The Effects of Vitamin D Supplementation on Metabolic and Oxidative Stress Markers in Patients With Type 2 Diabetes: A 6-Month Follow Up Randomized Controlled Study.

Vitamin D deficiency could play an important role in the pathogenesis of type 2 diabetes mellitus (T2DM) as it may alter several crucial processes in the development of diabetes and its complications, such as pancreatic insulin secretion, peripheral insulin resistance, persistence of systemic "sterile" inflammation and immune activation. Vitamin D may also have an antioxidant effect through the inhibition of free radicals generation. The reported study was designed with eligible consecutively recruited patients with T2DM on standard metformin therapy (n=130), randomized in 1:1 ratio, considered to have undergone Vitamin D supplementation according to the guidelines proposed by the Endocrine Society, or to have continued with metformin only. The potential benefit was monitored through the influence on glycemia level, glycated haemoglobin (HbA1c), insulin resistance index (calculated as homeostatic model assessment; HOMA-IR), Castelli Risk Index I and Tryglicerides/Thiobarbituric acid-reactive substances (TG/TBARS) Index in a 6-month follow up period. Our study indicates that oral daily doses of vitamin D improve HbA1c levels over the 3-month and 6-month period, followed by a significant decrease in advanced oxidation protein products levels over the 3-month period when higher vitamin D doses are given. The effect of vitamin D on HOMA-IR index, malondialdehyde levels and TG/TBARS index was not statistically significant. Further investigation should consider defining the doses of vitamin D in patients with T2DM which may attenuate the oxidative stress risk, the risk of metabolic syndrome and the risk of related cardiovascular events.

Data-Driven Cluster Analysis of Oxidative Stress Indexes in relation to Vitamin D Level, Age, and Metabolic Control in Patients with Type 2 Diabetes on Metformin Therapy.

Recent advances in vitamin D research indicate that patients with type 2 diabetes mellitus (T2DM) are suffering from vitamin D deficiency and increased oxidative stress to a variable extent, which could produce different health impacts for each individual. The novel multivariate statistical method applied in the present study allows metabolic phenotyping of T2DM individuals based on vitamin D status, metabolic control, and oxidative stress status in order to identify effectively different subtypes in our type 2 DM study population. Data-driven statistical cluster analysis was performed with 95 patients with T2DM, treated with metformin. Clusters were based on 12 variables-age, disease duration, vitamin D level, insulin, fasting glycemia (FG), glycated hemoglobin (HbA1c), high-density and low-density lipoprotein, total cholesterol (TC), triglycerides (TG), body mass index (BMI), and triglycerides/glucose index (TYG). The analysis revealed four unique clusters which differed significantly in terms of vitamin D status, with a mean 25 (OH) D level in cluster 1 (57.84 ± 11.46 nmol/L) and cluster 4 (53.78 ± 22.36 nmol/L), falling within the insufficiency range. Cluster 2 had the highest mean level of 25 (OH) D (84.55 ± 22.66 nmol/L), indicative of vitamin D sufficiency. Cluster 3 had a mean vitamin D level below 50 nmol/L (49.27 ± 16.95), which is considered deficient. Patients in the vitamin D sufficient cluster had a significantly better glycemic and metabolic control as well as a lower level of lipid peroxidation compared to other clusters. The patients from the vitamin D sufficient cluster also had a significantly higher level of vitamin D/MPO, vitamin D/XO, vitamin D/MDA, vitamin D/CAT, and vitamin D/TRC than that in the vitamin deficient and insufficient clusters. The vitamin D deficient cluster included significantly younger patients and had a significantly lower level of AOPP/TRC and albumin/TRC than the vitamin D sufficient cluster. The evidence from our cluster analysis in the context of separated T2DM demonstrates beneficial effects of optimal vitamin D status on metabolic control and oxidative stress in T2DM patients. Older T2DM patients require higher vitamin D levels in order to achieve good metabolic control and favorable antioxidant protection. Since protein damage is more pronounced in these patients, adding water-soluble antioxidant in addition to higher doses of vitamin D should be considered.

A novel mechanism of vitamin D anti-inflammatory/antioxidative potential in type 2 diabetic patients on metformin therapy.

