Red Cell Glycolysis Research Articles

Les anomalies de la glycolyse au cours de l'anémie hémolytique par déficit du globule rouge en pyruvate kinase

In two cases of hemolytic anemia with pyruvate kinase deficiency, we have studied the modifications of glycolysis in red blood cells and the kinetic behaviour of pyruvate kinase. Red cell glycolysis was markedly diminished. This alteration of the metabolic capacity coincides with an accumulation of phosphoenol pyruvate and phospho-glycerates and with a low level of hexose phosphates and triose phosphates. ATP content is very much decreased (35 % of normal values) ; 2,3-diphosphoglycerate is slightly increased. The level of pyridine nucleotides was found to be low, with predominance of reduced forms. These modifications are interpreted in relation to pyruvate kinase deficiency. The kinetic studies of the purified enzyme revealed two forms of pyruvate kinase in normal erythrocytes as well as in pyruvate kinase-deficient cells : — pyruvate kinase-B, with hyperbolic kinetics and K m app. (phosphoenol-pyruvate) = 3.6· 10 −5 M, insensitive to fructose diphosphate. — pyruvate kinase-A, with allosteric kinetics and K m app. (phosphoenol-pyruvate) = 19.3· 10 −5 M. The allosteric form is activated by fructose diphosphate at very low concentrations ( K m app. (fructose diphosphate) = 43·10 −9 M). In activated A-form, K m (phosphoenolpyruvate) is lowered at the same value as the B-form (= 2.8 · 10 −5 M). ADP is inhibitor at concentrations above 30 · 10 −5 M, and this action is overcome by fructose diphosphate. ATP inhibits at physiological concentrations. Pyruvate kinase-A and pyruvate kinase-B have identical V max and are interconvertible. The transition factors are discussed. In this study, no differences appeared in the molecular structure of pyruvate kinase in normal and deficient cells.

Glycolysis and glycolytic enzyme activity of aging red cells in man. Changes in hexokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and glutamic-oxalacetic transaminase.

SUMMARYHexokinase, aldolase, glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase and glutamic‐oxalacetic transaminase activities, and the rate of glycolysis have been measured in red cells from seven adult males responding to repeated phlebotomy or erythropheresis with iron replacement. The change in median age of the red cell populations of these men was estimated from the measured rates of red cell loss and erythropoietic replacement. These enzyme activities and glycolytic rates were measured also in a patient with aplastic anaemia in whom transfused red cells were aging at a known rate.Hexokinase, aldolase and transaminase activities in the youngest red cell populations were four times greater than the corresponding levels in normal aged cell populations. Pyruvate kinase activity was two or three times higher than normal, but glyceraldehyde‐3‐phosphate dehydrogenase activity was not significantly increased in the youngest cell populations. The rate of glycolysis in intact cells and of lactate formation from glucose or glucose‐6‐phosphate in coenzyme‐supplemented haemolysate showed a two‐fold increase above normal in the youngest cell populations. Subnormal enzyme activity in older cells was observed only for transaminase.The deterioration of hexokinase and aldolase activity with red cell aging is sufficiently rapid and the activity of these enzymes is normally sufficiently low to indicate a severe glycolytic limitation in old red cells. Such limitation in glycolysis may impair red cell viability and be the determining factor in normal red cell life span.Measurement of activity of hexokinase, aldolase, or glutamic‐oxalacetic transaminase in an unknown red cell population should indicate the median age of the cell population much sooner than such information can be obtained by measurement of red cell survival.

Glycogen concentration in working and nonworking ventricles of isolated dog hearts.

Glycogen utilization in working and nonworking ventricles was studied at high (over 70 mg/100 ml) and low (27 to 61 mg/100 ml) arterial glucose concentrations and after insulin or epinephrine addition in 16 isolated ventricle preparations of dog hearts. Coronary perfusion and hemodynamic determinants of right ventricular work were controlled, and the left ventricle was kept unloaded. Time courses of change in ventricular glycogen concentration were determined during monitoring of heart rate, workload, arterial oxygen saturation, and coronary perfusion pressure. Epicardial samples for glycogen analysis were taken from each ventricle, and glucose uptake from circulating blood was determined. Glycogen loss was greater in working right than in nonworking left ventricles. In spontaneously fibrillating hearts, this difference was not observed, and there was greater glycogenolysis than during coordinated contraction. Insulin administration early in experiments led to equivalent glycogen loss in working right and nonworking left ventricles. There was glycogen preservation in both ventricles of fibrillating hearts. Epinephrine augmented glycogen loss in fibrillating hearts; depletion was never complete. Myocardial glucose uptake, corrected for red cell glycolysis, was proportional to initial arterial glucose concentration.

