Erythrocyte Sorbitol Research Articles

Unraveling the therapeutic potential of Astilbe rivularis Buch.-Ham. ex D. Don in attenuation of diabetic neuropathy in laboratory rats

Ethnopharmacological relevanceAstilbe rivularis Buch.-Ham. ex D. Don is a rare medicinal plant, traditionally employed for treating several disorders. The juice, decoction or powder of the roots, rhizomes, leaves and even the entire plant, are used for managing peptic ulcer, diarrhoea, jaundice, sprains and muscular swellings, bone fracture and dislocation of joints, postpartum bleeding and other menstrual disorders. These conventional medicinal uses make Astilbe rivularis a promising candidate for further research. Aim of the studyThis study was designed to explore the neuroprotective potential of hydroethanolic extract of Astilbe rivularis (ARHE) in diabetic neuropathy (DN) in rats. Materials and methodsGC-MS analysis was used to identify the phytoconstituents present in the plant extract. DN was induced by administration of STZ (55 mg/kg, i.p.), 15 min after NAD (230 mg/kg, i.p.) injection. The rats with fasting blood glucose (FBG) level >250 mg/dl were included in the study. DN was assessed by estimating the level of FBG, lipid profile, and invitro and invivo oxidative stress parameters. Additionally, behavioural parameters like, mechanical hyperalgesia, hot and cold allodynia were estimated to assess diabetic neuropathy. Furthermore, the level of antioxidant enzymes like SOD, GSH, and TBARS in sciatic nerve and inflammatory markers like, TGF-β and IL-6 were measured. ResultsAltogether, 30 phytoconstituents were identified including heptafluorobutyric acid, hexadecanoic acid, and beta-sitosterol depicting antioxidant, antidiabetic, and anticancer properties, respectively. Administration of different doses (100, 200, and 400 mg/kg) of ARHE to diabetic rats attenuated elevated blood glucose level and restored lipid profile, body weight, food and water intake, and antioxidant level. Moreover, elevated level of inflammatory markers like, TGF-β and IL-6 was also found to be attenuated in sciatic nerve. Furthermore, ARHE attenuated the pain response assessed by mechanical hyperalgesia and hot and cold allodynia in diabetic neuropathy rats. ARHE also showed inhibitory activity on ALR enzyme and erythrocyte sorbitol accumulation, and ameliorated oxidative stress. Histopathological study indicated improvement in the architecture of sciatic nerve tissue in diabetic neuropathy rats with the treatment of ARHE. ConclusionsConclusively, hydroethanolic extract of Astilbe rivularis exhibited neuroprotective potential and ameliorated diabetic neuropathy in rats.

Quantification of total phenols in Coleus forskohlii and inhibition of aldose reductase by rosmarinic acid

Many complications of diabetes have been associated with the accumulation of intracellular sorbitol caused by excessive activity of aldose reductase (ALR2). ALR2 is a member of the aldo-keto reductases (AKR) superfamily and has gained significant attention due to its role in the development of diabetic complications. Inhibiting ALR2 could help prevent or delay some of these problems. Coleus forskohlii is a natural medicinal plant that is known for its medicinal effects and is also used as a beverage in several European countries. High-performance liquid chromatography (HPLC) was used to measure the polyphenol content of aqueous leaf extracts of C. forskohlii, and it was found that rosmarinic acid (RA) had the highest concentration (1.25 mg/g) compared to other polyphenols. In vitro bioassays were conducted to evaluate the inhibitory effect of RA on ALR2. RA significantly and dose-dependently reduced the build-up of sorbitol in human erythrocytes under high glucose conditions. Molecular docking studies suggest that RA, which is an essential polyphenol, could be a potential ALR2 inhibitor. This study is the first to show that RA has an IC50 value of 56 % to exhibit an inhibitory effect against ALR2.

