Biochem. J. (2006) 399
(513–524) (Printed in Great Britain)
Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo
Jacqueline DUNNE*1, Alexis CARON†1, Patrick MENU†, Abdu I. ALAYASH‡, Paul W. BUEHLER‡, Michael T. WILSON*, Radu SILAGHI-DUMITRESCU*, Beatrice FAIVRE† and Chris E. COOPER*2
*Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K., †Laboratoire Hématologie-Physiologie, Faculté de Pharmacie, Université Henri Poincaré, 5–7 rue Albert Lebrun, BP 403, F-54001 Nancy cedex, France, and ‡Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29, Room 112, Bethesda, MD 20892, U.S.A.
Key words: ascorbate, blood substitute, ferryl, free radical, haemoglobin, oxidative stress.
Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 µM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t1/2=1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.
Abbreviations used: DBBF, bis-(3,5-dibromosalicyl)fumarate; MAP, mean arterial pressure.
1These authors contributed equally to this manuscript.
2To whom correspondence should be addressed (email ccooper@essex.ac.uk).
Received 2 March 2006/19 June 2006; accepted 18 July 2006
Published as BJ Immediate Publication 18 July 2006, doi:10.1042/BJ20060341
The Biochemical Society, London ©2006
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