The Study of Glycative and Oxidative Stress in Type 1 Diabetes Patients in Relation to Circulating TGF-Beta1, VCAM-1 and Diabetic Vascular Complications

Vladimir Jakus1, Jana Kostolanska2, Dagmar Michalkova1 and Michal Sapak3 1Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Bratislava 2Children Diabetological Center of the Slovak Republic, 1st Department of Pediatrics, Comenius University and University Hospital for Children, Bratislava 3Institute of Medical Immunology, Faculty of Medicine, Comenius University, Bratislava Slovakia


Introduction
Type 1 diabetes mellitus (T1DM) is one of most frequent autoimmune diseases and is characterized by absolute or nothing short of absolute endogenous insulin deficiency which results in hyperglycemia that is considered to be a primary cause of diabetic complications (DC) (Rambhade et al., 2010). T1DM leads to various chronic micro-and macrovascular complications. Diabetic nephropathy is a major cause of morbidity and mortality in patients with DM. Microvascular disease is the main determinant in the development of late complications in DM. Persistent hyperglycemia is linked with glycation, glycoxidation, and oxidative stress (Aronson, 2008;Negre-Salvayre et al., 2009). During glycation and glycoxidation there are formed early, intermediate and advanced glycation products via Maillard reaction, glucose autoxidation and protein glycation. Accumulation of advanced glycation end products (AGEs) has several toxic effects and takes part in the development of DC, such as nephropathy (Kashihara et al, 2010), neuropathy, retinopathy and angiopathy (Peppa & Vlassara, 2005;Yamagishi et al., 2008;Goh & Cooper, 2008;Karasu, 2010). Higher plasma levels of AGEs are associated also with incident cardiovascular disease and all-cause mortality in T1DM (Nin et al., 2011). AGEs are believed to induce cellular oxidative stress through the interaction with specific cellular receptors (Ramasamy et al., 2005;Yamagishi, 2009;Mosquera, 2010). On the other side, carbonyl stress-induced tissue damage is caused by AGE precursors formed by hyperglycaemia, hyperlipidemia, nonenzymatic glycation, peroxidation of lipids and metabolic processes.
The Study of Glycative and Oxidative Stress in Type 1 Diabetes Patients in Relation to Circulating TGF-Beta1, VCAM-1 and Diabetic Vascular Complications 5 matrix components such as collagen-IV, fibronectin, proteoglycans (decorin, biglycan). TGF-beta1 may cause glomerulosclerosis and its one of the causal factor in myointimal hyperplasia after baloon injury of carotid artery. It mediates angiotensin-II modulator effect on smooth muscle cell growth. Besides profibrotic activity, TGF-beta1 has immunoregulatory function on adaptive immunity too. AGEs induce connective tissue growth factor-mediated renal fibrosis through TGF-beta1-independent Smad3 signalling (Zhou et al., 2004;Chung et al., 2010). The present study investigates the relationship between diabetes complications presence, diabetes control (represented by actual levels of HbA1c (HbA1cA) and mean of HbA1c during the last 2 years (HbA1cP)), early glycation products (fructosamine (FAM)), serum advanced glycation end products (s-AGEs), lipid peroxidation products (LPO), advanced oxidation protein products (AOPP), profibrotic cytokines and adhesive molecules in patients with T1DM. We wanted to find a relationship of DC to glycative and oxidative stress parameters, circulating (serum) TGF-beta1 and soluble VCAM-1. Further, we aimed to compare measured parameters in groups -DC, one with DR, with DR combined with another DC and one with only DC another than DR and their combinations. The further aim of the present study was to evaluate if monitoring of circulating FAM, HbA1c, s-AGEs, AOPP, LPO in patients with T1DM could be useful to predict the diabetic complications development.

Patients and design
The studied group consisted of 46 children and adolescents with T1DM regularly attending the 1 st Department of Pediatrics, Children Diabetological Center of the Slovak Republic, University Hospital, Faculty of Medicine, Comenius University, Bratislava. They had T1DM with duration at least for 5 years. One of children was obese (BMIc 97 percentile) and three of them were of overweight (BMIc about 90 percentile). The file was divided into two subgroups: 20 persons without DC (-DC) and 46 those with them (+DC). Then the file of +DC patients was divided into several subgroups according to particular complications: the patients only with retinopathy, those with neuropathy combined with another kinds of DC and those with other than retinopathy to compare the parameters of glycative and oxidative stress and cytokines in each mentioned subgroups. The urine samples in our patients were collected 3 times overnight, microalbuminuria was considered to be positive when UAER was between 20 and 200 microgram/min in 2 samples. No changes (fundus diabetic retinopathy) were found by the ophtalmologist examining the eyes in subject without retinopathy. Diabetic neuropathy was confirmed by EMG exploration using the conductivity assessment of sensor and motor fibres of peripheral nerves. The controls file consists of 26 healthy children. The samples of EDTA capillary blood were used to determine of HbA1c and serum samples were used to determine of FAM, s-AGEs, AOPP and VCAM-1. The samples of serum were stored in -18°C/-80°C.

Parameter analysis 2.2.1 Determination of UAER
UAER was determined by means of immunoturbidimetric assay (Cobas Integra 400 Plus, Roche, Switzerland), using the commercial kit 400/400Plus. The assay was performed as a part of patients routine monitoring in Department of Laboratory Medicine, University Hospital, Bratislava.

Determination of fructosamine
For the determination of fructosamine we used a kinetic, colorimetric assay and subsequently spectrophotometrical determination at wavelength 530 nm. We used 1-deoxy-1-morpholino-fructose (DMF) as the standard. Serum samples were stored at -79°C and were defrost only once. This test is based on the ability of ketoamines to reduce nitroblue tetrazolium (NBT) to a formazan dye under alkaline conditions. The rate of formazan formation, measured at 530 nm, is directly proportional to the fructosamine concentration. Measurements were carried out in one block up to 5 samples. To 3 ml of 0.5 mmol/l NBT were added 150 microliters of serum and the mixture was incubated at 37°C for 10 minutes. The absorbance was measured after 10 min and 15 min of incubation at Novaspec analyzer II, Biotech (Germany).

Determination of glycated hemoglobin HbA1c
HbA1c was determined from EDTA capillary blood immediately after obtained by the lowpressure liquid chromatography (LPLC) (DiaSTAT, USA) in conjunction with gradient elution. Before testing hemolysate is heated at 62-68°C to eliminate unstable fractions and after 5 minutes is introduced into the column. Hemoglobin species elute from the cationexchange column at different times, depending on their charge, with the application of buffers of increasing ionic strength. The concentration of hemoglobins is measured after elution from the column, which is then used to quantify HbA1c by calculating the area under each peak. Instrument calibration is always carried out when introducing a new column set procedure (Bio-RAD, Inc., 2003).

Determination of serum AGEs
Serum AGEs were determined as AGE-linked specific fluorescence, serum was diluted 20fold with deionized water, the fluorescence intensity was measured after excitation at 346 nm, at emission 418 nm using a spectrophotometer Perkin Elmer LS-3, USA. Chinine sulphate (1 microgram/ml) was used to calibrate the instrument. Fluorescence was expressed as the relative fluorescence intensity in arbitrary units (A.U.).

Determination of serum lipoperoxides
Serum lipid peroxides were determined by iodine liberation spectrophotometrically at 365 nm (Novaspec II, Pharmacia LKB, Biotech, SRN). The principle of this assay is based on the oxidative activity of lipid peroxides that will convert iodide to iodine. Iodine can then simply be measured by means of a photometer at 365 nm. Calibration curves were obtained using cumene hydroperoxide. A stoichiometric relationship was observed between the amount of organic peroxides assayed and the concentration of I 3 produced (El-Saadani et al., 1989).

Determination of serum AOPP
AOPP were determined in the plasma using the method previously devised by Witko-Sarsat et al. (1996), modified by Kalousova et al. (2002). Briefly, AOPP were measured by spectrophotometry on a reader (FP-901, Chemistry Analyser, Labsystems, Finland) and were calibrated with chloramine-T solutions that in the presence of potassium iodide absorb at 340 nm. In standard wells, 10 microliters of 1.16 M potassium iodide was added to 200 www.intechopen.com The Study of Glycative and Oxidative Stress in Type 1 Diabetes Patients in Relation to Circulating TGF-Beta1, VCAM-1 and Diabetic Vascular Complications 7 microliters of chloramine-T solution (0-100 micromol/l) followed by 20 microliters of acetic acid. In test wells, 200 microliters of plasma diluted 1:5 in PBS was placed to cell of 9 channels, and 20 microliters of acetic acid was added. The absorbance of the reaction mixture is immediately read at 340 nm on the reader against a blank containing 200 microliters of PBS, 10 microliters of potassium iodide, and 20 microliters of acetic acid. The chloramine-T absorbance at 340 nm being linear within the range of 0 to 100 micromol/l, AOPP concentrations were expressed as micromoles per liter of chloramine-T equivalents.

Determination of serum soluble form of adhesion molecule VCAM-1
For serum soluble form of VCAM-1 (sVCAM-1) estimating we used bead-based multiplex technology and Athena Multi-LyteTM Luminex 100 xMAP (multi-analyte profiling) analyser. We used RnD systems manufacturer kits: "Human Adhesion Molecule MultiAnalyte Profiling Base Kit" and "Fluorokine® MAP Human sVCAM-1/CD106 Kit". Analyte-specific antibodies are pre-coated onto color-coded microparticles. Microparticles, standards and samples are pipetted into wells and the immobilized antibodies bind the analytes of interest. After washing away any unbound substances, a biotinylated antibody cocktail specific to the analytes of interest are added to each well. Following a wash to remove any unbound biotinylated antibody, streptavidin-phycoerythrin conjugate (Streptavidin-PE), which binds to the captured biotinylated antibody, is added to each well. A final wash removes unbound Streptavidin-PE and the microparticles are resuspended in buffer and read using the Luminex analyzer. One laser is microparticle-specific and determines which analyte is being detected. The other laser determines the magnitude of the phycoerythrin-derived signal, which is in direct proportion to the amount of analyte bound (R&D Systems, Inc. 2010).

Statistical analysis
Shapiro-Wilk test was performed to the test the distribution of all continuous variables. The variables with normal distribution were compared by one way ANOVA test followed by Bonferroni´s post-test and the results was expressed as mean ± SD. Since the evaluated variables did not have normal distribution, we compared them with Kruskal-Wallis nonparametric analysis of variance (ANOVA) followed by Bonferroni´s post-test and the results was expressed as median (1st quartile, 3rd quartile). The Fisher´s test was used to compare the subgroups in regard to diabetic retinopathy and other complications presence/absence. Pearson´s test with correlation coefficient r or Spearman´s one with Spearman's rank correlation coefficient R in case of small count of variables were then used to evaluate the association between parameters described within the text, in all studied patients and in diabetic and non-diabetic subgroups. P values less than 0.05 were accepted as being statistically significant. All statistical analyses were carried out using Excel 2003, Origin 8 and BioSTAT 2009.

Results
Clinical and biochemical characteristics of the patients with T1DM without and with diabetic complications and controls (CTRL) are reported in Table 1 As seen, HbA1c and FAM were significantly elevated in both diabetic groups in comparison with controls and also in +DC vs. -DC those. Serum AGEs were significantly elevated in +DC compared to -DC and also to controls, but the difference between -DC and controls was not significant. The levels of AOPP were evidently higher in +DC compared to controls, but the difference was not significant. The levels of LPO were significantly elevated in +DC vs. controls, the differences between both diabetic groups and between -DC vs. controls were not significant. The levels of TGF-beta1 similarly to s-AGEs were significantly elevated in +DC compared to -DC and also to controls, but the difference between -DC and controls was not significant (Fig. 1). In terms of the VCAM-1 values, only between +DC and controls there were found significant difference there (Fig. 2). The levels of TGF-beta1 are significantly elevated in +DC compared to -DC (10.49 ± 4.55 vs. 5.9 ± 4.14 ng/ml, p<0.05) and also to controls (10.49 ± 4.55 vs. 3.30 ± 3.41ng/ml, p<0.05), but the difference between -DC and controls (5.9 ± 4.14 vs. 3.30 ± 3.41ng/ml, p>0.05) was not statistically significant.

The subgroup of patients with T1DM with diabetic complications
The relationships characterized by Pearson´s correlation coefficient r or Spearman´s coefficient R between the parameters described within the text are reported in Table 3.
www.intechopen.com  Moderate relationships were found also between TGF-beta1 and oxidative stress parameters, but those were not statistically significant.

Conclusion
Our results suggest the relation of glycation and oxidation to profibrotic cytokines, vascular molecules and diabetic complications. Serum AGEs were connected with complications other than retinopathy more than just with retinopathy, nevertheless, some relation of retinopathy and s-AGEs was found (p-values were only slightly higher than 0.05). Lipoperoxides showed some relation to DR since higher in patients with retinopathy than in those with other DC, whereas AOPP did not show any relation to any DC. It seems that in our patients TGF-beta1 and VCAM-1 are linked with the development of DC, but only TGF-beta1 showed some linkage to diabetic retinopathy. We ought to keep in mind the fact our investigation concerns the children and adolescents. Maybe the study of older patients with T1DM would show more, especially about VCAM-1 and its relation to glycative and oxidative stress and consequently to development of retinopathy/other complications.  This book is intended as an overview of recent progress in type 1 diabetes research worldwide, with a focus on different research areas relevant to this disease. These include: diabetes mellitus and complications, psychological aspects of diabetes, perspectives of diabetes pathogenesis, identification and monitoring of diabetes mellitus, and alternative treatments for diabetes. In preparing this book, leading investigators from several countries in these five different categories were invited to contribute a chapter to this book. We have striven for a coherent presentation of concepts based on experiments and observation from the authors own research and from existing published reports. Therefore, the materials presented in this book are expected to be up to date in each research area. While there is no doubt that this book may have omitted some important findings in diabetes field, we hope the information included in this book will be useful for both basic science and clinical investigators. We also hope that diabetes patients and their family will benefit from reading the chapters in this book.