Formation of Nitroso Compounds in the Reaction of Nitrite and Hemoglobin.

Nitrite is being studied as a therapeutic agent for several pathologies, largely due to the demonstration that infusions of nitrite into the human forearm results in increased blood flow. Thus, nitrite may serve as a storage pool for the endothelium derived vasodilator, nitric oxide (NO). Nitrite is known to react with deoxyhemoglobin to form NO, which would need to diffuse from the red blood cells to the smooth muscle cells in order to activate its target enzyme soluble guanylyl cyclase. However, hemoglobin scavenges NO extremely quickly so that NO formed in the red blood cell cannot get out. The export of nitrite-derived NO activity is likely to require the formation of either a diffusible intermediate or alternative species for the transport of NO. One of the suggested intermediates is S-nitrosohemoglobin, small amounts of which have been reported to have formed during nitrite infusion studies. Other investigators recently reported that S-nitrosohemoglobin is directly formed in the reaction of nitrite and hemoglobin via the formation of a quasi-stable Fe(III)NO-hemoglobin intermediate. We have used absorption spectroscopy, electron paramagnetic resonance spectroscopy, and several chemiluminescence-based assays to the nitrite/hemoglobin reaction, focusing on the formation of S-nitrosohemoglobin and other nitroso species such as S-nitrosoglutathione (GSNO). We have also developed a sensitive chemiluminescence-based assay to explore Fe(III)NO-Hb formation. We applied these techniques to a variety of conditions, varying oxygen saturation and reactant concentrations in cell-free and red cell encapsulated hemoglobin. Based on our results, we conclude (contrary to several other recent reports) that little or no nitroso species are formed in the reaction of nitrite and hemoglobin at neutral pH (7.4) and a quasi-stable Fe(III)NO-Hb intermediate is also not formed. We find that, under thoroughly deoxygenated conditions, one nitrite molecule results in the formation of one Fe(II)NO-Hb and one MetHb species, with some nitrite bound MetHb being formed subsequently. Importantly, we find that at pH 6.5, significant nitrosation does occur. These data suggest that much or all of the nitroso species measured in nitrite therapeutics may be formed in metabolically active muscle or other tissues of mild acidity.