تويت
Hemoglobin-Based Oxygen Carriers
Stroma free hemoglobin has been produced for some time, yet significant renal toxicity has heretofore prevented its widespread use. Hemoglobin is a tetrameric protein of approximately 64,000 daltons; outside of its red blood cell milieu, the hemoglobin molecule rapidly dissociates into dimers composed of an alpha and a beta subunit. In addition to rendering the hemoglobin non-functional, these dimers are then filtered by the kidney, and the interaction of these hemoglobin residua with minute amounts of cell wall pieces in the renal glomeruli results in rapid acute tubular necrosis and renal failure. Development of a suitable stroma free hemoglobin molecule therefore depends on the development of a stable, functional tetramer of hemoglobin which would not dissociate into dimers upon infusion. This problem has been solved in several novel ways.
Prevention of dissociation of the hemoglobin tetramer in plasma is accomplished by binding the hemoglobin protein subunits together to prevent dissociation. Binding of the hemoglobin tetramer has been approached both chemically and genetically. Chemical binding of the tetramer involves binding of the alpha subunits by a so-called bifunctional agent, such as diaspirin, which links the hemoglobin molecules and thus stabilizes them. These polyhemoglobins are now undergoing clinical trials as potential blood substitutes.
A second significant problem is the lack of 2,3-DPG associated with the stroma free hemoglobin; the resulting stroma free hemoglobin, although polymerized with bifunctional agents, will not be functional at physiologic levels of tissue oxygenation. The P50 of native stroma free hemoglobin in solution is approximately 17 mmHg. This has been overcome chemically by the binding of pyridoxal phosphate to the hemoglobin molecule. The resulting polymerized, pyridoxi-lated stroma free hemoglobin has a P50 of approximately 32 mmHg (as compared to native, RBC associated hemoglobin P50 of approximately 27 mmHg) (Figure 4). Therefore, chemically altered stroma free hemoglobin are functionally superior to native hemoglobin.
Figure 4. A comparison of the oxy-hemoglobin dissociation curves of native ("wild-type" or A1) hemoglobin contained with the normal red blood cell milieu ("RBC-Enclosed Native Hemoglobin"), native or "wild-type" hemoglobin after removal from a red blood cell ("Stroma Free Native Hemoglobin"), and typical hemoglobin based oxygen carrier solutions.
Another approach to hemoglobin modification has been genetic engineering. The structure and amino acid sequence of wild-type hemoglobin is known. Therefore, by genetically altering the native hemoglobin by the addition of a single amino acid, it is possible to covalently bind two alpha subunits, thus preventing the dissociation of the hemoglobin tetramer. A single point mutation in the beta subunits produces a hemoglobin with a P50 of approximately 32 mmHg. Thus specific mutations in the hemoglobin molecule result in a functional, stable stroma free hemoglobin. Insertion of this engineered hemoglobin into E. coli plasmids results in the production of large quantities of hemoglobin.7 Purification of the hemoglobin would be similar to those processes used currently for other genetically engineered substances, such as insulin.
Xenograft material can also be used to produce stroma free hemoglobin. Bovine hemoglobin can be used after polymerization, as bovine hemoglobin does not require 2,3-DPG or other ligands to modify its oxy-hemoglobin dissociation.8 A ready supply of this hemoglobin is available, and chemical sterilization of this protein possible, although the prospect of zoonotic infection must be considered, particularly with concern for prion disease.
Benefits Of Hemoglobin-Based Oxygen Carriers
All blood substitutes utilizing chemical sterilization involve the reclamation of human blood cells from outdated red blood cell products. Questions regarding the ability to chemically sterilize these products sufficiently to avoid infectious disease transmission have been largely answered; however, production of this product involves a ready supply of outdated blood in a time when voluntary donations are decreasing. Genetically produced hemoglobin from E. coli does not suffer from supply problems associated with the use of polymerized human hemoglobin. The use of bovine hemoglobin should be in ready supply theoretically for the foreseeable future. These approaches may prove more effective in satisfying the predicted high demand for these products.
Use of these products is predicated on knowledge of the serum half-lives of these preparations. In general, poly-hemoglobin preparations will increase in plasma half-life as their size is enlarged; a limit to the size is the viscosity and oncotic effects of the larger hemoglobin molecules. Most preparations will be retained in the plasma for half-lives of 8-30 hours.
