Focus
Artificial Blood Designing blood substitutes for emergency care requires methods to measure their oxygen transport activity and possible toxicity
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t sounds like something you'd use for Halloween, but artificial blood is actually receiving high R&D priority at the National Institutes of Health (NIH) and the Department of Veterans Affairs (VA). The incidence of AIDS transmission through blood transfusions in the United States (some estimates place it at > 9000 reported cases in the past 10 years) and the ongoing risk of hepatitis transmission have put an already threatened blood supply under suspicion as unsafe to use. Blood substitutes can be made by modifying outdated blood or by de novo synthesis. Those that aren't derived from human blood products and can be mass produced may be the best hope for eventually solving the problems of availability and sterility. When a patient loses a great deal of blood, fluids with high oncotic pressure (plasma volume expanders) or isotonic saline can be used to replace the lost volume, but they lack the key attribute for an effective blood substitute: provision of oxygen transport. Without that, a patient
may suffocate even while breathing normally, because the oxygen has no way to get from the lungs to the tissues where it's needed. In natural blood, oxygen is transported by hemoglobin (Hb), a tetramer of four subunits, each with an iron-containing porphyrin function (heme) that binds a molecule of 02. The subunits have a synergistic or "cooperativity" effect on one another; as one subunit binds oxygen, it makes it easier for the others to follow suit. As a result, the oxygen affinity of Hb changes with its environment—as red blood cells (RBCs) pass through the oxygenated tissue of the lungs, the Hb they contain binds oxygen tightly but then releases it easily in the oxygen-poor environment of other tissues. Learning how native and modified hemoglobins function and determining the potential toxicity problems for proposed substitutes—whether Hb-based or not—in situations requiring large volumes of blood substitute are some of the analytical challenges involved in the design of artificial blood.
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Focus
Non-Hb substitutes
Although it would be cheaper and simpler to produce small compounds than Hb ge netic mutants as artificial blood substi tutes, attempts to use non-Hb oxygenat ing agents such as perfluorocarbon emul sions have met with limited success. Perfluorocarbons, which aren't oxygen carriers themselves, increase the solubil ity of oxygen in blood when administered in an emulsion. Because of problems with emulsifier excretion and insufficient oxy genation when a patient breathes normal ( ~ 20% 02) air, they aren't very effective as blood substitutes; instead, they are used for perfusing donor organs such as kidneys to keep them oxygenated before transplantation surgery. Current strategies for creating an effec tive blood substitute include modifying Hb via recombinant gene technology so it can function outside an RBC or designing a totally different (and preferably much simpler) oxygen carrier. Monitoring the effectiveness of oxygen transport, reten tion of the blood substitute in the circula tion, toxicity, and blood substitute inter ference with clinical analytical methods are part of the rational drug design ap proach.
amino acids and positions most affect the protein's function. Spectrophotometry can be used to monitor Hb kinetics because as the pro tein changes oxygenation states, its opti cal absorption spectrum changes mark edly between 480 and 640 nm. Oxygen ated, deoxygenated, and dysfunctional Hbs have distinct spectral profiles that can be used to calculate percent oxygen satu ration in blood or other solutions. Simple clinical "oximeters" for analyzing patient blood samples calculate oxygen saturation by monitoring the wavelength that corre sponds to the point where equimolar oxyand deoxy-Hb have the same absorbance (the isosbestic point) and the wavelength where the molar absorbance coefficients of the two species differ most. More so phisticated oximeters also monitor wave-
Pulse oximeters noninvasively measure oxygen in the capillanes dunng surgery
Hemoglobin activity
The fine points of how the structure of na tive Hb affects its oxygen-binding prop erties are still not well understood, accord ing to Robert Noble of the State Univer sity of New York at Buffalo. "Up to now, it's been hit-or-miss," he says. "We're trying to get some predictive knowledge about the amino acids and how they affect Hb oxygen affinity." He and collaborators from six other universities are working on a series of genetic modifications of the protein in which specific amino acids are systematically replaced with alanine using recombinant DNA techniques. Alanine has a neutral sidechain that is small enough to avoid distorting the overall 3D structure of the Hb subunits, he explains, but large enough to prevent any of the protein backbone from rotating, as glycine substitution would allow. The research teams observe structural changes in the genetic variants under high- and low-02 affinity conditions by X-ray crystallography and perform spectrophotometric oxygenbinding kinetic assays to determine which
lengths for dysfunctional Hbs and other in terferences such as dyes, turbidity, and abnormal proteins. All of the amino acid substitutions are located at the interface "hinge region" be tween the a.x and β2 subunits, where the two αβ dimers are connected to each other. Noble says that conformation changes at this junction are associated with the high- and low-02 affinity states. In addition to the use of X-ray crystallographic methods for structural elucida tion and spectroscopic kinetics assays for oxygen binding, which Noble's group per forms, other members of the collabora tion are using resonance Raman spectros copy to assess the effects of modifying the amino acids at the hinge on structural changes to the heme porphyrin in each subunit. The stretching frequency of the bond between the heme iron center and nearby hystidyl residues changes as the interface between subunits is altered. (See also Anal. Chem. 1993, 65, 201 A210 A.)
