Metabolism of leukotriene B4 by cultured human keratinocytes. Formation of glutathione conjugates and dihydro metabolites.
Six previously unidentified leukotriene (LT) B4 metabolites formed during incubation of LTB4 with human keratinocytes in primary culture indicate the importance of the 12-hydroxyeicosanoid dehydrogenase pathway in LTB4 metabolism. The ultraviolet absorption spectra obtained for all keratinocyte metabolites revealed the presence of a conjugated diene structural moiety rather than the conjugated triene structure of LTB4. Metabolites were characterized using fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the pentafluorobenzyl ester, trimethylsilyl ether derivatives and specific degradation reactions. The previously identified 10,11-dihydro-LTB4 and 10,11-dihydro-12-epi-LTB4 were observed as well as 20-OH-10,11-dihydro-LTB4, consistent with the reductase pathway of LTB4 metabolism. This pathway involves initial formation of 12-oxo-LTB4 catalyzed by 12-hydroxyeicosanoid dehydrogenase followed by reduction by delta 10-reductase. The most lipophilic metabolite of LTB4 was identified as 10-hydroxy-4,6,12-octadecatrienoic acid which could result from beta-oxidation reactions of LTB4 following reduction of the 10,11-double bond. One of the most abundant metabolites was characterized as 3,7,14-trihydroxy-8,10,16- docosatrienoic acid which could result from fatty acid elongation following reduction of the 10,11-double bond. Additional abundant LTB4 metabolites were identified that result from glutathione conjugation of 12-oxo-LTB4. These were characterized using fast atom bombardment-mass spectrometry and by chemical degradation using hypochlorous acid as well as transpeptidases. These metabolites were identified as 5,12-dihydroxy-6-glutathionyl-7,9,14-eicosatrienoic acid (c-LTB3), 5,12-dihydroxy-6-cysteinyl-glycyl-7,9,14-eicosatrienoic acid (d-LTB3) and 5,12-dihydroxy-6-cysteinyl-7,9,14-eicosatrienoic acid (e-LTB3). We propose that these metabolites result from a 1,8 Michael-type addition of glutathione to the 12-oxo-LTB4 intermediate.
Wheelan
P
P
Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
Zirrolli
J A
JA
Morelli
J G
JG
Murphy
R C
RC
D001711:Biotransformation; D002478:Cells, Cultured; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000378:metabolism*; D007975:Leukotriene B4 / Q000031:analogs & derivatives* / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry; D012867:Skin / Q000166:cytology / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment
C064945:5,12-dihydroxy-6,8,14-eicosatrienoic acid; D007975:Leukotriene B4; D005978:Glutathione