The effects of acute phosphate supplementation in subjects of different aerobic fitness levels.

Six trained cyclists (high-fitness group) and six untrained individuals (low-fitness group), performed a 20-min cycle ergometer exercise test at 70% of maximum oxygen consumption (VO2max) followed by a 30-min rest period and then an incremental ride to exhaustion on two occasions, 1 week apart. Ninety minutes prior to exercise subjects consumed a drink containing either 22.2 g dibasic calcium phosphate (DCP; treatment) or calcium carbonate (placebo). Blood was drawn prior to drink ingestion, during submaximal exercise, during recovery and at exhaustion for determination of blood 2,3-DPG, blood ATP, plasma lactate, plasma phosphate, haemoglobin and haematocrit. Throughout exercise, cardiorespiratory variables [oxygen uptake (VO2), minute ventilation, (VE), respiratory exchange ratio, heart rate and oxygen pulse] were monitored, and ratings of perceived exertion obtained. Although there was a trend for the low-fitness group to have a higher plasma phosphate concentration prior to treatment ingestion, no treatment effects on plasma phosphate were noted at any sample time in either group. 2,3-DPG, VO2, oxygen pulse, VE, time to exhaustion and VO2max were significantly higher in the high-fitness group; however, no differences in these variables were observed as a result of phosphate ingestion. Plasma lactate was significantly lower in the high-fitness group during the submaximal exercise and the recovery period, but again phosphate ingestion had no effect. These results suggest that acute DCP supplementation is not effective as an ergogenic aid and that aerobic fitness level does not affect the response to phosphate supplementation.

Erythrocyte 2,3-diphosphoglycerate depletion associated with hypophosphatemia detected by routine arterial blood gas analysis.

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 parenteral 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-DPG 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 oxygen affinity which reduces the oxygen release to the tissues.

Effects of multibuffer supplementation on acid-base balance and 2,3-diphosphoglycerate following repetitive anaerobic exercise.

The purpose of this investigation was to determine the effects of a 3.5-day dietary multibuffer supplement (containing predominantly inorganic phosphate, or Pi, along with bicarbonate and carnosine, i.e., PhosFuel) on repetitive (four trials separated by 2 min rest) Wingate test (WT) performances and whole blood 2,3-diphosphoglycerate (2,3-DPG) concentrations in 10 recreationally trained road cyclists (T) and 10 normally active but untrained (UT) men. A 2-week washout period was utilized between experimental sessions. Venous blood samples were obtained via cannula once before exercise (baseline), immediately post each WT, and 3 min after the final WT (recovery). The data indicate that this supplement does not affect acid-base status with following intense anaerobic exercise and does not improve repetitive WT performance. However, the supplement does enhance post-exercise levels of 2,3-DPG and the 2,3-DPG/Hb ratio in recreationally trained cyclists while improving acute recovery of peak power in these men.

Autotransfusion after open heart surgery: the oxygen delivery capacity of shed mediastinal blood is maintained.

Autotransfusion of mediastinal shed blood after open heart surgery has become a common and accepted procedure in reducing the need for homologous transfusion during the last 15 years. The objectives of the present study were to investigate the oxygen delivery capacity of autotransfused shed mediastinal blood, compared to patient-blood, during cardiopulmonary bypass and in the postoperative period. Ten consecutive patients undergoing elective cardiac surgery were studied. Mediastinal shed blood was collected in the cardiotomy reservoir and retransfused during the first 18 postoperative hours. The oxygen delivery capacity of the blood to the tissues was calculated by use of the oxygen status algorithm (OSA 2.0) programme and measurement of the 2,3-diphosphoglycerate (2,3-DPG) concentration. Autotransfusion volume ranged from 450-1530 ml per patient (median 824 ml). Shed blood had a mean haemoglobin level of 8.8 g/dl and 7.4 g/dl at 1 h and 6 h of autotransfusion, respectively. There were no significant changes of 2,3-DPG concentration in the patient-blood during cardiopulmonary bypass or after autotransfusion compared to preoperative values. P50 for oxygen (3.6 and 3.6 kPa) and 2,3-DPG concentrations (5.3 and 5.1 mikromol/ml erythrocyte) in shed mediastinal blood (1h and 6h postoperatively) were not significantly different compared to patient-blood. The results demonstrate that the oxygen delivery capacity of shed mediastinal blood is maintained and that the oxygen affinity of patient-blood is not influenced by autotransfusion.

The effects of KBT-3022, a new anti-platelet agent, on hemorheological properties in guinea pigs.

