Search PubMed⌕ Search

Biomedical subjects

V W Macdonald

Publications and source records attributed to V W Macdonald.

At least 19 recordsLinked to original sources
PubMed2004

White particulate matter found in blood collection bags consist of platelets and leukocytes.

BACKGROUND: In late January 2003, some blood centers and hospitals throughout the US voluntarily sus-pended the use of some RBC and plasma units for trans-fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets. STUDY DESIGN AND METHODS: All AS-5 nonleuko-reduced RBCs and plasma units were visually inspected for WPM by placing the bags on a flat counter, undisturbed, for approximately 10 minutes and then perform-ing a visual examination for particles. Particles were isolated and placed on microscope slides or in plastic tubes for further analysis. Electron microscopy, bright field microscopy, differential interference contrast microscopy, infrared spectroscopy, and flow cytometry procedures were performed to establish the nature of the particulate matter. In addition, leukoreduction filters and blood transfusion sets were used on RBCs units with WPM. RESULTS: The particles were mostly composed of PLTs and WBCs, and fragments of these cells. All macroscopic WPM was removed from RBCs with leukoeduction and transfusion filters. CONCLUSIONS: WPM originated from PLTs and WBCs. Foreign matter (e.g., plastic) was not observed in any of the units. Leukoreduction and transfusion filters can be used to remove macroscopic WPM.

Blood Platelets↗
PubMed2001

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 degrees C of acellular and liposome-encapsulated hemoglobin (LEH) crosslinked between alpha chains with bis (3,5-dibromosalicyl) fumarate (alphaalphaHb). 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 alphaalphaHb and LEH samples, respectively. Similar oxidation measurements with alphaalphaHb 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 alphaalphaHb 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.

Aspirin↗
PubMed2000

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.

Animals↗
PubMed2000

Virus photoinactivation in stroma-free hemoglobin with methylene blue or 1,9-dimethylmethylene blue.

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 microM 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 microM MB phototreatment but were not changed by 1 microM DMMB phototreatment. The migration of hemoglobin during electrophoresis 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.

Animals↗
PubMed1999

Affinity reagents for cross-linking hemoglobin: bis(phenoxycarbonylethyl)phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP).

Synthesis and properties of two new macrobiomolecular cross-linking reagents, bis(phenoxycarbonylethyl) phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP), have been reported. The reagents were successfully employed to cross-link human hemoglobin under oxygenated conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and fast protein liquid chromatography (FPLC) analyses of the reaction products indicated that the cross-link was intramolecular in nature, and that it was between the two beta subunits of hemoglobin in each case. The products were purified by DEAE-cellulose chromatography, and the purified material was employed for oxygen-binding assessments. The oxygen equilibrium curve of the cross-linked material, in each case, was right-shifted toward lower oxygen affinity as desired. The sigmoidal shapes of oxygen curves, in each case, suggested retainment of oxygen-binding cooperativity, although considerably lower than that of the native hemoglobin

Chromatography, High Pressure Liquid↗
PubMed1998

Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP): a novel affinity reagent for the beta-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.

Chromatography, High Pressure Liquid↗
PubMed1997

An improved process for the production of sterile modified haemoglobin solutions.

The process for manufacturing bulk quantities of sterile solutions of human haemoglobin (Hb) cross-linked between the alpha chains (alphaalphaHb) with bis(3,5-dibomosalicyl) fumarate (DBBF) was modified to: (1) improve product purity; (2) increase product yield; (3) eliminate non-United States Pharmacopoeia materials; (4) reduce reagent costs; and (5) reduce production time. These process modifications were the result of increased scientific understanding of the Hb cross-linking chemistry and were in the form of engineering and procedure controls that reflect current good manufacturing practices (cGMP). Purity, as reflected in the fractional yield of the desired alphaalphaHb product, has increased from 60% to 90+% of total Hb, and uncross-linked Hb was virtually eliminated. Impurities such as pyrogens, methaemoglobin, phospholipid, and free iron were reduced. The net yield of alphaalphaHb was increased from 33% to 58% of starting Hb content. Production time, the use of overtime, the consumption of expensive reagents and filters, and losses because of contamination have all been reduced. As a result, cost per gram of alphaalphaHb produced has decreased 60%. With this improved process, efficient production of very pure alphaalphaHb is possible.

