arachidonic acid, 9a,lla-peroxido-15(5)-hydroperoxy-prosta-5-cw, 13-frans-dienoic acid (trivial name, prostaglandin G 2, PGG 2 ) (Fig. 1). The two remaining double bonds of arachidonic acid give rise to the name of the series of compounds originating in this parent molecule, namely the 2-series. The 1-series, originating in dihomo-7-lino-

3 OH OH leukotriene B; Leukotriene A; Glutathione OH CHCONHCH 2 NHCOCH 2 CH 2 CH JDehydrase Leukotriene C4 HOO 5-Lipoxygenase 12-Hydroperoxyeicosatetraenoic acid 12-Lipoxygenase 15-Lipoxygenase OOH 15-Hydroperoxyeicosatetraenoic acid Prostaglandin endoperoxide, PGH, q. < HO' OH Prostaglandin E2 -- ^^ OH Thromboxane A2 9", HO' 6H Prostacyclin, PGI2 FIG. 1. Simplified scheme of arachidonic acid metabolism, showing some products of the cyclooxygenase and lipoxygenase catalyzed pathways.

4 62 BIOCHEMISTRY OF EICOSANOIDS FIG. 2. Structures of prostanoic acid (left) and thrombanoic acid (right). lenic acid, analogously has only one double bond, A 13 trans, and the 3-series from timnodonic acid has three double bonds, A 5 cis, A 13 trans, and A 17 cis. Prostaglandin G 2 is then converted into the corresponding 15-hydroxy compound by an endoperoxide peroxidase. This product is named prostaglandin H 2 (PGH 2 ; Fig. 1). So far it has not been possible to separate the two enzyme activities, the cyclooxygenase and the peroxidase. It is thus currently believed that these two activities are catalyzed by the same enzyme, called prostaglandin endoperoxide synthetase. The two endoperoxides are highly unstable in aqueous medium (half-life about 5 min at 37 C and ph 7.4) and are biologically very potent compounds. Among their biological activities are vasoconstriction, constriction of airways, and induction of platelet aggregation. It is however uncertain how much of their registered effects can be attributed to the endoperoxides themselves, since they are rapidly converted into other compounds, both enzymatically by most tissues and nonenzymatically. The earliest discovered products of endoperoxide metabolism are the "classical" prostaglandins, PGE, PGF, and PGD (Fig. 1). In fact, these compounds of the 1-series were the first ones to be identified in this entire field (4). The prostaglandins originating in arachidonic acid are PGE 2 (9-keto-lla,15(5)-dihydroxyprosta-5-cw, 13-frans-dienoic acid), PGF2c (9a,lla,15(S)-trihydroxyprosta-5-cw, 13-fran.s-dienoic acid) and PGD 2 (ll-keto-9a,15(s)-dihydroxyprosta-5-c«,13- trans-dienoic acid, an isomer of PGE 2 ) (Fig. 1). At least PGE 2 and PGD 2 are formed enzymatically from the endoperoxides by the action of specific isomerases. The enzymatic formation of PGF 2c< is less certain: the existence of an endoperoxide reductase catalyzing this reaction has not been demonstrated unequivocally. All these three compounds can also be formed nonenzymatically during spontaneous degradation of the endoperoxides in aqueous solution. A large number of important biological activities have been attributed to the prostaglandins. PGE 2, for example, is a potent vasodilator and lowers the blood pressure, inhibits the secretion of acid in the gastric mucosa, and also acts as a "cytoprotective" agent in the gastrointestinal tract; PGF 2a is a powerful stimulant of the myometrium and also induces regression of the corpus luteum in many animal species; and PGD 2 is a potent inhibitor of platelet aggregation. For a recent review on the biochemistry and physiological effects of prostaglandins, see ref. 1.

