Biomaterials, artificial cells, and immobilization biotechnology : official journal of the International Society for Artificial Cells and Immobilization Biotechnology最新文献

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.

New fluorophilic and hydrophilic, cost efficient telomeric surfactants derived from tris(hydroxymethyl)acrylaminomethane were synthesized in 2 steps in 80% yield with respect to the perfluoroalkylated telogen. They demonstrate better ability to emulsify fluorocarbons than Pluronic f-68. The biological tolerance of these new surfactants is remarkable, the perfluorohexyl derivative was tolerated at doses of 4g/kg bw after i.v. injection in mice. None of the perfluoroalkylated THAM derivatives induces hemolysis of human red blood cells at concentrations up to 200g/l in physiological solutions.

In view of the potential application for hemoglobin-based oxygen carriers (HBOCs) in organ perfusion under hypothermic conditions, we examined the temperature dependence of oxygen equilibrium curves (OECs) at 15-37 degrees C of three HBOCs: HbA-FMDA and HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. OECs for HbA-DBBF, HbA-FMDA and HbBv-FMDA at 37 degrees C were right shifted (P50 = 24.5, 17 and 35 torr, respectively). van't Hoff's rule gave HbA-DBBF (-12.2 +/- 2.8), HbA-FMDA (-12.0 +/- 2.0), HbBv-FMDA (-10.5 +/- 1.8); these values do not significantly differ from that for native HbAo (-11.5 +/- 2.4). Among the hemoglobins included in this study, HbBv-FMDA had the most favorable oxygenation characteristics at low temperatures (a P50 of 6.0 torr at 15 degrees C as compared to only 2-3 torr for the other hemoglobins in the study). Recently, however, a human hemoglobin crosslinked with bis-pyridoxyl tetraphosphate was reported to have a P50 of 15 torr at 16 degrees C (Keipert et al, Transfusion 1989; 29: 768-773). Therefore, precise knowledge of the oxygen delivering capacity of any potential HBOC should be explored under hypothermic conditions as performance under these conditions may determine its usefulness as an organ perfusate.

We examined how changes in oxygen affinity brought about by different chemical modifications of hemoglobins affect their oxidation-reduction reactions. The three modified hemoglobins studied were HbA-FMDA, HbBv-FMDA, produced by the reaction of human or bovine oxyHb with fumaryl mono-dibromoaspirin; and HbA-DBBF, produced by the reaction of human deoxyHb with bis(3,5-dibromosalicyl) fumarate. Exposure of oxyHb to H2O2 causes generation of free radicals capable of cleaving dimethylsulfoxide (Me2SO) to produce formaldehyde (HCHO). Relative to the reaction rate for HbAo (630 +/- 130 M/min) the rates of HCHO formation were roughly 70% for HbA-DBBF, 50% for HbA-FMDA and 16% for HbBv-FMDA. Exposure to H2O2 also caused spectral changes at varied rates for the HBOCs analyzed. Although these rates were not directly correlated with the rates of free radical formation, addition of mannitol or thiourea slowed both the rate of spectral changes and HCHO formation. The relative ability of the ferric derivatives of the HBOCs to participate in free radical reactions was monitored by assays of non-enzymatic NADPH oxidation and aniline hydroxylation. HbBv-FMDA showed significantly slower rates than the other HBOCs in both assays. The observed differences between HBOCs in these assays indicate differences in their ability to generate or interact with free radicals.

Pyridoxalated hemoglobin polymerized with glutaraldehyde has been proposed as a hemoglobin based blood substitute. The preparations contain significant amounts of unpolymerized hemoglobin. We have prepared polymerized pyridoxalated hemoglobin labelled with 14C by reductive methylation free of unpolymerized hemoglobin and pyridoxalated hemoglobin labelled with 3H by reductive methylation to compare the handling of the two forms after infusion into dogs. Four dogs were examined sequentially. After three hours, 52.4 +/- 8.9% of the 3H label had disappeared from plasma whereas 21.7 +/- 5.8 of the 14C label had disappeared. The decrease of both labels occurred in a very close to linear fashion over the time period examined. From radioactivity in collected urine, it was calculated that 30.7 +/- 6.3% of the 3H and 9.0 +/- 2.7 of the 14C that had been cleared from plasma appeared in urine. The ratio of the specific radioactivity in tissue to the specific radioactivity of plasma indicated that extravascular accumulation of 3H label from unpolymerized hemoglobin occurred in kidney, heart and liver, with the kidney cortex exhibiting a very high concentration of the label. The specific radioactivity of both 3H and 14C label in liver suggested the substantial involvement of the reticuloendothelial system in the removal of both unpolymerized and polymerized hemoglobin from the circulation.

