The Autoxidation of αα Cross-Linked Hemoglobin: A Possible Role in the Oxidative Stress to Endothelium

Abstract

The aim of the present study was to investigate the role of hemoglobin autoxidation in the induction of endothelial heme oxygenase (HO), an inducible “stress” protein which is responsible for heme catabolism. Porcine aortic endothelial cells were incubated for six hours in the presence of 60 μM unmodified hemoglobin (HbA₀), hemoglobin cross-linked between the a chains with bis-(3,5-dibromosalicyl) fumarate (ααHb) or cyanomet-αα-hemoglobin (CNmetααHb). Microsomal HO content increased 4.1-fold in the presence of ααHb, 2.7-fold with HbA₀ and 1.8-fold with CNmetααHb over the control value. The rates of methemoglobin formation exhibited a linear relationship over the time of incubation (r = 0.94) and the apparent rate constant was 1.8-fold higher for ααHb (0.023 h^-1) than HbA₀ (0.013 h^-1). In addition, a linear relationship was obtained by plotting the rates of autoxidation of hemoglobins versus the HO activity (r = 0.99). When cells were incubated with 100% methemoglobin, HO activity increased 5.0-fold and 4.7-fold for HbA₀ and ααHb, respectively. Intracellular heme concentration, measured after 24 hours of incubation, was also significantly greater in the presence of ααHb (52.6% over baseline) compared to HbA₀ (10.8%) and CNmetααHb (15.3%) groups (p<0.05). However, lactate dehydrogenase (LDH) release, measured as an index of endothelial cell injury, increased in all the hemoglobins examined: ααHb, 33.8±1.1 U/l; HbA₀, 38.5±3.5 U/l; CNmetααHb, 41.9±4.0 U/l; (control group, 19.4±2.8 U/l). We conclude that: 1) the higher rate of oxidation of ααHb renders the molecule more susceptible to induce endothelial oxidative stress (HO induction); 2) the accelerated methemoglobin formation is directly correlated to intracellular HO content and endothelial heme uptake; 3) persistent cell injury suggests that other factors besides heme release may contribute to the hemoglobin-mediated cytotoxicity.