5 BIOCHEMISTRY OF EICOSANOIDS 63 THROMBOXANES Several other products are also formed from the endoperoxides. One biologically very potent endoperoxide metabolite is formed in large amounts from platelets, lung, and spleen, and also by a number of other tissues but in smaller quantities: thromboxane A (Fig. 1). The basic structure of this compound is different from that of the prostaglandins; i.e., it is not a derivative of prostanoic acid but of thrombanoic acid (Fig. 2): it contains an oxane-oxetane ring structure instead of the normal prostaglandin cyclopentane ring. The structures of the side chains are however identical with the prostaglandins. Thromboxane A 2 from arachidonic acid is even more unstable (half-life about 30 sec) and biologically potent than its precursor endoperoxides. Among its activities are vasoconstriction, bronchoconstriction, and induction of platelet aggregation. The compound is rapidly hydrolyzed into a biologically almost inactive substance, thromboxane B 2 (TXB 2 ). Thromboxane biosynthesis is usually detected either by rapid bioassay of the vasoactive or proaggregatory effects of TXA 2 or by detection by other methods of its degradation product, TXB 2. For a recent review of the thromboxanes, see, e.g., ref. 5. PROSTACYCLINS Another potent endoperoxide metabolite is found only in the 2- and 3-series of prostanoids, namely PGI. The structure of PGI 2 from arachidonic acid ("prostacyclin") is 9-deoxy-6,9a-epoxy-ll,15-dihydroxyprosta-5,13-dienoic acid (Fig. 1). The A 5 double bond is a necessary part of its structure, and no PGl! can be formed from 8,11,14-eicosatrienoic acid. Prostacyclin has some features in common with TXA 2. It is very unstable in aqueous medium, particularly at acid ph, and is hydrolyzed with a half-life of a few minutes to a biologically almost inactive product, 6-keto-PGF la. However, the biological activities of this potent compound are, interestingly, opposite those of TXA 2 : inhibition of platelet aggregation and relaxation of vessels. Due to these effects, the compound itself (or a chemically stable analog) has a considerable clinical potential, and prostacyclin has been successfully employed in the treatment of a number of thrombotic disorders. For a recent review on prostacyclins, see, e.g., ref. 6. CYCLOOXYGENASE INHIBITORS All compounds described so far are metabolites of the prostaglandin endoperoxides; i.e., they have the first step of the biosynthesis, the cyclooxygenase step, in common. An inhibitor of this enzyme thus blocks the biosynthesis of all "distal" compounds. A number of such inhibitors are known and have in fact been known

6 64 BIOCHEMISTRY OF EICOSANOIDS since long before their mechanism of action was understood the nonsteroidal antiinflammatory drugs, such as aspirin, indomethacin, etc. These compounds have many biological effects in common: they are, for example, analgesic, antipyretic, and antiinflammatory. Some side effects also occur with several of these drugs, such as gastric erosion, salt and water retention, prolongation of the bleeding time due to inhibition of platelet aggregation, and so on. These effects can all be explained by the inhibition of the cyclooxygenase: some prostaglandins can induce fever and hyperalgesia, several are mediators of the inflammatory response, some prostanoids inhibit the gastric acid secretion, are natriand diuretic, or induce platelet aggregation. A recent review of the mode of action of aspirin-like drugs can be found in ref. 7. The observation of certain prostaglandin effects as well as of effects of cyclooxygenase inhibitors has given some insight into the roles of prostaglandins in certain disorders. One of the earliest to attract attention was dysmenorrhea: several prostaglandins are strong stimulants of myometrial contractility, and they are also known to be biosynthesized by the uterus and in particularly large amounts during an attack of dysmenorrhea. Aspirin has also long been used to alleviate menstrual pain. A large number of other diseases are also believed to involve an altered prostaglandin biosynthesis or metabolism, although this derangement is seldom thought to be the true cause of the disease, but rather involved in the pathogenesis of the symptoms. These diseases include thrombotic and bleeding disorders, various inflammatory diseases, gastric ulcer, asthma, and many others. The interest in certain of these conditions has increased considerably in recent years, when it became clear that also other pathways in the metabolism of polyunsaturated fatty acids lead to potent compounds. LIPOXYGENASE PRODUCTS The cyclooxygenase introduces oxygen at carbon-11 of the fatty acid molecule. Other enzymes, namely the various lipoxygenases, may instead introduce oxygen at carbon-5, carbon-8, carbon-9, carbon-12, and carbon-15 of arachidonic acid. The initial products of these pathways are hydroperoxy fatty acids, which can then be converted by several different pathways into a large and heterogenous group of compounds. Figure 1 shows some products formed via the 5-, 12-, and 15-lipoxygenase pathways. The biologically most interesting group of compounds is formed by the action of a 5-lipoxygenase. The initially formed 5-hydroperoxy fatty acid (5-hydroperoxy- 6-frani-8,ll,14-c/\s-eicosatetraenoic acid from arachidonic acid) is converted by a dehydrase into a 5,6-epoxy compound. This metabolite formed from arachidonic acid is called leukotriene A4 (LTA») and is the first compound with the characteristic conjugated triene structure, typical for this group of substances. (The first part of the name, "leuko," is derived from one source of these substances, leukocytes;

