Warm storage of whole blood for 72 hours.

BACKGROUND: In field emergency medicine, fresh whole-blood units are stored at room temperature up to 24 hours or occasionally longer. Few data exist on the integrity and in vitro functional properties of whole blood stored warm beyond 24 hours. STUDY DESIGN AND METHODS: Ten citrate phosphate dextrose solution whole-blood units were collected and divided into two equal volumes. One-half of each unit was stored at 19 degrees C and the other half was stored at 25 degrees C, encompassing the accepted range for room temperature storage. At 6, 24, 48, and 72 hours, aliquots were collected from each unit and whole blood analyzed for cell counts, gases, and clotting function with thromboelastography, red cells for intracellular analytes, platelet (PLT)-rich plasma for aggregometry, and the supernatant for hemoglobin, potassium, glucose, lactate, and plasma clotting studies. RESULTS: Whole-blood units stored at room temperature maintained cellular counts and coagulation activity for up to 72 hours. Units stored at 19 degrees C demonstrated greater RBC adenosine triphosphate and 2,3-diphosphoglycerate (DPG) content and stronger responses in PLT aggregation studies when compared with 25 degrees C storage. No significant hemolysis was observed, and no bacterial growth was detected. CONCLUSION: Storage of whole blood at room temperature for 72 hours leads to marked reductions in pH and DPG, but the observed reduction in PLT function and plasma coagulation factor activity was surprisingly modest compared to literature values. These findings should prompt additional investigation, given their potential importance for whole blood processing and field-expedient transfusion.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

Diaspirin cross-linked hemoglobin: tissue distribution and long-term excretion after exchange transfusion.

This report describes the tissue distribution and long-term (14-day) excretion of hemoglobin cross-linked between the alpha-chains (alpha alpha Hb) with carbon 14-labeled bis(3,5-dibromosalicyl)fumarate. Fully conscious, chronically cannulated rats (n = 40) were treated with a 50% isovolemic exchange transfusion (ET) with solutions of 14C-labeled alpha alpha Hb (8.0 gm/dl) and were then monitored for as long as 14 days. Thirteen tissue types were analyzed for radioactivity by liquid scintillation counting. The highest concentration of label was found in the kidney and in tissues of the reticuloendothelial system (i.e., spleen, bone marrow, and liver). The 14C-labeled alpha alpha Hb did not appear to cross the blood-brain barrier, because radioactivity in the brain was barely detectable. The dose of 14C-labeled alpha alpha Hb (2.4 gm Hb/kg) produced an initial plasma Hb level of 4.6 gm/dl, with a half-life in the plasma of 5.0 hours. The peak concentration in kidney, spleen, and liver occurred at 24 hours after ET, when at least 92% of the 14C-labeled alpha alpha Hb in plasma had been cleared. At 48 hours, red casts were seen in a tiny number of renal tubules in some rats. By 14 days, up to 64% of the injected radioactivity had been recovered in urine and about 10% was recovered in feces. Most excretion occurred 24 to 48 hours after ET. This study demonstrated that 2 weeks were required for the metabolic degradation and elimination of a large dose of alpha alpha Hb in rats.

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.

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.

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.

Acute changes in systemic blood pressure and urine output of conscious rats following exchange transfusion with diaspirin-crosslinked hemoglobin solution.

This report describes acute changes in systemic blood pressure and urine output observed after a 50-percent isovolemic exchange transfusion (ET) with diaspirin-crosslinked hemoglobin (alpha alpha Hb). Stroma-free Hb was crosslinked between the alpha chains by using a 14C-labeled diaspirin, bis(3,5-dibromosalicyl)fumarate. Forty conscious, chronically cannulated rats underwent ET with 14C-labeled alpha alpha Hb solution (8.0 g/dL [80 g/L]). This resulted in systemic hypertension for 3 to 4 hours after ET (mean arterial pressure rose from 120 to 145 torr at 1 to 2 hours after ET) and mild bradycardia for 2 to 3 hours (heart rate decreased from 420 to 335 beats/min [bpm] before stabilizing at 360 +/- 10 bpm). This was accompanied by significant diuresis immediately after ET (5- 6-fold increase in urine output, which normalized after 12 hours), and mild hemoglobinuria. The total amount of Hb recovered in the urine was < 5 percent of the injected dose. Reversed-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the presence of crosslinked alpha alpha Hb molecules in the urine. Renal excretion of radioactivity was significantly greater, with 20 percent of total radioactivity being eliminated within 24 hours. The plasma half-life for alpha alpha Hb was 5 hours (administered dose, 2.4 g Hb/kg body weight). Thus, infusion of alpha alpha Hb caused a transient systemic hypertension, and intramolecular crosslinking alone was not enough to exclude completely the filtration of alpha alpha Hb by the kidneys.