Chemistry, Pharmaceutical↗
PubMed1997

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.

Albumins↗
PubMed1997

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.

Chromatography, DEAE-Cellulose↗
PubMed1996

Hemoglobin-induced contraction of pig pulmonary veins.

The effects of hemoglobin Ao (HbAo), alpha alpha cross-linked hemoglobin (alpha alphaHb), cyanomet alpha alpha cross-linked hemoglobin (cyanomet alpha alphaHb), and human serum albumin (HSA) were compared under basal conditions and during relaxation with acetylcholine (ACh), sodium nitroprusside (SNP), and papaverine (PAP) in porcine pulmonary veins. Isometric tension changes were recorded in isolated rings (3 to 4 mm) that were suspended in Krebs solution bubbled with 95% O2/5% CO2. Increasing concentrations of HbAo and alpha alphaHb (10(-9) - 3 x 10(-6) mol/L) caused concentration-dependent increases in tension that reached a maximum of 4.20 +/- 0.3 gm and 3.78 +/- 0.6 gm, respectively. Cyanomet alpha alphaHb and HSA (10(-9) - 3 x 10(-6) mol/L) did not cause significant increases in tension. The maximum responses to HbAo and alpha alphaHb were significantly increased during relaxation with ACh and SNP but not with PAP. In contrast, SNP (10(-4) mol/L) and PAP (10(-5) mol/L), but not ACh, reversed contractions induced by HbAo and alpha alphaHb. These studies support the concept that hemoglobin-induced vascular contraction is primarily mediated by inactivation of the vasodilator nitric oxide in vitro. We suggest that this mechanism is common to acellular hemoglobins in which the ligand binding site is unimpaired and in which the heme iron is in the ferrous (+2) state.

Animals↗
PubMed1994

Increased vascular resistance with hemoglobin-based oxygen carriers.

PURPOSE: To compare the effects of resuscitation with hemoglobin-based oxygen-carriers and conventional resuscitation fluids on hemodynamics, oxygen transport, and oxygen consumption in an animal model of the use of these fluids in the treatment of hemorrhagic shock. PROTOCOL: Twenty-eight immature swine were surgically prepared, allowed to recover five days, water deprived for 48 hours, hemorrhaged of 25 ml/kg over one hour, resuscitated promptly with 1) Ringer's lactate, 75 ml/kg, 2) 7% albumin in Ringer's acetate, 25 ml/kg, 3) 9% unmodified hemoglobin in Ringer's acetate, 25 ml/kg, or 4) 9% alpha alpha-crosslinked hemoglobin in Ringer's acetate, 25 ml/kg, and observed with three hours of hemodynamic and oxygen transport measurements. RESULTS: Systemic and pulmonary vascular resistance were increased in hemoglobin-treated animals to more than twice the levels seen in crystalloid- or colloid-treated controls. Oxygen consumption and the rate of correction of lactic acidosis were not increased in hemoglobin-treated animals. CONCLUSIONS: Increased vascular resistance limits the oxygen transport benefit of cell-free-hemoglobin-based oxygen carriers. Cell-free-hemoglobin-induced increases in vascular resistance may place animals' hearts on an unfavorable portion of the Frank-Starling curve as well as complicate further medical treatment by reducing the animals' tolerance to increases in blood viscosity.

Animals↗
PubMed1994

Vasoconstrictor effects in isolated rabbit heart perfused with bis(3,5-dibromosalicyl)fumarate cross-linked hemoglobin (alpha alpha Hb).

