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Biomedical subjects

M D Scott

Publications and source records attributed to M D Scott.

At least 37 records · Page 2Linked to original sources
PubMed1993

Role of oxygen and carbon radicals in hemoglobin oxidation.

We investigated the role of free radicals in hemoglobin (Hb) oxidation and denaturation. To generate free radicals, we used two azocompounds, the hydrophilic 2,2'-azobis(2-amidinopropane hydrochloride and the hydrophobic 2,2'-azobis(2,4-dimethylvaleronitrile) and a drug of the quinone family, phenazine methosulfate. The radical species involved were analyzed by direct EPR and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide, and N-t-butyl-alpha-phenyl-nitrone. The free radicals generated by the azocompounds were carbon radicals and, in the presence of molecular oxygen, peroxyl/alkoxyl radicals. The reaction of phenazine with Hb produced a nitrogen-centered semiquinoid radical detectable by EPR only under N2 and reactive oxygen species (O2-. and H2O2) in the presence of molecular oxygen. Azocompounds oxidized Hb to methemoglobin, hemichromes, and choleglobin while phenazine produced methemoglobin and ferrylhemoglobin. For all three drugs, low oxygen tensions (pO2 = 62 mm Hg) increased the formation of Hb oxidation products, whereas high oxygen tensions (pO2 = 540 mm Hg) reduced Hb oxidation. The formation of irreversible Hb oxidation products (irreversible hemichromes and Hb cross-linking) was observed only with the azocompounds and was reduced at high pO2. Spin traps and thiourea protected Hb from the oxidative damage induced by the azocompounds, whereas enzymes scavenging reactive oxygen species, such as superoxide dismutase and catalase, affected Hb oxidation induced by phenazine and that induced by the hydrophobic azocompound. These results indicate distinct patterns of oxidation and denaturation with each agent. Damage induced by phenazine was dependent on the formation of reactive oxygen species, whereas the damage induced by the azocompounds was due mainly to carbon-centered radicals with some involvement by reactive oxygen species only for the hydrophobic azocompound. The preferential interaction of Hb with drug radicals scavenged by molecular oxygen indicates that this protein may be more reactive under hypoxic conditions and led to the view that a good supply of oxygen can provide an important defense against drug-induced Hb oxidation.

Amidines↗
PubMed1993

Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes.

While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-hemoglobin chains, purified alpha-hemoglobin chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-hemoglobin chain autoxidation and appearance of membrane-bound globin. In addition, alpha-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-hemoglobin chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-hemoglobin chains. In summary, entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-hemoglobin chains can be examined.

Amitrole↗
PubMed1992

Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high-performance liquid chromatography in plasma and cardiac tissue.

A rapid ion-pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous detection of creatine, phosphocreatine, hypoxanthine, inosine, adenosine, AMP, ADP, ATP, 8-azaguanine, 2-chloroadenosine, and 2'-O-methyladenosine. This method has proven useful for measuring changes in nucleotide concentrations in both heart tissue and plasma samples. Separation of the compounds of interest is achieved in less than 8 min with re-equilibration in 7 min, making the total run time 15 min. Separation is performed on a 3-microns Ultrasphere ODS column employing tetrabutylammonium phosphate as the ion-pair agent and dipotassium hydrogenphosphate as the counter ion. The accuracy, rapid separation, and re-equilibration time make this method particularly useful for the routine analysis of a large number of samples.

Adenosine↗
PubMed1992

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes as a model for human beta thalassemia.

Entrapment of purified alpha-hemoglobin chains within normal erythrocytes resulted in structural and functional changes very similar to those observed in human beta thalassemic erythrocytes (Table 1). Membrane proteins and reactive thiol groups were decreased in a pattern similar to that observed in vivo in beta thalassemia. In addition, the alpha-chain loaded cells exhibited evidence of enhanced oxidant stress. Functionally, entrapment of alpha-chains resulted in the loss of cellular and membrane deformability, an important pathologic characteristic of the beta thalassemic erythrocytes. These results also demonstrate that the loss of membrane proteins and thiols as well as the functional loss of cellular and membrane deformability characteristic of the beta thalassemic cell occur very rapidly in the presence of soluble alpha-chains. Utilizing this model of the thalassemic erythrocyte, it is now possible to directly investigate the mechanisms underlying the cellular pathophysiology induced by excess alpha-chains. An understanding of these mechanisms may allow for the development of therapeutic interventions that would improve effective erythropoiesis and prolong erythrocyte survival in the peripheral circulation of individuals with beta thalassemia. Successful therapeutic interventions would diminish the frequency and/or necessity of blood transfusions and chelation therapy in beta thalassemia.

Erythrocyte Deformability↗
PubMed1992

Activated oxygen generation by a primaquine metabolite: inhibition by antioxidants derived from Chinese herbal remedies.

