Age-related changes in glycolysis and levels of modulators of oxygen transport function of hemoglobin in human erythrocytes

The glycolysis rate and the amount of the acid-transport hemoglobin function modulators (diphosphoglycerate (2,3-DPG), ATP, glutathione, chlorides) in human erythrocytes are studied under the effect of age changes. It is shown that the glycolysis rate and the content of ATP decrease under ageing by 16% and 21%, respectively. The concentration of 2,3-DPG lowers insignificantly (by 8%). At the same time it is established that the amount of reduced glutathione in erythrocytes of middle-aged and old people enhances by 25-29% and that of chlorine ions by 23-24%. The revealed changes in the concentration of modulators of the hemoglobin affinity to oxygen with ageing are, probably, one of reasons of the oxyhemoglobin dissociation increase.     

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     

Comparative effects of phosphoenolpyruvate on selected mammalian erythrocytes

1. Incubation of human, rat, cow, sheep, dog, rabbit and monkey erythrocytes with phosphoenolpyruvate (PEP) resulted in increased intracellular 2,3-diphosphoglycerate (2,3-DPG). 2. Physiologic temperature (37 degrees C) and a pH less than 6.5 were required for transport and metabolism of PEP in rat and monkey erythrocytes. 3. Although erythrocytes from all species (except pig) exhibited PEP transport and metabolism, hemoglobin oxygen affinity (HOA) was affected only in species whose hemoglobins are sensitive to 2,3-DPG. 4. These results suggest that the effect of PEP incubation on HOA is mediated through 2,3-DPG.     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

A possible new mechanism of oxygen affinity modulation in mammalian hemoglobins

Bovine red cells do not contain appreciable amounts of 2,3-diphosphoglycerate (2,3-DPG). Bovine hemoglobin, however, has a particular sensitivity to chloride ions and as a result it can attain oxygen affinity values lower than those measured for human hemoglobin in the presence of 2,3-DPG. The interaction of bovine hemoglobin with anions is modulated by the hydrophobic characteristics of the protein. Comparison of the hydropathy plots of primate and ruminant hemoglobins indicates constant regions of opposite hydrophobicity, which have fixed amino acid differences. A model is proposed for explaining the regulation of oxygen affinity by chlorides, as an alternative to the classic modulation by 2,3-DPG.     

Seasonal changes of heart weight and erythrocytes in the Djungarian hamster, Phodopus sungorus

Djungarian dwarf hamsters (Phodopus sungorus) show an annual cycle in weight-specific metabolic rate with a high level during winter. These seasonal changes in oxygen demand are met by hematological adjustments, primarily based on an increased number of erythrocytes, but a decreased erythrocyte volume during winter. Subsequently, the diffusion area for blood gas exchange is increased during this time of high metabolic capabilities. Blood oxygen capacity (hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) does not change with the season. However, seasonal changes in heart weight suggest changes in cardiac output, causing an increased blood flow per unit tissue weight during winter. This increase in circulatory efficiency, as well as changes in erythrocyte surface, are primarily controlled by photoperiod, since it occurred in hamsters living indoors at thermoneutrality but subjected to seasonal changes in photoperiod to the same extent as in hamsters living outdoors.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.     

Postnatal changes in 2,3-diphosphoglycerate (2,3-DPG) content of calf erythrocytes

