5%). Results of the SST were available to the treating physicians. Free cortisol was not directly tested.

We estimated its concentration using the free cortisol index (FCI) and the calculated free cortisol (cFC). FCI is the ratio between total cortisol (nmol/L) and transcortin (mg/L).[23] cFC was derived using Coolens’ equation: U2K(1 + N) + U[1 + N + K(T − C)] − C = 0, where U represents the molar concentration of unbound cortisol, C is the molar concentration of total cortisol, T is the concentration of trancortin, and K is the affinity of transcortin for cortisol at 37°C. N is the ratio of albumin bound to free cortisol, and 1.74 is the MAPK inhibitor value conventionally used.[24] Baseline plasma renin activity (patient in supine position for at least 1 hour), plasma concentrations of vasopressin, norepinephrine, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and serum levels of nitric oxide, total serum cholesterol and triglyceride, and high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol were determined at inclusion. Plasma renin activity and plasma concentration of vasopressin and norepinephrine were assessed Z-VAD-FMK order by radioimmunoassay (Clinical Assays,

Cambridge, MA; Bühlman Laboratories, Basel, Switzerland, and CAIBL Laboratories, Hamburg, Germany, respectively). To measure serum levels of nitrates and nitrites (NO2− and NO3−), samples were ultrafiltered (PL-10 Ultrafree-MC centrifugal filter units; Millipore, Bedford, MA) at 1,200g for 1 hour to remove proteins before analysis. Filtered serum was refluxed in glacial acetic acid containing sodium iodide. Under these conditions NO2− and NO3− are reduced to NO, which, after reacting with ozone, can be quantified by a chemiluminescence detector (Nitric Oxide Analyzer, NOA 280, Sievers Instruments, Boulder, CO). IL-6 and TNF-α

were measured by enzyme-linked immunosorbent assay (Medgenix Diagnostics, Fleurus, Belgium). Serum levels of total and HDL cholesterol and triglyceride were measured by the enzymatic colorimetric methods (ADVIA 2400 Chemistry System; Bayer Health Care). LDL cholesterol was calculated using the Friedewald formula. Normal values in our laboratory are: transcortin 25-55 μg/mL, plasma renin activity: 1.4 ± 0.9 ng/mL*h, norepinephrine: 136-364 pg/mL, vasopressin: 上海皓元医药股份有限公司 1.5-3.3 ng/L, IL-6: < 5 pg/mL, TNF-alpha: < 20 pg/mL, nitrates and nitrites: 37 ± 14 nMol/mL, total cholesterol: 148-247 mg/dL, triglycerides: 50-150 mg/dL, HDL cholesterol: >40 mg/dL, and LDL cholesterol: <180 mg/dL. FCI above 12 represents sufficient adrenal function.[19] All patients were managed following standard protocols for each clinical decompensation. Patients were followed during hospitalization and monthly up to 3 months. Any significant new clinically relevant event was recorded including bacterial infections, gastrointestinal bleeding, hepatic encephalopathy, and HRS.