**1. 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; Boulanger et al., 2006; 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

(Zhou et al., 2004; Chung et al., 2010).

**2. Study design and methods** 

**2.1 Patients and design** 

**2.2 Parameter analysis 2.2.1 Determination of UAER** 

Hospital, Bratislava.

development.

in Relation to Circulating TGF-Beta1, VCAM-1 and Diabetic Vascular Complications 5

matrix components such as collagen-IV, fibronectin, proteoglycans (decorin, biglycan). TGFbeta1 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

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

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

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

and VCAM-1. The samples of serum were stored in -18°C/-80°C.

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 cellular macromolecules (DNA, lipids and proteins).

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 decreased antioxidant defenses, or alterations in enzymatic pathways.

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 et al, 2000).

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 matrix components such as collagen-IV, fibronectin, proteoglycans (decorin, biglycan). TGFbeta1 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.
