Biosynthesis of 14,15-hepoxilins in human l1236 Hodgkin lymphoma cells and eosinophils.
Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA(3) 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB(3) 13(R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA(3)-D). Incubation of L1236 cells with synthetic 14,15-HxA(3) 11(S) also led to the formation of 14,15-HxA(3)-C and 14,15-HxA(3)-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA(3) to 14,15-HxA(3)-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA(3) 11(S) and 14,15-HxB(3) 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.
Department of Medicine, Karolinska Institutet and Karolinska University Hospital Solna, Stockholm, Sweden.
Asa
A
Brunnström
Mats
M
Hamberg
William J
WJ
Griffiths
Bengt
B
Mannervik
Hans-Erik
HE
Claesson
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000378:metabolism; D004804:Eosinophils / Q000166:cytology / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006689:Hodgkin Disease / Q000201:enzymology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis*; D013237:Stereoisomerism
D004151:Dipeptides; D006893:Hydroxyeicosatetraenoic Acids; D001093:Arachidonate 15-Lipoxygenase; D015126:8,11,14-Eicosatrienoic Acid; D005978:Glutathione