Erythrocytes Of Diabetic Patients Research Articles

Red Blood Cell Morphology in Diabetic Patients: A Case-control Study

Introduction: According to earlier studies, patients suffering from diabetes frequently experience haematological changes. India is the diabetes capital, with 69.1 million residents suffering from the disease. Aim: To study Red Blood Cell (RBC) morphological changes and grading on percentage of cells that differ in size and shape from normal erythrocytes in diabetic patients. Materials and Methods: The case-control study was conducted at Division of Central Clinical Laboratory in Department of Pathology in coordination with Department of General Medicine at Jawaharlal Nehru Medical College, Wardha, Maharashtra, India, from October 2019 to October 2021. Total 90 known cases of diabetes mellitus, aged between 18-80 years and 90 subjects (control group) with normal blood glucose levels were included for the study. Demographic details, anthropometric, parameters i.e, Fasting Plasma Glucose (FPG), Random Blood Sugar (RBS), Glycated haemoglobin (HbA1c) and morphology of blood smear were assessed. Results: The mean age of study group was 54.98±2.31 years and control group was 50.45±17.94 years (p-value= 0.0004). The mean random blood sugar of study group was 163.92±91.31 mg/dL and of control group was 96.92±22.31 mg/dL (p-value=0.0001). Mean HbA1c for the study group was 7.73±1.71%, while in control group was 5.24±0.62% (p-value=0.0001). In the study group, 9 (10%) cases showed a slight anisocytosis (1+) and 3 (3.3%) cases showed moderate anisocytosis (2+), 2 (2.2%) cases showed marked anisocytosis (3+) (variation in size of RBCs with respect to normal) in respect to Mean Corpuscle Volume (MCV), and 11 (12.2%) cases showed MCV <80 fL, 2 (2.2%) cases showed MCV >80 fL, while majority of the cases; 77 (85.5%) had normal sized RBCs, had MCV between 80-99 fL. Conclusion: The findings of the study suggest that there is need for some simple and effective techniques like ectocytometry, micropore filtration for routine haematological tests in type 2 diabetes mellitus.

Impact of Diabetes Mellitus on Human Erythrocytes: Atomic Force Microscopy and Spectral Investigations.

Diabetes mellitus (DM) is a common metabolic disease indicated by high sugar levels in the blood over a prolonged period. When left untreated, it can lead to long-term complications, such as cardiovascular disease, stroke, and diabetic retinopathy or foot ulcers. Approximately 415 million people (about 8.3% of the world’s population) had diabetes worldwide in 2015, with 90% of the cases classified as Type 2 DM, which is caused by insulin resistance that arises mostly from being overweight and from a lack of exercise. DM affects every part of the body, including the erythrocytes. The aim of the present report is to gain insight into the damage done to the erythrocytes of patients classified with pre-diabetes and diabetes (plenty are found in the Kingdom of Saudi Arabia, a country where young people encompass a large segment of the population). The study presents results on the morphological analysis of erythrocytes by atomic force microscopy (AFM) and molecular investigations by fluorescence spectroscopy (FS). Our results indicate significant differences (in the morphology, size, and hemolytic end products) between the erythrocytes of diabetic patients (HbA1C, glycated hemoglobin, levels of 8–10%) and normal controls. It is well-known that DM and smoking are two major contributory factors for cardiovascular diseases (CVDs), and our observations presented in this study suggest that diabetes plays a relatively less damaging role than smoking for CVD.

Anti-Diabetic Effect of Balanites Aegyptiacea Leaves Extract (Heglig) by Regulation of Erythrocyte Glucose Uptake in Diabetic Patients Type 2 in Vitro

