New Methods to Evaluate Endothelial Function: A Search for a Marker of Nitric Oxide (NO) In Vivo: Re-evaluation of NOx in Plasma and Red Blood Cells and a Trial to Detect Nitrosothiols

Abstract

Abstract. Although plasma NOx (NO 2 -and NO 3 -) has been used as an index of nitric oxide (NO) formation in vivo, many unreasonable results appeared even after active elimination of NOx contamination from laboratory ware. For example, plasma NOx concentrations did not increase during vasodilation mediated by the NO / cGMP pathway or after organ perfusion. A possible shift of NOx from plasma to erythrocytes (RBCs) as a cause of these phenomena has been excluded, leaving the destination of NOx (after leaving plasma) unknown. Kinetic analyses have revealed that steady state NOx concentrations in plasma and whole blood did not correlate with the NOx formation rate, but rather with the NOx elimination rate. Therefore, the supposition that the NO status is directly reflected by plasma NOx concentrations appears untenable. As nitrosothiols (R-SNOs), possible carriers of NO bioactivity, have been flagged as alternative indices of NO status in vivo, efforts have been made to detect these substances. When interference by ultrafiltration was eliminated, low molecular weight R-SNOs such as nitrosocystein and nitrosogluthathione were undetectable. However, a high-molecular weight R-SNO, nitrosoalbumin, was detected in human blood. Further research is required into the significance and practical use of nitrosoalbumin as a marker of NO in vivo.

Photoinstability of S-Nitrosothiols during Sampling of Whole Blood: A Likely Source of Error and Variability in S-Nitrosothiol Measurements

“…Hence, this light effect may explain the wide discrepancy of mean reference range values reported in the literature, because additional photoinduced losses can occur during the extra sample preparation steps (e.g., preparing plasma) that are required for all other RSNO detection methods. Indeed, some researchers have found LMW RSNO species to be "undetectable" in plasma samples (11 ).…”

Arteriovenous Differences in NO2- Kinetics in Anesthetized Rabbits

“…1) Early studies that attempted to use plasma nitrate (NO 3 Ϫ ) or NO x (nitrite [NO 2 Ϫ ] plus NO 3 Ϫ ) as stable metabolites of NO 2,3) encountered too high a degree of interference, resulting from numerous confounding factors (including contamination), for their results to be applied practically. [4][5][6] In addition, as-yet unexplained and paradoxical decreases in NO x following increases in NO formation have been reported. 7,8) In contrast, plasma NO 2 Ϫ has been the source of much interest lately as a promising indicator of NO production following reports that approximately 70-90% of circulating plasma NO 2 Ϫ is derived from endothelial nitric oxide synthase (eNOS) activity in humans and animals.…”

Long-Term Treatment With Imidapril but Not With Nifedipine Enhances Plasma NOx Concentration in Patients With Essential Hypertension

“…To avoid contamination of such NOx in our NOx measurement, we minimized the time of exposure of blood samples to the air and used ultrapure water purified with the Milli-Q system (Millipore Corporation, Billerica, MA, USA) when all solutions were prepared (21). NOx from laboratory ware is also a major source of NOx contamination (22). Indeed, when we substituted the plasma samples by freshly prepared ultrapure water purified with the Milli-Q system throughout whole procedure from blood sampling to NOx quantification, we detected a low level of NOx (6.8 ± 0.7 µmol / L, n = 8).…”

“…However, whether NOx is a reliable marker of NO formation in vivo is still controversial, and indeed there are conflicting facts regarding plasma NOx level in hypertensive individuals: two studies reported that plasma NOx concentrations were significantly reduced in patients with essential hypertension compared to those in normotensive healthy subjects (12,25) whereas one study showed that plasma NOx levels were similar in refractory hypertensive patients and normotensive healthy subjects (26). In addition, it has been suggested that NO 2 − , but not NOx, is a useful marker for assessing NO production (22,27). However, we have recently demonstrated that plasma NOx concentration as well as urinary NOx excretion are significantly lower in accordance with the number of disrupted NOS isoforms in the order of singly, doubly, and triply NOS −/ − mice (28).…”

“…These results suggest that NOx can be of use as a marker of systemic NO production to some extent. At least, when long-term response, but not short-term response, to therapy is monitored in individual patients, NOx is thought to be reliable as a marker of NO formation in vivo (22,23).…”