NO detection in biological samples: differentiation of 14 NO and 15 NO using infrared laser spectroscopy.

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between (14)NO and (15)NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples. With the cavity leak-out spectroscopy setup (CALOS) a differentiation between (14)NO and (15)NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of (14)NO/N(2) and (15)NO/N(2) were used to calibrate the system. (14)NO2- and (15)NO2- of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation. The correlation received for both methods for the detection of NO in the gas phase was r=0.999, p<0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for (14)NO2- 0.3 pmol and (15)NO2- 0.5 pmol; correlation (14)NO: p<0.0001, r=0.975, (15)NO: p<0.0001, r=0.969). The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.

Recent methodological advances in the analysis of nitrite in the human circulation: nitrite as a biochemical parameter of the L-arginine/NO pathway.

Nitric oxide (NO) plays a pivotal role in the modulation of multiple physiological processes. It acts as a messenger molecule within the cardiovascular system. NO is a highly unstable free radical in circulating blood and is oxidized rapidly to nitrite and nitrate. Recent studies suggest that nitrite has the potential to function as a surrogate of NO production under physiological and pathophysiological conditions and could therefore be of high relevance as a biochemical parameter in experimental and clinical studies. Under hypoxic conditions nitrite is reduced to bioactive NO by deoxyhemoglobin. This mechanism may represent a dynamic cycle of NO generation to adapt the demand and supply for the vascular system. Because of these potential biological functions the concentration of nitrite in blood is thought to be of particular importance. The determination of nitrite in biological matrices represents a considerable analytical challenge. Methodological problems often arise from pre-analytical sample preparation, sample contamination due to the ubiquity of nitrite, and from lack of selectivity and sensitivity. These analytical difficulties may be a plausible explanation for reported highly diverging concentrations of nitrite in the human circulation. The aim of this article is to review the methods of quantitative analysis of nitrite in the human circulation, notably in plasma and blood, and to discuss pre-analytical and analytical factors potentially affecting accurate quantification of nitrite in these human fluids.

Flavanols and cardiovascular health: effects on the circulating NO pool in humans.

Atherosclerosis is the major cause for chronic vascular diseases. The key event in the pathogenesis of atherosclerosis is believed to be dysfunction of the endothelium and disruption of endothelial homeostasis, leading to vasoconstriction, inflammation, leukocyte adhesion, thrombosis, and proliferation of vascular smooth muscle cells. Endothelium-derived nitric oxide (NO) plays a major role in vascular homeostasis and a decrease in NO-bioavailability accelerates the development of atherosclerosis. Given that endothelial dysfunction is at least in part reversible, the characterization of endothelial function and therapeutical approaches have gained much attention over the past years. Recent studies demonstrated that especially the consumption of plant-derived foods rich in certain flavonoids can improve endothelial function in both compromised and healthy humans. Furthermore, various physiologic and biochemical measures have been used previously as biomarkers for the assessment of the proposed beneficial effects of flavonoids in this context. More recently, the analysis of plasma nitros(yl)ated species (RXNOs), referred to as the circulating NO pool, has gained recognition, especially as a marker for endothelial function. This review is aimed at evaluating the suitability of quantifying this NO pool as a biomarker for cardiovascular function in humans, in particular during dietary interventions with flavonoid-rich foods.

Positive effects of nitric oxide on left ventricular function in humans.

AIMS: The myocardial effect of tonically released nitric oxide (NO) in humans is still not known. We tested the hypothesis that low-dose NO exerts positive effects on left ventricular (LV) function. METHODS AND RESULTS: Twelve healthy volunteers, 26+/-4 years, were enrolled in this study. Magnetic resonance imaging was used to precisely measure the direct effects of NO on stroke volume index (SVI). The NO pool was monitored by chemiluminescence. We reduced endogenous NO levels with intravenous infusion of the NO synthase-inhibitor N(G)-monomethyl-l-arginine. Replenishment of the NO pool was achieved with the NO donor S-nitrosoglutathione (GSNO) (0.5 micromol iv). To differentiate load-dependent from the direct effects of NO on LV function, changes in SVI in response to GSNO were compared with changes in the NO-independent vasodilator dihydralazine (2.5 mg iv) at matched arterial pressure and heart rate. Inhibition of NO synthesis was followed by reduction in SVI. Subsequent replenishment of the circulating NO with GSNO significantly increased SVI (39+/-8 to 54+/-7 mL m(-2); P=0.001), whereas no significant changes were observed with the NO-independent vasodilator dihydralazine (39+/-8 to 46+/-8 mL m(-2); P=0.0626). CONCLUSION: Inhibition of endogenous NO release reduces, whereas replenishment with exogenous NO increases LV function, pointing towards a positive effect of tonically released NO on LV function in healthy humans.