Background: in Diabetes, the increase in the oxidative stress and decrease in the antioxidant defense may elevate he susceptibility of diabetic patients to many pathological complications, oxidative induced cell damage has been proposed to play an important role in the etiology of numerous pathological conditions. So, the aim of the present study was to investigate the antioxidant potential of Alcoholic Leaves extract of Balanites aegyptiacea (Heglig) due to the presence of phenolic and flavonoids compounds on uptake of glucose in vitro by erythrocytes of diabetic patients. Results: in hyperglycemic patients, erythrocytes malondialdehyde level was highly significantly increased (P<0.0001) than that of control. However, the erythrocytes glutathione content was highly significantly decreased (P< 0.0001) when compared to that of corresponding control values. The glucose uptake by erythrocytes of diabetic patients was highly significantly decreased (P < 0.0001) with increasing hyperglycemia (Fasting Blood glucose), while it was highly significantly elevated (P< 0.0001) after addition of Balanites aegyptiacae leaves extract to the incubation medium. On the other hand, the malondialdehyde concentration was highly significantly reduced (P < 0.0001) on adding the extract. So, it could be concluded that , an appreciate support for enhancing Antioxidant supply from natural sources such Balanites aegyptiace leaves extract may help control blood glucose levels and prevent pathological complications of diabetes

Diabetes screening by telecentric digital holographic microscopy.

Diabetes is currently the world's fastest growing chronic disease and it is caused by deficient production of insulin by the endocrine pancreas or by abnormal insulin action in peripheral tissues. This results in persistent hyperglycaemia that over time may produce chronic diabetic complications. Determination of glycated haemoglobin level is currently the gold standard method to evaluate and control sustained hyperglycaemia in diabetic people. This measurement is currently made by high-performance liquid chromatography, which is a complex chemical process that requires the extraction of blood from the antecubital vein. To reduce the complexity of that measurement, we propose a fully-optical technique that is based in the fact that there are changes in the optical properties of erythrocytes due to the presence of glucose-derived adducts in the haemoglobin molecule. To evaluate these changes, we propose to perform quantitative phase maps of erythrocytes by using telecentric digital holographic microscopy. Our experiments show that telecentric digital holographic microscopy allows detecting, almost in real time and from a single drop of blood, significant differences between erythrocytes of diabetic patients and healthy patients. Besides, our phase measurements are well correlated with the values of glycated haemoglobin and the blood glucose values.

Erythrocyte phospholipid and polyunsaturated fatty acid composition in diabetic retinopathy.

BackgroundLong chain polyunsaturated fatty acids (LCPUFAs) including docosahexaenoic acid and arachidonic acid are suspected to play a key role in the pathogenesis of diabetes. LCPUFAs are known to be preferentially concentrated in specific phospholipids termed as plasmalogens. This study was aimed to highlight potential changes in the metabolism of phospholipids, and particularly plasmalogens, and LCPUFAs at various stages of diabetic retinopathy in humans.Methodology and Principal FindingsWe performed lipidomic analyses on red blood cell membranes from controls and mainly type 2 diabetes mellitus patients with or without retinopathy. The fatty acid composition of erythrocytes was determined by gas chromatography and the phospholipid structure was determined by liquid chromatography equipped with an electrospray ionisation source and coupled with a tandem mass spectrometer (LC-ESI-MS/MS). A significant decrease in levels of docosahexaenoic acid and arachidonic acid in erythrocytes of diabetic patients with or without retinopathy was observed. The origin of this decrease was a loss of phosphatidyl-ethanolamine phospholipids esterified with these LCPUFAs. In diabetic patients without retinopathy, this change was balanced by an increase in the levels of several phosphatidyl-choline species. No influence of diabetes nor of diabetic retinopathy was observed on the concentrations of plasmalogen-type phospholipids.Conclusions and SignificanceDiabetes and diabetic retinopathy were associated with a reduction of erythrocyte LCPUFAs in phosphatidyl-ethanolamines. The increase of the amounts of phosphatidyl-choline species in erythrocytes of diabetic patients without diabetic retinopathy might be a compensatory mechanism for the loss of LC-PUFA-rich phosphatidyl-ethanolamines.

