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Bacterial endotoxin (lipopolysaccharide) has affinity for a number of cations, including iron. Previous investigations have demonstrated that lipopolysaccharide can affect the oxidation rate of iron; heme-bound ferrous iron in hemoglobin is oxidized to ferric iron when hemoglobin binds lipopolysaccharide. In the present study, we directly examined the interaction between lipopolysaccharide and iron. Lipopolysaccharide caused a concentration-dependent increase in the rate of iron oxidation, with up to a 23-fold increase in oxidation in the presence of 200 microg/ml Escherichia coli lipopolysaccharide. This effect was seen both with several carbohydrate-rich smooth lipopolysaccharides and also with carbohydrate-poor rough lipopolysaccharide. Extensively deacylated rough lipopolysaccharide had no effect, suggesting a role of the fatty acid components of lipopolysaccharide in this process. Purified lipid A produced inconsistent results: some preparations stimulated iron oxidation and others did not. A series of sugars, starches and a preparation of purified O-chain polysaccharide (the carbohydrate portion of the lipopolysaccharide macro-molecule) had no effect on the rate of iron oxidation, whereas phospholipid-enriched brain tissue extracts (similar to the lipid A component of lipopolysaccharide) stimulated oxidation. We conclude that the lipid moiety of bacterial lipopolysaccharide is responsible for the stimulation of iron oxidation. This process may contribute to the ability of lipopolysaccharide to cause oxidation of heme-bound iron in hemoglobin.

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The heme oxygenase-1 gene, HO-1, induced by heme, ischemia, and heat shock, metabolizes heme to biliverdin, free iron, and carbon monoxide. Though the distribution of HO-1 has been described in normal rat brain, little is known about how extracellular heme proteins in the subarachnoid space distribute in brain. To address this issue, hemoglobin was injected into the cisterna magna of adult rats. Expression of HO-1 in these animals was compared with saline-injected, BSA-injected, and uninjected controls. Western blot analysis showed that 24 hours after injection oxyhemoglobin increased HO-1 levels approximately four- to fivefold in all brain regions studied compared with saline-injected and BSA-injected controls. In the brain, HO-1 immunoreactivity was evident at 4 hours and peaked at 24 hours after oxyhemoglobin injections, returning to control levels by 4 to 8 days. This HO-1 induction was detected mainly in cells with small, rounded somas bearing two to four truncated processes, a morphology consistent with that of microglia. These cells were double-stained with the microglial marker, OX42, in every brain region examined. It is proposed that subarachnoid hemoglobin may be taken up into microglia wherein heme induces HO-1.

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Many researchers are trying to develop a blood substitute based on chemically modified human hemoglobin. In the process of making such solutions, we were faced with the problem of determining the best storage conditions to minimize oxidation of the solutions between the time of manufacture and use. Samples of stroma-free hemoglobin, purified A0 hemoglobin, and various cross-linked hemoglobins were stored for 8-12 months at +4 degrees C -20 degrees C, and -80 degrees C and were analyzed periodically for formation of methemoglobin (MetHb). Various suspending solutions were evaluated for their effects on the rate of MetHb formation, and the approximate rates of MetHb production per month were calculated. Short-term storage of hemoglobin solutions (< 14 days) can be done at +4 degrees C, but extended storage should be done at -80 degrees C with quick thawing. Salts minimize the hemoglobin oxidation during the stress of freeze-thaw operations. Storage at -20 degrees C. presents further problems and should be avoided.