Inhibitory Potential of Murraya Koenigii (L.) and Ficus Carica L. Extracts Against Aldose Reductase (ALR), Advanced Glycation End Products (AGEs) Formation and Sorbitol Accumulation

Abstract Introduction: Murraya koenigii (L.) and Ficus carica L. are traditionally used plants with significant medicinal and nutritional values. Aim and Objective: The present study was focused on the evaluation of hydro-alcoholic and aqueous extracts of M. koenigii (L.) leaves [MKHA (M. koenigii (L.) hydro-alcoholic extract) and MKAQ (M. koenigii (L.) aqueous extract)] and dried fruits of F. carica L. [FCHA (F. carica L. hydro-alcoholic extract) and FCAQ (F. carica L. aqueous extract)] in the attenuation of markers of microvascular complications associated with diabetes mellitus which can be further used to investigate the pharmacological activity of these plants in treatment of diabetes and its complications. Material and Method: The attenuating effect of the extracts was evaluated by calculating the ALR1 enzyme inhibition in a kidney of Wistar rat, anti-glycation activity in bovine serum albumin (BSA) and erythrocyte sorbitol accumulation inhibition in heparinized human blood. Results: A significant inhibitory effect (IC50 6.47μg/ml,7.26μg/ml,8.93 μg/ml and 9.66μg/ml) was observed with different concentrations of extracts (MKHA, MKAQ, FCHA and FCAQ) respectively, against ALR enzyme. After the 4th week of incubation, the inhibition of AGEs formation by MKHA, MKAQ, FCHA and FCAQ (500μg/ml) was found to be 82.58%, 78.58%, 74.39% and 69.56% respectively. MKHA, MKAQ, FCHA and FCAQ were found to exhibit significant inhibition against the accumulation of sorbitol in RBCs with IC50 188.88 μg/ml, 247.74μg/ml, 291.94μg/ml and 345.34μg/ml, respectively. Conclusion: The administration of different concentrations of MKHA, MKAQ, FCHA and FCAQ significantly attenuated ALR, AGEs and sorbitol accumulation; hence, it can provide a basis for identification and development of new inhibitors of these biomarkers.

Study of Epalrestat in Diabetic Distal Symmetric Polyneuropathy and correlation of its therapeutic efficacy with erythrocyte sorbitol levels : A step towards Precision Medicine

Aims : Diabetic Distal Symmetric Polyneuropathy (DSPN), despite being the most common and a disabling diabetic complication, remains difficult to treat. It has led to rekindle our interest in Epalrestat which has the potential to alter the natural history of the disease. The present study was designed to evaluate the efficacy of Epalrestat in DSPN and to corelate its therapeutic efficacy with baseline erythrocyte sorbitol levels. Methods : 100 patients with duration of diabetes more than five years and Diabetic Neuropathy Symptom Score (DNSS) ≥ 1 were included. They were divided into two groups of 50 patients each : Group 1 (received Tablet Epalrestat 150 mg once a day), Group 2 (received placebo). Baseline Diabetic Neuropathy Symptom Score (score out of 4), Numeric Pain Intensity Scale (score out of 10), Monofilament score (score out of 10), Vibration Perception Threshold (VPT) by Sensitometer and erthyrocyte sorbitol levels (by ELISA) were recorded. Same parameters were repeated at three months follow up visit. Results : Epalrestat was overall more effective than placebo in improving the symptoms as well as in improving the quantitative sensory nerve function measured by sensitometer The improvement in all the parameters positively correlated with baseline erythrocyte sorbitol levels. Conclusions : Epalrestat offers a ray of hope in the treatment of DSPN patients who have high baseline erythrocyte sorbitol levels and thus can be a useful tool in predicting the response to drug.

Triglyceride-lowering effect of the aldose reductase inhibitor cemtirestat-another factor that may contribute to attenuation of symptoms of peripheral neuropathy in STZ-diabetic rats.

