Glycolysis In Human Erythrocytes Research Articles

Regulation of human erythrocyte metabolism by insulin: Cellular distribution of 6-phosphofructo-1-kinase and its implication for red blood cell function

Human erythrocytes are highly specialized cells whose function is oxygen transport. These cells’ sole metabolic source of energy is the fermentation of glucose via glycolysis. They contain an active insulin receptor and respond to insulin by increasing phosphorylation of tyrosine residues in several proteins. However, no metabolic effects have yet been associated with activation of this receptor in human erythrocytes. Here, we show that insulin increases the rate of glycolysis in human erythrocytes. Lactate production increased 56 and 173% in the presence of 10 and 100 nM insulin, respectively. A higher insulin concentration (1000 nM) partially reversed the stimulation of glycolysis. These effects occur through activation of the key glycolytic enzyme 6-phosphofructo-1-kinase, which exhibits the same pattern of modulation by insulin as seen for glycolytic flux. This modulation also occurs physiologically since ex vivo experiments revealed 50% stimulation of 6-phosphofructo-1-kinase (PFK) activity following a high carbohydrate meal. Insulin increases phosphorylation of PFK and redistributes the enzyme in red blood cells, causing it to detach from the erythrocyte membrane: upon insulin stimulation, the amount of enzyme associated with the plasma decreases by 86%. Detachment is a common mechanism of enzyme activation. As a consequence, insulin prevents up to 68% of red cells hemolysis. These results show that insulin regulates erythrocyte glycolysis and viability and suggest that this regulation is associated to other erythrocyte functions such as oxygen transport. Finally, we suggest that this regulatory mechanism might be compromised in patients with diabetes mellitus.

Human red blood cells targeted metabolome analysis of glycolysis cycle metabolites by capillary electrophoresis using an indirect photometric detection method

A capillary electrophoretic (CE) method with UV indirect spectrophotometric detection for determination of the main compounds of glycolysis in human erythrocytes has been elaborated. Blood samples for electrophoretic analysis were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration with a centrifuge equipped with filter devices: M r cut off 5000. Using 20 mM 2,6-pyridinedicarboxylic acid (PDC) as a highly UV absorbing carrier electrolyte and 4 mM cetyltrimetylammonium bromide (CTAB), at the resulting pH 12.3, nine of the glycolysis intermediates were separated and characterized. The repeatability and linearity of the method was assessed with percent relative standard deviation (%R.S.D.) for migration time ranging from 0.3 to 1.9% and correlation coefficients of 0.991–0.999 for the studied concentration range. Limits of detection (LOD) for the analyzed metabolites were in the range of 6.25 × 10 −6 to 5.0 × 10 −5 M. The optimized CE method was used to compare metabolome content of red blood cells of 22 healthy volunteers. Mean metabolite concentrations in erythrocytes ranged from 49.6 μM for fructose-6-phosphate (F-6-P) to 3.1 mM for 2,3-diphospho- d-glyceric acid (2,3-DPG). The method can be readily applied in clinical, pathophysiological and epidemiological studies.

Lactoferrin-protector against oxidative stress and regulator of glycolysis in human erythrocytes.

