Red Cell Glycolysis Research Articles

Accelerating effect of 2,4,6-trinitrophenol on the glycolytic rate of human red cells.

A number of instantaneous changes occurred when picrate was added to a suspension of human red cells in steady state with respect to glycolysis and ion distribution across the membrane at pH 7.40. The rate of glycolysis increased, without change in glycolytic quotient, to a new steady-state value, the effect reaching a maximum of 1.75 times the rate of the control at 0.5 mM picrate. Inorganic phosphate (P(i)) was released at a relatively constant rate, increasing with picrate concentration to 1.0 mmol P(i)/liter cells x h at 5-6 mM picrate. The steady- state concentrations of ATP and 1,3-diphosphoglycerate (1,3-DPG) decreased to new stable values within 15-45 min after the addition of picrate. The ATP level was affected only at picrate concentrations of 1 mM or more, and the level of ATP stabilized at 75 percent of the control values at 4 mM of picrate. In contrast, 1,3-DPG concentrations decreased to 40 percent of the control value of 0.5 mM picrate. Higher concentrations of picrate resulted in only a small additional decrease in the stationary concentration of 1,3-DGP. A net efflux of cellular potassium at constant rate took place. This net efflux was an almost linear function of picrate concentration in the range of 0.1-3 mM. At the latter concentration the net efflux amounted to about 2.7 meq/liter cells x h and a further increase in picrate concentration caused only a minor increase in the potassium efflux. Possible mechanisms for the effects of picrate on human red cell glycolysis are discussed.

Glycolysis of red cells suspended in solutions of impermeable solutes. Intracellular pH and glycolysis.

The glycolytic rate human red cells suspended in a sucrose medium of low or physiological pH was higher than that of the cells suspended in Ringer's medium of the same. pH. The medium pHP-glycolytic rate curve of red cells suspended in soucrose media shifted to the acidic side by about one unit compared with that of cells suspended in Ringer's medium. Similarly, the pattern of glycolytic intermediates in red cells suspended in a sucrose medium resembled that in cells suspended in Ringer's solution of about one unit higher pH. These phenomena could be ascribed to the change of intracellular pH, which was measured by the 5,5'-dimethyl-oxazolidine-2,4-dione method. A similar stimulation of glycolysis was observed when sodium citrate was added to red cells suspended in Ringer's solution at constant pH. These observations indicate that membrane-impermeable non-electrolytes or anions stimulate glycolysis of red cells by elevation ofthe intracellular pH. Red cell glycolysis is influenced mainly by the intracellular pH rather than by the pH of the suspending medium.

The effect of epinephrine on the in vivo concentration of erythrocyte glycolytic intermediates.

SummaryErythrocyte adenosine triphos-phate (ATP) concentration was found to increase in adult rhesus monkeys which have been injected with 3.5 mg of epinephrine. The average increase in red cell ATP concentration was 19.5% (significant at the 1% probability level). Epinephrine injection also increased the concentration of the early red cell glycolytic intermediates, glucose 6-phosphate (G6P), fructose 6-phos-phate (F6P), and dihydroxyacetone phosphate (DHAP). Respiratory alkalosis, which might have been induced by the epinephrine, is not responsible for the glycolytic changes. This effect of epinephrine on rhesus red cell glycolysis is compatible with an in vivo stimulation of red cell hexokinase by epinephrine or some substance elicited by epinephrine and suggests the possibility that such a stimulation may have a physiologic role in the mammalian erythrocyte.The authors wish to thank John A. Faulkner and Daniel H. Ringler for their help in the experiments involving the injection of monkeys with epinephr...

Some evidence for aldosterone action on 2,3-diphosphoglycerate level in human red cells

In healthy male subjects aldosterone excretion and plasma renin activity were reduced by a 4–6 hr head-out immersion in thermoindifferent water baths (35.5 ± 0.1°C). The red cell 2,3-diphosphoglycerate (DPG) concentration before and throughout immersion period was positively correlated both with aldosterone excretion in 2 hr pooled urine ( r = +0.69; 2 p < 0.001) and with renin activity ( r = +0.54; 2 p < 0.001) despite a concomitant increase of cubital venous pH and inorganic phosphate concentration. These findings furnish evidence for a regulatory role of aldosterone in DPG metabolism, possibly by a direct influence on red cell glycolysis.

The effect of red cell 2,3-DPG changes induced by diabetic ketoacidosis on parameters of the oxygen dissociation curve in man.