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Another heme-based assay the collabo rators perform is to create hybrid Hb mol ecules containing non-oxygen-binding metal-substituted porphyrins in certain subunits. Because they don't bind oxygen, the inactivated subunits maintain the physical environment of the nominally in tact tetramer without producing cooperativity effects. That way, the oxygenbinding kinetics of an individual subunit with a functional heme can be observed spectrophotometrically. This strategy may help the researchers to discover how the subunits act on each other aside from binding oxygen themselves. The rela tionship between any two functional subunits can also be studied by substituting inactive porphyrins in the other two. Oxygen transport in vivo
Standard spectroscopic binding kinetics studies may help to determine how well a modified Hb transports oxygen in a cellfree medium, but measuring oxygen trans port in living tissue is more complicated. Noninvasive methods include the use of pulse oximeters, which attach to a fin ger, toe, or earlobe and can be used to measure oxygen in vivo in the capillaries. These instruments are used during sur gery and, although they suffer interfer ences from tissue thickness, they have the advantage over conventional oximeters or general spectrophotometers that no precautions need to be taken to prevent ac cidental oxygenation of the blood after sampling. However, they are only useful for measuring oxygen in blood vessels, not in tissues, because Hb generally is ex cluded from the tissues. Microelectrodes have been used to measure oxygen con centration in tissues near blood vessels, but this method requires anesthesia as well as isolation of the electrodes and tis sue from environmental oxygen. To measure oxygen saturation in the tissues of a living animal, Marcos Intaglietta of the University of California, San Di ego (UCSD), uses a palladium-contain ing porphyrin compound bound to serum albumin as an oxygen probe and injects it into an awake hamster. The hamster has very thin cheek pouch tissues, and the vessels and capillaries can be observed un der a microscope. Blood flow can be mon itored with a video camera as oxygen is determined spectroscopically. When the
cheek tissue is epi-illuminated with a pulsed xenon arc lamp, the Pd-porphyrin phosphoresces, but the signal is quenched on binding oxygen. The intravascular oxygen concentration depends only on the time-dependent features of the phosphorescence signal, not on the emission intensity, so the method can measure oxygen in microvessels and tissues. It can be used to measure the effectiveness of blood substitutes in the test animals, but it may not be an easy way to evaluate human subjects in clinical trials or to monitor patients during medical treatment. Hb toxicity
Genetically modified Hb, even with good oxygen transport and even when administered in a suitably buffered solution, still poses problems as a blood substitute. Normally, Hb is contained in RBCs, which also contain enzymes that restore its function if the hemes become oxidized. Outside these cells, Hb behaves differently, binds oxygen more tightly, and is more susceptible to degradation. Although it might be easier to produce single subunits or dimers in bulk than the full Hb tetramer, renal function assays in test animals have shown that the smaller proteins are cleared by the kidneys in a single pass, so those types of blood substitutes would have to be replaced in a matter of a few hours. Aside from the problem of ineffective treatment, the massive clearance of heme-containing protein poses the risk of kidney damage. To prevent early clearance or degradation, Hbbased products have to be large and stabilized. Some pharmaceutical companies have tried to polymerize Hb, but Noble says this type of product behaves less predictably as an oxygen carrier than the Hb products that are either genetically or chemically cross-linked to hold the subunits together. Some groups have modified Hb to enhance oxygen binding, increase retention, and decrease toxicity. However, Robert Christenson of the University of Maryland says these modified Hbs can cause platelets and RBCs to aggregate and be removed from circulation. This in turn can lead to blood clotting problems and exsanguination (uncontrollable bleeding). After several test animals died from these effects, his group developed an in vitro