In 82 calves, 22 adult cows and 10 fetuses the 2,3-DPG level was determined in the erythrocytes. The lowest level was found in cows (1.13 microM/g haemoglobin, on the average), and in calves aged 35 days or more. In the foetuses the mean 2,3-DPG concentration in the erythrocytes was 4.98 microM/g Hb and it was higher that that in the erythrocytes of cows, the oldest foetuses and calves during the first two days of postnatal life. During 5 weeks of postnatal life the changes taking place in 2,3-DPG concentration could be divided into two periods: period I or the period of increase covering the first 6 days of life, with a characteristic rise in the concentration of this component from 1.37 to 15.80 microM/g Hb, and period II or the period of decrease lasting from the 6th to the 35th day of life. In period II two phases could be discerned, the first phase lasting from the 6th to the 10th day with a steep fall of 2,3-DPG level from 15.80 to 4.58 microM/g Hb, and the second phase from the 10th to the 35th day of life in which the level of 2,3-DPG reached slowly the value found in adult cows. A comparison of oxygen affinity of haemoglobin in calves aged 6 days, which was composed of about 80% of fetal haemoglobin and about 20% of adult haemoglobin, and in adult cows, which contained exclusively adult haemoglobin, showed that the oxygen-binding capacity of haemoglobin was lower in calves.     

Interaction of zinc and hemoglobin: binding of zinc and the oxygen affinity.

Stripped human hemoglobin was shown to have a high apparent zinc association constant of 1.3 X 10(7) M-1 with a stoichiometry of one zinc for every two hemes. The saturation of this site produces a dramatic 3.7-fold increase in the oxygen affinity. The effect of zinc on the oxygen affinity is interrelated with the interaction of 2,3-diphosphoglyceric acid (2,3-DPG) and hemoglobin. Thus, a smaller zinc effect is observed in the presence of added 2,3-DPG. Information about the location of the zinc-binding site responsible for the increased oxygen affinity has been obtained by comparing the binding of zinc to various hemoglobins. Blocking the beta93 sulfhydryl group decreases the apparent zinc association constant by an order of magnitude. The substitution of histidine-beta143 in hemoglobin Abruzzo [beta143 (H21) His leads to Arg] and hemoglobin Little Rock [beta143 (H21) His leads to Gln] decreases the apparent zinc association constant by two orders of magnitude. The substitution of histidine-beta143 by other amino acids and the reaction of the beta93 sulfhydryl group are known to produce dramatic increases in the oxygen affinity. The binding of zinc to one or both of these amino acids can, therefore, explain the zinc-induced increase in the oxygen affinity.     

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.     

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.     

Unchanged in vivo P50 at high altitude despite decreased erythrocyte age and elevated 2,3-diphosphoglycerate

We measured hematological and erythrocyte O2 transport parameters in whole blood and density-separated erythrocytes in 11 mountaineers before and during 5 days of exposure to high altitude (4,559 m). We determined the in vivo (arterial pHblood and PCO2) and standard (pHblood = 7.4, PCO2 = 40 Torr) O2 tension at 50% O2 saturation of hemoglobin and (P50,vv and P50,st) and Bohr coefficients (BC) for fixed acid (H+) and CO2 and examined the contribution of the altered average age of circulating erythrocytes due to the stimulation of erythropoiesis on whole blood 2,3-diphosphoglycerate (2,3-DPG) and P50,st. At altitude, whole blood P50,vv remained almost unchanged, whereas P50,st and 2,3-DPG increased significantly (+4 Torr; 3.5 mumol/g hemoglobin). BCCO2 was elevated significantly at altitude. Serum erythropoietin increased transiently fourfold, iron utilization increased, and serum iron decreased by 66%. Reticulocyte counts increased, but other hematological parameters were unchanged. In density-separated erythrocytes, P50,st and 2,3-DPG increased with decreasing cell density but were higher in fractions with comparable reticulocyte counts in cells prepared at altitude than in those from control studies. Our data show that, despite the increase in 2,3-DPG and the decrease in average erythrocyte age, the in vivo hemoglobin-O2 affinity remains unchanged. P50,st values reflect the elevation of 2,3-DPG, and approximately 50% of the increase in both parameters can be ascribed to the increase in the number of reticulocytes and young erythrocytes.     

Regulation of Hemoglobin Function in Mammals

This survey of hemoglobin function in mammals reveals a broadrange in oxygen affinity. The concentration of red cell 2,3-DPGvaries widely among groups of mammals. Those animals (feloidsand ruminants) that have very low levels of this intracellularmediator have hemoglobins of intrinsically low oxygen affinitywhich fail to respond to the addition of 2,3-DPG. Mammals whichhave adapted to various types of hypoxia tend to have increasedoxygen affinity, primarily mediated through reduced levels ofred cell 2,3 DPG. In contrast mammals who are experimentallysubjected to low oxygen tensions develop decreased oxygen affinityowing to increased red cell 2,3-DPG. Mammals employ one of threedifferent mechanisms for the maintenance of higher oxygen affinityof fetal red cells, compared to maternal red cells. Many of these phenomena can be satisfactorily explained at themolecular level but their adaptational significance is lessclear.     

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 bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).     

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.     

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.     

Inhibition of the gelation of extracellular and intracellular hemoglobin S by selective acetylation with methyl acetyl phosphate

Methyl acetyl phosphate binds to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin and selectively acetylates three amino groups at or near that site. The subsequent binding of 2,3-DPG is thus impeded. When intact sickle cells are exposed to methyl acetyl phosphate, their abnormally high density under anaerobic conditions is reduced to the density range of oxygenated, nonsickling erythrocytes. This change is probably due to a combination of direct and indirect effects induced by the specific acetylation. The direct effect is on the solubility of deoxyhemoglobin S, which is increased from 17 g/dL for unmodified hemoglobin S to 22 g/dL for acetylated hemoglobin S at pH 6.8. Acetylated hemoglobin S does not gel at pH 7.4, up to a concentration of 32 g/dL. The indirect effect could be due to the decreased binding of 2,3-DPG to deoxyhemoglobin S within the sickle erythrocyte, thus hindering the conversion of oxyhemoglobin S to the gelling form, deoxyhemoglobin S.     

Role of the state of erythrocyte cytoplasm in the change of hemoglobin affinity for oxygen

The dimensions and refractive properties of erythrocytes and the affinity of their hemoglobin for oxygen have been assessed using laser interference microscopy and Raman spectroscopy. Lowering the pH and addition of 2,3-diphosphoglycerate cause comparable changes in the cell shape and the state of cytoplasm, as well as in the oxygen affinity of hemoporphyrin, but the mechanisms of these effects appear to be different. It is shown that acidification mainly decreases the hemoporphyrin oxygen-binding capacity, whereas 2,3-diphosphoglycerate increases the ability of hemoporphyrin to release oxygen.     

Effect of covalent labeling of dextran-benzenehexacarboxylate on hemoglobin

The covalent fixation of benzenehexacarboxylate (BHC) onto dextran was carried out according to several reaction schemes. The polyanionic polymers thus synthesized were capable of decreasing the oxygen affinity of hemoglobin by specifically interacting with the 2,3-diphosphoglycerate (2,3-DPG) binding site of the protein. The intensity of this effect was correlated to both the chemical structure of the polyanionic polymers and the BHC content in polymer. The polyanionic polymer, containing 0.035 mol BHC/g and presenting no cross-linking between its polymer chains, possessed the best effector properties. These properties were used to direct the covalent fixation of the dextran-benzenehexacarboxylate onto the phosphate binding site of the protein. The resulting hemoglobin was mainly substituted at the same time by one or more linked BHC onto both dimers in the vicinity of the 2,3-DPG site. Thus, the modification of hemoglobin led to an increase in the hydrodynamic volume of each dimer sufficient to limit the diffusion of the conjugates through the kidney membrane, even if the conjugates had dissociated into dimers. Compared to that of free hemoglobin, the oxygen affinity of the conjugates was significantly decreased. This type of covalent conjugate exhibited general properties quite suitable for use as blood substitutes.Abbreviations used Hb hemoglobin - 2,3-DPG 2,3-diphosphoglycerate - BHC benzenehexacarboxylate - IHP inositolhexaphosphate - EDCI 3-ethyl-1-(3-dimethylaminopropyl) carbodiimide hydrochloride