The inhibition of anaerobic glycolysis in red cells by leukaemic leucocytes.

SUMMARYRates of anaerobic glycolysis were determined simultaneously in the red cells and leucocytes of patients with malignant haematologic disorders. The disappearance of glucose and the evolution of CO2 from bicarbonate were measured in the same Warburg flasks over the same period of time.In four patients with lymphogenous leukaemia and in one with acute leukaemia of disputed type but probably myelogenous, the leucocytes suppressed the glycolytic activity of the red cells. This abnormal finding appeared with exacerbation of the disease and disappeared with remission. It was present during the terminal phase in four patients, but could not be attributed to the presence of blast cells in the peripheral blood.The leukaeinic leucocytes exerted the same inhibitory effect on normal donor cells as on those of the patient. The plasma and the supernatant fluid from strongly inhibited reactions were without effect, as were aqueous and hypertonic extracts of the leucocytes. In all Warburg reactions the rate was constant, with no evidence of a cumulative effect. The inhibition was reversed by removal of the leucocytes.The characteristics of this phenomenon resemble molecular behaviour and suggest an equilibrium of some kind. Its kinetic properties closely resenible those of stoichiometric inhibition of an enzyme system. It is conjectured that an intercellular stress, perhaps physicochemical in nature, may have been exerted between the leucocytes and red cells, temporarily interfering with some event in thc glycolytic cycle of the latter.

Pyruvate kinase deficiency and related disorders of red cell glycolysis

Pyruvate kinase deficiency and related disorders of red cell glycolysis

FACTITIAL HYPERKALEMIA DUE TO ICING BEFORE ANALYSIS

Both adult and newborn blood plasma potassium concentration rose significantly when whole blood was kept on ice for periods of 5 hours. This appears to be due to inhibition of red cell glycolysis. The rise in plasma potassium concentration of blood from newborn infants is significantly greater than that occurring in blood from adults; this difference is a function of the neonate's increased red cell volume. Meaningful interpretation of potassium concentration, particularly in the newly born, demands that the plasma be separated immediately. The plasma potassium concentration of normal newborns at birth does not differ significantly from adults; in this study the mean value in 20 infants was 4.81 mEq/l.

Effect of deoxygenation of intracellular hemoglobin on red cell glycolysis.

Effect of deoxygenation of intracellular hemoglobin on red cell glycolysis.

862. Copper poisoning in calves: Todd, J. R. & Thompson, R. H. (1965). Studies on chronic copper poisoning. IV: Biochemistry of the toxic syndrome in the calf. Br. vet. J.121, 90

Red cell glycolysis, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and methaemoglobin reductase activities, reduced glutathione and the production of methaemoglobin under oxidative stress have been determined in 6 patients with burns ranging from 12 to 50 per cent. None developed methaemoglobinaemia. Periods of increased glycolysis, decreased response to ascorbate stimulation, lowered activity of glucose-6-phosphate dehydrogenase and increased activity of methaemoglobin reductase were found in 5 out of 6 patients. Additionally, reduced glutathione was low.Timing and magnitude of these findings suggest that individuals whose pre-injury glycolytic enzyme activities are in the upper normal range develop only a subclinical state. Oxidative stresses do not lead to acute haemolysis. The theoretical significance of the findings is discussed.

The influence of ammonium ions on glycolysis in red cells

The effect of different ions on red cell metabolism has been examined. Ammonium ions have been found to have a specific effect on the hexose monophosphate shunt. This effect has been observed by adding ammonium chloride and ammonium hydroxide to the cells. The stimulative effect of ammonium on the pentose phosphate pathway is an independent phenomenon, not related to increased alkalinity of the medium. Increasing alkalinity, by addition of sodium hydroxide and potassium hydroxide stimulates mainly the anaerobic pathway of glycolysis. The observed effects were found to be not due to changes in ionic strength or in osmolarity of the media.

Small volume perfusion system of the isolated rat liver

A perfusion system utilizing 25 ml of heparinized whole blood as a perfusate has been developed to study the metabolism of the isolated rat liver. Changes in the composition of the plasma of the perfusate were studied by circulation of the whole blood for 3 hours in the presence and absence of the liver. The discharge of glucose by the liver is marked by a pronounced initial hyperglycemia, which is countered slightly by red cell glycolysis. Despite continuous carbohydrate metabolism, a fall in sodium and a rise in potassium concentration occurs, ascribable chiefly to alteration of red blood cells. The hepatic glycogen content decreased slightly during perfusion. Urea production and bile flow were well preserved. Note: (With the Technical Assistance of Donna Cary and Veronica Mena) Submitted on June 26, 1959

The separation of cation exchange and glycolysis in human red cells exposed to non-ionizing radiations.

