The pre- and postoperative 2,3-diphosphoglycerate concentration of erythrocytes in children with cyanotic heart disease

The behaviour of 2,3-diphosphoglycerate concentration (2,3-DPG) of red blood cells of babies and children with cyanotic heart diseases is studied before and after shunt operations. In babies with cyanotic heart diseases at the age of up to 6 months an increase of 2,3-DPG-level and haematocrit (HCT) is seen. Later, the compensation of chronic hypoxia is effected by further increase of HCT at unchanged high 2,3-DPG-level. The 2,3-DPG concentration which is still increased after successfull shunt operations as compared with the normal value is considered as an effective adaptation mechanism to the residual hypoxia presenting only a small load on the circulatory system. The 2,3-DPG concentration alone does not represent a criterion for the assessment of chronic hypoxia.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

Effect of a tissue oxygenator (almitrine + raubasine) on the metabolism of 2,3-diphosphoglycerate of rabbits submitted by hypoxia

In normoxic rabbits, the intravenous injection (1 mg/kg) of the product (almitrine + raubasine) do not modify the erythrocytic level of 2,3-DPG. But, after hypoxia (the rabbits being submitted to an oxygen pressure of 7,8 kPa during 20 minutes) the same dose of this product induce a durable rise of erythrocytic 2,3-DPG level which remain, 24 hours latter, + 15% above normal.     

Anti-inflammatory and neuroprotective effects of magnolol in chemical hypoxia in rat cultured cortical cells in hypoglycemic media

Our previous studies demonstrated that magnolol protects neurons against chemical hypoxia by KCN in cortical neuron-astrocyte mixed cultures (14). In the present study, we examined whether the neuroprotective effect of magnolol involve modulating inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), induced by KCN (hypoxia) or KCN plus lipopolysaccharide (LPS). In glucose-absent (hypoglycemia) media, KCN or KCN plus LPS induced increases in lactate dehydrogenase (LDH) activity by 32% and 34%, and PGE2 production by 12% and 32%, respectively. Both LDH and PGE2 increases were suppressed by 100 microM magnolol. In addition, although KCN or LPS alone did not increase NO generation, KCN plus LPS increased NO generation. This increase was reduced by 100 microM magnolol or 10 microM L-NAME, but the LDH increase and PGE2 production were not reduced by L-NAME. These findings suggest that the protective effects of magnolol against brain damage by KCN or KCN plus LPS in hypoglycemic media may involve inhibition of PGE2 production, but inhibition of NO generation may not be important.     

A procedure for decreasing the level of 2,3-bisphosphoglycerate in red cells in vitro

The physiological adaptation to anemia and other hypoxic states includes an increase in the level of 2,3-bisphosphoglycerate (2,3-DPG) in the red cell. We suggest that the high level of 2,3-DPG may have adverse effects in vivo. It has been found that red cells incubated with glycolate lose 2,3-DPG at a rapid rate relative to controls. ATP is stable. Net 2,3-DPG synthesis is observed after the glycolate is removed from the cells suggesting that they are not harmed. The effect appears to be specific for glycolate since lactate, glyoxylate, glycerate, acetate, and citrate were without effect. This procedure could be used to assess the effects of decreasing the 2,3-DPG level to normal in the erythrocytes of sickle cell and other anemias.     

2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate

The binding of inorganic vanadate (Vi) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [Vi]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 X 10(-6) M. The relevance of this result to other studies which have shown that the Vi-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [Vi] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 microM Vi has little effect on the phosphatase activity. However, in the presence of 25 microM Vi, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed.     

Effect of long-term training and acute physical exercise on red cell 2,3-diphosphoglycerate

A statistically significant 10% increase (p less than 0.005) in mean red cell 2,3-diphosphoglycerate (2,3-DPG) concentration, concomitantly with a mean 16% increase (p less than 0.001) in the predicted maximal oxygen uptake (VO2max) was observed in 29 recruits, who were studied during 6 months of physical training in military service. The increase in 2,3-DPG was higher, the lower the initial 2,3-DPG and VO2max levels. The mean initial 2,3-DPG level was higher in the subjects with a higher initial VO2max. A strenuous but highly aerobic 21-km marching exercise elicited a mean 9% increase (p less than 0.005) in red cell 2,3-DPG concentration. A significantly greater response of 2,3-DPG to marching exercise was observed in subjects with a lower pre-test VO2max than in those with a higher pre-test VO2max. During another more competitive march 2,3-DPG remained almost unchanged and was associated with a tendency towards a negative correlation with the acccompanying lactate response (r = -0.60, p less than 0.05). Red cell 2,3-DPG response to a standardized exercise is considered to be a suitable indicator for evaluating the effect of training on an individual.     

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.     

Altitude-induced erythrocytic 2,3-DPG and hemoglobin changes in rats of various ages.

Rats of various ages (2, 12, 24, and 40 mo of age) were exposed for 4 wk to either a simulated high altitude of 23,000 ft or to a Peoria, Ill., altitude of 650 ft above sea level. Hematocrit ratios, hemoglobin, and erythrocytic 2,3-diphospho-glycerate (2,3-DPG) concentrations were measured. Hematocrit and hemoglobin determinations revealed a decrease in erythrocytic content with increasing age, and the augmented erythropoietic response was seen in all age groups of animals as a result of altitude exposure. The maximal erythrocytic content of hemoglobin in the 40-mo-old animals was significantly lower than that of all other age groups. Erythrocytic 2,3-DPG levels were significantly changed by aging alone. In the 40-mo-old group there was a 35% increase over the next highest sea-level value. However, while erythrocytic 2,3-DPG content increased significantly in all other age groups following altitude exposure, it decreased 46% in the 40-mo-old group.     

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.     