Using guinea pigs, a study was conducted on the effects of KBT-3022, a new anti-platelet agent, on hemorheological properties in various tests including blood filterability, blood viscosity, shear stress-induced red blood cell (RBC) deformability and contents of ATP and 2,3-diphosphoglycerate (2,3-DPG). Oral administration of KBT-3022 at 1 and 10 mg/kg significantly increased blood filterability, and significantly reduced blood viscosity at 10 mg/kg without changing the hematocrit, plasma fibrinogen concentration or plasma viscosity. KBT-3022 (10 mg/kg, p.o.) improved RBC deformability in response to shear stress, which was evoked by passing the blood through a thin tube. This dose of KBT-3022 also increased the contents of ATP and 2,3-DPG in RBC. These findings indicate that KBT-3022 may reduce blood viscosity as a sequel to improvement of RBC deformability through direct action on RBC. The increase in the intracellular levels of ATP and 2,3-DPG was considered to be involved in this improvement of hemorheological properties. These hemorheological effects of KBT-3022 appear to be promising for the treatment of patients with ischemic vascular disease.

[The effect of exogenous iron on levels of adenosinetriphosphate and 2,3-diphosphoglycerate in erythrocytes of men during extreme physical exertion]. / Wplyw egzogennego zelaza na stezenie adenozynotrifosforanu I 2,3-difosfoglicerynianu w erytrocytach mezczyzn przy duzych obciazeniach fizycznych.

Nine men were examined during a three-week training requiring much physical effort. They were given nutrient, "LIVEX", enriched with iron. Hematological parameters as well as concentration of erythrocyte ATP and 2,3-DPG were determined before and after the experiment. Hematological parameters were determined using standard methods while Boehringer's test (Germany) was used for determining ATP and 2,3-DPG. The level of reticular cells was statistically higher after the experiment, and the increase in ATP and 2,3-DPG concentration was insignificant. A positive adaptation of energy metabolism after exogenous iron administration during physical effort was discussed.

Alterations in plasma phosphorus, red cell 2,3-diphosphoglycerate and P50 following open heart surgery.

OBJECTIVES: To evaluate changes in and the correlation between plasma phosphorus, red cell 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP), and P50 in children following heart surgery. EXPERIMENTAL DESIGN: Prospective, observational study with factorial design. SETTING: A pediatric intensive care unit in a university hospital. PATIENTS: Twenty children undergoing open heart surgery for congenital heart defects. INTERVENTIONS: None. MEASURES: Red cell 2,3-DPG and ATP, P50, plasma phosphorus, and arterial lactate were obtained before and at 1, 8, 16, 24, 48, and 72 hours after surgery. The amount of intravenous fluid and glucose administered, and age of blood utilized were documented. Variables were analyzed by repeated measure analysis of variance followed by paired t-tests. To investigate the relationship between variables at each time point, scatterplot matrices and correlation coefficients were obtained. RESULTS: There was a reduction in plasma phosphorus, red cell 2,3-DPG, and P50 and an increase in arterial lactate at 1, 8, 16, 24, 48, and 72 hours after surgery. Red cell 2,3-DPG correlated with P50 at 1, 8 and 16 hours. The decrease in the plasma phosphorus correlated with the amounts of intravenous fluid and glucose administered on the day of surgery and on the first and second postoperative days. The age of the blood utilized correlated with the decrease in red cell 2,3-DPG on the day of surgery. CONCLUSIONS: Reduction in red cell 2,3-DPG, P50, and plasma phosphorus occurs after open heart surgery in children. These changes can potentially contribute to impaired oxygen utilization in the postoperative period, when adequacy of tissue oxygenation is critical.

Ergogenic effects of phosphate loading: physiological fact or methodological fiction?

Inorganic phosphate (Pi) supplementation has been reported to provide ergogenic benefits, though the research findings are inconsistent. Several confounding influences may explain these results, including mode of exercise, exercise intensity, dietary intake, daily biological fluctuations, type of supplement, administered dose of the supplement, washout period, length of loading period, fitness level of subjects, blood volume alterations, and sample collection and analysis techniques. This review provides a critical analysis of the methodological difficulties that may contribute to the equivocal research findings pertaining to Pi supplementation.

Increased postprandial energy expenditure in obese women after peroral K- and Mg-phosphate.

Energy expenditure (EE) was studied in 15 obese (BMI > 27.5, median 29.2) and 15 lean (BMI < 22.9, median 21.7) healthy, postmenopausal women before and after peroral orange juice with or without K- and Mg-phosphate supplementation. The women, 56-58 years old, were studied in a double-blind, crossover experiment on two separate days. They received 100 ml of orange juice (10 g of carbohydrates) with or without a supplement of K+ (35 mmol), Mg2+ (17 mmol) and HPO4(2-) (39 mmol). Measurements were made before and 30 and 60 min after the stimuli. Intake of juice only had no effect on EE in either the lean or the obese subjects. When the minerals were added, EE (+6.3%; p < 0.001) increased significantly in the obese group with in a maximum 30 min after stimulus, while no such increase was seen in the lean group. The difference between the groups was significant (p < 0.05) and so was the difference in the obese group with or without minerals (p < 0.01). We conclude that an addition of potassium/magnesium-phosphate to glucose increases the postprandial thermogenesis in obese postmenopausal women, but not in lean ones.

