Further investigation of the use of dimethyl methylphosphonate as a 31P-NMR probe of red cell volume.

We have refined a method for measuring erythrocyte volume using the 31P-NMR spectrum of a probe molecule, dimethyl methylphosphonate. This compound, when added to an erythrocyte suspension, gives rise to two 31P-NMR resonances, and the frequency separation between them is linearly dependent on the intracellular haemoglobin concentration. If, for a given cell sample (under standard conditions), the separation of the two dimethyl methylphosphonate peaks has been measured and an independent estimation of the mean cell haemoglobin content and concentration has been obtained, then changes in the mean cell volume due to altered experimental conditions may be estimated from the peak separation measured under the new conditions. Although the peak separation was independent of extracellular pH, it did vary with (i) a range of extracellular suspension media, (ii) temperature, (iii) dimethyl methylphosphonate concentration, (iv) haemoglobin ligand state and (v) different blood donors.

Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure.

Methylphosphonate in conjunction with 31P-NMR spectroscopy was used for the measurement of transmembrane delta pH in human erythrocytes stored at 4 degrees C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (Pi). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2'-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including Pi. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of Pi.

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system.

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 mumol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [14C]pyruvate to haemoglobin and the rate of efflux of [14C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent Km and Vmax for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.

Evaluation of an electrochemical model of erythrocyte pH buffering using 31P nuclear magnetic resonance data.

When erythrocytes are suspended in a solution of known composition the resultant values of such basic cell parameters as volume and pH are difficult to predict. To facilitate such predictions, we developed a mathematical model describing the passive transmembrane distribution of permeant species; three simultaneous equations were produced. Certain essential data required for the model were determined experimentally; these included the pH dependence of the charge on the hemoglobin molecule and the variation of the osmotic coefficient of hemoglobin with cell volume. Finally, cells were added to various solutions, and then titrated to produce a wide pH range (pH 6-8). We measured the resultant cell volume, cellular and extracellular pH using both conventional and 31P NMR methods. The expected equilibrium values of these electrochemical parameters were also calculated by solving (numerically) the three model equations. The accuracy of the model simulations was evaluated by direct comparison of calculated and experimentally determined values.

Supernatant hemoglobin determinations after prolonged blood storage.

Measurement of extracellular hemoglobin is useful for assessing the relative success of different blood storage strategies. The test is now also included in routine quality assurance procedures. With the recent trend towards transfusion of concentrated red cells resuspended in "additive" solutions, a safe and efficient method for the estimation of hemoglobin in these units is required. We have developed a method suitable for this purpose, based on the formation of cyanomethemoglobin. Tetramethylbenzidine (TMB) methods were also investigated and shown to detect hemoglobin and hemoglobin derivatives quantitatively when present in packed cell supernatants. A close correlation was recorded between the estimates of supernatant hemoglobin using the 2 techniques with 68 stored red cell concentrates. Because of its simplicity, adequate sensitivity and avoidance of carcinogenic benzidine derivatives, we recommend the cyanomethemoglobin method for routine use in the measurement of supernatant hemoglobin in stored modified red cell concentrates.

Red cells suspended in a phosphate-fortified additive: biochemical enhancement and longer storage.

When compared to our current storage regime, an additive solution, with 75 mM inorganic phosphate, improved certain biochemical and morphological parameters measured in suspensions of packed red cells throughout 49 days of liquid storage. In particular, the mean ATP level of 20 donations stored with the high phosphate additive was consistently and significantly higher than in standard (n = 6) suspensions. The increased amount of inorganic phosphate and the higher pH of the new additive stimulated ATP production and provided better pH buffering than the standard solution currently in use. Although survival study results indicated adequate 24h survivals for erythrocytes stored for 42 days with the new solution, after 49 days storage, the mean 24h survival of autologous erythrocytes was only 58 +/- 7% (n = 6).

Effect of intracellular magnesium on calcium extrusion by the plasma membrane calcium pump of intact human red cells.