The effect of natural dicarbonyls on activity of antioxidant enzymes in vitro and in vivo

Natural dicarbonyls, which may be accumulated during oxidative stress in atherosclerosis (e.g. malondialdehyde) or carbonyl stress in diabetes mellitus (glyoxal and methylglyoxal) effectively inhibited activities of commercial preparations of the antioxidant enzymes: Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and Se-contained glutathione peroxidase from human and bovine erythrocytes, and also rat liver glutathione-S-transferase. After incubation of human erythrocytes with 10 mM of each investigated dicarbonyls the decrease of intracellular Cu,Zn-SOD was observed. The decreased activity of erythrocyte Cu,Zn-SOD was also detected in patients with diabetes mellitus type 2 with carbohydrate metabolism impairments but effective sugar-lowered therapy was accompanied by the increase of this enzyme activity. The increase of erythrocytes Cu,Zn-SOD activity in diabetic patients treated with metformin (which may utilize methylgly-oxal) was higher than in erythrocytes of diabetic patients subjected to traditional therapy.

In vitro upregulation of erythrocytes glucose uptake by Rhaphnus sativa extract in diabetic patients

In diabetes, both the increase in the oxidative stress and the decrease in the antioxidant defense may elevate the susceptibility of diabetic patients to many pathological complications. So, the aim of the present study was to investigate the effect of superoxide dismutase (SOD) like activity protein, partially purified from radish (Rhaphnus sativa) on uptake of glucose in vitro by erythrocytes of diabetic patients. In hyperglycemic patients, erythrocytes malondialdehyde level was highly significantly increased (P < 0.0001) than that of the control. However, the erythrocytes glutathione content and glutathione reductase activity, were both highly significantly decreased (P < 0.0001) compared to that corresponding control values. The glucose uptake by erythrocytes of diabetic patients was highly significantly decreased (P < 0.0001) with increasing hyperglycemia, while it was highly significantly elevated (p < 0.0001) after addition of the partially purified SOD like activity protein. On the other hand, the malondialdehyde concentration was highly significantly reduced (p < 0.001) on adding the partially purified protein. It thus can be concluded that, an appropriate support for enhancing antioxidant supply, such as SOD like activity protein from natural sources, may help control blood glucose level and may prevent clinical complications of diabetes.

Eryptosis and oxidative damage in type 2 diabetic mellitus patients with chronic kidney disease

It has been suggested that oxidative stress may participate in the progression of diabetes and its complications. Long-term complications of type 2 diabetes mellitus (T2DM) include retinopathy, atherosclerosis, shortened life span of erythrocytes, nephropathy, and chronic kidney disease (CKD). Oxidative damage has been associated with erythrocyte apoptosis induction in other pathological conditions. Our aim was to study the presence of eryptosis and its possible relationship with oxidative damage in patients with T2DM without CKD (T2DM/CKD(-)) and in patients with T2DM and CKD (T2DM/CKD(+)).Oxidative damage of lipids erythrocytes were increased in diabetic patients. The highest lipoperoxidation was found in T2DM/CKD(+). Likewise, the lower plasma total antioxidant capacity, GSH/GSSG ratio, and GSH in erythrocytes were found in T2DM/CKD(+) patients. A negative correlation was found between plasma total antioxidant capacity and oxidative damage. Phosphatidylserine (PS) externalization was measured in erythrocytes to evaluate eryptosis. Annexin binding in erythrocytes of T2DM/CKD(+) patients was higher than in healthy subjects and T2DM/CKD(-) patients. A positive correlation between lipoperoxidation and PS externalization in erythrocytes was found. This work showed that the erythrocytes of diabetic patients have increased oxidative damage, a reduction of antioxidant systems and more erythrocyte PS externalization. The duration of diabetes and the presence of CKD increase both oxidative damage and eryptosis. It is possible that a longer time of evolution induces an increase in erythrocyte oxidative damage and the consumption of blood antioxidant systems, adding to the osmotic stress in CKD and so contributes to an increase in PS externalization in diabetic patients.

In vitro nonenzymatic glycation of guanosine 5′-triphosphate by dihydroxyacetone phosphate

Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.