INTRODUCTION: The performed study focused on determining the effect of vitamin D supplementation on enzymes involved in both inflammation and reactive oxygen species (ROS) production and ROS degradation in patients with type 2 diabetes mellitus (T2DM). MATERIAL AND METHODS: The 6-month follow-up, randomized, controlled study included 140 patients with T2DM, ≥ 30 years old, with good metabolic control, treated with metformin and lifestyle advice only. All patients were randomly assigned to two groups (70 each). Patients from the first group (Intervention group) were assigned to receive vitamin D3 50 000 IU or 14 000 IU regarding their vitamin D baseline levels. Patients from the second (Metformin) group continued to receive only metformin during the 6-month study period. RESULTS: After 6 months, the myeloperoxidase activity was significantly lower and gradually decreased in the Intervention group by about 40%, compared to the baseline measurement (p = 0.015) and compared to the Metformin group (p = 0.001). After 6 months, the xanthine oxidase (XO) activity decreased significantly in the Intervention group compared to the baseline and 3rd month levels (p < 0.001). In the Metformin group there was also a significant decrease in XO after 6 months compared to baseline (p < 0.001) and the 3rd month (p = 0.003). The catalase activity significantly increased within the Intervention group only when comparing the 3rd and 6th month (p = 0.027). CONCLUSIONS: Our study showed that vitamin D may improve endothelial dysfunction in patients with T2DM on metformin therapy by influencing two important factors implicated in the pathogenesis of diabetic complications - ROS production and inflammation, which can additionally contribute to a stable metabolic control during metformin therapy.

Author Correction: Clinical Benefit of Basal Insulin Analogue Treatment in Persons with Type 2 Diabetes Inadequately Controlled on Prior Insulin Therapy: A Prospective, Noninterventional, Multicenter Study.

In the original publication, values of the doses of insulin glargine, the most commonly used basal insulin analogue under the 'Discussion' section was incorrectly published.

Clinical Benefit of Basal Insulin Analogue Treatment in Persons with Type 2 Diabetes Inadequately Controlled on Prior Insulin Therapy: A Prospective, Noninterventional, Multicenter Study.

INTRODUCTION: Basal insulin analogues offer persons with type 2 diabetes mellitus (T2DM) adequate glycemic control combined with a favorable safety profile. BASAL-BALI-a prospective, noninterventional, multicenter disease registry-assessed the effectiveness and safety of basal insulin analogues in adult Serbians with T2DM previously inadequately controlled on other insulin types. METHODS: The primary objective was to assess the reduction in glycated hemoglobin (HbA1c) from basal insulin analogue initiation to the end of a 6-month observation period. Data collection was performed at three study visits: baseline, 3 months, and 6 months. All treatments and procedures were performed at the physicians' discretion. RESULTS: In total, 460 subjects were included. Mean diabetes duration was 11.6 ± 6.6 years. Late complications of diabetes were present in 67% of subjects and comorbidities in 85%. After 6 months, the mean reduction in HbA1c was 1.8% (p < 0.01 vs. baseline); body weight (mean reduction of 0.9 kg, p < 0.01), waist circumference (1.5 cm, p < 0.01), and BMI (0.2 kg/m2, p < 0.01) were also reduced. A total of 49.1% of subjects reached their individualized HbA1c treatment target, and 42.0% met the composite HbA1c and fasting plasma glucose (FPG) target. The incidence of symptomatic hypoglycemia was reduced from 96.3% in the 6 months prior to initiating basal insulin analogues to 15.4% over the 6-month treatment period. CONCLUSION: Introducing basal insulin analogues in persons with T2DM previously inadequately controlled on other insulin types can significantly improve glycemic control and reduce the risk of hypoglycemia, without adversely affecting body weight. FUNDING: Sanofi, Serbia.

Cross-talk between the dipeptidyl peptidase-4 and stromal cell-derived factor-1 in stem cell homing and myocardial repair: Potential impact of dipeptidyl peptidase-4 inhibitors.

Dipeptidyl peptidase-4 (DPP-4), glycyl-prolyl-naphthylamidase, is a serine protease that catalyzes the hydrolysis of various proline-containing polypeptides. It is involved in the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), having in this way a profound influence on glucose metabolism. During organ damage, stromal and endothelial cells produce a chemokine known as stromal cell-derived factor-1 (SDF-1), a powerful chemoattractant of stem/progenitor cells. SDF-1 binds to a specific α-chemokine receptor (CXCR4) and can be degraded by proteases, including matrix DPP-4/CD26, presented in the circulation, or activated in injured tissues. DPP-4 inhibition has received considerable attention because of its significant therapeutic benefits in the regulation of insulin secretion and tissue insulin sensitivity, the regulation of tumor growth and metastasis, angiogenesis, tissue repair, especially after myocardial infarction, and regulation of endocrine function. Inhibition of circulating proteases appears to maintain the optimal endogenous SDF-1 concentration and may enhance homing of endothelial progenitor cells. In the present article, we present an overview of some basic facts about the role of DPP-4 in glucose homeostasis, the mechanism of its inhibition, and a brief summary of available DPP-4 inhibitors. Furthermore, since protection against the overactivity of proteases is important for restorating cardiac function and repair after myocardial damage, necrosis and apoptosis, we propose that administration of a DPP-4 inhibitor may also be beneficial following myocardial infarction by the prevention of cleavage of stem cell chemoattractant cytokine SDF-1.

