Jan Škrha

Gene polymorphisms of superoxide dismutases and catalase in diabetes mellitus

BackgroundReactive oxygen species generated by hyperglycaemia modify structure and function of lipids, proteins and other molecules taking part in chronic vascular changes in diabetes mellitus (DM). Low activity of scavenger enzymes has been observed in patients with DM. Protective role of scavenger enzymes may be deteriorated by oxidative stress. This study was undertaken to investigate the association between gene polymorphisms of selected antioxidant enzymes and vascular complications of DM.ResultsSignificant differences in allele and genotype distribution among T1DM, T2DM and control persons were found in SOD1 and SOD2 genes but not in CAT gene (p < 0,01). Serum SOD activity was significantly decreased in T1DM and T2DM subjects compared to the control subjects (p < 0,05). SOD1 and SOD2 polymorphisms may affect SOD activity. Serum SOD activity was higher in CC than in TT genotype of SOD2 gene (p < 0,05) and higher in AA than in CC genotype of SOD1 gene (p < 0,05). Better diabetes control was found in patients with CC than with TT genotype of SOD2 gene. Significantly different allele and genotype frequencies of SOD2 gene polymorphism were found among diabetic patients with macroangiopathy and those without it. No difference was associated with microangiopathy in all studied genes.ConclusionThe results of our study demonstrate that oxidative stress in DM can be accelerated not only due to increased production of ROS caused by hyperglycaemia but also by reduced ability of antioxidant defense system caused at least partly by SNPs of some scavenger enzymes.

Comparison of the effects of atorvastatin or fenofibrate on nonlipid biochemical risk factors and the LDL particle size in subjects with combined hyperlipidemia

BACKGROUND Combined hyperlipidemia (CH) is an increasingly prevalent risk factor for premature heart disease, and its treatment is troublesome. The aim of this study was to compare the effects of atorvastatin and fenofibrate on nonlipid biochemical risk factors and the low-density lipoprotein (LDL) particle size in subjects with CH. METHODS Twenty-nine middle-aged men with CH were randomly assigned to open-label therapy with atorvastatin (10 mg daily) or micronized fenofibrate (200 mg daily); they were sequentially treated with both drugs, with crossover of medication after 10 weeks. RESULTS Atorvastatin was more efficient in the reduction of total cholesterol, whereas fenofibrate was more efficient in the reduction of triglycerides. Only atorvastatin led to a significant reduction of LDL cholesterol and apolipoprotein B. Only fenofibrate increased high-density lipoprotein cholesterol. Neither drug influenced lipoprotein(a). Mean LDL particle size increased both after fenofibrate (3.08%) and atorvastatin (1.77%). Fenofibrate increased serum homocysteine (HCY) by 36.5%. Atorvastatin had no effect on HCY. Only atorvastatin increased fibrinogen by 17.4%. Only fenofibrate reduced C-reactive protein by 51.7%. Neither drug influenced HOMA (homeostasis model assessment) index of insulin resistance. The plasma level of thiobarbituric acid reactive substances, an index of oxidative stress, decreased after both treatments. CONCLUSIONS Both atorvastatin and fenofibrate had similar beneficial effects on LDL particle size and on oxidative stress. The effects of both drugs on other parameters such as triglycerides, total and high-density lipoprotein cholesterol, fibrinogen, or HCY differed significantly. These differences, together with the risk profile of a patient, should be considered during selection of a particular lipid-lowering modality.

Insulin action and fibrinolysis influenced by vitamin E in obese Type 2 diabetes mellitus

Increased oxidative stress, hypofibrinolysis and insulin resistance are present in obese Type 2 diabetic patients. It is supposed that treatment with antioxidant alpha-tocopherol (vitamin E) could not only decrease free radical production, but also ameliorate insulin action. We evaluated the effect of 3 months administration of vitamin E (600 mg daily) on insulin action examined by hyperinsulinemic clamp in 11 obese Type 2 diabetic patients. Oxidative stress and fibrinolysis were also determined. The administration of vitamin E caused a decrease of glucose disposal rate (26.6 +/- 9.5 vs 21.3 +/- 7.5 micromol/kg/min, P < 0.02) and of metabolic clearance rate of glucose (3.7 +/- 1.6 vs 2.9 +/- 0.8 ml/kg/min. P < 0.02). A decrease of insulin receptor number was observed on erythrocytes after vitamin E (284 +/- 84 vs 171 +/- 59 pmol/l, P < 0.01). Significantly higher plasma malondialdehyde (MDA) concentration documented an increased oxidative stress in diabetic patients as compared with healthy persons (3.13 +/- 0.68 vs 1.89 +/- 0.18 micromol/l, P<0.001). An inverse relationship was found between MDA concentration and insulin sensitivity expressed by glucose disposal rate (r = -0.73). Vitamin E further worsened the hypofibrinolysis documented by a decrease of tissue plasminogen activator (P < 0.01) without changes in its inhibitor PAI-1. In conclusion. our results demonstrate that higher doses of vitamin E may further deteriorate insulin action and fibrinolysis in obese Type 2 diabetic patients.

