Effects of the medium on the filterability of human red blood cells

Red blood cell filterability as an appropriate method for evaluating red blood cell deformability was studied with different suspending media of erythrocytes. Comparison of autologous plasma and buffer suspensions of healthy subjects' erythrocytes showed no significant difference in filterability. Albumin alone resulted in a dose-dependent increase, while fibrinogen caused a decrease in red cell filterability. In the presence of fibrinogen, albumin showed controversial effects. The results suggest that measurements of red blood cells in their original surroundings give more accurate information about the microcirculation because plasma components may have a crucial influence on erythrocyte deformability.     

Effects of hyperglycaemia and sorbitol accumulation on erythrocyte deformability in diabetes mellitus.

Erythrocyte deformability was studied in a total of 83 poorly controlled diabetics (mean blood glucose 12.2 mmol/l) who were divided into three groups, each with matched healthy controls. There was no appreciable difference between diabetics and matched controls regarding the filtration of erythrocytes through 3 micron diameter straight channel pores (25 diabetics) or tortuous channel pores (28 diabetics), or for the measurement of erythrocyte elongation over a range of osmolalities in the Ektacytometer (30 diabetics). When erythrocytes from 17 additional diabetics and 17 healthy controls were incubated for two hours at 37 degrees C in hyperglycaemic (50 mmol glucose/l) buffer, however, there was a considerable reduction in erythrocyte filterability for both diabetics and controls in parallel with an increase in erythrocyte sorbitol concentration. This loss of filterability was prevented by the addition of an aldose reductase inhibitor (Sorbinil). High glucose concentrations (congruent to 50 mmol/l) impair the filterability of erythrocytes through 3 micron pores, and the intracellular accumulation of sorbitol in poorly controlled outpatients is therefore unlikely to have a major adverse effect on erythrocyte rheology in diabetes mellitus.     

Rheological properties of young and aged human erythrocytes

Summary Rheological properties were studied of young and old human erythrocytes from healthy adults. Viscosity measurements of packed erythrocyte suspensions as well as filtration of cells through polycarbonate sieves show that young cells are more flexible than aged ones. Since deformability of erythrocytes is the product of cell shape, flexibility of the membrane and fluidity of the intracellular hemoglobin, we studied the manner in which these factors are relevant to the diminished flexibility of aged erythrocytes. The biconcave cell shape is maintained during the process of aging. The viscosity of packed ghost suspensions from aged erythrocytes is increased versus that of young ones. The diminished flexibility of old ghosts correlates well with their smaller cell volume. The fluidity of the hemoglobin in the interior of the cells is decreased as indicated by an increased hemoglobin content of the isolated ghosts. We conclude that aged erythrocytes loose their deformability as a result of both a decreased fluidity of the intracellular hemoglobin and a diminished flexibility of the membrane.This work was supported by the Deutsche Forschungsgemeinschaft     

Extracellular fluid volume and composition changes during sodium pentobarbitone anaesthesia in the dog

Serial measurements of extracellular fluid (ECF), and plasma volumes were evaluated in dogs before and during general anaesthesia with sodium pentobarbitone and under controlled conditions of arterial pH, pO2, pCO2, and blood pressure. Sodium pentobarbitone anaesthesia caused an early, significant rise in ECF volume with a fall in haematocrit, plasma protein, and plasma potassium concentrations. Plasma osmolality and sodium concentrations were unchanged. The lack of change in ECF sodium concentration suggests that the total ECF sodium content increased in parallel with the expansion of this compartment. Sodium bound to macromolecules in the interstitial space or to bone is suggested as a possible source of sodium ions. It is unlikely that intracellular sodium stores contribute to a significant extent in these changes. During prolonged anaesthesia plasma volume progressively increased while total ECF volume returned towards control values. This work clarifies previous observations and suggests that major fluid movements occur during sodium pentobarbitone anaesthesia primarily associated with altered cell membrane properties and generalised haemodynamic changes.     

