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.
The concentration of testosterone in whole saliva is significantly increased (by 9%) after toothbrushing. In ultrafiltrates of saliva collected at the same time as the whole saliva, testosterone concentrations after toothbrushing were unchanged. In 88% of the 162 whole-saliva specimens, but not in the ultrafiltrates, we also measured higher hemoglobin concentrations after toothbrushing. We conclude that the increase of testosterone in whole saliva after toothbrushing can be attributed to a protein-bound fraction. For analytes that are bound to serum proteins, salivary measurements can give spurious results. This problem can be avoided by using as a diagnostic medium an ultrafiltrate of saliva collected directly in the mouth.
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