Fidarestat (SNK-860), a potent aldose reductase inhibitor, normalizes the elevated sorbitol accumulation in erythrocytes of diabetic patients

Sorbitol accumulation in nerves has been regarded as one of the major causes of diabetic neuropathy. In this study, fidarestat (SNK-860; 1 mg daily), a potent new aldose reductase inhibitor (ARI), or the commercially available ARI epalrestat (150 mg daily), was administered for 4 weeks to 58 Type 2 diabetic patients. Treatment with these drugs had no effect on glycemic control, judging from plasma glucose and HbA 1c levels. However, fidarestat treatment normalized the elevated sorbitol content of erythrocytes under fasting as well as postprandial conditions. In contrast, the effect of epalrestat was minimal. There were no major side effects with fidarestat. Thus, fidarestat is considered to be a potent and promising ARI, possibly useful for both preventing and treating diabetic neuropathy. Further studies are needed to clarify how much the occurrence and progression of diabetic neuropathy are inhibited by normalizing sorbitol elevation with fidarestat treatment.

Properties of erythrocyte light refraction in diabetic patients

Since hyperglycaemia changes the erythrocyte cell membrane fluidity and impairs cell deformity, our goal was to characterize hemoglobin and red blood cell (RBC) light refractive property changes in diabetic patients. Microscopic investigation was carried out on intact and fixed RBCs. To determine the refractive index (RI): smears of peripheral blood were air dried and fixed for 3 min in methanol. Mixtures of polyvinylpyrolidine and buffer of different pH (1:1) were used as embedding media. Intact RBCs were mixed with a buffered embedding medium, placed on a slide and overlaid with a coverslip. Interference microscopy was used for RI measurements at 18 different pH (pH=2-13). The results showed that curves of the RI of diabetic patients and of a control group were of similar configuration, with one branch in the acidic portion of the pH scale, a maximum and two minima in the neutral (middle) portion, and one branch in the alkaline portion. The curves of the individuals from the control group overlapped each other. To the contrary, the curves of the diabetic patients were not uniform in the neutral portion and the alkaline portion. The curves of the diabetic patients in the neutral zone were shifted towards the alkaline end of the pH scale, and the RBC RI curves were lower in comparison to the control curves. The center maximum of the curves of diabetic patients corresponded to pH=6.6 whereas the central maximum of the control group curves was at pH=6.2-6.8. Contrary to in the diabetic group, intact RBC RI curves in the control group revealed only one significantly different minimum at pH of 7.2 in the neutral zone. Using this method it is possible to show phenotypic differences between uniform type intact and fixed cells, erythrocytes of diabetic patients and of healthy donors.

Impaired erythrocyte transmembrane potential in diabetes mellitus and its possible improvement by resorcylidene aminoguanidine

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vitro transmembrane potential and the possible effect of resorcylidene aminoguanidine (RAG) on its modulation in erythrocytes of diabetic subjects. The transmembrane potential was assessed in RAG-treated and untreated erythrocytes, respectively, using a fluorescent dye (3,3′-dipropylthiadicarbocyanine iodide [DiSC 3(5)]). We confirmed earlier findings that the transmembrane potential of diabetic erythrocytes is significantly increased compared with control ( P<0.01). The membrane hyperpolarization found in diabetic cells seems to be a result of oxidative stress present in diabetes mellitus. On one hand, the RAG treatment induced decrease in abnormal transmembrane potential values in diabetic erythrocytes ( P<0.01), presumably via its antioxidant and antiglycation activity. On the other hand, RAG moderately hyperpolarized the control erythrocytes ( P<0.05). We suggest that the drug-induced transient membrane expansion leads to an intracellular potassium loss and a subsequent change of the transmembrane potential. However, if controlled by an appropriate dosage, RAG can eliminate certain types of erythrocyte membrane damage induced by diabetes mellitus.

Epalrestat, an aldose reductase ihibitor, reduces the levels of Nepsilon-(carboxymethyl)lysine protein adducts and their precursors in erythrocytes from diabetic patients.

