PubMed Health⌕ Search

Biomedical subjects

K D Vandegriff

Publications and source records attributed to K D Vandegriff.

At least 19 recordsLinked to original sources
PubMed2001

The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers.

We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA(0) and alpha alpha-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes-Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a 'blood substitute' based on mimicking O(2) transport by RBCs to prevent autoregulatory changes in blood flow and pressure.

Algorithms↗
PubMed2000

Haemoglobin-based oxygen carriers.

Haemoglobin-based oxygen carriers are being developed for use in blood replacement therapies, either for perioperative haemodilution or for resuscitation from haemorrhagic blood loss. There is a high demand for these products because of risks associated with blood transfusions and pending worldwide blood shortages. Development of these products has required new technologies in protein engineering; since the haemoglobin is cell-free in solution, the molecule must be modified to be retained within blood circulation. Three classes of haemoglobin are under development: intramolecular cross-linked, intermolecular polymerised and surface conjugated with polyethylene glycol. Two products based on cross-linking chemistry have been discontinued because of serious adverse events and/or increased mortality rate in Phase III clinical trials. Three products based on polymerisation chemistry are in ongoing Phase III clinical trials. A new product based on surface conjugation is in preclinical evaluation. Although cross-linked and polymerised products have shown to be safe in preclinical and early Phase I/II clinical trials, they have had difficulty in proving efficacy. The primary adverse effect for the majority of cross-linked or polymerised products is a haemodynamic response, leading to increased vascular resistance to blood flow. The physiological mechanisms are still incompletely understood, so that safety and efficacy cannot be completely dissociated. New understandings on the mode of action of these products will help to define their utility and application. New products are under development, designed specifically to maximise blood flow and tissue perfusion and therefore, oxygenation.

Blood Banks↗
PubMed1998

Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide.

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (.NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in .NO scavenging reactions. All hemoglobins studied exhibited a value of 30 microM-1 s-1 for both .NO bimolecular association rate constants and the rate constants for .NO-induced oxidation in vitro. Only the .NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pM and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter .NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of .NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.

Animals↗
PubMed1998

Hemoglobin-oxygen equilibrium curves measured during enzymatic oxygen consumption.

A rapid, new method to measure hemoglobin-oxygen equilibrium curves is described using the protocatechuic acid/protocatechuic acid 3,4-dioxygenase system [C. Bull and D.P. Ballou (1981) J. Biol. Chem. 256, 12673-12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2 microelectrode. Multicomponent analysis allows the determination of fractions of oxyhemoglobin, deoxyhemoglobin, and high-spin and low-spin methemoglobins in each spectrum collected as the reaction proceeds. Fractional saturation as a function of oxygen partial pressure is calculated as the ratio of oxyhemoglobin to oxy- plus deoxyhemoglobin. Several advantages are offered by this method: (i) Hemoglobin-O2 binding curves are obtained rapidly and reproducibly; (ii) the speed of the reaction limits methemoglobin formation by autooxidation; (iii) there is no gas-liquid interface, eliminating protein denaturation at the surface; and (iv) direct calculations of fractional saturation are made using spectral analysis, thus avoiding the assumption of a linear transition between deoxy- and oxyhemoglobin.

Evaluation Studies as Topic↗
PubMed1998

Vascular resistance and the efficacy of red cell substitutes in a rat hemorrhage model.

We have compared polyethylene glycol-modified bovine hemoglobin (PEG-Hb; high O2 affinity, high viscosity, high oncotic pressure) and human hemoglobin cross-linked between the alpha-chains (alpha alpha-Hb; low O2 affinity, low viscosity, low oncotic pressure) with a non-O2-carrying plasma expander (pentastarch, high viscosity and oncotic pressure) after a 50% (by volume) exchange transfusion followed by a severe (60% of blood volume) hemorrhage. Mean arterial pressure and systemic vascular resistance rose significantly in the alpha alpha-Hb but not in the PEG-Hb animals. Two-hour survival was greater in the PEG-Hb animals (93%) than in control (35%), pentastarch (8%), or alpha alpha-Hb (6%) animals. In the PEG-Hb animals, there was no disturbance of acid-base balance, significantly less accumulation of lactic acid, and higher cardiac output than in the other groups. The data suggest that the rise in vascular resistance that follows alpha alpha-Hb exchange transfusion offsets the additional O2 transport provided by the cell-free hemoglobin. When resistance does not rise, as with PEG-Hb, even relatively small amounts of cell-free hemoglobin appear to be a very effective blood replacement.

