Correction of Metabolic Indicators of Erythrocytes and Myocardium Structure with Ozonized Red Blood-Cell Mass

The early posttransfusion period after acute blood loss using erythromass without ozone was characterized by decreased electrophoretic mobility of erythrocytes (EPME), ATP, 2,3-diphosphoglycerate (2,3- DPG) concentration, Na⁺-K⁺-ATPase activity, and increased concentration of malonic dialdehyde (MDA) and catalase activity in erythrocytes. Most parameters gradually were restored over 5 days, but a reduced concentration of 2,3-DPG was observed throughout the entire experiment with transfusion of erythromass without ozone. Transfusion of the ozonized erythrocyte mass 1 h after its administration caused an increase in 2,3- DPG concentration in erythrocytes circulating in the vascular bed. Twenty-four hours later, increased catalase activity, EPPE, and ATP concentration were registered, and, after 5 days, enhanced Na⁺-K⁺-ATPase activity was registered. Their values were maintained until the end of the experiment at a higher level than in animals that had undergone transfusion of the nonozonized erythromass. Ozonized erythrocyte mass improves the functional metabolic state of erythrocytes, promotes an earlier recovery of the oxygen transport in blood, and limits the damage to the microcirculatory bed of the myocardium and cardiomyocytes during blood loss.     

Organic phosphate binding to hemoglobin in intact human erythrocytes determined by 31P nuclear magnetic resonance spectroscopy.

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to estimate the percent of 2,3-diphosphoglycerate and ATP bound to hemoglobin in intact human erythrocytes at 37 degrees C. Binding was assessed by comparing the chemical shifts (delta) of 2,3-diphosphoglycerate and of ATP observed in intact cells with the delta values of these organic phosphates determined in model solutions closely simulating intracellular conditions, in which percent binding was directly evaluated by membrane ultrafiltration. The results showed that the percent of bound 2,3-diphosphoglycerate in intact cells varied with pH, the state of oxygenation, and 2,3-diphosphoglycerate concentration. The values ranged from 33% in cells incubated with glucose in air at an intracellular pH of 7.2 to 100% in cells incubated with inosine in N2 at a pH of 6.75. At the same 2,3-diphosphoglycerate concentration, a greater percentage of the compound appeared to be bound in erythrocytes than in the closely simulated model system. ATP was not significantly bound to hemoglobin under any condition examined, but appeared to be strongly complexed to Mg2+ inside the erythrocyte. The binding percentages for both 2,3-diphosphoglycerate and ATP in intact cells estimated by 31P NMR spectroscopy were lower than those calculated by others from individual association constants determined for the binding of different ligands to hemoglobin.     

Relationships between hemorheology, erythrocyte metabolism, and von willebrand factor in athletes and patients with peripheral arterial disease

The relationship between hemorheology, erythrocyte ATP and 2,3-diphosphoglycerate (2,3-DPG) concentrations, and von Willebrand factor antigen was studied in athletes and peripheral arterial disease patients. Lower blood viscosity, mainly due to a higher erythrocyte deformability, was found in athletes compared to control subjects. Higher 2,3-DPG/Ht levels in athletes were correlated with blood viscosity, erythrocyte deformability, the rigidity index, and erythrocyte suspension viscosity at low shear stress. It is suggested that these relationships might be determined by the predominance of immature erythrocytes in the blood circulation of the athletes. In the group of patients, a decrease in ATP/Ht was related to increased erythrocyte aggregation and a higher erythrocyte suspension viscosity. Moreover, the concentration of von Willebrand factor was positively correlated with the erythrocyte aggregation index, erythrocyte suspension viscosity, and plasma viscosity. The results show that alterations in erythrocyte and plasma rheology may be involved in the modification of the functional state of the vascular endothelium and the development of atherosclerosis.     

A liver-type mutation in a case of pronounced erythrocyte phosphofructokinase deficiency without clinical expression

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.     

Cyclic AMP-dependent phosphorylation of erythrocyte variant pyruvate kinase

Cyclic AMP-dependent phosphorylation of a variant erythrocyte pyruvate kinase (PK; EC 2.7.1.40) was studied. This variant PK shows a faster electrophoretic mobility than the normal enzyme. The decreased enzyme activity observed in this variant is associated with a quantitative decrease of enzyme protein. Other parameters are within normal ranges. The partially purified variant PK is phosphorylated with a subsequent increase of k0.5s (phosphoenolpyruvate) similar to the normal control, suggesting that the structural abnormality of the variant enzyme has no influence on the phosphorylation-deactivation mechanism. On the other hand, the variant PK in the erythrocyte was less extensively phosphorylated than PK in normal erythrocytes. This may be the result of abnormal metabolism in the patient's red cells, including increased 2,3-diphosphoglycerate and decreased adenosine triphosphate levels.     

