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Every year, more than 6 million people are diagnosed with and more than 4 million people die from cancer all over the world, which is approximately 10% of the total mortality. The incidence of cancer in Ukraine shows a gradual increase, with the number of newly diagnosed patients 304-308 per 100 thousand people. Bladder cancer ranks second in the world among oncological diseases of the urinary system after prostate cancer. In the later stages of diseases, their course may be associated with malignant neoplasm anemia. The purpose of the study was to study the content of 2,3-diphosphoglycerate in erythrocytes of peripheral venous blood of patients with malignant neoplasm anemia with urothelial bladder cancer and prostate cancer, depending on the severity of anemia; to evaluate the possible diagnostic and prognostic value of the studied indicator. Materials and methods. 96 patients (64 men and 32 women) with urothelial bladder cancer were examined. There were 39 patients (28 men and 11 women) among them, the course of their underlying disease was not accompanied by the presence of anemia (first (I) observation group) and 57 patients (36 men and 21 women), the course of their underlying disease was aggravated by malignant neoplasm anemia (second (II) observation group). Also, 48 men with prostate cancer whose course of the disease was not aggravated by malignant neoplasm anemia (19 men) (third (III) observation group) and 29 men (fourth (IV) observation group) were diagnosed with malignant neoplasm anemia. The age of the patients under the survey was from 22 to 79 years old. All patients were examined after verifying the diagnosis and before starting any treatment. The content of 2,3-diphosphoglycerate in erythrocytes of peripheral venous blood was determined by the method of I. S. Luganova, M. N. Blinov (1975). Results and discussion. The content of 2,3-diphosphoglycerate in peripheral venous erythrocytes of patients with malignant neoplasm anemia due to urothelial bladder cancer and prostate cancer has been studied. It was found that malignant neoplasm anemia in patients with urothelial bladder cancer and prostate cancer is associated with an increase in the studied indicator (p

Background: Alzheimer’s Disease (AD) features the accumulation of β-amyloid in erythrocytes. The subsequent red cell damage may well affect their oxygen-carrying capabilities. 2,3- diphosphoglycerate (2,3-DPG) binds to the hemoglobin thereby promoting oxygen release. It is theorized that 2,3-DPG is reduced in AD and that the resulting hypoxia triggers erythropoietin (EPO) release. Methods & Objective: To explore this theory, we analyzed red cell 2,3-DPG content and EPO in AD, mild cognitive impairment, and the control group, subjective cognitive impairment. Results: We studied (i) 2,3-DPG in red cells, and (ii) circulating EPO in AD, and both markers were unaffected by dementia. Disturbances of these oxygen-regulatory pathways do not appear to participate in brain hypoxia in AD.

Four phosphonate derivates of 2,3-diphosphoglycerate (2,3-DPG), in which the phosphate group is replaced by a methylene or difluoromethylene, were successfully synthesized for use as allosteric modulators of hemoglobin (Hb) O2 affinity. The syntheses were accomplished in four steps and the reagents were converted to their potassium salts to allow for effective binding with Hb in aqueous media. O2 equilibrium measurements of the chemically modified Hbs exhibited P50 values in the range 8.9–12.8 with Hill coefficients in the range of 1.5–2.4.

BACKGROUND Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares with stored blood and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs and in patients' blood after transfusion. METHODS Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood bags before transfusion and in blood samples drawn from patients before and after transfusion. RESULTS The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by approximately 90%; P < 0.0001) and a decreased P50 (by approximately 30%; P < 0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by approximately 20% (P < 0.05) in postoperative blood sampled from patients who received 1 to 3 stored RBC units and by approximately 30% (P < 0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over 3 postoperative days in patients who received stored RBCs. CONCLUSIONS Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left-shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to 3 postoperative days in patients who receive stored RBCs but are unchanged in those who receive only ASB RBCs.

A consistent and organized transition from embryonic to fetal to adult hemoglobin (Hgb) occurs during human fetal development. Hgb concentrations gradually increase, averaging 18 g/dL (180 g/L) by 40 weeks' gestation. The ability to deliver oxygen to tissues in the fetus and neonate is primarily determined by the percentage of fetal versus adult Hgb and the concentration of 2,3 diphosphoglycerate (2,3-DPG). Studies continue to evaluate the relationship between Hgb concentrations and oxygen delivery in neonates to determine what Hgb concentrations best meet the needs of a wide variety of clinical situations from the critically ill extremely low-birthweight infant to the stable growing preterm infant. Biochemical interactions between nitric oxide (NO) and Hgb beyond the production of methemoglobin do occur and may be a source of deliverable NO to the microcirculation under hypoxic conditions.

