Influence of bicarbonate on the content of 2,3-DPG (bisdihydroglycerophosphate) in preserved erythrocytes]

Both whole blood in ACD-adenine-guanosine solution (PCV about 36%) and resuspensions of erythrocytes from EDTA-blood (PCV about 80%) were stored at 4 degrees C with an admixture of sodium bicarbonate. To the resuspensions simultaneously were added xylitol and inorganic phosphate. The erythrocyte concentration of 2,3-disphosphoglycerate in whole blood will be preserved the longer the more bicarbonate is added. Compared with whole blood in resuspensions of erythrocytes the preservation of 2,3-DPG is significantly prolonged, probably because xylitol was added. From these results and from literature data it is concluded that bicarbonate is fixed to metabolites of the pentose metabolism. The products of the fixation of bicarbonate may influence the 2,3-DPG metabolism of the erythrocytes.     

Decreased level of 2,3-diphosphoglycerate and alteration of structural integrity in erythrocytes infected with Plasmodium falciparum in vitro

2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis.     

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.     

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.     

Purification and regulatory properties of pigeon erythrocyte pyruvate kinase

1. Pigeon erythrocyte pyruvate kinase (PK) was purified 22,000 fold by successive column chromatography on Sephadex DEAE A-50 and Red Agarose. The resulting enzyme preparation had a specific activity of 815.3 U/mg protein and an overall yield of 18.5%. 2. The molecular weight, as determined by gel filtration on Sephadex G-200 was 152,000. 3. Isoelectric focusing in the pH range of 3-10 showed that pigeon erythrocyte contained at least 3 PK isozymes with isoelectric points of 5, 5.7 and 6. 4. The variation of activity of PK at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. The Km values for ADP and PEP were 0.40 and 0.46 mM respectively. 5. The enzyme was activated by FDP, and inhibited by ATP, highly phosphorylated inositol derivatives and 2,3-DPG: 6. It was activated by K+ and Mg2+ ions. 7. Phosphorylated hexoses and Pi stimulated the activity of PK. 8. The regulatory role of PK of pigeon erythrocytes, which lack the typical 2,3-DPG bypass, is discussed.     

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 procedure for decreasing the level of 2,3-bisphosphoglycerate in red cells in vitro

The physiological adaptation to anemia and other hypoxic states includes an increase in the level of 2,3-bisphosphoglycerate (2,3-DPG) in the red cell. We suggest that the high level of 2,3-DPG may have adverse effects in vivo. It has been found that red cells incubated with glycolate lose 2,3-DPG at a rapid rate relative to controls. ATP is stable. Net 2,3-DPG synthesis is observed after the glycolate is removed from the cells suggesting that they are not harmed. The effect appears to be specific for glycolate since lactate, glyoxylate, glycerate, acetate, and citrate were without effect. This procedure could be used to assess the effects of decreasing the 2,3-DPG level to normal in the erythrocytes of sickle cell and other anemias.     

Effects of whole body UV-irradiation on oxygen delivery from the erythrocyte

In 16 healthy caucasian volunteers (mean age: 22.2 years) the influence of whole body UV-irradiation on the oxygen transport properties of erythrocytes was investigated. Four hours after irradiation with UV (using the minimal erythema dose, MED) no variation of haemoglobin concentration, hematocrit, mean corpuscular haemoglobin concentration, pH or standard bicarbonate could be found, whereas inorganic plasma phosphate (Pi), calcium, the intraerythrocytic 2,3-diphosphoglycerate (2,3-DPG), the activity of erythrocytic phosphofructokinase (PFK) and pyruvatekinase (PK) increased significantly. The half saturation tension of oxygen (P50-value) tended to increase. The increase of Pi causes--via a stimulation of the glycolytic pathway--an increase in 2,3-DPG concentration and thus results in a shift of the oxygen dissociation curve. It is therefore possible to enhance tissue oxygenation by whole body UV-irradiation.     

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).     

