Cell Sorbitol Research Articles

Modulation of osmolytes in MDCK cells by solutes, inhibitors, and vasopressin

MDCK cells accumulate organic osmolytes in response to hyperosmotic NaCl-supplemented medium. We examined time course and inhibitor sensitivity of myo-inositol, sorbitol, and glycerophosphorylcholine (GPC) accumulation in MDCK cells exposed to hyperosmotic NaCl-, D-glucose-, or mannitol-supplemented media. In NaCl medium, cells preferentially accumulated inositol and GPC. In comparison, in glucose medium cells preferentially accumulated sorbitol and GPC. Inositol demonstrated a late (72-96 h) accumulation in glucose medium, although less than in NaCl medium. Mannitol medium did not significantly stimulate accumulation of any of these three osmolytes at 24 h, suggesting that hyperosmolality alone is not sufficient stimulus for their accumulation in this time frame. GPC accumulation was very rapid in glucose medium, and fell to the level induced by NaCl medium at 96 h (approximately 50 nmol/mg protein). Inositol and sorbitol accumulated more gradually, each reaching greater than 400 nmol/mg protein after 96 h. Sorbitol was still accumulating at 96 h, whereas inositol plateaued at 72-96 h. Phlorizin or sorbinil blocked accumulation of inositol or sorbitol, respectively. Sorbitol and GPC accumulation in glucose medium were partially inhibited in absence of serum or in presence of 1 microM vasopressin. Thus NaCl and glucose appear to stimulate specific cellular mechanisms responsible for accumulation of inositol, sorbitol, and GPC in MDCK cells. This accumulation is also modulated by constituents of serum.

Effects of NaCl, glucose, and aldose reductase inhibitors on cloning efficiency of renal medullary cells

To analyze the effects of sorbitol accumulation on the survival and growth of epithelial cells from rabbit renal inner medulla, cloning efficiency (an index of cell viability) was measured at normal and high glucose and NaCl concentrations and when sorbitol accumulation was prevented by Tolrestat and Sorbinil, which inhibit aldose reductase. With PAP-HT25 cells grown to near confluence, high NaCl increases aldose reductase activity, causing enough rise in cell sorbitol concentration to balance most of the increased osmolality of the high extracellular NaCl. Inhibition of aldose reductase prevents both the increased enzyme activity and sorbitol accumulation in a dose-related manner. Paralleling this, colony-forming efficiency is not affected by the inhibitors at a normal NaCl concentration but is greatly reduced when extracellular NaCl is high. On the other hand, high glucose levels, as occur in diabetes, increase sorbitol content well above the concentration required for osmotic balance and inhibit colony-forming efficiency. Under those conditions, aldose reductase inhibitors lower cell sorbitol and reverse (at 300-350 mosmol/kgH2O) or reduce (at 500-550 mosmol/kgH2O) the decrease in colony-forming efficiency caused by high glucose. Thus sorbitol accumulation is necessary for osmoregulation when induced by high osmolality but is harmful when induced by high glucose.

Reduction of Red Blood Cell Sorbitol by Ascorbic Acid in Diabetic Patients

Reduction of Red Blood Cell Sorbitol by Ascorbic Acid in Diabetic Patients

Red cell sorbitol concentration in relation to short- and medium-term variation of plasma glucose.

To evaluate the time course of changes in red cell sorbitol (RCS) concentration in relation to variations of plasma glucose levels, RCS was determined in 7 normal subjects during i.v. glucose infusion (IVGTT); in 6 hyperglycemic insulin-dependent diabetic subjects while glycemia was normalized with Biostator GC 115 and in 4 diabetic patients in previously poor metabolic control, in whom normal glycemia was obtained in 8-10 days by intensive insulin therapy. During IVGTT, plasma glucose levels increased with significant differences from baseline at 5, 10, 16, 25, 60, 100 and 160 min and returned to basal levels after 3h; RCS concentration showed small and insignificant increases. During i.v. insulin infusion, plasma glucose fell to almost normal levels within roughly 3h; RCS levels showed a gradual reduction becoming significant at 180 min. In the third study, decrease in plasma glucose was always associated with a fall in RCS level which became significant between the 2nd and the 3rd day of the study. Thus, RCS levels were not affected by very short-term variations of glycemia but by a previous hyperglycemic crisis that lasted a few hours. There were therefore medium-term variations of RCS level. In conclusion, RCS determination is not useful as an index of metabolic control in diabetes.