To study the mechanism by which cell-free hemoglobin preparations may alter coronary vascular reactivity, we investigated the effect of human hemoglobin cross-linked between alpha chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) on the vasomotor response to acetylcholine (ACh) in isolated perfused rabbit hearts. Dose-response curves were generated by monitoring the increase in coronary pressure during serial addition of 0.2-10 microM ACh before, during and after 20 min infusion of three test solutions: a) 0.1 g/dl alpha alpha Hb (62 microM heme); b) 0.1 g/dl alpha alpha Hb plus 60 microM deferoxamine (DFO); c) 50 microM NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide (NO) synthase. We found that the sensitivity to ACh-induced vasoconstriction was significantly potentiated in the presence of alpha alpha Hb and L-NAME. In addition, this response was only partially reversed after removal of alpha alpha Hb, except when DFO was simultaneously infused with the alpha alpha Hb solution. These findings are consistent with the idea that both NO binding to hemoglobin and iron-mediated oxygen free radical generation contribute to an altered coronary vasomotor responsiveness induced by cell-free hemoglobin.

Acetylcholine↗
PubMed1993

Cell-free hemoglobin potentiates acetylcholine-induced coronary vasoconstriction in rabbit hearts.

Cell-free hemoglobin (Hb) preparations have been shown to alter vascular tone in vitro and in vivo. The high affinity of Hb for nitric oxide, the putative endothelium-derived relaxing factor (EDRF), may be primarily responsible for this activity, but the contribution of tissue-damaging oxygen-derived free radicals has not been established. We investigated the effects of human Hb interdimerically cross-linked with bis-(3,5-dibromosalicyl)fumarate (alpha alpha Hb) on the coronary vasomotor response to acetylcholine (ACh) in isolated perfused rabbit hearts. Infusion of 0.1 g/dl alpha alpha Hb altered the dose-dependent response to ACh, decreasing the calculated IC50 (ACh concn at which coronary pressure is 50% of its maximal value) from 3.96 +/- 0.34 to 0.85 +/- 0.06 microM (P < 0.01). This augmented sensitivity to ACh was only partially reversed upon washout of alpha alpha Hb (IC50 1.93 +/- 0.13 microM). Simultaneous infusion of 60 microM deferoxamine mesylate with alpha alpha Hb attenuated this response (IC50 decreased from 3.86 +/- 0.27 to 1.73 +/- 0.38 microM), which was completely reversed after removal of alpha alpha Hb (IC50 3.41 +/- 0.17 microM). NG-nitro-L-arginine methyl ester (50 microM) and cross-linked cyanomethemoglobin (CNmet alpha alpha Hb, 0.1 g/dl) induced a significant (P < 0.05) increase in ACh-induced vasoconstriction accompanied by a reduction in myocardial functions in the same range as that caused by alpha alpha Hb. Infusion of deferoxamine mesylate (60 microM) with CNmet alpha alpha Hb completely prevented the reduction in IC50 elicited by the infusion of CNmet alpha alpha Hb alone. These data demonstrate that alpha alpha Hb can alter coronary vasomotor responsiveness and suggest the involvement of at least two mechanisms, one that is related to an accessible ferrous heme and is reversible and another that does not require an open heme site and is irreversible.

Acetylcholine↗
PubMed1992

Pharmacokinetic studies in the rat on a o-raffinose polymerized human hemoglobin.

