Serum Myostatin Levels in Liver Disease

Within the first 72 h after admission, blood samples were taken at 8.00 a.m. in fasting conditions and were immediately frozen at −20 °C. Serum myostatin levels were determined by quantitative sandwich enzyme immunoassay technique (R&D Systems, Abingdon Science Park, Abingdon, UK). Each serum sample needed a pretreatment with acid hydrolysis and posterior adjustment to PH 7.4 with HEPES buffer to cleave the active peptide (myostatin) of the original propeptide. We have strictly followed the recommendations of the ELISA manufacturer (R&D Systems, Abingdon Science Park, Abingdon, UK). The yellow final complex was measured at 450 nm in a microplate spectrophotometer reader (Spectra MAX-190, Molecular Devices, Sunnyvale, CA 94089, USA). The calibration curve was prepared with authentic myostatin standards. The coefficient of correlation of the standard curve was 0.997. The detection limit of this assay was established at 2.25 pg/mL; the intra and interassay CV were 3.23% and 4.23%, respectively. The antibody used to coat wells of the plate in this ELISA kit is a human specific monoclonal one. This assay recognizes natural and recombinant mature myostatin. No significant cross-reactivity or interference was observed with other peptides as activin RIIA, activin RIIB, decorin, GASP-2, GDF-11, and GDF-15. The molecules listed previously were prepared at 50 ng/mL in calibrator diluent and assayed for cross-reactivity.
Routine laboratory testing was done for all patients and controls. We recorded prothrombin activity, bilirubin, and albumin, and further calculated the Child–Pugh score. Mean corpuscular volume (MCV) and gamma glutamyl transferase (GGT) were considered “markers” of ethanol consumption.