The surface activity of PVP and other polymers and their antihemolytic capacity

The polymeric cryoprotective agents polyvinylpyrrolidone, dextran, and hydroxyethyl starch do not penetrate the cell membrane and are not present in high osmotic concentrations. Thus, they can exert little of the "antifreeze" behavior generally attributed to glycerol or dimethyl sulfoxide, and must protect cells from freezing injury by some action external to the cell surface. Surface energy measurements of droplets of hemoglobin solution immersed in solutions of cryoprotective polymers indicate that these polymers lower the surface energy of the solution below that of the hemoglobin droplets and form a stable interface. In injured cells, these polymers will therefore hide membrane defects by forming an interface across which hemoglobin cannot easily pass. When freezing is slow, the polymers have little if any true cryoprotective effect but interfere with hemoglobin release as an assay of injury.     

Study of chemically modified hemoglobin as an artificial oxygen carrier in the model of hemorrhagic shock in dogs]

Hemodynamic and gas-transporting properties of the chemically modified hemoglobin solution have been studied on the model of hemorrhagic shock in dog. It has been shown that the polymerized hemoglobin solution exerts the hemodynamic action just as the plasma substitute "polyglucin" does. However, in contrast to the latter, polyhemoglobin circulating in the vascular bed for a prolonged period of time increases the blood oxygen capacity and oxygen delivery to tissues with the resultant increase in body total oxygen taking-up.     

The conversion of variance and the evolutionary potential of restricted recombination

Genetic recombination is usually considered to facilitate adaptive evolution. However, recombination prevents the reliable cotransmission of interacting gene combinations and can disrupt complexes of coadapted genes. If interactions between genes have important fitness effects, restricted recombination may lead to evolutionary responses that are different from those predicted from a purely additive model and could even aid adaptation. Theory and data have demonstrated that phenomena that limit the effectiveness of recombination via increasing homozygosity, such as inbreeding and population subdivision and bottlenecks, can temporarily increase the additive genetic variance available to these populations. This effect has been attributed to the conversion of nonadditive to additive genetic variance. Analogously, phenomena such as chromosomal inversions and apomictic parthenogenesis that physically restrict recombination in part or all of the genome may also result in a release of additive variance. Here, we review and synthesize literature concerning the evolutionary potential of populations with effectively or physically restricted recombination. Our goal is to emphasize the common theme of increased short-term access to additive genetic variance in all of these situations and to motivate research directed towards a more complete characterization of the relevance of the conversion of variance to the evolutionary process.     

Autoxidation of hemoglobin enhanced by dissociation into dimers.

Autoxidation as a function of hemoglobin concentration indicates a 17-fold increase in the rate of autoxidation from 0.25 (%/h) to 4.3 (%/h) when tetrameric oxyhemoglobin dissociates into dimers. As a result of this large enhancement, a contribution of dissociation to the autoxidation is evident even at relatively high concentrations of hemoglobin for which it is usually considered that dissociation can be neglected. The mechanism for this phenomenon is attributed to alterations in the ligand pocket which occur when constraints due to subunit contacts within the R-state are eliminated.     

Changes in erythrocyte 2,3 diphosphoglycerate in women following short term maximal exercise.

15 untrained women were subjected to a walking treadmill test to determine the influence of maximal exercise upon synthesis of erythrocyte 2,3 DPG. Although there was a 9.8% increase in the 2,3 DPG content following exercise, there was a concomitant 9.4% increase in the hemoglobin level; therefore, when 2,3 DPG is expressed as a ratio to hemoglobin (See Article), there was no significant change as a result of exercise stress. It was suggested that three additive factors produced during strenuous exercise; decreased pH; increased hemoglobin concentration; and increased CO2 production result in by-product inhibition of 2,3 DPG synthesis. It is concluded that 2,3 DPG does not provide a physiologic benefit in the adaptation of the oxygen transport system to exercise.     

Vascular response to infusions of a nonextravasating hemoglobin polymer.

