**2.2.7 Determination of TGF- beta1**

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

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

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

Serum AGEs were determined as AGE-linked specific fluorescence, serum was diluted 20 fold 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

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 I3 produced (El-Saadani et al.,

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

expressed as the relative fluorescence intensity in arbitrary units (A.U.).

**2.2.2 Determination of fructosamine** 

**2.2.3 Determination of glycated hemoglobin HbA1c** 

column set procedure (Bio-RAD, Inc., 2003).

**2.2.5 Determination of serum lipoperoxides** 

**2.2.6 Determination of serum AOPP** 

**2.2.4 Determination of serum AGEs** 

1989).

II, Biotech (Germany).

Quantitative detection of TGF- beta1 in serum was done by enzyme linked immunosorbent assay, using human TGF-beta1 ELISA-kit (BMS249/2, Bender MedSystem).

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.

### **2.2.8 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).

#### **2.2.9 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

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

Fig. 1. Comparison of TGF-beta1 levels of patients with T1DM and controls

Fig. 2. Comparison of VCAM-1 levels of patients with T1DM and controls

ng/ml, p>>0.05).

The levels of VCAM-1 are significantly elevated in +DC compared to controls (17.4 ± 3.3 vs. 12.6 ± 3.7 ng/ml, p<0.05). The values of VCAM-1 in –DC subgroup differ obviously from those in controls, but the difference is non statistically significant (17.1 ± 3.1 vs. 12.6 ± 3.7 ng/ml, p>0.05). There are similar levels in both diabetic subgroups ((17.4 ± 3.3 vs. 17.1 ± 3.1

in Relation to Circulating TGF-Beta1, VCAM-1 and Diabetic Vascular Complications 9

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.