To clarify the role of the polyol pathway in the intracellular formation of advanced glycation end products in human tissues, we examined the effects of epalrestat, an aldose reductase inhibitor, on the level of Nepsilon-(carboxymethyl)lysine (CML) along with 3-deoxyglucosone (3-DG) and triosephosphates in erythrocytes from diabetic patients. Plasma thiobarbituric acid-reactive substances (TBARS) were also determined as indicators of oxidative stress. Blood samples were collected from 12 nondiabetic volunteers, 38 untreated type 2 diabetic patients, and 16 type 2 diabetic patients who had been treated with 150 mg epalrestat/day. Blood samples were also collected from 14 of the untreated type 2 diabetic patients before and after the administration of epalrestat for 2 months. The amount of erythrocyte CML was determined by a competitive enzyme-linked immunosorbent assay, and 3-DG was measured by high-performance liquid chromatography In diabetic patients not treated with epalrestat, the erythrocyte CML level was significantly elevated above levels seen in nondiabetic individuals (49.9 +/- 5.0 vs. 31.0 +/- 5.2 U/g protein, P < 0.05) and was significantly lower in patients receiving epalrestat (33.1 +/- 3.8 U/g protein, P < 0.05). Similar results were observed with 3-DG. The treatment of patients with epalrestat for 2 months significantly lowered the level of erythrocyte CML (46.2 +/- 5.6 at baseline vs. 34.4 +/- 5.0 U/g protein, P < 0.01) along with erythrocyte 3-DG (P < 0.05), triosephosphates (P < 0.05), fructose (P < 0.05), sorbitol (P < 0.05), and plasma TBARS (P < 0.05) without changes in plasma glucose and HbA(1c) levels. A positive correlation was evident between the erythrocyte CML and sorbitol (r = 0.49, P < 0.01) or fructose (r = 0.40, P < 0.05) levels in diabetic patients. The results indicate that epalrestat administration lowers CML and associated variables and that polyol metabolites are correlated with CML in the erythrocytes of diabetic patients. The observed results suggest that aldose reductase activity may play a substantial role in the intracellular formation of CML in the mediation of reactive intermediate metabolites and oxidative stress.

Analysis of glutathionyl hemoglobin levels in diabetic patients by electrospray ionization liquid chromatography–mass spectrometry: effect of vitamin E administration

By using electrospray ionization liquid chromatography–mass spectrometry, we demonstrated that glutathionyl hemoglobin (Hb)β-chain levels are markedly increased in the erythrocytes of diabetic patients as compared with healthy subjects. The administration of vitamin E to the diabetic patients for 8 weeks significantly decreased the levels of glutathionyl Hbβ, whereas it did not affect the levels of HbA1c, glycated Hbβ or glycated Hbα. Glutathionyl Hb levels can be used as a new clinical marker of oxidative stress.

Glycated hemoglobin and lipid peroxidation in erythrocytes of diabetic patients

In diabetes, glycation and subsequent browning (or glycoxidation) reactions are enhanced by elevated glucose concentrations. It is unclear whether the diabetic state per se also induces an increase in the generation of oxygen-derived free radicals (OFRs). However, there is some evidence that glycation itself may induce the formation of OFRs. OFRs cause oxidative damage to endogenous molecules, including cholesterol. 7-Oxocholesterol is known to be one of the major products of cholesterol oxidation. The level of cholesterol peroxidation products was assessed in erythrocyte membrane lipid by monitoring the peak height ratio of 7-oxocholesterol, one of the products of cholesterol peroxidation, to cholesterol with gas chromatography/mass spectrometry (GC/MS). The peak height ratio of 7-oxocholesterol to cholesterol was used as a biomarker of lipid peroxidation. The hemoglobin A1c (HbA1c) value, an index of glycemic stress, was measured by high-performance liquid chromatography. We examined the relationship between the levels of cholesterol peroxidation products and HbA1c in erythrocytes of diabetic and healthy subjects. There was a significantly increased ratio of 7-oxocholesterol to cholesterol in diabetic erythrocytes compared with control erythrocytes. The ratio of 7-oxocholesterol to cholesterol was significantly correlated with the level of HbA1c. This suggests that glycation of hemoglobin via chronic hyperglycemia is linked to cholesterol peroxidation in erythrocytes of both diabetic and healthy subjects.

Human erythrocyte bisphosphoglycerate mutase: inactivation by glycation in vivo and in vitro.

2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.

