Erythrocyte pH Research Articles

Erythrocyte organic phosphates and whole blood oxygen affinity in patients on continuous ambulatory peritoneal dialysis.

Whole blood oxygen affinity, erythrocyte pH and organic phosphates were studied in five anaemic untransfused patients with end stage renal disease undergoing continuous ambulatory peritoneal dialysis. Decreased whole blood oxygen affinity with increased adenosine triphosphate and normal 2.3 diphosphoglycerate (DPG) values were observed. Normal and stable serum phosphate and permanent mild metabolic acidosis may be important factors contributing to maintain DPG levels within the normal range despite anaemia. Continuous dialysis avoids cyclic fluctuation of blood oxygen affinity as described during and after dialysis sessions in patients on maintenance haemodialysis.

Systemic effects of NaHCO3 in experimental lactic acidosis in dogs.

Lactic acidosis is characterized by metabolic acidosis due to accumulation of H+ ions from lactic acid with blood lactate of at least 5 mM. The standard treatment is intravenous NaHCO3, with resultant mortality in excess of 50%. Despite the high mortality, the metabolic and systemic effects of NaHCO3 used in the treatment of lactic acidosis have not been extensively studied. The present experiments in diabetic dogs were designed to address these questions. Dogs with phenformin-induced lactic acidosis (blood lactate above 5 mM, arterial pH below 7.20) were treated with equimolar amounts of either NaCl or NaHCO3 or received no therapy. Intravenous NaHCO3 resulted in a decline of cardiac output and intracellular pH (pHi) of liver and erythrocytes, whereas treatment with NaCl did not. With NaHCO3 but not with NaCl infusion gut lactate production increased almost stoichiometrically, with no change in arterial pH or bicarbonate but with a doubling of lactate. Bicarbonate also resulted in a decrease of hepatic portal vein blood flow. The mean survival time and percent mortality were similar in NaCl- vs. NAHCO3(-) treated animals. Although both groups lived longer than did animals receiving no therapy, the differences were not significant. Thus, treatment of experimental lactic acidosis with either NaCl or NaHCO3 or with no therapy results in no change of blood pH and bicarbonate and in a similar mortality. In terms of systemic effects, however, NaHCO3 results in significant decrements of liver and erythrocyte pHi, hepatic portal vein blood flow, and cardiac output and in significant increments of gut lactate production, whereas NaCl does not. The data suggest that the rationale for therapy of lactic acidosis with NaHCO3 should probably be reevaluated.

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

A method is described for determining the intracellular pH of intact erythrocytes by 1H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bonded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from 1H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the 1H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-of-saturation by cross-relaxation method.

Interaction of added amphiphilic lipids with the membrane of intact human erythrocytes to induce change in the cell shape.

Addition of an appropriate amount of amphiphilic lipid, such as fatty acid, lysophospholipid and medium-chain phospholipid, into a suspension of human erythrocytes (pH 7.4) at 37 degree C resulted in their incorporation into the membrane and induction of a cell shape change of crenation (echinocyte-spherocyte) type without causing hemolysis. The extent of the shape change was dependent on the amount of the lipid incorporated and the crenation disappeared on removing the incorporated molecules from the membrane. The crenation induced by acidic lipids was further altered drastically by resuspending the treated cells in media of pH 6, 7, and 8, whereas that induced by choline-phospholipid or -lysophospholipid was not so pH-dependent. Based on these results, the mechanism of this shape change is discussed.

Reduced erythrocyte membrane protein methylation in sickle cell anemia.

The methylation of erythrocyte membrane components in sickle cell anemia has been studied and found to differ considerably from that of normal erythrocytes. When sickle erythrocytes were incubated under physiological conditions (pH = 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine or S-adenosyl-L-[methyl-3H] methionine, a 50% decrease in the protein-carboxyl methylation was observed compared to the normal erythrocyte. This reduction in degree of methylation was reflected in all of the major methylated protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since this methylation is catalyzed by the cytosolic protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC 2.1.1.24), the in vitro substrate capability of sickle erythrocyte ghosts and pH 11 treated ghosts were tested by incubation with purified protein methylase II and S-adenosyl-L-[methyl-14C]methionine. Both types of sickle cell ghost preparations showed the same 50% decrease in methylation as was seen in the intact cells. Since it was also shown that the protein methylase II and methyl acceptor membrane protein levels in the sickle erythrocytes are the same as the normal control, the data suggest that the observed reduced methylation may be due to an altered membrane conformation. The methylation of phospholipid was also studied and found to be decreased in sickle cell erythrocytes. However, this methylation was extremely minor in comparison to protein-carboxyl methylation which represents the bulk of the membrane methylation.

