Detection of Human Red Blood Cell-bound Nitric Oxide

Abstract

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...

Assessment of nitric oxide signals by triiodide chemiluminescence

“…The same sample was assayed by triiodide (41-45): 0.2 M FeCN and 0.2 M KCN was used to eliminate FeNO; the sample was desalted on a G25 spin column to remove nitrite; Hg was used to displace NO from SNO, 0.5% SAA/1 M HCl was added to verify nitrite/SNO. Substantial sample precipitation leading to losses during desalting could not be avoided, as previously noted (56). The SNO-Hb yield by triiodide (i.e., NO that survives FeCN and SAA and is reactive to Hg) was 430 nM or 8.4% of SNO measured by photolysis (Fig.…”

“…The SNO-Hb yield by triiodide (i.e., NO that survives FeCN and SAA and is reactive to Hg) was 430 nM or 8.4% of SNO measured by photolysis (Fig. 3D) (56).…”

Measurement of Nitric Oxide Levels in the Red Cell

“…Meanwhile, this assay has been validated by numerous other laboratories (24 -28) and is gaining greater acceptance over the photolysis method. Nevertheless, its merit continues to be challenged by proponents of the photolysis technique, who claim that the tri-iodide assay underestimates NO measurements because the "harsh chemical pretreatment" with acidified sulfanilamide, designed to remove nitrite prior to assaying for other NO-related species, destroys S-nitrosothiols (1,29,30). To date, however, no experimental results have been presented to substantiate this claim.…”

Acute whole body UVA irradiation combined with nitrate ingestion enhances time trial performance in trained cyclists

“…Plasma samples were stored for a maximum of 4 months at -80 o C for later analysis of nitrite via ozone-based chemiluminescence (Rogers et al, 2005). We have described the procedures for the determination of nitrite previously (Muggeridge et al, 2013).…”