Relationship of oxidative stress and fibrinolysis in diabetes mellitus

This study attempted to verify the existence of a relationship between oxidative stress documented by malondialdehyde (MDA) and superoxide dismutase (SOD) and fibrinolysis analysed by tissue plasminogen activator (tPA) and its inhibitor (PAI‐1) in diabetes mellitus. Forty‐seven patients with Type 1 (n = 27) and Type 2 (n = 20) diabetes were examined together with 20 non‐diabetic controls. The following were analysed: plasma MDA concentration, SOD activity in erythrocytes, tPA activity and antigen, PAI‐1 activity and antigen, fasting blood glucose, fructosamine, glycated haemoglobin (HbAlc), and urine albumin. SOD activity was decreased in patients with diabetes. This contrasted with an increased plasma MDA concentration especially in Type 2 diabetes as compared with Type 1 or healthy persons (p < 0.001). tPA activity was increased in both groups of patients with diabetes as compared to healthy persons (p < 0.001), PAI‐1 activity was higher in Type 2 diabetes with vascular changes than in the remaining subgroups (p < 0.001). Multivariate analysis revealed a significant positive relationship between plasma MDA concentrations and PAI‐1 antigen (r = 0.53, p < 0.001) and a negative relationship between SOD and tPA activities (r = −0.53, p < 0.01). We conclude that oxidative stress may modulate fibrinolytic properties in diabetes mellitus.

Comparison of laser-Doppler flowmetry with biochemical indicators of endothelial dysfunction related to early microangiopathy in Type 1 diabetic patients

The aim of this study was to compare biochemical markers of endothelial activation with microcirculation measured by laser-Doppler flowmetry in Type 1 diabetic patients with or without microangiopathy. A total of 44 Type 1 diabetic patients were subdivided into those with (n=24) and without (n=20) microangiopathy according to ophthalmological findings and the presence or absence of microalbuminuria. The control group consisted of 25 healthy people of comparable age, sex, and body mass index. Postocclusive reactive hyperemia (PORH) and thermal hyperemia (TH, at 44 degrees C) were measured at the forearm. Serum N-acetyl-beta-glucosaminidase (NAG) activity, serum E-selectin, and ICAM-1 concentrations were used as biochemical markers of endothelial dysfunction. A significantly lower velocity of perfusion increase during postocclusive hyperemia (PORH(max) x t(1)(-1)) and during thermal hyperemia (TH(max) x t(2)(-1)) (P<.01) were accompanied by higher serum NAG activity (20.9+/-4.6 vs. 16.3+/-2.5 U l(-1), P<.01) in diabetic patients with microangiopathy as compared to healthy persons. An inverse relationship was found between PORH(max) x t(1)(-1) and NAG (r=-.33) results in diabetic patients. In addition, higher mean values of serum NAG activity, E-selectin, and ICAM-1 concentrations were associated with significantly lower values of microcirculation parameters (PORH(max) x t(2)(-1) and TH(max) x t(2)(-1)) in six patients without microangiopathy who had at least one of the above biochemical markers higher than mean+2 S.D. range. We suggest that serum NAG activity, E-selectin, and ICAM-1 concentrations may be used together with laser-Doppler flowmetry in Type 1 diabetic patients as early indicators of vascular changes in very early stage of diabetic microangiopathy.

