Grace Simoni

Protective effect of selenium on hemoglobin mediated lipid peroxidation in vivo.

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)

Control of Oxidative Reactions of Hemoglobin in the Design of Blood Substitutes: Role of the Ascorbate–Glutathione Antioxidant System

Uncontrolled oxidative reactions of hemoglobin (Hb) are still the main unresolved problem for Hb-based blood substitute developers. Spontaneous oxidation of acellular ferrous Hb into a nonfunctional ferric Hb generates superoxide anion. Hydrogen peroxide, formed after superoxide anion dismutation, may react with ferrous/ferric Hb to produce toxic ferryl Hb, fluorescent heme degradation products, and/or protein-based free radicals. In the presence of free iron released from heme, superoxide anion and hydrogen peroxide might react via the Haber-Weiss and Fenton reactions to generate the hydroxyl radical. These highly reactive oxygen and heme species may not only be involved in shifting the cellular redox balance to the oxidized state that facilitates signal transduction and pro-inflammatory gene expression, but could also be involved in cellular and organ injury, and generation of vasoactive compounds such as isoprostanes and angiotensins. It is believed that these toxic species may be formed after administration of Hb-based blood substitutes, particularly in ischemic patients with a diminished ability to control oxidative reactions. Although varieties of antioxidant strategies have been suggested, this in vitro study examined the ability of the ascorbate-glutathione antioxidant system in preventing Hb oxidation and formation of its ferryl intermediate. The results suggest that although ascorbate is effective in reducing the formation of ferryl Hb, glutathione protects heme against excessive oxidation. Ascorbate without glutathione failed to protect the red blood cell membranes against Hb/hydrogen peroxide-mediated peroxidation. This study provides evidence that the ascorbate-glutathione antioxidant system is essential in attenuation of the pro-oxidant potential of redox active acellular Hbs, and superior to either ascorbate or glutathione alone.

Reaction of Human Endothelial Cells to Bovine Hemoglobin Solutions and Tumor Necrosis Factor

Human umbilical vein endothelial cells (HUVEC) were incubated for 24 hours with 0.1 mM or 0.3 mM of: [A] unmodified (U) Hb-FeIIO2; [B] UHb-FeIII; [C] UHb-FeIV-OH; [D] polymerized low molecular weight Hb (< 400 kDa); [E] polymerized high molecular weight Hb (< 1,020 kDa); [F] polymerized low molecular weight Hb + Endotoxin (2.5 EU/mL); [G] rTNF alpha 100 pg/mL; [H] rTNF alpha 400 pg/mL; [I] rTNF alpha 800 pg/mL. The medium of the incubation was tested for LDH (index of cell injury), and for cytokines GM-CSF and IL-1 alpha released by the cells. The data suggests that oxidation status of the iron in the Hb molecule and concentration of Hb play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mM of UHb-FeIV-OH (ferryl-Hb) and no toxicity with 0.3 mM of Hb-FeIII (ferric-Hb). The direct stimulation of EC by Hb for the production of IL-1 was limited, related only to high molecular weight Hb polymers or to Hb+E, however GM-CSF expression was increased by almost all Hb forms. TNF induced dose-related injury (R2 = 0.986), and dose-related release of IL-1 (R2 = 0.977). A different EC reaction was observed on the release of GM-CSF. Intermediate levels of TNF (400 pg/mL) increased the expression of this cytokine, while high levels (800 pg/mL) blocked its release.

A novel hemoglobin-adenosine-glutathione based blood substitute : Evaluation of its effects on human blood ex vivo

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 PGF2&agr;) 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-&agr; and IL-1&bgr; 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 PGF2&agr; 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 PGF2&agr;, 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.

Intrinsic Toxicity of Hemoglobin: How to Counteract It

The development of safe and effective blood substitutes is of great importance in both civilian and military medicine. The currently tested hemoglobin (Hb)-based oxygen carriers, however, have toxicity and efficacy problems. A number of unwanted effects have been observed in human trials, creating doubts about their clinical usefulness. In some subjects, vasoconstriction and decreased blood flow to the vital organs, heart attack, stroke, systemic inflammation, organ damage, and even death, have been attributed to the transfusion of these experimental products. Hb is a well-known pressor agent and strong oxidant, although the full understanding of its intrinsic toxicity is yet to be uncovered. In particular, the complete mechanism of Hb-induced vasoconstriction needs full elucidation. Knowledge of the biological events that trigger the induction of genes upon treatment with redox-active Hb, as well as its catabolism, is still incomplete. It seems that our limited knowledge of free Hb effects in vivo is the main reason for not yet having a viable substitute of human blood. The future for universal red cell substitutes is in the new-generation products that address all of Hbs intrinsic toxicity issues.