1. The effect of varying the concentration of intracellular magnesium on the Ca(2+)-saturated Ca(2+)-extrusion rate through the Ca2+ pump (phi max) was investigated in human red blood cells with the aid of the divalent cation ionophore A23187. The aim was to characterize the [Mg2+]i dependence of the Ca2+ pump in the intact cell. 2. The initial experimental protocol consisted of applying a high ionophore concentration to obtain rapid sequential Mg2+ and [45Ca]CaCl2 equilibration, prior to measuring phi max at constant internal [MgT]i by either the Co2+ block method or by ionophore removal. With this protocol, competition between Ca2+ and Mg2+ through the ionophore prevented Ca2+ equilibration at high [Mg2+]o. To provide rapid and comparable Ca2+ loads and maintain intracellular ATP within normal levels it was necessary to separate the Mg2+ and the Ca2+ loading-extrusion stages by an intermediate ionophore and external Mg2+ removal step, and to use different metabolic substrates during Mg2+ loading (glucose) and Ca2+ loading-extrusion (inosine) periods. 3. Intracellular Co2+ was found to sustain Ca2+ extrusion by the pump at subphysiological [Mg2+]i. Ionophore removal was therefore used to estimate the [Mg2+]i dependence of the pump at levels below [MgT]i (approximately 2 mmol (340 g Hb)-1), whereas both ionophore removal and Co2+ block were used for higher [MgT]i levels. 4. [Mg2+]i was computed from measured [MgT]i using known cytoplasmic Mg(2+)-buffering data. The phi max of the Ca2+ pump increased hyperbolically with [Mg2+]i. The Michaelis parameter (K 1/2) of activation was 0.12 +/- 0.04 mmol (1 cell water)-1 (mean +/- S.E.M.). Increasing [MgT]i and [Mg2+]i to 9 mmol (340 g Hb)-1 and 2.6 mmol (1 cell water)-1, respectively, failed to cause significant inhibition of the phi max of the Ca2+ pump. 5. The results suggest that within the physiological and pathophysiological range of [Mg2+]i, from 0.3 mmol (1 cell water)-1 in the oxygenated state to 1.2 mmol (1 cell water)-1 in the deoxygenated state, the Ca(2+)-saturated Ca2+ pump remains unaffected by [Mg2+]i at normal ATP levels.

Measurement of the distribution of anion exchange function in normal human red cells.

1. The aim of the present work was to investigate cell-to-cell variation in anion exchange turnover in normal human red cells. Red cells permeabilized to protons and K+ dehydrate extremely rapidly by processes that are rate-limited by the induced K+ permeability or by anion exchange turnover. Conditions were designed to render dehydration rate-limited by anion exchange turnover. Cell-to-cell variation in anion exchange function could then be measured from the distribution of delay times required for dehydrating cells to attain resistance to haemolysis in a selected hypotonic medium. 2. Red cells were suspended at 10% haematocrit in a low-K+ solution and, after a brief preincubation with 20 microM SITS at 4 degrees C, were warmed to 24 degrees C, and the protonophore CCCP was added (20 microM) followed 2 min later by valinomycin (60 microM). Delay times for cells to become resistant to lysis were measured from the instant of valinomycin addition by sampling suspension aliquots into thirty volumes of 35 mM NaCl. After centrifugation the per cent lysis was estimated by measuring the haemoglobin concentration in the supernatant. Typical median delay times with this standardized method were 4-5 min. 3. The statistical parameters of the delay time distributions report the population spread in the transport function that was limiting to dehydration. In the absence of SITS and CCCP, dehydration was limited by the diffusional Cl- permeability (PCl). Delay time distributions for PCl- and anion exchange-limited dehydration were measured in red cells from three normal donors. For both distributions, the coefficients of variation ranged between 13.0 and 15.2%, indicating a high degree of uniformity in PCl and anion exchange function among individual red cells.

Refinement and evaluation of a model of Mg2+ buffering in human red cells.

The total Mg2+ content of human red cells ([Mg]T,i) is partitioned between free and bound forms. The main cytoplasmic Mg2+ buffers are ATP and 2,3 bisphosphoglycerate. Haemoglobin binds free ATP and bisphosphoglycerate, preferentially in the deoxygenated state. Thus, the free ionized Mg2+ concentration ([Mg2+]i) oscillates with the oxy-deoxy condition of the cells. The binding reactions are also modulated by the pH changes that accompany the oxygenation-deoxygenation transitions. The complex interactions between Mg2+, its ligands and Hb can be encoded in a set of equilibrium equations representing all the known binding reactions of the system. To develop a comprehensive understanding of the Mg2+ homeostasis of intact red cells it is necessary to correct and refine the equations and parameters of the model by systematic comparisons between model predictions and measured cytoplasmic Mg2+ buffering curves under a variety of experimental conditions. Earlier models largely underestimated total Mg2+ binding in intact cells. We carried out experiments in which [Mg]T,i and [Mg2+]i were controlled over a wide range ([Mg]T,i between 0.1 and 23 mM) by the use of the ionophore A23187, under diverse metabolic conditions, and the results were used to interpret the adjustments required for good model fits. By the inclusion of low-affinity Mg2+ binding to ATP and bisphosphoglycerate, and also binding of Mg2+ to haemoglobin (four ions per tetramer) with an apparent dissociation constant of 45 mM we were able to realistically model, for the first time, all the experimentally observed changes in [Mg2+]i in human red cells under diverse metabolic conditions.

Distribution of chloride permeabilities in normal human red cells.

