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.
Subject(s)
Colorimetry , Enzyme-Linked Immunosorbent Assay/economics , Hydroxyeicosatetraenoic Acids/analysis , Reproducibility of Results , Sensitivity and Specificity , Vasoconstrictor Agents/analysisABSTRACT
Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogen's inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the renin-angiotensin vasoconstrictor/vasodilator system is to promote the antihypertensive effect.
Subject(s)
Animals , Female , Rats , Estrogens/metabolism , Renin-Angiotensin System/physiology , Angiotensins/analysis , Angiotensins/drug effects , Aorta, Thoracic/enzymology , Estrogen Replacement Therapy , Estrogens/pharmacology , Haplorhini , Kidney/enzymology , Ovariectomy , Peptidyl-Dipeptidase A , Plasma/enzymology , Renin-Angiotensin System/drug effects , Vasoconstrictor Agents/analysisABSTRACT
Este trabalho teve por finalidade avaliar: a) os critérios utilizados pelos Cirurgiöes-Dentistas, Clínicos-Gerais, no emprego dos anestésicos locais em pacientes hipertensos controlados ou näo; b) cuidados que devem ser tomados pelos Profissionais na anamnese e no exame clínico para o diagnóstico desta patologia, bem como os cuidados adotados durante procedimentos clínicos de rotina. Questionários, padronizados em número aproximados de 200 (duzentos), foram preenchidos pelo pesquisador que entrevistou Cirurgiöes-Dentistas da regiäo Zona-Leste da grande Säo Paulo. Os resultados deste estudo mostraram que 75,0 por cento dos Cirurgiöes-Dentistas habitualmente näo realizam tomada da pressäo arterial. Para pacientes hipertensos, 60,38 por cento dos Cirurgiöes-Dentistas preferem utilizar anestésicos locais sem vasocontrictores