The effect of hemodialysis on the transport of sodium in erythrocytes from chronic renal failure patients maintained on hemodialysis.

Studies were undertaken to evaluate the modulatory effect of maintenance hemodialysis on ouabain sensitive (OS) and ouabain insensitive (OIS) 22Na(+) uptake in erythrocytes (E) of 8 chronic renal failure patients of both sexes. Following the receipt of informed consent, the blood samples were obtained just before and after Dialysis. The % 22Na(+) uptake of the total 22Na(+) present in the assay media was determined in the purified E just before and after Dialysis. The assay medium was composed of 100 mM NaCl, 5 mM KCl, 10 mM trisbase, 10 mM MOPS, 10 mM D-glucose and 60 mM sucrose, pH 7.4 with and without ouabain. Five different concentrations of E, ranging from 0.75 to 2.00 x 10(9)/mL were used for this study. We observed a linear relationship between the 22Na(+) uptake and E concentrations in both of the assay systems (OS and OIS). The mean total 22Na(+) uptake per 6.5 x 10(9) E/mL in OS and OIS before and after hemodialysis were 3.28 +/- 0.4 (OS) and 3.26 +/- 0.42 (OIS), and 3.42 +/- 0.54 (OS) and 3.42 +/- 0.68 (OIS) respectively. The relative % differences between pre- and post-Dialysis were 4 and 5%, which were statistically not significant. From this study, we conclude that hemodialysis does not affect E membrane properties influencing 22Na(+) transport.

Acetylcholinesterase activity in chronic renal failure.

Twenty healthy subjects and 39 Chronic Renal Failure patients (CRF-patients) maintained on chronic hemodialysis were used in this investigation to study the changes in acetylcholinesterase (AChE) activity of red blood cells (RBCs). The CRF-patients were all undergoing hemodialysis treatment. AChE activity from the CRF-patients was determined before and after dialysis. An additional objective was to study the effect of chronic renal failure on human red blood cell aging. Blood samples were drawn from controls and CRF-patients in tubes containing EDTA or sodium heparin as an anticoagulant. Red blood cells were purified to avoid interference with monocytes, reticulocytes and leukocytes. The purified RBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinous percoll gradient. The mean +/- SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB), purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) in CRF-patients were 31.2+/-3.43, 29.3+/-3.26, 30.4+/-3.91, 25.1+/-5.25, 17.1+/-6.02 in females and 29.8+/-5.39, 28.8+/-5.29, 28.7+/-5.29, 23.7+/-5.39 and 16.0+/-5.60 in males. AChE activity from CRF-patients were higher than that found in the control subjects. The aging of human RBCs in both the controls and CRF-patients showed a progressive reduction in AChE activity. AChE activity of RBCs from female CRF-patients were significantly higher (p < 0.05) than that of the female control subjects. The RBCs isolated from male CRF-patients showed a higher AChE activity than control males, but a significant difference was only observed with the mid-age-cells. These studies further indicate that AChE activity remained insignificantly different in the various density based age subfractions of RBCs of both CRF-patients and controls.

Acetylcholinesterase: an enzymatic marker of human red blood cell aging.

The purpose of this investigation was to determine whether acetylcholinesterase (AChE) can be used as a marker of cell aging in human red blood cells (RBCs). This study used consented subjects; both males and females in an age range of 21-42 years. The blood samples (8-9 mL) were drawn in tubes containing sodium heparin or EDTA as anticoagulants. To avoid contamination with other cells, (lymphocytes, monocytes and reticulocytes), RBCs were purified (PRBC) by Hypaque-Ficoll gradient technique. The PRBCs were subfractionated into young (y) (1.08-1.09), mid (m) (1.09-1.11) and old (o) (1.11-1.12) percoll density (g/mL) fractions using a discontinuous percoll gradient. The mean +/- 1 SD AChE per gram hemoglobin (U/g Hgb) activities in whole blood (WB) purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) were 27.4 +/- 2.98, 26.0 +/- 2.33, 25.5 +/- 1.64, 20.3 +/- 3.84, 14.6 +/- 3.42 in males and 26.3 +/- 4.44, 24.8 /- 4.83, 26.4 +/- 4.59, 24.0 +/- 5.50 and 12.4 +/- 7.09 in females respectively. Although there was variation in the data, the results indicated that old human red blood cells showed significantly (p<.05) lower AChE activity compared to young human red blood cells of both sexes. These preliminary but novel observations suggest that AChE can be an excellent enzymatic marker for RBC aging in man.

Sodium-22 uptake in erythrocytes can differentiate between the essential and the secondary hypertensive patient.

