Human Erythrocyte Metabolism Research Articles

In Vivo Effects of Meloxicam, Celecoxib, and Ibuprofen on Free Radical Metabolism in Human Erythrocytes

One of the major groups of chemical mediators involved in the inflammatory response is the prostaglandins, which are synthesized from arachidonic acid by the enzyme cyclooxygenase. The aim of this study is to compare the in vivo effects of celecoxib, meloxicam, and ibuprofen on the activities of catalase (CAT), glutathione peroxidase (GSHPx), superoxide dismutase (SOD) as well as malondialdehyde (MDA), and antioxidant potential levels (AOP) in human erythrocytes. Patients diagnosed as osteoarthritis were included in the study. Patients were treated with Celecoxib (200 mg/d) (n = 12), Meloxicam (15 mg/d) (n = 12), and Ibuprufen (1200 mg/d) (n = 9) for 21 days. SOD, CAT, GSHPx activities, MDA, and AOP levels were investigated in human erythrocyte haemolysates. SOD activity and AOP levels were significantly decreased in all NSAID groups when we compared the values before and after 21 days of celecoxib, meloxicam, ibuprofen treatment. There were no significant difference in CAT, GSHPx activities, and MDA levels before and after treatment in each group. Decreased SOD activities are thought to be related with the increased superoxide anion. Decreased AOP levels may indicate impairment in the total antioxidant defence system. These NSAIDs have similar effects on free radical metabolism on human erythrocytes; despite some difference in action mechanisms.

Glyceraldehyde metabolism in human erythrocytes in comparison with that of glucose and dihydroxyacetone.

Metabolism of D-glyceraldehyde in human erythrocytes in comparison with that of glucose and dihydroxyacetone was studied. Both trioses were metabolized to produce L-lactate at rates comparable to that of L-lactate formation from glucose. Almost complete inactivation of glyceraldehyde-3-phosphate dehydrogenase by treatment of cells with iodoacetate resulted in a 95% decrease in L-lactate formation from the ketotriose as well as from glucose, whereas L-lactate formation from the aldotriose was only partially reduced (60%). D-Lactate was produced faster from either the aldotriose or the ketotriose than from glucose, but the ability of the two trioses to produce D-lactate was far lower than that to produce L-lactate. Almost complete inhibition of aldehyde dehydrogenase by disulfiram and of both aldose reductase and aldehyde reductase II by sorbinil, had no effect on L-lactate formation from D-glyceraldehyde. The present study suggests that D-glyceraldehyde is metabolized via two or more pathways including the glycolytic pathway after its phosphorylation by triokinase, and that neither oxidation to D-glyceric acid nor reduction to glycerol is a prerequisite for D-glyceraldehyde metabolism.

Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: computer simulation and Metabolic Control Analysis

This is the third of three papers [see also Mulquiney, Bubb and Kuchel (1999) Biochem. J. 342, 565-578; Mulquiney and Kuchel (1999) Biochem. J. 342, 579-594] for which the general goal was to explain the regulation and control of 2,3-bisphosphoglycerate (2,3-BPG) metabolism in human erythrocytes. 2,3-BPG is a major modulator of haemoglobin oxygen affinity and hence is vital in blood oxygen transport. A detailed mathematical model of erythrocyte metabolism was presented in the first two papers. The model was refined through an iterative loop of experiment and simulation and it was used to predict outcomes that are consistent with the metabolic behaviour of the erythrocyte under a wide variety of experimental and physiological conditions. For the present paper, the model was examined using computer simulation and Metabolic Control Analysis. The analysis yielded several new insights into the regulation and control of 2,3-BPG metabolism. Specifically it was found that: (1) the feedback inhibition of hexokinase and phosphofructokinase by 2, 3-BPG are equally as important as the product inhibition of 2,3-BPG synthase in controlling the normal in vivo steady-state concentration of 2,3-BPG; (2) H(+) and oxygen are effective regulators of 2,3-BPG concentration and that increases in 2,3-BPG concentrations are achieved with only small changes in glycolytic rate; (3) these two effectors exert most of their influence through hexokinase and phosphofructokinase; (4) flux through the 2,3-BPG shunt changes in absolute terms in response to different energy demands placed on the cell. This response of the 2,3-BPG shunt contributes an [ATP]-stabilizing effect. A 'cost' of this is that 2, 3-BPG concentrations are very sensitive to the energy demand of the cell and; (5) the flux through the 2,3-BPG shunt does not change in response to different non-glycolytic demands for NADH.