Animals↗
PubMed1997

Colloid osmotic properties of modified hemoglobins: chemically cross-linked versus polyethylene glycol surface-conjugated.

Colloid osmotic pressures of hemoglobin solutions containing unmodified, intramolecularly cross-linked, intermolecularly polymerized, or polyethylene glycol (PEG) surface-conjugated hemoglobin have been measured to determine their macromolecular solution properties. Tetrameric and polymeric hemoglobins show nearly ideal solution behavior: whereas, hemoglobins conjugated to PEG have significantly higher colloid osmotic activity and exhibit solution non-ideality. From these studies, the average calculated molecular weights are 65.300 +/- 3500 for unmodified and intramolecularly cross-linked hemoglobin tetramers, 156,000 for ring-opened raffinose polymerized human hemoglobin, 97,000 for pyridoxalated human hemoglobin conjugated to a carboxy-PEG polymer, and 117,000 for bovine hemoglobin conjugated to a methoxy-PEG polymer. The calculated radius of gyration for tetrameric hemoglobins is 2.9 +/- 0.2 nm compared to 4.9 nm for the polymerized hemoglobin, and 7.2 and 14.1 nm for the human and bovine PEG-conjugated hemoglobins, respectively. Exclusion volumes are calculated to be 823 +/- 148 nm3 for tetramers, 4000 nm3 for polymers, and 13,000 nm3 and 94,000 nm3 for human and bovine PEG-conjugated hemoglobins, respectively. These studies show that polyethylene glycol conjugated to surface amino groups greatly increases the effective macromolecular size of hemoglobin in solution.

Animals↗
PubMed1997

Blood volume and cardiac index in rats after exchange transfusion with hemoglobin-based oxygen carriers.

We have measured plasma volume and cardiac index in rats after 50% isovolemic exchange transfusion with human hemoglobin cross-linked between the alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) and with bovine hemoglobin modified with polyethylene glycol (PEGHb). alpha alpha Hb and PEGHb differ in colloid osmotic pressure (23.4 and 118.0 Torr, respectively), oxygen affinity (oxygen half-saturation pressure of hemoglobin = 30.0 and 10.2 Torr, respectively), viscosity (1.00 and 3.39 cP, respectively), and molecular weight (64,400 and 105,000, respectively). Plasma volume was measured by Evans blue dye dilution modified for interference by plasma hemoglobin. Blood volumes in PEGHb-treated animals were significantly elevated (74.0 +/- 3.5 ml/kg) compared with animals treated with alpha alpha Hb (49.0 +/- 1.2 ml/kg) or Ringer lactate (48.0 +/- 2.0 ml/kg) or with controls (58.2 +/- 1.9 ml/kg). Heart rate reduction after alpha alpha Hb exchange is opposite to that expected with blood volume contraction, suggesting that alpha alpha Hb may have a direct myocardial depressant action. The apparently slow elimination of PEGHb during the 2 h after its injection is a consequence of plasma volume expansion: when absolute hemoglobin (concentration x plasma volume) is compared for PEGHb and alpha alpha Hb, no difference in their elimination rates is found. These studies emphasize the need to understand blood volume regulation when the effects of cell-free hemoglobin on hemodynamic measurements are evaluated.

Animals↗
PubMed1995

Effects of bacterial endotoxin on human cross-linked and native hemoglobins.

Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.

Bacterial Toxins↗
PubMed1994

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.

Amino Acid Sequence↗
PubMed1994

Assessment of hemoglobin-dependent neurotoxicity: alpha-alpha crosslinked hemoglobin.

