SIMULTANEOUS DETERMINATION OF LEUKOTRIENES B(4) AND E(4) IN WHOLE-BLOOD AND OF LEUKOTRIENE E(4) IN URINE OF RABBIT BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Sulfidopeptide leukotrienes E(4) (LTE(4)) and B-4 (LTB(4)) were simultaneously extracted from rabbit whole blood with acetonitrile. LTC(4) and LTD(4) were converted to LTE(4) by gamma-glutamyl transpeptidase and leucine-amino peptidase before extraction. LTE, was extracted from urine with C,, Sep-Pak cartridges. The compounds were resolved and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with a diode-array detector; in selected cases the collected fractions were assayed for LTB(4) and LTE(4) by specific enzyme immunoassay (EIA). The correlation factor of the measured increase in LTE(4) concentrations and addition of incremental amounts of LTC(4) to blood was r = 0.998; slope of 1.05 +/- 0.01 (mean +/- S.D.). Concentrations of LTE, measured by HPLC correlated with those obtained with EIA (r = 0.996; slope = 0.98 +/- 0.03 and r = 0.991; slope = 0.97 +/- 0.04 in blood and urine, respectively). For blood LTB(4) the correlation of HPLC versus EIA was r = 0.990; slope = 1.12 +/- 0.04. The method described is accurate and reproducible, allowing measurement of both LTB(4) and LTE(4) in whole blood after a single extraction procedure. Simultaneous measurement of these metabolites after specific stimulation or in pathological conditions is recommended for in vivo investigations of LTs production.