Hyperglycemia is considered a key risk factor for development of diabetic complications including neuropathy. There is strong scientific evidence showing a primary role of aldose reductase, the first enzyme of the polyol pathway, in the cascade of metabolic imbalances responsible for the detrimental effects of hyperglycemia. Aldose reductase is thus considered a significant drug target. We investigated the effects of cemtirestat, a novel aldose reductase inhibitor, in the streptozotocin-induced rat model of uncontrolled type 1 diabetes in a 4-month experiment. Markedlyincreased sorbitol levels were recorded in the erythrocytes and the sciatic nerve of diabetic animals. Osmotic fragility of red blood cells was increased in diabetic animals. Indices of thermal hypoalgesia were significantly increased in diabetic rats. Tactile allodynia, recorded in diabetic animals in the early stages, turned to mechanical hypoalgesia by the end of the experiment. Treatment of diabetic animals with cemtirestat (i) reduced plasma triglycerides and TBAR levels; (ii) did not affect the values of HbA1c and body weights; (iii) reversed erythrocyte sorbitol accumulation to near control values, while sorbitol in the sciatic nerve was not affected; (iv) ameliorated indices of the erythrocyte osmotic fragility; and (v) attenuated the symptoms of peripheral neuropathy more significantly in the middle of the experiment than at the end of the treatment. Taking into account the lipid metabolism as an interesting molecular target for prevention or treatment of diabetic peripheral neuropathy, the triglyceride-lowering effect of cemtirestat should be considered in future studies. The most feasible mechanisms of triglyceride-lowering action of cemtirestat were suggested.

Evaluation of effect of alcoholic extract of heartwood of Pterocarpus marsupium on in vitro antioxidant, anti-glycation, sorbitol accumulation and inhibition of aldose reductase activity

Rising popularity of phytomedicines in various diseased conditions have strengthened the significance of plant-research and evaluation of phytoextracts in clinical manifestations. Pterocarpus marsupium Roxb., a medicinal plant, known for its anti-oxidant and anti-diabetic activity is a rich source of phytochemicals with antihyperglycemic and antihyperlipidemic activities. However, its possible role in diabetic complications is not evaluated yet. The present study explores the possible role of alcoholic extract of heartwood of P. marsupium in the treatment of long-term diabetic complications. The alcoholic extract of P. marsupium was evaluated for advanced glycation-end-products formation, erythrocyte sorbitol accumulation and rat kidney aldose reductase enzyme inhibition at the concentration of 25–400 μg/ml using in-vitro bioassays. Also the phytoextract at the concentration of 10–320 μg/ml was evaluated for its antioxidant potential by in-vitro antioxidant assays which includes, determination of total phenol content; reducing power assay; nitric oxide scavenging activity; superoxide radical scavenging activity; total antioxidant capacity; total flavonoid content; DPPH scavenging activity; and hydrogen peroxide scavenging activity.The alcoholic extract of P. marsupium across varying concentrations showed inhibitory effect as evident by IC50 on advanced glycation-end-products formation (55.39 μg/ml), sorbitol accumulation (151.00 μg/ml) and rat kidney aldose reductase (195.88 μg/ml). The phytoextract also exhibited high phenolic and flavonoid contents with promising antioxidant potential against the antioxidant assays evaluated. The present investigation suggests that the phytoextract showed prominent antioxidant, antiglycation property and, inhibited accumulation of sorbitol and ALR enzyme, thus promising a beneficial role in reducing/delaying diabetic complications.

Vegetables' juice influences polyol pathway by multiple mechanisms in favour of reducing development of oxidative stress and resultant diabetic complications.

Objective:Hyperglycemia induced generation of free radicals and consequent development of oxidative stress by polyol pathway is one of the crucial mechanisms stirring up development of diabetic complications. We evaluated influence of ten vegetables’ juice on polyol pathway along with their antioxidant and antioxidative stress potentials.Materials and Methods:Aldose reductase activity was determined utilising goat lens and human erythrocytes. In goat lens, utilization of nicotinamine adenine dinucleotide phosphate (NADPH) and aldose reductase inhibition was assayed. In human erythrocytes, sorbitol formation was measured as an index of aldose reductase activity under normoglycemic and hyperglycemic conditions. Ability of juices in inhibiting oxidative damage to deoxyribose sugar and calf thymus DNA and inhibitory activity against hydrogen peroxide induced hemolysis of erythrocytes was also analysed. Phytochemical contents like total polyphenol, total flavonoid and total protein were measured to find their influence on biological activities.Results:Vegetables’ juice displayed varying degrees of inhibitory potentials in mitigating NADPH dependent catalytic activity of aldose reductase in goat lens, accumulation of sorbitol in human erythrocytes under different glucose concentrations; Fenton-reaction induced oxidative damage to deoxyribose sugar, and calf thymus DNA. Substantial variations in vegetables phytochemicals content were also noticed in this study.Conclusions:Vegetables’ juice possesses potent activities in influencing polyol pathway by various mechanisms in favour of reducing development of oxidative stress independent of their inherent antioxidative properties. Juice of ivy gourd followed by green cucumber and ridge gourd were among the most potent for they displayed strong activities on various parameters analysed in this study. These vegetables’ juice may become part of mechanism-based complementary antioxidant therapy to prevent development of diabetic complications.