[P50, 2,3-diphosphoglycerate and sodium as well as potassium in red blood cells in the perioperative period after hemodilutional autotransfusion].

The changes of the P50, 2,3-DPG and sodium as well as potassium of red blood cells in perioperative period were studied in 12 patients who underwent hemodilutional autotransfusion (HAT). P50 and 2, 3-DPG did not show remarkable changes before the third day after operation, but increased significantly on 5th and 7th day. There was a positive correlation between P50 and 2,3-DPG. Potassium of red blood cell increased significantly except on the first day. But sodium of red blood cell showed no remarkable change. In conclusion, our data demonstrate that the tissue oxygen supply from the red blood cell is maintained, because P50 did not decrease. Sodium in the red blood cell does not show remarkable changes, and therefore the membrane function of the red blood cell should be kept intact.

A comparison of effect of temperature on phosphorus metabolites, pH and Mg2+ in human and ground squirrel red cells.

1. 31P NMR spectra were obtained at temperatures ranging from 2 to 30 degrees C from freshly drawn human (cold-sensitive) and ground squirrel (cold-tolerant) red cells. The concentration of ATP was also determined by luciferin-luciferase assay over the same temperature range. 2. The concentration of ATP as determined by NMR or by the luciferin-luciferase assay did not change with temperature in either species. The absolute concentration of ATP in human cells determined by NMR was not significantly different from the total ATP determined enzymatically. 3. The concentration of 2,3-diphosphoglycerate was higher and that of pyridine nucleotides lower in human than in ground squirrel red cells. This species difference was independent of temperature. 4. Intracellular pH, as determined from the positions of the NMR peaks of 2- and 3-phosphates of diphosphoglycerate, became more alkaline as the temperature was lowered. 5. Free intracellular magnesium, determined from the difference in the positions of the peaks for alpha- and beta-phosphorus of ATP, increased in the ground squirrel red cells and decreased in the human red cells with cooling from 30 to 2 degrees C. Total magnesium, as determined by atomic emission spectroscopy, did not change with temperature in red cells of either species. 6. The intensities of all phosphorus metabolite signals from the ground squirrel cells increased with decreasing temperature, while those from the human cells were unaffected. Since chemical shift anisotropy in the presence of magnesium is a powerful spin-lattice relaxation mechanism for phosphates, this is additional evidence for the temperature dependence of free magnesium concentration in the ground squirrel cells. 7. We conclude that there is no difference in phosphorus metabolites or intracellular pH which could account for the differential cold sensitivity in human and ground squirrel red cells. We suggest that, in the cold-tolerant red cells from the ground squirrel, magnesium is released from binding sites as the temperature is lowered. The change in free intracellular Mg2+ may account at least in part for the unusually low temperature sensitivity of the Na(+)-K+ pump in the red cells of this species.

Phosphate metabolism in the red cell of sick prematures.

The effect of different amounts of phosphorus supplementation on phosphate metabolism was studied in two groups of sick preterms (B and C) during the first week of life. Group B included prematures that started formula milk feeding from day 3 and group C neonates on total parenteral nutrition with phosphate supplementation for 7 days. Twelve healthy preterm neonates on formula milk feeding from day 2 (group A) were used as controls. On the 7th day of life all phosphate parameters measured in group C (plasma and red cell inorganic phosphate and erythrocyte ATP and 2,3-diphosphoglycerate) were found significantly lower than in the controls (1.60 +/- 0.07; 0.60 +/- 0.08; 1.45 +/- 0.09 and 4.42 +/- 0.18 vs. 2.05 +/- 0.10; 0.94 +/- 0.11; 1.69 +/- 0.07 and 6.24 +/- 0.42 mmol/l, respectively). The renal tubular reabsorption of phosphate was also lower in this group (86.8 +/- 3.2 vs. 97.3 +/- 1.0%). No significant differences were observed between groups B and A. Higher phosphorus intake that was better achieved through early milk feeding prevented phosphate depletion and the decline of 2,3-diphosphoglycerate in the erythrocytes even of sick neonates which in turn might help to improve tissue oxygenation. The amount of parenteral phosphate supplementation that could have beneficial effects on red cell phosphate parameters needs further investigation.

Anemia of chronic renal failure in dogs.