Binding of lactoferrin (Lf) to its membrane receptors requires an electron for the reduction of Fe(3+)LF to Fe(2+)LF. It is possible that glyceraldehyde -3-phosphate dehydrogenase, a glycolytic enzyme part of the erythrocyte membrane, delivers that electron. Then Lf, obtaining an electron from the coenzyme NADH, might stimulate glycolysis, which requires the oxidised state of the coenzyme NAD+. Such possibility is supported by the finding that another extracellular e- acceptor--potassium ferricyanide activates glycolysis by the similar mechanism. Present results show that ferricyanide inhibited the specific 59Fe-lactoferrin binding to its erythrocyte membrane receptors. It may be assumed that ferricyanide competes with lactoferrin for an electron which leads to decrease of the binding of 59Fe-lactoferrin to its receptors. Lactoferrin (50 and 100 nM), similar to ferricyanide, increased the accumulation of lactate (respectively by 25% and 30%). These results support the assumption that ferricyanide and lactoferrin are final acceptors of a common electron transport chain connected with the regulation of glycolysis. We established an antioxidative effect of lactoferrin on erythrocytes, which was expressed as: a) an influence on content and on activity of intracellular antioxidants--namely an enhancement of the content of reduced glutathione; b) a decreased content both of products of lipid peroxidation (thiobarbituric acid reactive substances) and hemolysis under normal conditions and oxidative stress. Lactoferrin is capable to bind metal ions and thus to block their catalytic participation in the oxidative disturbances of the membrane. In most of our experiments there were no metal ions in the incubation mixtures (except those stimulating oxidative stress). Our results showed that Lf limited both the generation of thiobarbituric acid reactive substances and hemolysis in the absence of metal ions in the media, as well as in their presence. These facts suggest that probably the antioxidative property of lactoferrin is glycolysis stimulation, leading to increased formation of ATP, which is necessary to maintain the ion gradient, membrane potential and morphology of the erythrocyte.

Analysis of metabolites of glucose pathways in human erythrocytes by analytical isotachophoresis

The aim of this study was to establish an isotachophoretic (ITP) method for the determination of the main compounds of glycolysis in human erythrocytes in order to analyze the influence of different glucose concentrations (mimicking the situation in diabetes mellitus) on this pathway. Samples for ITP were prepared by isolation of erythrocytes, lysis of the cells by heating in double-distilled water and subsequent ultrafiltration ( M r cut-off: 5000). All the main compounds of glycolysis were characterized by ITP. The influence of different glucose concentrations on the main compounds of the energy metabolism (ATP, ADP, lactate, pyruvate) and 2,3-diphosphoglycerate were analyzed in short- and long-time incubations.

4-Hydroxy-2-Nonenal Hardly Affects Glycolysis

4-Hydroxy-2-nonenal (HNE), one of the major products of lipid peroxidation, inactivated the rate-limiting enzymes (from animal sources) of the glycolytic pathway and the pentose phosphate pathway when incubated at 37°C for 1 h in the absence of glutathione (GSH). The HNE concentration for half-maximal inactivation of 6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase was 3–10 μM; and that value for pyruvate kinase, glucose-6-phosphate dehydrogenase, and hexokinases I and II was 0.15-0.6 mM. In the presence of 5 mM GSH, however, only PFK, irrespective of the source (muscle, liver, or erythrocyte), was inactivated by 40–-50% when incubated with 0.1 mM HNE for 1 h. Even PFK was not inactivated in the presence of both GSH and its substrate, ATP (2 mM). Glycolysis in human erythrocytes was not affected by treatment of cells with 0.1 mM HNE at 37°C for 30 min. The results suggest that HNE, at concentrations observable under physiological and pathological conditions, hardly affects glycolysis in cells.

Separation and Analysis of 4′-Epimeric UDP-Sugars, Nucleotides, and Sugar Phosphates by Anion-Exchange High-Performance Liquid Chromatography with Conductimetric Detection

An anion-exchange HPLC method coupled with conductimetric detection was developed for the analysis of UDP-sugars, nucleotides, and sugar phosphates, each in a single chromatographic run. The analysis was applicable to concentrations over 50 pmol. The utility of this technique was demonstrated by the measurement of UDP-sugar 4′-epimerase activity of cell-free extracts from wild-type and mutant Neisseria meningitidis serogroup B strains, Additionally, the method has been applied to the analysis of the intermediates of glycolysis in human erythrocytes.

Control of metabolism by dynamic macromolecular interactions.

This chapter discusses the control of metabolism by dynamic macromolecular interactions. Metabolic pathways are controlled and directed by pacemaker, bottleneck, and key enzymes. In general, no single enzyme is responsible for the control of a whole metabolic pathway. In pursuing the pacemaker theory, attempts are made to quantify metabolic regulation, and one studies each enzyme in a sequence separately in situ , determines its kinetic properties in the greatest possible detail and accuracy, and then seeks to determine how it works when it is in the intact cell. In prokaryotes and in eukaryotes the largest macrocompartment is the cytoplasm, containing quantities of soluble enzymes and is full of membranes associated with a great variety of organelles. A theoretical analysis of glycolysis in human erythrocytes has been provided, implicitly assuming that it proceeds in a homogeneous bulk solution without any interaction among the participating enzymes.