Effects of sodium and potassium salts with anions other than chloride on renin secretion in the dog

Intrarenal arterial infusions of sodium and potassium salts with anions other than chloride were given to evaluate the role of the chloride ion in influencing renin secretion (RS). The studies were conducted in dogs with thoracic caval constriction. Sodium lactate increased renal venous plasma sodium concentration (RVPNa) from 142 to 166 meq/liter (n, 6); RS decreased from 3,070 to 1,510 ng angiotensin/min (P less than 0.005). Arterial blood pressure and renal blood flow were not changed appreciably. Sodium excretion (ENa) increased, whereas chloride excretion (EC1) fell during the first three 15-min infusion periods. Potassium lactate increased renal venous plasma potassium concentration from 4.1 to 6.2 meq/liter (N, b). RS decreased during the first three 15-min periods of infusion (from 3,470 to 2,180 ng angiotensin/min, P less than 0.01). ENa and EC1 increased during the infusion. Potassium sulfate also decreased RS, and EC1 was usually increased. The results with sodium lactate favor a role for sodium compared with chloride in mediating the decreased renin release, but there are other possible interpretations which have been discussed. Additional studies are needed to resolve the role of chloride during potassium infusion.     

Retention of water and potassium by erythrocytes prevents calcium-induced membrane rigidity.

Modest increases in intracellular calcium concentrations, in association with ATP depletion, cause the appearance of pathologic changes in erthrocyte shape and deformability. The loss of erythrocyte ATP and simultaneous increase in cellular calcium have previously been considered the sole requisites for the appearance of erythrocyte membrane rigidity. We report that red cells suspended in high-potassium buffers may be simultaneously loaded with calcium (through exposure to the divalent cation ionophore A23187) and depleted of ATP without incurring drastic changes in shape or in membrane stiffness. Incubation of erythrocytes under these conditions effectively blocks both water and potassium loss normally caused by calcium accumulation. However, the high external potassium has no influence on either the ionophore-induced accumulation of calcium or on the the concomitant hydrolysis of cellular ATP. These results suggest the involvement of at least one further parameter, ie, changes in cell water and cation content, in the development of calcium-induced erythrocyte rigidity.     

Oxpentifylline and cetiedil citrate improve deformability of dehydrated sickle cells.

Erythrocytes from 14 patients with homozygous sickle cell anaemia were treated with the calcium ionophore A23187 to induce loss of cellular potassium and water. The dehydrated cells showed a decrease in filterability (loss of deformability) through pores of 5 micron diameter. Oxpentifylline and cetiedil citrate, which preserve erythrocyte cation and water content, had a significant (p less than 0.01) protective effect against loss of deformability at a concentration of 1 mumol/l. Oxpentifylline showed no adverse effect on the rheology, morphology, or haemolysis of sickle cells at concentrations up to 500 mumol/l. Drugs that act on the erythrocyte membrane to maintain cell hydration are of potential rheological benefit in sickle cell anaemia.     

Peripheral effects of thyroid hormones: Alteration of intracellular Na-concentration,ouabain-sensitive Na-transport,and Na-Li countertransport in human red blood cells

Summary To investigate the effect of thyroid hormones on erythrocyte cation transport systems and intracellular electrolyte content we have measured the activity of Na-K ATPase, Na-Li countertransport, as well as red cell sodium and potassium contents in patients with hyperthyroidism and in euthyroid controls. Intracellular Na- and K-concentrations were determined in erythrocytes washed three times in isotonic MgCl₂ solution. Ouabain-sensitive Na-transport was estimated as the increase of Na before and after addition of ouabain in an erythrocyte suspension in isotonic Na-free medium. Na-Li countertransport was measured according to the method described by Canessa et al. [2]. The patients with hyperthyroidism exhibited a significantly elevated intracellular sodium content as well as a highly increased Na-K ATPase activity. Intracellular potassium content was not altered in the hyperthyroid subjects, but Na-Li countertransport was markedly decreased as compared to the controls.The results indicate that different ion transport systems of the erythrocyte membrane are influenced by thyroid hormones. We suggest that the elevation of Na-K ATPase activity might be due to the increased intracellular sodium concentration which is caused by the diminished countertransport pathway. Furthermore, the activity of Na-K ATPase, Na-Li countertransport, and intracellular sodium content in erythrocytes might be a useful peripheral indicator of thyroid hormone excess.Supported by the Bundesministerium für Forschung und Technologie (MMT 27)     

The effects of pentoxifylline on rat erythrocytes of different age

Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug-induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca2+; (2) membrane protein phosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5-2.5 mM) reduced intracellular Ca2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0-5.0 mM) increased Ca2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inhibition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca2+ entry into the cell.     