The Influence of Sodium Phosphate Supplementation on VO2max, Serum 2,3-diphosphoglycerate Level and Heart Rate in Off-road Cyclists The main objective of the work was to evaluate the influence of a six day supplementation with sodium phosphate on circulatory and pulmonary variables, the level of 2,3-diphosphoglycerate (2,3-DPG) and the concentration of inorganic phosphates in blood serum of elite off-road cyclists. The research material included 19 cyclists which were randomly divided into a experimental group, supplemented with sodium phosphate and a control group receiving a placebo The subjects in the experimental group ingested sodium diphosphate in a dose of 50mg/kg of fat free mass per day. The supplement was ingested in even doses, four times per day. The control group received 4g of glucose in gelatin capsules (500mg), which were also divided into 4 even portions. During the experiment a significant (p2max), maximal minute ventilation (VEmax), as well as oxygen pulse (O2/HR). Also a significant decrease in resting and maximal exercise heart rate occurred. This was also true for each exercise load. A significant (p

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb. The level of intermediates is quantitated by the excess deoxyHb consumed over the concentrations of the final products formed. The dominant intermediate has a spectrum that does not correspond to that of Hb(III)NO formed when NO reacts with methemoglobin (MetHb), but is similar to metHb resulting in the spectroscopic determinations of elevated levels of metHb. It is a delocalized species involving the heme iron, the NO, and perhaps the beta-93 thiol. The putative role for red cell reacted nitrite on vasodilation is associated with reactions involving the intermediate. (1) The intermediate is less stable with a 10-fold excess of nitrite and is not detected with a 100-fold excess of nitrite. This observation is attributed to the reaction of nitrite with the intermediate producing N2O3. (2) The release of NO quantitated by the formation of Hb(II)NO is regulated by changes in the distal heme pocket as shown by the 4.5-fold decrease in the rate constant in the presence of 2,3-diphosphoglycerate. The regulated release of NO or N2O3 as well as the formation of the S-nitroso derivative of hemoglobin, which has also been reported to be formed from the intermediates generated during nitrite reduction, should be associated with any hypoxic vasodilation attributed to the RBC.

Six male well trained cross-country skiers were exposed to 6 d of training 4 h/d at an altitude of 2700 m. Living quarters were at 2000 m. Measurements of serum-erythropoietin concentration ([EPO]), erythrocyte- 2,3 diphosphoglycerate (DPG), haemoglobin (HB), packed cell volume (PCV), body mass (BM) and resting heart rate (HR) were made before, during and after moderate-altitude training. Total body haemoglobin (THB) was determined before and after altitude training. Plasma volume (PV) was calculated from THB, PCV and HB. The daily EPO production was calculated from [EPO] and PV. Moderate-altitude EPO production at days 2, 3 and 6 was significantly higher than prealtitude levels. DPG increased significantly at moderate altitude. Pre-altitude and moderate-altitude EPO production vs DPG were correlated. Moderate-altitude levels of BM decreased, PV increased and HB decreased (NS). During moderate-altitude training, DPG exerts a certain feedback regulation of EPO production. The temporal courses of [EPO] and EPO production at moderate altidude are not affected by variations in PV.

Recently we demonstrated an increased 2,3-diphosphoglycerate (2,3-DPG) erythrocyte concentration in rat pups subjected to nucleotide-enriched artificial feeding.The present study was carried out to test the hypothesis that a possible increase in 2,3-DPG concentration can also be obtained in human neonates who are fed nucleotide-enriched formula. Preterm neonates born or referred to the neonatal intensive care unit of the G. Gaslini Hospital, Genoa University, with a gestational age30 weeks and37 weeks were enrolled in our randomized trial. Recruitment took place within 48-72 hours from birth. Only newborns of mothers deciding not to breast-feed were eligible to be randomized for the supplemented group (FN) or non-supplemented group (RF). Breast-fed newborns were considered the control group (C). The study window (for supplementation and blood samples) was restricted to the first two weeks following birth (from the 2nd (t1) to the 16th (t2) day of life). At the end of our study, only 21 neonates were eligible for statistical analysis.The stimulating action of dietary nucleotides on 2,3-DPG concentration failed to be demonstrated; increases in 2,3-DPG concentration that were observed in newborns fed with nucleotide supplemented formula (FN) were comparable to those observed in newborns fed with regular formula (RF) and breast-fed newborns.The EC recommendation for the amount of nucleotides allowed in formula milk does not seem to be high enough to have positive effects on 2,3-DPG synthesis. Whether this possible 'pharmacological' effect can be achieved by a higher intake of ingested nucleotides and/or a change in the proportions of single nucleotides contained in milk formulas remain interesting end points to be elucidated.

