5-hydroxy-6-8-11-14-eicosatetraenoic-acid and Leukemia

A validated method is described for the simultaneous analysis of PGE2, 11-, 12-, and 5-HETEs from cultured cells using HPLC negative electrospray ionization tandem mass spectrometry (LC/MS/MS). This method permits quantification of selected individual arachidonic acid metabolites from cell extracts without derivatization, multiple purification steps, or lengthy separation times required by traditional GC-MS- or HPLC-UV -based methods. Accuracy assessments of values calculated using this method showed deviations from nominal values were < or =15%. An average relative deviation of 7% of mean calculated values was observed for values taken on separate days. The lower limit of detection for all metabolites was 1.3 pg. The method was used to quantify arachidonic acid metabolites present in various cancer cell lines after incubation with arachidonic acid and the selective cyclooxygenase-2 inhibitor celecoxib. Results showed that the presence of celecoxib in lung cancer A549 cells reduced production of both PGE2 and 11-HETE in a concentration-dependent manner.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Celecoxib; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Leukemia; Lipoxygenase; Lung Neoplasms; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Spectrometry, Mass, Electrospray Ionization; Sulfonamides; Tumor Cells, Cultured

This study examines the effects of oxidized low density lipoprotein (ox-LDL) on mononuclear cell arachidonic acid (AA) metabolism. U937 and HL60 cells, employed as models for mononuclear cells and promyelocytic cells, respectively, were exposed to ox-LDL (20 micrograms protein/ml) for 24 hours. HPLC analysis of 1-14C-AA metabolites indicates that ox-LDL increases U937 and HL60 cell production of 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Northern analysis indicates that ox-LDL increases U937 cell FLAP transcript levels 10-times control levels but did not appear to alter 5-lipoxygenase (5-LO) mRNA levels. In contrast, ox-LDL increases HL60 cell transcript levels for FLAP and 5-LO 1.5 times and 10 times control levels, respectively. Thus, we propose that ox-LDL plays an important role in the up-regulation of the 5-LO pathway in mononuclear cells. Such activation may explain, in part, the mechanisms by which ox-LDL promotes atherogenesis.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blotting, Northern; Carrier Proteins; Cell Line; Enzyme Activation; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia; Lipoproteins, LDL; Lymphoma, Large B-Cell, Diffuse; Malondialdehyde; Membrane Proteins; Oxidation-Reduction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured

Utilizing a variety of fatty acids, differing in chain length, degree and position of unsaturation, we investigated the substrate specificity for the enzymatic production of biologically active slow reacting substances (SRS) and of the other leukotrienes. A cell-free enzyme system obtained from RBL-1 cells was used in this study. The primary structural requirement observed for the conversion by this lipoxygenase enzyme system was a delta 5,8,11 unsaturation in a polyenoic fatty acid. Such fatty acids as 20:4 (5,8,11,14) 20:5 (5,8,11,14,17), 20:3 (5,8,11), 19:4 (5,8,11,14) and 18:4 (5,8,11,14) were readily converted to compounds that comigrated with 5-HETE and 5,12-DiHETE and to biologically active SRS. Chain length did not have an influence on the formatin of these hydroxyacids. Fatty acids with the initial unsaturation at delta 4, delta 6, delta 7, or delta 8 were a poor substrate for the leukotriene enzyme system. Therefore, this lipoxygenase pathway in leukocytes is quite different from the lipoxygenase in platelets which does not exhibit this specificity.

Topics: Animals; Arachidonic Acids; Autacoids; Basophils; Cells, Cultured; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Ileum; Leukemia; Leukotriene A4; Leukotriene B4; Lipoxygenase; Rats; Substrate Specificity