As early as in 19 39 Guest and Rapoport reported a striking decrease of the red cell 2,3-diphosphoglycerate (2,3-DPG) concentration in patients suffering from severe diabetic ketoacidosis (1). This observation has been amply confirmed in more recent years (2,3,4,5), when interest was resurrected to investigate the regulation of red cell 2,3-DPG after Benesch and Benesch (6) and Chanutin and Curnish (7) had shown the effect of 2,3-DPG on the oxygen affinity of hemoglobin. 2,3-DPG is formed as an intermediary product of red cell glycolysis; increase in hydrogen-ion concentration decreases red cell glycolysis mainly through inhibition of the phosphofructokinase step (8) and it has been widely accepted that the remarkable low erythrocyte 2,3-DPG levels in diabetic ketoacidosis — as little as 2o per cent of the normal concentration is observed — are due to this mechanism and an additional inhibition of di-phosphoglycerate mutase at lower pH (9).

Pyruvate kinase in malaria host-parasite interaction.

THE mammalian malaria host–parasite systems seem particularly useful for studies aimed at elucidating the biochemical mechanisms of such interactions. During the vertebrate phase of their life cycles the malaria organisms are intracellular parasites of the red cell. The mature mammalian host red cell is also relatively simple metabolically; its energy metabolism is solely that of Embden–Meyerhof glycolysis and it also lacks the capacity for protein synthesis. We therefore studied several aspects of red cell glycolysis in monkeys heavily infected with Plasmodium knowlesi and mice heavily infected with P. berghei. In both these systems we have found an increase in red blood cell adenosine triphosphate (ATP) and a decrease in red cell 2,3-diphosphoglycerate (DPG). We also have evidence that these malaria parasites introduce a pyruvate kinase isozyme into their host red cells in amounts sufficient to alter red cell glycolysis. First, glycolytic intermediate data demonstrate an in vivo increase in pyruvate kinase activity in infected red cells; second, there is an increase in pyruvate kinase Vmax activity in infected cells; and third, gel electrophoretic patterns show a new pyruvate kinase isozyme in infected cells. We suggest that this alteration in red cell glycolysis is in a direction favourable to the parasite because it will increase red cell ATP at the expense of red cell DPG.

The Binding of Phosphorylated Red Cell Metabolites to Human Hemoglobin A

Abstract The binding affinity to hemoglobin of 2,3-bisphosphoglycerate, ATP, and some other phosphorylated red cell metabolites has been measured at pH 7.2 in 0.12 m KCl to simulate physiological conditions. The dissociation constant with 2,3-bisphosphoglycerate is independent of hemoglobin concentration in the range 0.06 to 4 mm. There is one binding site per deoxyhemoglobin tetramer. Phosphorylated compounds bind weakly to one site per tetramer of oxyhemoglobin. Dissociation constants determined for 2,3-bisphosphoglycerate are 4.22 x 10-5 m at 22° and 1.03 x 10-4 m at 37° with deoxyhemoglobin and ∼2.5 x 10-3 m at 22° and 4.75 x 10-3 m at 37° with oxyhemoglobin. Dissociation constants for ATP are 8.54 x 10-5 m at 25° with deoxyhemoglobin and 2.55 x 10-3 m for oxyhemoglobin at 25°. Binding to ADP, 1,3-bisphosphoglycerate and glucose-1,6-P2 was measured. The dissociation constant for magnesium complexes of 2,3-bisphosphoglycerate to deoxyhemoglobin was too weak to measure (g 10-3 m at 37°); magnesium complexes of ATP have an apparent dissociation constant of 1.15 x 10-3 m at 25°. The effects of the differential binding of cellular intermediates on red cell glycolysis and 2,3-bisphosphoglycerate metabolism are discussed.

Glucose- and mannose-1,6-P 2 as activators of phosphofructokinase in red blood cells

Glucose-1,6-P 2 and mannose-1,6-P 2 are concluded to be important activators from “reconstruction” experiments showing that the other known effectors of phosphofructokinase poise it at ∼ 0.1% of its V max, compared with the in vivo rate of ∼ 1%. These activators may explain the relative insensitivity of red cell glycolysis to fructose-1,6-P 2. Glucose-1,6-P 2 is elevated more than two-fold in pyruvate kinase deficient cells but not in cells from patients with alkalosis although both have increased levels of the inhibitor, glycerate-2,3-P 2.

