Iron nitrosyl hemoglobin formation from the reaction of hydroxylamine and hemoglobin under physiological conditions

Abstract Sickle cell disease patients receiving hydroxyurea (HU) therapy have shown increases in the production of nitric oxide (NO) metabolites, which include iron nitrosyl hemoglobin (HbNO), nitrite, and nitrate. However, the exact mechanism by which HU forms HbNO in vivo is not understood. Previous studies indicate that the reaction of oxyhemoglobin (oxyHb) or deoxyhemoglobin (deoxyHb) with HU are too slow to account for in vivo HbNO production. In this study, we show that the reaction of methemoglobin (metHb) with HU to form HbNO could potentially be fast enough to account for in vivo HbNO formation but competing reactions of either excess oxyHb or deoxyHb during the reaction reduces the likelihood that HbNO will be produced from the metHb–HU reaction. Using electron paramagnetic resonance (EPR) spectroscopy we have detected measurable amounts of HbNO and metHb during the reactions of oxyHb, deoxyHb, and metHb with excess hydroxylamine (HA). We also demonstrate HbNO and metHb formation from the reactions of excess oxyHb, deoxyHb, or metHb and HA, conditions that are more likely to mimic those in vivo. These results indicate that the reaction of hydroxylamine with hemoglobin produces HbNO and lend chemical support for a potential role for hydroxylamine in the in vivo metabolism of hydroxyurea.