Oxyhemoglobin dissociation curve and 2,3-diphosphoglycerate in chronic hypoxemia

The measurement of the oxyhemoglobin dissociation curve (ODC) and 2,3-diphosphoglycerate (2,3-DPG) in patients with chronic hypoxemia is important from the view point of tissue oxygenation. However, there have been no consistent results that explain the relation among chronic hypoxemia, 2,3-DPG and P50, which is oxygen pressure at an oxygen saturation of 50 percent. The aim of this study is to clarify what factors affect P50 and 2,3-DPG. 1) Patients with chronic hypoxemia, who showed PaO2 less than 60 Torr, had significantly higher P50 than normal subjects. 2) The concentration of Hb showed significant negative correlation with both P50 and 2,3-DPG. 3) Arterial blood pH showed significant positive correlation with both P50 and 2,3-DPG. 4) In a group with normal levels of Hb and pH, there was significant negative relationship between PaO2 and P50. 5) In a group with normal levels of Hb and pH, there was significant positive relationship between PaCO2 and P50. 6) In a group with normal levels of Hb, pH and PaCO2, there was significant negative relationship between PaO2 and 2,3-DPG. In conclusion, P50 and 2,3-DPG are affected largely by Hb concentration or blood pH, with or without hypoxemia. However there is a mechanism by which P50 and 2,3-DPG are increased by hypoxemia itself in a group with normal levels of Hb, pH and PaCO2.     

Mechanism of red cell 2,3-diphosphoglycerate increase in neonatal lambs

The tenfold increase in red cell 2,3-diphosphoglycerate (DPG) concentration that occurs during the first 5 days of life in lambs is an important adaptation to extrauterine life. In lambs, DPG reduces hemoglobin oxygen affinity by the Bohr effect. Our data on 10 neonatal lambs suggest that the biochemical mechanism underlying this DPG increase involves the following: (1) a rise in plasma glucose from 40 to 100 mg/dl in the first 48 hr of life, which allows for increased glucose consumption in the highly glucose-permeable neonatal RBC; (2) a transitory rise in blood pH begins at birth, peaks at about 20 hr, and falls slightly; (3) the pH increase coincides with a threefold increase in RBC fructose-1,6-diphosphate (FDP) concentration due, we believe, to pH activation of phosphofructokinase; (4) glycolytic intermediates after the glyceraldehyde-3-phosphate dehydrogenase (GAPD) step do not rise in the first 24 hr of life, possibly due to insufficient inorganic phosphate (Pi), a substrate of GAPD; (5) plasma Pi increases from about 7 mg/dl at birth to 11 mg/dl at 72 hr, activates the GAPD, and FDP levels decline; and (6) the in vitro activity of the DPG synthetic enzyme, DPG mutase, is increased 12-fold in neonatal compared to adult RBC. We conclude that the postnatal rise in DPG is explained at least in part by the sequential effects of these metabolic changes.     

Interlaboratory comparison of red-cell ATP, 2,3-diphosphoglycerate and haemolysis measurements

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) storage systems are licensed based on their ability to prevent haemolysis and maintain RBC 24-h in vivo recovery. Preclinical testing includes measurement of RBC ATP as a surrogate for recovery, 2,3-diphosphoglycerate (DPG) as a surrogate for oxygen affinity, and free haemoglobin, which is indicative of red cell lysis. The reproducibility of RBC ATP, DPG and haemolysis measurements between centres was investigated. MATERIALS AND METHODS: Five, 4-day-old leucoreduced AS-1 RBC units were pooled, aliquotted and shipped on ice to 14 laboratories in the USA and European Union (EU). Each laboratory was to sample the bag twice on day 7 and measure RBC ATP, DPG, haemoglobin and haemolysis levels in triplicate on each sample. The variability of results was assessed by using coefficients of variation (CV) and analysis of variance. RESULTS: Measurements were highly reproducible at the individual sites. Between sites, the CV was 16% for ATP, 35% for DPG, 2% for total haemoglobin and 54% for haemolysis. For ATP and total haemoglobin, 94 and 80% of the variance in measurements was contributed by differences between sites, and more than 80% of the variance for DPG and haemolysis measurements came from markedly discordant results from three sites and one site, respectively. In descending order, mathematical errors, unvalidated analytical methods, a lack of shared standards and fluid handling errors contributed to the variability in measurements from different sites. CONCLUSIONS: While the methods used by laboratories engaged in RBC storage system clinical trials demonstrated good precision, differences in results between laboratories may hinder comparative analysis. Efforts to improve performance should focus on developing robust methods, especially for measuring RBC ATP.     

