title: Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo.
abstract: 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 microM 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 (t(1/2) = 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.
authors.affiliation: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Dunne
authors.forename: Alexis
authors.initials: A
authors.lastname: Caron
authors.forename: Patrick
authors.initials: P
authors.lastname: Menu
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
authors.forename: Paul W
authors.initials: PW
authors.lastname: Buehler
authors.forename: Michael T
authors.initials: MT
authors.lastname: Wilson
authors.forename: Radu
authors.initials: R
authors.lastname: Silaghi-Dumitrescu
authors.forename: Beatrice
authors.initials: B
authors.lastname: Faivre
authors.forename: Chris E
authors.initials: CE
authors.lastname: Cooper
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology* / Q000627:therapeutic use; D001802:Blood Substitutes / Q000493:pharmacokinetics / Q000633:toxicity*; D002474:Cell-Free System; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005078:Exchange Transfusion, Whole Blood; D005609:Free Radicals; D006418:Heme / Q000378:metabolism; D006438:Hemodilution; D006441:Hemoglobin A / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood; D007501:Iron / Q000097:blood / Q000378:metabolism* / Q000633:toxicity; D008297:Male; D008706:Methemoglobin / Q000378:metabolism*; D008708:Methemoglobinemia / Q000139:chemically induced / Q000517:prevention & control*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D010108:Oxyhemoglobins / Q000378:metabolism*; D010545:Peroxides / Q000097:blood; D011817:Rabbits; D014168:Transferrin / Q000378:metabolism
chemical_list: D001802:Blood Substitutes; D005609:Free Radicals; D010108:Oxyhemoglobins; D010545:Peroxides; D014168:Transferrin; C066317:ferryl iron; D006418:Heme; C028881:hemoglobin A(0); D008706:Methemoglobin; D006441:Hemoglobin A; D006861:Hydrogen Peroxide; D007501:Iron; D001205:Ascorbic Acid