Poor performance in racehorses is often due to subclinical respiratory disorders that are likely associated to hypoxia even in resting conditions. During hypoxia, the concentration of 2,3-diphosphoglycerate (2,3DPG) in blood increases, facilitating the release of oxygen to tissues. Thus, 2,3DPG is a candidate biomarker for detection of respiratory origin following a poor performance episode. The objective of this study was to determine the 2,3DPG concentrations in blood from resting horses with poor performance due to respiratory disorders and assess its diagnostic utility. To this aim, 2,3DPG was measured in blood from 11 standardbred horses with inflammatory airway disease (IAD), 5 with exercise-induced pulmonary haemorrhages (EIPH), and 7 control horses. IAD and EIPH were diagnosed by cytology and bacteriology on bronchoalveolar lavage fluids. The diagnostic sensitivity, specificity, and the likelihood ratio of 2,3DPG increases were also evaluated. 2,3DPG (μmol/g Hb) was higher in horses with EIPH (33.77±5.03) and IAD (27.69±7.37) compared to controls (18.23±4.70). This increase was associated with augmented haemoglobin and haematocrit. The optimal cut-off to discriminate horses with respiratory disorders from controls was 21.78 μmol DPG/g Hb. These results demonstrate that 2,3DPG is increased in blood from horses with IAD or EIPH, likely due to the presence of hypoxia and the measurement of 2,3DPG may have clinical utility in the diagnosis of poor performance in horses: elevated 2,3DPG levels may be used to suspect respiratory origin for poor performance in horses before resorting to more expensive and invasive techniques.

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.

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.

Sickle cell disease is a hereditary blood disorder characterized by chronic manifestations of progressive organ failure. To control a disease of this type, it is important to study the factors which affect the course of the disease. Diphosphoglycerate [2,3 DPG] is the most common metabolite generated by the glycolysis. It is a potent modifier for hemoglobin function. To clarify the role of 2,3 DPG in suckling, its concentration was measured in 15 normal health individuals and in 12 sickle cell patients at steady state and during crisis. Hematological indices and partial oxygen tension [PO2] were also determined for sicklers and controls. Sickle cell patients during crisis were found to have a significant higher levels of 2,3 DPG [9.17+5.36 mold/L] [p < 0.05], and significantly low PO2 [8.41 +1.82 Kpa, p < 0.01] than controls [8.54 + 0.72 mmol/L for 2,3 DPG and 12.44 + 0.7 Kpa for PO4]. The increase in 2,3 DPG concentration during crisis may be due to its increased production via glycolysis, which in turn increased as a result of decreased oxygen tension during crisis. The increased 2,3 DPG concentration increases and stabilized the deoxygenated HbS leading to its precipitation, sickling. And hemolysis of red blood cells. In conclusion, the factors which can increase 2,3 DPG or decrease PO4 may lead to the sickling crisis and increase the phenotypic severity of the disease.

The position of the oxygen dissociation curve (ODC) is modulated by 2,3-diphosphoglycerate (2,3-DPG). Decreases in 2,3-DPG concentration within the red cell shift the curve to the left, whereas increases in concentration cause a shift to the right of the ODC. Some earlier studies on diabetic patients have reported that insulin treatment may reduce the red cell concentrations of 2,3-DPG, causing a shift of the ODC to the left, but the reports are contradictory. Three groups were compared in the present study: 1) nondiabetic control individuals (N = 19); 2) insulin-dependent diabetes mellitus (IDDM) patients (on insulin treatment) (N = 19); 3) non-insulin-dependent diabetes mellitus (NIDDM) patients using oral hypoglycemic agents and no insulin treatment (N = 22). The overall position of the ODC was the same for the three groups despite an increase of the glycosylated hemoglobin fraction that was expected to shift the ODC to the left in both groups of diabetic patients (HbA1c: control, 4.6%; IDDM, 10.5%; NIDDM, 9.0%). In IDDM patients, the effect of the glycosylated hemoglobin fraction on the position of the ODC appeared to be counterbalanced by small though statistically significant increases in 2,3-DPG concentration from 2.05 (control) to 2.45 mol/ml blood (IDDM). Though not statistically significant, an increase of 2,3-DPG also occurred in NIDDM patients, while red cell ATP levels were the same for all groups. The positions of the ODC were the same for control subjects, IDDM and NIDDM patients. Thus, the PO2 at 50% hemoglobin-oxygen saturation was 26.8, 28.2 and 28.5 mmHg for control, IDDM and NIDDM, respectively. In conclusion, our data question the idea of adverse side effects of insulin treatment on oxygen transport. In other words, the shift to the left reported by others to be caused by insulin treatment was not detected.

Two haemoglobin components have been identified and purified from fallow-deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same α chain, while two different β chains are detected in the two components. The β chains differ by 14 residues, even though they both have 145 amino-acid residues, which account for a molecular mass of 16 023 and 16 064 Da, respectively, while α chain has 141 residues, yielding a molecular mass of 15 142 Da. Compared with human Hb, the N-terminal region of both β chains shows deletion of Valβ1 and the replacement of Hisβ2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3-diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between β chains of the two haemoglobin components.

