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Conversion of tritiated arachidonic acid (AA) into metabolites of the cyclo- and lipoxygenase pathways by bovine fetal placental tissue (200 mg) and fetal plus maternal placental tissue (400 mg) of Days 255, 265, 275 of gestation and at parturition (n = 5) during a 30 min incubation was measured using reverse-phase high pressure liquid chromatography. Fetal placental tissue produced 13,14-dihydro-15-keto-prostaglandin E2 (PGEM) as the major metabolite, the synthesis of which increased from Day 265 to Day 275 and parturition by 150% and 475%, respectively. In tissues collected at parturition, PGE2 synthesis was also detected. On Day 275 and at parturition fetal placental tissue synthesized the metabolite 12-hydroxyheptadecatrienoic acid (HHT), and throughout the experimental period the lipoxygenase product 15-HETE was detected with synthesis rates increasing over time of gestation. In addition, an unidentified metabolite was regularly found in the radiochromatograms which eluted at 1 h and 1 min (U101), between HHT and 15-HETE. The synthesis of this metabolite decreased as pregnancy progressed. Furthermore, various other polar and nonpolar metabolites pooled under the heading UNID were eluted, the production of which increased over time of gestation. The presence of maternal placental tissue did not influence the synthesis of PGEM, 15-HETE and U101, but the production of HHT was decreased when maternal tissue was present. Also, as pregnancy progressed, maternal placental tissue seemed to contribute to the pool of unidentified metabolites. In conclusion, fetal placental tissue seems to be the major source of the AA metabolites when compared with maternal placental tissue, and AA metabolism by bovine placental tissue is markedly increased throughout the last month of pregnancy, suggesting a role for AA metabolites in mechanisms controlling parturition.

Rat elicited polymorphonuclear leucocytes (PMNs), when exposed to the ionophore A23187, release three isomers of leukotriene B4. The three isomers have been purified and tested for their ability to induce the chemokinesis of human PMNs in vitro, the aggregation of rat PMNs in vitro and changes in vascular permeability in rabbit skin in vivo in the presence of PGE2. The results demonstrate that all three isomers are biologically active and that the enzymatically produced isomer, in which the conjugated triene contains one cis and two trans double bonds, is more potent than the two diastereoisomers of LTB4 which contain all trans double bonds in the conjugated triene and which are produced by non-enzymatic hydrolysis.

Separation of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C18 reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-14C]arachidonic acid was quantitative. HPLC also resolved several metabolites that were not detected by scanning of TLC separations.

5(S)-hydroxy-6 trans-8,11,14 cis-eicosatetraenoic acid (5-HETE) is the major product of arachidonic acid metabolism via the 5-lipoxygenase pathway. A limiting factor in the quantitation of 5-HETE by GC-MS analysis is the availability of a stable isotope analog for use as an internal standard. In this report, we detail procedures for selective chemical synthesis of multimilligram quantities of octadeuterated (+/-)-5-HETE from octadeuterated arachidonic acid. The octadeuterated (+/-)-5-HETE is suitable for use as an internal standard for GC-MS quantitation of 5-HETE. Preparation of the octadeuterated analog of 5-HETE can be readily performed in most laboratory settings.

Rat peritoneal monocytes and macrophages when exposed to the ionophore A23187 release products of the lipoxygenase pathway of arachidonic acid metabolism which cause the aggregation and chemokinesis of polymorphonuclear leucocytes suspensions. The major biologically active compound released was leukotriene B which accounted for greater than 80% of the activity. The remaining biological activity was due to the release of a more polar as yet unidentified compound. In addition rat macrophages release 5, 12 and 15-HETE but these mono-HETEs do not significantly contribute to the biological activity.

We have utilized HPLC to develop optimal conditions for assaying the transformation of arachidonic acid in thrombin-treated human platelets. In the presence of increasing amounts of albumin, the total amount of radioactivity released from thrombin-treated platelets pre-labeled with 3H-arachidonic acid is first enhanced and then inhibited. Maximal release, reflecting primarily enhanced amounts of free labeled arachidonic acid, occurs at a final albumin concentration of 0.5 mg/ml. Calcium promoted the release of all radiolabeled metabolites, but it specifically enhanced HETE formation and release. Magnesium was without effect. Cyclo-oxygenase derived products constituted the bulk of released label at short time intervals, but after ten minutes exposure to thrombin in the presence of albumin (0.5 mg/ml) and 3 mM calcium, radioactivity in the released products was equally distributed among cyclo-oxygenase derived products (TXB2 + PGD2 + HHT), HETE and free arachidonic acid.

The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metabolites of arachidonic acid: Techsphere 5-C18 column, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pH 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify lipoxygenase products released from mouse macrophage cells stimulated with gamma-hexachlorocyclohexane. Detection limits between 1 and 10 ng were obtained.

We have developed a technique for the rapid separation and quantitative collection of thromboxane B2 (TXB2), PGE2, PGD2, PGF2 alpha, 12-hydroxy-5,8,10 heptadecatrienoic acid (HHT), 12-L-hydroxy-5,8,10,14 eicosatetraenoic acid (HETE), and arachidonic acid released from thrombin treated human platelets. Platelets were pre-labeled with 3H-arachidonic acid and then isolated by gel filtration. They were then exposed to thrombin for various intervals and separated by centrifugation. Aliquots of the cell-free medium were applied directly to a high pressure liquid chromatograph containing a fatty acid column as the stationary phase. A quarternary solvent system containing tetrahydrofuran (THF), acetonitrile (CH3CN), water and acetic acid (HOAC) resolved and eluted the arachidonic acid metabolites within 30 minutes. Since no sample preparation is required and since the solvent system does not quench the counting efficiency of a standard liquid scintillation fluor the technique permits rapid separation and quantitation of radiolabeled arachidonic acid and its metabolites.