HISTORY OF THE FIELD
A solution that can be used instead of blood for transfusion is a goal as old as the understanding of how blood circulates. The vigor with which the goal has been pursued over the centuries has been a reflection of the development of the blood transfusion industry. Until modern blood banks were established in the immediate post World War II era, many substitutes were tried including exotic candidates like milk, wine and various vegetable extracts. As technology for collection of blood matured so did efforts to purify the oxygen-carrying component, hemoglobin, from red blood cells. Although other oxygen carriers have been studied, such as perfluorocarbons (PFC’s), hemoglobin has the greater appeal because its oxygen capacity is much higher under the conditions normally found in the circulation.
The purification of hemoglobin from red blood cells is itself not a simple task. Red cells must be broken open and then the hemoglobin must be separated from the ruptured cells. The resultant solution is often called “stroma-free hemoglobin,”or just SFH (the word “stroma” refers to membrane material). The procedures for preparation of SFH have also matured over the past 40 to 50 years. Initially, cells are mixed with water, causing them to swell and burst. A significant problem has been development of methods that are not only efficient in separating hemoglobin from ruptured cell membranes, but which do not introduce toxic materials, such as organic solvents. Furthermore, the inner surfaces of cell membranes contain certain molecules, phospholipids, which can be toxic when exposed to other cells in the body. Some of the companies developing hemoglobin-based solutions have included patented or proprietary procedures into their production processes which allow for a high degree of purity of SFH.
Having discovered methods to produce purified SFH, the next problem to be faced was the toxicity of the hemoglobin molecule itself. This is a complex issue; hemoglobin is contained within cells in all but the lowest life-forms. Absent containment within a cell hemoglobin would rapidly leave the circulation and the body could not produce enough of it to maintain life. Hemoglobin is a very complex protein made up of 4 subunits (2 alpha and 2 beta chains), each of which contains an iron-containing group, heme. One molecule of oxygen binds to each of the iron groups, so a molecule of hemoglobin carries 4 molecules of oxygen. When hemoglobin is outside of the red blood cells, its component subunits tend to fall apart, and the much smaller subunits are removed very efficiently as they pass through the kidney. A large amount of this material, when presented to the kidney, may cause severe toxicity, even kidney failure.
To summarize, developers of cell-free hemoglobin solutions faced the following problems until approximately 1965:
• Purification of SFH and removal of stroma contamination
• A very short retention time in the circulation
• Production of free radical molecules that are themselves toxic
In the middle 1960’s hemoglobin biochemists found ways to chemically crosslink the hemoglobin subunits, which prevented its rapid clearance by the kidney. This discovery opened the door to many commercial efforts to produce blood substitutes, and some of the technology developed at that time is still being pursued today. Examples of crosslinking technology include reaction of hemoglobin with molecules like glutaraldehyde, a tissue fixative that produces polymers of hemoglobin of various sizes. These products were among the first to be tested in humans.
With the production of highly purified hemoglobin solutions, and modified hemoglobins that were retained for longer times in the circulation, another problem came to light: hypertension. Actually, Amberson first noted the ability of hemoglobin solutions to raise the blood pressure in humans in studies as early as the late 1940’s. This was initially thought to be a beneficial feature of hemoglobin solutions, since blood transfusions are often given to patients in shock where blood pressure is low. However, in more recent decades Dr. Winslow’s research helped make clear that the mechanism of hypertension involved vasoconstriction, a narrowing of the blood vessels, and that increased blood pressure per se is not beneficial if it is accompanied by decreased flow of blood to the tissues. It was this understanding that led to the development of Sangart’s technologies.
The purification of hemoglobin from red blood cells is itself not a simple task. Red cells must be broken open and then the hemoglobin must be separated from the ruptured cells. The resultant solution is often called “stroma-free hemoglobin,”or just SFH (the word “stroma” refers to membrane material). The procedures for preparation of SFH have also matured over the past 40 to 50 years. Initially, cells are mixed with water, causing them to swell and burst. A significant problem has been development of methods that are not only efficient in separating hemoglobin from ruptured cell membranes, but which do not introduce toxic materials, such as organic solvents. Furthermore, the inner surfaces of cell membranes contain certain molecules, phospholipids, which can be toxic when exposed to other cells in the body. Some of the companies developing hemoglobin-based solutions have included patented or proprietary procedures into their production processes which allow for a high degree of purity of SFH.
Having discovered methods to produce purified SFH, the next problem to be faced was the toxicity of the hemoglobin molecule itself. This is a complex issue; hemoglobin is contained within cells in all but the lowest life-forms. Absent containment within a cell hemoglobin would rapidly leave the circulation and the body could not produce enough of it to maintain life. Hemoglobin is a very complex protein made up of 4 subunits (2 alpha and 2 beta chains), each of which contains an iron-containing group, heme. One molecule of oxygen binds to each of the iron groups, so a molecule of hemoglobin carries 4 molecules of oxygen. When hemoglobin is outside of the red blood cells, its component subunits tend to fall apart, and the much smaller subunits are removed very efficiently as they pass through the kidney. A large amount of this material, when presented to the kidney, may cause severe toxicity, even kidney failure.
To summarize, developers of cell-free hemoglobin solutions faced the following problems until approximately 1965:
• Purification of SFH and removal of stroma contamination
• A very short retention time in the circulation
• Production of free radical molecules that are themselves toxic
In the middle 1960’s hemoglobin biochemists found ways to chemically crosslink the hemoglobin subunits, which prevented its rapid clearance by the kidney. This discovery opened the door to many commercial efforts to produce blood substitutes, and some of the technology developed at that time is still being pursued today. Examples of crosslinking technology include reaction of hemoglobin with molecules like glutaraldehyde, a tissue fixative that produces polymers of hemoglobin of various sizes. These products were among the first to be tested in humans.
With the production of highly purified hemoglobin solutions, and modified hemoglobins that were retained for longer times in the circulation, another problem came to light: hypertension. Actually, Amberson first noted the ability of hemoglobin solutions to raise the blood pressure in humans in studies as early as the late 1940’s. This was initially thought to be a beneficial feature of hemoglobin solutions, since blood transfusions are often given to patients in shock where blood pressure is low. However, in more recent decades Dr. Winslow’s research helped make clear that the mechanism of hypertension involved vasoconstriction, a narrowing of the blood vessels, and that increased blood pressure per se is not beneficial if it is accompanied by decreased flow of blood to the tissues. It was this understanding that led to the development of Sangart’s technologies.