An 11-year-old female crossbred dog showed signs of polyuria, polydipsia, vomiting, posterior weakness and ataxia. Clinical and laboratory findings suggested the diagnosis of polycythaemia vera. The haematological values shown over a six-month period are presented. In four samples some aspects of erythrocyte function (glucose-6-phosphate dehydrogenase [G6PD] and pyruvate kinase [PK] activities, 2,3 diphosphoglycerate [2,3 DPG] concentration, osomotic fragility and intracellular sodium and potassium concentrations) were studied. Variable activities of G6PD and PK, probably related to different reticulocyte number, were detected together with normal osmotic fragility and intracellular sodium and potassium concentrations. 2,3 DPG concentration was higher than normal in all four samples. This could be interpreted as a response to a low tissue perfusion rather than a higher content of 2,3 DPG in red blood cells from the polycythaemic dog.

The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS-Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pKa and the relative surface accessibility of the nucleophilic residues, calculated from the X-ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.

Few data are available on the activities of canine erythrocyte enzymes and on 2,3 diphosphoglycerate (2,3DPG) concentrations in pathological conditions other than heritable erythrocyte defects. Because some diseases might affect erythrocyte metabolism and oxygen transport, we evaluated these parameters in 10 healthy dogs and in dogs with symptomless dirofilariosis (n = 9), mild (n = 13) and severe (n = 8) cardiac failure, and haemolytic anaemia (n = 8). To evaluate possible membrane damage, the osmotic fragility of the red cells was measured. No haematological abnormalities were found in the dogs with mild cardiopathy or in those with symptomless dirofilariosis. Severe anaemia and neutrophilic leucocytosis were found in the dogs with haemolytic anaemia and, to a lesser degree, in those with severe heart failure. In dogs with these two diseases, elevated values obtained were, respectively: pyruvate kinase (PK) 17.5 +/- 10.3 U/g haemoglobin (Hb) (P0.001) and 11.6 +/- 7.5 U/g Hb (P0.01); glucose-6-phosphate dehydrogenase (G6PDH) 8.9 +/- 5.4 U/g Hb (P0.001) and 5.6 +/- 4.2 U/g Hb; 2,3DPG 21.8 +/- 4.9 U/g Hb (P0.001) and 22.5 +/- 4.1 U/g Hb (P0.001). The increased 2,3DPG concentrations may have been due to diminished oxygen availability but the observed enzymatic changes were attributed mainly to the presence of young red blood cells: there was a positive correlation between nucleated red blood cells and PK activity, G6PDH activity and 2,3DPG concentration and a negative correlation between mature erythrocytes and PK activity, G6PDH activity and 2,3DPG concentration. This was supported by the derivative curve of the fragiligram, which showed two or three peaks corresponding to different erythrocyte populations and by the negative correlation between the maximum haemolytic NaCl concentration and the reticulocyte number. The measurement of PK and G6PDH activity and of the 2,3PG concentration, together with information provided by the fragiligram, would seem to be of value in defining the clinico-haematological picture in clinical heart diseases and haemolytic anaemia.

The binding of two strong allosteric effectors (2,3 Diphosphoglycerate D.P.G., and Bezafibrate, Bzf) to both adult and the three human embryonic haemoglobins, either individually or in combination, have been studied in detail. The adult protein exhibits one binding site for D.P.G and two for Bzf. When both effectors are present simultaneously their effects are simply additive. The same qualitative pattern of binding is observed in the case of the three human embryonic haemoglobins, although with different binding constants. The lack of synergism between these effectors and the different binding affinity expressed by these proteins are discussed in terms of the known amino acid sequence differences.

A fluorescent analogue of diphosphoglycerate (DPG), hydroxy-pyrenetrisulfonate (HPT), was used as a probe of the allosteric equilibrium of methemoglobin. Like DPG, HPT binds, one per tetramer, with a higher affinity to deoxyHb than to oxyHb. Once bound, the HPT fluorescence is quenched by energy transfer to the hemes. HPT can thus serve as a probe of the conformational state of the hemoglobin tetramer: a higher quenching indicates a stronger binding and therefore, more of the deoxy conformation. Since HPT binds to the same site as DPG, it can be displaced by DPG in order to determine the fluorescence intensity of the free HPT under the same conditions, to correct for the inner filter effect. The high spin ferric ligands, such as water and F, showed less fluorescence (more of the deoxy state) than low spin cyano-metHb. The aquo-metHb samples showed a reversion to the oxyHb conformation above pH 7, as expected due to the acid–alkaline transition forming hydroxy-metHb. Effectors such as bezafibrate, which do not bind to the same site as DPG, show an increase in the deoxy-like characteristics.