Modified Hemoglobin Solution, with Desired Pharmacological Properties, Does not Activate Nuclear Transcription Factor NF-kappa B in Human Vascular Endothelial Cells

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 Hbs 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 Hbs 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, was shown to be non-toxic to the endothelium and promises to be an effective free-Hb based blood substitute.

Improved blood substitute: evaluation of its effects on human endothelial cells.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

An improved blood substitute. In vivo evaluation of its hemodynamic effects.

The purpose of the present study was to assess the ability of an improved free hemoglobin based blood substitute to serve as a resuscitative fluid in the treatment of hemorrhagic shock. Comparison studies were performed by using blood autotransfusion as a positive control. The hemodynamic parameters studied included cardiac index, mean arterial pressure, pulse pressure, heart rate, stroke volume index, and total peripheral resistance. Tissue oxygenation was measured in the biceps femori muscle by polarography. Hemorrhagic shock (at 40% of the total blood volume) in anesthetized rats caused severe disturbances in hemodynamic parameters and tissue oxygenation. Shock was characterized by a 66% drop in cardiac index, a 67% drop in mean arterial pressure with a significant increase in total peripheral resistance, and a 78% reduction in tissue oxygenation, all lasting 30 min. Resuscitation from shock with the blood substitute was effective in restoring hemodynamic parameters, producing vasodilation, and improving tissue oxygenation. Autotransfusion with blood also restored hemodynamics. However, lower tissue oxygenation and lack of vasodilation were noted. Therefore, the modified hemoglobin solution yielded better results than blood in the resuscitation of rats after hemorrhagic shock. The vasodilatory activity and the reduction of vasoconstriction that followed hemorrhage can be primarily linked with adenosine, which possesses vasodilatory and anti-inflammatory properties, and is used in our technology as an intermolecular cross linking reagent and hemoglobin surface modifier.

Evidence for the direct inhibition of endothelin-1 secretion by hemoglobin in human endothelial cells.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hbs inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.

An Improved Blood Substitute: In Vivo Evaluation of Its Renal Effects

Nephrotoxicity of free hemoglobin (Hb) based blood substitutes still awaits full elucidation. Previous reports attributed Hb passage through the renal glomeruli to a tendency of the Hb tetramer to dissociate into dimers. Now it has become more evident that the Hb tetramer is able to extravasate. It appears that the electrical charge of proteins plays an important role, with electronegativity and a low isoelectric point favoring intravascular persistence. This effect was utilized in the development of an improved blood substitute, comprising Hb reacted with o-ATP and o-adenosine, to form an intra- and intermolecularly cross linked product, which is reduced with glutathione. The modification reagents possess the desired pharmacologic activities and produce an increase in the electronegative charges on the Hb surface. All Hb polymers and chemically modified tetramers present in this solution have a uniform electronegative charge, with a pl of 6.1–6.2. In this present study, unmodified bovine Hb and an improved blood substitute were used for the replacement of 40% of the total blood volume in rats. The nephrotoxic effect was investigated by the determination of urinary output, glomerular filtration rate (GFR), fractional excretion of sodium (FENa), potassium (FEK), and chloride (FEC1), urine/plasma osmolality ratio, and urine N-acetyl-β-D-glucosaminidase (NAG) level. The free Hb and non heme protein contents in the urine were analyzed by using isoelectric focusing and size exclusion liquid chromatography methods. The results indicate that unmodified Hb is nephrotoxic. An initially elevated urinary output was followed by a significant oliguria associated with decreased GFR, FEK, and FEC1 and elevated FENa and NAG. Severe hemoglobinuria was associated with proteinuria. Analysis of urine from unmodified Hb treated rats revealed the presence of Hb tetramers. Histopathological examination of the kidneys showed cytoplasmic vacuolization of proximal tubular epithelium. On the contrary, an improved blood substitute did not produce any nephrotoxic reactions. It was found that this Hb solution did not pass through the renal glomerular barrier and was not present in urine samples. In