Erythrocyte 2,3-DPG,ATP and oxygen affinity in hemodialysis patients

Patients on a chronic hemodialysis regimen were studied with respect to their erythrocyte adaptation to anemia. Erythrocyte 2,3-diphosphoglycerate (DPG) concentration was suboptimal compared with that of anemic patients who were not uremic. In uremic patients erythrocyte 2,3-DPG correlated poorly with hemoglobin level but more strongly with plasma pH. Differences between observed levels of erythrocyte 2,3-DPG and the values predicted using data from other anemic patients also correlated with pH. Gradual correction of plasma pH with oral sodium bicarbonate resulted in a substantial increase in erythrocyte 2,3-DPG and a decrease in oxygen affinity. Therefore, maintenance of normal pH in uremic subjects may improve tissue oxygenation. On the other hand, the rapid correction of acidosis during dialysis resulted in increased oxygen affinity. This response was due to the direct effect of pH on oxygen affinity in the absence of a significant change in erythrocyte 2,3-DPG or adenosine triphosphate (ATP) during hemodialysis. Erythrocyte ATP but not 2,3-DPG correlated with serum inorganic phosphate in uremic subjects. A 21% reduction of serum phosphate produced by ingestion of aluminum hydroxide gel had no significant effect on these variables.     

Effect of 2,3-diphosphoglycerate concentration on the alkaline fragility of phosphofructokinase-deficient canine erythrocytes

1. Erythrocytes in whole blood samples from dogs with phosphofructokinase (PFK) deficiency had lower 2,3-diphosphoglycerate (2,3-DPG) concentrations, higher ATP concentrations, and were more alkaline fragile than normal canine erythrocytes. 2. Reticulocytes from a PFK-deficient dog contained nearly three times the ATP concentration of normal canine erythrocytes, and had 2,3-DPG concentrations similar to normal canine erythrocytes. 3. PFK-deficient reticulocytes are not alkaline fragile. 4. The erythrocyte 2,3-DPG concentration in whole blood samples from PFK-deficient dogs was increased to normal by in vitro incubation with dihydroxyacetone, pyruvate and phosphate. This incubation resulted in only a slight increase in ATP concentration. 5. The alkaline fragility of these 2,3-DPG replenished PFK-deficient erythrocytes was normal. 6. Findings in this study indicate that the increased alkaline fragility of canine PFK-deficient erythrocytes is the result of decreased intracellular 2,3-DPG concentration.     

Pharmacological correction of microcirculation in rats suffering from traumatic brain injury

We investigated the effect of mexicor on functional indices of erythrocytes and the structure of myocardial microcirculation in rats suffering from traumatic brain injury (TBI). At 3, 7, and 12 days after TBI, we measured the concentration of 2,3-diphosphoglycerate (2,3-DPG) and the degree of erythrocyte aggregation and their electrophoretic mobility (EPME) in the blood of rats, as well as analyzing sections of the left ventricular myocardium. The first day after the TBI, we observed a decrease in EPME, an increase of erythrocyte aggregation, and an increase of 2,3-DFG concentration in erythrocytes as compared with intact animals. Intraperitoneal injection of mexicor led to an increase of EPME and 2,3-DPG level and reduced the aggregation of erythrocytes, which was most pronounced during the 3–7 days of the post-traumatic period. Improved functional parameters of erythrocytes were accompanied by the dynamics of regenerative processes in the heart. Intraperitoneal injection of mexicor restrained architectonic damage of microvasculature and cardiomyocytes ultrastructure of the left ventricular myocardium of the heart.     

Influence of whole-body gamma-irradiation upon rat erythrocyte: lipid peroxidation and osmotic fragility

The effects of whole-body gamma-irradiation of rats (8 Gy) on erythrocyte enzymes and biochemical components involved in lipid peroxidation were studied. Decreased superoxide dismutase and glutathione reductase activities, and lowered concentrations of reduced glutathione, were found to be the main factors responsible for the observed increase in lipid peroxidation in the erythrocytes of irradiated rats. This increased lipid peroxidation did not result in a greater tendency to hemolysis in hypotonic media; on the contrary, the mean osmotic fragility was decreased at days D + 1 and D + 3 after irradiation. The behavior of the erythrocyte populations towards hemolysis in hypotonic media appeared to be most homogeneous at days D + 4 and D + 8 after irradiation, which correspond to maxima of malonic dialdehyde concentrations in erythrocytes. Such a synchrony of variations suggests that crosslinking of primary amino groups of proteins or phospholipids by malonic dialdehyde might produce a rigidification in erythrocyte membranes, possibly leading to a more homogeneous behavior of the erythrocyte populations towards hemolysis in hypotonic media.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape

Na⁺ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na⁺ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the ³¹P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg²⁺/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

Recovery from radiation-induced decrease in cell membrane charge by added adenosine triphosphate and its modification by colchicine or cytochalasin B.