Polyol profile as an early diagnostic and prognostic marker in natural product chemoprevention of hepatocellular carcinoma in diabetic rats

AimDiabetes mellitus (DM) is a risk factor for hepatocellular carcinoma (HCC). It directs glucose to sorbitol and fructose in polyol pathway (PP). To pursue contribution of PP in hepatocarcinogenesis. MethodsWe utilized ascorbic acid (AA) and diallyl sulfide (DAS) in experimental DM and HCC against control. HCC was induced by diethyl nitrosamine (DENA, one intraperitoneal (IP) dose 125mg/kg), DM, by streptozotocin (STZ, IP dose 65mg/kg). AA was given as 7.4g/kg/d, I.P., DAS 200mg/kg/d, orally. All animals were killed after 10 weeks. ResultsDENA elevated serum AFP, erythrocyte sorbitol (ES), neoplastic changes in liver, lowered blood glucose, increased hepatocyte aldose reductase (AR) and sorbitol dehydrogenase (SDH), significantly alleviated by DAS/AA combination. DM elevated ES activating AR, inhibiting SDH, improved by DAS and AA. ConclusionCo-induction of DM and HCC increased liver tissue lesion, serum AFP, ES, liver AR and SDH. Co-administration of DAS/AA reduced ES, AR without changing SDH. DAS/AA co-therapy lowered ES by depressing AR without affecting SDH, meaning that AR is activated by cancer and DM in different ways. PP is early marker for HCC detection and response to chemoprevention. DAS/AA combination is promising cost effective chemopreventive and anti-diabetic combination.

Elevated Serum Sorbitol and not Fructose in Type 2 Diabetic Patients

Reductions in fasting serum fructose or erythrocyte sorbitol have been proposed as markers for early proof of mechanism in clinical development of aldose reductase (AR) inhibitors. However fructose is significantly impacted by meals and evaluation of erythrocyte sorbitol poses technical challenges. To more accurately assess the performance of these markers in biological samples, a gas chromatography-mass spectrometry assay was modified and validated. Serum was collected on three consecutive days from 13 healthy volunteers (HV) and 14 patients with type 2 diabetes mellitus (T2DM), and assayed for sorbitol and fructose using this assay. Serum fructose and sorbitol were relatively constant across the three days. Fasting fructose levels were comparable between the two groups (T2DM: 1.48 +/- 0.49 mg/L; HV: 1.39 +/- 0.38 mg/L, mean +/- standard deviation, P = 0.61), but fasting sorbitol levels were significantly higher in diabetics (T2DM: 0.280 +/- 0.163 mg/L; HV: 0.164 +/- 0.044 mg/L, P = 0.02). Feeding resulted in a 5-6 fold increase in serum fructose levels, but only a 5%-10% increase in sorbitol. Only sorbitol remained significantly elevated pre- and post feeding in T2DM patients relative to HV. These data suggest that serum sorbitol may be a robust proof of mechanism biomarker and facilitate dose selection for clinical development of AR inhibitors.

Protective Effects of Dietary Chamomile Tea on Diabetic Complications

Matricaria chamomilla L., known as "chamomile", has been used as an herbal tea or supplementary food all over the world. We investigated the effects of chamomile hot water extract and its major components on the prevention of hyperglycemia and the protection or improvement of diabetic complications in diabetes mellitus. Hot water extract, esculetin (3) and quercetin (7) have been found to show moderate inhibition of sucrase with IC50 values of 0.9 mg/mL and 72 and 71 microM, respectively. In a sucrose-loading test, the administration of esculetin (50 mg/kg body weight) fully suppressed hyperglycemia after 15 and 30 min, but the extract (500 mg/kg body weight) and quercetin (50 mg/kg body weight) were less effective. On the other hand, a long-term feed test (21 days) using a streptozotocin-induced rat diabetes model revealed that the same doses of extract and quercetin showed significant suppression of blood glucose levels. It was also found that these samples increased the liver glycogen levels. Moreover, chamomile extract showed potent inhibition against aldose reductase (ALR2), with an IC50 value of 16.9 microg/mL, and its components, umbelliferone (1), esculetin (3), luteolin (6), and quercetin (7), could significantly inhibit the accumulation of sorbitol in human erythrocytes. These results clearly suggested that daily consumption of chamomile tea with meals could contribute to the prevention of the progress of hyperglycemia and diabetic complications.

