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 isovolemic 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 were observed in rats after 30 minutes infusion with the polymerized protein. Chemical polymerization to increase the size of cross-linked hemoglobin did not blunt its pressor effects nor improved its renal hemodynamic effects under partial exchange transfusion, a more clinically relevant indication for a hemoglobin-based blood substitute. Data transpired from this project have recently been published in J. Lab .Clin. Med. (1997;129:603-610). The pro and anti-oxidant effects of NO appear to be dependent on the chemistry it undergoes in a biological system, and that under certain conditions the balance between NO/02.-may be channeled by way of peroxynitrite (ONOO-), a byproduct of NO reactions with O2.-. Reactions of native and chemically modified hemoglobins with ONOO- leads 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 ONOO- with these hemoglobins. Using colorimetric and chemiluminescent immunodetection techniques we were able to show that tyrosine nitration by ONOO- can indeed occur with both native and modified hemoglobins. A mechanism describing the reaction of ONOO-with a number of chemically modified hemoglobins has just been accepted for publication in Arch. Biochem. Biophysics. Treatment of the hemodynamic imbalance due to the over-production of NO, in patients undergoing septic shock with hemoglobin was recently suggested. We studied the effects of hemoglobin on lipopolysaccharide (LPS) interactions with endothelial cells. Preliminary studies indicate that the increased toxicity of the Hb/LPS complex involves an oxidative mechanisms, since Trolex, antioxidant, can resolve the cytotoxicity due to the LPS/Hb complex but not the cytotoxicity due to LPS or hemoglobin alone. We are currently exploring the mechanism of the chemical interaction between LPS and hemoglobin in order to verify that these reactions are indeed driven by free radical mechanisms.
