FREE-RADICAL IN BLOOD - A MEASURE OF HEMOGLOBIN AUTOXIDATION IN-VIVO

The EPR spectrum of whole human or animal blood, measured at 77 K, exh ibits a free radical signal identical to that observed when purified m ethaemoglobin (metHb) reacts with H2O2. This signal is usually attribu ted to a globin-based radical formed as a result of two electron oxida tion of metHb. We have recently proposed a mechanism to account for th e formation of these globin radicals in blood. H2O2 is produced in sma ll amounts via dismutation of O-2(.-) formed during normal autoxidatio n of haemoglobin and this H2O2 then reacts with metHb to form the radi cal. This mechanism allows us to explain the variability (fluctuations ) in metHb and free radical concentrations in apparently identical sam ples of frozen blood. In the present work we further confirm that the fluctuations are caused by sample freezing. We also show that the rang e of fluctuations of both metHb and free radical EPR signals in human venous blood decreases with increasing time of incubation at room temp erature under aerobic conditions, and the fluctuations are completely absent after three hours. This may be understood as an effect of incre asing the oxygenation of the venous blood. When fully oxygenated, haem oglobin autoxidation is suppressed; therefore O-2(.-) is not produced in significant amounts and H2O2 is not formed on sample freezing. To c onfirm this interpretation we have studied venous and arterial rat blo od and found that the free radical concentration is low and does not f luctuate (neither did metHb) in the oxygenated arterial blood, while v enous rat blood shows the inverse fluctuations similar to those observ ed previously in human venous blood. We therefore conclude that the in tensity of the free radical and metHb signals in frozen blood samples can be used as a measure of haemoglobin autoxidation and O-2(.-) produ ction in blood.