Phthalates Exposure in Type 2 Diabetic Patients and Diuretic Therapy

* Total concentrations of MEHP, MEOHP and MEHHP will be quantified, in the laboratories of the Institute of Clinical Physiology, National Research Council, Pisa, in a spot morning urine sample by ultra-HPLC coupled with electrospray ionization/quadrupole time-of-flight mass spectrometry (Agilent UHPLC 1290 infinity coupled to an Agilent 6540 MS-QTOF, Santa Clara, CA) using stable isotope labeled substrates, i.e. MEHP (ring-1,2-13C2, dicarboxyl-13C2), MEHHP, MEHHP 13C4, MEOHP and MEOHP 13C4 that will be purchased from Cambridge Isotope Laboratories (Tewksbury, MA). * Urinary creatinine concentrations will be measured to adjust urinary concentrations of DEHP metabolite (Beckman Coulter AU400, Brea, CA), thus minimizing the influence of urine volume. * Serum and urinary inflammatory markers and adipocytokines will be quantitatively determined using sandwich enzyme-linked immunosorbent assays kits according to the manufacturer's instructions. Optical density will be measured using a microplate reader. * Serum and urinary markers of oxidative stress will be measured by gold standard techniques. In detail, MDA will be quantified by TBARS reactive substances measured by optical density; GSH-Px by a specific assay kit according to the manufacturer's instruction; SOD activity will be determined using a specific SOD kit; urinary 8-isoprostane concentration will be measured by a specific affinity sorbent. (Cayman Chemical, Ann Harbor, MI, USA) according to the manufacturer's instructions. * To analyze mitochondrial DNA we will apply a triplex design previously reported to amplify mitochondria loci located within the MinorArc and MajorArc, respectively. To assess nuclear DNA, we will use RNase P Copy Number Reference. * The phthalates-free diet will be self-administered by the individuals under intervention, following a set of instruction and rules provided by the physicians based on the current literature data.