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. A mathematical model of glycolysis in normal and pyruvate-kinase-deficient red blood cells

A mathematical model of glycolysis in human erythrocytes is proposed to study the influence of a pyruvate kinase deficiency on the energy metabolism. The model takes into account the main regulatory properties of the non-equilibrium enzymes and the magnesium-complex formation by the adenine nucleotides and by 2,3-bisphosphoglycerate. In the normal case (no enzyme defect) the calculated flux rates and metabolite concentrations are in a good agreement with experimental data. It is shown that a severe pyruvate kinase deficiency manifested in a tenfold diminished activity of that enzyme leads to a remarkable decrease of the glycolytic flux and the ATP concentration of about 50% of the normal values. On the other hand a lowering of the pyruvate kinase activity to half of the normal value, characteristic for the heterozygotes, gives no significant alterations of the metabolite concentrations and the flux rates compared with the normal case which is in accordance with the lack of clinical symptoms for a metabolic disease of these probands. For three patients with known alterations of their pyruvate kinase mutants the calculated metabolite concentrations and the control characteristics permit estimation of the degree of disorder of the glycolytic pathway. The resulting classification corresponds well to other independent experimental and clinical findings. In particular, the calculation demonstrates that there is no simple correlation between the lowered enzyme activity and the reduced flux rate through the affected pathway.

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [ 14C]pyruvate to haemoglobin and the rate of efflux of [ 14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent K m and V max for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.

The Regulation of Glycolysis in Human Erythrocytes

According to the mathematical model, the dependence of the rate of glycolysis on the ATP concentration can be represented by the bell‐shaped curve with the descending part at the concentration of ATP close to physiological values. The existence of the descending part is a result of the strong inhibition of phosphofructokinase by ATP and hexokinase by glucose 6‐phosphate and provides stabilisation of the intracellular concentration of ATP. Using arsenate as an uncoupler of the oxidation of glucose and ATP synthesis, the dependence of the rate of glycolysis on the ATP concentration in erythrocytes was measured. This dependence (glycolysis characteristic) is represented by a bell‐shaped curve. The normalized glycolysis characteristics are the same for all the donors investigated. The increase of permeability of the erythrocyte membrane to monovalent cations by the polyene antibiotic levorin leads to an increase of glycolytic rate in erythrocytes (40–70%) and to a decrease in the ATP concentration (15%) and glucose 6‐phosphate (25–60%). The data obtained with levorin, and also other results on Na, K‐ATPase activation are in a good agreement with the characteristic obtained using arsenate. In all cases, inhibition of Na+, K+‐ATPase of erythrocytes by ouabain leads to the decrease of the rate of lactate production (10–20%) and increase of the concentration of glucose 6‐phosphate (14–40%). The ATP concentrations remains almost unchanged or is only slightly increased. The rate of glucose utilisation in erythrocytes from different donors show two types of behaviour. In the first type of erythrocyte it drops proportionally to the decrease in the lactate production rate; in the second type of erythrocyte it remains unchanged. The experimental results presented are in qualitative agreement with the predictions of the mathematical model.

Relationships among purine nucleoside metabolism, adenosine triphosphate catabolism, and glycolysis in human erythrocytes.

In human erythrocytes incubated with both naturally occurring purine nucleosides and with a variety of purine nucleoside analogs, ATP catabolism was accelerated and lactate accumulation was increased. Tubercidin was a particularly potent inducer of ATP catabolism. In cells incubated with tubercidin, the major route of adenylate metabolism was deamination, whereas in cells incubated with deoxyglucose, the major pathway was dephosphorylation.

