Dependency on acid-base status of oxyhemoglobin dissociation and 2,3-diphosphoglycerate level in human erythrocytes. II. In vivo studies.
01 Aug 1970-Scandinavian Journal of Clinical & Laboratory Investigation (Taylor & Francis)-Vol. 26, Iss: 1, pp 47-52
TL;DR: It was demonstrated that high concentrations of inorganic phosphate in serum influence the intraerythrocytic 2,3-diphosphoglycerate concentration and this effect should be taken into account when evaluating the increase at hypoxemic and anemic hypoxia.
Abstract: The intraerythrocytic level of 2,3-diphosphoglycerate and the oxygen affinity of hemoglobin in the red cell are shown to be strongly dependent on the intraerythrocytic pH. This effect should be taken into account when evaluating the increase in 2,3-diphosphoglycerate found at hypoxemic and anemic hypoxia. It was also demonstrated that high concentrations of inorganic phosphate in serum influence the intraerythrocytic 2,3-diphosphoglycerate concentration.
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TL;DR: Findings indicate that the rate of 2,3-DPG synthesis becomes enhanced in deoxygenated cells and the relief of product inhibition of DPG mutase brought about by a greater binding of 2.,3- DPG to deoxyhemoglobin seems to be of minor importance.
Abstract: Hypoxia induces in rats a rather rapid increase of the concentration of 2,3-DPG in red blood cells. This increase is reversed when the animals are returned to normal conditions. Pigeons do not respond to hypoxia with an increase of inositol hexaphosphate concentration in their erythrocytes. In rats exposed for 24 h to gas mixtures containing low oxygen and in addition 5% CO2 the hypoxia induced rise of 2,3-DPG concentration is abolished. The hemoglobin concentration in whole blood is negatively correlated to red cell 2,3-DPG levels in normal as well as in anemic or polycythemic rats. The rate of 2,3-DPG decomposition in human erythrocytes incubated without glucose is the same in the presence and in the absence of oxygen. The incorporation of32P into 2,3-DPG proceeds faster in deoxygenated than in oxygenated human red blood cells and exceeds considerably the concomitant acceleration of the glycolytic flux rate. These findings indicate that the rate of 2,3-DPG synthesis becomes enhanced in deoxygenated cells. This is mainly due to an elevation of the intracellular pH; the relief of product inhibition of DPG mutase brought about by a greater binding of 2,3-DPG to deoxyhemoglobin seems to be of minor importance. The regulation of 2,3-DPG concentration by the intraerythrocytic pH as well as by the oxygenation state of hemoglobin and the significance of these regulatory mechanisms in inducing and limiting the changes of red cell 2,3-DPG during hypoxia and anemia are discussed.
109 citations
TL;DR: The model was examined using computer simulation and Metabolic Control Analysis and it was found that the feedback inhibition of hexokinase and phosphofructokinase by 2, 3-B PG are equally as important as the product inhibition of 2,3-BPG synthase in controlling the normal in vivo steady-state concentration of 2-bisphosphoglycerate.
Abstract: 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.
106 citations
TL;DR: Eels acclimated 2 weeks to hypoxia have blood with significantly higher O2 capacity and O2 affinity than normoxic control eels and a marked decrease in red cell ATP concentration attended the changes in HbO2 binding.
85 citations
TL;DR: The shape and position of the oxyhemoglobin dissociation curve contribute to efficient transport of oxygen from the lungs to the tissues as well as mediating alterations in the affinity of blood for oxygen.
85 citations
TL;DR: The Donnan induced intracellular acidification causes a gradual impairment of red cell metabolism as the 2, 3-DPG concentration increases, thus imposing a selflimitation to the accumulation of 2,3-DPGs in the presence of IPP and leading to an inhibition of glucose consumption in IPP-pretreated cells.
Abstract: The 2,3-DPG content of human red blood cells can be elevated in vitro to a maximum of 24 μMoles/g by incubating the cells in media containing inosine, pyruvate and inorganic phosphate (=IPP media). The rate of accumulation depends on the extracellular phosphate level. The concentrations of organic phosphate fractions other than 2,3-DPG also increase during the initial phase of incubation in IPP media, but rediminish thereafter. As a consequence of these changes, the total concentration of acid-soluble organic phosphates in the red cells rises from 14 to 55 μMoles P/g red cells. This increase of non-penetrating anions produces a shifting of the Donnan ratio H
e
+/H
i
+ to lower values and thereby diminishes progressively the intracellular pH of the red cells during incubation in IPP media. The extent of these changes can be calculated on the basis of equations derived by van Slyke. The Donnan induced intracellular acidification causes a gradual impairment of red cell metabolism as the 2,3-DPG concentration increases, thus imposing a selflimitation to the accumulation of 2,3-DPG in the presence of IPP and leading to an inhibition of glucose consumption in IPP-pretreated cells. The findings are discussed with respect to their metabolic and biophysical basis and in view of possible implications for the regulation of red cell metabolism and function.
72 citations
Cites background from "Dependency on acid-base status of o..."
...This fundamental discovery has initiated a great number of investigations concerning the enzymatic formation and degradation of 2,3-DPG [17,18,19,23, 29, 42,43], its concentration changes under various conditions of hypoxia in vivo [4, 20, 28, 33, 38] and the implications of its decomposition in stored blood [11,13,14,47]....
