The identification and formation of 20-aldehyde leukotriene B4.
Microsomes of human polymorphonuclear leukocytes (PMN) in the presence of 100 microM NADPH converted 0.6 microM leukotriene B4 (LTB4) to 20-OH-LTB4 (retention time = 18.0 min) and to two additional compounds designated I (retention time = 16.8 min) and II (retention time = 9.6 min) as analyzed by reverse-phase high performance liquid chromatography (HPLC). Compounds I and II were also formed from the reaction of 1.0 microM 20-OH-LTB4, PMN microsomes, and 100 microM NADPH; the identity of compound II was confirmed as 20-COOH-LTB4 by gas chromatography-mass spectrometry. Equine alcohol dehydrogenase in the presence of 100 microM NAD+ in 0.2 M glycine buffer (pH 10.0) converted 20-OH-LTB4 to 20-aldehyde (CHO) LTB4, which coeluted with compound I on reverse-phase HPLC. In the presence of 100 microM NADH in 50 mM potassium phosphate buffer (pH 6.5), equine alcohol dehydrogenase reduced both 20-CHO-LTB4 and compound I to 20-OH-LTB4, indicating the identity of compound I as 20-CHO-LTB4. Gas chromatography-mass spectrometry of trideuterated O-methyl-oxime trimethylsilyl ether methyl ester derivative of 3H-labeled compound I definitively established compound I as 20-CHO-LTB4. Addition of immune IgG to cytochrome P-450 reductase or 1.0 mM SKF-525A completely inhibited the formation of 20-CHO-LTB4 from 20-OH-LTB4, indicating that the reaction was catalyzed by a cytochrome P-450. LTB5 (3.0 microM), a known substrate for cytochrome P-450LTB and a competitive inhibitor of LTB4 omega-oxidation, completely inhibited the omega-oxidation of 1.5 microM 20-OH-LTB4 to 20-CHO-LTB4, indicating that the cytochrome P-450 was P-450LTB. Conversion of 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 by PMN microsomes was also dependent on NADPH and inhibited by antibody to cytochrome P-450 reductase, 1.0 mM SKF-525A, or 5.0 microM LTB5, indicating that this reaction was also catalyzed by cytochrome P-450LTB. These results identify the novel metabolite 20-CHO-LTB4 and indicate that cytochrome P-450LTB catalyzes three sequential omega-oxidations of LTB4 leading to the formation of 20-COOH-LTB4 via 20-OH-LTB4 and 20-CHO-LTB4 intermediates.
Soberman
R J
RJ
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
Sutyak
J P
JP
Okita
R T
RT
Wendelborn
D F
DF
Roberts
L J
LJ
Austen
K F
KF
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006736:Horses; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D007975:Leukotriene B4 / Q000031:analogs & derivatives* / Q000378:metabolism*; D013058:Mass Spectrometry; D008861:Microsomes / Q000378:metabolism; D009249:NADP / Q000378:metabolism
D007527:Isoenzymes; C056168:20-aldehyde leukotriene B4; D007975:Leukotriene B4; D009249:NADP; C031571:5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D000426:Alcohol Dehydrogenase