Subclinical hypothyroidism: association with cardiovascular risk factors and components of metabolic syndrome.

The aim of this cross-sectional study was to evaluate the cardiovascular risk in patients with subclinical hypothyroidism (SH) and metabolic syndrome (MetS) components. The study included 60 patients with SH and a control group of 60 healthy volunteers, gender and age matched, with normal thyroid-stimulating hormone (TSH) and free thyroxin (FT4) concentration. The following measurements were made in all participants: TSH, FT4, thyroid peroxidase antibodies, anti-thyroglobulin antibodies, body mass index (BMI), waist circumference, blood pressure, fasting plasma glucose, total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides (TG), TC/HDL cholesterol and LDL/HDL cholesterol ratio, basal insulin level and homeostatic model assessment insulin resistance (HOMA-IR) index. MetS was diagnosed according to the National Cholesterol Education Program Adult Treatment Panel III criteria. The results showed that the following indices were statistically significantly higher in the SH group: BMI (p < 0.05), diastolic blood pressure (p < 0.001), TC (p < 0.05), TG (p < 0.05) and basal insulin level (p < 0.05). Although MetS parameters were present in a higher per cent in the SH group, there was a significantly higher number of patients with hypertension and decreased HDL cholesterol (p < 0.05). More frequently, MetS was diagnosed in SH patients (46.67%) than in the control group (33.33%), although the difference was not statistically significant. These results indicated that the traditional cardiovascular risk factors were more frequently present in SH patients as compared to euthyroid participants. Our results did not confirm significantly higher presence of MetS in SH patients in comparison with euthyroid respondents.

The hypertriglyceridemic waist phenotype and metabolic syndrome by differing criteria in type 2 diabetic patients and their relation to lipids and blood glucose control.

BACKGROUND: Metabolic syndrome (MetS) describes clustering of obesity, dyslipidemia, hyperglycemia and hypertension and increases risk for cardiovascular disease and type 2 diabetes. The 'hypertriglyceridemic waist' phenotype (HTGW) represents a simple approach to identifying individuals with increased risk. The aim of the study was to determine the prevalence of HTGW and MetS in type 2 diabetic patients, and to examine their relation to lipids and blood glucose control. MATERIAL AND METHODS: 300 type 2 diabetic patients were analysed, and their history of diabetes, anthropometric measures, measurements of blood pressure (BP), lipids and glycemic control parameters were taken. RESULTS: In type 2 diabetic patients, the prevalence of MetS was 71.0% by the AHA/NHLBI definition and 75.33% by the IDF definition. The prevalence was 62.58% and 66.45% in men, and 80% and 84.83% in women by the same definitions, respectively. There were 41.33% of patients with HTGW (42.76% among women and 40% among men). There were statistically significant differences of age, fasting plasma glucose (FPG) and postprandial glucose (PPG) in women with and without MetS according to both definitions, and of total and LDL cholesterol with and without MetS according to AHA/NHLBI (but not IDF). In men, there were statistically significant differences of total cholesterol and of HbA(1c) with and without MetS according to AHA/NHLBI (but not IDF). Women with HTGW had higher levels of total and LDL cholesterol, systolic and diastolic BP. Men with HTGW had higher levels of total cholesterol, diastolic BP, HbA(1c), FPG and PPG. CONCLUSIONS: Determining MetS or HTGW helps identify those with increased cardiovascular risk.

Circulating ribonucleic acids and metabolic stress parameters may reflect progression of autoimmune or inflammatory conditions in juvenile type 1 diabetes.