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The binding of carbon dioxide to human hemoglobin cross-linked between Lys alpha 99 residues with bis(3,5-di-bromosalicyl) fumarate was measured using manometric techniques. The binding of CO2 to unmodified hemoglobin can be described by two classes of sites with high and low affinities corresponding to the amino-terminal valines of the beta and alpha chains, respectively (Perrella, M., Kilmartin, J. V., Fogg, J., and Rossi-Bernardi, L. (1975b) Nature 256, 759-761. The cross-linked hemoglobin bound less CO2 than native hemoglobin at all CO2 concentrations in deoxygenated and liganded conformations, and the ligand-linked effect was reduced. Fitting the data to models of CO2 binding suggests that only half of the expected saturation with CO2 is possible. The remaining binding is described by a single affinity constant that for cross-linked deoxyhemoglobin is about two-thirds of the high affinity constant for deoxyhemoglobin A and that for cross-linked cyanomethemoglobin is equal to the high affinity constant for unmodified cyanomethemoglobin A or carbonmonoxyhemoglobin A. The low affinity binding constant for cross-linked hemoglobin in both the deoxygenated and liganded conformations is close to zero, which is significantly less than the affinity constants for either subunit binding site in unmodified hemoglobin. Comparing the low affinity sites in this modified hemoglobin to native hemoglobin suggests that cross-linking hemoglobin between Lys alpha 99 residues prevents CO2 binding at the alpha-subunit NH2 termini.

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Erythrocytes, suspended in a glucose-containing buffer, catalyzed the partial reduction of extracellular methemoglobin. Physiological concentrations of ascorbic acid or dehydroascorbic acid greatly enhanced the rate of reaction and the ultimate extent of reduction. The relationship between erythrocyte concentration and initial reaction rate was nonlinear, which suggested that the rate limiting factor was not an erythrocyte membrane enzyme. Also, significant dehydroascorbate-stimulated reduction occurred even when the erythrocytes and methemoglobin were separated by a dialysis membrane. The above observations indicate that the transfer of reducing equivalents across the erythrocyte membrane and reduction of extracellular methemoglobin can be accomplished by release and recycling of ascorbic acid.

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Pyridoxylated hemoglobin derivatives have been studied by many investigators. In this study hemoglobin A0 rather than stroma-free hemoglobin was used as a starting material in order to reduce the number of proteins to A0 and A1c. Derivatives were characterized using a Synchropak Q300 strong anion-exchange column, a PolyCAT A weak cation-exchange column and a VYDAC reversed-phase high-performance liquid chromatographic column. Resulting peak profiles of these two ion-exchange separations demonstrated enhanced resolution and showed the presence of pyridoxylated hemoglobin products not previously described. We compared products from the reduction of these Schiff base derivatives using either sodium borohydride or sodium cyanoborohydride reduction procedures. The sodium cyanoborohydride reduction method produced a lower percentage of products with multiple-site pyridoxylation modifications than the sodium borohydride reduction process.

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Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5-phosphate (PLP) per heme and reduced with either NaCNBH3 or NaBH4. A separate sample was pyridoxylated and passed through a mixed-bed ion exchange column without reduction. All three preparations had a P50 of 29 +/- 2 torr and a cooperativity of n = 2.4 +/- 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4 degrees C and at room temperature. The polymerization invariably reduced the P50 to 18 +/- 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH3, all retained the PLP in differing amounts (2-3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the alpha- and beta-chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4 degrees C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH3 gave products with a lower P50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma-free hemoglobin, but a large number of molecular species were still formed.

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Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P50 of 30 +/- 2 torr and a Hill coefficient of 2.3 +/- 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen-ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14C pyridoxal 5-phosphate (PLP) shows three major bands for the cyanoborohydride-reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed-bed resin, contains 0.4 equivalents of PLP per heme, and has a P50 of 30 +/- 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse-phase chromatography indicates that the addition of PLP to HbAo is directed solely to the beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.

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A procedure is presented for the preparation of a purified fraction of adult human hemoglobin (HbAo) from one unit of outdated blood. The entire process requires less than 16 h and gives a sterile, endotoxin-free solution of HbAo (approximately 30 g) in a yield of 50%. The solutions are isoionic with a conductivity of less than 15 mu mhos and less than 2 mmol total phosphorus per mol heme. The methemoglobin content is less than 1% and on storage at 4 degrees C rises less than 1% per month.