7 BIOCHEMISTRY OF EICOSANOIDS 65 cf. thromboxanes from thrombocytes. The index, 4, again refers to the number of double bonds in the molecule.) Leukotriene A 4 subsequently undergoes at least two different metabolic fates. It can be enzymatically hydrolyzed to form a 5,12-dihydroxy compound leukotriene B 4 (Fig. 1) or converted into a peptido-leukotriene, LTC 4, by addition of one molecule of glutathione at carbon-6. This latter reaction is catalyzed by glutathione 5-transferase. Leukotriene C 4 is then further converted into smaller molecules, LTD 4 and LTE 4, by sequential hydrolysis of the glutathione moiety. Thus, in LTD 4, the substituent at carbon-6 is cysteinylglycine and, in LTE 4, cysteine. This compound can be further metabolized into LTF 4, in which the substituent is 7-glutamylcysteine. These so-called peptido-leukotrienes constitute the well-known entity, "slow reacting substance of anaphylaxis," and are now believed to be the compounds mainly responsible for the symptoms of human bronchial asthma. Their potent biological effects include induction of long-lasting bronchoconstriction, extravasation of fluid, and increased mucus secretion in airways. The dihydroxy acid, LTB 4, is also a biologically active compound: it is a potent inducer of chemokinesis, chemotaxis, and aggregation of leukocytes. The leukotrienes are thus believed to be important mediators in inflammation and allergy, probably far more important than the cyclooxygenase products. Considerable efforts are at present going into development of specific lipoxygenase inhibitors for the treatment of a large number of diseases belonging to this category. Recent reviews on the biochemistry and biological importance of the leukotrienes may be found elsewhere (8,9). Several other compounds with biological effects have been identified from the lipoxygenase pathways, although less is known about their roles in the body. Among the latest identified substances are the lipoxins, trihydroxytetraenoic acids from arachidonic acid, in which the four double bonds are arranged as a conjugated tetraene. Two such compounds are known to date: lipoxin A (LXA), 5,6, 15-trihydroxy-7,9,ll,13-eicosatetraenoic acid, and lipoxin B (LXB), 5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid. Among their biological effects are stimulation of superoxide anion generation, degranulation of neutrophils and inhibition of human natural killer cell activity (10,11). ACKNOWLEDGMENT This review was supported by the Swedish Medical Research Council (proj. no. 03P-5804). REFERENCES 1. Oliw E, Granstrom E, Anggard E. The prostaglandins and essential fatty acids. In: Pace-Asciak C, Granstrom E, eds. Prostaglandins and related substances. New comprehensive biochemistry, volume 5, Amsterdam: Elsevier, 1983:1 44.

8 66 BIOCHEMISTRY OF EICOSANOIDS 2. Van den Bosch H. Intracellular phospholipases A. Biochim Biophys Ada 1980;604: Irvine RF. How is the level of free arachidonic acid controlled in mammalian cells? Biochem J 1982;204: Bergstrom S, Carlson LA, Weeks JR. The prostaglandins: A family of biologically active lipids. Pharmacol Rev 1968;20:l-^8. 5. Granstrom E, Diczfalusy U, Hamberg M. The thromboxanes. In: Pace-Asciak C, Granstrom E, eds. Prostaglandins and related substances. New comprehensive biochemistry, volume 5, Amsterdam: Elsevier, 1983: Pace-Asciak C, Gryglewski R. The prostacyclins. In: Pace-Asciak C, Granstrom E, eds. Prostaglandins and related substances. New comprehensive biochemistry, volume 5, Amsterdam: Elsevier, 1983: Moncada S, Vane JR. Mode of action of aspirin-like drugs. Adv Intern Med 1979;24:l Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 1983;220: Hansson G, Malmsten C, Radmark O. The leukotrienes and other lipoxygenase products. In: Pace-Asciak C, Granstrom E, eds. Prostaglandins and related substances. New comprehensive biochemistry, volume 5, Amsterdam: Elsevier, 1983: Serhan CN, Hamberg M, Samuelsson B. Lipoxins: Novel series of biologically active compounds formed from arachidonic acid in human leukocytes. Proc Natl Acad Sci USA 1984;81: Ramstedt U, Ng J, Wigzell H, Serhan CN, Samuelsson B. Action of novel eicosanoids lipoxin A and B on human natural killer cell cytotoxicity: effects on intracellular camp and target cell binding. J Immunol 1985;135: