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.     

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.     

Intracellular free magnesium and phosphorylated metabolites in hexokinase- and pyruvate kinase-deficient red cells measured using 31P-NMR spectroscopy

The erythrocyte metabolism of two patients with nonspherocytic hemolytic anemia caused by a hexokinase deficiency, and a pyruvate kinase deficiency, respectively, were studied with NMR. The complexing of ATP and 2,3-diphosphoglycerate (2,3-DPG) with Mg2+ and hemoglobin (Hb) was determined using 31P-NMR on oxygenated and deoxygenated cells to investigate the influences of these enzyme defects on intracellular magnesium distribution and on Hb oxygen dissociation. In the pyruvate kinase-deficient red blood cells, the 2,3-DPG concentration was almost twice the normal value and the ATP concentration was near the lower limit of the normal range. In the hexokinase-deficient red cell population, the predominance of young cells masked the deficiency. Therefore, reticulocyte control cells were included in this study. In the oxygenated pyruvate kinase-deficient cells, the fraction of ATP that is complexed to magnesium as well as the free Mg2+ concentration were normal, despite the abnormal concentration of 2,3-DPG. In the deoxygenated cells the free Mg2+ concentration was lower than in normal cells. The fraction of Hb complexed with 2,3-DPG was higher than normal in both oxygenated and deoxygenated pyruvate kinase-deficient cells, in accordance with the high p50 of the oxygen-hemoglobin dissociation curve. In hexokinase-deficient cells, two major abnormalities are found: when the cells were deoxygenated, the concentration of ATP and 2,3-DPG fell. This was not observed for any other sample and could, therefore, be a consequence of the hexokinase deficiency. Despite almost normal levels of magnesium-binding metabolites, the free Mg2+ concentration in oxygenated and deoxygenated cels is much lower than in normal cells. This could be a cell-age-related phenomenon, since lower free Mg2+ concentrations were also found in reticulocyte control cells.     

Relationship of phosphate administration to serum and red cell phosphate concentration, erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and blood P50 in the hyperglycemic dog

In vitro studies indicate that acute increases in intracellular phosphate concentration decrease red blood cell 2,3-diphosphoglycerate levels (G. Momsen, B. Vestergaard-Bogind, Arch Biochem Biophys 190:67, 1978). We have examined the relationship in vivo of serum phosphate concentration, red cell phosphate, 2,3-DPG and blood P50 in hyperglycemic dogs infused alternately with phosphate or chloride (control) solutions. During the 8-hr insulin infusion, serum phosphate (Pi) fell 40% in the chloride-treated animals and rose 71% in the phosphate-treated dogs (P less than 0.001, phosphate vs. control). RBC Pi concentration declined in the controls and rose significantly in the phosphate-infused dogs (P less than 0.02). Serum Pi and RBC Pi were correlated in the phosphate-managed animals (r = 0.76, P less than 0.02), but not in the controls. RBC 2,3-DPG failed to rise in either group during insulin infusion and regression analysis showed a negative correlation between serum Pi and 2,3-DPG (r = -0.90, P less than 0.005) and between RBC Pi concentration and 2,3-DPG (r = -0.84, P less than 0.02). P50 failed to change in either group during insulin treatment and for up to 24 hr after initiation of the 8-hr infusion of insulin.     

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.     

Consequences of 6 weeks of strength training on red cell O2 transport and iron status

Effects of endurance training on O2 transport and on iron status are well documented in the literature. Only a few data are available concerning the consequences of strenuous anaerobic muscular exercise on red cell function. This study was performed to test the influence of strength training alone on parameters of red cell O2 transport and iron status. Twelve healthy untrained males participated in a strength-training programme of 2-h sessions four times a week lasting 6 weeks. After 6 weeks a small but significant reduction of haemoglobin (Hb; -5.4 g.l-1) was found (p less than 0.05). Mean red cell volume did not change, but a pronounced decrease of mean cell Hb concentration (from 329.2 g.l-1, SE 2.5 to 309.8 g.l-1, SE 1.2; p less than 0.001) and mean corpuscular Hb (from 29.6 pg, SE 0.4 to 27.7 pg, SE 0.3; p less than 0.01) was observed. Serum ferritin decreased significantly by 35% (p less than 0.01); transferrin, serum iron and iron saturation of transferrin were unaltered. Serum haptoglobin concentration was diminished significantly by 30.5% (p less than 0.01). The reticulocyte count had already increased after 3 weeks of training (p less than 0.05) and remained elevated during the following weeks. Strength training had no significant influence on the O2 partial pressure at which Hb under standard conditions was 50% saturated, red cell 2,3-diphosphoglycerate and ATP concentration as well as on erythrocytic glutamate-oxalacetate transaminase activity. The data demonstrate that mechanical stress of red cells due to the activation of large muscle masses led to increased intravascular haemolysis, accompanied by a slightly elevated erythropoiesis, which had no detectable influence on Hb-O2 affinity. Training caused an initial depletion of body iron stores (prelatent iron deficiency). Although Hb had decreased by the end of the training phase a true "sports anaemia" could not be detected.     

