*2.5. Carbonyl Residues (CO)*

Carbonyl residues were determined following the method of Correa-Salde and Albesa [15] with a few modifications. Serum samples (100 μL) were treated for 1 h at room temperature with 900 μL of 0.1% dinitrophenylhydrazine in 2 M HCl and precipitated with 400 μL of 10% trichloroacetic acid (TCA) before being centrifuged for 20 min at 4 ◦C at 10,000× *g*. The pellets were extracted with 500 μL of ethanol:ethyl acetate mixture (1:1) and centrifuged for 3 min at 4 ◦C at 10,000× *g*, three times and then dissolved in 15 mL of 6 M guanidine HCl in 20 mM potassium phosphate buffer (PBS), pH 7.5. The solutions were incubated at 37 ◦C for 30 min and insoluble debris was removed by centrifugation. The absorbance was measured at 370 nm.

Carbonyl content was calculated using a molar absorption coefficient of 22,000 M−<sup>1</sup> cm<sup>−</sup><sup>1</sup> and expressed as nmol/mg of proteins. Protein content was estimated by using the Bio-Rad DC protein assay kit (Bio-Rad, Segrate, Milan, Italy).

#### *2.6. Thiobarbituric Acid Reactive Substances (TBARS)*

TBARS were evaluated as an index of lipid peroxidation according the method by Dietrich-Muszalska et al. [16]. A total of 100 μL of serum was first deproteinized by adding 100 μL of TCA, then 160 μL was added to 32 μL of 0.12 M thiobarbituric acid (Sigma-Aldrich, Milan, Italy) in TRIS 0.26 M, and heated for 15 min at 100 ◦C. The reaction was stopped by placing the vials in an ice bath for 10 min and after centrifugation (at 1600× *g* at 4 ◦C for 10 min), the absorbance of the supernatant was measured at 532 nm (Perkin Elmer Wallac 1420 Victor3 Multilabel Counter).

TBARS content was calculated using a molar absorption coefficient of 1.56 × 10−<sup>5</sup> M−<sup>1</sup> cm<sup>−</sup><sup>1</sup> and expressed as μM.

## *2.7. Advanced Glycated End-Products (AGEs)*

AGEs were determined by exploiting the characteristic autofluorescence of the large part of AGEs as described by Cournot and Burillo [17]. A total of 100 μL of 1:5 diluted in PBS serum, were placed in a 96-well plate and the fluorescence intensity was read at 460 nm, after excitation at 355 nm. Results were expressed as arbitrary units (AU).