Chloride transport in the thick ascending limb of Henle's loop: potassium dependence and stoichiometry of the NaCl cotransport system in plasma membrane vesicles

Cells were isolated from the thick ascending limb of Henle's loop of rabbit kidney outer medulla and a plasma membrane fraction was prepared by differential centrifugation. Sodium and rubidium uptake into the plasma membrane vesicles were determined by a rapid filtration technique. In the presence of a 100 mM KCl gradient and 0.5 mM sodium the vesicles took up 252±82 pmoles sodium/mg protein x 15s; 52% of the uptake was dependent on the presence of chloride or inhibited by 10^?3 M bumetanide. If KCl was stepwise replaced by choline chloride, sodium uptake decreased in the absence of bumetanide but was only insignificantly altered in the presence of bumetanide. Potassium exerted a halfmaximum stimulation at 22.3±9.7 mM. In tracer exchange experiments under zero salt gradient conditions, sodium uptake was also strongly reduced in the absence of potassium. Rubidium uptake into the same membrane fraction was highest in the presence of a NaCl gradient, and decreased 41% when sodium was replaced by choline or when 10^?3 M bumetanide was present. Replacement of sodium chloride by sodium nitrate also inhibited rubidium uptake. When the sodium, chloride and potassium dependence of the bumetanide sensitive sodium uptake was investigated in more detail, Hill coefficients for sodium of 1.0±0.03, for chloride of 1.8±0.2 and for potassium of 0.98±0.03 were obtained. These results are consistent with the presence of a Na?Cl?K cotransport system in the medullary thick ascending limb of Henle's loop which may operate with a stoichiometry of 1/2/1.     

Diabetes and heterozygotic sickle-cell anemia: study of erythrocyte deformability

The erythrocyte filterability of 25 diabetics, 6 patients with sickle cell trait ( HbAS ) and diabetes mellitus, and 25 controls was studied. Erythrocyte filterability was estimated by a filtration technique with washed erythrocytes. Red cell filterability was significantly decreased in diabetic patients compared to control subjects and this abnormality was correlated with the extent and severity of vascular complications. The values of erythrocyte filterability in diabetics + HbAs and diabetic patients were in the same range. These results suggest that HbAS does not influence the abnormalities of erythrocyte filterability observed in diabetes mellitus.     

Calcium-induced erythrocyte rigidity: the roles of cellular metabolism,hydration, and ionic balance.

Previous investigations have shown that incubation of human erythrocytes with the ionophore A23187 and calcium causes accumulation of the cation, losses in potassium, water, and cellular volume, hydrolysis of adenosine triphosphate (ATP), conversion of biconcave discocytes to echinocytes and spheroechinocytes, and marked increases in erythrocyte resistance to micropipette aspiration. Subsequent studies demonstrated that prevention of water and potassium loss blocked the influence of calcium loading on erythrocyte stiffness without affecting calcium uptake by the cells or hydrolysis of ATP. In the present study erythrocytes were exposed to conditions that permitted individual or coordinate manipulation of cellular ATP, water, potassium, and calcium in order to determine which factors developing as a result of calcium loading were responsible for the calcium-induced changes in erythrocyte viscoelastic properties. Results of the study demonstrate that volume loss, ATP hydrolysis, and potassium depletion do not individually or in combination cause increases in erythrocyte stiffness. However, all of these changes are essential and must develop in conjunction with calcium loading in order for erythrocytes to develop diminished deformability and elasticity.     

Effect of amiloride on sodium transport of frog skin

Summary The effect of Amiloride on intracellular sodium content of the isolated frog skin was investigated. The maximal effect on intracellular exchangeable sodium concentration (–10 meq/kg cell water) was found at concentrations of Amiloride in the incubation solution between 0.3 and 1.0×10^–4 M/l, and at an incubation time of 6–15 min. Since total intracellular sodium concentration is also reduced by approximately 10 meq/kg cell water, it follows that the intracellular non-exchangeable sodium concentration was not affected by Amiloride. Water content, extracellular volume, and intracellular potassium concentration remained constant. The short circuit current reached a new steady state within a few seconds after addition of Amiloride. It is concluded that Amiloride affects a sodium transport pool, which contains about 10% of the exchangeable intracellular sodium.Supported by the Deutsche Forschungsgemeinschaft.     