model to screen for toxic effects and evaluate the protective properties of additives such as buffers and anticoagulants. One fairly recent discovery is that in high concentration, cell-free Hb also appears to cause constriction of the blood vessels—perhaps as part of the normal physiological mechanism that stops bleeding. Some commercially developed cell-free Hb products have posed problems during clinical trials because this effect caused hypertension in patients. Kim Vandegriff of UCSD says the vasoconstriction may be caused by Hb binding NO (also known as "endothelium-derived relaxing factor"), which activates the enzyme guanylate cyclase. Some genetic mutation studies are geared toward designing an Hb that provides oxygen transfer without this interaction.
He notes that one strategy used in Phase I (safety testing) clinical trials for U.S. Food and Drug Administration premarket approval is to perform dose tolerance tests to assess the safety of modified cell-free Hb and its clearance from the subjects' plasma. The modified cell-free human Hb has very similar spectral and gel electrophoretic properties to the subjects' native Hb that may be released from RBCs. Christenson says more work appears to be necessary because bleeding effects need to be minimized in any blood substitute that progresses to clinical trials. However, because the cell-free product his group is evaluating is chemically modified with polyoxyethylene groups, it could be distinguishable from native Hb by SEC. Future solutions
Noble says the various problems involved in cell-free Hb blood substitution may not be solved by any single Hb structure. Some groups are exploring the encapsulation of Hb products inside a liposome package that mimics the RBC and prevents both degradation and harmful side effects. However, says Noble, the problems with liposome impermeability to protons and other critical ions, which proteins in the RBC membranes transport acChristenson says that the high concen- tively, could mean further complications in blood substitute design. He says, "You tration of cell-free Hb in the proposed don't want to end up redesigning the red blood substitutes also interferes signifiblood cell." Four orfivepharmaceutical cantly with a number of standard toxicology tests used to monitor its in vivo effects. companies are testing polymerized or cross-linked Hb products in Phase I clini"When we replaced 50% of the blood in a test animal with an 8-g/100 mL solution of cal trials. modified Hb," he says, "the plasma fracOther groups, some commercial, are tion was opaque." Several of the cardiac, trying to design more effective fluorocarliver, and renal function tests are coloribon compounds for organ perfusion and metric immunoassays with which free Hb blood substitution. And a few researchis known to interfere at high concentraers are exploring the possibility of using tion. To cope with these problems, his nonprotein oxygen carriers such as hemegroup measured the effect of free-Hb incontaining cyclodextrin compounds in terference on each method for a particular artificial blood. analyte as a function of concentration, seWhether any of these methods can be lected the least affected method for each made to do the work of human blood withanalyte, and diluted the samples accordout compromising safety remains to be ingly. Success was mixed and analyte- seen. Other analytical issues such as stadependent, he says. "For example, the bility, purity, and shelf life must also be reimmunoassay for creatine kinase-MB [a solved before any artificial blood substicardiac enzyme] was interfered with least tutes come to market. What is clear at this by Hb, but the assay for bilirubin [a point is that new analytical tools may breakdown product of heme] was comneed to be invented along with the new pletely useless on any instrument." blood substitutes. Deborah Noble Analytical Chemistry, Vol. 67, No. 1, January 1, 1995 3 3 A