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.     

Effect of irradiation and/or leucocyte filtration on RBC storage lesions

Red blood cell (RBC) storage lesions have been shown to be associated with some adverse reactions; numerous studies have focused on the lesions caused by storage, and few data on the RBC storage lesions caused by prestorage treatments of leucocyte filtration and irradiation. In this study, we examined the changes related with the RBC storage lesions, including 2,3-diphosphatidylglyceric acid (2,3-DPG), pH, free hemoglobin (Hb), supernatant free K+ and Na+ concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH). Along with the increasing storage time, decreases in 2, 3-DPG levels, pH and Na+ concentration, increases in K+ and free Hb concentrations, and significant morphological changes in RBC in all groups were found. The changes in the groups of irradiation, leucocyte filtration and the combined irradiation and leucocyte filtration were more significant than those in the untreated group. Meanwhile, the MCV levels of the three treated groups were significantly lower than those in the untreated group, while the MCH variations were significantly higher. Our results suggest that irradiation and leucocyte filtration before storage may aggravate blood storage lesions.     

Viability, ATP and 2,3-DPG content of resuspended erythrocytes during several-week storage in cold

Leukocyte-and thrombocyte-poor packed red cells obtained from ACD or. ACD-AG blood were resuspended to a hematocrit of about 55% and stored at 4 degrees C. The resuspension solution consisted of xylitol, inorganic phosphate, bicarbonate, adenine (A) and guanosine (G) solved in water. In one case glucose, citrate and sucrose were also added, in another one, sorbitol. The 2,3-DPG and the ATP level remained for a longer period in the sorbitol-xylitol-medium than in the glucose-xylitol-medium. The ATP content in the red cell suspension was higher than in packed cells. Higher ATP values were obtained in red blood cells from whole blood with adenine and guanosine. The survival rate of resuspended red blood cells in glucose-AG-citrate-sucrose medium was about 80--85% after 3 weeks of storage and 77% after 6 weeks with a higher range.     

Erythrocyte adenosine triphosphate depletion during voluntary hyperventilation

Chronic hypophosphatemia in humans is associated with a slow depletion of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes, combined with shape alteration, impaired deformability, and viability of the cells. Likewise, incubation of erythrocytes in alkaline solution is associated with ATP depletion. Since in hyperventilation both hypophosphatemia and alkalosis are present, we have investigated red cell organic phosphates, shape, deformability, and osmotic fragility before, during, and after 20 min of voluntary hyperventilation. On the average, red cell ATP decreased by 42%, the blood pH increased by 0.2 units, and plasma inorganic phosphorus decreased by 46% compared with the initial values. Red cell 2,3-DPG, shape, deformability, and osmotic fragility remained unchanged. After the end of hyperventilation ATP increased rapidly to control values in parallel with the normalization of the blood pH, whereas inorganic plasma phosphorus remained at the low level observed during hyperventilation. It is concluded that the combined effects of hypophosphatemia and alkalosis in acute hyperventilation lead to an isolated fall of red cell ATP, which occurs as rapid as after total inhibition of red cell glycolysis in vitro.     

Adaptation of red cell enzymes and intermediates in metabolic disorders.

The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.     

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.     