We have studied the plasma half-life (T 1/2), oxygen-binding affinity (P50), organ distribution, and excretion of the individual molecular weight (MW) components of human hemoglobin polymerized with periodate-oxidized, ring-opened raffinose (oR poly-Hb), following transfusion in the rat. The model was an isovolemic 50% exchange transfusion in the conscious, chronically catheterized rat. Total plasma Hb levels yielded a (T 1/2) of 10 to 11 hr for oR poly-Hb. The T 1/2 values of individual MW components of the poly-Hb as determined by size-exclusion HPLC were approximately: 4 hr for the monomeric fraction (Hb)1, 9 hr for the dimer (Hb)2, and 15 hr for the fraction representing trimers to nanomers (Hb)3-9. The P50 values of plasma samples containing oR poly-Hb (collected from 0-24 hr after exchange) remained unchanged at 28 +/- 3 mmHg. oR stabilized and polymerized Hb were not excreted via the kidneys. Hepatic and renal distribution as well as plasma and renal clearance were determined by liquid scintillation counting using individual tritium [3H] labelled MW components purified from [3H]-oR poly-Hb: (Hb)1/2, (Hb)1, (Hb)2, (Hb)3&4, and (Hb) greater than 9. In kidney, uptake (determined by the relative concentration of radioactivity) decreased with increasing MW of the labelled component. Conversely, in liver, uptake increased with increasing MW. Plasma and renal clearance results were consistent with those obtained by HPLC analysis. Hematocrit levels returned from a 20% post-transfusion level to normal pre-transfusion levels (44%) within 10 days after the exchange.

Animals↗
PubMed1992

The role of the kidneys in the excretion of chemically modified hemoglobins.

Stroma-free hemoglobin (SFHb) can be chemically modified to prolong the intravascular retention (prevent renal filtration), and to improve oxygen delivering capability for use as a red cell substitute. Hb derivatives radioactively tagged with tritium [3H] or 14C were used to study their metabolic fate following clearance from the circulation. Fully conscious, chronically cannulated rats were treated by exchange transfusion (ET). Hb solutions tested were: PLPHb (Hb monovalently reacted with pyridoxal 5'-phosphate); HbXL (Hb crosslinked beta-beta with 2-nor-2-formylpyridoxal 5'-phosphate, or with bis-pyridoxal tetraphosphate); alpha alpha Hb (Hb cross-linked between the alpha-chains using bis-3,5-dibromosalicyl fumarate); and polyHb (polymerized with glutaraldehyde or o-raffinose). Plasma retention (T1/2) was significantly affected by dose and the degree of cross-linking. Urine flow rates all increased transiently above normal. In rats treated with any 64 kDa interdimerically cross-linked Hb, mild hemoglobinuria was evident and kidney tissue had the highest label concentration at all time points (1, 5, 10, 24, 48 hr, 7 d, and 14 days post-ET). For polymerized Hb derivatives, the amount of radioactivity in urine and kidneys was inversely related to the MW of the polyHb molecules. In all rats, regardless of the Hb derivative tested, the majority of radioactivity (dpm's) was excreted in urine. About 75% of all renal excretion of radioactivity occurred from 12-60 hours post-ET. This provided evidence that catabolism of cross-linked Hb's began early, and that the kidneys are primarily responsible for excreting smaller degradation fragments.

Animals↗
PubMed1990

Coronary vasoconstrictor activity of purified and modified human hemoglobin.

Unpurified stroma-free hemoglobin (SFH) from water-lysed human red blood cells, hemoglobin Ao (HbAo), and hemoglobin cross-linked between alpha chains with 3,5-bis-dibromosalicyl-fumarate (HbXLDBBF) were infused into isolated perfused rabbit hearts. Vasoactivity and myocardial performance were determined using an isovolumic Langendorff preparation. With constant coronary flow, infusion of SFH (55 mg/dl) resulted in a 56% increase in aortic pressure as opposed to 29% and 11% increases with HbAo and HbXLDBBF, respectively. Rates of aortic pressure increase were over 6 times greater with SFH than with either HbAo or HbXLDBBF and exhibited concentration dependence only with SFH. Myocardial function remained normal. With constant coronary pressure, coronary flow decreased by 36% with SFH accompanied by a 23% decline in left ventricular developed pressure indicative of ischemia. With HbAo or HbXLDBBF, coronary flow decreased by only 1/3 that with SFH, while developed pressure declined by 4% and 2% with HbAo and HbXLDBBF, respectively. These data suggest that purification of HbAo to a single component can eliminate the dominant of at least two distinct vasoactive factors normally found in SFH. Furthermore, the physiological significance of the vascular response to HbAo is minimal. Preparations of HbXLDBBF accomplish this same result.

Animals↗