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.
Highlights
Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods
The addition of K3FeIII(CN)6 to the cysteine/cuprous chloride reagent totally inhibited the detection of GSNO; the addition of K3FeIII(CN)6 to the triiodide reagent had no influence on the signals derived from NaNO2Ϫ (Fig. 1A), GSNO (Fig. 1B), or plasma samples
The broadening of signals derived from NaNO2Ϫ or GSNO in the presence of red blood cell lysate or purified Hb was inhibited following the addition of K3FeIII(CN)6 to the triiodide reagent (Figs. 6 and 7 respectively)
Summary
Chemicals and Standards—All chemicals were purchased from Sigma, except for GSNO, which was from Alexis, and glacial acetic acid, from Fischer. Samples were injected into 4-ml EDTA collection tubes, which were centrifuged at 600 ϫ g for 10 min at 4 °C Both the red cell fraction and plasma were snap frozen in liquid nitrogen and stored at Ϫ80 °C for subsequent analysis. Red blood cell samples were subsequently lysed 1:4 in EDTA (0.5 mM; pH corrected to 7.0) and incubated for 5 min on ice. Purified Hb was acquired by passing 500 l of the red blood cell lysate through a prewashed (with high pressure liquid chromatography NO2Ϫ-free water) Sephadex G25 column, which takes ϳ3 min at room temperature (20 – 22 °C). One red blood cell or Hb sample was injected into each assay mix before being replaced to prevent saturation of Hb and exhaustion of the denitrosating reagent. Reactions of the triiodide reagent with other redoxactive agents could not be controlled; the effect of endogenous redox-active agents on triiodide have previously been investigated and found not to interfere with the denitrosation process [28]
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