Erythrocyte sorbitol level as a predictor of the efficacy of epalrestat treatment for diabetic peripheral polyneuropathy

The relationship between the effect of aldose reductase inhibitors (ARIs) on the activation of the polyol pathway and on diabetic neuropathy has not been fully established. To address this issue, we investigated the effect of epalrestat (150 mg/day), an ARI, on erythrocyte sorbitol levels as an index of polyol activation and on nerve function test results in 43 patients with diabetic peripheral polyneuropathy. After 6 months of epalrestat administration, erythrocyte sorbitol levels did not decrease in patients as a whole. However, a decrease in erythrocyte sorbitol levels during epalrestat administration was significantly correlated with baseline erythrocyte sorbitol levels ( ρ=−.47, P<.01): The higher the level at baseline, the greater the decrease after epalrestat treatment. Moreover, the mean sorbitol level during epalrestat treatment was associated with the beneficial effect of epalrestat on vibration sensitivity as measured with a C-128 tuning fork ( ρ=−.66, P<.01) and/or a pallesthesiometer TM-31A ( ρ=.53, P<.05). On the other hand, erythrocyte sorbitol levels did not reflect the prognosis of nerve conduction velocity. These findings at least partly suggest a causal relationship between polyol activation and the development of diabetic neuropathy. Aldose reductase inhibitor treatment may be clinically useful in the control of polyol activation, especially in patients with excessive accumulation of sorbitol.

Quantitative determination of endogenous sorbitol and fructose in human erythrocytes by atmospheric-pressure chemical ionization LC tandem mass spectrometry

Evaluation of different extraction methods for quantification of endogenous sorbitol and fructose in human red blood cells (RBCs) and matrix effects in ESI and APCI showed that protein-precipitation followed by mixed-mode solid-phase extraction was more effective extraction method and APCI more effective ionization method. Then the LC/APCI-MS/MS method was fully validated and successfully applied to analysis of clinical RBC samples. The concentrations of endogenous sorbitol and fructose were determined using calibration curves employing sorbitiol- 13C 6 and fructose- 13C 6 as surrogate analytes. The method has provided excellent intra- and inter-assay precision and accuracy with a linear range of 50.0–10,000 ng/mL (correlation coefficient >0.999) for sorbitol- 13C 6 and 250–50000 ng/mL (correlation coefficient >0.999) for fructose- 13C 6 in human RBCs.

Nitric oxide prevents aldose reductase activation and sorbitol accumulation during diabetes.

Increased glucose utilization by aldose reductase (AR) has been implicated in the development of diabetes complications. However, the mechanisms that regulate AR during diabetes remain unknown. Herein we report that several nitric oxide (NO) donors prevent ex vivo synthesis of sorbitol in erythrocytes obtained from diabetic or nondiabetic rats. Compared with erythrocytes of nondiabetic rats, the AR activity in the erythrocytes of diabetic rats was less sensitive to inhibition by NO donors or by AR inhibitors-sorbinil or tolrestat. Treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, enhanced AR activity and sorbitol accumulation in tissues of nondiabetic rats. Application of transdermal nitroglycerin patches or treatment with L-arginine did not inhibit AR activity or sorbitol accumulation in the tissues of nondiabetic animals. Treatment with L-NAME increased, whereas treatment with L-arginine or nitroglycerine patches decreased AR activity and sorbitol content in tissues of diabetic rats. These observations suggest that NO maintains AR in an inactive state and that this repression is relieved in diabetic tissues. Thus, increasing NO availability may be a useful strategy for inhibiting the polyol pathway and preventing the development of diabetes complications.

