**2. Study design and methods**

#### **2.1 Patients and design**

4 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

It has been suggested that the chronic hyperglycaemia in diabetes enhances the production of reactive oxygen species (ROS) from glucose autoxidation, protein glycation and glycoxidation, which leads to tissue damage (Son, 2007). Also, cumulative episodes of acute hyperglycaemia can be source of acute oxidative stress. A number of studies have summarized the relation between glycation and oxidation (Boyzel et al., 2010). The overproduction of ROS leads to oxidative modification of biologically important compounds and damage of them. Uncontrolled production of ROS often leads to damage of

Some oxidation products or lipid peroxidation products may bind to proteins and amplify glycoxidation-generated lesions. Lipid peroxidation of polyunsaturated fatty acids, one of the radical reaction in vivo, can adequately reflect increased oxidative stress in diabetes. Advanced oxidation protein products (AOPP) are formed during oxidative stress by the action of chlorinated oxidants, mainly hypochlorous acid and chloramines. In diabetes the formation of AOPP is induced by intensified glycoxidation processes, oxidant-antioxidant imbalance, and coexisting inflammation (Piwowar, 2010a, 2010b). AOPP are supposed to be structurally similar to AGEs and to exert similar biological activities as AGEs, i.e. induction of proinflammatory cytokines in neutrophils, as well as in monocytes, and adhesive molecules (Yan et al., 2008). Accumulation of AOPP has been found in patients with chronic kidney disease (Bargnoux, et al., 2009). Further possible sources of oxidative stress are

Diabetes is associated also with inflammation (Navaro & Mora, 2006; Wautier et al., 2006; Devaraj et al., 2007; Hartge et al., 2007; Fawaz, et al., 2009 ; Van Sickle et al., 2009; Nobécourt et al., 2010). ROS are implicated also in the pathogenesis of the inflammatory response to ischemic-reperfusion which is exacerbated in diabetes. Oxidative stress during reperfusion is markedly balanced in diabetes and this appears to results from increased leukocyte recruitment and a higher capacity of diabetic leukocytes to generate ROS in response to stimulation. Several adhesion molecules are expressed on endothelial cells and participate in leukocyte adhesion to the endothelium. These molecules are important for monocyte– endothelium interaction in the initiation and progression of atherosclerosis. The monocytemacrophage is a pivotal cell in atherogenesis. Cellular adhesion molecules mediate attachment and transmigration of leukocytes across the endothelial surface and are thought to play a crucial role in the early steps of atherogenesis (Seckin et al., 2006). Adhesion molecule VCAM-1 is not expressed under baseline conditions but is rapidly induced by proatherosclerotic conditions in rabbits, mice, and humans, including in early lesions. Initially, it is unclear whether VCAM-1 is simply a marker for atherogenesis or whether it acts in this disease pathway. AGEs promote VCAM-1 expression and atheroma formation in rabbits (Vlassara et al., 1995) and in cultured human endothelial cells (Schmidt et al., 1995). These results suggest the involvement of AGEs in the accelerated coronary atherosclerosis on diabetes (Zhang et al., 2003). Plasma concentrations of VCAM-1 are elevated also in T1DM patients with microalbuminuria and overt nephropathy (Schmidt et al., 1996; Clausen

Diabetic nephropathy is characterized by specific morphological changes including glomerular basement membrane thickening, mesangial expansion and glomerular and tubulointersticial sclerosis. The first clinical manifestation of diabetic nephropathy is microalbuminuria, defined as a urinary albumin excretion rate of 20 to 200 microgram/min. Growth factor TGF-beta1 is one of profibrotic cytokines and is important mediator in the pathogenesis of diabetic nephropathy (Goldfarb & Ziyadeh, 2001; Schrijvers et al., 2004; Wang et al., 2005; Wolf & Ziyadeh, 2007). TGF-beta1 stimulates production of extracellular

cellular macromolecules (DNA, lipids and proteins).

et al, 2000).

decreased antioxidant defenses, or alterations in enzymatic pathways.

The studied group consisted of 46 children and adolescents with T1DM regularly attending the 1st 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.

#### **2.2 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.

The Study of Glycative and Oxidative Stress in Type 1 Diabetes Patients

**2.2.7 Determination of TGF- beta1** 

(R&D Systems, Inc. 2010).

**2.2.9 Statistical analysis** 

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.

Quantitative detection of TGF- beta1 in serum was done by enzyme linked immunosorbent

Brief description of the method: into washed, with anti-TGF-beta1 precoated microplate were added prediluted (1:10) sera (100 microliters) and "HRP-Conjugate" (50 microliters) as a antihuman-TGF-beta1 monoclonal antibody and incubated for 4 hour on a rotator (100rpm). After microplate washing (3 times) "TMB Substrate Solution" (100 microliters) was added and was incubated for 10 minutes. Enzyme reaction was stopped by adding "Stop Solution" (100 microliters). The absorbance of each microwell was readed by HumaReader spectrophotometer (Human) using 450 nm wavelength. The TGF-beta1 concentration was determined from standard curve prepared from seven TGF-beta1 standard dilutions. Each sample and TGF-beta1 standard dilution were done in duplicate.

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

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

assay, using human TGF-beta1 ELISA-kit (BMS249/2, Bender MedSystem).

**2.2.8 Determination of serum soluble form of adhesion molecule VCAM-1** 

## **2.2.2 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).