A Possible Role of Adenylate Metabolism in Human Erythrocytes. 2. Adenylate Metabolism is Able to Improve the Erythrocyte Volume Stabilization

We constructed and studied a mathematical model that describes the control of cell volume, ion balance, energy and adenylate metabolism in human erythrocytes. According to the model, adenylate metabolism can provide an effective stabilization of the cell volume over relatively large changes of cell parameters. For example, the steady-state value of the cell volume remains almost unchanged when the cell membrane permeability increases by 15-fold. The cell volume also changes only slightly over large changes in the parameters of energy metabolism. The relaxation time for the cell volume changes is about 100 hr over changes in these parameters. In other words, the volume stabilization operates most effectively against long-term slow perturbations.

Human erythrocyte metabolism is modulated by the O 2-linked transition of hemoglobin

The metabolic behaviour of human erythrocytes has been investigated with particular regard to the effect of their oxygenation state. Experiments performed at high phosphate concentration (80 mM) within the pH range 7.0–7.8 on erythrocytes at high (HOS) and low (LOS) oxygen saturation showed that at any pH value: (1) glucose consumption was independent of the oxygenation state; (2) pentose phosphate pathway (PPP) flux was about 2 times higher in the HOS than in the LOS state. At low phosphate concentration (1.0 mM) the PPP flux doubled in HOS as well as in LOS erythrocytes, whereas the decrease in glucose consumption was more marked in the HOS state. Metabolism of LOS erythrocytes approached that of HOS erythrocytes under the following conditions: (1) erythrocytes having band 3 modified by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid; (2) CO-saturated erythrocytes. These data support the hypothesis of a modulation of the relative rates of PPP and glycolysis achieved through competition between deoxy-hemoglobin (deoxy-Hb) and glycolytic enzymes for the cytoplasmic domain of band 3.

A Possible Role of Adenylate Metabolism in Human Erythrocytes: Simple Mathematical Model

A simplified mathematical model of cell metabolism describing ion pump, glycolysis and adenylate metabolism was developed and investigated in order to clarify the functional role of the adenylate metabolism system in human erythrocytes. The adenylate metabolism system was shown to be able to function as a specific regulatory system stabilizing intracellular ion concentration and, hence, erythrocyte volume under changes in the permeability of cell membrane. This stabilization is provided via an increase in adenylate pool in association with ATPases rate elevation. Proper regulation of adenylate pool size might be achieved even in the case when AMP synthesis rate remains constant and only AMP degradation rate varies. The best stabilization of intracellular ion concentration in the model is attained when the rate of AMP destruction is directly proportional to ATP concentration and is inversely proportional to AMP concentration. An optimal rate of adenylate metabolism in erythrocytes ranges from several tenths of a percent to several percent of the glycolytic flux. An increase in this rate results in deterioration of cell metabolism stability. Decrease in the rate of adenylate metabolism makes the functioning of this metabolic system inefficient, because the time necessary to achieve stabilization of intracellular ion concentration becomes comparable with erythrocyte life span.

THE POSSIBLE CONSEQUENCES OF LARGE-SCALE ENZYME ALTERATIONS ON THE METABOLIC EFFICIENCY OF RED BLOOD CELLS AS STUDIED ON THE BASIS OF A MATHEMATICAL MODEL

Based on a comprehensive mathematical model of the energy and redox metabolism of human erythrocytes we calculated stationary metabolic states of the cell varying the activity of the participating enzymes by several orders of magnitude. To compare the computational results with clinical and experimental data reported for various enzymopathies of the red cell, the three essential metabolic variables ATP, GSH and osmolarity were included in a “homeostasis function” which allows us to estimate the range of enzyme activities in which the metabolic alterations should be either tolerable, associated with non-chronic or chronic hemolytic diseases, or letal.

