Acetylation of human hemoglobin by methyl acetylphosphate. Evidence of broad regio-selectivity revealed by NMR studies.

The development of chemical modification agents that reduce the tendency of sickle hemoglobin (HbS) to aggregate represents an important chemotherapeutic goal. Methyl acetylphosphate (MAP) has been reported to bind to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin, where it selectively acetylates residues, resulting in increased solubility of HbS. We have prepared [1-(13)C]MAP and evaluated the adduct formation with hemoglobin using (1)H-(13)C HMQC and HSQC NMR studies. These spectra of the acetylated hemoglobin adducts showed 10-11 well resolved adduct peaks, indicating that the acetylation was not highly residue specific. The chemical shift pattern observed is in general similar to that obtained recently using [1'-(13)C]aspirin as the acetylating agent (Xu, A. S. L., Macdonald, J. M., Labotka, R. J., and London, R. E. (1999) Biochim. Biophys. Acta 1432, 333-349). Blocking the 2, 3-DPG binding site with inositol hexaphosphate (IHP) resulted in a selective reduction in intensity of adduct resonances, presumably corresponding to residues located in the 2,3-DPG binding cleft. The pattern of residue protection appeared to be identical to that observed in our previous study using IHP and labeled aspirin. Pre-acetylation of hemoglobin using unlabeled MAP, followed by acetylation with [1'-(13)C]aspirin indicated a general protective effect, with the greatest reduction of intensity for resonances corresponding to acetylated residues in the 2,3-DPG binding site. These studies indicated that both MAP and aspirin exhibit similar, although not identical, acetylation profiles and target primarily the betaLys-82 residue in the 2,3-DPG binding site, as well as sites such as betaLys-59 and alphaLys-90, which are not located in the beta-cleft of hemoglobin.     

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.     

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.     

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     

Hemoglobin Kariya [alpha 40 (C5) Lys leads to Glu]: a new hemoglobin variant with an increased oxygen affinity

A new abnormal hemoglobin, Hb Kariya [alpha 40 (C5) Lys leads to Glu], with an amino acid substitution at the alpha 1 beta 2 contact was discovered in a young Japanese man. This variant migrated to the anode faster than Hb A, being nearly the same as Hb I in electrophoretic mobility. It amounted to about 6% of the total hemoglobin of the hemolysate. This hemoglobin showed an increased oxygen affinity, decreased heme-heme interaction and a lowered 2,3-DPG effect.     

A comparative study of the temperature dependence of the oxygen-binding properties of mammalian hemoglobins.

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.     

1H and 31P nuclear magnetic resonance investigation of the interaction between 2,3-diphosphoglycerate and human normal adult hemoglobin

High-resolution 1H and 31P nuclear magnetic resonance spectroscopy has been used to investigate the binding of 2,3-diphosphoglycerate to human normal adult hemoglobin and the molecular interactions involved in the allosteric effect of the 2,3-diphosphoglycerate molecule on hemoglobin. Individual hydrogen ion NMR titration curves have been obtained for 22-26 histidyl residues of hemoglobin and for each phosphate group of 2,3-diphosphoglycerate with hemoglobin in both the deoxy and carbonmonoxy forms. The results indicate that 2,3-diphosphoglycerate binds to deoxyhemoglobin at the central cavity between the two beta chains and the binding involves the beta 2-histidyl residues. Moreover, the results suggest that the binding site of 2,3-diphosphoglycerate to carbonmonoxyhemoglobin contains the same (or at least some of the same) amino acid residues responsible for binding in the deoxy form. As a result of the specific interactions with 2,3-diphosphoglycerate, the beta 2-histidyl residues make a significant contribution to the alkaline Bohr effect under these experimental conditions (up to 0.5 proton/Hb tetramer). 2,3-Diphosphoglycerate also affects the individual hydrogen ion equilibria of several histidyl residues located away from the binding site on the surface of the hemoglobin molecule, and, possibly, in the heme pockets. These results give the first experimental demonstration that long-range electrostatic and/or conformational effects of the binding could play an important role in the allosteric effect of 2,3-diphosphoglycerate on hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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).     

Orally administrated cerium chloride induces the conformational changes of rat hemoglobin, the hydrolysis of 2,3-DPG and the oxidation of heme-Fe(II), leading to changes of oxygen affinity

