All subjects underwent a complete work-up, including medical history, clinical examination, anthropometric measurements, laboratory tests, and liver biopsy. A 12-hour overnight fasting blood sample was obtained on the morning of the liver biopsy in all subjects to assess fasting blood glucose (mg/dL), total cholesterol (mg/dL), high-density lipoprotein cholesterol (mg/dL), triglycerides (mg/dL), aspartate aminotransferase (IU/L), alanine aminotransferase
(IU/L), gamma-glutamyl transpeptidase (IU/L), alkaline phosphatase (IU/L), blood urea nitrogen (mg/dL), creatinine (mg/dL), serum calcium (mg/dL), and phosphorus (mg/dL). The following laboratory tests were performed to rule out other causes of liver disease: hepatitis B surface antigen (HBsAg) positivity in patients with CHC; HBsAg and anti–hepatitis C virus (HCV) positivity in patients with NAFLD; and anti-HIV positivity, anti-nuclear antibody titer ≥1:80, anti–smooth muscle antibody titer ≥1:40, anti-mitochondrial antibody at any titer, reduced ceruloplasmin or α1-antitrypsin, and transferrin saturation ≥45%, in both groups. Biochemical assessments were performed using
standard laboratory methods. Insulin (μU/mL) was measured via radioimmunoassay (ADVIA Insulin Ready Pack 100; Bayer Diagnostics, Milan, Italy), with intra- and interassay coefficients of variation <5%. Plasma adiponectin concentrations were measured using an RIA kit (reference range, 1.5-100 ng/mL; Linco Research, St. Louis, MO) with intra-
and interassay coefficients of variation of 4.5% and 3%, respectively. The degree of insulin resistance was estimated by means of homeostasis model assessment of insulin resistance (HOMA-IR). Vitamin D status in our population was evaluated measuring serum 25(OH)D3, the most stable circulating form of this molecule.26 25(OH)D3 (nmol/L) was measured by a validated colorimetric CYTH4 method (selleckchem LAISON, DiaSorin) on sera frozen immediately after separation and stored at −25°C for less than two months. Liver biopsies undertaken for clinical purposes were obtained via percutaneous echo-assisted method by the same expert hepatologist. Subjects in the comparison group underwent intraoperative liver biopsy during surgery. Liver fragments were fixed in buffered formalin for 2-4 hours and embedded in paraffin with a melting point of 55°C-57°C. Three- to 4-μm sections were cut and stained with hematoxylin and eosin and Masson’s trichrome stains. A single pathologist blinded to each patient’s identity, history, and biochemistry read all of the slides. A minimum biopsy specimen length of 15 mm or at least the presence of 10 complete portal tracts was required.27 Liver biopsy samples were classified according to the presence of NASH by Brunt definition.