To determine the Na+/K+ ATPase independent 22Na+ influx into erythrocytes (E) from normal subjects (N), from essential hypertensive patients (EH), and from renal failure patients with secondary hypertension (SH), these studies involved two assay conditions. The erythrocyte suspensions were incubated for 30 minutes at 37 degrees C under two assay conditions: (i) with assay buffers containing 5mEq/L KC1 and varying amounts of NaCl (5 to 100 mEq/L) and (ii) with assay buffers with a range of KC1 (5 to 100 mEq/L) and a constant amount of 5 mEq/L NaCl. The ouabain insensitive percent uptake of 22Na+ in 2 x 10(9) E/mL from N, EH, and SH were 2.77 +/- 0.34, 4.37 +/- 0.83, and 3.72 +/- 0.60, respectively, when the assay media contained equimolar amounts of NaCl and KC1 (5 mEq/L each). The 22Na+ uptake was decreased gradually by progressive increasing concentrations of NaCl or of KC1 in the assay media. When erythrocytes were incubated in assay buffers containing either 50mEq/L NaCl with 5 mEq/L KC1 and 50 mEq/L KC1 with 5 mEq/L NaCl, the relative percentages of 22Na+ uptake in erythrocytes became significantly increased [65% increases in EH, 17% increases in SH over that in N subjects, 41% increases in EH over that of SH subjects; and, 49% increases in EH, 23% increases in SH other than in N subjects, 21% increases in EH over that of SH subjects, respectively]. None of the other assay media showed such differences in the 22Na+ uptake values.(ABSTRACT TRUNCATED AT 250 WORDS)

How extracellular potassium affects intracellular sodium pool in human erythrocytes.

In order to evaluate the regulation of intracellular sodium and potassium balance, we investigated the Na+/K(+)-ATPase independent 22Na+ uptake and concentrations of Na+ in erythrocytes from eleven normal subjects. The experiments were performed with the purified erythrocyte suspensions in different assay buffers containing (i) 5 mEq/L KCl and varying amounts of NaCl (5 to 100 mEq/L); and (ii) a range of KCl (5 to 100 mEq/L) and a constant amount of NaCl (5 mEq/L). These erythrocyte suspensions were incubated at 37 degrees C for 30 minutes to assess ouabain insensitive 22Na+ uptake. Erythrocytes (2.0 x 10(9)/mL) showed an uptake of 2.03 to 0.88%, and 2.00 to 1.15% of the total 22Na+ present in the media under these experimental conditions, respectively. The 22Na+ uptake by erythrocytes was decreased by a gradual increase of either NaCl or KCl in the assay buffers. Erythrocytes in the experimental condition (i) showed an increase in intracellular sodium [Na+]i from 8.29 to 10.06 mEq/L. However in the condition (ii), KCl up to 20 mEq/L extracellularly caused a limited inhibition of [Na+]i accumulation (8.29 to 8.23 mEq/L), however, when KCl was raised extracellularly greater than 20 mEq/L it enhanced [Na+]i slowly (8.23 to 9.19 mEq/L). When NaCl 20, 50 and 100 mEq/L were replaced by an equivalent amount of KCl in the assay buffers, this extracellular K+ prevented 7, 6 and 10% [Na+]i accumulation, respectively. We also found that bicarbonate induced ouabain resistance 22Na+ influx was both inhibited and stimulated depending upon the amount of KCl in the assay media.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte carbonic anhydrase: a major intracellular enzyme to regulate cellular sodium metabolism in chronic renal failure patients with diabetes and hypertension.

Changes in carbonic anhydrase (CA) activity have been associated with metabolic diseases like diabetes mellitus and hypertension. To explore the exchange of H+ for Na+ and 22Na+, the sodium pool, CA activity and H2O content in erythrocytes from the two groups of diabetic chronic renal failure (CRF) patients with and without hypertension before dialysis were studied. The results were compared with those from the normotensive controls. The CA activity was determined spectrophotometrically, the sodium pool by ouabain insensitive 22Na+ influx and the percent H2O content gravimetrically. The 22Na+ influx in CRF patients with hypertension was significantly higher (p less than 0.025) than in the normotensive CRF patients and the controls. The levels of CA activity (U/min/mL) and the percent H2O content were significantly different in the hypertensive and the normotensive CRF patients from the control group (2.24 +/- 0.69 and 67.11 +/- 1.33, 1.95 +/- 0.63 and 66.43 +/- 1.51, 1.44 +/- 0.07 and 63.61 +/- 1.72, respectively). The present study implies a relationship between the 22Na+ influx and CA activity in CRF patients with hypertension. The variation of CA activity may thus result in changes in H+ production and ultimately in the intracellular Na+ pool.