The occurrence of abnormal levels of AI(III) connected with human pathologies and with encephalopathies experimentally induced in experimental animals is reviewed and critically evaluated in molecular terms. It is assumed that phosphate-bearing biomolecules are elected targets for the biochemical action of Al(III) and the reactivity of the metal centre towards both inorganic phosphates and nucleotides, diphosphoglycerate, phosphorylated proteins, phospholipids and myo-inositol-phosphates is thoroughly reviewed. All these data are evaluated in the light of the potential relevance of higher Al(III) levels to the etiopathology of dialysis dementia and of Alzheimer's disease.

Objective: to describe a clinical case where an extremely low erythrocyte 2,3-diphosphoglycerate concentration (2,3-DPG) was discovered by routine blood gas analysis supplemented by computer calculation of derived quantities. The finding of a low 2,3-DPG revealed a severe hypophosphatemia.Design: open uncontrolled study of a patient case.Setting: intensive care observation during 41 days.Patient: A 44 year old woman with an abdominal abscess.Interventions: Surgical drainage, antibiotics and parented nutrition.Measurements and results: daily routine blood gas analyses with computer calculation of the hemoglobin oxygen affinity and estimation of the 2,3-DPG. An abrupt decline of 2,3-DPG was observed late in the course coincident with a pronounced hypophosphatemia. The fall in 2,3-DFG was verified by enzymatic analysis.Conclusion: 2,3-DPG may be estimated by computer calculation of routine blood gas data. A low 2,3-DPG which may be associated with hypophosphatemia causes an unfavorable increase in hemoglobin...

As the available hemoglobin A1 at birth ranges from 20 to 30% a possible mechanism to favor oxygen release to the tissues could be a decrease of hemoglobin A1 affinity to oxygen. This may be accomplished by an increase in blood pH soon after birth and by an elevation in red cell 2,3-diphosphoglycerate (2,3-DPG). This hypothesis is supported by Valleri and Hirsch, who described a rapid 2,3-DPG recovery of transfused depleted 2,3-DPG red cells. That being so, we carried out this current study by assaying the 2,3-DPG of cord blood from 22 newborns and at 6, 24 and 72 hours after birth, as well as those enzymes assumed to be envolved in the 2,3-DPG levels regulation. 2,3-DPG (nmoles g-1 Hb) demonstrated the following values: cord blood: 9,770 +/- 1,026; 6h: 12,773 +/- 1,726; 72 h: 11,990 +/- 728, unveiling a distinct behavior of a sharp increase of 30% by the sixth hour. This confirmed our hypothesis. Regarding the metabolic mechanisms which can account for the 2,3-DPG increase, besides the rise of blood pH, we detected a significant decrease of the 2,3-DPG phosphatase activity, which might diminish the 2,3-DPG breakdown.

The literature on the response of erythrocyte 2,3-diphosphoglycerate (2,3-DPG) following exercise is replete with inconsistencies, and recent studies have shown that the time of blood sampling during and following exercise, as well as the duration of exercise, are important in evaluating the response of 2,3-DPG. Experiments were designed to measure the response of 2,3-DPG following short-term strenuous exercise in two groups of untrained men. Twelve men, 19-22 years old (study 1), exercised on a bicycle ergometer at 122.5 W for 10 min and red blood cell (RBC) 2,3-DPG was measured at 0 and 50 min following exercise. The level of 2,3-DPG (mumol.ml-1 RBC) increased after exercise (P < 0.05), but this increase was not significant when 2,3-DPG was expressed as mol.mol-1 hemoglobin (Hb). However, following 50 min of rest, 2,3-DPG (mol.mol-1 Hb) decreased significantly. In a second group (study 2), nine other men, aged 18-19 years, exercised at the same workload for 15 min and 2,3-DPG was measured at 0, 30, 60, 180, and 330 min respectively after exercise, and no significant mean changes in the level of the phosphate were observed. Findings from these studies suggest that 2,3-DPG does not provide a compensatory adjustment to facilitate oxygen delivery in the hypoxia of short-term strenuous exercise in untrained males immediately following exercise and when recovery intervals of up to 330 min are also examined. It is suggested that 2,3-DPG be reported as mol.mol-1 Hb, since the phosphate exists on Hb in an equimolar ratio in normal physiological states.