Comparison of N-acetyl-β-glucosaminidase and albuminuria with clinical finding of microangiopathy in type I diabetes mellitus

N-acetyl-beta-Glucosaminidase activity in serum and urine and microalbuminuria were measured in 70 type-I diabetics and compared with glycated serum protein as well as with the finding of diabetic retinopathy. A significantly increased N-acetyl-beta-glucosaminidase activity in serum correlated positively with glycated protein but not with the development of retinopathy. Urinary N-acetyl-beta-glucosaminidase activity and albuminuria were significantly increased in diabetics with (p less than 0.001) or without (p less than 0.01) retinopathy as compared to healthy controls. A significant positive correlation was observed between urinary N-acetyl-beta-glucosaminidase activity and albuminuria (r = 0.73, p less than 0.01) as well as between blood pressure and albuminuria (r = 0.51, p less than 0.05).

Plasma Malondialdehyde and Obesity: Is there a Relationship?

Accelerated oxidative stress has been repeatedly found in Type 1 and Type 2 diabetes mellitus. It plays an important role in the development of diabetic vascular complications but it also modulates some physiological processes, such as fibrinolysis in diabetes (1, 2). The aim of this study was to evaluate how the oxidative stress measured by plasma malondialdehyde (MDA) concentration is influenced by diabetes control, body weight and serum triglyceride concentration in Type 1 and Type 2 diabetic patients. We examined 44 Type 1 (mean age 35±11 years, body mass index (BMI) 24.2±2.7 kg.m–2) and 40 Type 2 (mean age 55±11 years, BMI 29.9±4.2 kg.m–2) diabetic patients. Control group consisted of 20 non-obese (mean age 38±12 years, BMI 24.6±2.6 kg.m–2) and 16 obese (mean age 53±11 years, BMI 30.4±4.7 kg.m–2) healthy persons. Plasma malondialdehyde concentrations were measured by the fluorimetric method and glycated hemoglobin was determined by HPLC (3, 4). Serum triglycerides were evaluated by a routine enzymatic method on Hitachi 717 analyzer (Hitachi, Tokyo, Japan). The mean BMI was significantly higher in Type 2 than in Type 1 diabetic patients (p<0.001) and therefore two control groups of comparable age and BMI were used for comparison of the results. The mean MDA (2.09±0.38 vs. 1.82±0.37 μmol.l–1 , p<0.02) and triglyceride (2.49±1.0 vs. 1.35±0.41 mmol.l–1, p<0.001) concentrations were higher in obese than in non-obese healthy subjects. Significantly higher plasma MDA was observed in Type 2 as compared with Type 1 diabetic patients (2.30±0.39 μmol.l–1 vs. 1.84±0.34 μmol.l–1, p<0.01) but diabetes control was comparable in both groups (HbA1c 8.14±1.75 vs. 7.97±1.43%, n.s.). A significant relationship was observed between plasma MDA and HbA1c in Type 1 (y=0.10x + 1.1, r=0.40, p<0.05) and Type 2 (y=0.11x + 1.4, r=0.55, p<0.02) diabetic patients. The mean serum triglyceride concentration was higher in Type 2 than in Type 1 diabetic patients (2.45±1.27 vs. 1.28±0.45 mmol.l–1, p<0.001) but no significant relationship was found between plasma MDA and serum triglycerides in the whole cohort of patients (r=0.44, p=0.055). A subgroup of 18 Type 2 diabetic patients with normal serum triglycerides (1.43±0.34 mmol.l– 1) had the mean plasma MDA concentration (2.36±0.46 μmol.l–1) comparable to that of 22 hypertriglyceridemic patients (2.26±0.33 μmol.l–1, n.s.) who had serum triglyceride concentration significantly higher (3.65±1.25 mmol.l–1, p<0.001). No difference in HbA1c was observed between normotriglyceridemic and hypertriglyceridemic Type 2 diabetic patients. Plasma MDA concentrations significantly correlated with BMI in both Type 1 and Type 2 diabetic patients (Figure 1). No relationship was observed between serum triglycerides and BMI in separate groups of subjects. Multiple regression analysis confirmed that BMI and HbA1c were significant predictors of plasma MDA concentration in diabetic patients whereas triglycerides had less power in this relationship. Serum triglycerides were previously considered as an important variable influencing plasma MDA concentrations (5, 6). We demonstrated that both MDA and triglyceride concentrations were higher in Type 2 than in Type 1 diabetic patients but a weak relationship between both variables was present only in non-obese Type 1 diabetic patients (r=0.45, p<0.03). No such relationship was found in Type 2 diabetic patients (r=0.20, p>0.05). On the contrary, BMI correlated significantly with MDA concentration in the whole cohort of persons as well as in separately evaluated Type 1 and Type 2 diabetic patients and healthy subjects. We conclude that plasma MDA concentration as an indicator of oxidative stress may be higher in Type 2 than in Type 1 diabetic patients with a comparable diabetes control. This difference is related to BMI. Obesity may therefore contribute, by an unknown mechanism, to higher oxidative stress expressed by MDA.