Temperature and acid-base status of human blood at constant and variable total CO 2 content

The influence of temperature on the acid-base status of normal human blood was studied in closed systems (constant C CO 2 ) and open systems (variable C CO 2 ). pHT coefficients of true plasma and erythrocytes in closed systems were similar to coefficients for water (dpH/dt = −0.017 u/°C at 25°C). Between 26 °C and 42 °C there was no significant variation in the relative alkalinities [OH −]/[H +], the charge state of proteins in plasma and erythrocytes or the proton Donnan ratio. The equations established enabled calculation of the pH of true plasma and erythrocytes and of blood P CO 2 and temperature, using only one of these four parameters. Under open-system conditions, temperature was shown to cause a rise in the apparent buffer power of whole blood non-bicarbonate systems (28.8 and 34.8 mM · 1 −1 · u −1 at 26 °C and 42 °C respectively). These results show (1) that erythrocytes in closed systems seem very well able to maintain proton distribution regardless of temperature fluctuations and (2) when blood temperature rises, it cannot be excluded that vital organs are better protected against respiratory disturbances.

A phosphorus-31 nuclear magnetic resonance investigation of intracellular environment in human normal and sickle cell blood

Intracellular pH and 2,3-diphosphoglycerate concentration in sickle cell amenia and normal human blood samples were measured by means of phosphorus-31 nuclear magnetic resonance spectroscopy. To monitor the concentrations of various internal phosphorylated metabolites of intact red blood cells, heparinized blood samples were used and were incubated at 37 degrees C with 5.6% C92, 25% O2, and 69.4% N2. The 31P chemical shifts of phosphorylated compounds, such as 2,3-diphosphoglycerate, adenosine 5′-triphosp-ate, and inorganic phosphate, depend on pH, and by using an appropriate calibration curve, the intracellular pH of intact erythrocytes can be obtained. The intracellular pH values in fresh sickl cell blood and normal blood were found to be 7.14 and 7.29, respectively. However, the whole-blood pH, as measured by a standard pH meter, was found to be 7.54 for both types of blood. The initial concentration of 2,3-diphosphoglycerate in sickle cell blood was about 30% higher, but it was depleted much faster during incubation than that in normal blood. The difference in intracellular pH between these two types of blood samples remained constant during incubation, even after depletion of 2,3-diphosphoglycerate. These results suggest that there are differences in intracellular environment between normal and sickle cell blood. Thus, 31P nuclear magnetic resonance spectroscopy provides a fast, direct, continuous, and noninvasive way to monitor the intracellular environment of intact erythrocytes.

A Phosphorus-31 Nuclear Magnetic Resonance Investigation of Intracellular Environment in Human Normal and Sickle Cell Blood

AbstractIntracellular pH and 2,3-diphosphoglycerate concentration in sickle cell anemia and normal human blood samples were measured by means of phosphorus-31 nuclear magnetic resonance spectroscopy. To monitor the concentrations of various internal phosphorylated metabolites of intact red blood cells, heparinized blood samples were used and were incubated at 37C with 5.6% CO2, 25% O2, and 69.4% N2. The 31P chemical shifts of phosphorylated compounds, such as 2,3-diphosphoglycerate, adenosine 5'-triphosphate, and inorganic phosphate, depend on pH, and by using an appropriate calibration curve, the intracellular pH of intact erythrocytes can be obtained. The intracellular pH values in fresh sickle cell blood and normal blood were found to be 7.14 and 7.29, respec tively. However, the whole-blood pH, as measured by a standard pH meter, was found to be 7.54 for both types of blood. The initial concentration of 2,3-diphosphoglycerate in sickle cell blood was about 30% higher, but it was depleted much faster during incubation than that in normal blood. The difference in intracellular pH between these two types of blood samples remained constant during incubation, even after depletion of 2,3-diphosphoglycerate. These results suggest that there are differences in intracellular environment between normal and sickle cell blood. Thus, 31P nuclear magnetic resonance spectroscopy provides a fast, direct, continuous, and noninvasive way to monitor the intracellular environment of intact erythrocytes.

Secondary polycythaemia in chronic respiratory insufficiency.