These hemoglobin products, however, do not require a supraphysiologic oxygen tension to be effective in delivering oxygen. Indeed, these compounds will most likely be more effective than native hemoglobin in delivering oxygen to tissues at physiologic arterial oxygen tensions (Figure 4). Thus hemoglobin-based oxygen carriers have an advantage over perfluorocarbons in this respect.
Hemoglobin-based oxygen carriers have some advantages over allogeneic red blood cell transfusions. The lack of iso-agglutinating antigens, due to the absence of a red cell membrane, obviates blood typing and screening and eliminates the most common morbidity and mortality of allogeneic and autologous transfusions, mismatching of blood units and the transfusion recipient. The lack of cross-matching requirements also allows virtually immediate availability of an oxygen carrier in critical periods of trauma or hemorrhage. However, there may be issues with administration of free hemoglobin in potentially septic situations.9
Disadvantages Of Hemoglobin-Based Oxygen Carriers
Plasma hemoglobin is not a true blood substitute; hemoglobin can replace only the oxygen transport capacity of whole blood, without the coagulation or immunologic aspects normally present in blood. While allogeneic blood may not supply these functions either, the plasma half-life of cross-matched allogeneic red blood cells is several fold greater than that of plasma hemoglobin. Thus, hemoglobin-based oxygen carriers will not replace blood, allogeneic whole blood, or allogeneic red blood cells completely. Thus use of these products may be limited to specific applications or in conjunction with specialized techniques, such as cardiopulmonary bypass with extracorporeal circulation or acute normovolemic hemodilution with harvesting of autologous whole blood for later reinfusion.
Free hemoglobin avidly binds nitric oxide. It is unknown whether this in vivo binding is of clinical significance, although binding of nitric oxide has been implicated as the cause of hypertension commonly seen with hemoglobin infusion. It remains to be determined what effects stroma free hemoglobin has on regional autoregulation of blood flow, and whether the hypertension associated with hemoglobin infusion has pathophysiologic consequences. At present, little data are available in large animal or clinical studies utilizing these compounds to elucidate the importance of this phenomenon.
Metabolism of plasma free hemoglobin-based oxygen carriers is identical to native hemoglobin released as a red blood cell is destroyed. Bilirubin levels will rise as hemoglobin is metabolized. Amylase levels also rise and some degree of lipase increase occurs; the pancreas appears to be the source of these increases in amylase and lipase, although no clinical evidence of pancreatitis has been documented in patients receiving hemoglobin-based oxygen carriers. The administration of these hemoglobin thus may cause significant alterations in laboratory values, potentially masking serious clinical consequences. Additionally, the consequences of metabolism of hemoglobin-based oxygen carriers may be similar to those of multiple transfusions, namely hemosiderosis and chronic iron overload.
Clinical Utility Of Blood Substitutes
Current blood substitutes have been demonstrated to be safe when administered in small quantities to volunteers. Both perfluorocarbon and hemoglobin based oxygen carriers have undergone clinical trials designed to determine the safety of these compounds when given to otherwise healthy patients. These preliminary studies have shown that a clinical useful dose of a blood substitute can be infused to patients. However, further information regarding the effectiveness and clinical usefulness of these compounds is in short supply at present.
The short plasma half-life of these compounds limits the usefulness of blood substitutes to short periods of time. Ultimately, the blood substitute will be sequestered or metabolized, and decreased oxygen carrying capacity will reappear as the plasma oxygen carrying capacity diminishes. Thus, if no longer acting agents are available, it is likely that these blood substitutes will merely delay an allogeneic transfusion, rather than avoiding exposure, when used in place of conventional allogeneic red blood cell transfusions.
In order to effectively use these compounds, special techniques should be considered. One technique which theoretically should optimize blood substitute utility is acute normovolemic hemodilution. Aggressive harvesting of potentially several units of autologous fresh whole blood is possible when the solution to replace the harvested blood is capable of transporting oxygen. Coupling of blood substitutes with acute normovolemic hemodilution has been successful in small clinical trials; whether this mode of using blood substitutes will result in substantial clinical and economic benefits await larger clinical trials.
Summary
Blood substitutes are currently undergoing preliminary clinical trials to determine their safety. Two distinctly different classes of oxygen carriers are being developed, each capable of transporting and delivering oxygen to peripheral tissues. The delivery of oxygen by these two methodologies may have both benefits and risks which are unique to its class. Early clinical trials have been promising; however, effective use of these blood substitutes may involve using them in conjunction with other techniques such as normovolemic hemodilution to effectively reduce or eliminate the need for transfusions in certain instances. However, this first generation of clinically safe blood substitutes will not replace allogeneic blood transfusions as a means of treating many types of anemia.
