Sensitive and specific methodology for detection of labile NO-ferroheme complexes in vitro and in blood

Nitric oxide (NO) is formed via the oxidation of l-arginine in a reaction catalyzed by the NO synthase enzymes or via reduction of inorganic nitrite (NO₂^-) by deoxygenated hemoproteins and molybdopterin enzymes. We have recently demonstrated that NO can form a stable, labile ferrous heme-nitrosyl complex (NO-ferroheme) that protects NO from scavenging reactions in blood and has potent vasodilatory and platelet signaling activity. To enable future in vivo and in vitro studies establishing physiological NO-ferroheme formation, transport, and signaling, accurate and sensitive detection methods specifically for NO-ferroheme in biological milieu must be developed and validated. NO-heme complexes can be oxidized to release NO into the gas-phase for ozone-based chemiluminescence detection, which has been used for detection of iron-nitrosylated hemoglobin. In the current studies, we extend classical assays such as acidic potassium triiodide - with and without acidified sulfanilamide (AS) pre-treatment to eliminate NO₂^- and mercury(II) chloride (HgCl₂) pretreatment to eliminate S-nitrosothiols - to detect and quantify NO-ferroheme, S-nitrosothiols, and nitrite. We also developed a new potassium ferricyanide/cyanide-based assay for sensitive and specific NO-ferroheme detection. All assays are sensitive and specific for NO-ferroheme to concentrations as low as 5 nM, with validated detection in buffer, plasma, whole blood and in vivo studies in mice. Additionally, we detect and quantify in vivo plasma NO-ferroheme formation and levels in mice after treatment with lipopolysaccharide (LPS), modeling pathological sepsis. These studies validate highly sensitive and specific assays for NO-ferroheme quantification and for the first time demonstrate plasma NO-ferroheme formation in vivo in a sepsis model.