Hemolysis by Detergents and their Effects on Glycolysis in Human Erythrocytes

As a part of the study on safety evaluation of detergents, the hemolytic activity and the effect on glycolysis in human erythrocytes were investigated.Fifty per cent hemolysis by various detergents occurred in the range from 7 to 73ppm in low blood concentration, but the hemolytic activity in high blood concentration (50v/v%) was decreased to about one-hundredth. The order of hemolytic activity was almost parallel to the lowering activity of surface-tension. In general, the hemolysis by anionic detergents was more potent than that of nonionic ones.The glycolysis in erythrocytes was not influenced by treatment with various detergents at the concentration of one-fourth of 50% hemolytic value. However, the glycolysis in burst erythrocytes was inhibited from 50 to 70% by the anionic and stimulated from 30 to 100% by the nonionic, at 10-3M concentration of detergents. From the studies with some glycolytic enzymes, it was suggested that the inhibition of glycolysis by anionic detergents was due to the formation of the enzyme–detergent complex, and the stimulation by nonionic detergents was attributed to the stimulation of aldolase activity.

Glycolysis in human erythrocytes containing elevated concentrations of 2,3-P2-glycerate

In studies on the mechanism of the inhibitory effect of 2, 3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained: 1) Glucose consumption and lactate production are reduced by 70 and 40 per cent relative to normal erythrocytes in red blood cells containing five times the normal amount of 2, 3, -P2-glycerate ("high-diphosphoglycerate" cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the "high-diphosphoglycerate" cells. 2) About 50 per cent of the inhibition of glycolysis in "high-diphosphoglycerate" cells can be accounted for by the 2, 3-P2-glycerate-induced decrease of the red-cell pH. This fall of the red-cell pH which occurs as a conswquence of the Donnan effect of the non-pentrating 2, 3-P2-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinase. 3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2, 3-P2-glycerate of glycolytic enzymes. From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2, 3-P2-glycerate concentration in "high-diphosphoglycerate" cells suggests that also the enzyme pyruvate kinase is inhibited by 2, 3-P2-glycerate. 4) The dependencies of net-change of 2, 3-P2-glycerate concentration on the red-cell pH are identical in normal and "high-diphosphoglycerate" cells indicating that the balance between formation and decomposition of 2, 3-P2-glycerate is the same in erythrocytes with normal and very high compositions of 2, 3-P2-glycerate.

Influence of cyclic 3′, 5′-AMP on glycolysis in human erythrocytes

Small increases in lactate production and in glucose utilization were observed when human red cells were incubated in Tris-Ringer's medium with 5 mM adenosine 3′, 5′-monophosphate (cyclic 3′, 5′-AMP) or its dibutyryl analogue. 5 mM caffeine facilitated this response whereas 5′-AMP, cyclic 2′, 3′-AMP, and guanosine 3′, 5′-AMP had no effect.

Effect of oxygen tension on glycolysis in human erythrocytes.

Effect of oxygen tension on glycolysis in human erythrocytes.

Book Reviews

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Control of glycolysis in human erythrocytes by inorganic phosphate and sulfate.

Control of glycolysis in human erythrocytes by inorganic phosphate and sulfate.

Potassium transport and control of glycolysis in human erythrocytes.

Potassium transport and control of glycolysis in human erythrocytes.

Erythrocyte magnesium metabolism in intact and cold-inhibited glycolysis

Continuation of glycolysis in human erythrocytes by incubation of heparinized whole blood at 37°C is followed by a decrease in the Mg-content of red cells. On inhibition of glycolysis by cold (4°C), the Mg-content of erythrocytes remains unaltered or, often, slightly increases. These actions are in clear contrast to the behaviour of potassium ions under the same conditions.

Phosphate uptake and glycolysis in human erythrocytes heated in vitro.

AbstractThis work was supported by Contract H-1142 (C-4) with the Department of Health, Education, and Welfare, U. S. Public Health Service, National Institutes of Health, Bethesda, Maryland, and by Contract DA 49–007-MD-435 with the Department of the Army, Office of the Surgeon General, Medical Research and Development Board, Washington, D. C.