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Biomedical subjects

R M Winslow

Publications and source records attributed to R M Winslow.

At least 19 recordsLinked to original sources
PubMed2006

Current status of oxygen carriers ('blood substitutes'): 2006.

An alternative to blood transfusion, based on oxygen-carrying solutions, has been sought for over a century. The present 'first-generation' haemoglobin-products were based on observations that crosslinking with, for example, glutaraldehyde, overcame subunit dissociation and renal toxicity. Experience with these solutions has shown that they can be vasoactive, sometimes increasing blood pressure, sometimes decreasing tissue perfusion and sometimes both. Clinical trials have been disappointing because of unexpected toxicity. The 'second-generation' products are based on a better understanding of the mechanisms of this vasoconstriction. Such products may seem counterintuitive by traditional standards, but it is hoped that they will be less toxic, more beneficial to patients, and more economical to produce.

Blood Substitutes↗
PubMed2003

Current status of blood substitute research: towards a new paradigm.

For many reasons, haemoglobin, modified to prolong its circulation time, seems to be the optimal choice for a cell-free O2 carrier (blood substitute) because of its capacity to reversibly bind O2 in the lung and release it in tissue. After refining methods to prepare highly purified haemoglobin solutions and to chemically or genetically modify haemoglobin to overcome renal toxicity and to prolong retention time, a number of unwanted effects were observed in human clinical trials. These included symptoms referable to the GI tract, elevated pancreatic enzymes and hypertension, presumed to be the result of vasoconstriction. Studies on the mechanism of vasoconstriction induced by haemoglobin, using new techniques to investigate the microcirculation have led to a surprising new paradigm for the design of safe and effective solutions. These include increased O2 affinity (low P50) and increased viscosity and oncotic pressure. These second-generation solutions hold greater promise for clinical development.

Blood Substitutes↗
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

Blood substitutes.

Red cell substitutes are a group of oxygen carriers designed to temporarily replace transfused blood. Each product is unique in its limitations and advantages. Research and development has been slow because of the far-reaching consequences of replacing an oxygen carrier outside of the red cell. Nevertheless, a number of products are in advanced clinical trials and nearing the market. When they are available for use it is likely that development will accelerate and even better products will substantially alleviate the world-wide shortage of blood for transfusion and enable the delivery of medical care to underserved populations. An important consequence of the development of these products has been a better understanding of how oxygen is delivered to tissues.

Animals↗
PubMed2000

alphaalpha-crosslinked hemoglobin: was failure predicted by preclinical testing?

In 1998, Baxter Healthcare announced that it was abandoning its product, diaspirin-crosslinked hemoglobin (DCLHb), the first 'blood substitute' to complete all phases of human trials. The company announced that the phase III (pivotal) trials in humans had resulted in an unexpectedly high survival in a group of patients serving as controls for those who received their product in a trauma setting. It is not possible to quantitate the time, efforts and money that were expended in the course of developing this product, from 1985 to 1998. It is rumored that the giant healthcare company had expended more than a half billion dollars on this product, not to mention the investment in the same product by the US Army, the National Institutes of Health and many independent university-based scientists. The disappointment was profound and far-reaching. Although the threat of HIV transmission by banked blood has all but disappeared in the developed world, still the bulk of the world's population faces blood shortages, which this product and its future generations might have helped alleviate. Only Baxter and the Food and Drug Administration may forever know key elements of the history of development of this product. However, because the US Army decided to make its version of the product widely available to scientists, there is a substantial published record, contributed to by both Baxter and independent scientists. Examination of this record leads to the conclusion that there is no single reason for failure. However, it shows that the characteristic hemodynamic response caused by alphaalphaHb increased vascular resistance, and probably eliminates its potential as a red cell substitute. Newer solutions that overcome this limitation should fare better in clinical development when this problem is overcome.

Aspirin↗
PubMed1999

New transfusion strategies: red cell substitutes.

Red cell substitutes are solutions that can potentially be used in emergencies or during surgery when rapid expansion of the blood volume with an oxygen carrier is needed. The three main types of products in development are based on cell-free hemoglobin, perfluorocarbon emulsions, or liposome-encapsulated hemoglobin. None is currently approved for clinical use, but several are in advanced clinical trials. Outside the red blood cell, hemoglobin is subject to degradation and heme loss. It readily diffuses in the plasma space and effectively scavenges nitric oxide. These properties must be understood and controlled if hemoglobin-based products are to fulfill their promise. The development of red cell substitutes affords us a deeper insight into how oxygen is delivered to tissues in the microcirculation and how blood-flow distribution is regulated within and between organs. As red cell substitutes become available to clinicians and scientists, clinical applications are expected to expand.

Blood Circulation↗
PubMed1999

Blood substitutes: a review of the literature 1997-1998.

Blood substitutes are oxygen-carrying plasma expanders intended to be used instead of blood. Their development is a long-standing quest and seen as being one of the several most important prizes of biotechnology. Their value, both in the clinic and market place, is virtually limitless. Most of the emphasis today, both in research and development, is on hemoglobin-containing solutions, although modern perfluorocarbon emulsions have considerable advantage over the first-generation products.