Cell electrophoretic mobility of rat erythrocyte decreased with time after 3000 R X-irradiation without spontaneous recovery. On addition of 10^?4M ATP to the irradiated cells, recovery was observed within 10 minutes. Washing out of ATP and subsequent incubation for 1 hr resulted in the return of mobility to the low level. Preincubation with 0.1 μg/ml colchicine for 15 minutes or 1 μg/ml cytochalasin B for 30 min completely blocked the reversible effect of ATP on electrophoretic mobility. These results suggest the existence of tubulin-like polymerizing protein in the cytoplasmic membrane and changes in its conformation induced both by X-irradiation and by added ATP.     

Human erythrocyte 2,3-diphosphoglycerate metabolism. Influence of 1,3-diphosphoglycerate and Pi. In vitro studies at low pH with computer simulations.

The kinetics of 2,3-diphosphoglycerate (2,3-DPG) net breakdown was examined in intact human erythrocytes incubated at pH 7.00 and 37 °C. The concentrations of 2,3-DPG, 1,3-diphosphoglycerate (1,3-DPG), 3-phosphoglycerate, ATP, P_i, glucose, and lactate were determined during 10 to 12 h. Since the concentration of 1,3-DPG has been suggested to be the main regulating factor with respect to the rate of 2,3-DPG net breakdown the interdependence between the concentration of 1,3-DPG and pH was determined in the range of pH 6.9 to 7.4. It was found that the stationary level of 1,3-DPG decreased strongly with decreasing pH within this range. Qualitatively, the net breakdown of 2,3-DPG observed at pH 7.00 can be explained by the lowered level of 1,3-DPG. The influence of the concentration of P_i upon the rate of net degradation of 2,3-DPG at pH 7.00 was studied at low cell volume fraction (0.04), where given concentrations of P_i could be maintained for several hours. A marked increase in the rate of 2,3-DPG net breakdown by P_i was demonstrated. Computer simulations showed that activation of diphosphoglycerate phosphatase by the increasing concentration of P_i and decrease of degree of inhibition of the diphosphoglycerate mutase by the decreasing concentration of 2,3-DPG may well keep the rate of the degradation balanced at the time constant value observed. On the basis of the observed kinetics and a computer simulation, the flux through the phosphoglycerate bypass was estimated to be 10 to 15% of the total glycolytic flux at physiological conditions.     

Glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes. Kinetic mechanism and competitive substrate inhibition by glyceraldehyde 3-phosphate

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was isolated from human erythrocyte ghosts by a simple procedure utilizing ammonium sulfate precipitation and affinity chromatography on NAD⁺-Sepharose 4B. The purified enzyme had a specific activity of 98 units/mg protein. The kinetic mechanism of GAPD was studied by product and deadend inhibition using NADH, α-glycerophosphate, nitrate, and 2,3-diphosphoglycerate. The results indicated that the human erythrocyte GAPD-catalyzed reaction follows an ordered ter bi mechanism characterized by the sequential addition of NAD⁺, glyceraldehyde 3-phosphate (GAP), and phosphate to the enzyme and the sequential release of 1,3-diphosphoglycerate and NADH from the enzyme. This contrasts with the mechanism (rapid equilibrium random ter bi) proposed by Oguchi (1970, J. Biochem. (Tokyo)68, 427–439) who based his conclusion on the initial rate data alone. Since the Michaelis-Menten kinetics were not applicable to this enzyme because of the competitive substrate inhibition by GAP, we devised a new kinetic approach for determining the parameters of the GAPD-catalyzed reaction. Results of this study indicate that the GAPD-catalyzed reaction is regulated by both ATP and GAP. We propose that GAP acts as an “amplifier” for the feedback inhibition effect of ATP. We discuss the effect this may have played in causing controversy over the regulatory role of this enzyme in glycolysis.     

A reassessment of the phosphoglycerate bypass enzymes in human erythrocytes.

Dissociation of the human erythrocyte into cytoplasmic and membranous components, shows that all of the cell's intrinsic 2,3-diphosphoglycerate phosphatase activity is associated with the soluble component. Further fractionaction of the cytoplasm on DEAE cellulose illustrates that both 1,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities occur coincidently within one peak. Thermal denaturation of the peak proteins at 60° results in a parallel loss in phosphatase and mutase activity. The identical phenomenon is observed in the presence of the 2,3-diphosphoglycerate phosphatase activator, 2-phosphoglycolate. Homogeneous 1,3-diphosphoglycerate mutase, which quantitatively accounts for all of the intrinsic 2,3-diphosphoglycerate phosphatase within the red cell, also exhibits thermal instability at 60°. These findings suggest that the phosphoglycerate bypass in erythrocytes is under the control of a single, bifunctional enzyme.     