Adult human hemoglobin A0 (HbA0) has been shown to be neurotoxic, and we wish to report on similar studies conducted using a modified hemoglobin, which has been crosslinked between the alpha subunits (alpha-alpha Hb). Cortical cell cultures were prepared from fetal Swiss-Webster mice at 15-16 days gestation. Mature cultures (days in vitro, 12-16) were exposed to alpha-alpha Hb in a defined medium for 24-48 hours at 37 degrees C. Low micromolar amounts of alpha-alpha Hb were neurotoxic in a concentration-dependent fashion. This toxicity was attenuated by the antioxidants Trolox and U-74500A and by the iron chelator deferoxamine. The hemoglobin-binding protein, haptoglobin, also completely blocked alpha-alpha Hb-dependent neurotoxicity. The latter result was unexpected because complex formation between alpha-alpha Hb and haptoglobin was not detected using assays of haptoglobin fluorescence and hemoglobin peroxidase activity.

Adult↗
PubMed1994

A comparison of rates of heme exchange: site-specifically cross-linked versus polymerized human hemoglobins.

The stability of the heme-globin interaction of chemically modified human hemoglobin (Hb) was tested by measuring rates of heme loss from methemoglobin. Heme transfer from methemoglobin to human serum albumin was measured by rapid-scanning spectrophotometry, and the resulting absorption matrices were analyzed by singular value decomposition. Unmodified human HbA0, hemoglobin cross-linked between beta subunits with either 2-nor-2-formylpyridoxal 5'-phosphate or 3,5-(dibromosalicyl)fumarate (DBBF), hemoglobin cross-linked between alpha subunits with DBBF, and pyridoxalated hemoglobin polymerized with either glycolaldehyde or glutaraldehyde were tested. Initial rates were evaluated by fitting the time courses to a biexponential equation using a matrix least squares curve-fitting algorithm. Reaction rates fell into two classes: (1) HbA0 and the site-specifically cross-linked hemoglobins, with biphasic rates of heme loss of 0.02 and 0.004 min-1, and (2) polymerized hemoglobins, with 10-20-fold higher rates at 0.5 and 0.03 min-1. The total fitted amplitudes of the reaction depended upon the specific modification: beta beta-cross-linked Hbs < alpha alpha-cross-linked Hb approximately glycolaldehyde polymerized Hb < glutaraldehyde polymerized Hb < HbA0.

Blood Substitutes↗
PubMed1994

Encapsulation of hemoglobin in non-phospholipid vesicles.

The efficiency of encapsulating hemoglobin in non-phospholipid liposomes by rapidly mixing hemoglobin with lipids heated above their solid-liquid phase transition temperature was examined. Human hemoglobin was mixed at 55-60 degrees C with a lipid solution containing polyoxyethylene-2 cetyl ether and cholesterol (molar ratio, 3:1) at 60-65 degrees C. Repeated mixing was carried out through a high-shear orifice, followed by rapid cooling and additional mixing. Lipid vesicles were heterogeneous in size, with diameters from approximately 300 nm to 10 microns. The non-encapsulated aqueous phase was removed by centrifugation, and total hemoglobin was determined spectrophotometrically. Encapsulation efficiency was calculated as the percentage of hemoglobin associated with the liposome phase (i.e., encapsulated) as a function of hemoglobin concentration and the aqueous:lipid hydration ratio. Hemoglobin concentrations were varied from 1 to 10 nM (in heme). Aqueous:lipid ratios of 8:1 and 4:1 were tested. Percent encapsulation varied from 13-30%, with the greatest efficiency, i.e., 30%, at a 4:1 hydration ratio of hemoglobin:lipid at 5.6 mM hemoglobin.

Blood Substitutes↗
PubMed1992

Regeneration of functional hemoglobin from iron(III) hemoglobin by reduction with hydrogen and a heterogeneous catalyst.