The clinical utility of cross-linked tetrameric hemoglobin solutions is limited by peripheral vasoconstriction thought to be due to scavenging of nitric oxide. In addition, transfusion of crude preparations of hemoglobin polymers can cause arterial hypertension. We tested the hypothesis that eliminating low-molecular-weight components from the polymer solution would prevent extravasation and its associated pressor response. A zero-link polymer of bovine hemoglobin was developed without chemical linkers left between the tetramers. Transfusion of unprocessed preparations of these polymers in rats resulted in appearance of the polymer in the renal hilar lymph. However, eliminating the low-molecular-weight components with a 300-kDa diafiltration resulted in an average hydrodynamic radius of 250 A and in undetectable levels of polymer in hilar lymph. Exchange transfusion in anesthetized rats and cats and in awake cats produced no increase in arterial pressure. In anesthetized cats, exchange transfusion with an albumin solution reduced hematocrit from 30 to 18%, increased cerebral blood flow, and dilated pial arterioles. In contrast, reducing hematocrit by transfusing the diafiltered polymer did not increase cerebral blood flow as pial arterioles constricted. These results are consistent with the hypothesis that the increase in arterial pressure associated with cell-free hemoglobin transfusion depends on hemoglobin extravasation. Constriction observed in the cerebrovascular bed with a nonextravasating hemoglobin polymer at low hematocrit is presumably a regulatory response to prevent overoxygenation at low blood viscosity.     

Ligand binding and the gelation of sickle cell hemoglobin.

The solubility equilibrium between monomer and polymer which has been shown to exist in deoxyhemoglobin S solutions is examined in solutions partially saturated with carbon monoxide. The total solubility is found to increase monotonically with increasing fractional saturation. At low fractional saturations the increase is nearly linear, amounting roughly to an increase of 0.01 g cm^?3 in solubility for each 10% increase in fractional saturation. Linear dichroism measurements on the spontaneously aligned polymer phase are used to examine the composition of the polymer as a function of the fractional saturation of the corresponding solution phase. The dichroism experiments show that the polymer phase contains less than 5% of CO-liganded hemes even at supernatant fractional saturations in excess of 70%. The polymer selects against totally liganded hemoglobin molecules by a minimum factor of 65 and against singly liganded molecules by a factor of at least 2.5. Consequently, polymerized hemoglobin S has a ligand affinity which is significantly lower than that of monomeric hemoglobin S in the deoxy quaternary structure.The kinetics of the polymerization reaction in the presence of CO are similar to those observed in pure deoxyhemoglobin S solutions. The polymerization is preceded by a pronounced delay, the duration of which, t_d, is proportional roughly to the 30th power of the solubility. At low fractional saturations, this amounts to a tenfold increase in t_d for each 10% increase in the fractional saturation.These results show that the polymerization reaction is nearly specific for deoxyhemoglobin. Models for the dependence of the solubility and the polymer saturation on ligand partial pressure demonstrate the importance of solution phase non-ideality in determining the solubility of mixtures. The results require selection against partially liganded species which is significantly greater than is predicted by the two-state allosteric model. The data are compatible with either sequential or allosteric models in which the major polymerized component is the unliganded hemoglobin molecule.     

Cerebrovascular response to decreased hematocrit: effect of cell-free hemoglobin,plasma viscosity,and CO2

