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.
Sulfidopeptide leukotrienes E4 (LTE4) and B4 (LTB4) were simultaneously extracted from rabbit whole blood with acetonitrile. LTC4 and LTD4 were converted to LTE4 by gamma-glutamyl transpeptidase and leucine-amino peptidase before extraction. LTE4 was extracted from urine with C18 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 LTB4 and LTE4 by specific enzyme immunoassay (EIA). The correlation factor of the measured increase in LTE4 concentrations and addition of incremental amounts of LTC4 to blood was r = 0.998; slope of 1.05 +/- 0.01 (mean +/- S.D.). Concentrations of LTE4 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 LTB4 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 LTB4 and LTE4 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.
Celardo
A
A
Istituto di Ricerche Farmacologiche, Mario Negri, G. Bizzozero Laboratory of Platelet and Leukocyte Pharmacology, Santa Maria Imbaro, Italy.
Dell'Elba
G
G
Eltantawy
Z M
ZM
Evangelista
V
V
Cerletti
C
C
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D007124:Immunoenzyme Techniques; D007931:Leucyl Aminopeptidase / Q000737:chemistry; D007975:Leukotriene B4 / Q000097:blood*; D017999:Leukotriene E4 / Q000097:blood* / Q000652:urine*; D008297:Male; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D005723:gamma-Glutamyltransferase / Q000737:chemistry
D007975:Leukotriene B4; D017999:Leukotriene E4; D005723:gamma-Glutamyltransferase; D007931:Leucyl Aminopeptidase