Physical exercise appears to affect both blood magnesium status and erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration. Concentrations of erythrocyte and plasma magnesium (ErMg and PlMg) and erythrocyte 2,3-DPG were measured three times over a period of 2 months in a group of 11 athletes who were training for a marathon. The concentration of 2,3-DPG was found to be significantly increased at the end of the 1st month (P < 0.05) compared to its level at the beginning of the study. However, at the end of the 2nd month, it was significantly lower (P < 0.05) than at the end of the 1st month. This decrease might have been due to the reduction in the intensity of training, despite an increase in the training distance. Both ErMg and PlMg did not change significantly after the 1st and 2nd months of training. However, the decrease of total circulating magnesium, i.e., whole blood magnesium was significant, after both the 1st and 2nd months. This decrease may have been due to an increased loss of magnesium or to a shift of magnesium from the blood to other compartments. We observed a significant negative correlation between ErMg and 2,3-DPG after the 1st month: r = -0.59, P < 0.05. We hypothesized that this inverse relationship might have been due to the sympathetic stimulation secondary to physical stress. Furthermore, in view of the mechanism of binding ErMg and 2,3-DPG by haemoglobin, the negative correlation between ErMg and 2,3-DPG might have been due to the relative tissue hypoxia that accompanies aerobic exercise.

The haemoglobin-2,3-diphosphoglycerate complex structure has been solved at 2.5 A resolution using crystals grown from low-salt solutions. The results show some important differences with the precedent haemoglobin-2,3-diphosphoglycerate high-salt structure solved by Arnone. First, we observe a loss of symmetry in the binding site, secondly both of the lysine residues 82 beta interact with 2,3-diphosphoglycerate at the same time, each making two contacts. This level of interaction is in agreement with the functional behaviour of natural haemoglobin mutants with mutations at the 2,3-diphosphoglycerate binding site.

Red blood cells can be stored in liquid suspension in approved additive solutions for periods up to 6 weeks with 0.4% hemolysis, 84% 24-h in vivo recovery, and normal subsequent survival of the cells that persist in the circulation for at least 24 h. However, while they are stored, the red cells undergo changes including the loss of adenosine triphosphate, diphosphoglycerate, and potassium, oxidative injury to proteins, lipids, and carbohydrates, loss of shape and membrane, increased adhesiveness, decreased flexibility, reduced capillary flow, and decreased oxygen delivery. Deaths have been reported related to the high potassium and lysophospholipids, but are rare.

A novel method was developed to quantify the intermediates involved in cyclic 2,3-diphosphoglycerate metabolism, i.e. cyclic 2,3-diphosphoglycerate, 2,3-bisphosphoglycerate, 2-phosphoglycerate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate and phosphate. The method consists of an ion-chromatographic separation followed by conductivity and ultraviolet detection. In a single run (41 min) all compounds were readily resolved, except for 2- and 3-phosphoglycerate. Concentrations down to 10-20 μM and up to 1.0-1.5 mM can be accurately determined. The method was suitable for the analysis of cell-free extract and for the determination of enzymic conversions of the compounds involved. It was found to be reliable and faster than isotachophoresis or enzymic determination.

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (Ki=150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.

We have established that 2,3-diphosphoglycerate (2,3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (csat) of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic csat assay system, a steep dependence of csat on pH in the physiologic range 7.0 to 7.6 was shown for both stripped (Hb) and DPG-saturated deoxy-Hb S (Hb-DPG). The solubility-pH profile for Hb under near-physiologic buffer conditions also showed that csat increased steeply in the same pH range (6.8 to 7.6). The effect of 2,3- DPG on csat under near-physiologic conditions was evaluated separately. At pH 7.20, the pH of the human red blood cell, csat values for Hb and Hb-DPG were 19.56 +/- 0.14 and 17.95 +/- 0.45 g/dL, respectively, indicating that the solubility of Hb-DPG is lower than that of Hb by 8.2% +/- 2.3%. Thus, binding of 2,3-DPG in the beta-cleft promotes the polymerization of deoxy-Hb S, the ultimate determinant of cell sickling. Furthermore, because of the abnormal Bohr effect of sickle blood (approximately double that of normal blood), the intracellular pH of deoxygenated sickle erythrocytes should be approximately 0.28 pH unit higher than that of oxygenated cells (ie, 7.41 v 7.13). At the higher pH, the corresponding csat for Hb-DPG is 20.22 g/dL, which is the best estimate of the intrinsic solubility of T-state Hb S under conditions that approximate closely those of pH, temperature, ionic strength, and 2,3-DPG saturation in the fully desaturated sickle erythrocyte.

