Free radical in blood: a measure of haemoglobin autoxidation in vivo?

The EPR spectrum of whole human or animal blood, measured at 77 K, exhibits a free radical signal identical to that observed when purified methaemoglobin (metHb) reacts with H2O2. This signal is usually attributed to a globin-based radical formed as a result of two electron oxidation of metHb. We have recently proposed a mechanism to account for the formation of these globin radicals in blood. H2O2 is produced in small amounts via dismutation of O-2(.-) formed during normal autoxidation of haemoglobin and this H2O2 then reacts with metHb to form the radical. This mechanism allows us to explain the variability (fluctuations) in metHb and free radical concentrations in apparently identical samples of frozen blood. In the present work we further confirm that the fluctuations are caused by sample freezing. We also show that the range of fluctuations of both metHb and free radical EPR signals in human venous blood decreases with increasing time of incubation at room temperature under aerobic conditions, and the fluctuations are completely absent after three hours. This may be understood as an effect of increasing the oxygenation of the venous blood. When fully oxygenated, haemoglobin autoxidation is suppressed; therefore O-2(.-) is not produced in significant amounts and H2O2 is not formed on sample freezing. To confirm this interpretation we have studied venous and arterial rat blood and found that the free radical concentration is low and does not fluctuate (neither did metHb) in the oxygenated arterial blood, while venous rat blood shows the inverse fluctuations similar to those observed previously in human venous blood. We therefore conclude that the intensity of the free radical and metHb signals in frozen blood samples can be used as a measure of haemoglobin autoxidation and O-2(.-) production in blood.