Correction of the permeability defect in hereditary stomatocytosis by dimethyl adipimidate.

The effect of imidoesters on the extraordinarily increased cation permeability of hereditary stomatocytes was evaluated in erythrocytes from a splenectomized boy with persistent hemolytic anemia. Reaction of stomatocytes with dimethyl adipimidate reduced ouabain-associated potassium loss from 15 to 1.7 and sodium gain from 22 to 2.5 meq per liter of red cells per hours. Red-cell volume, cation concentration, and deformability, previously abnormal, rapidly became normal after stomatocytes were reacted with dimethyl adipimidate. Instead of stomatocytes, normal red cells and target cells were noted. The survival (half-time) of stomatocytes treated with 51Cr-labeled dimethyl adipimidate infused into rats rendered tolerant to human erythorocytes by pretreatment with ethyl palmitate and cobra-venom factor was double that of untreated stomatocytes. Chemical modification of the defect in vitro allowed stomatocytes to regain many properites of normal erythrocytes and favorably influenced the subsequent survival of these cells in vivo.     

Effect of acute metabolic acidosis on transmembrane electrolyte gradients in individual renal tubule cells

We studied the effect of acute metabolic acidosis on potassium, sodium and chloride gradients across the apical membrane of proximal and distal tubule cells by determining electrolyte concentrations in individual cells and in tubule fluid employing electron microprobe analysis. Cellular measurements were performed on freeze-dried cryosections of the renal cortex, analysis of tubule fluid electrolyte concentrations on freeze-dried microdroplets of micropuncture samples obtained from proximal and from early and late distal collection sites. Acidosis (NH₄Cl i.v. and i.g.) induced a substantial rise in plasma potassium concentration without significant effects on cell potassium concentrations. Potassium concentrations along the surface distal tubule were also unaltered; thus the chemical driving force for potassium exit from cell to lumen was not affected by acidosis. In all but intercalated cells acidosis markedly increased cell phosphorus concentration and cell dry weight indicating cell shrinkage and thus diminution of cell potassium content. Because the increase in intracellular chloride concentration exceeded the increase in plasma chloride concentration, the chemical chloride gradient across the contraluminal membrane was markedly depressed by acidosis.     

Decreased red cell filterability in patients with acute myocardial infarction

The red cell filterability was decreased in patients with acute myocardial infarction (AMI) when compared with healthy controls, 14.6 (12.2-16.3) units and 16.9 (15.6-17.4) units respectively, P50 (P25-P75), p less than 0.001). No significant correlations could be seen within the AMI group between the decrease in filterability and the levels of serum aspartate aminotransferase or serum lactate dehydrogenase. The erythrocyte filterability, however, correlated to the serum concentrations of hepatic enzymes in AMI. The addition of sodium lactate in vitro in physiological concentrations (0.9-3.6 mM/l final concentration) lowered the erythrocyte filterability markedly to 2.7 (0-9.8) units in a dose-dependent manner, supporting the hypothesis that the decrease in erythrocyte filterability in AMI might be caused by an increase in the lactate concentration.     

Impaired filterability of erythrocytes from patients with chronic hepatitis C and effects of eicosapentaenoic acid on the filterability

Although erythrocyte filterability plays a key role in microcirculation, it is unknown whether the filterability of erythrocytes from patients with chronic hepatitis C (CH-C) is impaired. This study aimed to investigate erythrocyte filterability in CH-C patients in relation to medical treatment. The mean erythrocyte filterability (%) for all 24 patients with CH-C (69.2 +/- 10.8%) was significantly lower than that for 5 normal controls (80.5 +/- 1.7%, P < 0.03). In 8 patients, the combination therapy of ribavirin (RBV) and interferon improved liver function but caused anemia. The filterability after treatment (57.8 +/- 12.8%) was lower than that before treatment (70.8 +/- 9.7%, P < 0.05). Decreased filterability showed no correlation with the mean corpuscular volume or mean corpuscular Hb concentration during treatment, suggesting that the decrease in filterability mainly arises from changes in erythrocyte membrane properties. We investigated the protective effects of eicosapentaenoic acid (EPA) on the RBV-induced anemia. Filterability in 7 responders was markedly improved from 68.4 +/- 4.6% to 77.4 +/- 2.4% (P < 0.001), but not in 3 nonresponders. In the responders, the progression of anemia was restrained. In conclusion, we found an obvious impairment of the filterability of erythrocytes from CH-C patients, further impairment of the filterability induced by oxidative membrane damage caused by RBV leading to hemolytic anemia, and amelioration of the filterability caused by the antioxidative effects of EPA.     