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A solution of hemoglobin has several potential applications as a blood substitute. However, because of high oxygen affinity (P50 approximately 14 mm Hg) and short vascular retention time of hemoglobin (plasma half-disappearance time approximately 3.5 hr), a solution of hemoglobin presents limitations for its general use in blood replacement therapy. To overcome these limitations crystalline hemoglobin was modified by pyridoxalation and subsequent polymerization. Pyridoxalation yielded a product with a P50 ranging from 23 to 26 mm Hg. The pyridoxalated hemoglobin was then polymerized with glutaraldehyde and the final modified hemoglobin showed a P50 of 19 to 22 mm Hg. The modified hemoglobin was tested in vitro for coagulation activities. The results indicated that no adverse coagulant activity was demonstrated by the modified products. In vivo studies in the rat have shown that pyridoxalated-polymerized hemoglobin has a plasma half-disappearance time of about 25 hr. The data demonstrated that a solution of pyridoxalated-polymerized hemoglobin, because of its lower oxygen affinity and longer vascular retention than unmodified hemoglobin, has significant potential as a basis for an efficient resuscitation solution.

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The development and evaluation of an effective Hb solution as a blood substitute are important not only for the care of casualties resulting from mass disasters, but also for eventual use in other special clinical situations. Substantial improvements have been made in recent years in the quality of Hb solutions. Solutions of unmodified Hb, although with certain limitations as indicated, potentially could be useful in several applications. The limitations presented by unmodified Hb can be overcome by a modification of the Hb molecule. In oxygen transport, not only the flow of the vascular fluid but also the vascular retention time, the oxygen affinity, and the concentration of free circulating Hb are important. A solution of Pyr-Poly Hb possesses positive characteristics in regard to the last three of these factors and, if the possibility of using bovine Pyr-Poly Hb is considered, the problem of supply for the material needed for the stockpiling of large quantities of this resuscitating solution can be eliminated. Products obtained by such modification must be evaluated also for their safety so that no potential adverse clinical effects would develop when administered to human patients.

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A solution of pyridoxalated and polymerized hemoglobin has been used for total blood exchange in the rat to test its effectiveness in vivo. Two groups of eight rats each were transfused to 93 to 95 per cent blood replacement with hemoglobin, control group, or with modified hemoglobin, experimental group, solution. All of the rats in the experimental group survived and became hematologically and physiologically normal at eight days after transfusion, whereas the rats in the control group died at approximately five hours after transfusion. Immediately after transfusion, the circulating fluid in the two groups of rats showed the same oxygen carrying capacity. At three and five hours after transfusion, differences in the oxygen capacity were observed with values in the experimental group of 34 and 103 per cent higher, respectively, than in the control group. The P50 of the vascular fluid in the experimental rats was 47 to 49 per cent greater than the corresponding value in the control group at zero, three and five hours after transfusion. The disappearance of hemoglobin from plasma was faster in the control than in the experimental group with a plasma half-disappearance time of 3.5 and 25 hours, respectively. The differences observed in the rate of disappearance of plasma hemoglobin were reflected in the rate of accumulation of hemoglobin in the urine. A solution of pyridoxalated and polymerized hemoglobin appears to be beneficial in blood replacement, since it promotes survival after massive transfusions.

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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.

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Fresh human blood and hemoglobin solution were mixed in different proportions to simulate hemodilution volumes occurring when blood is replaced by hemoglobin solution. Oxygen dissociation curves, P50 and hematocrit value of blood, hemoglobin solution and mixtures of blood and hemoglobin solution were determined. Total hemoglobin and oxygen content of the samples and the contribution to the total content by the two components in the mixtures were also measured. From these data, calculations were made of the oxygen release, at different pO2, by blood, hemoglobin solution and mixtures of blood and hemoglobin solution with contribution by each of two components. An in vitro analysis of static equilibrium between hemoglobin and oxygen demonstrates that the contribution of hemoglobin to the total oxygen released is affected by three factors, the left shift of the oxygen dissociation curve, the pO2 at the tissue level and the concentration of the hemoglobin in the solution used for hemodilution.

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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.

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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.

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