Pharmacological Properties of Fidarestat, a Potent Aldose Reductase Inhibitor, Clarified by Using Sorbitol in Human and Rat Erythrocytes

We examined the effect of fidarestat on the increase in sorbitol content in erythrocytes from healthy volunteers in vitro. Fidarestat inhibited the increase with an IC₅₀ value of 18 nmol/l. A subsequent experiment showed that fidarestat had a similar inhibitory effect on the increase in sorbitol content in erythrocytes from diabetic patients. On the other hand, epalrestat, the only aldose reductase inhibitor used clinically, inhibited increase in sorbitol content at a concentration over 500-fold higher than fidarestat. Although the IC₅₀ value of fidarestat was not affected by fasting plasma glucose, HbA_1C, age, aldose reductase content or gender, there was a significant positive relationship between the IC₅₀ value of epalrestat and fasting plasma glucose. In addition, in fidarestat (0.25–2 mg/kg)-treated diabetic rats, the inhibitory rate for erythrocyte sorbitol accumulation was well correlated with that for nerve sorbitol accumulation, which indicates that erythrocyte sorbitol is available for assessing the state of sorbitol pathway flux in target tissue after fidarestat administration. These results suggest that fidarestat potently inhibits the increase in sorbitol pathway flux in diabetic patients independent of various factors and that erythrocyte sorbitol is useful for its estimation.

Portfolios of Alternative Assets

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.

Inhibition of aldose reductase in human erythrocytes by vitamin C

Ascorbic acid, or vitamin C, has been reported to lower erythrocyte sorbitol concentrations, and present studies were performed to determine the mechanism of this effect. Incubation of erythrocytes with increasing concentrations of glucose (5–40 mM) progressively increased erythrocyte sorbitol contents, reflecting increased flux through aldose reductase. At extracellular concentrations of 90 μM, both ascorbic acid and its oxidized form, dehydroascorbate, decreased intracellular sorbitol by 25 and 45%, respectively. This inhibition was not dependent on the extracellular glucose concentration, or on erythrocyte contents of free NADPH or GSH. To test for a direct effect of ascorbate on aldose reductase, erythrocyte hemolysates were prepared and supplemented with 100 μM NADPH. Hemolysates reduced glucose to sorbitol in a dose-dependent manner that was inhibited with a K i of 120 μM by the aldose reductase inhibitor tetramethylene glutaric acid. Above 100 μM, ascorbic acid also lowered hemolysate sorbitol generation by about 30%. Studies with ascorbic acid derivatives showed that the reducing capacity of ascorbic acid was not required for inhibition of sorbitol production from glucose in erythrocyte hemolysates. These results show that high, but physiologic, concentrations of ascorbic acid can directly inhibit erythrocyte aldose reductase, and provide a rationale for the use of oral vitamin C supplements in diabetes.

Influence of interindividual variability of aldose reductase protein content on polyol-pathway metabolites and redox state in erythrocytes in diabetic patients.

To clarify the influence of interindividual difference in the level of aldose reductase on the polyol pathway-related metabolism in diabetic patients. The enzyme protein content was determined by a two-site enzyme-linked immunosorbent assay using monoclonal and polyclonal antibodies to recombinant human aldose reductase in erythrocytes from 35 diabetic patients and 11 healthy volunteers. Patients were stratified into two groups by the median of aldose reductase content, and the erythrocyte sorbitol level, the fructose level, and the lactate-to-pyruvate ratio were compared between the two groups. We also examined the correlation of the enzyme content with these metabolic parameters. The group of patients whose enzyme content was above the median showed a significant increase in the levels of sorbitol (34.7 +/- 4.9 vs. 20.4 +/- 2.0 nmol/g Hb, P < 0.05) and fructose (99.8 +/- 17.2 vs. 45.9 +/- 4.6 nmol/g Hb, P < 0.05), along with an elevated lactate-to-pyruvate ratio (28.6 +/- 6.1 vs. 11.7 +/- 1.2, P < 0.05), compared with patients with low enzyme levels. The aldose reductase content in erythrocytes was well correlated with its activity, and there was a significant correlation between the enzyme content and the erythrocyte sorbitol (r = 0.58, P < 0.001) or fructose (r = 0.57, P < 0.001) levels as well as between the enzyme level and the lactate-to-pyruvate ratio (r = 0.38, P < 0.05). These results suggest that the interindividual variability of aldose reductase content may contribute tangibly to the polyol-pathway flux and cytoplasmic redox alteration in diabetic patients.