Levels of lipid peroxidation product and glycated hemoglobin A 1c in the erythrocytes of diabetic patients

In diabetes, the glycation and subsequent browning (or glycoxidation) reactions are enhanced by elevated glucose concentrations. It is unclear whether or not the diabetic state per se also induces an increase in the generation of oxygen-derived free radicals (OFRs). There is some evidence, however, that glycation itself may induce the formation of OFRs. OFRs could cause oxidative damage to endogenous molecules. We examined the relationship between the levels of lipid peroxidation and the levels of glycated hemoglobin A 1c (GHbA 1c) in erythrocytes of diabetic and healthy subjects. Lipid peroxidation was assessed in erythrocyte membrane lipids by monitoring peak height ratios of conjugated linoleic acid (CLA), one of the products of lipid peroxidation, to linoleic acid (LA) using gas chromatography–mass spectrometry (GC/MS). CLA is a collective term used to designate a mixture of positional and geometric isomers of LA in which the double bonds are conjugated. The peak height ratio of CLA to LA was used as a biomarker of lipid peroxidation. GHbA 1c, an index of glycemic stress, was measured by high-performance liquid chromatography. There were significantly increased ratios of CLA to LA in diabetic erythrocytes compared with control erythrocytes. These ratios of CLA to LA were also significantly correlated with GHbA 1c values. This suggests that glycation via chronic hyperglycemia links lipid peroxidation in the erythrocytes of both diabetic and healthy subjects.

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.

Immunohistochemical detection of imidazolone, a novel advanced glycation end product, in kidneys and aortas of diabetic patients.

To investigate the role of the Maillard reaction in the pathogenesis of diabetic complications, we produced several clones of monoclonal antibodies against advanced glycation end products (AGEs) by immunizing mice with AGE-modified keyhole limpet hemocyanin, and found that one clone (AG-1) of the anti-AGE antibodies reacted specifically with imidazolones A and B, novel AGEs. Thus, the imidazolones, which are the reaction products of the guanidino group of arginine with 3-deoxyglucosone (3-DG), a reactive intermediate of the Maillard reaction, were found to be common epitopes of AGE-modified proteins produced in vitro. We determined the erythrocyte levels of imidazolone in diabetic patients using ELISA with the monoclonal anti-imidazolone antibody. The imidazolone levels in the erythrocytes of diabetic patients were found to be significantly increased as compared with those of healthy subjects. Then we studied the localization of imidazolone in the kidneys and aortas obtained from diabetic patients by immunohistochemistry using the antibody. Specific imidazolone immunoreactivity was detected in nodular lesions and expanded mesangial matrix of glomeruli, and renal arteries in an advanced stage of diabetic nephropathy, as well as in atherosclerotic lesions of aortas. This study first demonstrates the localization of imidazolone in the characteristic lesions of diabetic nephropathy and atherosclerosis. These results, taken together with a recent demonstration of increased serum 3-DG levels in diabetes, strongly suggest that imidazolone produced by 3-DG may contribute to the progression of long-term diabetic complications such as nephropathy and atherosclerosis.

Effect of aldose reductase inhibition on glutathione redox status in erythrocytes of diabetic patients

Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in non-insulin-dependent diabetic patients. For this purpose, we investigated the effect of polyol pathway inhibition on the glutathione redox status in these patients. We measured GSH and GSSG levels in erythrocytes of non-insulin-dependent diabetic patients (n = 15) before and after 1 week of treatment with placebo, followed by 1 week of treatment with an aldose reductase inhibitor (tolrestat 200 mg/d). We found lower GSH levels (7.7 ± 1.4 μmol/g hemoglobin [Hb]), higher GSSG levels (0.35 ± 0.09 μmol/g Hb), and lower GSH/GSSG ratios (23.9 ± 7.7) in diabetics compared with controls (n = 15; 9.8 ± 0.8 μmol/g Hb, P < .001; 0.17 ± 0.02, P < .001; and 58.3 ± 9.1, P < .001, respectively). We did not demonstrate any statistical difference after 1 week of treatment with placebo. In contrast, the treatment with tolrestat induced a significant increase in GSH (8.9 ± 0.7 μmol/g Hb, P < .01), a decrease in GSSG (0.25 ± 0.06 μmol/g Hb, P < .02), and an increase in the GSH/GSSG ratio (37.3 ± 8.4, P < .01). These data strongly support the hypothesis that the polyol pathway plays an important role in the impairment of the glutathione redox status in diabetic patients.