Characterization of monohydroxylated lipoxygenase metabolites of arachidonic and linoleic acid in rabbit peritoneal tissue.

Rabbit peritoneal tissue contains a lipoxygenase which converts arachidonic acid preferentially into 15-hydroxy-5,8,11,13-eicosatetraenoic acid. Stereochemical analysis of the menthyloxycarbonyl derivative of this metabolite by means of a high-pressure liquid chromatography method, involving the use of a Ag+ -loaded cation-exchange column, indicated that it has mainly the 15-Ls-hydroxy configuration. The biosynthesis of 15-hydroxy-5,8,11,13-eicosatetraenoic acid could be confirmed during examination of the monohydroxy acids obtained without addition of fatty acids, thus formed from endogenously released substrate. However, the 9-and 13-hydroxy derivatives of linoleic acid were also formed and in quantities exceeding those of 15-hydroxy-5,8,11,13-eicosatetraenoic acid.

15-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) formation by rabbit peritoneal tissue.

The capacity of rabbit peritoneal tissue to produce lipoxygenase metabolites from exogenous arachidonic acid has been studied. The lipoxygenase pathway of arachidonic acid in this tissue is mainly directed to 15-HETE. Preliminary experiments with peritoneal macrophages indicate that these cells, just like PMN leukocytes, may contribute to the 15-HETE formation observed in peritoneal tissue.

Study of the two pathways for arachidonate oxygenation in blood platelets.

During collagen-induced blood platelet aggregation, arachidonic acid is set free from membrane phospholipids and subsequently converted into 12-hydroxyeicosatetraenoic acid by arachidonate lipoxygenase and into thromboxane A2, 12-hydroxyheptadecatrienoic acid (HETE) and malondialdehyde by cyclooxygenase and thromboxane synthase. Lipoxygenase and cyclooxygenase have optimal activity at neutral to basic pH, while the thromboxane synthase is pH-independent between 5 and 9. These enzymes are membrane-bound. The cyclooxygenase is rapidly inactivated upon membrane disruption by nonionic detergents or phospholipid degradation with phospholipase A2. It was found that platelet phospholipase A2 preferentially splits off fatty acid with four double bonds. Eicosatetraynoic acid was used to investigate the physiological function of the arachidonate lipoxygenase during collagen-induced aggregation of rat blood platelets. This fatty acid is a more efficient inhibitor of lipoxygenase than of cyclooxygenase. At an inhibitor concentration of 0.6 microgram/ml, platelet aggreation, 12-hydroxyeicosatetraenoic acid production as well as 15-hydroxytryptamine release are completely inhibited, while there is an apparent stimulation of the cyclooxygenase. These results indicate that arachidonate lipoxygenase is essential for irreversible blood platelet aggregation.

Determination of prostaglandins and other products of arachidonate oxygenation in perfusates and during platelet aggregation.

Some analytical aspects of prostaglandin research are discussed. We present our latest results of the determination of the various products of arachidonate oxygenation in biological fluids and during platelet aggregation. These products were determined by (GLC) in combination with electron-capture detection or mass spectrometry (multiple-ion detection). The major component released from isolated perfused heart and lung was prostacyclin, determined as its hydrolysis product 6-oxo-PGF1alpha. Spleen released much thromboxane and monohydroxy acids. Normal rat platelets formed also much hydroxy acids during aggregation but at essential fatty acid deficiency, only very small amounts of product were detected.