Effects of 2,3-diphosphoglycerate on the mechanical properties of erythrocyte membrane

Investigation by Schindler et al and Sheetz and Casaly have indicated that high (approximately 10 mmol/L) concentrations of 2,3- diphosphoglycerate (2,3-DPG) have a destabilizing effect on erythrocyte membrane and the membrane skeleton. We have investigated changes in the membrane mechanical properties that occur at elevated 2,3-DPG levels in both intact cells and ghosts. The membrane shear modulus, viscoelastic recovery time constant, critical force, "plastic" viscosity, and material relaxation time constant were measured by standard micropipette and flow channel techniques. Intact cells showed no change in properties at physiologic ionic strength and 2,3-DPG concentrations of about 20 mmol/L, except for an increase in membrane viscosity resulting from an increased cellular hemoglobin concentration that occurs when the 2,3-DPG concentration is elevated. At ionic strengths 20% below physiologic and 2,3-DPG concentrations of approximately 20 mmol/L, decreases in membrane shear modulus and membrane viscosity were observed. In ghosts, no changes in these properties were observed at a 2,3-DPG concentration of 10 mmol/L and ionic strengths as low as 25% below physiologic, but a decrease in the force required to form tethers (critical force) was observed at physiologic ionic strength. The decrease in membrane shear modulus and viscosity of intact cells and the reduced critical force in ghosts are consistent with the results of other investigators. However, the difference in the effects of 2,3-DPG on ghosts and intact cells indicates that the effects of 2,3-DPG depend strongly on the conditions of the experiment. It appears unlikely that 2,3-DPG affects erythrocyte membrane material properties under physiologic conditions.     

Red cell 2,3-diphosphoglycerate and adenosine triphosphate in patients with shock

Summary . 2,3-Diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP) were studied in 12 patients with shock. Observations in the first 6 hr revealed a mean value of 2,3-DPG higher than normal with a range above and below the normal limits. After this period the mean value was lower. In the presence of metabolic acidosis, the values were low. In two patients, the improvement in clinical state was associated with a rise in the level of these compounds. The mean value for ATP was slightly higher than normal in the first 6 hr and remained the same later; there was no correlation with 2,3-DPG or with pH. Our data failed to reveal any definite relation between shock and 2,3-DPG levels in the red cells. The metabolite varied independently of ATP and did not correlate with red cell age. There was a significant correlation of the levels of 2,3-DPG with the hydrogen ion concentration of arterial blood.     

Effect of adenosine on P50 and its relation to 2,3-diphosphoglycerate

The effects of increasing doses of intravenous adenosine upon the dissociation haemoglobin curve (DHC) and its relation to the intraerythrocytic level of 2,3-diphosphoglyceric acid (2,3-DPG), were studied in 17 anesthetized dogs. The DHC moved significantly to the left in all dogs except at the dose of 120 micrograms/kg/min which induces a displacement to the right. These changes in the DHC were parallel to the intraerythrocytic levels of 2,3-DPG. We conclude that adenosine modifies the DHC, shifting it generally to the left, and that this effect seems to be related to a change in the intraerythrocytic level of 2,3-DPG.     

Physiologic features of hemolysis axxociated with altered cation and 2,3-diphosphoglycerate content

A hemolytic disorder characterized by altered RBC cation composition (increases Na, decreases K), reduced monovalent cation content (decreased Na + K/liter RBC), and decreased levels of 2,3- diphosphoglycerate (2,3-DPG) is described. The etiology of these RBC abnormalities was not elucidated following extensive laboratory evaluation, although two important physiologic principles were manifested by this case: (1) Hemolysis was relatively well compensated (41% hematocrit) despite a significantly decreased RBC survival (51 Cr t 1/2 = 10.5 days). This effect presumably was due to reduced 2,3-DPG content (1.9 mumol/ml RBC) and the associated increase in whole blood oxygen affinity (P50 = 19.6 mm hg). (2) RBC size and water content were normal in spite of marked cation depletion. This anomaly was thought to reflect the osmotic effects of reduced polyvalent anion (2,3-DPG) content.     