Magnesium deficiency lowers endurance capacity of untrained rats by an as yet unknown mechanism. We tested the hypothesis that low plasma and blood Mg concentrations would reduce blood 2,3-diphosphoglycerate (2,3-DPG) concentrations, thus diminishing the oxygen delivery system of the Mg-deficient rat. Untrained male Osborne-Mendel rats (95 g) were studied at rest or following exhaustive exercise after three weeks of dietary treatment (400 or 100 micrograms Mg/g diet; deionized or high Mg water [greater than 85 micrograms Mg/ml]). Magnesium deficiency resulted in lowered plasma and red blood cell (RBC) Mg concentrations and reduced endurance capacity compared with controls. Magnesium supplementation normalized these parameters. 2,3-DPG was elevated by 10% in rats acutely exercised compared with sedentary rats; however, 2,3-DPG levels were not affected by dietary Mg intake. Acute exercise tended to increase erythrocyte Mg concentrations in control rats and lower erythrocyte Mg levels in deficient rats. In conclusion, the reduced endurance capacity of Mg-deficient rats is not due to impaired 2,3-DPG production.

To investigate the claim that equine erythrocytes released from the spleen are older cells than those found at rest in the circulation, the 2,3, diphosphoglycerate (2,3 dpg ), adenosine triphosphate ( atp ) and creatine concentration of erythrocytes before and following splenic emptying were examined. Normal values for thoroughbreds (43) and ponies (10) at rest were established. Following either exercise or intravenous injection of adrenaline in six thoroughbreds, there was an increase in erythrocyte creatine content and a decrease in atp concentration. Exercise produced a slight increase in 2,3 dpg while no change occurred with adrenaline. In two splencctomised ponies adrenaline only produced a decrease in erythrocyte atp , with no change in creatine or 2,3 dpg content. It was concluded that erythrocytes released from the spleen are not aged cells. However, the stimuli used produced a decrease in atp content which may affect the properties of the erythrocytes.

Cyclic 2,3-diphosphoglycerate (cDPG), a metabolite exclusively present in methanogenic bacteria, was separated from other phosphate-containing compounds in extracts of Methanobacterium thermoautotrophicum by means of isotachophoresis. The detection limit was 100 pmol per injected sample. Quantification of intracellular concentrations of cDPG with the isotachophoretic assay and with a spectrophoto-metric cDPG assay gave comparable results, but the isotachophoretic assay was faster, less laborious and more sensitive. A possible involvement of cDPG in the energy metabolism was indicated by studies of Mb. thermoautotrophicum in batch and continuous cultures.

Two abnormal hemoglobins (Hbs) with a substituted amino acid residue at the 2nd position of the B chain, a part of the 2,3-diphosphoglycerate (2,3-DPG) binding site, have been reported: Hb Deer Lodge (His+Arg) (1) and Hb Okayama (His-Gln) (2). In 1985, we found a new abnormal Hb during assay of Hb A1c and Hb A1 by high performance liquid chromatography (HPLC) in a 38-year-old Japanese female patient suffering from diabetes mellitus. This abnormal Hb was designated Hb Fukuoka [B2(NA2)HisTyr] after the name of the city where the patient lived. Herein, we describe the structural analysis and functional properties of this Hb.

The blood red cell contains millimolar amounts of 2,3-diphosphoglycerate, yet no metal ion stability constants appear to have been determined. We show that in the free ligand the 2-phospho group is about six times more basic than the 3-phospho group. We have determined the stability constants for the binding of Zn 2+ which occurs at about 0.2 mM in the red cell, and of Al 3+ which reduces red cell size and inhibits haemoglobin synthesis. At physiological pH both metal ions occur predominantly as MA 2 complexes. The free Zn 2+ concentration permitted within the red cell by the 2:1 complex is about 0.3 μm. 2,3-Diphosphoglycerate binds Al 3+ strongly enough so that at equilibrium there should be comparable concentrations of Al 3+ on both sides of the red cell membrane.

INTRODUCTION: The concentration of 2,3-diphosphoglycerate (2,3-DPG/Hct) increases as a physiological occurrence to pH increase and hyperventilation. This response was tested in patients with severe traumatic brain injury (TBI). METHOD: The concentration of 2,3-DPG/Hct was measured daily for six days in eleven patients with severe TBI in need of optimized hyperventilation because of intracranial hypertension. RESULTS:There was correlation between pH and the concentration of DPG/Hct. The concentration of 2,3-DPG/Hct remained predominantly within normal levels with slight increase in the sixth day of the study. The concentration of 2,3-DPG/Hct correlated significantly with measured partial pressure of oxygen that saturates 50% the hemoglobin of the blood (P50st), confirming the consistency of our data. CONCLUSION: The expected physiological response of a progressive increase in concentration of 2,3-DPG/Hct to hyperventilation was not observed. This fact may be explained by the intermittent and not sustained hyperventilation as dictated by the protocol of optimized ventilation. INTRODUÇÃO: A concentração de 2,3-difosfoglicerato nos eritrócitos (2,3-DPG/Hct) aumenta como ocorrência fisiológica ao aumento do pH e à hiperventilação. Esta resposta foi testada em pacientes com traumatismo craniencefálico (TCE) grave. MÉTODO: A concentração de 2,3-DPG/Hct foi medida diariamente, durante seis dias, em 11 pacientes com TCE grave necessitando de hiperventilação otimizada por causa da hipertensão intracraniana. RESULTADOS: Houve correlação entre o pH e a concentração de 2,3-DPG/Hct. A concentração de 2,3-DPG/Hct permaneceu predominantemente dentro da normalidade, com ligeira tendência à elevação no sexto dia de estudo. Houve correlação entre a concentração de 2,3-DPG/Hct e a pressão parcial de oxigênio que satura 50% da hemoglobina (P50st), confirmando a consistência dos dados. CONCLUSÃO: A esperada resposta fisiológica de aumento progressivo da concentração de 2,3-DPG/Hct não foi observada. Este fato pode ser explicado pela aplicação intermitente e não sustentada de hiperventilação, como ditado pelo protocolo clínico de ventilação otimizada.