Red blood cell insulin receptors in health and disease.

CONTENTS: Structure and characteristics of erythrocyte insulin receptor. Red blood cell age and insulin receptors. Insulin receptors in human disease states. Obesity. Chronic renal failure. Acanthosis nigricans. Miscellaneous disease states. Insulin receptors in children. Insulin receptors in women during pregnancy. Insulin binding and other hormones. Comparison of biosynthetic insulin, pancreatic human insulin and porcine insulin binding to erythrocytes. Effect of exercise on insulin binding to red blood cells of normal human volunteers. Miscellaneous insulin binding studies. Insulin internalization and degradation. Insulin and erythrocyte metabolism. Summary and conclusion.

Changes in carbonic anhydrase may be the initial step of altered metabolism in hypertension.

Carbonic anhydrase (CA) is a well characterized pH regulatory enzyme in most of the tissues in the body. Changes in activities of CA have been associated with altered metabolism, especially in diabetes mellitus. Insulin resistance and hyperinsulinemia are common in hypertension. To investigate the possible role of CA, we measured the CA activity spectrophotometrically using p-nitrophenyl acetate as a substrate and acetazolamide, the specific inhibitor, in erythrocytes from normotensive and essential hypertensive subjects. Further, to evaluate the insulin action on CA, we used two different hemolysates; (i) insulin applied into hemolysate and (ii) hemolysate from insulin treated erythrocytes in vitro before the determination of CA activity. Two different levels of CA activities were obtained in these patients. CA activities were much lower (mean +/- SD, 0.88 +/- 0.19 U/min/mL) and higher (mean +/- SD, 1.77 +/- 0.23 U/min/mL) in patients than the normotensive controls (mean +/- 1 SD, 1.41 +/- 0.1 U/min/mL). These differences in both the groups were statistically significant (p less than 0.001). Similarly, total esterase activities in patients were (1.41 +/- 0.27 U/min/mL) that was 30% less in low activity group and (2.47 +/- 0.25 U/min/mL) that was 22% more in higher activity group in comparison with those from normotensives (2.02 +/- 0.17 U/min/mL). The relative percent of CA activities of insulin treated erythrocytes from normotensives and hypertensives were 11% and 18% higher than without insulin (p less than 0.05). No difference was observed when insulin was applied in the hemolysate. We conclude that essential hypertensive patients are associated with altered CA activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation and internalization of bound insulin in human erythrocytes.

Internalization and degradation of insulin by human erythrocytes were studied. Erythrocytes were incubated with 125I-insulin at 4 degrees C, 15 degrees C, and 37 degrees C for varying time intervals. These erythrocytes were then subjected to a low pH wash to release bound insulin followed by TCA precipitation. After 4, 22, and 24 hours of insulin binding at 4 degrees C, 92 to 95% of the bound 125I-insulin was dissociable and 92 to 98% of the extractable insulin was undegraded. After 3.5 hours of incubation at 15 degrees, 82% of the bound insulin was dissociable and 60% of this was intact. However, after 60, 90, 120, and 180 minutes of incubation at 37 degrees C, only 42, 34, 24, and 37%, respectively, of the bound insulin was dissociable. The undissociated insulin in the 37 degrees C studies was considered to be intracellular. With increasing time of incubation at 37 degrees C, the extractability of cell bound insulin and the proportion of undegraded dissociable insulin were decreased. When 125I-insulin binding was 95% blocked by preincubating the erythrocytes with anti-insulin receptor antibody, 95% of the degradation of 125I-insulin was also blocked. These studies indicate that mature human erythrocytes degrade internalized insulin and this process is time, temperature, and insulin receptor dependent.

Sodium-22 influx into erythrocytes from diabetic hypertensive patients on maintenance hemodialysis.

We have studied the percentage of 22Na+ uptake in cell suspensions; 0.4 to 2.0 x 10(9) erythrocytes/mL from diabetic uremic patients with secondary hypertension and from normal subjects. Suspensions from diabetic uremic patients with secondary hypertension 0.42 +/- 0.06 to 2.05 +/- 0.28; normal subjects showed a percentage uptake of 22Na+ of 0.27 +/- 0.05 to 1.28 +/- 0.22. The uptake of 22Na+ in 2.0 x 10(9) cells/mL was 60% more (P less than .05) in diabetic uremic patients than in the controls. These studies indicate that 22Na+ influx determinations may be used to distinguish secondary hypertensive patients from normal subjects.

The sodium-22 influx in erythrocytes from black males and females.