RED CELL TRIOKINASE DEFICIENCY - ANOTHER BIOCHEMICAL CHARACTERISTIC OF THE FETAL ERYTHROCYTE

The erythrocytes of normal adults have the capacity to incorporate and metabolize dihydroxyacetone (DHA). This substrate has been found to be useful for the maintenance of 2, 3-diphosphoglycerate (DPG) in stored red cells. The metabolism of DHA by cord blood erythrocytes was examined in an attempt to find a substrate for DPG synthesis that bypassed the early steps in red cell glycolysis. When the cells from adults and infants were incubated in the presence of DHA (10 mM), pyruvate (10 mM), and inorganic phosphate (10 mM), the DPG level increased by 2.34 μmoles/ml RBC's in the adult samples but only increased by 0.1 μmoles/ ml in the cells of infants during a period of 2 hours. DHA consumption averaged 3.76 μmoles/ml RBC's/hr in the cells from adults but was only 1.16 μmoles/ml RBC's/hr in the erythrocytes of the newborn. Assays of triokinase, the enzyme responsible for the phosphorylation of DHA, were performed in the hemolysates from newborn and adult erythrocytes. Triokinase activity averaged 4.56 ± 0.69 units/100 ml RBC's in adults and was only 2.97 ± 0.54 units in the cells from newborns (p <.01). Relative triokinase deficiency appears to be another biochemical characteristic of the red cells of the newborn infant.

A NEW VARIANT OF GLUCOSEPHOSPHATE ISOMERASE (GPI) DEFICIENCY WITH CONGENITAL HEMOLYTIC ANEMIA

A new variant of red cell GPI deficiency (Type Nordhorn) with congenital nonspherocytic hemolytic anemia is described. The propositus suffers since birth from a severe anemia. GPI activity is decreased to 22% of the normal. The parents exhibit activities between 36 and 47 %. The thermal stability of the mutant enzyme is decreased in the propositus and in all affected maternal relatives. The propositus is not homozygous, but double-heterozygous for two abnormal alleles. The heterozygous mother contributes an allele which produces a thermolabile enzyme of decreased activity and abnormal electrophoretic mobility, whereas the father contributes an inactive gene product. The enzyme defect becomes also manifest in the leucocytes of the propositus. The thermolability is also evident in the leucolysates of the mother. Compared with erythrocyte populations of similar mean cell age the rate of red cell glycolysis is reduced in the propositus. A premature inactivation of the enzyme during maturation of the erythrocytes is suggested.

Hemoglobin function in stored blood. 8. Further effects of phosphate on red cell ATP and 2,3-DPG.

Abstract : Inorganic phosphate which is known to stimulate red cell glycolysis is present in one of the preservatives for storing whole blood, citrate-phosphate-dextrose (CPD), but not the other, acid-citrate-dextrose (ACD). Both of these preservatives for liquid storage were developed before 2,3-diphosphoglycerate (2,3-DPG) was found to be necessary for normal hemoglobin function. In a recent study we have shown that very high concentrations of phosphate (10, 15, and 20 mM) were deleterious for maintaining 2,3-DPG. In the present study a lower range of phosphate concentrations (2, 4, 6, and 9 mM) was studied for maintenance of 2,3-DPG and ATP during storage under blood banking conditions. The lowest concentration, 2 mM, which corresponds to CPD was found to be the best concentration for maintaining 2,3-DPG and thus hemoglobin function. Four mM phosphate were not quite as good but better than no phosphate. Six and 8 mM phosphate were considerably worse. (Author)

Pathogenesis of leukemia.

Pathogenesis of leukemia.

The selective inhibitory effect of hyperthermia on the metabolism and growth of malignant cells.

Hyperthermia (42° C.) exerted an inhibitory effect on the O(2) uptake of rabbit VX2 carcinoma cells in vitro, and led to a decrease in viability and growth potential of the cells, as measured by their ability to produce tumours in rabbits. Anaerobic glycolysis of the tumour cells was unaltered by hyperthermia. Respiration and anaerobic glycolysis of normal rabbit liver, kidney and red blood cells were unaffected by the elevated temperature. Local heat was applied to established VX2 tumours in vivo, with a subsequent reduction in tumour size in all cases, the most effective therapy regime being 3 one-hour applications of heat within the mean cell generation time of the tumour. Following heating there was rapid and widespread tumour cell necrosis and lysis, with subsequent replacement of the tumour architecture by connective tissue. There was a prolongation of survival time in 50% of the treated animals, which are still alive 18 months after therapy; all the control animals died within 10 weeks. The selective inhibitory effects of hyperthermia on cancer cells, and its application to human neoplasms, are discussed.