Effect of a New Aldose Reductase Inhibitor, Ponalrestat, on Reduction of Red Blood Cell Sorbitol Level in Diabetics

Effect of a New Aldose Reductase Inhibitor, Ponalrestat, on Reduction of Red Blood Cell Sorbitol Level in Diabetics

Red Blood Cell Sorbitol Lowering Effects and Tolerance of Single Doses of AL 1576 (HOE 843) in Diabetic Patients

The safety and biochemical effects of AL 1576 (HOE 483), a recently developed aldose reductase inhibitor, were evaluated. In a double-blind, placebo-controlled, clinical trial, AL 1576 (HOE 483) was administered to diabetic patients for the first time. Four single, orally administered dose levels were tested, (2, 5, 10, and 20 mg). No clinically important adverse effects were seen in any of the patients. AL 1576 (HOE 483) suppressed red blood cell (RBC) sorbitol concentrations in a dose-related fashion. Also found were statistically significant inverse correlations between the plasma drug concentration and both RBC sorbitol concentrations as well as RBC sorbitol/serum glucose ratios. In single doses up to 20 mg, AL 1576 (HOE 483) is well tolerated and decreases RBC sorbitol, a biochemical marker of pharmacologic activity, in diabetic patients.

Cone electroretinograms and visual acuities of diabetic patients on Sorbinil treatment

Photopic, 30-Hz, and foveal electroretinograms were measured in 19 diabetic patients in an experimental study of the effects of short-term Sorbinil (an aldose-reductase inhibitor) on retinal function. Patients were assigned in double-blind fashion to Sorbinil (250 mg/day) or placebo groups and were tested at the outset and after four weeks of therapy. Comparisons (t-test) between the Sorbinil and placebo groups failed to show significant effects of treatment on electroretinograms, although there was a significant correlation within the Sorbinil group between foveal recordings and red cell sorbitol at the end of treatment. Analysis showed that increased foveal electroretinograms were found in patients with low initial retinopathy but not in those with greater retinopathy. Eight patients continued Sorbinil treatment for one year. Again patients improving their foveal measurements had less initial retinopathy than those not improving. This difference was significant after one year of treatment but not at four weeks.

Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol flux.

Renal medullary extracellular NaCl concentration is high during antidiuresis. To compensate, the cells accumulate large amounts of nonperturbing, osmotically active solutes (organic "osmolytes"), including sorbitol. GRB-PAP1 is a continuous line of epithelial cells from rabbit inner medulla. These cells accumulate sorbitol when medium NaCl concentration is elevated. The accumulation involves increase in aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate control of cell sorbitol once aldose reductase was induced. We measured cell sorbitol, cell-to-medium sorbitol flux, and aldose reductase in cells grown in medium made hyperosmotic (600 mosmol/kg) with added NaCl and at intervals after medium osmolality was reduced to 300 mosmol/kg. In the hyperosmotic medium, cell sorbitol averaged 990 mmol/kg protein (approximately 260 mM), and its flux into the medium was 740 mmol.kg cell protein-1.day-1 (permeability less than 2 X 10(-9) cm/s). Within 5 min after return to isosmotic medium, sorbitol efflux increased greater than 150-fold. By the end of 1 day, cell sorbitol fell 77% but aldose reductase decreased only 10%. Aldose reductase then fell slowly to low levels over 2 wk. Thus renal medullary cells, chronically adapted to high NaCl, reduced their sorbitol level on return to isosmotic conditions by at least two mechanisms: 1) rapid increase in sorbitol flux into the medium, and 2) slow changes in the amount of aldose reductase.