The structure and oxygen affinity of hemoglobin from erythrocytes of CeCl(3) fed Wistar rats in the dose range of 0.2-20.0 mg/kg body weight/day were investigated by means of various spectroscopic methods. The changes in oxygen saturation curves of hemoglobin are dependent upon both feeding dose and feeding time. After 40 days feeding with 20 mg CeCl(3)/kg body weight/day, the curve changed to a double sigmoid shape and the oxygen affinity in low oxygen pressure increases. It regained the sigmoid form after 80 days feeding, but the degree of oxygen saturation in higher oxygen pressure became higher than that in the control. These results indicate that CeCl(3) can increase the oxygen affinity of hemoglobin of rat erythrocytes. This effect is further demonstrated by the analysis of M?ssbauer spectra of erythrocytes. Increase of hemoglobin content in erythrocytes was found in rats fed with CeCl(3). It might be the offset response to the poor oxygen-releasing capability of the hemoglobin. CD and FT-IR deconvoluted spectra indicate that secondary structures of hemoglobin have remarkable changes, characterized by a gradual decrease of alpha-helix content, in a dose- and feeding time-dependent fashion. Meanwhile, the 31P NMR spectra demonstrate that the level of 2,3-diphosphoglyceric acid (2,3-DPG) in erythrocytes, an allosteric regulator of oxygen release from hemoglobin, decreases due to its hydrolysis. In addition, the M?ssbauer and ESR spectra show clearly that a fraction of the heme-iron changes from Fe (II) to Fe (III) in CeCl(3) fed rats. The results indicate that the oral administration of CeCl(3) leads to a microenvironment changes of heme in intracellular hemoglobin. Oxygen affinity changes might be attributed to a series of events triggered by the binding of Ce (III) to hemoglobin and 2,3-DPG, including conformational changes of hemoglobin and 2,3-DPG hydrolysis, respectively and also the partial transformation from heme-Fe (II) to heme-Fe (III).     

P50 values in hypoxic albino rats of first and second generation.

We determined the "in vivo" (arterial pH and PCO2) and standard (pH = 7.4, PCO2 = 40 mm Hg) PO2 at 50% O2 saturation of hemoglobin (P50, vv and P50, st) in Wistar albino rats when living in a normobaric hypoxic environment. Two generations of hypoxic rats were observed for changes in their P50, vv, P50, st, (n50) 2,3-diphosphoglycerate (2,3-DPG), hemoglobin (Hb) and DPG-Hb ratio: the first generation (H1) and the second generation (H2). A few hours after birth, the H1 rats were placed and raised in a normobaric hypoxic environment (10% O2 in N2). The H2 rats were born from hypoxic parents of first generation and were raised in the same hypoxic environment. The control group had a normoxic environment. The P50, st was significantly higher in H1 rats than both H2 and controls. P50, st was similar in H2 and control rats. The P50, vv was significantly higher in H1 rats than both H2 and controls but it was significantly lower in H2 when compared with both controls and H1. Hb and 2,3-DPG had values significantly greater for both H1 and H2 when compared with their controls. However, the values of H2 were significantly lower than H1. The effectiveness of an increase in Hb-O2 affinity as an adaptive mechanism in H2 rats is discussed.     

The deoxygenation kinetic properties of human fetal hemoglobin: effect of 2,3-diphosphoglycerate

The deoxygenation kinetics of isolated adult and fetal hemoglobin are measured. The results demonstrate that significant functional differences exist between the two tetrameric hemoglobins. It is pointed out that these functional differences closely parallel the differences in similar properties of beta and gamma chains. It is also shown that 2,3-diphosphoglycerate (2,3-DPG) has no significant effect on the deoxygenation rate of fetal hemoglobin. This result appears to be consistent with the reported weaker binding of 2,3-DPG to the oxygen linked groups of fetal hemoglobin.     

Red cell 2,3-DPG, ATP, and mean cell volume in highly trained athletes. Effect of long-term submaximal exercise

20 male elite long distance runners were compared to a control group of blood donors to determine the effect of training on red blood cells. The acute effects of exercise on red cells were investigated in 11 of the runners following a race of 15-30 km. The runners had elevated resting values of red cell 2,3-DPG (P less than 0.05) and mean cell volume (P less than 0.01); blood Hb and ATP were not different from concentrations in the control group. The red cell status of the athletes may be explained by an increased proportion of young erythrocytes in runners. No statistically significant changes in red cell 2,3-DPG, ATP, mean cell volume or blood Hb were found post exercise.     

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.     

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.     

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 Ln³⁺ binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln³⁺ 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 Ln³⁺ 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 Ln³⁺ concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln³⁺ binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln³⁺ concentration. At the range of [Ln³⁺]/[Hb]<2, the marked increase of oxygen affinity (P₅₀ decrease) with the Ln³⁺ concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln³⁺ concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln³⁺ binding. This was indicated by the changes in secondary structure characterized by the decrease of α-helix content.     

Domain-specific effector interactions within the central cavity of human adult hemoglobin in solution and in porous sol-gel matrices: evidence for long-range communication pathways

The water-filled central cavity of human adult hemoglobin (Hb A) is the binding or interaction site for many different allosteric effectors. Oxygen binding titrations reveal that pyrenetetrasulfonate (PyTS), a fluorescent analogue of 2,3-diphosphoglycerate, behaves like an allosteric effector. The ligation state, pH, and concentrations of other effectors (IHP, L35, and chloride) alter PyTS fluorescence for both solution-phase and sol-gel-encapsulated Hb samples. These conditions also alter the resonance Raman spectra and rates of geminate recombination of CO-ligated Hb. Together, these results demonstrate that there are conformational and functional consequences resulting from interactions between specific domains of the central cavity and individual effectors as well as from long-range synergistic effects that are mediated through the central cavity.     

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.     

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.