Primaquine is an important antimalarial drug which causes hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PDH) deficiency, probably due to oxidant generation by its metabolites. One of primaquine's metabolites, 5,6-dihydroxy-8-aminoquinoline (AQD), was found to cause chemiluminescence (CL) in vitro when incubated in the presence of luminol. This CL is inhibited by catalase and deferoxamine, unaffected by mannitol, and stimulated by superoxide dismutase (SOD), suggesting that it is mediated by H2O2. Three antioxidants (daphnetin, ferulate, and maltol), derived from Chinese herbal remedies, inhibited AQD- and H2O2-mediated CL, whereas a fourth, anisodamine, had no effect. Daphnetin also potently inhibited H2O2-mediated lipid peroxidation as measured by the production of thibarbituric acid reacting substances (TBARS). Thus, the possibility is raised that an antioxidant might be able to mitigate the oxidant hemolytic effects of primaquine.

Aminoquinolines↗
PubMed1992

Alpha- and beta-haemoglobin chain induced changes in normal erythrocyte deformability: comparison to beta thalassaemia intermedia and Hb H disease.

The alpha- and beta-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess alpha- and beta-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-containing alpha- and beta-chains were entrapped within normal erythrocytes. Entrapment of purified alpha-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in beta-thalassaemia intermedia. The decreased deformability was correlated with alpha-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane alpha-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the alpha-chain loaded cells. While both beta-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the alpha-chain loaded and beta-thalassaemic cells and may arise from either the increased intracellular viscosity of the beta-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.

Blood Proteins↗
PubMed1991

NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by the loss of NADPH and enhanced erythrocyte oxidant sensitivity. Historically, it has been theorized that the elevated oxidant sensitivity of G6PD-deficient erythrocytes arises as the direct consequence of decreased intracellular glutathione (GSH) concentrations. To directly investigate the basis of G6PD deficiency oxidant sensitivity, the effects of altered GSH and NADPH concentrations were examined in normal and G6PD-deficient erythrocytes. The results of this study demonstrated that GSH depletion, by 1-chloro-2,4-dinitrobenzene (CDNB), had no effect on hemoglobin oxidation in response to hydrogen peroxide (H2O2) generating systems (phenazine methosulfate and menadione bisulfite) in either normal or G6PD-deficient cells. Furthermore, a fourfold to sixfold increase in intracellular GSH concentration also did not protect against H2O2-generating systems in the normal or G6PD-deficient erythrocytes. Conversely, introduction of an NADPH-generating system (purified G6PD) into G6PD-deficient cells resulted in a significant decrease in oxidant sensitivity and an ability to cycle GSH. Further experiments demonstrated that the reduced oxidant sensitivity of the G6PD-reconstituted erythrocytes was not due to the maintenance of GSH levels because CDNB-mediated depletion of GSH did not alter this protective effect. Analysis of these results demonstrated a direct correlation between NADPH, but not GSH, concentration and hemoglobin oxidant sensitivity.

Dinitrochlorobenzene↗
PubMed1991

Inflammatory pseudotumor: a gallium-avid mobile mesenteric mass.

An 8-yr-old boy with a 1-mo history of culture-negative fever and anemia underwent gallium, ultrasound, and computed tomography studies as part of the evaluation of a fever of unknown origin. These studies revealed a mobile gallium-avid solid abdominal mass subsequently proven to be an inflammatory pseudotumor of the mesentery, a rare benign mass. This report documents the gallium-avid nature of this rare lesion and discusses associated characteristic clinical, pathologic, and radiographic features.

Child↗
PubMed1991

Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase.

To investigate the relative importance of catalase and glutathione in erythrocyte oxidant defense, human and mouse (normal and acatalasemic) erythrocytes were reversibly lysed and resealed in the presence of exogenous catalase or glutathione. This resulted in an increase in intracellular catalase activity or glutathione concentration in the resealed erythrocytes while normal cellular structure, hemoglobin concentration, cell volume, cellular deformability, and adenosine triphosphate concentration were maintained. Resealing alone had no effect on oxidant sensitivity. In human cells, a threefold increase in catalase activity resulted in the maintenance of glutathione levels in response to hydrogen peroxide (H2O2) challenge. Reconstitution of congenitally acatalasemic mouse erythrocytes, which were extremely sensitive to even micromolar concentrations of H2O2 with purified catalase resulted in complete protection against H2O2. Indeed, the catalase-reconstituted acatalasemic cells were less sensitive to H2O2-mediated damage than were normal, catalase-replete mouse cells. In contrast, alteration of the glutathione status of human and mouse (normal and acatalasemic) cells had no significant effect on oxidant sensitivity. Even a five-fold increase in intracellular glutathione concentration (greater than 30 micromoles glutathione per gram of hemoglobin) in normal or catalase-deficient (azide-treated or acatalasemic) red blood cells had no protective effect against H2O2-mediated lipid peroxidation or methemoglobin generation. Similarly, depletion of glutathione by 1-chloro-2,4-dinitrobenzene also had no effect on erythrocyte H2O2 sensitivity. These results suggest an important role for catalase in protection against H2O2-mediated damage at physiologic levels and that catalase is as at least as important as glutathione in cellular defense against H2O2.