Multiple peaks in human nerve action potentials: [formula omitted], E. Stålberg and F. Zarola (Dept. of Clin. Neurophysiology, University Hospital, Uppsala, Sweden)

Three clinical trials to evaluate the efficacy of the aldose reductase inhibitor sorbinil in improving or preventing diabetic neural function have either been completed or are currently in progress. In the first study from Seattle and Chicago, motor and sensory nerve conduction velocities (NCV) were evaluated in 39 insulin- and noninsulin-dependent, glycemic-stable diabetic patients in a randomized, double-blind, crossover trial. During the 9 weeks of treatment with 250 mg/d of sorbinil, there was a faster nerve conduction velocity of all 3 nerves tested when compared with the placebo period: peroneal motor NCV (+0.70 ± 0.24 m/s; x ± SEM; P < 0.008), median motor NCV (+0.66 ± 0.27 m/s; P < 0.005), and median sensory NCV (+1.16 ± 0.50 m/s; P < 0.035). Conduction velocity for all 3 nerves declined significantly within 3 weeks following cessation of the drug. These effects of sorbinil were unrelated to glycemic control, which was constant during the study. Although the effects of sorbinil in improving nerve conduction velocity were small, the findings suggest that the polyol-pathway activity contributes to slowed nerve conduction velocity in diabetics. The second study is a seven-center, double-blind, randomized, 12-month trial of 210 to 280 diabetic patients with clinical signs, symptoms, and objective measurements of neuropathy. The trial has a common-core protocol with end-point evaluations of scored neural signs, symptoms, and neural measurements. Two unique neural tests were designed and validated for use in this trial: thermal and tactile perception thresholds of the fingers and toes. Thresholds were determined using a two-alternative, forced-choice procedure on the Opticon Tactile Tester and Thermal Sensitivity Tester. Data analysis showed that there was no significant difference as a result of gender, dominant v nondominant side, age, digit temperature, room temperature, presence of heavy callouses, or technologists. In validation studies, diabetics without clinical evidence (history and examination) of diabetic neuropathy were not significantly different from normals, but diabetics with clinical evidence of diabetic neuropathy had elevated finger and toe thermal and tactile perception thresholds (all P < 0.001). There was no significant difference in the test results from normals from each of the seven centers. Thus, these tests were well suited for multicenter trials. Special protocols in each of the seven centers allowed for a broad spectrum of ancillary studies including the following: evaluation of autonomic nervous system integrity, white blood cell sorbitol and myo-inositol levels, impotence evaluation, single-fiber electromyography, sural nerve biopsy, and sensory evoked potentials. This trial is designed to address the question of whether sorbinil could improve diabetic neuropathy. Using the core protocol and the special studies for each center, a wide spectrum of neurologic end-points are evaluated in a bouble-blind fashion. The third protocol is a 12-center, double-blind, randomized, 212-year study of 600 to 720 diabetic patients with minimal or no diabetic complications. There is a common-core protocol with end-point measures of retinopathy, nephropathy, and neuropathy. This trial is unique in that (1) the placebo data adds valuable information into the natural history of diabetic complications, and (2) there is a unique collaborative arrangement between the national Institutes of Health and a pharmaceutical company, representing a first of its kind. This trial is designed to address the question of whether sorbinil could prevent diabetic complications (the primary end-point is retinopathy; the secondary end-points are nephropathy and neuropathy). In summary, these three trials are landmark trials representing advances in the basic pathophysiology of diabetic neuropathy, resulting in the development of new clinical tools and establishing a precedence for collaboration in important clinical trials.

Induction of aldose reductase and sorbitol in renal inner medullary cells by elevated extracellular NaCl.

