In vivo evaluation of pyridoxalated-polymerized hemoglobin solution.

Previous evaluations of stroma free hemoglobin solutions (SFHS) revealed two problems, a low P50 (13 to 15 torr) which theoretically limits oxygen off-loading to tissues, and a short intravascular half time of only 3.5 hours. To correct these problems the hemoglobin molecule was pyridoxalated and polymerized to raise the P50 (20 to 22 torr) and to increase the plasma half-disappearance time to 25 hours. In the current investigation, rats exchange transfused to 75 per cent of their initial blood volume with SFHS exhibited centrilobular liver necrosis 12 and 24 hours after the transfusion as a result of the hypovolemic state induced by the hemoglobin diuresis. In contrast, animals administered pyridoxalated polymerized hemoglobin solution (Pr-Pl-Hb) displayed a normal hepatic morphologic finding. Renal morphologic findings in the two groups were similar except for the smaller quantities of hemoglobin observed in the proximal and distal tubules of Pr-Pl-Hb animals. This finding may be a result of the slower degradation of Pr-Pl-Hb in the intravascular compartment. Renal function in Pr-Pl-Hb remained normal after exchange transfusion, while the BUN in SFHS treated animals increased significantly due to the state of dehydration. Thus, Pr-P1-Hb represents an improvement over the previously tested SFHS in both P50 and intravascular half-time and, because of lowered osmotic and oncotic activities, it may be used in higher concentration to improve oxygen carrying capacity.

Viscosity of human blood hemodiluted with crystalline hemoglobin solution.

Hemoglobin solution has been proposed as a blood substitute and, when administered intravenously, causes hemodilution that affects the viscosity of the circulation fluid. To quantitate the changes in viscosity, hemodilutions were made by mixing freshly drawn human blood with a 7-g/dl hemoglobin solution in different proportions. Viscosity measurements were made with a micro-cone plate viscosimeter at various shear rates. The results demonstrate that even at low or moderate hemodilutions with hemoglobin solution, the viscosity of blood decrease considerably at each shear rate investigated. The decrease of viscosity is greater with increasing hemodilution. A shear thinning effect is observed with whole blood and with each hemodiluted sample. The viscosity-hematocrit relationship, which could be demonstrated not only by cone-plate but also by the Ostwald viscosimeters at a fixed shear rate, shows that the concentration of red blood cells significantly affects the viscosity of blood. Hemodilution of blood with hemoglobin solution not only reduces the viscosity but also may improve the blood flow.

Extending the limits of hemodilution on cardiopulmonary bypass using stroma-free hemoglobin solution.

Ten swine were subjected to exchange transfusion to a hematocrit level of 5% with either stroma-free hemoglobin solution (SFHS) or 7% albumin solution. Myocardial performance, oxygen kinetics, and myocardial metabolism were subsequently examined using a perfused, in situ, right heart bypass, swine heart model with control of preload, afterload, and heart rate. Animals were tested during a control period (hematocrit = 30%) and following exchange transfusion with either solution to a hematocrit level of 5%. We found that myocardial performance following albumin solution exchange could not be sustained on right heart bypass, and these animals had a stroke volume of zero at a left ventricular end-diastolic pressure of 14 torr. SFHS animals had a significant drop in stroke volume at 14 torr following exchange (20 +/- 3 versus 10 +/- 4, p < 0.025), but this 50% performance level could be sustained. Coronary blood flow rose and myocardial oxygen consumption fell in both groups, although the statistically nonsignificant mean differences were less with SFHS. Arterial-coronary sinus oxygen difference fell significantly (p < 0.05) with albumin solution (7.3 +/- 0.8 versus 2.2 +/- 0.2) and nonsignificantly with SFHS (5.6 +/- 0.4 versus 4.1 +/- 0.7). Lactate production occurred in both groups, but was greater with albumin (34% +/- 6%) than with SFHS (3% +/- 16%). No changes in myocardial tissue gasses were noted in either group. Although myocardial performance decreased and some lactate production occurred with SFHS, we believe these comparative results provide promise in the eventual utilization of an oxygen-carrying agent such as SFHS to extend the limits of hemodilution to a hematocrit value of 5% or less.

Lyophilization of crystalline hemoglobin solution and exchange transfusions with lyophilized, reconstituted hemoglobin.

Crystalline hemoglobin solution was lyophilized following deoxygenation or addition of several compounds, or both, to establish protective conditions for obtaining freeze-dried hemoglobin chemically and functionally unaltered and clinically suitable as a blood substitute. Glucose and sucrose were most active in protecting the hemoglobin molecule from deterioration. The results of stability studies demonstrated that lyophilized hemoglobin maintained at 4 degrees C. did not show any significant alteration in structure and function for a period of nine months. Freeze-dried hemoglobin samples stored at room temperature were unchanged for six months, but after this time, a progressive increase in methemoglobin content and a decrease in P50 were observed. The effectiveness of lyophilized hemoglobin in vivo was investigated by transfusions in rats exchanged to blood replacements of 75 or 95 per cent, using lyophilized hemoglobin reconstituted soon after lyophilization or after seven months of storage at room temperature. The data show that lyophilized, reconstituted hemoglobin is effective in restoring or maintaining, or both, vital signs. In rats transfused to a 75 per cent blood replacement, several hematologic and physiologic parameters change soon after transfusion but return to normal pretransfusion levels within seven days after transfusion.

Total and partial blood exchange in the rat with hemoglobin prepared by crystallization.

Hemoglobin, prepared by crystallization, has been used as a blood substitute in total (91 to 93%) and partial (70 to 76%) blood replacement studies. Exchange transfusions have been carried out in laboratory animals to a total blood replacement of 91 to 93 per cent with hemoglobin or with albumin solutions. When albumin was used, all animals died at approximately ten minutes after transfusion was completed, whereas all animals transfused with hemoglobin survived for five hours and displayed normal activity during this time. In these studies the plasma half-disappearance time of hemoglobin was 3.5 hours and body distribution of 51Cr-labeled hemoglobin, as a percentage of initial levels, has shown six per cent in the kidney, six per cent in the liver, 10.5 per cent in the marrow and 13 to 14 per cent in the urine at three hours after transfusion. Survival was obtained with all animals transfused with hemoglobin or albumin solutions to a partial blood replacement of 70 to 76 per cent. However, the oxygen capacity of the circulating fluid in the hemoglobin transfused animals was about three times greater than that found in the corresponding albumin-transfused controls. Values of hemoglobin, hematocrit, platelets, and P50 returned to normal pretransfusion levels within five to seven days.

Characteristics of stroma-free hemoglobin prepared by crystallization.

Stroma-free hemoglobin was prepared from outdated human red cells by crystallization. After hemolysis with water and toluene and low speed centrifugation, the solution was dialyzed against 2.8M phosphate buffer. Hemoglobin crystals formed within the dialysis casing and were washed with phosphate buffer. After being dissolved in water, dialyzed against kidney dialysis fluid, and sterilized by Millipore filtration, the hemoglobin solution obtained had normal serum potassium, sodium, and osmolality. Spectral maxima and minima were characteristic for oxyhemoglobin, and cellulose acetate electrophoresis showed a sharp, well-defined hemoglobin band and slight contamination with carbonic anhydrase. The crystallized hemoglobin solution showed no coagulant activity, and preparation from group A cells showed no blood group A activity by hemagglutination inhibition. Methemoglobin was less than 0.28 gm./dl. and did not increase with storage at refrigerated temperature for a period up to 6 months. The P50 ranged between 15 and 18 mm. Hg at pH 7.4 and n values were normal.