Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation

The reactions of nitric oxide with hemoglobin play an important role in explaining the vascular biology of this free radical. It is perhaps surprising that the level of nitrosylhemoglobin (HbNO) in which NO is bound to the ferrous hemoglobin heme in whole human blood under basal and stimulated conditions is a matter of some controversy, with measurements ranging from <1 nm to close to 10 mum. In order to examine HbNO levels in human blood by using EPR spectroscopy, we have developed a regression-based spectral analysis technique that has a detection level of about 200 nm HbNO. We have utilized this methodology to detect the level of HbNO under basal conditions and during NO inhalation. The major findings of this study are as follows. (i) HbNO can be accurately detected and quantified in whole blood with a detection limit of approximately 200 nm. (ii) By using regression analysis, levels of HbNO as low as 0.5-1 mum can be deconvoluted into component species. (iii) HbNO is present at less than 200 nm at basal conditions in both arterial and venous blood and is formed at a level of 0.5-2.5 mum upon inhalation of 80 ppm NO. (iv) The levels of HbNO detected by EPR are remarkably close (within a factor of 2) to those detected by tri-iodide-based chemiluminescence and much smaller than those detected by photolysis chemiluminescence. (v) The half-time of HbNO in vivo is approximately 40 min.