The separation of cation exchange and glycolysis in human red cells exposed to non-ionizing radiations.

Abnormalities of Carbohydrate Metabolism of Red Cells in Hereditary Spherocytosis

By a technique for investigating glycolysis of red cells, further insight was gained into the nature of the disturbances in metabolism associated with hereditary spherocytosis. A defect in intracellular glycolysis was found in cells from patients with hereditary spherocytosis. The abnormalities persist after splenectomy. The abnormalities in carbohydrate metabolism are correlated with the clinical features of the disease. The susceptibility of red cells to hemolysis may be associated with an inadequate supply or utilization of energy within the cells. Spherocytes possessing this abnormality when trapped within the spleen may be faced with relative starvation, making them more susceptible to hemolysis. When the spleen is removed, such cells may survive normally because of more ready access to the plasma constituents, despite the continued presence of the metabolic defects in glycolysis. Thus the beneficial effects of splenectomy in hereditary spherocytosis may be more understandable.

STUDIES ON THE PRESERVATION OF BLOOD: III. REVERSIBLE INHIBITION OF GLYCOLYSIS IN BLOOD BY OXALATE

Oxalate was found to inhibit glycolysis in red blood cells during storage at 5 °C. The inhibition of glycolysis in blood cells by oxalate is reversible. The glycolytic activity of the oxalated cells can be restored by washing the cells, or by precipitating the oxalate with an equivalent concentration of added calcium ions. However, with increased duration of storage prior to removal of oxalate the rate of glycolysis in the erythrocytes was found to be decreased on removal of the oxalate.

STUDIES ON THE PRESERVATION OF BLOOD: III. REVERSIBLE INHIBITION OF GLYCOLYSIS IN BLOOD BY OXALATE

Oxalate was found to inhibit glycolysis in red blood cells during storage at 5 °C. The inhibition of glycolysis in blood cells by oxalate is reversible. The glycolytic activity of the oxalated cells can be restored by washing the cells, or by precipitating the oxalate with an equivalent concentration of added calcium ions. However, with increased duration of storage prior to removal of oxalate the rate of glycolysis in the erythrocytes was found to be decreased on removal of the oxalate.

Rates of glycolysis in human red cells in relation to energy requirements for cation transport.

THE extrusion of sodium and accompanying potassium accumulation by red cells against electrochemical gradients is linked to the energy of cell glycolysis1,2. Active cation transport ceases at low temperatures, in the presence of iodoacetate or fluoride, and in the absence of glucose. Per contra, the process is unrelated to red-cell respiration, since transport is not influenced by cyanide, azide, carbon monoxide, methylene blue, pyruvate or malonate.

GLYCOLYSIS AND PHOSPHATE FRACTIONS OF RED BLOOD CELLS

GLYCOLYSIS AND PHOSPHATE FRACTIONS OF RED BLOOD CELLS

The In Vitro Metabolism of Plasmodium Gallinaceum

Within the past few years the metabolism of malarial parasites has been studied in several laboratories. Christophers and Fulton' measured the respiration of Plasmodium knowlesi and Maier and Coggeshall2 have demonstrated the utilization of glucose and other substrates during respiration of the same organism. The latter workers also found that the rate of respiration was a function of the size of the parasite, i.e., it increased with the development of the parasite from merozoite to segmenter. Wendel,3 also working with P. knowlesi, has demonstrated that both the respiration and glycolysis of infected red blood cells are greater than those of unparasitized erythrocytes.4 Velick5 has reported that the respiratory quotient of P. cathemerium increases when nuclear division of the parasite occurs. In all these experiments the intact red cells containing the parasite were studied. However, Christophers and Fulton' have obtained P. knowlesi free from red cells by saponin cytolysis and report that such organisms also respire in the presence of glucose. The present report deals with the glucose metabolism of P. gallinaceum. This organism exhibits little synchronism, but has particular advantages in that its natural host, the chicken, is readily available and infections can be obtained which yield large amounts of parasitic material. The study of this organism is made difficult by its presence within the nucleated red blood cell of the host. It would be desirable to lyse the cell and then to study the parasite suspended in a medium of known composition. In view of our lack of any convenient way of doing this, we have been forced to study the organism together with the red cell, hoping by the use of proper controls to arrive at a picture of the activities of the organism itself. A large portion of our experiments, therefore, are concerned with parasitized red blood cells suspended in an inorganic medium containing glucose.