Single-step organic extraction of leukotrienes and related compounds and their simultaneous analysis by high-performance liquid chromatography.
A method for the simultaneous single-step organic extraction from biological matrices of peptido- and dihydroxyleukotrienes as well as 5-hydroperoxy- and 5-hydroxyeicosatetraenoic acid followed by separation and quantitation in a single run on reversed-phase high-performance liquid chromatography was evaluated. Using an extraction system comprising 400/1200/4800 (v/v/v) aqueous phase/isopropanol/dichloromethane, pH 3.0, absolute recoveries of 82.3 +/- 2.0, 89.7 +/- 1.0, 93.7 +/- 1.4, 92.8 +/- 1.4, 90 +/- 4, and 90 +/- 4% for prostaglandin B1 (PGB1), leukotriene C4 (LTC4), leukotriene B4 (LTB4), leukotriene D4 (LTD4), 5-hydroperoxyeicosatetraenoic acid (5-HETE), respectively, were achieved. Separation and quantitation of products were performed on a Nucleosil 100 C18 column (5 microns, 4.6 X 250 mm) using, at pH 6.0, a gradient system comprising 72/28/0.02 (v/v/v) methanol/water/glacial acetic acid from 0 to 15 min, followed by a convex gradient to 76/24/0.02 (v/v/v) methanol/water/glacial acetic acid, followed by a 10-min hold at this methanol concentration. The method was used to investigate the profile of leukotrienes synthesized by rat hepatocyte homogenates from 5-HPETE or leukotriene A4 in absence or presence of glutathione (GSH). During a 5-min incubation with 100 microM 5-HPETE, 9.6 ng LTB4/mg protein and 2.2 micrograms 5-HETE/mg protein were formed in the absence of GSH. In the presence of 0.4 mM GSH, 3.7 ng LTB4/mg protein and 11.0 micrograms 5-HETE/mg protein were formed. Using 20 microM LTA4 as a substrate, 17.3 and 324.0 ng LTC4/mg protein X min and 14.3 and 19.3 ng LTB4/mg protein X min were formed in the presence of 0.4 and 10 mM GSH, respectively.
Huwyler
J
J
Department of Pharmacology, Biocenter of the University, Basel, Switzerland.
Gut
J
J
D000433:1-Propanol; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005978:Glutathione; D006863:Hydrogen-Ion Concentration; D006893:Hydroxyeicosatetraenoic Acids / Q000302:isolation & purification; D017572:Leukotriene A4; D007975:Leukotriene B4 / Q000302:isolation & purification; D015289:Leukotrienes / Q000302:isolation & purification*; D008099:Liver / Q000378:metabolism; D008297:Male; D008752:Methylene Chloride; D011456:Prostaglandins B / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013189:SRS-A / Q000302:isolation & purification
D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D015289:Leukotrienes; D011456:Prostaglandins B; D013189:SRS-A; D007975:Leukotriene B4; C034467:prostaglandin B1; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D008752:Methylene Chloride; C025348:arachidonic acid 5-hydroperoxide; D000433:1-Propanol; D005978:Glutathione