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The aim of the present study was to evaluate the role of hemoglobin (Hb) and the contribution of chemically modified Hb solutions on the activation of nuclear transcription factor. NF-kappa B, and propagation of oxidative stress within human vascular endothelial cells. The activation of an oxidative stress-sensitive NF-kappa B can be linked with the propagation of an inflammatory state via rapid induction of genes for several pro-inflammatory mediators. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips or cell culture plates to confluence. Then, the cells were incubated for up to 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and test agents in a concentration of 0.1 and 0.2 mmol: 1) unmodified bovine Hb (UHb): 2) modified Hb solution polymerized with glutaraldehyde (GLUT-Hb), and 3) a novel modified Hb solution (Hb-PP-GSH) prepared according to our patented procedure (U.S. Patent No. 5,439,882). The positive control for the NF-kappa B activation study included a treatment of the cells with: I) endotoxin: IL-1; TNF; and H2O2. Results indicate that Hb's pro-oxidant potential was influenced by the type of chemical modification procedure. The GLUT-Hb autoxidation rate, peroxidase-like activity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxidant potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of the HCAECs was found to be the Hb modification-type and concentration dependent. Although the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH was associated with 0.2 mmol of GLUT-Hb, the Hb-PP-GSH did not produce significant changes when compared to the control cells. The UHb generated a moderate oxidative stress to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the induction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapidly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuclear induction of NF-kappa B. However, Hb-PP-GSH did not activate NF-kappa B directly. In this study, the positive control cells treated with endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whereas H2O2 caused only partial induction. In conclusion, nuclear translocation of NF-kappa B by Hb solutions might be dependent on Hb's pro-oxidant potential and extent of Hb-mediated endothelial oxidative stress. Besides the low oxidative potential of Hb-PP-GSH, the observed lack of NF-kappa B activation by this Hb solution can be also related to the anti-inflammatory properties of adenosine which is used in our novel modification procedure. In this study, only the Hb-PP-GSH, cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathiore, ...

Chemically modified hemoglobin (Hb) solutions are under current investigation as potential red cell substitutes. Researchers at Texas Tech University have developed a novel free Hb based blood substitute product. This blood substitute is composed of purified bovine Hb cross-linked intramolecularly with o-adenosine-5'-triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione (GSH). In this study, we compared the effects of our novel blood substitute and unmodified (U) Hb, by using allogenic plasma as the control, on human blood components: red blood cells (RBCs), platelets, monocytes (Mo), and low-density lipoproteins (LDLs). The pro-oxidant potential of both Hb solutions on RBCs was examined by the measurement of osmotic and mechanical fragility, conjugated dienes (CD), lipid hydroperoxides (LOOH), thiobarbituric acid reactants (TBAR-S), isoprostanes (8-iso PGF2alpha) and intracellular GSH. The oxidative modification of LDLs was assessed by CD, LOOH, and TBAR-S, and the degree of apolipoprotein (apo) B cross-linking. The effects of Hb on platelets have been studied by monitoring their responses to the aggregation agonists: collagen, ADP, epinephrine, and arachidonic acid. Monocytes were cultured with Hb solutions or plasma and tested for TNF-alpha and IL-1beta release, then examined by electron microscopy. Results indicate that native UHb initiates oxidative stress of many blood components and aggravates inflammatory responses of Mo. It also caused an increase in RBC osmotic and mechanical fragility (p < 0.001). While the level of GSH was slightly changed, the lipid peroxidation of RBC increased (p < 0.001). UHb was found to be a stimulator of 8-iso PGF2alpha synthesis, a potent modulator of LDLs, and an effective potentiator of agonist induced platelet aggregation. Contrarily, our novel blood substitute did not seem to induce oxidative stress nor to increase Mo inflammatory reactions. The osmotic and mechanical fragility of RBCs was similar to that of the control. Such modified Hb failed to alter LDLs, increase the production of 8-iso PGF2alpha, but markedly inhibited platelet aggregation. The effect of this novel blood substitute can be linked with the cytoprotective and anti-inflammatory properties of adenosine, which is used as a cross-linker and surface modifier, and a modification procedure that lowers the hemoglobin pro-oxidant potential.