In order to establish a standard for sodium influx in erythrocytes for the black population, 22Na+ uptake was measured in 29 normotensive black volunteers. Nineteen males and 10 females during the follicular phase of the menstrual cycle were studied utilizing the procedure of Gambhir et al. (1). In the males, cell concentrations ranging from 0.64 to 2.0 X 10(9)/ml showed an influx of 0.42 to 1.34% of the total 22Na+ added, and in the females, using the same erythrocyte concentrations, the 22Na+ influx ranged from 0.37 to 1.1%; these differences were not significant. Intraassay variation of the 22Na+ data was 4.8%. Interassay variations have been explained elsewhere (1). These data provided a range of observed values for 22Na+ uptake in erythrocytes from a subpopulation of normotensive black males and females for comparison with hypertensive patients.

Potassium efflux: a simple method to determine intactness of erythrocytes.

This article describes a simpler approach to checking the integrity of erythrocyte membrane and internal milieu of the human erythrocyte. Potassium (K+) and hemoglobin (Hb) effluxes were studied in erythrocytes incubated with buffer G (pH 7.4) at varying time intervals and temperatures. Potassium in the supernatant was measured by direct ion selective electrode method and Hb by cell counter following incubation for 0 to 3 hours at 4 degrees to 55 degrees C. Basal adenosine triphosphate levels were also determined during varying time intervals at 37 degrees, 50 degrees, and 55 degrees C. At 4 degrees, 25 degrees, and 37 degrees C, insignificant (P greater than .05) amounts of K+ and Hb were released; amounts were significantly higher (P less than .0005) after 2 hours of incubation at 45 degrees for K+ and 50 degrees C for Hb. After 30 minutes at 55 degrees C, maximum K+ and Hb concentrations were found in the supernatant; concentrations remained the same up to 3 hours. Basal levels of adenosine triphosphate in the erythrocytes decreased significantly with time at higher temperatures. We conclude that K+ efflux, being more sensitive than Hb and more erythrocyte concentration-dependent, can be used as an effective method to confirm the intactness or viability of red blood cells.

A simpler procedure to study sodium-22 uptake (ouabain insensitive) in human erythrocytes.

We report a modification of the technique of Mahoney et al. (Blood 1982; 59: 439) for the determination of sodium-22 (22Na+) uptake in human erythrocytes. This modification facilitates the separation of 22Na+ taken up by erythrocytes from the free 22Na+ in the buffer by the addition of dibutyl phthalate, which forms an immiscible layer between the two. To further improve the sensitivity of 22Na+ uptake, we incubated a range of known numbers of erythrocytes with 22Na+ as opposed to the single cell suspension of known hematocrit used in Mahoney's et al. procedure (1). Erythrocytes are incubated in KCI buffer containing 2627 Bq (0.071 microCi) 22Na+ in a total volume of 0.5 mL for 0.5 h at 37 degrees C. Incubation is terminated by placing the tubes in ice for 10 min and the amount of 22Na+ taken up by the erythrocytes determined. We observe a linear relationship between erythrocyte concentrations (0.5 to 2.5 X 10(9) cells/mL) and percent uptake of 22Na+ (0.37 +/- 0.06 (1 SD) to 1.85 +/- 0.27 (1 SD) of the total 22Na+, respectively). The procedure is simple and sensitive, and can be used in clinical laboratories for the routine evaluation of 22Na+ uptake in erythrocytes.

Characterization of an intracellular insulin-degrading enzyme in human erythrocytes.

Using conventional techniques of ammonium sulfate fractionation and Sephadex gel column chromatography, insulin-degrading enzyme was partially purified from lysate of human erythrocytes. The enzymatic activity was measured by the trichloroacetic acid precipitation method. Compared to trypsin, the enzyme was highly specific for insulin. The apparent molecular weight of the enzyme was 160,000 Da, the optimum pH was the 7.4 to 7.8 range, and the Km value for insulin for the partially purified enzyme was 162 nM. Bacitracin and N-ethylmaleimide were potent inhibitors, while chloroquine, ethylenediaminetetraacetate, antipain, and soybean trypsin inhibitor failed to inhibit the activity of the enzyme. Like most nucleated cells, human erythrocytes not only have the membranal insulin receptors, but also possess the cytosolic specific insulin-degrading enzyme. Insulin internalization and degradation are shown to be due to the receptor and the enzyme acting in concert as in many nucleated cells. Anucleated erythrocytes have both these entities for possible internalization and degradation of insulin.

Comparison of human erythrocyte insulin binding and adenosinetriphosphatase activity.