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467 citations
TL;DR: The data strongly suggest that with anoxia, the observed rise in organic phosphate content of the red cell is responsible for increased availability of oxygen to tissues.
Abstract: The relationship between oxygen dissociation and 2,3-diphosphoglycerate (2,3-DPG) in the red cell has been studied in subjects moving from low to high altitude and vice versa. Within 24 hr following the change in altitude there was a change in hemoglobin affinity for oxygen; this modification therefore represents an important rapid adaptive mechanism to anoxia. A parallel change occurred in the organic phosphate content of the red cell. While this study does not provide direct evidence of a cause-effect relationship, the data strongly suggest that with anoxia, the observed rise in organic phosphate content of the red cell is responsible for increased availability of oxygen to tissues.
391 citations
TL;DR: In eight subjects exposed to hypoxia at high altitude the oxyhemoglobin dissociation curve was shifted to the right, the half saturation oxygen tension at pH 7.40 being changed from 26.7 to 30.2 mm Hg on the average, and the decreased affinity of hemoglobin for oxygen appeared within 12 hours after exposure to Hypoxia.
Abstract: In eight subjects exposed to hypoxia at high altitude the oxyhemoglobin dissociation curve was shifted to the right, the half saturation oxygen tension at pH 7.40 being changed from 26.7 to 30.2 mm Hg on the average. The decreased affinity of hemoglobin for oxygen appeared within 12 hours after exposure to hypoxia. The shift was confirmed in short exposure experiments using simulated high altitude in a low pressure chamber. At prolonged carbon monoxide exposure the expected shift of the oxygen dissociation curve to the left occurred, but no adaptive changes were found. The physiological implication of these differences are discussed. It is suggested that the rapid shift to the right at high altitude may be a physiological regulation.
344 citations
TL;DR: 2,3-Diphosphoglycerate decreased the binding of ATP to deoxygenated haemoglobin and inorganic phosphate up to 10 mM had no effect on either compound.
Abstract: The binding of 2,3-diphosphoglycerate and adenosine triphosphate to human haemoglobin was studied in solutions of a high haemoglobin concentration (3 mM) and varying amounts of 2,3-diphosphoglycerate, ATP, inorganic phosphate, magnesium and hydrogen ions. Both phospho compounds were found to be bound by both deoxygenated and oxygenated haemoglobin; the affinity of the former was at physiological conditions about twice that of the latter. At least two binding sites were found for 2,3-diphosphoglycerate; these sites show strong cooperation. Binding of both phospho compounds decreased at higher hydrogen ion concentration. Inorganic phosphate up to 10 mM had no effect on either compound. 2,3-Diphosphoglycerate decreased the binding of ATP to deoxygenated haemoglobin.
113 citations
TL;DR: Observations suggest that pathological variations in human red cell DPG may significantly effect oxygen transport, and the erythrocytes of individuals homozygous for sickle hemoglobin have significalntly higher levels of DPG; this probably accounts for the lower oxygen affinity of the intact cells.
Abstract: The central role of 2,3-diphosphoglycerate (DPG) in the metabolism and function of most mammalian red cells is only beginning to become apparent. 2,3-Diphosphoglycerate, a glycolytic intermediate present in high concentration only in the red cell, has been said to function as a storage source for phosphate.1 It may also act as a metabolic regulator, since DPG has been found to exert significant inhibitory effects on several red cell enzymes, such as transaldolase, transketolase,2 and hexokinase.3 Most recently, the findings of Benesch and Benesch,4 Benesch et al.,5 and Chanutin and Curnish6 show that DPG (and adenosine triphosphate (ATP), which is quantitatively less important) combines reversibly with deoxyhemoglobin, and, in normal concentrations, greatly decreases the oxygen affinity of hemoglobin, shifting the oxygen dissociation curve to the right. Although this shift increases the oxygen pressure required for oxygenation of the hemoglobin, unloading can occur at higher tissue oxygen tensions. Although both sets of authors4-6 have emphasized the importance of this effect, Benesch et al.5 stated that variations in levels of red cell DPG within the normal human range will have little or no influence on the oxygen affinity of the whole blood. Part of the stimulus for the present study derives from observations which suggest that pathological variations in human red cell DPG may significantly effect oxygen transport. An inherited abnormality of pyruvic kinase in which the activities of this enzyme were elevated about twofold was present in a family reported by Zurcher et al.7 Individuals affected with this disorder had levels of red cell ATP about twice normal and levels of DPG about one-fourth normal. Although both ATP and DPG can decrease the oxygen affinity of hemoglobin, DPG is quantitatively much more important, since its normal concentration in the human red cell is three to four times that of ATP. The authors noted that affected individuals had an unexplained elevation of hemoglobin and hematocrit values of about 20 per cent. We now think it likely that the reason for this increase in blood values was a significant lessening of oxygen unloading in the tissues of affected individuals, due to the decrease in the amount of DPG in the red cell. The resulting tissue anoxia has been compensated by erythrocytosis. Secondly, a recent abstract8 dealt with the levels of DPG in the erythrocytes of individuals homozygous for sickle hemoglobin. The oxygen dissociation curve of intact red cells in sickle cell anemia, compared to that of normal cells, has been known to be shifted to the right. However, this shift is no longer apparent when studies are performed on dialyzed hemoglobin. These authors found that the erythrocytes of individuals homozygous for sickle hemoglobin have significalntly higher levels of DPG; this probably accounts for the lower oxygen affinity of the intact cells.
105 citations