Simultaneous determination of leukotrienes B4 and E4 in whole blood and of leukotriene E4 in urine of rabbit by reversed-phase high-performance liquid chromatography
Celardo, A.; Dell'Elba, G.; Eltantawy, Z.M.; Evangelista, V.; Cerletti, C.
Journal of Chromatography. B Biomedical Applications 658(2): 261-269
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-18 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-4 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.