Functional hemoglobin was regenerated from partially autoxidized hemoglobin by reduction with molecular hydrogen in the presence of a heterogeneous catalyst consisting of elemental platinum embedded in an electroactive polymer. The visible spectrum of the regenerated hemoglobin was identical to that of native iron(II) hemoglobin. The regenerated hemoglobin displayed highly cooperative oxygen-binding characteristics. P50 values for oxidized-regenerated hemoglobin samples were not different from native hemoglobin. The Hill coefficients for regenerated hemoglobin were slightly lower than the controls, possibly because of small amounts of irreversibly oxidized hemoglobin arising during the initial autoxidation. The advantages of the reduction system include: (1) the heterogeneous catalyst avoids the problem of protein adsorption onto bare platinum, (2) catalyst and reducing agent are easily removed from the protein, and (3) the by-product H+ is buffered easily.

Buffers↗
PubMed1991

Determination of the rate and equilibrium constants for oxygen and carbon monoxide binding to R-state human hemoglobin cross-linked between the alpha subunits at lysine 99.

The kinetics of O2 and CO binding to R-state human hemoglobin A0 and human hemoglobin cross-linked between the alpha chains at Lys99 residues were examined using ligand displacement and partial photolysis techniques. Oxygen equilibrium curves were measured by Imai's continuous recording method (Imai, K. (1981) Methods Enzymol. 76, 438-449). The rate of the R to T transition was determined after full laser photolysis of the carbon monoxide derivative by measuring the resultant absorbance changes at an isosbestic point for ligand binding. Chemical cross-linking caused the R-state O2 affinity of alpha subunits to decrease 6-fold compared with unmodified hemoglobin. This inhibition of O2 binding was the result of both a decrease in the rate constant for ligand association and an increase in the rate constant for dissociation. The O2 affinity of R-state beta subunits was reduced 2-fold because of an increase in the O2 dissociation rate constant. These changes were attributed to proximal effects on the R-state hemes as the result of the covalent cross-link between alpha chain G helices. This proximal strain in cross-linked hemoglobin was also expressed as a 5-fold higher rate for the unliganded R to T allosteric transition. The fourth O2 equilibrium binding constant, K4, measured by kinetic techniques, could be used to analyze equilibrium curves for either native or cross-linked hemoglobin. The resultant fitted values of the Adair constants, a1, a2, and a3 were similar to those obtained when K4 was allowed to vary, and the fits were of equal quality. When K4 was fixed to the kinetically determined value, the remaining Adair constants, particularly a3, became better defined.

Carbon Monoxide↗
PubMed1991

Carbon dioxide binding to human hemoglobin cross-linked between the alpha chains.

The binding of carbon dioxide to human hemoglobin cross-linked between Lys alpha 99 residues with bis(3,5-di-bromosalicyl) fumarate was measured using manometric techniques. The binding of CO2 to unmodified hemoglobin can be described by two classes of sites with high and low affinities corresponding to the amino-terminal valines of the beta and alpha chains, respectively (Perrella, M., Kilmartin, J. V., Fogg, J., and Rossi-Bernardi, L. (1975b) Nature 256, 759-761. The cross-linked hemoglobin bound less CO2 than native hemoglobin at all CO2 concentrations in deoxygenated and liganded conformations, and the ligand-linked effect was reduced. Fitting the data to models of CO2 binding suggests that only half of the expected saturation with CO2 is possible. The remaining binding is described by a single affinity constant that for cross-linked deoxyhemoglobin is about two-thirds of the high affinity constant for deoxyhemoglobin A and that for cross-linked cyanomethemoglobin is equal to the high affinity constant for unmodified cyanomethemoglobin A or carbonmonoxyhemoglobin A. The low affinity binding constant for cross-linked hemoglobin in both the deoxygenated and liganded conformations is close to zero, which is significantly less than the affinity constants for either subunit binding site in unmodified hemoglobin. Comparing the low affinity sites in this modified hemoglobin to native hemoglobin suggests that cross-linking hemoglobin between Lys alpha 99 residues prevents CO2 binding at the alpha-subunit NH2 termini.

Biological Transport↗