The effect of transfusing a nonextravasating, zero-link polymer of cell-free hemoglobin on pial arteriolar diameter, cerebral blood flow (CBF), and O2 transport (CBF x arterial O2 content) was compared with that of transfusing an albumin solution at equivalent reductions in hematocrit (approximately 19%) in anesthetized cats. The influence of viscosity was assessed by coinfusion of a high-viscosity solution of polyvinylpyrrolidone (PVP), which increased plasma viscosity two- to threefold. Exchange transfusion of a 5% albumin solution resulted in pial arteriolar dilation, increased CBF, and unchanged O2 transport, whereas there were no significant changes over time in a control group. Exchange transfusion of a 12% polymeric hemoglobin solution resulted in pial arteriolar constriction and unchanged CBF and O2 transport. Coinfusion of PVP with albumin produced pial arteriolar dilation that was similar to that obtained with transfusion of albumin alone. In contrast, coinfusion of PVP with hemoglobin converted the constrictor response to a dilator response that prevented a decrease in CBF. Pial arteriolar dilation to hypercapnia was unimpaired in groups transfused with albumin or hemoglobin alone but was attenuated in the largest vessels in albumin and hemoglobin groups coinfused with PVP. Unexpectedly, hypocapnic vasoconstriction was blunted in all groups after transfusion of albumin or hemoglobin alone or with PVP. We conclude that 1) the increase in arteriolar diameter after albumin transfusion represents a compensatory response that prevents decreased O2 transport at reduced O2-carrying capacity, 2) the decrease in diameter associated with near-normal O2-carrying capacity after cell-free polymeric hemoglobin transfusion represents a compensatory mechanism that prevents increased O2 transport at reduced blood viscosity, 3) pial arterioles are capable of dilating to an increase in plasma viscosity when hemoglobin is present in the plasma, 4) decreasing hematocrit does not impair pial arteriolar dilation to hypercapnia unless plasma viscosity is increased, and 5) pial arteriolar constriction to hypocapnia is impaired at reduced hematocrit independently of O2-carrying capacity.     

Effect of ethanol on functional properties of the human hemoglobin molecule

The effect of ethanol on the oxygenation of hemoglobin was studied by kinetic absorption spectroscopy. It was found that the efficiency of oxygen geminate rebinding decreased upon ethanol addition. At ethanol concentrations up to 4.5 M, its influence on the structure and functional properties of the hemoglobin molecule is determined by changes in the bulk dielectric constant of solution. The decrease in the rate constant of the bimolecular stage of rebinding k'4 was caused by an increase in the viscosity of solution, with k'4 being approximately 1/eta 0.5. Upon oxidation of hemoglobin to hemichrome initiated by ethanol, dramatic conformational changes in the region of the heme pocket took place. They lead to a more than twofold increase in the efficiency of exit of oxygen molecules from the protein matrix to the solution after photodissociation.     

Simulation of combined transfer of oxygen and heat through the skin using a capillary-loop model

A model of a microcirculatory unit has been developed to study oxygen exchange processes within the upper part of the skin. The model includes the loop-shaped capillary structure of stratum papillare, the nonlinear binding of oxygen by hemoglobin, and, in particular, the shift of the oxygen hemoglobin dissociation curve due to temperature variations. The corresponding nonlinear elliptic boundary value problem is defined and the existence of at least one solution assured. After describing the numerical procedure to calculate an approximation to the solution, results of several calculations representing different supply situations of the upper skin are presented and discussed.     

Oxidative stress during ex vivo hemodialysis of blood is decreased by a novel hemolipodialysis procedure utilizing antioxidants

The high cardiovascular mortality in patients receiving hemodialysis (HD) has been attributed, in part, to oxidative stress. Here we examined the effectiveness of antioxidants introduced by means of a novel hemolipodialysis (HLD) procedure in terms of reducing oxidative stress during ex vivo blood circulation. Oxidative stress was studied in a model HD system resembling the extracorporeal circulation of blood during clinical HD. Blood circulation produced an increase of up to 280% in free hemoglobin levels and an increase of 320% in electronegative LDL (LDL(-)) subfraction. A significant correlation between LDL(-) and free hemoglobin levels confirmed previous findings that LDL(-) formation during ex vivo circulation of blood can be mediated by the oxidative activity of free hemoglobin. These effects were significantly attenuated during HLD using a dialysis circuit containing vitamin E with or without vitamin C. By contrast, HLD with vitamin C alone had a marked pro-oxidant effect. TBARS, lipid hydroperoxides, vitamin E and beta-carotene content in LDL were not significantly altered by the HD procedure. These findings demonstrate the occurrence of oxidative stress in human plasma where lipoproteins are a target and indicate antioxidant-HLD treatment as a specific new approach to decreasing the adverse oxidative stress frequently associated with cardiovascular complications in high-risk populations of uremic patients.     

Hemoglobin charge dependence on hemoglobin concentration in vitro

Studies have been made of the dependence of the charge of the hemoglobin molecule on hemoglobin concentration in the concentration range between 3 and 11 mmolal. The charge has been determined by measuring the distribution of ⁴²K between a hemoglobin solution in a cellophane bag and an external solution. The pH was 6.6, the K concentration was 10 mM, and the temperature was 4°C. The charge decreased along a sigmoid curve from a value of +3 in the most dilute solutions to a value of +0.5 in the most concentrated solutions. The results were in excellent agreement with earlier studies of Gary-Bobo and Solomon in which Cl distribution was measured between human red cells and external solutions and thus give added support to the conclusion that the apparent anomalous osmotic behavior of human red cells may be attributed to concentration-dependent changes in the hemoglobin net charge. The present findings also support the view that the water in the red cell is solvent water for K and Cl and differs in no quantitatively important respect from bulk water in free solution.     

Hemoglobin and Escherichia coli, a Lethal Intraperitoneal Combination

Intraperitoneal injection into mice of approximately 8 x 10(6) washed cells of Escherichia coli suspended in a lysate of washed human red blood cells or an aqueous solution of crystalline hemoglobin was lethal. E. coli suspended in washed intact erythrocytes, whole blood, plasma, or saline was innocuous. Fractionation of non-hemoglobin proteins from hemoglobin in lysates showed that only hemoglobin promoted a lethal infection. Overwhelming intraperitoneal growth of E. coli was attained in about 12 hr in lethal infections. The polymorphonuclear leukocytic response was ineffective against this rapid growth. The lethal mechanism is hypothesized to center on a unique role for free hemoglobin in inhibiting peritoneal absorption and stimulating an intraperitoneal exudate which supports luxuriant bacterial growth. Death is attributed to a lethal intoxication from bacterial endotoxins. This role for hemoglobin involves neither enhanced bacterial virulence nor lowered host resistance, and it would be of importance not only in peritonitis but also in problems where hemolysis and infection coexist.     

A compartmental model for oxygen transport in brain microcirculation in the presence of blood substitutes

A compartmental model is developed for oxygen (O(2)) transport in brain microcirculation in the presence of blood substitutes (hemoglobin-based oxygen carriers). The cerebrovascular bed is represented as a series of vascular compartments, on the basis of diameters, surrounded by a tissue compartment. A mixture of red blood cells (RBC) and plasma/extracellular hemoglobin solution flows through the vascular bed from the arterioles through the capillaries to the venules. Oxygen is transported by convection in the vascular compartments and by diffusion in the surrounding tissue where it is utilized. Intravascular resistance and the diffusive loss of oxygen from the arterioles to the tissue are incorporated in the model. The model predicts that most of the O(2) transport occurs at the level of capillaries. Results computed from the present model in the presence of hemoglobin-based oxygen carriers are consistent with those obtained from the earlier validated model (Sharan et al., 1989, 1998a) on oxygen transport in brain circulation in the absence of extracellular hemoglobin. We have found that: (a) precapillary PO(2) gradients increase as PO(2) in the arterial blood increases, P(50 p) (oxygen tension at 50% saturation of hemoglobin with O(2) in plasma) decreases, i.e. O(2) affinity of the extracellular hemoglobin is increased, the flow rate of the mixture decreases, hematocrit decreases at constant flow, metabolic rate increases, and intravascular transport resistance in the arterioles is neglected; (b) precapillary PO(2) gradients are not sensitive to (i) intracapillary transport resistance, (ii) cooperativity (n(p)) of hemoglobin with oxygen in plasma, (iii) hemoglobin concentration in the plasma and (iv) hematocrit when accounting for viscosity variation in the flow; (c) tissue PO(2) is not sensitive to the variation of intravascular transport resistance in the arterioles. We also found that tissue PO(2) is a non-monotonic function of the Hill coefficient n(p) for the extracellular hemoglobin with a maximum occurring when n(p) equals the blood Hill coefficient. The results of the computations give estimates of the magnitudes of the increases in tissue PO(2) as arterial PO(2) increases,P(50 p) increases, flow rate increases, hematocrit increases, hemoglobin concentration in the plasma increases, metabolic rate decreases, the capillary mass transfer coefficient increases or the intracapillary transport resistance decreases.     