Erythrocyte deformability in peripheral occlusive arterial disease.

A rheological study of 32 patients with peripheral occlusive arterial disease (POAD), compared with 32 matched healthy controls, has shown no loss of erythrocyte deformability as measured by filtration methods (using initial flow rate and positive pressure instruments, polycarbonate and silver membranes, and 3 microns and 5 microns diameter pores) or by viscometry (using laser visco-diffractometric and high shear rate viscosity methods). Erythrocyte ATP concentration in POAD was also normal. Patients with POAD showed a small (4 fl) increase in mean erythrocyte volume, associated with a raised serum gamma-glutamyl transpeptidase concentration, which correlated with erythrocyte filtration and viscometric measurements. Previous reports of impaired blood filterability in POAD probably reflect the effects of accompanying leucocytosis, plasma hyperfibrinogenaemia, or an increase in erythrocyte size, but not an intrinsic loss of erythrocyte deformability.     

NKCC1 does not accumulate chloride in developing retinal neurons

GABA excites immature neurons due to their relatively high intracellular chloride concentration. This initial high concentration is commonly attributed to the ubiquitous chloride cotransporter NKCC1, which uses a sodium gradient to accumulate chloride. Here we tested this hypothesis in immature retinal amacrine and ganglion cells. Western blotting detected NKCC1 at birth and its expression first increased, then decreased to the adult level. Immunocytochemistry confirmed this early expression of NKCC1 and localized it to all nuclear layers. In the ganglion cell layer, staining peaked at P4 and then decreased with age, becoming undetectable in adult. In comparison, KCC2, the chloride extruder, steadily increased with age localizing primarily to the synaptic layers. For functional tests, we used calcium imaging with fura-2 and chloride imaging with 6-methoxy-N-ethylquinolinium iodide. If NKCC1 accumulates chloride in ganglion and amacrine cells, deleting or blocking it should abolish the GABA-evoked calcium rise. However, at P0-5 GABA consistently evoked a calcium rise that was not abolished in the NKCC1-null retinas, nor by applying high concentrations of bumetanide (NKCC blocker) for long periods. Furthermore, intracellular chloride concentration in amacrine and ganglion cells of the NKCC1-null retinas was approximately 30 mM, same as in wild type at this age. This concentration was not lowered by applying bumetanide or by decreasing extracellular sodium concentration. Costaining for NKCC1 and cellular markers suggested that at P3, NKCC1 is restricted to Müller cells. We conclude that NKCC1 does not serve to accumulate chloride in immature retinal neurons, but it may enable Müller cells to buffer extracellular chloride.     

Sampling of Nanoliter Volumes of Mammalian Subcutaneous Tissue Fluid and Ultra-micro Flame Photometric Analyses of the K and Na Concentrations

Local tissue fluid within the subcutaneous tissues of the hind legs of dogs was sampled with a liquid paraffin cavity method. The potassium and sodium concentrations of nanoliter samples of the tissue fluid were analyzed with an ultra-micro flame photometric method and compared to the plasma concentrations of these ions. The average potassium concentration in the tissue fluid was 4.67 meq/l and the average sodium concentration was 153.4 meq/l; the corresponding plasma values were 3.78 meq/l and 142.3 meq/l respectively. The average individual potassium tissue fluid: plasma ratio was 1.25 (P<0.001 vs. 1.0) and the sodium tissue fluid: plasma ratio 1.08 (P<0.001 vs. 1.0). Possible sources of error at sampling and analyses have been evaluated. The potassium and sodium content of the local tissue fluid was higher than expected of an ultrafiltrate of plasma according to the generally accepted Gibbs-Donnan constant (0.96) for these ions. The apparent deviation of the obtained values from the Gibbs-Donnan constant can be explained by assuming that charged anionic macromolecules in the ground substance of the tissues affect the cation distribution in the local tissue fluid.