The sensing of ribonucleic acids (RNAs) by the monocyte/macrophage system occurs through the TLR7/8 Toll-like receptor family, the retinoic acid-inducible protein I (RIG-I), and the melanoma differentiation-associated protein-5 (MDA-5). The aim of the present study was to evaluate the effect of circulating RNAs, isolated from juvenile type 1 diabetic patients and healthy control children, on the inflammatory, apoptotic, and antiviral response in human peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor. Obtained effects were compared to the effects of metabolic stress parameters (hyperglycemia, oxidative and nitrosative stress). Forty-eight patients with juvenile type 1 diabetes and control children were included in the study. By performing the chromatographic analysis of circulating RNAs, the peak at the retention time 0.645 min for diabetic and control RNA samples was identified. To determine whether circulating RNAs have an agonistic or antagonistic effect on the signaling pathways involved in inflammatory, apoptotic, and antiviral cascade, their effect on TLR8, RIG-I, MDA-5, MyD88, NF-KB, IRF-3, phosphoIRF-3, IRF-7, RIP, and p38 was evaluated. A significantly lower level was achieved by cultivating PBMCs with circulating RNAs isolated from type 1 diabetic children, compared to the intact PBMCs, in relation to TLR-8, MDA-5, NF-KB, phospho IRF-3, and RIP, while it was higher for Bax. All the metabolic stress conditions up-regulated NF-KB, Bcl-2, and Bax. The NF-êB determination seems to be the most sensitive parameter that may reflect disease processes associated with the progression of autoimmune or inflammatory conditions, while the IRF3/phosphoIRF3 ratio may suggest an insufficient antiviral response.

[New aspects about the impact of oxidative stress on development of chronic diabetic complications].

Oxidative stress is defined as a state when the free radical production ovecome their neutralisation via the antioxidative defense systems. Being extremely unstable, these molecules and ions tend to achieve a stable state by reacting with the surrounding molecules, which are immediately oxidised. Such chain reactions may have substantial effects on almost all cellular structures, including the lipids, the proteins and the nuclear DNA. LDL particles become atherogenic because their apoB is subjected to the oxidative modification, being recognized by the scavenger receptors. Type 2 diabetes represents a conformational disease in which proteins are subjected to the oxidative modification. The disease proceeds in several stages such as: the transition is the latent period where oxidatively modified proteins are deposited in the EPR structures, periods of accelerated redox stress when amyloid precursors are accumulated, accompanied with the insulin secretory deficiency. The process is aggravated by glycosylated proteins, which are the free radical producers. This complex cascade proceeds through the formation of early (Schiff bases and Amadori) products, while further glycation of proteins and lipids causes molecular rearrangements that lead to the generation of advanced glycation endproducts (AGEs). High glucose concentrations in non insulin-dependent tissues may follow the pathway of aldose reductase, when reduced nucleotides are used as the cofactors, leading to decreased reduced glutathione (GSH) content. Glucose fluctuations during postprandial periods exhibited a more specific triggering effect on free radical production. The superoxide overproduction is accompanied by an increased nitric oxide (NO) generation, a phenomenon favoring the formation of the stronger oxidant peroxynitrite. The increased free radical generation in accumulated fat causes dysregulated production of adipocytocines locally, leading to development of metabolic syndrome.

[Cardiovascular risk factors in patients with subclinical hypothyroidism].

BACKGROUND/AIMS: Overt hypothyroidism is disease associated with accelerated arteriosclerosis and coronary heart disease. Whether subclinical hypothyroidism (SH) is associated with increased cardiovascular risk is contraversial. As SH is a high prevalence thyroid dysfunction, specially in older women, it is important to evaluate cardiovascular risk factors in these patients and that was the aim of this study. METHODS: We examined 30 patients with SH and 20 healthy controls. Subclinical hypothireoidism was defined as an elevated thyrotropin (TSH) (> 4.5 mU/L) and normal free thyroxine (FT4) level. In all the participants we determined body mass index (BMI), blood pressure, TSH, FT4, antibodies to thyroid peroxidase, antibodies to thyroglobulin, total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, triglicerides, total cholesterol/HDL cholesterol ratio and LDL/HDL cholesterol ratio. RESULTS: Mean BMI in patients with SH was significantly higher (p < 0.05), as well as diastolic blood pressure (p < 0.01) compared with the controls. Average levels of total cholesterol (5.40 +/- 0.62 vs 5.06 +/- 0.19 mmol/l, p < 0.01) and triglycerides (2.16 +/- 0.56 vs 1.89 +/- 0.24 mmol/l, p < 0.05) were also significantly higher in the group with SH. Individual analysis revealed that the percentage of patients with SH having borderline elevated total cholesterol (63.33%), hypertrigliceridemia (43.33%) and elevated total cholesterol/HDL cholesterol ratio (26.67%) were significantly higher than the percentage in the controls. No significant correlation between TSH and lipid parameters was detected. CONCLUSION: Subclinical hypothyroidism was associated with higher BMI, diastolic hypertension, higher total cholesterol and triglicerides levels and higher total cholesterol/HDL cholesterols ratio. This might increase the risk of accelerated arteriosclerosis in patients with SH.