Alteration of the erythrocyte glutathione redox balance by N-acetylcysteine, captopril and exogenous glutathione

The effect of the thiol containing compounds N-acetylcysteine and captopril on glutathione metabolism in human erythrocytes has been investigated non-invasively using 1H spin echo NMR. N-Acetylcysteine was found to increase the reduced form of glutathione while captopril increased the oxidized form, but no changes in the total glutathione concentration were observed. Incubation of the cells with buthionine sulphoximine to inhibit de novo glutathione synthesis did not affect the response. The results show that these compounds act by altering the redox balance of glutathione rather than by stimulating its synthesis, and that their mechanisms of action are different, and not simply due to the presence of the thiol group.

Effect of thermal stress on glutathione metabolism in human erythrocytes

This is the first experiment to investigate the effect of heat and cold stress on glutathione metabolism in human erythrocytes. We immersed men at three different water temperatures for 10 min. At 39 degrees C, no remarkable changes were observed. Levels of glutathione (GSH) decreased from 2.44 (0.14) to 1.80 (0.10) mumol.ml red blood cells-1 [mumol.ml RBC-1; mean (SEM); P < 0.0005] and those of lipid peroxides increased from 1.87 (0.03) to 2.06 (0.04) nmol.ml RBC-1 (P < 0.01) after the immersion at 42 degrees C. In contrast, levels of GSH increased from 2.46 (0.17) to 2.91 (0.17) mumol.ml RBC-1 (P < 0.05) and those of lipid peroxides did not change after the immersion at 25 degrees C. The activities of glutathione peroxidase decreased from 35.90 (1.83) to 34.33 (1.66) IU.g Hb-1 (P < 0.01) after the immersion at 42 degrees C; however, these activities did not change after the immersion at 25 degrees C. The activities of glutathione reductase (both active and inactive forms) showed no changes at any temperatures. These changes indicate that heat stress causes oxidative stress in the human body; however, cold stress is thought to augment the activity of the antioxidative defence system. It is suggested that body exposure to hot environmental conditions should not be recommended for patients suffering from a damaged antioxidative defence system.

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2- 13C] d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has 13C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1- 13C]-, [2- 13C]-, and [3- 13C] d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.

Effects of the ionophore gramicidin D on energy metabolism in human erythrocytes.

Washed human erythrocytes were incubated in phosphate-buffered saline at 37 degrees C with different concentrations (2-80 mg l-1) of the monovalent cationophore gramicidin D. The ionophore induced a decrease of ATP content and energy charge (EC) and a concomitant increase in ADP and AMP contents. At a concentration of 10 mg l-1 of the ionophore the reduction of EC and ATP concentration was about 50%. Increasing the concentration of the ionophore resulted in a substantial haemolysis with no further effect on the nucleotide levels. Lowering the concentration of the ionophore to 5 mg l-1 (to avoid haemolysis) lead to a similar effect on the energy status. Ouabain at 0.5 mmol l-1 in incubation medium distinctly blunted the ionophoric effect on EC and adenine nucleotides. Addition of glucose on the contrary reversed these effects by about 80%. Hence, the glucose-induced increase in glycolytic flux as evidenced by an increase in lactate production was not sufficient to restore energy state.

Energy metabolism of human erythrocytes in psoriasis

1.1. Adenine nucleotide concentrations and metabolism in red blood cells (RBC)† and RBC ghosts from psoriatic patients and healthy subjects were compared.2.2. The ATP and total adenine nucleotide levels and the adenylate energy charge (EC) were elevated in the blood from psoriatic patients.3.3. The rate of glycolytic production of ATP by intact RBC was unchanged, but the Na+, K+-ATPase activity of RBC ghosts was decreased significantly in psoriasis.4.4. Results suggest that the defect in adenine nucleotide metabolism is a systemic manifestation of psoriasis, and that the quantification of adenine nucleotides in RBC and in whole blood samples may be of pathophysiological value in psoriatic lesion.