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.     

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.     

Human erythrocyte 2,3-diphosphoglycerate metabolism. Influence of 1,3-diphosphoglycerate and Pi. In vitro studies at low pH with computer simulations.

The kinetics of 2,3-diphosphoglycerate (2,3-DPG) net breakdown was examined in intact human erythrocytes incubated at pH 7.00 and 37 °C. The concentrations of 2,3-DPG, 1,3-diphosphoglycerate (1,3-DPG), 3-phosphoglycerate, ATP, P_i, glucose, and lactate were determined during 10 to 12 h. Since the concentration of 1,3-DPG has been suggested to be the main regulating factor with respect to the rate of 2,3-DPG net breakdown the interdependence between the concentration of 1,3-DPG and pH was determined in the range of pH 6.9 to 7.4. It was found that the stationary level of 1,3-DPG decreased strongly with decreasing pH within this range. Qualitatively, the net breakdown of 2,3-DPG observed at pH 7.00 can be explained by the lowered level of 1,3-DPG. The influence of the concentration of P_i upon the rate of net degradation of 2,3-DPG at pH 7.00 was studied at low cell volume fraction (0.04), where given concentrations of P_i could be maintained for several hours. A marked increase in the rate of 2,3-DPG net breakdown by P_i was demonstrated. Computer simulations showed that activation of diphosphoglycerate phosphatase by the increasing concentration of P_i and decrease of degree of inhibition of the diphosphoglycerate mutase by the decreasing concentration of 2,3-DPG may well keep the rate of the degradation balanced at the time constant value observed. On the basis of the observed kinetics and a computer simulation, the flux through the phosphoglycerate bypass was estimated to be 10 to 15% of the total glycolytic flux at physiological conditions.     

Pharmacological correction of microcirculation in rats suffering from traumatic brain injury

We investigated the effect of mexicor on functional indices of erythrocytes and the structure of myocardial microcirculation in rats suffering from traumatic brain injury (TBI). At 3, 7, and 12 days after TBI, we measured the concentration of 2,3-diphosphoglycerate (2,3-DPG) and the degree of erythrocyte aggregation and their electrophoretic mobility (EPME) in the blood of rats, as well as analyzing sections of the left ventricular myocardium. The first day after the TBI, we observed a decrease in EPME, an increase of erythrocyte aggregation, and an increase of 2,3-DFG concentration in erythrocytes as compared with intact animals. Intraperitoneal injection of mexicor led to an increase of EPME and 2,3-DPG level and reduced the aggregation of erythrocytes, which was most pronounced during the 3–7 days of the post-traumatic period. Improved functional parameters of erythrocytes were accompanied by the dynamics of regenerative processes in the heart. Intraperitoneal injection of mexicor restrained architectonic damage of microvasculature and cardiomyocytes ultrastructure of the left ventricular myocardium of the heart.     

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.     

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     

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.     

Response of erythrocytic 2,3-diphosphoglycerate to strenuous exercise.

Since increases of erythrocytic 2,3-diphosphoglycerate (2,3-DPG) have been shown to enhance the release of oxygen from hemoglobin, experiments were designed to evaluate the response of 2,3-DPG to two different work-loads in 13 fasted human subjects. No signficant mean change in 2.3-DPG was found following 16 min of strenuous exercise on a bicycle ergometer, but when the subjects were subjected later to a greater workload for 20 min, there was a significant mean decrease in 2.3-DPG despite much individual variation. In addition, there was a significant positive correlation of 2.3-DPG reduction with increases in postexercise lactate, and a significant inverse correlation of oxygen consumption during exercise with postexercise lactate. The data suggest that the 2.3-DPG mechanism may not be compensating in exercise when the workload requires a preponderance of anaerobic metabolism promoting lactacidaemia.     