Aldose reductase [aldehyde reductase 2; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21] catalyzes conversion of glucose to sorbitol. Although its activity is implicated in the progression of ocular and neurological complications of diabetes, the normal function of the enzyme in most cells is unknown. Both aldose reductase activity and substantial levels of sorbitol were previously reported in renal inner medullary cells. In this tissue, the extracellular NaCl concentration normally is high and varies considerably depending on the urine concentration. We report here on a line of renal medullary cells in which medium that is high in NaCl greatly increases both aldose reductase activity and intracellular sorbitol. In these tissue culture cells (and presumably also in the renal inner medulla), the intracellular sorbitol helps balance the osmotic pressure of elevated extracellular NaCl and thus prevents cellular dehydration.

P-6-19 Sorbitol in polymorphonuclear cells from diabetic patients

P-6-19 Sorbitol in polymorphonuclear cells from diabetic patients

Sorbitol in polymorphonuclear cells from diabetic patients

Sorbitol in polymorphonuclear cells from diabetic patients

Screening for red blood cell sorbitol dehydrogenase deficiency in patients with diabetes or cataracts.

Quantitative screening for red blood cell sorbitol dehydrogenase (RBC-SORD) deficiency in 111 patients with juvenile onset diabetes, 92 patients with adult onset diabetes, 42 patients with idiopathic cataracts and 192 professional blood donors was performed. A wide variability in RBC-SORD activity in controls and patients was observed. No significant differences in SORD activity either between patients with diabetes and patients with idiopathic cataracts or between diabetics with and without cataracts were observed. Whether or not there were carriers for either amorphous or hypomorphous alleles of the SORD locus in the population studied could not be defined in terms of enzymatic activity levels.

Applicability of red blood cell sorbitol measurements to monitor the clinical activity of sorbinil

Increased flux of glucose through the polyol pathway with resultant accumulation of tissue sorbitol is implicated in the pathogenesis of the chronic complications of diabetes. Sorbinil is a potent inhibitor of aldose reductase (the first enzyme in the polyol pathway) and has been shown to normalize sorbitol levels in relevant tissues (eg, nerve, kidney, lens) of experimentally-induced diabetic animals. The purpose of this study was to identify a relatively noninvasive method of monitoring the intrinsic (or biochemical) efficacy of sorbinil in diabetic man. Specifically, the objective was to identify a readily accessible tissue that would be reflective of polyol pathway activity and the activity of sorbinil in clinically relevant but less accessible tissues. Based on several previous studies, 1–3 which demonstrated that sorbitol accumulation in human red blood cells (RBCs) was a function of ambient glucose concentrations, either in vitro or in vivo, our investigations were conducted to determine if (1) RBC sorbitol accumulation would correlate with sorbitol accumulation in lens and nerve tissue of diabetic rats; (2) the effect of sorbinil in reducing sorbitol levels in lens and nerve tissue of diabetic rats would be reflected by changes in RBC sorbitol; and (3) sorbinil would reduce RBC sorbitol in diabetic man. 4

