Preparation of unaltered hemoglobin from human placentas for possible use in blood substitutes

Hemoglobin extracted from human placentas could be used as the basis of blood substitutes provided it could be prepared on a large scale with appropriate oxygen-binding properties. Unfortunately, the industrial conditions under which it is extracted, produce hemoglobin with high oxygen affinity and which is no longer influenced by the classical effectors. These characteristics were shown to be caused by a degradation of the alpha-chain brought about by an arginine carboxypeptidase present in the placental tissues and leading to the disappearance of the C-terminal arginine residue. This carboxypeptidase which is released from the tissues during the process of crushing the frozen placentas, degrades the protein during the chromatographic purification procedure. The addition of an inhibitor of this carboxypeptidase (for example, arginine) as soon as the placentas are thawed and during the chromatographic process, makes it possible to obtain placental hemoglobin with oxygen-binding properties quite similar to those of HbA prepared from peripheral venous blood.     

Bovine serum albumin-bovine hemoglobin conjugate as a candidate blood substitute

A bovine serum albumin-bovine hemoglobin conjugate was prepared using 3-maleimidobenzoic acid N-hydroxysuccinimide ester as cross-linker. The conjugate was purified using DEAE Sepharose. It had an M _r of 127 kDa. Its P₅₀ (half-saturated O₂ pressure) value and Hill coefficient were 27 mm Hg and 2, respectively.     

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCI2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the two a subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pi of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.     

Disuccinimidyl suberate cross-linked hemoglobin as a novel red blood cell substitute

Disuccinimidyl suberate (DSS) intramolecularly cross-linked hemoglobin (Hb) was developed as a novel red blood cell substitute. A multi-angle laser light scattering detector coupled with size exclusion HPLC was applied to determine the molecular weight of the modified Hb. SDS-PAGE was also used as a complement. It was proved that 83.8% of the product was intramolecularly cross-linked Hb with weight-average molecular weights (Mw) of 67.5 kD, 12% was dimeric Hb with Mw of 146.6 kD, and 4.2% was trimeric Hb with Mw of 306.4 kD. The tetramer structure of the cross-linked Hb was stable as shown in size-exclusion chromatography using a mobile phase containing 1 mol/L MgCl2. Analysis by LC-MS demonstrated that the reaction of DSS with Hb mainly took place between the twoα subunits within a Hb molecule, resulting in stabilization of the tetramer structure. However, the cross-linking was not site-specific. The P50 of the cross-linked Hb decreased from 21.8 mmHg to 14.3 mmHg, and the Hill coefficient decreased from 2.22 to 1.41. Result of isoelectric focusing showed that the pI of DSS cross-linked Hb was in the range of 4.6-5.2, similar to that of serum albumin. The safety of DSS cross-linked Hb was favored by animal tests on rats and guinea pigs. Exchange transfusion experiment with DSS cross-linked Hb using rats as a model indicated no pressor effect or other significant side effects. The characteristics and properties of DSS cross-linked Hb were also compared with that of diaspirin cross-linked Hb reported in the literature.     

Photopolymerization of bovine hemoglobin entrapped nanoscale hydrogel particles within liposomal reactors for use as an artificial blood substitute