Liposome-loaded phenylalanine or tryptophan as sickling inhibitor: a possible therapy for sickle cell disease

Phenylalanine or tryptophan entrapped in small unilamellar liposomes was used to transport Phe or Trp across the red blood cell membrane. The incorporation of Phe or Trp into RBCs via liposomes markedly inhibited and reversed the in vitro sickling of deoxy Hb S. Furthermore, normal and SS RBCs loaded with Phe or Trp did not exhibit significant change in osmotic fragility, mechanical fragility, autohemolysis, and glycolysis when compared to untreated RBCs. In addition, the oxygen affinity measured as the P50 and concentrations of 2,3-DPG and ATP were not affected by the incorporation of Phe or Trp into AA or SS RBCs. These results demonstrate that this liposomal transport system which transferred Phe and Trp into intact RBCs did not have any adverse effect on RBC metabolism and function, and may have therapeutic implications in the treatment of sickle cell disease.     

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.     

Iron status in runners of various running specialties.

The blood iron status of 44 male runners of various running specialties (18 sprinters, 13 middle- and 13 long-distance runners) is evaluated by measuring serum ferritin (SF), serum iron (Si), hemoglobin concentration (Hb), hematocrit (Ht), red blood cells content (RBC) and haptoglobin concentration (Hp). The results of these analyses (except Hp) are compared to those obtained in sedentary male subjects (control group) of the same mean age. Mean SF, SI, Hb and Ht measured in athletes are significantly lower than in control group. The remarkably low Hp values obtained in athletes suggests the occurrence of hemolysis. Using unpaired t test, it appears that the blood iron status of these runners does not depend on their running specialty.     

Adaptation to Hypoxia by Increased HbO<Subscript>2</Subscript> Affinity and Decreased Red Cell ATP Concentration

THERE is a decrease in the O₂ affinity of mammalian haemoglobin (Hb) as the levels of 2,3-DPG or ATP are increased, which is explained by an allosteric effect on the HbO₂ binding^1,2. Similar observations on amphibians³ and fish⁴, which have molar ratios of ATP to Hb similar to those of DPG to Hb in mammals, suggest that red cell organic phosphates modulate Hb function in all vertebrates. The adaptation of mammals to various hypoxic stresses involves reduced HbO₂ affinity^5–9, the attendant increase in O₂ “unloading” capacity being mediated by an increase in the concentration of red cell 2,3-DPG. We have found the opposite response in hypoxic fish and suggest that an increased O₂ affinity results in increased O₂ transport for the fish.     

Red cell function at extreme altitude on Mount Everest

As part of the American Medical Research Expedition to Everest in 1981, we measured hemoglobin concentration, red cell 2,3-diphosphoglycerate (2,3-DPG), Po2 at which hemoglobin is 50% saturated (P50), and acid-base status in expedition members at various altitudes. All measurements were made in expedition laboratories and, with the exception of samples from the South Col of Mt. Everest (8,050 m), within 2 h of blood collection. In vivo conditions were estimated from direct measurements of arterial blood gases and pH or inferred from base excess and alveolar PCO2. As expected, increased 2,3-DPG was associated with slightly increased P50, when expressed at pH 7.4. Because of respiratory alkalosis, however, the subjects' in vivo P50 at 6,300 m (27.6 Torr) was slightly less than at sea level (28.1 Torr). The estimated in vivo P50 was progressively lower at 8,050 m (24.9 Torr) and on the summit at 8,848 m (19.4 Torr in one subject). Our data suggest that, at extreme altitude, the blood O2 equilibrium curve shifts progressively leftward because of respiratory alkalosis. This left shift protects arterial O2 saturation at extreme altitude.     

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.     

31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes

Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.     

The effect of increasing glucose in CPD on the quality of stored blood

Changes in quality of blood units containing one and a half or double amounts of glucose, stored at +4 degrees C for three weeks were analysed. An experimental preservative containing glucose and fructose (1 : 1) was also used. No other additives (purine or purine-nucleoside) were applied. A standard CPD preservative of the National Inst. of Haematology and Blood Transfusion was used as control. The pH, plasma free haemoglobin, K+ content, red blood cell (RBC) ATP and 2,3-DPG content, and RBC fragility index were determined in each sample. Increase of glucose concentration, the addition of fructose had a beneficial effect on blood pH, and on plasma free haemoglobin and K+ concentration. 150% glucose improved the 2,3-DPG maintenance in stored blood.