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Stroma free hemoglobin has been produced for some time, yet significant renal toxicity has heretofore prevented its widespread use. Hemoglobin is a tetrameric protein of approximately 64,000 daltons; outside of its red blood cell milieu, the hemoglobin molecule rapidly dissociates into dimers composed of an alpha and a beta subunit. In addition to rendering the hemoglobin non-functional, these dimers are then filtered by the kidney, and the interaction of these hemoglobin residua with minute amounts of cell wall pieces in the renal glomeruli results in rapid acute tubular necrosis and renal failure. Development of a suitable stroma free hemoglobin molecule therefore depends on the development of a stable, functional tetramer of hemoglobin which would not dissociate into dimers upon infusion. This problem has been solved in several novel ways.
Prevention of dissociation of the hemoglobin tetramer in plasma is accomplished by binding the hemoglobin protein subunits together to prevent dissociation. Binding of the hemoglobin tetramer has been approached both chemically and genetically. Chemical binding of the tetramer involves binding of the alpha subunits by a so-called bifunctional agent, such as diaspirin, which links the hemoglobin molecules and thus stabilizes them. These polyhemoglobins are now undergoing clinical trials as potential blood substitutes.
A second significant problem is the lack of 2,3-DPG associated with the stroma free hemoglobin; the resulting stroma free hemoglobin, although polymerized with bifunctional agents, will not be functional at physiologic levels of tissue oxygenation. The P50 of native stroma free hemoglobin in solution is approximately 17 mmHg. This has been overcome chemically by the binding of pyridoxal phosphate to the hemoglobin molecule. The resulting polymerized, pyridoxi-lated stroma free hemoglobin has a P50 of approximately 32 mmHg (as compared to native, RBC associated hemoglobin P50 of approximately 27 mmHg) (Figure 4). Therefore, chemically altered stroma free hemoglobin are functionally superior to native hemoglobin.
Figure 4. A comparison of the oxy-hemoglobin dissociation curves of native ("wild-type" or A1) hemoglobin contained with the normal red blood cell milieu ("RBC-Enclosed Native Hemoglobin"), native or "wild-type" hemoglobin after removal from a red blood cell ("Stroma Free Native Hemoglobin"), and typical hemoglobin based oxygen carrier solutions.
Another approach to hemoglobin modification has been genetic engineering. The structure and amino acid sequence of wild-type hemoglobin is known. Therefore, by genetically altering the native hemoglobin by the addition of a single amino acid, it is possible to covalently bind two alpha subunits, thus preventing the dissociation of the hemoglobin tetramer. A single point mutation in the beta subunits produces a hemoglobin with a P50 of approximately 32 mmHg. Thus specific mutations in the hemoglobin molecule result in a functional, stable stroma free hemoglobin. Insertion of this engineered hemoglobin into E. coli plasmids results in the production of large quantities of hemoglobin.7 Purification of the hemoglobin would be similar to those processes used currently for other genetically engineered substances, such as insulin.
Xenograft material can also be used to produce stroma free hemoglobin. Bovine hemoglobin can be used after polymerization, as bovine hemoglobin does not require 2,3-DPG or other ligands to modify its oxy-hemoglobin dissociation.8 A ready supply of this hemoglobin is available, and chemical sterilization of this protein possible, although the prospect of zoonotic infection must be considered, particularly with concern for prion disease.
Benefits Of Hemoglobin-Based Oxygen Carriers
All blood substitutes utilizing chemical sterilization involve the reclamation of human blood cells from outdated red blood cell products. Questions regarding the ability to chemically sterilize these products sufficiently to avoid infectious disease transmission have been largely answered; however, production of this product involves a ready supply of outdated blood in a time when voluntary donations are decreasing. Genetically produced hemoglobin from E. coli does not suffer from supply problems associated with the use of polymerized human hemoglobin. The use of bovine hemoglobin should be in ready supply theoretically for the foreseeable future. These approaches may prove more effective in satisfying the predicted high demand for these products.
Use of these products is predicated on knowledge of the serum half-lives of these preparations. In general, poly-hemoglobin preparations will increase in plasma half-life as their size is enlarged; a limit to the size is the viscosity and oncotic effects of the larger hemoglobin molecules. Most preparations will be retained in the plasma for half-lives of 8-30 hours.
These hemoglobin products, however, do not require a supraphysiologic oxygen tension to be effective in delivering oxygen. Indeed, these compounds will most likely be more effective than native hemoglobin in delivering oxygen to tissues at physiologic arterial oxygen tensions (Figure 4). Thus hemoglobin-based oxygen carriers have an advantage over perfluorocarbons in this respect.