Journal Article↗
PubMed1998

The N-terminal sequence affects distant helix interactions in hemoglobin. Implications for mutant proteins from studies on recombinant hemoglobin felix.

The N-terminal 18-amino acid sequence of the beta-chain of hemoglobin, as far as the end of the A helix, has been replaced by the corresponding sequence of the gamma-chain of fetal hemoglobin with the remaining sequence of the beta-chain retained (helices B through H). The gamma-beta-chain had the correct mass, and its entire sequence was established by mass spectrometric analysis of its tryptic peptides; the alpha-chain also had the correct mass. This recombinant hemoglobin (named Hb Felix) retains cooperativity and has an oxygen affinity like that of HbA both in the presence and absence of the allosteric regulators, 2,3-diphosphoglycerate or chloride but differs from HbF in its 2,3-diphosphoglycerate response. However, Hb Felix has some features that resemble fetal hemoglobin, i.e. its significantly decreased tetramer-dimer dissociation and its circular dichroism spectrum, which measure the strength of the tetramer-dimer interface in the oxy conformation and its rearrangement to the deoxy conformation, respectively. Even though Hb Felix contains the HbA amino acids at its tetramer-dimer interface, which is located at a distance from the substitution sites, its interface properties resemble those of HbF. Therefore, the N-terminal sequence and not just those amino acids directly involved at the subunit interface contacts with alpha-chains must have a strong influence on this region of the molecule. The results reinforce the concept of fluid long range relationships among various parts of the hemoglobin tetramer (Dumoulin, A., Manning, L. R., Jenkins, W. T., Winslow, R. M., and Manning, J. M. (1997) J. Biol. Chem. 272, 31326-31332) and demonstrate the importance of the N-terminal sequence, especially in some mutant hemoglobins, in influencing its overall structure by affecting the relationship between helices.

Amino Acid Sequence↗
PubMed1998

Heme oxygenase-1-derived carbon monoxide contributes to the suppression of acute hypertensive responses in vivo.

The enzyme heme oxygenase, which exists in inducible (HO-1) and constitutive (HO-2) isoforms, catalyzes the degradation of heme to biliverdin and CO in mammalian tissues. CO has been implicated in the control of vascular tone in a manner similar to that for NO. In the present study, we investigated the contribution of the heme oxygenase/CO pathway to the modulation of acute hypertensive responses in vivo induced by (1) alphaalphaHb, a chemically modified hemoglobin known to scavenge NO, and (2) NG-nitro-L-arginine methyl ester (L-NAME), a competitive NOS inhibitor. Experiments were carried out in conscious rats in which femoral arteries and veins were surgically catheterized 1 or 5 days before treatment with the vasoconstrictor agents. Intravenous infusion of alphaalphaHb (8% solution) or L-NAME (30 micromol/kg) [corrected] produced an acute and significant increase in mean arterial pressure (P<0.05) in rats at 5 days after catheter implantation. In contrast, no change in blood pressure was observed when alphaalphaHb or L-NAME was infused 1 day after the surgical intervention. The suppression of the hypertensive response observed at 1 day after surgery correlated with a significant (P<0.05) HO-1 expression in aorta, heart, and liver as well as increased aortic CO production and cGMP levels. At 1 day after surgery, pretreatment of animals with the heme oxygenase inhibitor zinc protoporphyrin IX (50 micromol/kg IP) markedly decreased aortic CO and cGMP levels and completely restored the vasoconstrictor effects of both alphaalphaHb and L-NAME. These results provide evidence for a crucial role of the heme oxygenase/CO pathway in the regulation of blood pressure under stress conditions in vivo.

Acute Disease↗
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

Depression of endothelial and smooth muscle cell oxygen consumption by endotoxin.

An optical method based on the oxygen-dependent quenching of a phosphorescent probe (palladium-porphyrin) was used to investigate the effect of bacterial endotoxin [lipopolysaccharide (LPS)] on oxygen consumption (VO2) by vascular cells. Endothelial (EC) and smooth muscle (SMC) cells from pig aorta were suspended in culture medium in the presence of palladium-porphyrin and transferred to glass capillary tubes that were sealed to create a hypoxic environment. Measured PO2 changed as a function of time in a highly predictable fashion when cell suspensions were exposed to agents or treatment known to affect cellular metabolism. Both EC and SMC showed a significant decrease in VO2 as cell density increased, and SMC VO2 was significantly higher than EC (1.94 +/- 0.09 vs. 1.0 +/- 0.15 nmol . min-1 . 10(6) cells-1). Exposure to LPS (1 microg/ml) caused a decrease in VO2 of 46% and 15% for EC and SMC, respectively. Pretreatment of cells with N-acetyl-L-cysteine, a substrate for glutathione synthesis with antioxidant properties, restored VO2 to normal values after exposure to LPS. These data suggest that endotoxin impairs VO2 in cells derived from the vascular wall and indicate the importance of EC and SMC respiration in maintaining vascular homeostasis under conditions of sepsis.

Acetylcysteine↗
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↗