The hypertensive response to the infusion of hemoglobin has been attributed to the reaction between hemoglobin and the endothelial derived nitric oxide (NO). Systemic and renal responses to the infusion of unmodified, cross-linked and polymerized cross-linked hemoglobins were studied in anesthetized rats. Unmodified hemoglobin produced significant increase in the mean arterial blood pressure (MAP) throughout the 60 minute infusion. Cross-linked hemoglobin, on other hand, produced a more marked and prolonged increase in MAP over 120 minutes. Only a moderate increase in MAP was observed in rats after 30 minutes infusion with the polymerized protein. Data transpired from this project have recently been accepted for publication in J. Lab.Cli. Med. Treatment of the hemodynamic imbalance due to the overproduction of NO, in patients undergoing septic shock with hemoglobin was recently suggested. Since it is believed that hemoglobin preparations can enhance the toxicity of bacterial endotoxin, we studied the effects of hemoglobin on lipopolysccaharide with endothelial cells. Preliminary studies indicate that the increased toxicity of the Hb/endotoxin complex involves an oxidative mechanism, since Trolex, an antioxidant, can resolve the cytotoxicity due to the LPS/Hb complex but not the cytotoxicity due to LPS or Hb alone. When superoxide production exceeds nitric oxide formation, under ischemic conditions in the vascular wall, the vasorelaxing effects of NO may be suppressed, as result of peroxynitrite formation. Reaction of native and chemically modified hemoglobins with preoxynitrite (ONOO-) lead to rapid oxidation of the heme iron to the ferric form and possibly other oxidation products. Time courses obtained from rapid kinetic experiments showed a biphasic loss of the ferrous oxidation state on mixing peroxinitrite with these hemoglobins. Using colorimetric and chemiluminescent immunodetection techniques we were able to show that tyrosine nitration by peroxynitrite can occur with both native and modified hemoglobins. We have data to show that ONOO is cytotoxic to endothelial cells both directly and indirectly through peroxidation of serum components. The addition of unmodified hemoglobin specifically protects against the cytotoxicity due to peroxidation of serum components and doesn't protect against the direct action of preoxynitrite on the endothelial cells. In addition, we have measured nitrate/nitrite (a breakdown product of nitric oxide) and H2O2 (an enzymatic product of superoxide) in the media of endothelial both before and after an ischemic period in a new cell culture model. Both products were found in greater concentration after ischemia.