The relationship between polycythaemia, P50 and SaCO (saturation in carboxyhaemoglobin) has been studied in 50 patients who were hypoxaemic due to chronic respiratory insufficiency. These patients were divided into two groups according to their haemoglobin concentration and haematocrit: 21 polycythaemic patients with haemoglobin greater than or equal to 16 g/dl and haematocrit greater than or equal to 50% and 29 patients without polycythaemia. PaO2, PaCO2, plasma and erythrocyte pH, haemoglobin, haematocrit, and carbon monoxide saturation and intraerythrocytic 2-3 diphosphoglycerate concentration were measured during steady-state ventilation. All polycythaemic patients were smokers and their carbon level was significantly higher than that observed in patients without polycythaemia. Additionally, their P50 and 2-3 DPG concentration were significantly lower than in patients without polycythaemia. The correlations between P50 and HbCO and between Hb and HbCO were significant (r=--0-672; r=0-552 respectively: P less than 0-001). Eleven non-polycythaemic patients who were smokers had a high level of HbCO but normal P50. A group of 29 normoxic subjects was also studied, 14 non-smokers and 15 smokers with a high HbCO level. The mean value of P50 was lower in smokers and their haematocrit was higher although the difference was not significant for the latter. The HbCO increase by tobacco seems to be a factor in the occurrence of polycythaemia in patients with chronic respiratory insufficiency. The level of increase of HbCO and/or its duration and perhaps other individual factors and explain why all patients with high HbCO level and hypoxaemia were not polycythaemic.

Oxygen transport in congenital heart disease: influence of fetal hemoglobin, red cell pH, and 2,3-diphosphoglycerate.

In 48 individuals (age 1 day to 13 years) with congenital heart disease, blood oxygen transport function was studied in order to evaluate adaptive changes in shunt hypoxemia and to investigate the in vivo regulation of erythrocyte 2, 3-diphosphoglycerate concentration (RBC 2, 3-DPG) in the presence of fetal hemoglobin (HbF). Arterial pO2 and oxygen content, oxygen capacity, acid base status, oxygen affinity, HbF fraction, plasma pH, red cell pH, and RBC 2, 3-DPG were determined. During the first 50 days of life values of standard P50 (stdP50) (37, pH 7.4), actual in vivo P50 (actP50), RBC 2, 3-DPG, O2 capacity, arterial plasma pH, and red cell pH were scattered around the normal range, although tending to low values for stdP50 and arterial plasma pH and to high values for O2 capacity. After the third month, stdP50 actP50, RBC 2, 3-DPG, O2 capacity, and red cell pH were found to be elevated. Plasma pH and actP50 were scattered around the normal range (Figs. 1 and 2). Intraerythrocytic pH in hypoxemic infants was increased compared with normal children when related to plasma pH (Fig. 3). A close to normal intraerythrocytic pH was therefore found in the hypoxemic infants with low plasma pH, and an increased intraerythrocytic pH in the hypoxemic children with normal plasma pH (Fig. 1). A significant negative correlation exists between erythrocyte H+ ion and 2, 3-DPG concentration (Fig. 5); regression constants derived from data at high (mean 47%) and low (mean 9%) fractions of HbF are not significantly different (Regression Equations 8 and 11 in Table 1). Thus, the known difference in 2, 3-DPG binding to fetal or adult deoxyhemoglobin does not measurably influence the erythrocyte 2, 3-DPG concentration, indicating that in vivo the 2, 3-DPG synthesis in hypoxia is virtually regulated by the erythrocyte pH, which in turn is determined by plasma pH and the oxygenation state of hemoglobin.

EFFECTS OF THAM AND SODIUM BICARBONATE ON METABOLIC ACIDOSIS AND MINERAL METABOLISM

We caused in rabbits an acidosis by infusion of NH4Cl solution. Following we infused NaHCO3 solution, THAM solution pH 10.2 or THAM acetic acid solution pH 8.6. In correcting the metabolic acidosis the buffers had an equal efficacy. The correction of pH and base excess in erythrocytes was more alowly by use of NaHCO3 than by use of THAM solutions, Hematocrit, plasma K, Cl and phosphate decreased by infusion of buffer solutions. Na increased by use of NaHCO3, it-decreased by use of THAM solutions. Ca was not influenced. After the infusion of THAM followed by infusion of glucose solution in newborns we could see a fall of plasma electrolytes. Therefore we think that it is not sufficient to add minerals after 24 hours or later. but it is necessary to applicate minerals earlier. From the view of Na metabolism it is our opinion that we should not use NaHCO3 for the therapy of an acid osis in newborns.