Twelve healthy, non-obese male volunteers were selected to measure cellular magnesium (Mg(++)), calcium (Ca(++)), sodium/potassium (Na(+)/K(+)), and adenosinetriphosphatase (ATPase) activities. Measurements were performed using a crude hemolysate as well as a membrane fraction representing cytosolic pump activity. Binding-site data were subjected to Scatchard analysis for determination of receptor number and affinity.There were 432 ± 1.87 insulin-binding sites per cell. ATPase activity was measured in units of moles of inorganic phosphate (Pi) released per gram of hemoglobin (Hgb) every two hours (µmol of Pi/g of Hgb/2 h). Na(+)/K(+)-ATPase activity in the hemolysate and membrane fractions was 18 ± 0.99 µmol of Pi/g of Hgb/2 h and 11.0 ± 0.88 µmol of Pi/g of Hgb/2 h, respectively. Calcium-ATPase activity was 136 ± 1.92 µmol of Pi/g of Hgb/2 h in the hemolysate, and 82 ± 2.07 µmol of Pi/g of Hgb/2 h in the membrane. Magnesium-ATPase activity in these fractions measured 24 ± 1.09 and 16 ± 0.91, respectively.Linear regression analysis of binding capability vs pump activity failed to disclose a significant relationship. Given the narrow range of values in this small study group, a much larger sample size will be required before a relationship can be established.

The effect of citrate and polyamines on human benign prostate in organ culture.

A technique for short-term cultivation of slices of human benign prostate tissue in an organ culture environment is described. Minimum essential medium utilized was supplemented with 10 percent fetal bovine serum and 25 mg/mL of citrate, spermine, and spermidine. Spermine in the presence of testosterone in the culture medium induced morphological changes, enhancing the morphology of the tissue. The epithelium was well preserved morphologically. Citrate or spermidine together with testosterone does not enhance any morphological change. After seven days of culture, necrosis was developed. The spent culture medium showed a pattern of prostatic acid phosphatase production. When treated with spermine, the maximum level was reached at four days, after which it decreased. With spermidine or citrate, the maximum level is reached at two days of culture. Tissue ß-glucuronidase activity was enhanced by either spermidine, spermine, or citrate together with testosterone. The biochemical studies suggested that explants of human benign prostate can be well maintained in organ culture for a few days.

Blood pressure and urinary sodium in black American adolescents.

For 56 black American adolescents ranging in age from 13 to 19 years, 24-hour urinary sodium excretion was compared with blood pressure. The volunteers came from families of middle to low economic levels. Their body weight varied from 70 to 129 percent of ideal body weight. The average systolic pressure of all the subjects was 112.1 mmHg with a standard deviation of 9.5 mmHg; mean diastolic pressure was 69.6 mmHg with a SD of 8 mmHg. The average 24-hour urinary sodium excretion of the total population was 131.5 mEq/24 h with a SD of 59 mEq. For the 29 male subjects, the mean value was 137 mEq/24 h (range, 30 to 309 mEq) and for 27 female subjects, 126.3 mEq/24 h (range, 40 to 197 mEq). The average urinary excretion among these groups was not significantly different (P > .05) and the urinary Na/K ratio of the total group was 4.3 with a SD of 2.0. This ratio among various age and sex subgroups was not significantly different (P > .05). Urinary sodium excretion values were taken as a reflection of dietary salt intake among these volunteers. The data suggest that the black adolescents studied do not consume excess sodium and contradict the belief that blacks eat excess sodium. Further, these baseline data must be considered among the factors responsible for the development of hypertension.

Metabolism of insulin in erythrocytes from renal failure patients on maintenance hemodialysis.

Glucose tolerance does not improve to the normal level after dialysis; however, our studies showed that the insulin receptor binding to erythrocytes of nondiabetic patients with chronic renal failure (CRF) on hemodialysis was more than that in normal subjects. To understand this apparent anomaly in insulin receptor action and glucose metabolism, we investigated insulin degradation-a postreceptor event of insulin binding-in erythrocytes from CRF patients and compared it with that of normal subjects. We studied insulin degradation by erythrocytes from each of eight CRF patients and five normal subjects. The average hyperbolic insulin degradation curve for the CRF patients showed lower activity and a right-handed shift compared to the curve for the normal subjects. The average maximum degradation of insulin in the CRF patients was significantly lower than that of normal subjects. The number of erythrocytes required to produce 50% of maximum insulin degradation was significantly greater in these patients than that in the normal subjects. Furthermore, a linear correlation was observed between the duration of dialysis and maximum percent of insulin degradation in the CRF patients. Clinical implications of these findings are unclear at the present time. However, the insulin-degrading activity in erythrocytes may be reflective of that in other body tissues.