Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay.
Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.
Grates
Harry E
HE
Oxford Biomedical Research, P.O. Box 522, Oxford, MI 48371, USA.
McGowen
Richard M
RM
Gupta
Smiti V
SV
Falck
John R
JR
Brown
Thomas R
TR
Callewaert
Denis M
DM
Sasaki
Diane M
DM
D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics*; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis* / Q000097:blood / Q000378:metabolism / Q000652:urine; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014662:Vasoconstrictor Agents / Q000032:analysis*
D006893:Hydroxyeicosatetraenoic Acids; D014662:Vasoconstrictor Agents; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid