Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods. We chose to analyze triiodide chemiluminescence, drawn by the prospect of identifying why the most commonly employed assay in nitric oxide biology typically yielded lower metabolite values, compared with several other techniques. We found that the sensitivity of triiodide was greatly affected by the auto-capture of nitric oxide by deoxygenated cell-free heme in the reaction chamber. Potential contaminants and signal losses were also associated with standard sample purification procedures and the chemistry involved in nitrite removal. To inhibit heme nitric oxide auto-capture, we added potassium ferricyanide to the triiodide reagent, reasoning this would provide a more complete detection of any liberated nitric oxide. From human venous blood samples, we established nitric oxide levels ranging from 0.000178 to 0.00024 mol nitric oxide/mol hemoglobin. We went on to find significantly elevated nitric oxide levels in venous blood taken from diabetic patients in comparison to healthy controls (p < 0.0001). We concluded that the lack of signals reported of late by several groups using triiodide chemiluminescence for the detection of hemoglobin-bound nitric oxide may not represent levels on the border of assay sensitivity but rather underestimated values because of methodological limitations. We therefore stress the need for assay systems to be developed that differentiate between individual nitric oxide metabolite species and overcome the limitations we outline, allowing accurate conclusions to be drawn regarding physiological nitric oxide metabolite levels.Traditionally, endothelial-derived nitric oxide (NO) 1 has been viewed solely as a paracrine effector (1, 2), playing an important role in the local control of vascular tone and blood flow. However, more recent evidence has revealed an endocrine role for NO via the formation of metabolites that preserve bioactivity and contribute to blood flow regulation and oxygen delivery (3-7). Studies into the mechanisms for this effect have led to two alternative models that emphasize the significance of different metabolites, the ability of S-nitrosohemoglobin (SNOHb) within red blood cells to provide bio-available NO (3, 5) and the potential formation of NO from nitrite (NO 2 Ϫ ) mediated by the reductive potential of deoxyhemoglobin (8). Although NO 2 Ϫ reduction by deoxyhemoglobin has been shown to represent a potential source of NO (in conjunction with methemoglobin) (9), it has yet to be convincingly demonstrated that this NO could remain free to play a physiological role in vasodilation (10, 11). This would necessitate NO escaping the red blood cell and avoiding uptake/binding to ferrous heme, which is highly improbable considering the low oxygen conditions under which deoxyhemoglobin acts as a NO 2 Ϫ reductase (12, 13). Further detracting from a direct role for NO 2 Ϫ in hypoxic vasodilation is the fact that nitric-oxi...

A plethora of publications on techniques and methodologies for measuring nitric oxide (NO) or reaction products of NO (NO metabolites) has served in recent years to complicate and confuse the majority of researchers interested in this field. Here, we provide a practical approach and summarize the key issues and corresponding solutions regarding quantification with the use of ozone-based chemiluminescence, which is the most accurate, sensitive, and widely used NO detection method. We have drawn on the vast experience of leaders in the field to produce this consensus, but the views and implications presented herein represent our own, and we limit our advice to those techniques with which we have direct experience. Hopefully, this guide will allow authors to make more informed decisions regarding NO metabolite measurement methodology, without the need for each subsequent group to rediscover previously observed advantages and pitfalls.

Aortovascular medicine and surgery in relation to vascular health of the population requires input from a broad range of specialists and institutions throughout a patient life as well as integration with allied health care providers. This paper presents the essence of the novel clinical concept of the ‘Aortovascular Hub’ .

For the first time in human subjects across the resting coronary circulation we reveal significant re-apportionment of NO between metabolite species which correlate with haemoglobin oxygen saturation. These changes occur even within the transit time of blood across this single vascular bed. We demonstrate no net loss/gain of NO from the total metabolite pool across the coronary circulation even where NO biosynthesis is inhibited.