HEMATOLOGY: Irreversible oxidant injury in the erythrocytes of the newborn infant

It is generally recognized that the red cells of the newborn are more susceptible to injury, i.e. the appearance of Heinz bodies and glutathione instability, upon exposure to oxidant compounds. A more complete examination of the extent and nature of this injury was made. RBC's were incubated for 2 hours with and without glucose in the presence of acetylphenhydrazine (APH) or menadione (K3) and then reincubated in glucose media. Red cell hexokinase, phosphofructokinase, and glyceraldehyde-3-phosphate dehydrogenase activity, all SH containing enzymes, were irreversibly lost in the cells of the newborn when incubated in APH or K3 with no glucose in the medium. The cell's ability to consume glucose was reduced from 50 to 95%. The red cells of adults showed som fall in enzyme activity during drug exposure but full acitivity was restored and red cell glycolysis was unimpaired when reincubated in glucose. Incubation of the cells of the newborn in a carbon monoxide atmosphere during drug exposure prevented their adverse effects. In addition, in the presence of glucose, K3 increased the rate of fatty acid incorporation into adult cells and depressed the rate in newborn cells. Oxidant drugs apparently through alterations in the cells of the newborn and their use in the neonatal period appears contraindicated.

Reduced red cell glycolysis, 2, 3-diphosphoglycerate and adenosine triphosphate concentration, and increased hemoglobin-oxygen affinity caused by hypophosphatemia.

Abstract A marked reduction in red cell glucose utilization, lactate production, and 2,3-diphosphoglycerate and adenosine triphosphate concentration occurred in a patient with intractable diarrhea ...

Ergänzende versuche zur wirkung von menschlichem wachstumshormon auf die glykolyse menschlicher erythrocyten in vitro und aue die aktivität der phosphofruktokinase

We performed further studies of the influences on glycolysis in human red cells by purified human growth hormone by the method of Roos. We found that the observed inhibition of glucose consumption, decreased phosphofructokinase activity, low lactate production, decreasing effect on ATP concentration, and the relationship of pyruvate kinase, lactate dehydrogenase and pyruvate concentrations similar to the results obtained by using human growth hormone purified by the method of Raben. In both cases, the human growth hormone Raben and Roos we used have shown a noncompetitive inhibition of yeast phosphofructokinase.

A low Km phosphoenolpyruvate mutant in the Amish with red cell pyruvate kinase deficiency.

Summary. The biochemical heterogeneity of red cell pyruvate kinase (PK)‐deficiency was demonstrated in a study of six patients with this abnormality. Three siblings of an Amish kindred were found to possess a mutant enzyme with a decreased Km phosphoenolpyruvate. In this kindred the Km PEP ranged from 0.018 to 0.024 mM (normal 0.055–0.067 mm). Two deficient patients were found to have an enzyme with a normal Km PEP, while in the sixth patient the Km PEP was increased to 0.120 mM.In all patients the red cells possessed increased quantities of the glycolytic intermediates proximal to the PK step, reduced levels of ATP and reduced glycolysis for cell age.When reconstituted haemolysates from these patients were supplemented with PK, a correction of the glycolytic rate was observed. No acceleration of glycolysis occurred in PK‐supplemented normal or reticulocyte‐rich haemolysates.These studies re‐emphasize the heterogeneity of PK deficiency and demonstrate that this deficiency alone can result in an impairment in red cell glycolysis.

Oxygen-hemoglobulin dissociation curves: effect of inherited enzyme defects of the red cell.

The blood of a patient with a deficiency of hexokinase in the red cells and a decreased concentration of 2, 3-diphosphoglycerate in the red cells showed an increased affinity for oxygen, whereas a patient with a deficiency of pyruvate kinase and an elevated concentration of 2, 3-diphosphoglycerate in the red cells had blood with a decreased affinity for oxygen. Defects in red cell glycolysis may alter the oxygen affinity of blood by virtue of their effect on 2, 3-diphosphoglycerate concentrations in red cells.

Der einfluss von menschlichem wachstumshormon auf die glykolyse im menschlichen erythrocyten in vitro

The influence of human growth hormone on glucose consumption, and some enzymes and metabolites of glycolysis of human red cells were studied in vitro. It has been shown that human growth hormone caused: 1. 1. about 54% inhibition of the glucose consumption; 2. 2. correspondingly, diminished lactic acid production; 3. 3. accumulation of glucose-6-phosphate and fructose-6-phosphate due to inhibition of phosphofructokinase; 4. 4. using the glycolysis-activating effect of phosphate buffer, adenosine triphosphate production was depressed, but using the Krebs-Ringer-bicarbonate buffer, the diminution was lower than in the controls, without addition of the growth hormone; 5. 5. increased pyruvate production with less decreased pyruvate kinase and less increased lactic dehydrogenase activity. Possible connections in the course of the glycolytic process in human red cells are discussed.

Metabolism of 2,3-diphosphoglycerate and glycolysis in human red blood cells under the influence of dipyridamole and inorganic sulfur compounds

Metabolism of 2,3-diphosphoglycerate and glycolysis in human red blood cells under the influence of dipyridamole and inorganic sulfur compounds