One potential mechanism of toxicity of hemoglobin-based blood substitutes involves the reaction of hemoglobin with oxygen metabolites formed upon reperfusion of ischemic tissues. This could lead to a deleterious pathway in which oxidatively damaged hemoglobin releases its heme and subsequently, its toxic iron. We tested the susceptibility of a number of chemically modified hemoglobins to oxidative damage caused by hydrogen peroxide (H2O2). Treatment of hemoglobins with low levels of H2O2 produced soluble protein-bound heme products that were subsequently isolated using HPLC. Human hemoglobin cross-linked at the alpha subunits showed a typical propensity to oxidative modification than other forms of hemoglobin modifications. The heme derived adducts from the reaction of H2O2 with hemoglobins showed little or no oxidase activity in the NADPH-diaphorase methemoglobin reductase system. A similar approach is currently underway to assess the susceptibility of other hemoglobin-based red cell substitutes to oxidative damage in order to determine the molecular basis of heme and protein alterations. The potential for oxidative damage by hemoglobin solutions opens a critical field of study that will have to be well understood before any of the present generation of hemoglobin products will be successful as a reperfusion agent.