Clinical trials of sorbinil on nerve function

Three clinical trials to evaluate the efficacy of the aldose reductase inhibitor sorbinil in improving or preventing diabetic neural function have either been completed or are currently in progress. In the first study from Seattle and Chicago, motor and sensory nerve conduction velocities (NCV) were evaluated in 39 insulin- and noninsulin-dependent, glycemic-stable diabetic patients in a randomized, double-blind, crossover trial. During the 9 weeks of treatment with 250 mg/d of sorbinil, there was a faster nerve conduction velocity of all 3 nerves tested when compared with the placebo period: peroneal motor NCV (+0.70 ± 0.24 m/s; x ± SEM; P < 0.008), median motor NCV (+0.66 ± 0.27 m/s; P < 0.005), and median sensory NCV (+1.16 ± 0.50 m/s; P < 0.035). Conduction velocity for all 3 nerves declined significantly within 3 weeks following cessation of the drug. These effects of sorbinil were unrelated to glycemic control, which was constant during the study. Although the effects of sorbinil in improving nerve conduction velocity were small, the findings suggest that the polyol-pathway activity contributes to slowed nerve conduction velocity in diabetics. The second study is a seven-center, double-blind, randomized, 12-month trial of 210 to 280 diabetic patients with clinical signs, symptoms, and objective measurements of neuropathy. The trial has a common-core protocol with end-point evaluations of scored neural signs, symptoms, and neural measurements. Two unique neural tests were designed and validated for use in this trial: thermal and tactile perception thresholds of the fingers and toes. Thresholds were determined using a two-alternative, forced-choice procedure on the Opticon Tactile Tester and Thermal Sensitivity Tester. Data analysis showed that there was no significant difference as a result of gender, dominant v nondominant side, age, digit temperature, room temperature, presence of heavy callouses, or technologists. In validation studies, diabetics without clinical evidence (history and examination) of diabetic neuropathy were not significantly different from normals, but diabetics with clinical evidence of diabetic neuropathy had elevated finger and toe thermal and tactile perception thresholds (all P < 0.001). There was no significant difference in the test results from normals from each of the seven centers. Thus, these tests were well suited for multicenter trials. Special protocols in each of the seven centers allowed for a broad spectrum of ancillary studies including the following: evaluation of autonomic nervous system integrity, white blood cell sorbitol and myo-inositol levels, impotence evaluation, single-fiber electromyography, sural nerve biopsy, and sensory evoked potentials. This trial is designed to address the question of whether sorbinil could improve diabetic neuropathy. Using the core protocol and the special studies for each center, a wide spectrum of neurologic end-points are evaluated in a bouble-blind fashion. The third protocol is a 12-center, double-blind, randomized, 2 1 2 - year study of 600 to 720 diabetic patients with minimal or no diabetic complications. There is a common-core protocol with end-point measures of retinopathy, nephropathy, and neuropathy. This trial is unique in that (1) the placebo data adds valuable information into the natural history of diabetic complications, and (2) there is a unique collaborative arrangement between the national Institutes of Health and a pharmaceutical company, representing a first of its kind. This trial is designed to address the question of whether sorbinil could prevent diabetic complications (the primary end-point is retinopathy; the secondary end-points are nephropathy and neuropathy). In summary, these three trials are landmark trials representing advances in the basic pathophysiology of diabetic neuropathy, resulting in the development of new clinical tools and establishing a precedence for collaboration in important clinical trials.

The Relationship between Red Cell Sorbitol and Blood Glucose Concentration in Diabetes during Hyperglycaemic Clamp Studies and in Out-Patients

The Relationship between Red Cell Sorbitol and Blood Glucose Concentration in Diabetes during Hyperglycaemic Clamp Studies and in Out-Patients

Effect of tolrestat on red blood cell sorbitol levels in patients with diabetes

The effect of the aldose reductase inhibitor, tolrestat, on red blood cell (RBC) sorbitol levels was studied in 23 patients with diabetes after oral dosing with tolrestat, 25 or 100 mg b.i.d. The mean (+/- SE) RBC sorbitol levels (measured 12 hours after the preceding dose) after 3, 7, and 13 days of dosing decreased after both dose levels. After 25 mg tolrestat the RBC sorbitol levels fell from 25.1 +/- 4.0 to 20.0 +/- 5.7 nmol/gm hemoglobin (21%) and after 100 mg tolrestat the level fell from 26.7 +/- 3.7 to 11.4 +/- 1.7 nmol/gm hemoglobin (57%; P less than 0.001). This latter RBC sorbitol concentration is similar to levels in individuals without diabetes. At both dosage levels the maximum decrease in RBC sorbitol levels occurred after only 3 days of dosing. Tolrestat had no effect on plasma glucose or hemoglobin A1 concentrations. The overall mean plasma unbound drug concentration measured 12 hours after 100 mg tolrestat (11.7 +/- 3.0 ng/ml; 3.3 X 10(-8) mol/L) was similar to the median inhibitory level (3 X 10(-8) mol/L) of tolrestat for sorbitol accumulation in human RBCs incubated in a high-glucose medium. Our results demonstrate the systemic bioavailability of tolrestat and its aldose reductase inhibitory activity in erythrocytes of patients with diabetes.

