Au5800 system

Venous blood samples were collected after a fast for at least 8 h for measurements. Liver biochemical and metabolic parameters, including ALT, aspartate aminotransferase cellulase (AST), GGT, lactate dehydrogenase, choline esterase, leucine arylamidase, glutamate dehydrogenase, direct bilirubin (DBil), total bilirubin (TBil), total bile acid (TBA), lipid profile, fasting blood glucose (FBG), fasting insulin (FINS) and uric acid (UA) levels, were measured. The levels of liver enzymes were measured with the enzymatic-colorimetric method using a conventional automated analyzer (Biochemical analyzer from beckman coulter, Au 5800 System), and the cut-off values for ALT levels were set to 30 U/L for men and 19 U/L for women [14 (link)], while the cut-off values for GGT levels were set to 50 U/L for men and women [15 (link)]. The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as follows: FINS (μU/mL) × FBG (mmol/L)/22.5 [16 (link)]. A cut-off value of 2.69 was utilized to define insulin resistance (IR) [16 (link)]. Fibrosis-4 (FIB-4) index is a simple noninvasive test for liver fibrosis that produces results using the following formula: age (years) × AST (U/L)/(platelets (10⁹/L) × ALT (U/L)^1/2) [17 (link)]. An FIB-4 index < 1.45 in the context of NAFLD excludes clinically significant hepatic fibrosis [18 (link)].

Blood samples were centrifuged, and supernatants were stored at −85 °C until analysis. Plasma glucose concentrations were measured by the AU 5800 System (Beckman Coulter; inter-assay (CV) < 0.70%, intra-assay < 1.25%). Serum insulin, C-peptide, cortisol, and ACTH concentrations were determined by ElectroChemiLuminescence (ECL) immunoassay (Elecsys, cobas e 411 and 602, immunoassay-Systems, Roche Diagnostics, Germany; insulin: inter-assay coefficient of variation (CV) < 2.0%, intra-assay CV < 2.8%; C-peptide: inter-assay CV < 5.0%, intra-assay CV < 4.6%; cortisol: inter-assay CV < 2.8%, intra-assay CV < 1.7%; ACTH: inter-assay CV < 5.4%, intra-assay CV < 2.9%). Norepinephrine and epinephrine were measured by electrochemical detection (ECD) and an UV-detector for high-performance liquid chromatography (HPLC) by VWR Merck/Hitachi (norepinephrine inter-assay CV < 12.1%, intra-assay CV < 3.2%; epinephrine inter-assay CV < 9.9%, intra-assay CV < 1.8%).

Iron metabolism-related data were measured. The PF iron concentration and unsaturated iron binding capacity (UIBC) were determined using a colorimetric kit by the Ferene method (iron and total binding capacity (TIBC); Leadman, China) and measured using the Beckman–Coulter AU 5800 system (Beckman Coulter, USA). The ferritin concentration was measured using the immunoturbidimetric method (Abbott Laboratories, USA) by ARCHITECT-i2000 (Abbott Laboratories, USA). Transferrin was determined by the turbidimetric method using the Transferrin assay kit (Beckman Coulter Inc, USA) and the Immage® 800 Rate Nephelometer (Beckman Coulter Inc, USA). Transferrin saturation (TSAT) was calculated based on the iron concentration and TIBC (TSAT = Fe/TIBC × 100).