Lipogel particles encapsulating bovine hemoglobin (BHb) were synthesized via photopolymerization of poly(N-isopropylacrylamide) (pNIPA) and poly(acrylamide) (pAAm) monomers within liposomal reactors. Nanoscale hydrogel particles (NHPs) encapsulating bovine hemoglobin, which represent a hybrid between acellular and cellular hemoglobin based oxygen carriers, were formed upon solubilization of the lipid bilayer of lipogel particles encapsulating BHb. Lipogels and NHPs encapsulating BHb constitute a new class of blood substitute that prevents both dissociation of hemoglobin (Hb) and in vivo exposure of acellular Hb, while allowing oxygen transport through the polymer matrix. pNIPA and pAAm particles encapsulating BHb displayed oxygen affinities ranging from 9.9 +/- 1.9 to 14.4 +/- 0.1 mmHg for lipogels, methemoglobin levels ranging from 9.3 +/- 3.7% to 26.0 +/- 5.0% for lipogels and NHPs, and encapsulation efficiencies ranging from 34.2 +/- 3.4% to 97.4 +/- 15.8% for lipogels and NHPs. Interestingly, the methemoglobin level of pNIPA particles was reduced 61% by coencapsulating the reducing agent, N-acetylcysteine. Fractionation and light scattering results showed that lipogels and NHPs were spherical and exhibited narrow size distributions. The colloidal osmotic pressure of pNIPA and pAAm lipogels ranged from 3.71 +/- 0.02 to 206.87 +/- 0.42 mmHg, depending on UV-irradiation time, type of buffer, and polymer composition. These results demonstrate that hemoglobin can be encapsulated within hydrogel based particles for use as an artificial blood substitute.     

Dry storage of liposome-encapsulated hemoglobin: a blood substitute

We have previously demonstrated the stabilization of liposome-encapsulated hemoglobin (LEH) by lyophilization (Cryobiology 25, 277-284, 1988). In the present report, we examine the structural and functional recovery of LEH after 3 months in the dry state. We have investigated the incorporation of the protective carbohydrate trehalose in the production and preservation of lyophilized LEH. Vesicle size, retention of entrapped hemoglobin, oxygen-carrying capacity, and percentage methemoglobin were measured as a function of time stored in the dry state under vacuum at room temperature. The results indicate that 150-300 mM trehalose maintains LEH dry preparations with little change in their size or functional characteristics after 3 months in the dry state. These results are compared to those of LEH that has been stored hydrated at 4 degrees C for the same time period.     

Modification of human hemoglobin with polyethylene glycol: A new candidate for blood substitute

Human hemoglobin was modified with polyethylene glycols. The conjugates exhibited P₅₀ values of 10–15 mmHg, those are enough to deliver oxygen from the lungs to tissues. The most remarkable characteristic is their long half disappearance time from the circulation. The longest half disappearance time of these derivatives is about 180 minutes in contrast to 45 minutes of free hemoglobin. The half disappearance time shows a good corelation not to molecular weight but to the effective molecular size, which is determined by the elution time of HPLC on a gel permeation column.     

Arenicola marina extracellular hemoglobin: a new promising blood substitute

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.     

A natural compound (reuterin) produced by Lactobacillus reuteri for hemoglobin polymerization as a blood substitute

Stroma-free hemoglobin (Hb) has been modified by pyridoxylation and followed by polymerization with glutaraldehyde as a blood substitute. Nevertheless, the reaction rate of pyridoxylated Hb (PLP-Hb) with glutaraldehyde is too fast to control its molecular weight distribution. Additionally, it was reported that glutaraldehyde is cytotoxic even at low doses. To overcome these problems, another aldehyde, beta-hydroxypropionaldehyde (beta-HPA), was used in the study to polymerize hemoglobin (PLP-Hb). beta-HPA is a natural compound (reuterin) produced by Lactobacillus reuteri. It was found that the maximum degree of PLP-Hb polymerization by reuterin (RR-PLP-Hb) was approximately 40% if the formation of high molecular (> 500 kDa) polymers should be prevented. In contrast, at the same reaction condition, the glutaraldehyde-polymerized PLP-Hb solution became gel-like, due to overpolymerization. This indicated that the rate of PLP-Hb polymerization by reuterin was significantly slower than that by glutaraldehyde. With increasing the reaction temperature, PLP-Hb concentration, or reuterin-to-PLP-Hb molar ratio, the time to reach the maximum degree of PLP-Hb polymerization by reuterin became significantly shorter. Removal of unpolymerized PLP-Hb from the RR-PLP-Hb solution can be effectively achieved by a gel-filtration column. The P(50) value of the unmodified Hb solution was 14 torr, while that of the RR-PLP-Hb solution was 20 torr, an indication of lower oxygen affinity. Additionally, the oxygen-Hb dissociation curves for both test solutions had a sigmodial shape and a nearly 100% saturation at 100 torr. In the in vivo study, it was found that the animals treated with the RR-PLP-Hb solution all survived and remained healthy more than 3 months. In contrast, only one out of six rats survived for the control group treated with the unmodified Hb solution. Furthermore, it was found that the RR-PLP-Hb solution resulted in a significantly longer circulation time ( approximately 12 h) than the unmodified Hb solution ( approximately 1.5 h). These results suggest that the reuterin-polymerized PLP-Hb solution may be a new option in the development of blood substitutes.     