Impact of intensive insulin treatment on the development and consequences of oxidative stress in insulin-dependent diabetes mellitus.

BACKGROUND/AIM: The aim of this study, which included patients with insulin-dependent diabetes mellitus, was to determine the influence of the application of various treatment modalities (intensive or conventional) on the total plasma antioxidative capacity and lipid peroxidation intensity expressed as malondialdehyde (MDA) level, catalase and xanthine oxidase activity, erythrocyte glutatione reduced concentration (GSH RBC), erythrocyte MDA level (MDA RBC), as well as susceptibility of erythrocyte to H2O2-induced oxidative stress. METHODS: This study included 42 patients with insulin-dependent diabetes mellitus. In 24 of the patients intensive insulin treatment was applied using the model of short-acting insulin in each meal and medium-acting insulin before going to bed, while in 18 of the patients conventional insulin treatment was applied in two (morning and evening) doses. In the examined patients no presence of diabetes mellitus complications was recorded. The control group included 20 healthy adults out of a blood doner group. The plasma and erythrocytes taken from the blood samples were analyzed immediately. RESULTS: This investigation proved that the application of intensive insulin treatment regime significantly improves total antioxidative plasma capacity as compared to the application of conventional therapy regime. The obtained results showed that the both plasma and lipoproteines apo B MDA increased significantly more in the patients on conventional therapy than in the patients on intensive insulin therapy, most probably due to intensified xanthine oxidase activity. The level of the MDA in fresh erythrocytes did not differ significantly between the groups on intensive and conventional therapy. The level of GSH and catalase activity, however, were significantly reduced in the patients on conventional therapy due to the increased susceptibility to H2O2-induced oxidative stress CONCLUSION: The presented study confirmed positive effect of intensive insulin therapy on metabolic control expressed through glycemia level glycolysed hemoglobine (HbAlc) and fructosamine, as well as through antioxidative/prooxidative homeostasis. This is the confirmation that an adequate treatment choice can prevent numerous diabetes mellitus complications induced by free radicals.

Susceptibility to oxidative stress, insulin resistance, and insulin secretory response in the development of diabetes from obesity.

BACKGROUND/AIM: [corrected] Oxidative stress plays a critical role in the pathogenesis of various diseases. Recent reports indicate that obesity may induce systemic oxidative stress. The aim of the study was to potentiate oxidative stress as a factor which may aggravate peripheral insulin sensitivity and insulinsecretory response in obesity in this way to potentiate development of diabetes. The aim of the study was also to establish whether insulin-secretory response after glucagonstimulated insulin secretion is susceptible to prooxidant/antioxidant homeostasis status, as well as to determine the extent of these changes. METHODS: A mathematical model of glucose/insulin interactions and C-peptide was used to indicate the degree of insulin resistance and to assess their possible relationship with altered antioxidant/prooxidant homeostasis. The study included 24 obese healthy and 16 obese newly diagnozed non-insulin dependent diabetic patients (NIDDM) as well as 20 control healthy subjects, matched in age. RESULTS: Total plasma antioxidative capacity, erythrocyte and plasma reduced glutathione level were significantly decreased in obese diabetic patients, but also in obese healthy subjects, compared to the values in controls. The plasma lipid peroxidation products and protein carbonyl groups were significantly higher in obese diabetics, more than in obese healthy subjects, compared to the control healthy subjects. The increase of erythrocyte lipid peroxidation at basal state was shown to be more pronounced in obese daibetics, but the apparent difference was obtained in both the obese healthy subjects and obese diabetics, compared to the control values, after exposing of erythrocytes to oxidative stress induced by H2O2. Positive correlation was found between the malondialdehyde (MDA) level and index of insulin sensitivity (FIRI). CONCLUSION: Increased oxidative stress together with the decreased antioxidative defence seems to contribute to decreased insulin sensitivity and impaired insulin secretory response in obese diabetics, and may be hypothesized to favour the development of diabetes during obesity.

Protective effect of interferon-alpha on the DNA- and RNA-degrading pathway in anti-Fas-antibody induced apoptosis.