Red cell sorbitol and diabetic control.

There is a disagreement about the value of red cell sorbitol as an indicator of diabetic control. We studied 7 insulin requiring diabetics, one for 10 days and 6 for 24 hours. In the 10-day study, blood samples were obtained every 4-6 hours. Sequential analysis of plasma glucose and red cell sorbitol levels showed that simultaneous levels had the best correlation r = 0.534, P less than .001. In the 24-hour studies, blood samples were obtained at 1 to 4-hour intervals. Three of the 6 patients showed the best correlation between simultaneous plasma glucose and red cell sorbitol, with r = 0.727 to 0.957 and P less than .001 to P less than .07. When plasma glucose values were compared to red cell sorbitol levels 1, 2, 4, 8 and 12 h later, correlation coefficients were not as good as the simultaneous comparison. We conclude that the measurement of red cell sorbitol correlates well with simultaneous plasma glucose in some but not all diabetic subjects and provides no additional information to the clinician.

Sorbitol determination by liquid chromatography: application to red blood cells of diabetic rats.

This report describes an application of liquid chromatography to the determination of sorbitol in red blood cells. The chromatograph employed in the present study was made up of sub- and main-separation systems and a detector portion. The sub-separation system was for concentration of polyols and involved two small columns, each containing the same anion exchange resin. The first was a tiny column which, in borate form, served as the concentrator of polyols and sugars charged in a large volume, while the second, in acetate form, separated the carbohydrates from the borate. The main system was for the fine separation of each carbohydrate and employed cation exchange columns. The detector part utilized a flow fluorometric method comprising two successive reactions: periodate oxidation followed by the Hantzsch reaction. The resulting whole chromatographic system was applied to the determination of sorbitol in red blood cells obtained from normal rats and rats made diabetic by the administration of streptozotocin; a part of the latter group had also received an aldose reductase inhibitor. Our results supported the concepts that a prolonged duration of high blood glucose level induces an elevated level of sorbitol inside red blood cells and that aldose reductase inhibitors are effective in reducing this level.

Plasma and red cell sorbitol assay in diabetic subjects.

Plasma and red cell sorbitol concentrations, fasting plasma glucose, glycosylated hemoglobin (GHb) were evaluated in 30 diabetic patients and 42 normal subjects. Red cell sorbitol levels were evaluated in hemolysate and non-hemolyzed samples. Mean red cell sorbitol concentrations evaluated after hemolysis were 21.5 +/- 5 nmol/ml in diabetics and 12.2 +/- 4 nmol/ml in controls (p less than 0.001). Mean values of red cell sorbitol determined in non-hemolyzed samples were 13.9 +/- 3 nmol/ml in diabetics and 8.1 +/- 3 nmol/ml in controls (p less than 0.001). Mean plasma sorbitol concentrations were 8.4 +/- 3 nmol/ml in diabetics and 5.2 +/- 1 nmol/ml in controls (p less than 0.001). The within and between run reproducibilities evaluated in plasma, hemolysate and non-hemolyzed red cells gave the lowest values for hemolyzed red cell samples. The studies on stability showed that samples neutralized and stored at -20 degrees C gave reproducible values if assayed within 3 days. A positive correlation was found between red cell hemolysate sorbitol concentrations and fasting plasma levels in both diabetics (r = 0.56; p less than 0.001) and controls (r = 0.62; p less than 0.001). Red cell hemolysate sorbitol concentrations were also positively correlated to GHb in both diabetics (r = 0.64; p less than 0.001) and controls (r = 0.36; p less than 0.05). We believe the most effective sorbitol assay to be that obtained in hemolyzed red cells and that these values related to glycemic levels could be a useful index of metabolic control and an indicator of tissue sorbitol levels in humans.