Pathogenesis of angiopathy in diabetes

Abstract.Hyperglycaemia as a common feature of diabetes mellitus is a cause of different pathogenic mechanisms influencing endothelial function. Oxidative stress is one of the main causative factors inducing endothelial dysfunction and changes in plasma protein or platelet function. In type 2 diabetes mellitus, a combination of hyperglycaemia together with dyslipidaemia, obesity and other factors may accelerate the process of glycoxidation and lipid oxidation, causing an early impairment of the vessel wall or properties of circulating blood. This induces hypercoagulability characterised by impaired fibrinolysis and hyperaggregability. The initial functional changes are later substituted by morphologically impaired structure of the blood capillaries (microangiopathy) or arteries (macroangiopathy). The latter represent advanced atherosclerosis when typical plaques are formed. Failure of protective scavenger mechanisms is one possible explanation of vessel wall pathology in diabetes.

Serum α-tocopherol and ascorbic acid concentrations in Type 1 and Type 2 diabetic patients with and without angiopathy

BACKGROUND Alpha-tocopherol and ascorbic acid form a part of scavenger system influencing the level of oxidative stress in diabetes mellitus. The aim of this study was to evaluate serum concentrations of alpha-tocopherol and ascorbic acid in Type 1 and Type 2 diabetes mellitus and to compare them with the presence of vascular complications as well as with oxidative stress and endothelial dysfunction. METHODS A total of 38 Type 1 and 62 Type 2 diabetic patients were subdivided into those with and without angiopathy. Serum alpha-tocopherol and ascorbic acid concentrations were estimated in all patients and in 38 healthy persons. Their results were compared with diabetes control, with oxidative stress measured by plasma malondialdehyde and with endothelial dysfunction estimated by serum N-acetyl-beta-glucosaminidase activity. In addition, the differences in biochemical variables were compared between patients with and without angiopathy. RESULTS Serum alpha-tocopherol related to the sum of cholesterol and triglyceride concentrations (AT/CHT ratio) was significantly lower in diabetic patients with macroangiopathy than in those without vascular changes (p<0.05). Serum ascorbic acid levels were significantly lower only in Type 2 diabetic patients with macroangiopathy as compared with healthy controls as well as with patients without vascular disease (p<0.01). Positive relationship was observed between serum alpha-tocopherol and cholesterol or triglyceride concentrations in both Type 1 and Type 2 diabetic patients. The presence of oxidative stress together with endothelial dysfunction measured by N-acetyl-beta-glucosaminidase activity was accompanied by lower AT/CHT ratio (p<0.005) in Type 2 diabetic patients. CONCLUSION Diabetic patients with proven angiopathy or with advanced oxidative stress and endothelial dysfunction have significantly lower AT/CHT ratio and ascorbic acid concentration in serum. Their low concentrations may participate at the increased level of oxidative stress in these individuals.

A Six-year Follow-up of the Relationship Between N-Acetyl-β-glucosaminidase and Albuminuria in Relation to Retinopathy

Fifty patients with Type 1 diabetes mellitus were observed over 6 years. Serum and urinary N‐acetyl‐β‐glucosaminidase (NAG) activity, and albuminuria were measured in groups of patients subdivided according to ophthalmological findings. Significantly higher mean serum NAG activity was found at the beginning of the study in patients who later developed diabetic retinopathy in comparison with those who did not (geometric mean (2SD range) 19.7 (12.4–31.2) vs 14.4 (9.5–22.7) U l‐1 p < 0.01). Urinary NAG activity was significantly higher in all groups of diabetic patients than in healthy control subjects (p < 0.05). A significant increase in albumin: creatinine ratio during the study was found in patients with newly developed diabetic retinopathy compared with patients who did not (at 6 years 1.33 (0.40–4.43) vs 0.75 (0.24–2.31) g mol‐1, p < 0.01). No differences in either biochemical variable were found between hypertensive and normotensive diabetic patients at the end of the study. The results suggest that both serum NAG activity and albuminuria may serve as early functional indicators of diabetic retinopathy.