Cyclic 2,3-diphosphoglycerate metabolism in Methanobacterium thermoautotrophicum (strain ΔH): characterization of the synthetase reaction

The levels of cyclic 2,3-diphosphoglycerate (cDPG) in methanogenic bacteria are governed by the antagonistic activities of cDPG synthetase and cDPG hydrolase. In this paper we focus on the synthetase from Methanobacterium thermoautotrophicum. The cytoplasmic 150 kDa enzyme catalyzed cDPG synthesis from 2,3-diphosphoglycerate (apparent K_m=21 mM), Mg²⁺ (K_m=3.1 mM) and ATP (K_m=1–2 mM). In batch-fed cultures, the enzyme was constitutively present (6–6.5 nmol per min per mg protein) during the different growth phases. In continuous cultures, activity decreased in response to phosphate limitation. The synthetase reaction proceeded with maximal rate at pH 6 and at 65° C and was specifically dependent on high (>0.3M) K⁺ concentrations. The reaction conditions remarkably contrasted to those of cDPG degradation catalyzed by the previously described membrane-bound cDPG hydrolase.Abbreviations cDPG Cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-Diphosphoglycerate - 2-PG 2-Phosphoglycerate - 3-PG 3-Phosphoglycerate     

Effects of training on erythrocyte 2,3-diphosphoglycerate in normal men

The erythrocyte 2,3-diphosphoglycerate concentration (2,3-DPG) and the activity of red cell hexokinase, pyruvate kinase, glucose-6 phosphate dehydrogenase and glutathione reductase were studied in 27 normal volunteers before and after 2 and 4 months of physical endurance training. The 4 months of training increased maximal oxygen uptake and physical working capacity (PWC130) by 16% (p less than 0.001) and 29% (p less than 0.001) respectively. Resting heart rate was decreased (p less than 0.001) by 11 beats.min-1. With 2 months of training the erythrocyte 2,3-DPG concentration increased by 9% (p less than 0.001); with 4 months training the increase was only 4% (p less than 0.05). The training-induced increase in red cell 2,3-DPG was not accompanied by enhanced activity of erythrocyte hexokinase, pyruvate kinase, glucose-6 phosphate dehydrogenase or glutathione reductase. It is concluded that the rise in red cell 2,3-DPG induced by physical endurance training is not due to activation of red cell glycolytic enzymes or the enzymes involved in the pentose-phosphate cycle.     

Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the V_max of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher V_max of the enzyme from M. fervidus in the synthesizing direction.     

Erythrocyte 2,3-diphosphoglycerate concentration before and after a marathon in men

Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration was studied in 23 runners before and after a marathon race. Blood samples were drawn from an antecubital vein the morning before the race (baseline), at 3 p.m. 2 h before the start, on finishing, and 12 and 36 h later. Compared to the baseline values, erythrocyte 2,3-DPG concentration was increased (p less than 0.001) immediately after the marathon from 4.62 +/- 0.14 to 5.56 +/- 0.13 mumol.ml-1 RBC and remained elevated 12 h later (5.45 +/- 0.14 mumol.ml-1 RBC): it returned to prerace values 36 h after completion of the marathon.     

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.     

Regulatory properties of human erythrocyte hexokinase during cell ageing

Human red blood cell hexokinase exists in multiple molecular forms with different isoelectric points but similar kinetic and regulatory properties. All three major isoenzymes (HK Ia, Ib, and Ic) are inhibited competitively with respect to Mg.ATP by glucose 6-phosphate (Ki = 15 microM), glucose 1,6-diphosphate (Ki - 22 microM), 2,3-diphosphoglycerate (Ki = 4 mM), ATP (Ki = 1.5 mM), and reduced glutathione (Ki = 3 mM). All these compounds are present in the human erythrocyte at concentrations able to modify the hexokinase reaction velocity. However, the oxygenation state of hemoglobin significantly modifies their free concentrations and the formation of the Mg complexes. The calculated rate of glucose phosphorylation, in the presence of the mentioned compounds, is practically identical to the measured rate of glucose utilization by intact erythrocytes (1.43 +/- 0.15 mumol h-1 ml red blood cells-1). Hexokinase in young red blood cells is fivefold higher when compared with the old ones, but the concentration of many inhibitors of the enzyme is also cell age-dependent. Glucose 6-phosphate, glucose 1,6-diphosphate, 2,3-diphosphoglycerate, ATP, and Mg all decay during cell ageing but at different rates. The free concentrations and the hemoglobin and Mg complexes of both ATP and 2,3-diphosphoglycerate with hemoglobin in the oxy and deoxy forms have been calculated. This information was utilized in the calculation of glucose phosphorylation rate during cell ageing. The results obtained agree with the measured glycolytic rates and suggest that the decay of hexokinase during cell ageing could play a critical role in the process of cell senescence and destruction.