Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

Abstract

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.

Liquid chromatography-tandem mass spectrometry analysis of eicosanoids and related compounds in cell models

“…This restricts the amount of biological information obtained, and sample volume tends to be a limiting factor. Moreover, immunological assays lack selectivity, may be subject to cross-reactivity, and are commercially available for only few certain eicosanoids [17,18].…”

LC–MS/MS for the simultaneous analysis of arachidonic acid and 32 related metabolites in human plasma: Basal plasma concentrations and aspirin-induced changes of eicosanoids

“…Various analytical tools have been developed and validated for the separation, detection, and quantification of these eicosanoids, including gas chromatography-mass spectrometry (GC-MS) [21,22], liquid chromatography-mass spectrometry (LC-MS) [23,24], LC-fluorescence detection [25], electrophoresis [26], and radio-and enzyme immunoassays [26][27][28]. However, quantification of highly similar isomeric eicosanoid metabolites in complex biological matrices has limitations, including high cost, low specificity, limited sensitivity, cross-over and cross-talk effects, and time-consuming analysis.…”

Rapid, simultaneous quantitation of mono and dioxygenated metabolites of arachidonic acid in human CSF and rat brain

“…Multiple methods have been developed for the detection and quantification of these metabolites. Gas chromatography-mass spectrometry [27,28], liquid chromatography (LC) -mass spectrometry (MS) [29,30], LC-fluorescence detection [31], radioimmunoassays [32], electrophoresis [33], and enzyme immunoassays [34], have all aided in the quantitative analysis of these compounds from many different matrices. Although these assays are useful, limitations of these methods include high cost, limited sensitivity, cross-reactivity, and time-consuming analysis [35].…”