Correction of Metabolic Indicators of Erythrocytes and Myocardium Structure with Ozonized Red Blood-Cell Mass

The early posttransfusion period after acute blood loss using erythromass without ozone was characterized by decreased electrophoretic mobility of erythrocytes (EPME), ATP, 2,3-diphosphoglycerate (2,3- DPG) concentration, Na⁺-K⁺-ATPase activity, and increased concentration of malonic dialdehyde (MDA) and catalase activity in erythrocytes. Most parameters gradually were restored over 5 days, but a reduced concentration of 2,3-DPG was observed throughout the entire experiment with transfusion of erythromass without ozone. Transfusion of the ozonized erythrocyte mass 1 h after its administration caused an increase in 2,3- DPG concentration in erythrocytes circulating in the vascular bed. Twenty-four hours later, increased catalase activity, EPPE, and ATP concentration were registered, and, after 5 days, enhanced Na⁺-K⁺-ATPase activity was registered. Their values were maintained until the end of the experiment at a higher level than in animals that had undergone transfusion of the nonozonized erythromass. Ozonized erythrocyte mass improves the functional metabolic state of erythrocytes, promotes an earlier recovery of the oxygen transport in blood, and limits the damage to the microcirculatory bed of the myocardium and cardiomyocytes during blood loss.     

Hypophosphatemia Induced by Dietary Aluminium Hydroxide Supplementation in Growing Pigs: Effects on Erythrocytes,Myocardium, Skeletal Muscle and Liver

Three groups of pigs were studied during and after 10 weeks of treatment with either Al(OH)3 (Al[OH]3-group, n=8) to induce hypophosphatemia, A1P04 (AlP04-group, n=8, aluminium control without hypophosphatemia) or no addition to the feed (control group, n=8). Blood samples were taken at the start of the experiment and after 3, 6 and 10 weeks and were analyzed for phosphate, calcium and 2,3-diphosphoglycerate (2,3-DPG). Samples from myocardium, skeletal muscle and liver were obtained in connection with exsanguination and analyzed for glycogen, adenosine-tri-phosphate (ATP), creatine phosphate (CP), glucose-6-phosphate (G-6-P) and lactate. The Al(OH)3-group became hypophosphatemic and hypercalcémie with low levels of 2,3-DPG in erythrocytes within 3 weeks and showed a retarded growth rate. After 10 weeks the Al(OH)3-group had low levels of ATP in myocardium as compared with the control-group and low levels of G-6-P as compared with the AlP04-group. No disturbances on electro-cardiograms registered at rest could be documented. G-6-P concentration was low in the biceps muscle in the Al(OH)3-group as compared with the AlP04-group and in the liver low G-6-P concentration was seen in addition to high lactate concentration. The fibre type composition in M. Longissimus did not differ between groups, but the Al(OH)3-group had, due to retardation in growth, smaller mean fibre-areas than pigs in the AlP04-group. Hypophosphatemia gave rise to high serum calcium levels, low concentration of 2,3-DPG in erythrocytes and influenced G-6-P concentration in skeletal muscle, G-6-P and ATP in myocardium, G-6-P and lactate in liver. Retarded growth was one serious consequence of hypophosphatemia and the disturbed energy metabolism.     

Comparison of carbonic anhydrase activity and other haematological values of two Eutherian and three Australian marsupial mammals

1. Carbonic anhydrase activity and 2,3-diphosphoglycerate (2,3-DPG) concentration were determined in whole blood from humans (Homo sapiens), rabbits (Oryctolagus cuniculus), eastern grey kangaroos (Macropus giganteus), pademelons (Thylogale billardierii) and brush-tailed possums (Trichosurus vulpecula). 2. Marsupial blood carbonic anhydrase activity increased as species body size decreased. 3. T. billardierii haemoglobin was found to have a polymorphism which may be the same (beta 2 = histidine or glutamine) as that of M. giganteus. 4. The concentration of 2,3-DPG int e red cells of T. billardierii was approximately equal to that of the haemoglobin tetramer. Levels of 2,3-DPG in the other species were similar to those previously reported.