Victor W. Macdonald

Pulmonary hypertension and systemic vasoconstriction may offset the benefits of acellular hemoglobin blood substitutes.

OBJECTIVE We tested the hypothesis that the pharmacologic properties of a small volume of alpha alpha-cross-linked hemoglobin (alpha alpha Hb) could effectively resuscitate pigs subjected to hemorrhage. METHODS Fourteen pigs hemorrhaged to a mean arterial pressure (MAP) of 40 mm Hg for 60 minutes were treated with a 4-mL/kg 2-minute infusion of 10 g/dL alpha alpha Hb or 7 g/dL human serum albumin, an oncotically matched control solution. RESULTS The removal of blood (17 +/- 1.5 mL/kg) caused the typical physiologic responses to hemorrhagic hypovolemia. Infusion of alpha alpha Hb restored mean arterial pressure and coronary perfusion pressure, but cardiac output and mixed venous O2 saturation did not improve significantly. Pulmonary arterial pressure and pulmonary vascular resistance increased markedly and were higher than baseline levels after alpha alpha Hb. Infusion of human serum albumin produced only minor hemodynamic changes. Brain blood flow did improve to baseline values after alpha alpha Hb, but was the only tissue to do so. In the human serum albumin group, superior mesenteric artery blood flow recovered to baseline values, whereas brain blood flow did not. Blood flows to other tissues were similar in both groups. CONCLUSION Small-volume infusion of alpha alpha Hb restored mean arterial pressure and brain blood flow, but pulmonary hypertension and low peripheral perfusion may offset benefits for trauma patients.

Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium.

We investigated the toxicity of hemoglobin/myoglobin on endothelial cells under oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on microcarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of medium, and endothelial cell monolayers were depleted of their intracellular glutathione (GSH) by treatment with buthionine sulfoximine. Incubation of diaspirin cross-linked hemoglobin (DBBF-Hb) or horse skeletal myoglobin (Mb) with BAECs subjected to 3 h of hypoxia caused transient oxidation of the hemoproteins to the ferryl form (Fe(4+)). Formation of the ferryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 h of hypoxia. Optimal inhibition of ferryl formation, possibly due to the antioxidant action of NO, was achieved with 900 microM L-arginine. Addition of hydrogen peroxide to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly decreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment, which may explain the greater toxicity observed with Mb as opposed to DBBF-Hb. This model may be utilized to identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control heme-mediated injury under physiologically relevant conditions.

Virus Photoinactivation in Stroma-free Hemoglobin with Methylene Blue or 1,9-dimethylmethylene Blue

Abstract Photoinactivation of vesicular stomatitis virus (VSV) in stroma-free hemoglobin (SFH) was carried out using methylene blue (MB) or 1,9-dimethylmethylene blue (DMMB). The VSV was more sensitive to inactivation by 660 nm light with 1 μM DMMB than with the same concentration of MB. Under conditions that inactivated 6 log10 of VSV, the methemoglobin content (Met-Hb[%]) and P50 of hemoglobin were changed by 1 μM MB phototreatment but were not changed by 1 μM DMMB phototreatment. The migration of hemoglobin during electophoresis and the activity of superoxide dismutase were not changed by MB or DMMB phototreatment. In contrast to the results obtained with DMMB at 660 nm, 580 nm irradiation of SFH with DMMB resulted in a significant increase of Met-Hb(%) under conditions that only inactivated 1.19 log10 VSV. The 580 nm irradiation primarily activates the dimer and higher-order aggregates of the dyes, while 660 nm irradiation primarily activates the monomer. These results indicate that the monomer form of DMMB can effectively inactivate viruses without damage to SFH.

DIRECT MEASUREMENTS OF HEMOGLOBIN INTERACTIONS WITH LIPOSOMES USING EPR SPECTROSCOPY

Electron paramagnetic resonance (EPR) spectroscopy was used to compare the rates of autoxidation at 37°C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (ααHb). This method avoids the difficulties inherent in using conventional ultraviolet-visible (UV-vis) spectroscopy caused by the high turbidity of liposome suspensions. Rate constants of 0.039/h and 0.065/h were obtained for the ααHb and LEH samples, respectively. Similar oxidation measurements with ααHb using UV-vis spectroscopy gave a rate constant comparable to that obtained with EPR spectroscopy. Indirect measurement of the oxidation kinetics of LEH utilizing extraction of ααHb with chloroform from partially oxidized LEH samples was unreliable because the amount of extractable hemoglobin was inversely proportional to the degree of oxidation. EPR measurements showed a shift in the g value and substantial enhancement in the intensity of the bis-histidine low-spin B complex for the encapsulated hemoglobin, indicating a perturbation of this low-spin complex. We suggest that lipid-associated perturbations are responsible for the enhancement of the oxidation observed with the LEH samples compared to the unencapsulated material.

Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic Acid (BCCEP): A Novel Affinity Reagent for the β-Cleft Modification of Human Hemoglobin

The design, synthesis, and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP, 1) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglobin A0. It was synthesized in three steps commencing from 4-t-butoxycarbonylphenol. The tri-sodium salt of 1 was employed to cross-link human oxyHb. While SDS-PAGE analyses of the modified hemoglobin product pointed to the molecular mass range of 32 kDa, the HPLC analyse suggested that the cross-link had formed between the beta 1-beta 2 subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 degrees C showed significantly reduced oxygen affinity (P50 = 31.3 Torr) as compared with that of cell-free hemoglobin (P50 = 6.6 Torr). The sigmoidal shape of O2 curves of the modified Hb pointed to reasonable retainment of oxygen-binding cooperativity after the cross-link formation. Molecular dynamics simulation studies on the reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the-chains, and Lys-144 on the other. These predictions were consistent with the results of MALDI-MS analyses of the peptide fragments obtained from tryptic digestion of the cross-linked product.

Bis[2-(4-Carboxyphenoxy)Carbonylethyl]Phosphinic Acid (Bccep): A New Reagent for Hemoglobin Modification

The synthesis and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP; 2) has been reported. The reagent was synthesized in four steps from hydroxybenzoic acid. The tri-sodium salt of BCCEP was employed to cross-link oxyHb, and the product was purified by DEAE-cellulose chromatography. The purified material was analyzed by SDS-PAGE, IEF, and HPLC analyses, which clearly showed the formation of covalent, intramolecular cross-links. While SDS-PAGE analyses of individual bands pointed to the molecular weight range of 32 kDa, the HPLC analyses suggested that the cross-links had formed between beta 1-beta 2 subunits. The oxygen equilibrium measurements and the Hill plots were performed on the purified bands to assess oxygen affinity as well as cooperativity of oxygen binding of the modified hemoglobins. All bands corresponding to modified hemoglobins showed significantly reduced oxygen affinity as compared with that of cell-free hemoglobin, as desired. The modified hemoglobins, however, exhibited somewhat reduced oxygen-binding cooperativity as contrasted with human stroma-free hemoglobin. Molecular dynamics simulation studies (Insight II/Discover/Biosym) on the Reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are Lys82 or N-terminal Val1 on one of the beta chains, and Lys144 on the other.