- Study design.
The medical records of Hanyang University Hospital were collected for analysis, prospectively. Koreans are required to get health checkups every year or every two years for all adults, aged over 20 years, under the Basic Health Checkup Act. The costs associated with the checkups are covered by the state. The Institutional Review Board (IRB) of Hanyang University Medical Center approved this study protocol (IRB No No. 2018-12-020). The protocol was also registered at the Clinical Research Information Service. (https://cris.nih.go.kr/cris, Registration No. KCT0004462).
- Inclusion and exclusion criteria.
In this study, a total of 2,025 of the adult subjects, who underwent a health check-up at International Healthcare Service at Hanyang University Medical Center between March 2019 and June 2019 were initially recruited into the study. We collected the data, excluding foreigners (n = 339), subjects with insufficient remnant blood sample (n=53), subjects with missing data for baseline physical measurement including skeletal muscle mass (n = 20), abdominal SONO (n=48), for alcohol consumption (n = 28) or serum M2BPGi level (n=270). And then we further excluded the high risk liver disease population, who had the positive serologic markers of hepatitis B virus or hepatitis C or who was self-reported HBV carrier (n = 65) or subjects whose weekly alcohol consumption was greater than 210 g for men or greater than 140 g for women (n = 160). Finally, 1,073 subjects were included in this study (Figure. 1).
- Clinical variables and laboratory evaluations.
Personal medical and medication history, smoking history, exercise and alcohol consumption were collected through self-reported survey. Body weight and height were measured and body mass index (BMI) was calculated weight in kilograms (kg) divided by the height in meter squared (m2). Waist circumference (WC) was measured at the narrowest point between the iliac crest and the lower rib margin. Blood pressure was measured at rest in a sitting position. Body composition was analyzed by using Bioelectrical Impedance Analysis (BIA; InBody 720 body composition analysis). The skeletal muscle index (SMI) was calculated by dividing the total appendicular skeletal muscle (ASM), which is the sum of skeletal muscle in the bilateral upper and lower four limbs (kg), by the square of height (= total ASM/height2). The cutoff values for low muscle mass were defined by the hSMI (<6.58 kg/m2 for men and <4.56 kg/m2 for women).[12] In addition, the cutoff values for low muscle mass were defined by the wSMI (<29.1% for men and <23.0% for women).[12]
- Estimated hepatic fat and fibrosis formulae.
Fatty Liver Index (FLI) was calculated descriptive statistic analysis by using this equation (ey / (1 + ey) × 100 , where y = 0.953 × ln(Tg [mg/dL]) + 0.139 × BMI [kg/m2] + 0.718 × ln (GGT [U/L]) + 0.053 × WC [cm] – 15.745.[13] And the fibrosis-4 (FIB-4) index was calculated by using this equation. (age ⅹ AST [U/L] / platelet count [ⅹ 109/L] / ALT [U/L]).[6] FIB-4 grades are divided into 0, 1, 2 by using cut off value at 1.3 and 2.67. The NAFLD fibrosis score (NFS) was calculated by using this equation (–1.675 + 0.037 ⅹ 3 age [years] + 0.094 ⅹ BMI [kg/m2] + 1.13 ⅹ impaired fasting glycemia/diabetes mellitus [yes = 1, no = 0] + 0.99 ⅹ AST/ALT – 0.013 ⅹ platelet count (ⅹ 109/L) – 0.66 ⅹ Alb [g/dL]).[7] NFS grades are divided into 0, 1, 2 by using cut off value at -1.455 and 0.675. Grade 0,1 and 2 of FIB-4 index and NFS mean advanced fibrosis excluded, needing further examination and advanced fibrosis, respectively. Impaired fasting glucose (IFG) was defined as FBS of 110–125 mg/dL. The presence of diabetes mellitus (DM) was defined as FBS 126 mg/dL, HbA1c ≥ 6.5 %, or treatment with anti-diabetic drugs.
- Measurement of M2BP
M2BPGi quantification was based on a lectin antibody sandwich immunoassay performed using a fully automatic immune-analyzer (HISCL-2000i; Sysmex Co., Hyogo, Japan). The measured values of WFA1-M2BP conjugated to WFA were indexed with the obtained values using the following equation: Cutoff index(COI) = ([WFA+-M2BP]sample-[WFA+-M2BP]NC/([WFA+-M2BP]PC-[WFA+-M2BP]NC) where [WFA+-M2BP]sample was the WFA+-M2BP count for the serum sample, PC was the positive control, and NC was the negative control. The PC was supplied as a calibration solution preliminarily standardized to yield a COI value of 1.0.
- The definition of Metabolic syndrome and NAFLD
National Cholesterol Education Program’s Adult Treatment Panel III (NCEP-ATP III) criteria were followed, with the exception of abdominal obesity based on waist circumference for the diagnosis of metabolic syndrome.[14] The waist circumference used was ≥85 for women and ≥90 for men, which are the standard measurement for Korean.[15] Metabolic syndrome was diagnosed when at least three of the following five items were satisfied: (1) waist circumference: ≥85 for women and ≥90 for men (2) triglyceride: ≥150 mg/dL (3) high-density lipoprotein cholesterol: ≤50 mg/dL for women and ≤40 mg/dL for men (4) blood pressure: ≥130/85 mm Hg or taking a hypotensive agent and (5) fasting glucose: ≥100 mg/dL or taking a antidiabetic agents.
NAFLD was defined as people who have a liver fat, whose volume is greater than 5 % of total liver volume, without excessive alcohol consumption, viral or genetic liver disease. The degree of fatty liver was graded as normal, mild, moderate or severe fatty liver on a basis of sonographic hepatorenal index method.[16]
- Statistical analysis
Participants were divided into 2 groups (Abnormal M2BPGi/Normal groups) by using cut off 1.0 (>2SD). Continuous and categorical variables are presented as mean (SD) and number (percent), respectively. For comparing the mean values between the groups, T- test or ANOVA and Mann-Whitney U test or Kruskale Wallis test were used for continuous variables with and without a normal distribution respectively. Categorical variables were compared using chi-square tests. Fisher's Exact test was used for categorical variables, when 20% or more of the cells have an expected frequency of less than 5. Especially, odds ratios (OR) with 95% confidence intervals (CI) for risk factor analysis of high M2BPGi were calculated by using chi-square tests or Fisher's Exact test. For all analyses, p-values less than 0.05were considered statistically significant. Statistical analyses were performed using SPSS version 26 for Windows (SPSS Inc., Chicago, IL).