Mario Feola

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)

Nephrotoxicity of Hemoglobin Solutions

Nephrotoxicity is a problem of hemoglobin solutions (HbS) that still awaits full elucidation and correction. Therefore, a study was conducted using five HbS with different characteristics to replace 1/3 of blood volume in five groups of rabbits. All HbS contained bovine Hb, 6.5 g/dl, dissolved into a balanced electrolyte solution. HbS-I was Hb incompletely purified of stromal phospholipids and environmental bacterial endotoxins, and uncrosslinked; HbS-II was pure Hb non crosslinked; HbS-III was completely purified and crosslinked; HbS-IV was like HbS-III, but with pH 8.4; and HbS-V was like HbS-III, with the addition of mannitol. The effects of blood replacement with these solutions were studied on: (a) PAH clearance (expression of renal plasma flow); (b) endogenous creatinine clearance (expression of glomerular filtration); (c) fractional excretion of sodium and (d) urine/plasma osmolarity (expressions of tubular function). Histological changes were assessed after 24 hours. Significant alterations were observed in decrescent order following the administration of HbS-I, -II and -III, while HbS-IV and -V were well tolerated. These results suggest that the nephrotoxicity of Hb solutions can be prevented by the following steps: (1) complete purification of Hb; (2) complete crosslinking; and (3) protection of the kidney by alkalinization of the urine and/or the addition of mannitol.

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.

CYTOKINES AND PAF RELEASE FROM HUMAN MONOCYTES AND MACROPHAGES: EFFECT OF HEMOGLOBIN AND CONTAMINANTS

Monocytes [M] were isolated from venous blood of healthy volunteers and activated macrophage-leukocytes (Mø-L] were obtained from peritoneal fluid of patients with mild endometriosis. The M were incubated with pyrogen free CELLGRO culture medium [Control], and with 0.2 mM of [A] unmodified bovine hemoglobin (UHb), [B] Hb crosslinked to form polymers with M.W. < 400 kDa (LMWHb), [C] Hb crosslinked to form large polymers (< 1,020 kDa) (HMWHb), and Mø-L additionally with [D] UHb contaminated with endotoxin (Hb+E) (2.5 EU/mL), and [E] UHb contaminated with phospholipids (Hb+PLs). The Mø-L medium of incubation was tested for TNF alpha, IL-1 alpha, IL-6, GM-CSF and PAF after 6 and 24 hours, but M for TNF alpha and GM-CSF at 12, 24 and 36 hours. Mø-L were found more responsive than M colonies. The strongest reaction of Mø-L was to Hb+E, which produced levels of cytokines and PAF higher than Controls (p < 0.001). Hb+PLs induced smaller increases of TNF and IL-6, and a decrease in the levels of IL-1 and GM-CSF. However, the release of PAF was much greater with this Hb than with Hb+E. UHb caused an increase in TNF, as compared to control (p < 0.01). LMWHb generated a similar increase in TNF, but also a decrease in IL-1. Both polymerized Hb forms inhibited expression of GM-CSF. HMWHb induced high levels of TNF, IL-1 and PAF. UHb, LMWHb and HMWHb significantly increase levels of TNF in M cultures after 36 hours of incubation.

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.

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

Toxic factors in the red blood cell membrane.

The toxic effects of hemolysed RBCs have been studied for more than 100 years, but the specific factors involved have not been identified. This study focused on phosphatidylethanolamine (PE) and phosphatidylserine (PS), two aminophospholipids that normally reside on the cytoplasmic side of the red cell membrane. An in vitro experiment with murine peritoneal exudate macrophages showed that PE and PS: a) stimulated the production of H2O2, complement factor C3a, prostacyclin, and thromboxane at a dose of 5 micrograms/ml; b) produced cell injury, evidenced by release of lipid peroxides, LDH, and by morphologic changes on phase-contrast and electron microscopy at a dose of 50 micrograms/ml; and c) caused cell death in 50-66% of cells at a dose of 100 micrograms/ml. An in vivo experiment showed that PE and PS injected intravenously into various groups of rabbits: a) caused only transient hypotension at a dose of 0.05 mg/kg body weight; b) caused significant hypotension, cardiac arrhythmias, bronchospasm, activation of intravascular coagulation, complement, platelets, and leukocytes with release of histamine, serotonin, and thromboxane at a dose of 0.10 mg/kg; and c) caused cardiac arrest and death at a dose of 0.30 mg/kg. In contrast, the phospholipids of the outer cell membrane (phosphatidylcholine and phosphatidylinositol) caused minimal toxicity in vitro and none in vivo.

Chromatographic analysis of biopolymers distribution in “poly-hemoglobin”, an intermolecularly crosslinked hemoglobin solution

Abstract In order to eliminate the toxic effect introduced by the presence of large hemoglobin (Hb) polymers formed during intermolecular crosslinking, quality control of therapeutic-grade solution must include analysis of its molecular masses. Molecular mass distribution in glutaraldehyde polymerized Hb (“poly-Hb”) was evaluated using direct size exclusion chromatography (SEC). SEC separation revealed that type of packing materials, particle and pore sizes, column length and mobile phase composition are important factors for the precise analysis of poly-Hb molecular mass distribution. Computer analysis of the elution pattern showed that Asahipak® GFA-50F and Protein Pak™ 300SW columns were effecive only in the isolation of polymers with masses up to 204 kDa, without ability for complete separation of larger polymers. Using Asahipak® GFA-7M, poly-Hb was separated only into two fractions. Coupling is series three SEC columns (Asahipak GFA-7M, Protein Pak 300SW and Asahipak GFA-50F) significantly increased the separative resolution. It was found that glutaraldehyde, a nonspecific crosslinking agent, produces polymers with large molecular masses. About 42% of polymers have masses of 340–952 kDa and 5% have masses above 1020 kDa. Mobile phase contianing 300 mM NaCl improved the separation of polymers with molecular masses 340–952 kDa. Reaction of poly-Hb with 300 mM NaCl revealed the presence of electrostatic interactions between oppositely charged Hb polymers and formation of ca. 7% “pseudo-polymers”.

Evaluation of anion-exchange liquid chromatography for purification of hemoglobin from peptides and other proteins.

Abstract Solutions of hemoglobin (Hb) are under development for use as “red blood cell substitutes”. In order to reduce the presence of immunogenic and vasoactive factors in these solutions, the elimination of non-Hb proteins and peptides is necessary. The ability of anion-exchange liquid chromatography (LC) to purify Hb, using the Waters HPLC 600E system and five different columns (HP-PEI, MA7P, DEAE-5PW, MA7Q and Mono-Q HR) was evaluated. Bovine Hb, prepared by hyposmotic dialysis-ultrafiltration of isolated red blood cells, was subjected to chromatography by each column. The major fraction (HbAo) was analyzed by re-separation through the same column and by isoelectric focusing (IEF) with silver staining. The second chromatogram revealed HbAo to contain 2–4 non-Hb contaminants, some of which adherent to and some separate from hemoglobin. IEF revealed the full spectrum of Hb contamination, with 3–5 protein bands plus 10–15 peptide bands in association with HbAo. The results suggest that anion-exchange cannot completely purify hemoglobin and IEF with silver staining is more sensitive than LC.