Malaria studiesin vitro

The erythrocytic stages of strains of plasmodia sensitive to chloroquine (CQ) concentrate some three to six times more drug than CQ-resistant strains. A simple, but so far untested hypothesis is that the intracellular pH of drug resistant strains is higher than that for sensitive strains. This would result in a reduced accumulation of any weakly basic drug molecule (e.g. CQ) which is capable of passive diffusion through the cellular membran. The mean intracellular pH of erythrocytes parasitised with either CQ-sensitive or CQ-resistant strains of P. berghei was determined, therefore, from the distribution of 14C-5, 5-dimethyloxazolidine-2,4-dione between intra- and extracellular aqueous phases. The results obtained showed that over the range 6-8--7-2 the intracellular pH is governed directly by the extracellular pH and that the intracellular pH of CQ-sensitive parasites is slightly above that for resistant ones, neither being greatly different from that for uninfected erythrocytes. There was no evidence, therefore, to support, the suggested hypothesis. Elimination of this possibility adds weight to the opinion that changes in drug sensitivity of these plasmodia are the result of changes in the properties of drug-binding sites.

THE BLOOD OXYGEN TRANSPORT SYSTEM A numerical simulation of capillary‐tissue respiratory gas exchange

A theoretical model of the process of respiratory gas exchange between capillary and tissue is described, with special reference to the importance of variations in blood properties. The volume of tissue supplied with oxygen from a single vessel, as a function of the blood flowrate (u), hematocrit (h), and 2,3 diphosphoglycerate concentration (DPG), is calculated from a solution to the set of equations governing species distributions in the blood and tissue. The results, which are presented in the form of crossplots of the three blood parameters (u, h, and DPG) at a constant oxygen supply rate, show the possible significance of in vivo variations in the oxygen affinity of hemoglobin as a compensatory mechanism. Of further physiological interest is the sharp increase in venous erythrocyte pH in response to decreases in hematocrit, once the hematocrit is below a certain level. These results, and those relating DPG to hematocrit at constant O2 supply, are consistent with experimental observations of elevated DPG and pH levels in anemic individualts, and the dependence of erythrocyte DPG concentration upon pH.

Oxygenation dependent variations of the Bohr coefficient related to whole blood and erythrocyte pH. Effect of lactic and carbonic acid.

The Bohr effect related to whole blood (Be) and erythrocyte pH (Bi) was determined over a wide oxygen saturation range at an almost constant 2,3-diphosphoglycerate concentration. In two different sets of experiments acidification was caused either by fixed acid (lactic acid) or by CO2. Fixed acid-induced Bohr coefficients can be approximated by a second-order function of O2 saturation with a maximum at mid-saturation range. CO2-inducedBe andBi values yield a third-order relationship with highest results at low O2 saturation. The ratioBi/Be with respect to fixed acid-acidification exhibits a positive correlation with oxygenation, whereas the corresponding ratio referring to CO2 does not. For this different behavior a possibly more pronounced diminution of the Haldane effect, i.e. oxygen-linked proton release, in the presence of fixed acid is discussed. The physiological importance of the magnitude of CO2-inducedBi values at low O2 saturation is emphasized.

Determination of Intracellular pH by 31P Magnetic Resonance

Observation of the ^(31)P signal from various intracellular phosphates can provide a convenient, nondestructive technique for determining intracellular conditions such as pH. This procedure has been explored with particular reference to the erythrocyte. Both the chemical shift of intracellular inorganic phosphate relative to that of serum phosphate and the positions of, and more especially the difference between, the chemical shifts of 2,3-diphosphoglycerate have been used to monitor intracellular pH of erythrocytes whose hemoglobin has been liganded with carbon monoxide.

The relationship between pH and aerobic glycolysis in human and canine erythrocytes

1. 1. Canine erythrocytes contained less ATP and produced lactate at a lower rate than did human erythrocytes. 2. 2. The effect of pH, an increase in the glycolytic rate with increasing pH, was less pronounced in canine than in human erythrocytes. 3. 3. The hexokinase reaction was limiting in fortified hemolysates of both human and canine cells. Utilization of glucose and glucose-6-phosphate was pH dependent in both systems. 4. 4. The ATP content of human erythrocytes was lower at pH 7·6 than at pH 7·1.

