Arachidonate Metabolism

As we have seen in chapters 1 and 2, the metabolism of arachidonate begins with its mobilization from membrane phospholipids and consists mainly of an array of sequential oxidative reactions. Three main classes of oxygenase enzymes are implicated prostaglandin H-synthase, also called cyclooxygenase cytochrome P450 and a variety of different lipoxygenases. 

The first reaction catalyzed by all lipoxygenase enzymes is the addition of a hydroperoxide group to a select carbon atom in the arachidonate molecule, to form a chiral hydroperoxyeicosatetraenoic acid (HPETE). Thus, the primary product of mammalian 5-lipoxygenase is 5(S)-HPETE, that of sea-urchin 12-lipoxygenase, 

Cytochrome P450 catalyzes the monooxygenation of arachidonate to form a family of isomeric epoxyeicosatrienoic acids (EETs). These may be, in turn, transformed by the enzyme epoxide hydrolase to the corresponding diols, the dihydroxyeicosatrienoic acids (DHT). In addition, cytochrome P450 can also convert arachidonate into a family of nonchiral HETEs. 

In the following sections I will briefly outline each of these pathways, describing the enzymes involved, their molecular structures, cellular localizations and pharmacological inhibition. Before turning to our main subject, however, it may be useful to give a general introduction to the analytical strategies that are most frequently employed to study the biochemistry of arachidonate metabolism. 

After more than 30 years of intensive research on the eicosanoids, it is not surprising that a vast collection of analytical techniques are available to the investigator. Exhaustive reviews dealing with various methodological aspects of eicosanoid analysis may be found in the literature. In the next pages, I will give a few examples of these methods and provide a series of select references to serve as an introductory guide. 

---

Kimura Y, Hiromichi O, Shigeru A. Effects of stilbenes on arachidonate metabolism in leukocytes. Biochim Biophysica Acta 1985 834 275-278. 

Two other polycyclic hydrocarbons, 3-methylcholanthrene and 7,12-dimethylbenzanthracene are oxidized during arachidonate metabolism (21,26). Hydroxymethyl compounds that do not arise from arene oxides appear to be the products formed from 7,12-dimethylbenzanthracene. ... 

Matthews, N., Neale, M.L., Jackson, S.K., and Stark, J.M., 1987, Tumor cell kilhng by tumor necrosis factor inhibition by anearobiotic condition, free-radical scavengers and inhibitors of arachidonate metabolism. Immunology 62 153-155 Miller, M.G., Rodgers, A., and Cohen, G.M., 1986, Mechanisms oftoxicity of naphthoquinones to isolated hepatocytes. Biochem. Pharmacol. 35 1177-1184 Minko, T., Kopeckova, P., and Kopecek, J., 1999, Comparison ofthe anticancer effect of free and HPMA copolymer-bound adtiamycin in human ovarian carcinoma cells. Pharmaceut. Res. 16 986-996... 

Evans. Actions of cannabis constitu- CS238 ents on enzymes of arachidonate metabolism Anti-inflammatory... 

The cell damage associated with inflammation acts on cell membranes to cause leukocytes to release lysosomal enzymes arachidonic acid is then liberated from precursor compounds, and various eicosanoids are synthesized. As discussed in Chapter 18, the cyclooxygenase (COX) pathway of arachidonate metabolism produces prostaglandins, which have a variety of effects on blood vessels, on nerve endings, and on cells involved in inflammation. The lipoxygenase pathway of arachidonate metabolism yields leukotrienes, which have a powerful chemotactic effect on eosinophils, neutrophils, and macrophages and promote bronchoconstriction and alterations in vascular permeability. 

Previously, we reported the effects of Morus flavonoids on arachidonate metabolism in rat platelet homogenates, such as inhibition of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), thromboxane B2, and 12-hydroxy -5,8,10,14-eicosatetraenoic acid (12-HETE) [79,80]. As described in the... 

Moreover, the effects of chlorogenic acids on arachidonate metabolism in human peripheral neutrophils have been investigated [85], The results show that the formation of leukotriene B4 induced by calcium... 

Kimura Y, Okuda H, Kubo M. 1995 Effects of stilbenes isolated from medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes. J Ethnopharmacol 45 131-139. 

Hammamieh R, Sumaida D, Zhang X, Das R, Jett M. 2007. Control of the growth of human breast cancer cells in culture by manipulation of arachidonate metabolism. 

Crofford LJ (1997) COX-1 and COX-2 tissue expression implications and predictions. J Rheumatol 24, Suppl 49 15-19 Cromlish WA, Kennedy BP (1996) Selective inhibition of cyclogenase-1 and -2 using intact insect cell assays. Biochem Pharmacol 52 1777-1785 Evans AT, Formukong EA, Evans FJ (1987) Actions of cannabis constituents on enzymes of arachidonate metabolism anti-inflammatory potential. Biochem Pharmacol 36 2035-2037... 

Effects of Natural Products Isolated from Medicinal Plants on Arachidonate Metabolism and Degranulation in Platelets and Leukocytes. 

Fig. (33). Effects of polyacetylene compounds isolated from Saposhnikoviae Radix on arachidonate metabolism in human platelets.

Ginsenoside Rgi had no effect on arachidonate metabolism, but it did reduce the elevation of cytosolic free calcium concentration [Ca2+]i shown in the second phase (Ca2+ influx) induced by adrenaline and thrombin "Fig. (37)". The results suggest that ginsenoside Rgl in red ginseng roots may be effective as a drug for the treatment of arteriosclerosis and thrombosis. 

Fig. (45). Effects of resveratrol on arachidonate metabolism in leukocytes a Autoradiography of aracjodonate metabolism in leukocytes b arachiodonate metabolite HHT, Thromboxane B2, 5-IffiTE. Results are expressed as means S.E. of 4 experiments.

It was evident that both COX and LOX arms of arachidonate metabolism were inhibited, suggesting that these smkosaponins might provisionally be classified as dual inhibitors , with greater LOX than COX activity. 

Pathways of arachidonate metabolism in eicosanoid synthesis and their inhibitors. HPETE = Hydroperoxyeicosatetraenoic acid HETE = hydroxyeicosatetraenoic acid PG = prostaglandin PGI = prostacyclin TX = thromboxane. Conversions of arachidonic acid by various enzymes can be inhibited by analogues of the natural fatty acid, e g., the acetylenic analogue 5,8,11,14-eicosatetraynoic acid. 

Kontos HA. 1987. Oxygen radicals from arachidonate metabolism in abnormal vascular responses. Am Rev Respir Dis 136 474-477. 

Johnson, M., Carey, F., and McMillan, R. M., Alternative Pathways of Arachidonate Metabolism Prostaglandins, Thromboxanes and Leukotrienes, Lssays Biochem., 19 40-141, 1983. 

---