Transient response of encapsulated enzymes in hollow-fiber reactor

A mathematical model for the transient response of encapsulated enzymes is developed showing the effects of the outer boundary layer, the encapsulating membrane, the partition coefficient, and diffusion with reaction within the encapsulating medium. The model incorporates both first-order kinetics and Michaelis-Menten kinetics for the reaction rate. Using typical hollow-fiber or microcapsule parameters, the model shows that (a) the partition coefficient affects the overall rate only when the rate-limiting step is diffusion through the membrane, (b) the transient overall effectiveness factor rises sharply with time and approaches an asymptotic value for most situations, and (c) the first-order approximation to Michaelis-Menten kinetics is not valid when the initial outside bulk concentration is higher than the Michaelis constant and the overall rate is reaction limited. The model is compared with experimental data using uricase in a hollow-fiber enzyme reactor configuration. Batch assay and CSTUER (continuous-stirred ultrafiltration enzyme reactor) studies were conducted on the free enzyme to provide some of the parameters used in the model. The CSTUER data fit the case of substrate inhibition kinetics with the apparent Michaelis constant approaching zero. The hollow-fiber reactor was conducted with uricase dissolved in both a buffer solution and a concentrated hemoglobin solution. Diffusivities of the solute were measured in both solutions as was the osmotic pressure of the hemoglobin solution. While experimental data for uricase in buffer solution could easily be matched by the model, that in the concentrated hemoglobin solution could not.     

A thermodynamic model for gelation of sickle-cell hemoglobin

The two-step concept of gelation of sickle-cell hemoglobin (Minton, 1973) provides the basis for a thermodynamic model to account for the macroscopic solution properties of sickle-cell hemoglobin in terms of microscopic structure and interactions. Step 1, the formation of a rod-like microtubular array, is treated as thermodynamically equivalent to a precipitation. Step 2, the alignment of microtubules to form a nematic phase, is treated as an isotropic-nematic transition in a suspension of interacting rod-like particles. Upon combination of these two steps a qualitative temperature-composition phase diagram is obtained.The results of several experimental studies on sickle-cell hemoglobin solutions, including measurements of solubility, sedimentation equilibrium and viscosity, are reviewed and it is shown that the proposed model provides a unified interpretation of many of the observed physical properties of these solutions.     

Oxidation-reduction reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park

The kinetics and equilibrium of the redox reactions of hemoglobin A, hemoglobin M Iwate, and hemoglobin M Hyde Park using the iron (II) and iron (III) complexes of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA4-) as the reducing and oxidizing agents have been studied. With respect to the equilibrium it was found that hemoglobin M Iwate (where the beta chains were reduced) was more readily reduced than hemoglobin M Hyde Park (where the alpha chains are reduced). This difference was shown to be a result of a difference in the rate constant for reduction but not oxidation. The observed rate contants for the reduction of all three hemoglobins were shown to decrease with increasing pH. This was attributed to a decrease in the [T]/[R] ratio. The observed rate contants for the oxidation reaction were shown to increase with increasing pH. Accompanying this increase was a change in the kinetic profile for hemoglobin A from pseudo first order to one in which the rate increased as the extent of reaction increased. Inositol hexaphosphate had no effect on the rate of oxidation of deoxyhemoglobin A. This was a result of binding of FeCDTA2- or HCDTA3- to the protein. However, in the presence of inositol hexaphosphate, the reduction of methemoglobin A exhibited biphasic kinetics. This result was interpreted in terms of the production of a small amount of a conformation which was more readily reduced.     

Dynamics of oxygen transport in erythrocytes

Distribution dynamics of oxygen tension throughout the erythrocyte volume was calculated by means of a mathematical model describing the dynamics of oxygen transport in the erythrocyte, its shape, diffusion resistance of hemoglobin solution. The pattern of the dissociation curve of oxyhemoglobin being taken into account. The model is presented as a system of differential equations in partial derivatives. Its solution was performed on an electron computer by a net method. Sharp jumps of pO2 inside the erythrocyte at its fast movements in the media with different partial pressure of O2 were shown. A quantitative relationship was found between the rate of physico-chemical reactions of oxygen binding and yield by hemoglobin and the level of hemoglobin saturation with oxygen.