100 μL of samples of either serum or a standard solution or quality control sample, were added to 200 μL of a solution of ethanol containing tocopheryl acetate (4 μM) that was used as an internal standard. After stirring the mixture for 30 seconds, the CA4P vitamins were extracted with 1000 μL of hexane (2 min of stirring). The organic phase was evaporated under nitrogen and the residues dissolved in 200 Selleck Temsirolimus μL of methanol and 50 μL were injected into the chromatograph. All procedures were performed in a room with glass windows that prevented penetration of direct sunlight. GSTM1, GSTP1, GSTT1 and hOGG1 genotyping analysis DNA was extracted

by the phenol-chloroform method using an aliquot out of the 20 ml venous blood samples of the subjects. Determination of GSTM1, GSTP1 and GSTT1 polymorphisms in the 60 subjects was performed as previously described [17]. Analysis of CHIR-99021 purchase deletion polymorphism in GSTM1 and GSTT1 was performed by multiplex PCR and that of single nucleotide polymorphism in GSTP1 by a PCR-RFLP method as previously described [20]. In addition to these polymorphisms, subjects were also genotyped for the presence of either the serine or cysteine codon at position 326 (rs 1052133) of the hOGG1 gene by PCR-RFLP, using primers and conditions as previously described [21].

Briefly, the PCR amplification of the 293 bp fragment consisted of a 15-min denaturation at 95°C followed by 30 cycles of 95°C for 1 min, 50°C for 1 min and 72°C for 1 min. A final extension step of 72°C for 10 min was included. We used a simple RFLP method to identify the Ser 326 Cys by virtue of an Fnu 4HI restriction site. The hOGG1 PCR product was digested with Fnu 4HI overnight at 37°C. Recovery of two digested fragments (123/124bp

and 169/170bp) indicated presence of the Cys 326 allele, while an undigested amplicon indicated the Ser 326 allele. Statistical analysis All statistics and graphics have been performed with the SAS System release 9 (SAS Institute Inc., Cary, NC, USA). Distributions of 8-oxodG were normalised 3-mercaptopyruvate sulfurtransferase by logarithmic transformations. Mean values were compared by Student’s t-test or ANOVA and correlations between 8-oxodG and antioxidants were evaluated by Pearson correlation test. All statistical analyses were two-sided. Results Blood levels of 8-oxodG and vitamins A and E The mean serum concentrations of vitamin A were 2.77 μM and 2.74 μM, while those for vitamin E were 34.77 μM and 38.73 μM, in patients and controls respectively (Table 2). Table 2 Biochemical parameters of the study group Parameter Patients (mean ± s.d.) Controls (mean ± s.d.) P-value b patient vs. control 8-oxodG/10 6 2′dG c 7.2 ± 2.6 (n = 17) 4.9 ± 1.9 (n = 43) P < 0.001 Vitamin A (μM) 2.77 ± 0.94 (n = 15)a 2.74 ± 0.61 (n = 42)a P = 0.895 Vitamin E (μM) 34.77 ± 12.27 (n = 15)a 38.73 ± 9.47 (n = 42)a P = 0.204 PBMCs were collected and processed for measuring 8-oxodG. Vitamins were extracted from the serum samples for estimation.