Reduction of biological effluents in purge and trap micro reaction vessels and detection of endothelium-derived nitric oxide (edno) by chemiluminescence

Abstract

Various analytical approaches have been used to measure endothelium-derived nitric oxide (NO) [ 1, 3, 4, 7, 10, 12]. We have detected NO in perfusates with a sample size as low as 2 ml after acidification with 4 N HCl to pH < 2 at 25°C by using a Nitric Oxide Analyser (Sievers, Colorado) [ 7]. This procedure had the advantage that the detectable level of NO was enhanced by the self-decomposition of HNO 2 when the PH < pK a of NHO 2 (pKa = 3.15) and also the reaction temperature of 25°C substantially increased the half-line of NO [ 2]. Palmer, et al., [ 10] measured NO released by cultured porcine endothelial cells by chemiluminescence after passing cell effluents continyously at a rate of 5 ml/min into 75 ml of 1% sodium iodide in glacial acetic acid. The larger volumes involved in this method for continuous refluxing, made it less desirable for the detection of endothelium-derived nitric oxide. Feelisch et al. [ 1] utilized the activation of soluble guanylate cyclase, as well as, the quantitative oxidation of oxyhemoglobin to methemoglobin in aqueous solutions by NO as a means of measuring nitric oxide. We describe here a modification of our earlier micromethod which now enables us to detect NO after complete reduction with glacial acetic acid and sodium iodide. A comparison of the two procedures indicate that while freshly prepared NO standard solutions gave identical chemiluminescence response with and without reduction, effluents from bovine intrapulmonary artery under basal conditions gave substantially higher values upon reduction.