P Borgeat

A new metabolite of arachidonic acid, 5-D-(S),12-D-(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid, was found upon incubation of the fatty acid with a suspension of rabbit peritoneal polymorphonuclear leukocytes collected 4 h after injection of glycogen into the peritoneal cavity. The yield of the dihydroxy acid was 0.5 to 2%. The compound possesses three conjugated double bonds and was found to be stereochemically pure at C-5 and C-12. Incubation of the cells with 8,11,14-eicosatrienoic acid did not lead to the formation of the analogous triunsaturated dihydroxy acid.

Addition of arachidonic acid and homo-gamma-linolenic acid to a suspension of rabbit peritoneal neutrophils led to the synthesis of 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid and 8-L-hydroxy-9,11,14-eicosatrienoic acid, respectively. Both hydroxy acids were found to be the main metabolites of their respective unsaturated C-20 fatty acid precursor, constituting more than 50% of the total substrate conversion. The formation of the two metabolites was not inhibted bb indomethacin, indicating that the enzymes involved were unrelated to the prostaglandin synthetase system. The presence in the two compounds of a hydroxyl group alpha to a pair of conjugated cis/trans double bonds suggested that they were formed by action of lipoxygenease(s).

Arachidonic acid was incubated with rabbit peritoneal polymorphonuclear leukocytes (glycogen-induced) and compounds obtained from ether extractions were fractionated by silicic acid column chromatography. A fraction containing several unidentified metabolites of arachidonic acid was analyzed by reversed phase-high pressure liquid chromatography. The metabolites were esterified and further purified by silicic acid high pressure liquid chromatography. The structures of the pure compounds were elucidated by infrared and ultraviolet spectrometry, ozonolysis, and gas chromatography-mass spectrometry. The following novel compounds were identified: Compound 1, 5S, 12R-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 2, 5S, 12S-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 3, 5, 6-dihydroxy-7,9,11,14-eicosatetraenoic acid; Compound 4, a diastereoisomer of the latter. Evidence for the occurrence of the delta-lactone forms of the 5,12-dihydroxy acids is also presented.

An unstable intermediate was detected in the transformation of arachidonic acid into 5,6-dihydroxyicosatetraenoic acids (two isomers) and 5,12-dihydroxyicosatetraenoic acids (three isomers) in rabbit peritoneal (glycogen-induced) polymorphonuclear leukocytes. Addition of 10 vol of methanol, ethanol, or ethylene glycol to short-term incubations (30-45 sec) led to the formation of the corresponding 12-O-alkyl derivatives of the 5,12-dihydroxy acids. The time for 50% disappearance of the intermediate (37 degrees C), as measured by formation of 5-hydroxy-12-O-methylicosatetraenoic acids (two isomers) upon trapping with methanol, was about 1 min in live cell preparations (pH 7.4) and about 4 min in water/acetone (1:1), pH 7.4. At pH 6.0 or below, the hydrolysis of the intermediate was too rapid to be measured by the method employed. Data supporting both enzymatic and nonenzymatic hydrolysis of the intermediate into dihydroxy acids are presented. Incubation of the cells with arachidonic acid under an atmosphere of 18O2 led to incorporation of 18O into the 5,6-dihydroxy acids and 5,12-dihydroxy acids only at C-5. The 5-hydroxyicosatetraenoic acid was also labeled at C-5. Considering the chemical reactivity of the intermediate and the structures of the derivatives obtained, it is proposed that the intermediate is 5(6)-oxido-7,9,11,14-icosatetraenoic acid.