Hb Taradale [beta82(EF6)Lys--Arg] was initially detected as a split Hb A0 peak on Hb A1c, monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant beta chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide betaT-9. DNA sequencing confirmed the presence of a novel beta82(EF6)Lys--Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.

1. 1. A 6 hr time-course response of erythrocyte 2, 3-diphosphoglycerate was studied in rabbits following adrenaline administration. 2. 2. Eight female New Zealand white rabbits weighing about 3.6 Kg each were injected intra-peritoneally with a total of 0.97 mg/kg of adrenaline (0.56 mg/kg at time 0 min and 0.41 mg/kg at time 0.5 hr), and the venous level of red blood cell (RBC) 2,3-DPG was monitored at 0 hr, 1 hr, 3 hr, and 6 hr, respectively. As controls, the level of 2,3-DPG was also monitored in these rabbits weeks prior to the experiment. 3. 3. A significant (p

It is shown that in deep acute hypoxic hypoxia the 2,3-diphosphoglycerate content is reduced and the adenosine triphosphate content unchanged in the erythrocytes of newborn rats. Under the same conditions adult animals show an increase of 2,3-diphosphoglycerate and a drop in the adenosine triphosphate content in the erythrocytes. The importance of these changes is discussed in terms of the mechanisms regulating the oxygen-transporting function of erythrocytes during acute hypoxic hypoxia in newborn and adult animals.

We investigated whether 2,3-diphosphoglycer- ate (2,3-DPG) is altered in patients with low cardiac output and the influence of its concentration on the calculation of in vivo P50. Biochemical and blood gas analysis were per- formed along with the measurement of cardiac output and body temperature in 13 patients submitted to cardiopul- monary bypass surgeries without the use of donor blood. In vivo P50 was calculated using the measured (P50m) and the estimated 2,3-DPG (P50e). 2,3-DPG concentration was lower in these patients when compared to the values obtained in normal volunteers (6.9 ± 2.2 vs. 11.9 ± 2.4 mmol/gHb). P50m was lower than P50e (21.6 ± 1.1 vs. 30.1 ± 1.2 mm Hg) at all time points. Our data show that in patients with low cardiac output, 2,3-DPG concentration is reduced. Therefore, in these patients, the use of standard values for this variable may introduce an error in the cal- culation of in vivo P50. Key Words: 2,3-Diphosphoglycer- ate—In vivo P50.

Glucose metabolism is a cellular necessity for maintenance of the life process. The two important biochemical intermediates in glucose metabolism on red blood corpuscles (RBC) are adenosine triphosphate (ATP) and 2,3 diphosphoglycerate (2,3 DPG). ATP is responsible for maintenance of the size and shape of RBC and 2,3-DPG, and for the oxygen carrying capacity (the raison d'etre) of RBC.

A 2.16 A resolution structure of high-salt human carbonmonoxyhemoglobin crystallized at pH 6.4 is reported. The quaternary structure is similar to that of 'classic' R-state hemoglobin; however, subtle but significant tertiary structural changes are observed at the alpha(1)beta(2) and symmetrically equivalent alpha(2)beta(1) interfaces--these are the key subunit interfaces that govern the allosteric transition between the R and T states. Specifically, the movement and weakening of two important hydrogen bonds that are diagnostic for R-state structures, beta(2)His97-alpha(1)Thr38 and beta(2)Arg40-alpha(1)Thr41, have been observed. In addition, a phosphate molecule bound between the two beta-subunits (at the entrance to the central water cavity) has been identified and electron density indicates that this molecule occupies two alternate positions that are related by the dyad axis. Both positions superimpose on the 2,3-diphosphoglycerate binding site. One phosphate conformer interacts with beta(2)Asn139, beta(1)His143 and beta(1)His146, while the second interacts with symmetry-related counterparts (beta(1)Asn139, beta(2)His143 and beta(2)His146).