Systemic and pulmonary hypertension after resuscitation with cell-free hemoglobin.

Human hemoglobin (Hb) and Hb cross-linked between the alpha subunits with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) were used to treat hemorrhagic shock in water-deprived swine. Water was withheld for 48 h to induce a 10% loss of body mass, and 25 ml/kg of blood were removed in 1 h to produce circulatory shock. Swine were resuscitated with 1) Hb, 2) alpha alpha Hb, 3) human serum albumin, or 4) Ringer lactate. Mild high-output renal failure was observed in the non-cross-linked Hb-treated animals but not in other groups of animals. Swine treated with Hb and alpha alpha Hb had increases in plasma creatine kinase and lactate dehydrogenase activity that were resolved within 7 days. Both Hb- and alpha alpha Hb-treated swine displayed marked elevations of mean blood pressure in the systemic (39 +/- 6 Torr) and pulmonary (20 +/- 6 Torr) circulations that continued for 3 h and were associated with reduced cardiac output and a doubling of the systemic and pulmonary vascular resistances. Oxygen delivery was equivalent, and the rate of correction of the lactic acidosis was equal in all groups.

Oxygen delivery and myocardial function in rabbit hearts perfused with cell-free hemoglobin.

Isolated rabbit hearts were perfused with Krebs-Henseleit buffer that contained 1.5 g/dl hemoglobin Ao [HbAo; PO2 at which half-saturation of hemoglobin occurs = 12 Torr], human hemoglobin cross-linked between alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha-Hb; PO2 at which half-saturation of hemoglobin occurs = 30 Torr), or fatty acid-free bovine serum albumin (BSA). Myocardial performance and oxygen uptake were determined at different aortic PO2's [arterial PO2 (PaO2)] by use of an isovolumic Langendorff preparation. Function and oxygen uptake were comparable among the three different groups of hearts at an average mean PaO2 of 557 Torr. As PaO2 decreased, myocardial function was preserved better in hearts perfused with hemoglobin than in hearts perfused with Krebs-Henseleit buffer alone or with BSA. Hearts perfused with either HbAo or alpha alpha-Hb exhibited similar 10% decreases in left ventricular developed pressure and rate of change in left ventricular developed pressure at PaO2 of 141 Torr compared with a 58% decrease with BSA. However, corresponding venous PO2's were lower with HbAo (20 Torr) than with alpha alpha-Hb (35 Torr), and oxygen uptake decreased by 36% with HbAo but remained constant with alpha alpha-Hb. These data suggest that although myocardial function can be sustained over a fairly broad range of hemoglobin oxygen affinities, tissue oxygen gradients and myocardial oxygen uptake are maintained better by cell-free hemoglobin with an oxygen affinity in the normal physiological range.

Dehydration and shock: an animal model of hemorrhage and resuscitation of battlefield injury.

We have developed a porcine model of the anticipated military use of oxygen-carrying resuscitation solutions. The objective is to determine whether toxicity under adverse conditions will limit further development of hemoglobin-based products. Splenectomized immature female swine are used because of their extensive use in the evaluation of other resuscitation solutions. Five days prior to each experiment, central vascular catheters and a renal arterial flow probe are surgically placed in the animals. After recovery and weight gain has resumed, animals are placed in metabolic cages and deprived of water for 48 hours to produce hyperosmolar dehydration resulting in loss of approximately 7% of body weight. We remove 38% of estimated blood volume, 25 ml/kg, over one hour by a controlled logarithmic hemorrhage. Resuscitation is by administration of a fixed volume of test solution. Hemodynamic function is observed but not further therapy is given for three hours, a period corresponding to evacuation in the field. After this period, corresponding to arrival at a field hospital, the animals' blood is returned. Swine are then observed in metabolic cages for an additional 7 days while blood and urine are sampled daily. At the end of this period, animals are anesthetized, urinary catheters are implanted, and creatinine clearances are measured. Swine are than euthanized, and their tissues are examined. In a pilot study, resuscitation was performed with either Ringer's lactate, albumin, stroma-free hemoglobin, or cross-linked (alpha alpha Hb) hemoglobin. All animals survived.