**2.2.4 Determination of serum AGEs**

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

#### **2.2.5 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 I3 produced (El-Saadani et al., 1989).

#### **2.2.6 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 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.