1. The rate of dehydration of K+ permeabilized red cells is influenced by their Cl- permeability (PCl). In instances of pathological K+ permeabilization, cell-to-cell differences in PCl may determine which red cells dehydrate most. The present study was designed to investigate whether PCl differed significantly among red cells from a single blood sample. 2. Previously available methods measure only the mean PCl of red cell populations. We describe a 'profile migration' method in which dilute red cell suspensions in low-K+ media were permeabilized to K+ with a high concentration of valinomycin, rendering PCl the main rate-limiting factor for cell dehydration. As the cells dehydrated, samples were processed to obtain full haemolysis curves at precise times. Variations in PCl among cells would have appeared as progressive changes in the profile of their haemolysis curves, as the curves migrated towards lower tonicities. 3. Red cells from five normal volunteers showed no change in profile of the migrating haemolysis curves, suggesting that their PCl distributions were fairly uniform. Quantitative analysis demonstrated that intercell variation in PCl was less than 7.5%. 4. Results obtained with this technique were analysed using the Lew-Bookchin red cell model. The calculated PCl was within the normal range described in earlier studies.

Generation of normal human red cell volume, hemoglobin content, and membrane area distributions by "birth" or regulation?

Using flow cytometry and osmotic lysis measurements, we document here the means and coefficients of variation of the following red cell (RBC) properties: hemoglobin (Hb) content, volume, Hb concentration, and relative lytic tonicity distributions in populations of normal human RBCs, before and after density fractionation. The distributions showed a pattern characterized by much larger coefficients of variation of the Hb content and volume distributions than of the Hb concentration and relative lytic tonicity distributions. From analysis of the factors that determine those RBC properties, the patterns were interpreted as reflecting previously unrecognized statistical proportionalities between cell osmolyte content, Hb content, and membrane area. The possible origin of these statistical links was analyzed by considering alternative models with and without the participation of regulatory processes during cell maturation. A model was shown to be feasible in which mature RBC variability with proportional volume, area, and Hb content arises solely from cell size variability at the last erythroid cell division.

Intra- and extraerythrocyte pH at 37 degrees C and during long term storage at 4 degrees C: 31P NMR measurements and an electrochemical model of the system.

A 31P NMR method based on pH dependent variation of the chemical-shift-difference between the resonances of orthophosphate and methylphosphonate was used to measure simultaneously intracellular pH (pHi) and extracellular pH (pHc) during long term storage of erythrocytes; pH was determined at both 4 degrees C and 37 degrees C. An equation describing the equilibrium distribution of membrane-permeant ions was derived by consideration of the electrochemical and osmotic constraints in the RBC suspension. Calculations using the model-equation and the measured pHi yielded the Donnan ratio and therefore pHo; the relationship between experimentally determined pHi and pHo values was accurately predicted by the model. Sensitivity analysis of the model-equation revealed that the observed increase in transmembrane pH gradient during storage is principally due to the alteration of the total net charge of intracellular (poly)-anions.

Regeneration of phosphorylated metabolites in stored erythrocytes in an open perfusion system: studies using 31P NMR spectroscopy.

Human erythrocytes were maintained at high haematocrit in a metabolically functional state for several hours in a thermodynamically open perfusion apparatus. The concentrations of ATP and 2,3-bisphosphoglycerate (2,3-DPG) and pH were continuously monitored before and after metabolic perturbations by using 31P NMR; the monitoring was achieved with a 31P flow-through probe. Methylphosphonate was added to plasma perfusion medium as a phosphorus concentration standard and as a 31P NMR pH probe molecule. The rates of decline of ATP and 2,3-DPG levels in fresh cells in a glucose-free medium were measured as were the rates of reformation in response to a 'rejuvenation' medium. Also, rates of ATP and 2,3-DPG synthesis during perfusion with Krebs bicarbonate-0.5 mmol/l glucose and perfusion with pooled plasma were measured in cells that had been previously stored at 4 degrees C for 5 weeks.

Normal Ca2+ extrusion by the Ca2+ pump of intact red blood cells exposed to high glucose concentrations.

The ATPase activity of the plasma membrane Ca2+ pump (PMCA) has been reported to be inhibited by exposure of red blood cell (RBC) PMCA preparations to high glucose concentrations. It has been claimed that this effect could have potential pathophysiological relevance in diabetes. To ascertain whether high glucose levels also affect PMCA transport function in intact RBCs, Ca2+ extrusion by the Ca2+-saturated pump [PMCA maximal velocity (V(max))] was measured in human and rat RBCs exposed to high glucose in vivo or in vitro. Preincubation of normal human RBCs in 30-100 mM glucose for up to 6 h had no effect on PMCA V(max). The mean V(max) of RBCs from 15 diabetic subjects of 12.9 +/- 0.7 mmol. 340 g Hb(-1). h(-1) was not significantly different from that of controls (14.3 +/- 0.5 mmol. 340 g Hb(-1). h(-1)). Similarly, the PMCA V(max) of RBCs from 11 streptozotocin-diabetic rats was not affected by plasma glucose levels more than three times normal for 6-8 wk. Thus exposure to high glucose concentrations does not affect the ability of intact RBCs to extrude Ca2+.