Effects of 2,3-diphosphoglycerate and other organic phosphate compounds on oxygen affinity and intracellular pH of human erythrocytes.

The effects of changes of the 2,3-DPG content and of the total concentration of organic phosphates on the oxygen affinity and the intracellular pH of human erythrocytes were studied. The oxygen affinity as characterized by the P50 (oxygen tension at 50% O2 saturation) increases from 15 to 45 mm Hg when the 2,3-DPG concentration is elevated from 0.1 to 24 μmoles/g by incubation of erythrocytes in the presence of inosine, pyruvate and phosphate. In cells containing normal concentrations of 2,3-DPG, but accumulating high amounts of other organic phosphates during incubation with inosine and phosphate, the P50 was found to rise up to 36 mm Hg. This effect as well as a considerable part of the 2,3-DPG effect on the oxygen affinity of intact erythrocytes is due to a shift of the Donnan equilibrium induced by the accumulation of non-penetrating phosphate anions and consecutive changes of the intracellular pH, which in turn alter the oxygen affinity via the Bohr effect of hemoglobin. The intracellular pH is related to the intracellular concentration of organic phosphates (extracellular pH 7.40) by the equation: $${\text{pH}}_i {\text{ }} = {\text{ 7}}.306{\text{ }} - {\text{ }}0.0083{\text{ }} \cdot {\text{ }}P_{org} {\text{ }}(\mu moles organic P/g).$$ This dependency agrees closely with the theoretical relationship between the intracellular pH and the concentration of organic phosphates calculated from the osmolarities and the net charges of non-penetrating cell constituents. After correction of the oxygen affinities to a constant intracellular pH the P50 does not further increase in cells containing 2,3-DPG concentrations above 8 μmoles/g and remains unaltered in erythrocytes accumulating other organic phosphates.

Oxygen-linked hydrogen ion binding of human hemoglobin. Effects of carbon dioxide and 2,3-diphosphoglycerate. II. Studies on whole blood.

Acid-base titrations were performed on oxygenated and deoxygenated blood at constant PCO2. The BE-coefficient, - (δBlood-base excess)/(δHb-bound O2), is 0.3 mol/mol for normal blood, varying between 0 and 0.4 with variations in pH, PCO2, and 2,3-diphosphoglycerate concentration. The ratio between the BE-coefficient for a given plasma pH and the Haldane coefficient (-δHb-bound H+)/(δHb-bound O2)) for the corresponding erythrocyte pH is found to be 0.77. Comparison of the two coefficients indicates that (Ery-pH)=0.796 (Plasma-pH) + 1.356.

Intracellular pH in mature and immature rabbit red cells.

The intracellular pH of rabbit red cells was determined from the distribution of (2‐14C) DMO. Evidence is presented that the method provides a valid estimate of intracellular pH. In phosphate buffer at pH 7·40, the mean intracellular pH of the rabbit erythrocyte was 7·27. There was a close correspondence between the distribution ratios for hydrogen and chloride ions in normal rabbit blood. Values for intracellular pH, based on forty experiments in normal animals, were consistent with recent measurements of the membrane potential of mammalian red cells.The intracellular pH of the reticulocyte was deduced from the effect of acetylphenylhydrazide on the internal pH of rabbit red cells. At an external pH of 7·40, mean reticulocyte pH (7·32) was 0·06 pH units higher than mean erythrocyte pH (7·26).In preparations of reticulocytes, the distribution ratio for chloride ions was lower than the corresponding ratio for hydrogen ions. It is suggested that the presence in reticulocytes of small numbers of relatively alkaline mitochondria may be responsible for this phenomenon.

Intracellular pH and Electrolyte Metabolism in Chronic Stable Hypercapnia

Intracellular pH of whole body and erythrocytes was measured in patients with moderate chronic stable hypercapnia caused by chronic obstructive lung disease. These values were not significantly different from those of normal subjects or from normal values reported in the literature. Unlike acute hypercapnia, mean whole body intracellular pH values were maintained in a normal range despite arterial pH values which varied between 7.27 and 7.46. The percentage of total body water was not significantly different between hypercapnics and normal subjects. However, extracellular water level was higher and intracellular water level was lower. Although plasma chloride concentration [CI-] was significantly lower in hypercapnics than in normal subjects, total body chlorides per kilogram body weight was not significantly different between the groups. Plasma, muscle, and red blood cell sodium and potassium levels did not differ significantly between the hypercapnics and the normal group. The precise significance of the changes in acid-base