Abstract
We developed a procedure for serial measurement of fluorescent derivatives of eicosanoids in biological samples by HPLC. The 9-anthryldiazomethane (ADAM)-derivatized sample was first fractionated through SEP-PAK silica into fraction 1 (eluate of chloroform: toluene) and fraction 2 (eluate of acetonitrile:methanol). Both fractions were loaded separately onto an ODS column, and eluted with a step gradient of 85% and 95% acetonitrile for Fr-1 (HETE''s and arachidonic acid) and with 70% acetonitrile for Fr-2 (PG''s and LTB4). The method was applied to the arachidonate products of rat peritoneal leukocytes which were stimulated with A23187. The polymorphonuclear leukocytes (PMNL), which were collected after stimulation with casein, released mainly LTB4, 5-HETE, 6-K-PGF1α, but little arachidonic acid. In contrast to PMNL, rat macrophages, which were collected after peritoneal injection of soluble starch and bacto peptone, released 5-HETE, arachidonic acid, and 6-K-PGF1α, but no LTB4. These differences might be partly caused by the differential rates of uptake or turnover of arachidonic acid into their membrane phospholipids.