Inappropriately low red cell 2,3-diphosphoglycerate and p50 in transfused beta-thalassemia

The relationships among hemoglobin concentration (Hb), red cell 2,3- diphosphoglycerate (2,3-DPG), and p50 were studied in 20 chronically hypertransfused patients with thalassemia major. In the nontransfused control group, which included normal individuals as well as patients with sickle cell disease or iron deficiency anemia, the Hb correlated inversely with both 2,3-DPG concentration and p50, as is well established. In contrast, however, prior to transfusion, at the nadir of Hb, patients with thalassemia major had inappropriately low 2,3-DPG concentrations and p50s. These findings occurred in all patients, regardless of whether they had received packed, leukocyte-poor, or frozen-thawed red cells. The hypothesis that the time of blood storage was a factor was excluded by repeatedly transfusing one patient with packed red cells administered within 4 hr of collection in CPDA-1. A second hypothesis, that red cell function might be impaired by the iron- overloaded thalassemic environment, was excluded by studying a newly diagnosed, newly transfused patient with aplastic anemia. In both cases, the same inability to appropriately increase 2,3-DPG and p50 as the Hb fell during the intertransfusion interval was noticed. These data suggest that red cells of chronically transfused patients are unable to adapt to the decline in Hb that occurs during the intertransfusion interval.     

The oxyhemoglobin dissociation curve in health and disease. Role of 2,3-diphosphoglycerate

The shape and position of the oxyhemoglobin dissociation curve contribute to efficient transport of oxygen from the lungs to the tissues. In the past several years, factors affecting the position of the oxyhemoglobin dissociation curve in many disease states have been studied, and the important role of 2,3-diphosphoglycerate (2,3-DPG) in mediating alterations in the affinity of blood for oxygen has been repeatedly confirmed. At present, the effects of blood pH and blood oxygenation on levels of 2,3-DPG and on the oxygen affinity of blood are well established; on the other hand, precise prediction of the position of the oxyhemoglobin dissociation curve in complex disease states is not yet possible.     

Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects

Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.     

A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.     

Some evidence for aldosterone action on 2,3-diphosphoglycerate level in human red cells

In healthy male subjects aldosterone excretion and plasma renin activity were reduced by a 4–6 hr head-out immersion in thermoindifferent water baths (35.5 ± 0.1°C). The red cell 2,3-diphosphoglycerate (DPG) concentration before and throughout immersion period was positively correlated both with aldosterone excretion in 2 hr pooled urine (r = +0.69; 2p < 0.001) and with renin activity (r = +0.54; 2p < 0.001) despite a concomitant increase of cubital venous pH and inorganic phosphate concentration. These findings furnish evidence for a regulatory role of aldosterone in DPG metabolism, possibly by a direct influence on red cell glycolysis.     

Red cell 2,3-diphosphoglycerate and oxygen affinity of hemoglobin in patients with thyroid disorders

We measured red blood cell 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP), and the P50 value in vitro of the oxyhemoglobin dissociation curve, which is the oxygen tension at half saturation of hemoglobin, in order to quantitate red blood cell oxygen transport function in individuals who were diagnosed as hypothyroid, euthyroid, or hyperthyroid based on measurements of thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and their clinical status. Hypothyroid (mean T4 2.8 microgram/dl, T3 49 ng/dl, TSH 37 microU/ml) and hyperthyroid (mean T4 14 microgram/dl, T3 271 ng/dl, TSH less than 0.7 microU/ml) patients had normal red cell 2,3-DPG and ATP levels and normal P50 values in vitro. The known changes in oxygen consumption produced by alterations in thyroid hormone levels in patients with hypothyroidism or hyperthyroidism did not affect red blood cell oxygen transport function.