Hemoglobin-based oxygen carriers have some advantages over allogeneic red blood cell transfusions. The lack of iso-agglutinating antigens, due to the absence of a red cell membrane, obviates blood typing and screening and eliminates the most common morbidity and mortality of allogeneic and autologous transfusions, mismatching of blood units and the transfusion recipient. The lack of cross-matching requirements also allows virtually immediate availability of an oxygen carrier in critical periods of trauma or hemorrhage. However, there may be issues with administration of free hemoglobin in potentially septic situations.9
Disadvantages Of Hemoglobin-Based Oxygen Carriers
Plasma hemoglobin is not a true blood substitute; hemoglobin can replace only the oxygen transport capacity of whole blood, without the coagulation or immunologic aspects normally present in blood. While allogeneic blood may not supply these functions either, the plasma half-life of cross-matched allogeneic red blood cells is several fold greater than that of plasma hemoglobin. Thus, hemoglobin-based oxygen carriers will not replace blood, allogeneic whole blood, or allogeneic red blood cells completely. Thus use of these products may be limited to specific applications or in conjunction with specialized techniques, such as cardiopulmonary bypass with extracorporeal circulation or acute normovolemic hemodilution with harvesting of autologous whole blood for later reinfusion.
Free hemoglobin avidly binds nitric oxide. It is unknown whether this in vivo binding is of clinical significance, although binding of nitric oxide has been implicated as the cause of hypertension commonly seen with hemoglobin infusion. It remains to be determined what effects stroma free hemoglobin has on regional autoregulation of blood flow, and whether the hypertension associated with hemoglobin infusion has pathophysiologic consequences. At present, little data are available in large animal or clinical studies utilizing these compounds to elucidate the importance of this phenomenon.
Metabolism of plasma free hemoglobin-based oxygen carriers is identical to native hemoglobin released as a red blood cell is destroyed. Bilirubin levels will rise as hemoglobin is metabolized. Amylase levels also rise and some degree of lipase increase occurs; the pancreas appears to be the source of these increases in amylase and lipase, although no clinical evidence of pancreatitis has been documented in patients receiving hemoglobin-based oxygen carriers. The administration of these hemoglobin thus may cause significant alterations in laboratory values, potentially masking serious clinical consequences. Additionally, the consequences of metabolism of hemoglobin-based oxygen carriers may be similar to those of multiple transfusions, namely hemosiderosis and chronic iron overload.
Clinical Utility Of Blood Substitutes
Current blood substitutes have been demonstrated to be safe when administered in small quantities to volunteers. Both perfluorocarbon and hemoglobin based oxygen carriers have undergone clinical trials designed to determine the safety of these compounds when given to otherwise healthy patients. These preliminary studies have shown that a clinical useful dose of a blood substitute can be infused to patients. However, further information regarding the effectiveness and clinical usefulness of these compounds is in short supply at present.
The short plasma half-life of these compounds limits the usefulness of blood substitutes to short periods of time. Ultimately, the blood substitute will be sequestered or metabolized, and decreased oxygen carrying capacity will reappear as the plasma oxygen carrying capacity diminishes. Thus, if no longer acting agents are available, it is likely that these blood substitutes will merely delay an allogeneic transfusion, rather than avoiding exposure, when used in place of conventional allogeneic red blood cell transfusions.
In order to effectively use these compounds, special techniques should be considered. One technique which theoretically should optimize blood substitute utility is acute normovolemic hemodilution. Aggressive harvesting of potentially several units of autologous fresh whole blood is possible when the solution to replace the harvested blood is capable of transporting oxygen. Coupling of blood substitutes with acute normovolemic hemodilution has been successful in small clinical trials; whether this mode of using blood substitutes will result in substantial clinical and economic benefits await larger clinical trials.
Summary
Blood substitutes are currently undergoing preliminary clinical trials to determine their safety. Two distinctly different classes of oxygen carriers are being developed, each capable of transporting and delivering oxygen to peripheral tissues. The delivery of oxygen by these two methodologies may have both benefits and risks which are unique to its class. Early clinical trials have been promising; however, effective use of these blood substitutes may involve using them in conjunction with other techniques such as normovolemic hemodilution to effectively reduce or eliminate the need for transfusions in certain instances. However, this first generation of clinically safe blood substitutes will not replace allogeneic blood transfusions as a means of treating many types of anemia.
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