Animals↗
PubMed1990

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes. A model for beta thalassemia.

Altered membrane proteins have been previously described in beta thalassemia and are thought to play an important role in the shortened erythrocyte survival. To investigate the mechanism by which these changes occur, purified heme-containing alpha-hemoglobin chains were entrapped within normal erythrocytes by reversible osmotic lysis. These resealed cells exhibited normal hemoglobin concentration, cell volume, deformability, and no substantial modifications of membrane proteins. Incubation (37 degrees C; up to 20 h) of the alpha-chain-loaded cells resulted in increasing amounts of membrane-associated alpha-chains. This was associated with concurrent decreases in the protein concentrations and reactive thiol groups of spectrin, ankyrin, and actin as determined by gel electrophoresis. The decreases in membrane protein concentration and reactive thiol groups after 20 h of incubation were closely correlated (R2 = 0.947) in the alpha-chain-loaded cells. Indicative of increased oxidant stress within the alpha-chain-loaded erythrocytes, methemoglobin generation was also significantly increased in the alpha-chain-loaded erythrocytes. In addition, entrapment of alpha-chains led to a progressive and significant decrease in erythrocyte deformability. Thus, the entrapment of purified alpha-chains in normal erythrocytes resulted in structural and functional abnormalities very similar to that observed in beta-thalassemic erythrocytes in vivo. The model described provides a means by which the fate of excess alpha-chains, their pathophysiological effects, as well as possible therapeutic approaches to thalassemias can be examined.

Erythrocyte Deformability↗
PubMed1990

Antimalarial activity of diethyldithiocarbamate. Potentiation by copper.

The antimalarial activity of diethyldithiocarbamate (DDC) in vitro was potentiated by subtoxic concentrations of copper. DDC was also more potent in the presence of an intracellular source of copper, such as when parasites were grown in superoxide dismutase (SOD)-loaded erythrocytes. These data suggest that DDC forms a complex with copper, either intracellularly or extracellularly, which is toxic to malarial parasites. The exact cause of this toxicity is not known, but may be due to a membrane effect, since DDC and copper, in combination, exert a potent lytic effect on normal human erythrocytes.

Animals↗
PubMed1990

Parasite uptake of desferroxamine: a prerequisite for antimalarial activity.

Desferroxamine has been shown to exhibit potent antimalarial activity. However, it is unclear as to whether desferroxamine functions by the chelation of extracellular, intra-erythrocytic, or parasite-associated iron. In order to determine desferroxamine's site of action, we have employed a large molecular weight dextran derivative of desferroxamine (70 kDa) and a reversible osmotic lysis technique by which erythrocytes were intracellularly loaded with this chelator. The desferroxamine-dextran derivative has virtually identical iron-binding characteristics to desferroxamine but, unlike desferroxamine, it is unable to cross the erythrocyte membrane. As previously shown, desferroxamine added to culture media exhibited potent antimalarial activity (mean effective inhibitory dose (ED50) approximately 6 microM). However, extracellular desferroxamine-dextran showed antimalarial activity only at very high doses (ED50 greater than or equal to 180 microM), indicating that extracellular iron chelation is not involved in the antimalarial activity of desferroxamine. The intra-erythrocytic entrapment of the desferroxamine-dextran derivative also had no significant effect, except at very high concentrations, demonstrating that desferroxamine does not remove a non-haem iron source necessary for malarial replication. The results of this study clearly suggests that the antimalarial activity of desferroxamine is directly related to its ability to enter the parasitic compartment and not due to the chelation of extra- or intra-erythrocytic iron pools necessary for malarial growth.

Animals↗
PubMed1990

Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.

The fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.

Animals↗
PubMed1990

Effect of osmotic lysis and resealing on red cell structure and function.