Improved fluorometric enzymatic sorbitol assay in human blood

Samples for use in the fluorometric enzymatic assay of sorbitol in erythrocytes are normally prepared using HClO 4 and K 2CO 3. We have replaced these reagents with NaOH and ZnSO 4. Human whole blood, erythrocyte and plasma samples prepared with NaOH and ZnSO 4 are colorless and clear, while erythrocyte samples prepared with HClO 4 and K 2CO 3 are a pale yellow–brown color. The sorbitol dehydrogenase reaction in the supernatant of the mixture of NaOH and ZnSO 4 is inhibited, but ethylenediaminetetraacetate completely eliminates this effect. The sorbitol assay in erythrocytes prepared with NaOH and ZnSO 4 shows higher sensitivity and reproducibility than did that with HClO 4 and K 2CO 3. Recovery of sorbitol added to erythrocytes is similar in both assay methods. Concentrations of whole blood and erythrocyte sorbitol assayed by the present method are significantly higher in diabetics than in normals. Poorly controlled diabetics had higher whole blood and erythrocyte sorbitol than well-controlled diabetics. Whole blood sorbitol concentrations differed more between diabetic and normal subjects than did erythrocyte sorbitol concentrations.

Effects of fructose ingestion on sorbitol and fructose 3-phosphate contents of erythrocytes from healthy men.

To investigate the effect of fructose ingestion on sorbitol and fructose 3-phosphate (F3P) in erythrocytes, we administered 50 g fructose with and without treatment with an aldose reductase inhibitor, epalrestat, to seven healthy, normal-glucose-tolerant, male volunteers aged 20-43 years. The same subjects were given 50 g glucose on another day. The sorbitol and F3P contents in their erythrocytes increased significantly, reaching peak levels at 60 min and 180 min, respectively, following fructose in gestion. On the other hand, glucose ingestion did not cause any statistically significant change in sorbitol content in their erythrocytes, although it significantly elevated their F3P content. Treatment with epalrestat had no significant effect on incremental changes in erythrocyte sorbitol and F3P content following fructose ingestion. This suggests that oral fructose may be converted directly to sorbitol and F3P in erythrocytes instead of being converted via glucose. Thus, the dietary intake of fructose may affect the concentrations of sorbitol and F3P in erythrocytes in normal men.

Experimental and clinical studies on the reduction of erythrocyte sorbitol-glucose ratios by ascorbic acid in diabetes mellitus

In order to confirm the effect of ascorbic acid (AA) on human erythrocyte sorbitol accumulation and explore its mechanism of action, the effects of ascorbic acid in vitro on the sorbitol (S) and glucose (EG) content of human erythrocytes and in particular on the S EG ratio as a marker of aldose reductase (AR) activity were carefully observed. The results showed that both the accumulation of erythrocyte sorbitol and the S EG ratio were strongly reduced by the addition of AA. The sorbitol content in the erythrocyte and the S EG ratio were reduced by a maximum of 87.3% and 83.4% and 93.8% and 63.9% when the medium's AA concentration was at its peak with 5.6 mmol/l and 28 mmol/l glucose in the medium, respectively. The contents of erythrocyte glucose measured coincidentally revealed a positive correlation with the ascorbic acid concentration in the medium during incubation in 5.6 mmol/l glucose while at a higher glucose level (28 mmol/l) in the medium the correlation became negative. These results suggested that the polyol pathway could be inhibited effectively by AA through its direct action on AR. The results of a double-blind cross-over trial using AA tablets or inositol tablets in eight diabetic patients showed that the supplementation of 1000 mg AA/day for 2 weeks resulted in reductions of 12.2% and 21.8% in erythrocyte sorbitol and red cell sorbitol/plasma glucose ( S PG ) ratio, respectively ( P < 0.05), whereas the fasting plasma glucose levels measured coincidentally revealed no changes ( P > 0.05). This suggested that supplementation with moderate amounts of AA (1000 mg/day) might serve as a simple, safe and effective measure for prevention and amelioration of the chronic complications of diabetes.