The freeze-dried preservation of liposome encapsulated hemoglobin: a potential blood substitute

In this report, the ability of carbohydrates (trehalose, sucrose, and glucose) to preserve the blood substitute liposome-encapsulated hemoglobin (LEH) in the freeze-dried state is examined. The water-free stabilization of individual components of this blood substitute and LEH is reported. Lyophilization of hemoglobin solutions in the absence of carbohydrates results in significant oxidative degradation of Hb as measured by a large increase (approximately 60%) in methemoglobin. Hb samples lyophilized in increasing carbohydrate concentrations show reduced levels of methemoglobin, and at 0.5 M trehalose, sucrose, or glucose, these levels are reduced to nearly the same levels as unlyophilized controls. Storage of lyophilized Hb samples following rehydration at 4 degrees C shows the same rate of methemoglobin formation regardless of whether carbohydrates are present. This suggests that carbohydrates prevent Hb oxidation in the dry state but are less effective at retarding oxidative damage to Hb in solution. The addition of 0.25 M trehalose or sucrose to LEH results in the maintenance of liposomal size following lyophilization. In these experiments, glucose was least effective at inhibiting dehydration-induced LEH fusion. Lyophilization of LEH in 0.25 M trehalose or sucrose also results in significantly greater retention of the encapsulated hemoglobin following lyophilization and rehydration. These results suggest that the long-term stabilization of LEH in the dry state is a realizable goal.     

A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao

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.     

The use of Ultroser G as a serum substitute in the culture of human skin fibroblasts

Growth of human skin fibroblasts was dramatically enhanced when serum in the culture medium was replaced by Ultroser G. Compared to the same cells cultured in the presence of serum, alterations in glucose and lipid metabolism and an increase in the activity of prolidase (EC 3.4.13.9) and prolinase (EC 3.4.13.8) were also observed. Consequently, we advise extreme caution in the use of Ultroser G in metabolic studies, especially for periods of culture exceeding 10 days. However, Ultroser G can help to produce a large number of cells and so facilitate purification of the proteins produced during the stationary growth phase.     

Pooled umbilical cord blood as a possible universal donor for marrow reconstitution and use in nuclear accidents

Human umbilical cord blood has been shown to be an effective source of stem cells for marrow reconstitution in pediatric patients. Unfortunately, the quantity of stem cells obtained from an individual donor can be quite limited in both the total volume and the numbers of stem cells per ml of cord blood. HLA matching further limits the availability, but recent publications indicate close matching may be unnecessary. Therefore, if cord blood from different donors can be combined, larger numbers of stem cells can be available for clinical use provided pooling does not produce a negative effect. Storage of single cord blood specimens at 4 degrees C for 10-21 days in gas permeable bags produced an apparent increase in the percentage of immature cells (CD34, CD117, GPA) and mitotic activity (S+G2/M cells) over day 1. With similar storage of pooled specimens there was a further increase in the number of immature colonies cultured, CD34, CD117, GPA, S+G2/M cells. In addition, nucleated red blood cells increased over the mean values obtained from single cord blood samples. Our previous studies have indicated that large numbers of human mononuclear cells are necessary to reconstitute an irradiated animal model. By combining multiple samples of human cord blood, adequate numbers of stem cells could be pooled for use in adults and would provide cells for megadose therapy, including those patients that had accidentally received lethal irradiation.     

Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering nontoxic blood substitutes

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.