To attenuate hemoglobin's (Hb) intrinsic toxicity, Texas Tech University scientists developed a novel concept of "pharmacologic cross-linking" to formulate an effective oxygen carrier, HemoTech, which consists of purified bovine Hb cross-linked intramolecularly with ATP and intermolecularly with adenosine, and conjugated with reduced glutathione (GSH). In this composition, while ATP prevents Hb dimerization, adenosine permits the formation of homogeneous polymers. ATP also serves as a regulator of blood vessel tone via activation of the P2Y receptor, whereas adenosine counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine A2 and A3 receptors. GSH introduces electronegative charge onto the Hb surface that blocks Hb's transglomerular and transendothelial passage. Besides, GSH shields heme from nitric oxide and reactive oxygen species, thus enhancing vasodilation and lowering Hb prooxidative potential. HemoTech underwent favorable initial pre-clinical testing and proof of medical concept, and is under commercial development by HemoBioTech Inc. HemoTech has entered the regulatory process in the US. Several mandated requirements have already been met, including viral/transmissible spongiform encephalopathy (TSE) clearance validation studies and various pre-clinical pharmacological, pharmacokinetic, toxicological, genotoxicity and efficacy tests. These studies provided further evidence that "pharmacologic cross-linking" of the Hb molecule with ATP, adenosine and GSH, is useful for designing a viable Hb-based oxygen carrier.

The toxicity of hemoglobin (Hb) solutions is related, at least in part, to the generation of oxygen free radicals with consequent induction of lipid peroxidation. The present study was designed to examine whether selenium (Se) may prevent the oxidative damage observed after Hb administration. Three groups of rats were compared; (I) the negative control group receiving autotransfusion; (II) the positive control group with replacement of 40% total blood volume (TBV) with modified bovine Hb solution; and (III) the experimental group which received dietary supplemented selenium (Na2SeO3) in daily doses of 5 micrograms.kg body wt-1 in drinking water, 4 days before and 3 days after administration of Hb solution in the same volume as in group II. Three days after Hb injection, all animals were sacrificed. Oxidative stress was determined by measuring conjugated dienes (CD) and thiobarbituric acid reactants (MDA) in homogenates of the perfused liver, heart, lungs, kidney, brain and plasma. Additionally, the 45k x g supernatants of the organs homogenates and plasma were assayed for the antioxidant enzymes activity: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and the intracellular level of reduced glutathione (GSH). Also, a measurement of nonprotein bound intracellular free iron (Fe) and tissue Se concentrations was performed. Simultaneously, injury dysfunction of vital organs was assessed by the measurement of plasma LDH, SGPT, creatinine, blood PaO2 and by histopathological studies. Results indicate that the exchange transfusion with Hb solution introduced significant increases in CD and MDA formation, particularly in the liver and heart tissues, and in plasma. While the values of the SOD and CAT in the liver and heart tissue were generally altered, the SOD/CAT ratio was also increased. After the Hb injection, activity of GSH-Px remained unchanged and was associated with significant depletion of GSH. The plasma levels of SGPT and LDH were increased, but the creatinine and PaO2 was similar to that of the control and corresponded with histopathological findings. The liver and heart intracellular free Fe was found to be higher than that of control. Treatment with Se was very effective in the prevention of oxidative damage introduced by Hb. Full protection from MDA formation was noted in liver tissue (p < 0.001). Also, plasma levels of MDA, SGPT and LDH were significantly decreased and appeared similar to that of the control group (I). Treatment with Se increased liver (p < 0.05) and plasma (p < 0.1) level of GSH-Px.(ABSTRACT TRUNCATED AT 400 WORDS)