We have recently modified the dialysis tubing osmotic lysis and resealing method to examine the role of intracellular red blood cell (RBC) antioxidants. However, the potential effect of resealing on the RBC was not fully investigated. This study examined a number of cellular characteristics to determine the effects of physical lysis and resealing on the RBC. Following resealing, RBC exhibited normal morphology and at most only slight alterations in mean cell volume and mean cell hemoglobin concentration. RBC density distribution was significantly affected by resealing with increased populations of both light and dense cells, though the mean cell density was similar to that of control cells. Endogenous enzyme activities and adenosine triphosphate (ATP) concentration were unaffected by the resealing procedure. While reduced glutathione (GSH) concentration was decreased by 15%, RBC oxidant sensitivity was found to be unaltered. Cellular deformability of the resealed RBC was 80% to 90% that of the control cells. Membrane phospholipid and fatty acyl composition of the resealed RBC were unaffected when compared with matched control samples. Membrane transport, permeability, and Ca2(+)-mediated cellular vesiculation were minimally altered by resealing. Finally, entrapment of fluorescent compounds demonstrated that greater than 95% of the resealed RBC had incorporated exogenous agents. In summary, the osmotic lysis and resealing method described resulted in only minor changes in cellular characteristics while allowing for the efficient loading of compounds to which the RBC membrane is normally impermeable. Consequently, this method provides great potential for the selective modification of erythrocyte constituents in order to further define their roles within the RBC.

Adenosine Triphosphate↗
PubMed1990

Use of ektacytometry to determine red cell susceptibility to oxidative stress.

To define a more sensitive and reliable method to determine changes in the overall cellular characteristics of erythrocytes after oxidative damage, we used a viscodiffractometric method (ektacytometry) to measure the effect of oxidative stress. Erythrocytes were incubated in the presence of hydrogen peroxide, t-butyl hydroperoxide, or cumene hydroperoxide in phosphate buffer. This treatment resulted in decreased cellular deformability of the intact erythrocytes. In addition, deformability and fragility measurements of the erythrocyte ghost membranes indicated an increased membrane dynamic rigidity and altered-mechanical stability as a consequence of oxidant stress. These changes were observed before the onset of hemolysis. The observed decrease in deformability was accompanied by oxidation of hemoglobin, alterations of membrane proteins, and lipid peroxidation. To continuously measure the time course of the decrease in deformability in intact erythrocytes under oxidative stress, a new ektacytometric method was developed. Erythrocytes were oxidatively challenged within the viscometer at a constant osmolality and shear stress. The change in deformability was monitored and a typical range was defined for erythrocytes from normal individuals. Comparison of erythrocytes from patients with sickle cell disease with those from normal individuals demonstrated a higher susceptibility of sickle red cells toward oxidative stress.

Erythrocyte Deformability↗
PubMed1989

Enhancement of erythrocyte superoxide dismutase activity: effects on cellular oxidant defense.

To delineate further the role of superoxide dismutase (SOD) in red blood cell (RBC) oxidant defense, normal human erythrocytes were osmotically lysed and resealed in the presence of varying concentrations of exogenous SOD. This resulted in a dose-dependent increase in SOD activity in the resealed erythrocytes while maintaining nearly normal RBC hemoglobin concentration (less than 10% decrease from the control value), cell volume, and cellular deformability. Surprisingly, a five- or ninefold increase in SOD activity yielded no additional protection against superoxide-generating drugs (phenazine methosulfate or menadione sodium bisulfite). No significant differences were observed between the control and SOD-loaded RBCs in O2-driven methemoglobin formation or generation of thiobarbituric acid-reactive substances. In contrast, RBCs with elevated SOD activity pretreated with sodium azide (to block catalase activity) or 1-chloro-2,4-dinitrobenzene (to deplete reduced glutathione, GSH) showed significantly enhanced methemoglobin generation in response to superoxide generating drugs. No differential response was noted between the control, control-resealed, and SOD-loaded RBCs to oxidants other than superoxide. Based on our results and other data, we conclude that elevated SOD activity may imbalance cellular oxidant defense, resulting in enhanced oxidation due to the accelerated generation of H2O2, the product of O2- dismutation. This effect is significantly exacerbated under conditions in which H2O2 catabolism is altered.

Antioxidants↗
PubMed1989

Chlorpromazine inhibits vesiculation, alters phosphoinositide turnover and changes deformability of ATP-depleted RBCs.

To delineate further the underlying mechanism by which amphiphilic drugs can modulate vesicle release from human RBCs, we studied the effect of chlorpromazine on erythrocyte vesiculation induced by ATP depletion. This was correlated with turnover of the phosphoinositides as well as RBC deformability during the process since phosphoinositide metabolism may be involved in shape regulation of RBCs. Echinocytic shape transformation and subsequent vesiculation of RBCs, which commonly occur during ATP depletion, were inhibited by chlorpromazine. Furthermore, with a newly developed two-dimensional thin-layer chromatography separation of RBC membrane phospholipids, we showed that chlorpromazine significantly decreased the dephosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) in both ATP-depleted RBCs as well as in cells with partly maintained ATP levels. Concomitantly, there was a smaller increase in the relative amount of phosphatidylinositol. In addition, chlorpromazine also inhibited the decreased in RBC deformability as well as the shift of osmotic fragility that occurs during ATP depletion of erythrocytes.

Adenosine Triphosphate↗