Nucleotide metabolism in human erythrocytes: Purification and properties of specific pyrimidine 5'-nucleotidase

Nucleotide metabolism in human erythrocytes: Purification and properties of specific pyrimidine 5'-nucleotidase

Improved metabolic properties of hexokinase-overloaded human erythrocytes

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Anomeric specificity of d-glucose metabolism in human erythrocytes

The metabolism of d-[5- 3H]glucose and d-[U- 14C]glucose displays anomeric specificity in human erythrocytes. At close-to-physiological hexose concentrations, pure β- d-[5- 3H]glucose is metabolized at a higher rate than the corresponding pure α-anomer in most, but not all, subjects. This situation represents a mirror image of that found in rat erythrocytes. The anomeric preference is modulated by the extracellular concentration of d-glucose and may differ for distinct metabolic variables. The anomeric specificity of d-glucose metabolism remains operative, whether at low or normal temperature, even in erythrocytes exposed to equilibrated d-glucose.

Effects of synthetic incubation media on phosphate metabolism in human erythrocytes

citrate flux is similar to that found in renal brush border by Wright et al. (1982). The absence of a sodium-independent flux from the renal brush border membrane may implicate the sodium-independent flux component at the basolateral membrane. This proposal for citrate would parallel the hexose and amino acid systems, and would allow net absorption of citrate, which is known to take place (Newsome et al., 1983). We have found that in addition to the sodium dependence of one of the citrate fluxes, citrate significantly increases sodium influx. This increase in sodium influx has been previously seen with both the intestinal hexose and amino acid symport systems (Sepulveda et al., 1982). The pH dependence of citrate influx (Fig. Ib) led us to investigate the possibility that citrate elicits its effects on sodium influx via sodium-proton exchange. We have found a sodium-proton exchange system on the enterocyte membrane and have discovered that this system plays little part in the effects of citrate on sodium influx. We have also found that the citrate-induced increase in sodium influx is unaffected by the sodium-proton exchange inhibitor amiloride. It appears, therefore, that citrate increases the transcellular sodium flux by increasing sodium influx through a symport system. The sodium-independent citrate flux appears to play little part in the increase in sodium flux, and citrate does not indirectly use the sodium-proton exchange system available. Although we have found that the rotenone-poisoned cells do not metabolize citrate over at least 8min, i t has been suggested that citrate acts as a mucosal cell energy source in the dehydrated animal (Bywater, 1977). Therefore, in addition to increasing enterocyte sodium influx, citrate may also elevate depleted ATP pools and sodium ATPase activity, to aid further transcellular sodium flux.

Mechanism of o-aminophenol metabolism in human erythrocytes

o-Aminophenol was found to be rapidly metabolized to a brown compound in the presence of purified human oxy- and methemoglobin, coupled with the oxidation and reduction of these hemoglobins by o-amino-phenol. The final product of o-aminophenol was identified as 2-aminophenoxazine-3-one, by using spectrophotometry and HPLC. The metabolism of o-aminophenol was also observed in human erythrocytes. The production rates of 2-aminophenoxazine-3-one in the cells were very fast, but these were strongly decreased by bubbling carbon monoxide into the cell suspension when intracellular hemoglobin was in the ferrous state. The production of 2-aminophenoxazine-3-one from o-aminophenol in the cells was completely suppressed by cyanide and azide when intracellular hemoglobin was in the ferric state. These results suggest that oxy- and methemoglobin are involved in metabolism of o-aminophenol to 2-aminophenoxazine-3-one in human erythrocytes.

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages

Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes

AbstractThe human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent “futile cycle.” Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5′-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady- state labeling of erythrocyte ATP with 32P-phosphate was obtained, and the rate of synthesis of 32P-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with 32P; the transfer of 32P from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis.

Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes

The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent “futile cycle.” Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5′-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady- state labeling of erythrocyte ATP with 32P-phosphate was obtained, and the rate of synthesis of 32P-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with 32P; the transfer of 32P from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis.