Clinical assessment
A detailed clinical assessment was performed in each patient. Information on medical history, past and current drugs and alcohol consumption were collected. In this study, we analyzed alcohol consumption by asking the patient about the type and amount of alcohol consumption per day during the prior year, then estimating the mean alcohol intake per day. Anthropometric assessments were performed by well-trained nurses and included body mass index (BMI) and waist circumference (WC). Obesity was defined in patients that had BMI ≥ 30 kg/m2, and central obesity as WC >94 cm for male and >80 cm for female. Hypertension was defined if there was positive medical history or if the patient was taking anti-hypertensive drugs or if the average blood pressure after three repeated measures was ≥140/90 mmHg. Dyslipidemia was defined as positive medical history, using of lipid-lowering drugs, or if the serum total cholesterol level was ≥5.2 mmol/L, serum triglyceride (TG) level ≥1.7 mmol/L, serum high-density lipoprotein (HDL) cholesterol level <1.0 mmol/L for male or <1.3 mmol/L for female, or serum low-density lipoprotein (LDL) cholesterol level ≥3.4 mmol/L. The MetS was defined according to the International Diabetes Federation criteria that are including central obesity (waist circumference >80 cm for women and >94 cm for men) plus at least two of the following risk abnormalities: blood pressure ≥130/85 mmHg or drug treatment; previously diagnosed type 2 diabetes, or drug treatment or a fasting plasma glucose level ≥5.6 mmol/L; triglyceride levels >1.7 mmol/L and/or HDL-cholesterol <1.03 mmol/L for men and <1.29 mmol/L for women or drug treatment. An extensive laboratory characteristic was analyzed for each patient including and all necessary laboratory tests for exclusion of other CLD. Fasting blood samples for analyzing laboratory parameters were taken in the morning hours after overnight fasting at the day of TE and US examinations. Sleeping duration, coffee consumption and smoking were recorded using quantitate questionnaire. Sleeping duration was categorized in three groups: short (S) (<6h), moderate (M) (6-8h) and long (L) (>8h) sleep duration. Each patient was asked if he had drunk coffee in the previous year, and how many cups per day. Therefore, coffee drinking was categorized into no (0), moderate (1–2) and frequent (≥3) consumption (in cups/day, one cup equals 150 g). Smoking was categorized as yes vs. no. Those patients who reported as smoking at least sporadically during the last two year were defined as smokers. On the other hand, those who reported as having stopped smoking at least two year before their enrolment in our study and those who had not smoked during their life were defined as non-smokers.
Transient elastography and liver ultrasound
LSM and CAP were obtained by using Fibroscan® 502 (Echosens, Paris, France) by a single operator who had performed more than 2.000 examinations before the start of this study. In each patient a minimum of 10 seral measurements were done using the M- or the XL probe according to the instructions. The examination was defined as successful if there was ≥10 valid measurements with interquartile range (IQR)-to-median ratio of LSM was ≤0.3. Liver steatosis was defined by the mean CAP values of ≥238 dB/m (19). According to earlier published data, significant liver fibrosis (≥F2) was defined as an LSM ≥7kPa and advanced fibrosis (≥F3) if LSM was ≥9.6 kPa using the M probe or ≥9.3 kPa using the XL probe. On the other hand, cirrhosis was considered if the LSM was ≥ 11.5 kPa using the M probe or ≥ 11.0 kPa using the XL probe (20,21). Abdominal US was carried out by a certified and experienced gastroenterologist on Philips Affiniti (PC Best, Nederland). Diagnosis of steatosis was determined dichotomously according to the protocol of Hamaguchi et al (22), as presence or absence of a hyper-echogenic liver parenchyma.