, 2010) and a common R2* for all peaks were used in the modeling. The R2* parameter can be thought of as the peak width in frequency domain and can be used to detect liver iron deposition (positive correlation). In the present study, the R2* parameter was used as an additional MEK inhibitor biomarker of liver status. The liver fat content and R2* from the entire liver was analyzed by manual identification of the volume of interest and by fitting of a Gaussian function to the liver fat fraction and R2* histograms (see Fig. 1f and g). The center of the Gaussian function was used to sample robust estimates of liver fat content and

R2*. At termination blood was collected from the abdominal aorta in EDTA-treated tubes (Greiner bio-one, Frickenhausen, Germany) and centrifuged for 10 min to prepare plasma. Aliquotes were stored at −70 °C pending

biochemical analyses of the following circulating markers: triglycerides, cholesterol, and apolipoprotein A-I (apo A-I). The liver and the left perirenal fat pad (see Fig. 2) were dissected and weighed. The liver weight was used Ipilimumab chemical structure to calculate the liver somatic index (LSI, liver weight × 100/body weight). The analysis of cholesterol and triglycerides was a standard laboratory technique and was performed on an Architect C 8000 analyzer (Abbott Laboratories, Abbott Park, IL, USA) and reported using SI units. Analysis of protein apo A-I: Prior to western blot 1 μl of plasma from rats of all groups (W; n = 12, F; n = 12, BPA 0.025 mg/L; n = 11, BPA 0.25 mg/L; n = 8 and BPA 2.5 mg/L; n = 9) were separated on SDS-polyacrylamide gradient gels (T = 5–20%,

C = 1.5%) with stacking gels (T = 5%, C = 1.5%) for 1 h (180 V, 60 mA) in electrode buffer (0.15% (w/v) Tris, Meloxicam 0.72% (w/v) glycine, 0.05% (w/v) SDS) using a Mini Protean II electrophoresis cell (Bio Rad). Samples were diluted in sample cocktail (4% (w/v) SDS, 200 mM DTT, 20% (w/v) sucrose) and boiled for 3 min. Plasma proteins separated by SDS PAGE were transferred to a PVDF membrane. After blocking 1 h (5% milk in TBS) and incubation over night with primary antibodies 1:1000 (2% milk in TTBS) against apo A-I (rabbit anti rat apoA-I, polyclonal, Ab 20453, Abcam, UK), the membrane was incubated for 1 h with goat anti-rabbit HRP-conjugated secondary antibodies 1:40 000 (2% milk in TTBS). Proteins were visualized using an ECL plus western blotting detection system. Gel images were evaluated using Image Lab 3.0.1 (Bio Rad, Hercules, CA) and apo A-I levels were determined as intensity/mm2. Differences between the fructose control group and the three BPA plus fructose exposed groups were evaluated by factorial ANOVA. When the three BPA groups were analyzed vs the fructose control group one by one, a Bonferroni adjustment for 3 tests was used and p < 0.0167 considered significant (p = 0.05/3 = 0.0167). In the secondary analysis, when the water control group was compared with the fructose control group p < 0.05 was considered as significant.