Seventeen dogs with chronic renal failure (CRF) were studied to evaluate the incidence, type, and etiology of anemia in CRF. A nonregenerative, normochromic, normocytic anemia was seen in 12 of 17 dogs (70.6%). There was a direct correlation between the degree of anemia and the extent of CRF as assessed by serum creatinine concentrations (P = .0386, r = .50923). Erythrocyte concentrations of 2,3-diphosphoglycerate (DPG) were significantly increased in anemic animals and showed a close correlation to the degree of anemia. The high DPG concentrations may compensate for the anemia by decreasing the hemoglobin-oxygen affinity and thereby facilitating tissue oxygenation at low hematocrits. Serum concentrations of erythropoietin (Epo) were in the low to normal range, despite mild to moderate anemia, documenting a deficiency of Epo in dogs with CRF. The nonregenerative nature of the anemia supports impaired hematopoiesis as a significant etiologic factor. Other factors, such as increases in serum parathyroid hormone and phosphorus, were not found to correlate significantly with the degree of anemia, although there were significant differences between their concentrations in anemic compared with non-anemic dogs. There was no change in erythrocyte osmotic fragility with uremia. The documentation of a nonregenerative, normochromic, normocytic anemia, with failure of an appropriate increase in Epo production, supports the therapeutic use of Epo in the management of the anemia seen in CRF in the dog.

31P NMR spectroscopy of the human heart at 4 T: detection of substantially uncontaminated cardiac spectra and differentiation of subepicardium and subendocardium.

31P NMR spectroscopy of the human heart was undertaken at 4 T to investigate whether spectra localized exclusively to the myocardium can be obtained. Utilizing a Fourier series window approach to spectroscopic imaging, we find that at least two layers across the anterior left ventricle wall can be detected, with voxel sizes of about 8 cm3.

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.

13C and 31P NMR studies of glucose and 2-deoxyglucose metabolism in normal and enzyme-deficient human erythrocytes.

The flux of 13C-labeled glucose through the Embden-Meyerhof and pentose phosphate pathways was studied by 13C NMR in intact erythrocytes isolated from normal subjects or from patients suffering of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency. Similar rates of glucose catabolism and similar fluxes of the 13C-label into 2,3-bisphosphoglycerate and lactate were found, under basal conditions, in normal and in G6PD-deficient erythrocytes incubated in the presence of either [1-13C]- or D[6-13C]glucose. Exposure to oxidative stress by preincubation with tert-butylhydroperoxide induced in normal, but not in G6PD-deficient erythrocytes, a significant enhancement of glucose consumption, as well as a substantial reduction in 13C-label transfer from C1-glucose into lactate. It was also possible, by 31P NMR, to evaluate the conversion of 2-deoxyglucose to its phosphate-containing metabolites. The oxidation and subsequent decarboxylation of 2-deoxyglucose-6-phosphate was assessed in reconstituted systems and could subsequently be evidenced also in ethanolic extracts from normal (but not from G6PD-deficient) erythrocytes which had been exposed to oxidative stress. The results indicate that, in terms of glucose flux through the glycolytic pathway, there is little or no difference between normal and G6PD-deficient erythrocytes, regardless of previous exposure to oxidative stress. Faster consumption of either glucose or 2-deoxyglucose is induced, only in normal cells, by treatment with tert-butylhydroperoxide, essentially as a consequence of the activation of the pentose-phosphate pathway.

Reestimation of the effects of inorganic phosphates on the equilibrium between oxygen and hemoglobin.

In a previous paper, published in this journal, we showed that the data obtained in patients with severe ketoacidosis suggest that inorganic phosphates (K2HPO4) can increase their P50 and therefore enhance tissue oxygenation without concomitant alteration of the 2,3 diphosphoglycerate (DPG). In order to test the hypothesis that K2HPO4 could influence the oxyhemoglobin dissociation curve (ODC) by a mecanism which was not DPG mediated we have measured the total ODC on whole blood with and without addition of 13-80 mmol/l of inorganic phosphates. On average, the level of DPG remained unchanged when the P50 with K2HPO4 was significantly higher (p greater than 0.001) (P50 = 29.9 +/- 3.7 mmHg) than when phosphates were not administered (P50 = 25.5 +/- 2.8 mmHg). The relationship between P50 (mmHg) and K2HPO4 (= X mmol/l) was delta P50 = -2.97 10(-3)(X)2 +0.26(X)-0.42 (r = 0.78). Seeing that phosphates have an immediate action on the ODC, we calculated in our ketoacidosis patients, the relationship between the P50, the inorganic phosphates (P(i) in mg%) and the DPG in mumol/gHb. Both factors exert a highly significant effect (p less than 0.001) on the P50, according to the following equation: P50 = 0.35 DPG +0.26 P(i) + 18.92 (r = 0.73). Our data are important in two points. First it is useful to add inorganic phosphates to the treatment of patients with severe ketoacidosis in order to enhance their tissue oxygenation. Second they recall that the ODC is not only determined by the classical effects of temperature, pH and DPG but also by inorganic anions, like phosphates as described by Benesh and Benesh in their pioneering work.