FDC/FTPA (7:3) emulsions stabilized by procsanol (Emulsion 1) and by procsanol with yolk phospholipids (Emulsion 2) were incubated with the donor plasma. After the incubation during 6 hours of Emulsions 1 and 2 with plasma the 36% and 50% decrease of the cholesterol content in plasma was found. Analysis of the lipid content of lipoproteins after the Emulsion 2 administration to rats (2.5 ml/100 g of weight) revealed the 50% decrease of the cholesterol amount in the HDL fraction at 3 and 24 hours posttransfusion. The ratio cholesterol/total phospholipids in the erythrocyte membrane diminished up to 50% as well. The equal degree of the cholesterol adsorption by emulsion from plasma, HDL of rats and erythrocyte membrane is an evidence of nonspecific interaction of PFC particles with the blood components containing cholesterol.

The purpose of this study was to compare the cardiopulmonary, hematologic, and immunologic responses of unanesthetized sheep to single, "topload", intravenous infusions of either 10 mL/Kg or 40 mL/Kg of Diaspirin Cross-Linked Hemoglobin, 10 mL/Kg or 40 mL/Kg of a Human Serum Albumin (HSA) solution oncotically adjusted with human serum albumin to approximately match the oncotic pressure of the DCLHb, or 10 mL/Kg of Erythrocyte Hemolysate solution prepared in a manner similar to that commonly described in the literature and referred to as "stroma free hemoglobin". Solutions were infused at a rate of 1 mL/Kg/minute and animals were monitored for 72 hours after infusion. These studies demonstrated that in sheep infusion of either DCLHb or HSA solutions was well tolerated and did not produce a significant increase in plasma C3a levels, an increase in the plasma concentration of thromboxane B2, or unexpected fluid shifts. In contrast, infusion of the Erythrocyte Hemolysate produced a greater than 10-fold increase in plasma C3a concentrations, a greater than 6000-fold increase in plasma TxB2 concentration, significant fluid shifts, and changes in a variety of other parameters consistent with induction of a dramatic inflammatory response. These results indicate that appropriately prepared and purified DCLHb solutions do not elicit an inflammatory reaction in sheep.

To assess the potential immunogenicity of human diaspirin cross-linked hemoglobin (DCLHb) solution, repetitive doses of this material were given intravenously to rhesus monkeys at monthly intervals and the immune response to this challenge was assessed. Serum samples collected at multiple intervals throughout the study showed no evidence of DCLHb specific IgG or IgM production. Intradermal skin tests performed one month after the final DCLHb infusion were also negative. These data demonstrate that DCLHb is not antigenic when administered intravenously to rhesus monkeys. In addition, screening of a panel of normal human sera for pre-existing anti-DCLHb IgG antibodies was negative, suggesting that this modified hemoglobin is unlikely to be antigenic in humans.

The effects of modified hemoglobin (Hb) solutions on the coronary vasculature were studied. Hearts were perfused according to Langendorff with constant flow of Tyrode solution. The solutions studied were stroma- free Hb, prepared by lysis of red blood cells in water (SFHb-lys), or prepared by swelling of red blood cells in hypotonic phosphate buffer (SFHb). The increase in coronary vascular resistance at a dose of 200 mg Hb/dl was 68% for SFHb-lys and 13% for SFHb, respectively. Addition of the modified Hb solutions HbNFPLP and polyHbNFPLP produced an increase in coronary resistance of 11% and 8%, respectively. The left ventricular developed pressure (LVDP) (control value 72 +/- 12 mm Hg) increased by 18 and 12 mm Hg, respectively, for a dose of 250 mg Hb/dl. When HbNFPLP was converted to its met-Hb form the increase in LVDP was reduced to 3 mmHg and the increase in perfusion pressure to 6 mm Hg. We conclude that elimination of stromal contamination from Hb solutions can diminish vasoconstrictor effects. The increase in cardiac pressure development and in coronary vascular resistance found for dilute modified Hb solutions is partly due to an improved oxygen transport to the heart.

We examined effects of perfusion of the kidney with pyridoxalated hemoglobin polyoxyethylene conjugate(PHP) solution on rat renal vascular responsiveness to norepinephrine(NE), angiotensin-II (AN-II), acethylcholine(ACh) and nitroglycerine(NG). The rat kidney was perfused with hydroxyethylstarch(HES) or PHP at a constant flow rate, using a pump. Perfusion pressure was monitored by a pressure transducer. Changes in PP induced by NE, AN-II, ACh and NG was examined. NE and AN-II applied intra-arterially induced a dose-related increase in PP in rat kidney perfused with both HES and PHP perfused groups. ACh and NG produced a dose dependent decrease in PP in both HES and PHP-perfused groups. There was no significant difference in response to ACh and NG between both groups. Results suggests that in rat vascular beds, PHP dose not interfere vascular relaxation caused by release of EDRF induced by ACh.