AIM: Fas membrane-associated polypeptide antigen is a receptor molecule responsible for apoptosis-mediated signals. In animal models of acute viral hepatitis, apoptosis of hepatocytes is mediated by Fas-death receptors; therefore, the aim of this study was to evaluate the effect of interferon (IFN)-alpha on apoptotic markers and nuclease activity against different coding and non-coding single and double stranded RNAs during Fas-induced liver apoptosis. METHODS: An in vivo experiment was performed with simultaneous administration of anti-Fas (CD95) antibodies and IFN-alpha, and an in vitro experiment was performed in hepatocyte cultures treated with anti-Fas antibodies and IFN-alpha. RESULTS: Detection of apoptosis using Annexin V-FITC/propidium iodide, Bcl-2 and Bax expression in hepatocyte cultures confirmed the appearance of early apoptotic events and progression toward late apoptosis after anti-Fas antibody treatment. IFN-alpha had a tendency to retard the apoptosis process in Fas-induced apoptosis by increasing the number of viable cells and decreasing the number of cells in late apoptosis, by increasing the percentage of Bcl-2 positive cells, by decreasing the percentage of Bax positive cells, and by decreasing the nuclease activity compared to the anti-Fas antibody treated group. Total DNA and RNA concentration was much reduced in the Fas group and DNA fragmentation assay provided evidence for increased DNA degradation. Enhanced nuclease activity against DNA, rRNA, poly(A), poly(C), poly(U), poly(I:C), and poly(A:U) was manifested in the anti-Fas antibody treated group, except for the inhibitory-bound alkaline RNase. CONCLUSIONS: The results demonstrate that the RNA-degrading pathway in Fas-induced apoptosis can accelerate the liberation of the latent enzyme from the inhibitor complex. IFN-alpha prevented enormous, Fas-ligand induced degradation of all the substrates used in this experimental study, most probably due to similarities in the signal transduction pathways. Investigations of death receptor-induced apoptosis may lead to novel treatment combinations for patients with acute or chronic liver diseases.

Possible impact of plasma RNase activity on immune dysfunction in juvenile diabetes mellitus.

AIM: The ribonuclease (RNase) family represents important enzymes used widely in biomedical and biotechnological applications, as well as for diagnostic and therapeutic purposes. This study was undertaken to test the possibility that plasma alkaline RNase (free or inhibitory bound) determination may be useful in studying the dysregulation of nucleic acid and oligonucleotide metabolism as a possible pathogenetic mechanism in development of immune dysfunction in juvenile diabetes mellitus. PATIENTS AND METHODS: Children with type 1 diabetes (n=32, age group of 5--14 yr), together with age-matched control subjects (n=35), were enrolled in the study. None had microvascular complications. According to the metabolic regulation of the disease and the hemoglobin A1c (HbA1c) level, all patients were divided into two groups (HbA1c<7.5% and HbA1c>7.5%). According to the duration of diabetes, diabetic children were divided into two groups: duration of diabetes less than 1 yr and duration of diabetes greater than 1 yr. The control group consisted of age-matched subjects (n=35; 15 girls and 20 boys) who were clinically healthy. The activity of free and inhibitory-bound RNase and the level of acid soluble nucleotides were measured in heparinized plasma. RESULTS: The inhibitory-bound enzyme activity was higher in diabetic children, followed by sharply decreased free enzyme, especially in the group with the level of HbA1c above 7.5%. Recent-onset diabetic patients had lower free RNase activity compared with those with longer duration of the disease. The amount of pre-existing acid-soluble oligonucleotides was significantly increased in diabetic children, especially in those with poor metabolic control. CONCLUSION: Our observed preliminary results may suggest a hypothesis that a persistent increase of oligonucleotide fragments, most probably due to insufficient RNase activity, may lead to T-cell hyperactivity in type 1 diabetes through the activation of toll-like receptors (TLRs). The measurement of RNase(s) activity (free, inhibitory-bound, or specific toward different substrates), together with the well-known immunobiochemical parameters of diabetes, may help further efforts in identifying a disease-specific early biological marker of immunity dysfunction in juvenile diabetes.

Sodium nitroprusside and peroxynitrite effect on hepatic DNases: an in vitro and in vivo study.

BACKGROUND: It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mg2+- and caspase-dependent) and acid-DNase. The aim of this study was to examine an in vivo and in vitro possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite. RESULTS: The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after in vivo treatment with either SNP or peroxynitrite. A difference observed between the in vivo and in vitro effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased. CONCLUSION: Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis.