Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid.
This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicosatetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachidonic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, was added to samples, and the lipids were extracted and labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. P-450 metabolites were separated on a C18 reverse-phase HPLC column. Coelution and gas chromatography-mass spectrometry studies confirmed the identity of the 20-HETE peak. The 20-HETE peak can be separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids. Known amounts of 20-HETE were used to generate a standard curve (range 1-10 ng, r(2) = 0.98). Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was <5%. Levels of 20-HETE were measured in 100 microliter of urine and renal interstitial fluid or 0.1 mg of renal tissue. The assay was evaluated by studying the effects of 1-aminobenzotriazole (ABT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE by >65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field.
Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
K G
KG
Maier
L
L
Henderson
J
J
Narayanan
M
M
Alonso-Galicia
J R
JR
Falck
R J
RJ
Roman
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis* / Q000378:metabolism / Q000652:urine; D007202:Indicators and Reagents; D007672:Kidney Cortex / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology*; D051381:Rats; D011918:Rats, Inbred SHR; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence / Q000379:methods; D014230:Triazoles / Q000494:pharmacology
D006893:Hydroxyeicosatetraenoic Acids; D007202:Indicators and Reagents; D014230:Triazoles; C033020:1-aminobenzotriazole; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System