Abstract

It has been shown that infusion of nitrite into humans results in vasodilation. The goal of this study was to investigate the mechanism of nitrite-induced vasodilation, which may be due to NO production on nitrite reduction by deoxyhemoglobin (deoxyHb). We found that nitrosylhemoglobin (nitrosylHb) and methemoglobin (metHb) are formed in anaerobic deoxyHb/nitrite incubates and S-nitrosohemoglobin is produced on aeration. The efficiency of NO transfer from the heme to CysÝ93 of hemoglobin varies with the initial deoxyHb/nitrite ratio and with the amount of metHb present. Higher S-nitrosohemoglobin (HbSNO) yields were observed at close to the physiological nitrite to heme ratio and the yield decreased with increasing metHb. Also, hydrogen peroxide depressed the HbSNO yield whereas antioxidant enzymes promoted NO transfer from the heme to CysÝ93. These results indicate that NO transfer requires that Hb undergo its T[arrow right]R allosteric transition, that oxygen be present presumably as an oxidant and HbSNO formation is inhibited by metHb. Sequential extraction, HPLC/fluorescence and HPLC/MS/MS methods were developed to detect nitrite, adenosine and epoxyeicosatrienoic acids (EETs) in human interstitial fluid with high sensitivity, precision and accuracy. These methods were used to determine the interactions and compensatory responses amongst these endothelium-derived vasodilators. Nitrite and adenosine were found to increase with exercise level but EETs were below the level of detection (10 nM). Blockade of nitric oxide and prostaglandin formation resulted in diminished nitrite levels but the adenosine levels remain unchanged.