Effect of 2,3-diphosphoglycerate on the phosphorylation of protein 4.1 by protein kinase C.

We have previously shown that 2,3-diphosphoglycerate (2,3-DPG) inhibits the phosphorylation of erythrocyte membrane cytoskeletal proteins by endogenous casein kinases. Here, we report that 2,3-DPG stimulates the phosphorylation of protein 4.1 by protein kinase C. Studies with red cell membrane preparations showed that while the phosphorylation of most of the membrane proteins by endogenous membrane-bound kinases and purified kinase C was inhibited by 2,3-DPG, the phosphorylation of protein 4.1 was slightly enhanced by the metabolite. The effect of 2,3-DPG was further examined using purified protein 4.1 preparations. Our results indicate that 2,3-DPG stimulates both the rate and the extent of phosphorylation of purified protein 4.1 by kinase C. The amount of phosphate incorporated was found to double to 2 mol of phosphate per mole of protein 4.1 in the presence of 10 mM 2,3-DPG. The increase in phosphorylation was distributed over all phosphorylation sites as revealed by an analysis of the labeling patterns of phosphopeptides resolved by high performance liquid chromatography, but a significantly higher incorporation was detected in two of the phosphopeptides. The stimulatory effect of 2,3-DPG on the phosphorylation of protein 4.1 was observed only with kinase C. Phosphorylation by the cytosolic erythrocyte casein kinase and the cyclic AMP-dependent protein kinase was inhibited by 2,3-DPG. Moreover, the stimulatory effect of 2,3-DPG seemed to be unique to the phosphorylation of protein 4.1 since a similar effect had not been observed with other protein kinase C substrates. Our results suggest that 2,3-DPG may play an important role in the regulation of cytoskeletal interactions.     

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.     

Regulation of red cell 2,3-DPG and Hb-O2-affinity during acute exercise

Reports from the literature and our own data on red cell 2,3-DPG and its importance for unloading O2 from Hb to the tissues during exhaustive exercise are contradictory. We investigated red cell metabolism during incremental bicycle ergometry of various durations. Furthermore changes in blood composition occurring during exercise were simulated under in vitro conditions. The effect of a moderate (11.2 mmol X l-1 lactate, pH = 7.127) and severe (18 mmol X l-1 lactate, pH = 6.943) lactacidosis on red cell 2,3-DPG concentration was compared with the effect of similar acidosis induced by HCl. Our data indicate that the concentration of 2,3-DPG in red cells depends on the degree of lactacidosis, but not on the duration of exercise. During moderate lactacidosis red cell 2,3-DPG remains unchanged. This can be explained by an interruption of red cell glycolysis on the PK and GAP-DH step caused by a lactate and pyruvate influx into the erythrocyte, as well as an intraerythrocytic acidosis and a drop in the NAD/NADH ratio. During severe lactacidosis and HCL-induced acidosis a decrease in 2,3-DPG due to an inhibition of 2,3-DPGmutase and other glycolytic enzymes can be found. Mathematical correction of the observed P-50 value for the decrease in 2,3-DPG occurring during severe lactacidosis showed that a decrease in Hb-O2-affinity during strenuous exercise depends on the degree of lactacidosis and temperature elevation.     

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.     

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.     

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.     

Comparison of carbonic anhydrase activity and other haematological values of two Eutherian and three Australian marsupial mammals

1. Carbonic anhydrase activity and 2,3-diphosphoglycerate (2,3-DPG) concentration were determined in whole blood from humans (Homo sapiens), rabbits (Oryctolagus cuniculus), eastern grey kangaroos (Macropus giganteus), pademelons (Thylogale billardierii) and brush-tailed possums (Trichosurus vulpecula). 2. Marsupial blood carbonic anhydrase activity increased as species body size decreased. 3. T. billardierii haemoglobin was found to have a polymorphism which may be the same (beta 2 = histidine or glutamine) as that of M. giganteus. 4. The concentration of 2,3-DPG int e red cells of T. billardierii was approximately equal to that of the haemoglobin tetramer. Levels of 2,3-DPG in the other species were similar to those previously reported.     

Diurnal changes of the 2,3-diphosphoglycerate concentration in human red cells and the influence of posture.

The 2,3-diphosphoglycerate (2,3-DPG) concentration, oxygen half saturation pressure at pH 7.4 (P50), pH in plasma and red cells, and mean corpuscular hemoglobin concentration (MCHC) of venous blood were determined during unrestricted daily activity (series I) throughout 24 hrs as well as during prolonged bed rest until noon (series II). In series I almost synchronous dirunal behavior of P50 2,3-DPG, and plasma pH as well as red cell pH became significantly apparent with highest values in the afternoon. The [2,3-DPG] yielded most pronounced alterations, which made up to 13.5% of the average day value. During prolonged recumbency the [2,3-DPG] showed a nonsignificant tendency to decline; the P50 remained unchanged throughout that period. The possible reason for the missing [2,3-DPG] increase is a reduced change of red cell pH in series II. An influence of a posture dependent aldosterone secretion either directly on the 2,3-DPG metabloism of indirectly via mediating the red cell pH and thus ruling the formation of this organic PHOSPHORIS COMPOUND IS DISCUSSED.     

The effect of pH and pO2 changes in the blood on the energy metabolism of human erythrocytes in old age

Changes in pH and pO2 of the blood have been studied for age peculiarities of their effect on the glycolysis rate and the content of ATP and 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes (in vitro). The fresh venous blood of practically healthy young (aged 20-29) and old (aged 75-85) people was used. Acidosis was shown to induce inhibition of glycolysis and decrease of the ATP and 2.3-DPG concentrations in erythrocytes, while alkalosis and hypoxemia-an increase of the glycolysis rate and 2.3-DPG content. In the both cases changes in the indices studied were considerably lower in old people as compared to young ones.     

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²