Clinical implications of serum Mac-2-binding protein (M2BPGi) as novel biomarkers of advanced hepatic fibrosis in diabetes CURRENT STATUS:

Background Appropriate strategy for screening, identification, and linkage to care of patients with advanced fibrosis in general population is unsolved issue. The aim of this study was to find reference value and clinical role of Mac2 binding protein glycan isomer (M2BPGi) in health check-up setting. Methods Adult subjects (n=1,073) who underwent a health check-up at the healthcare center were finally included for analysis, except for 952 subjects with risk factors for liver disease and insufficient data. Body composition analyzed by using the Bioelectrical Impedance Analysis (BIA). M2BPGi quantification based on a lectin antibody sandwich immunoassay. Fatty liver diagnosed by using abdominal sonography. Results Reference value of M2BPGi was 0.5~1.0 C.O.I in the average risk group. Serum M2BPGi showed a positive correlation with metabolic parameters as well as age. Prevalence of abnormal M2BPGi (> 1.0) was higher in low muscle mass (4.7%, vs 17.4%, p=0.002), metabolic syndrome (14.2% vs. 30.4%, p=0.003) and hypertension (21.8%, vs. 58.7%, p<0.001) compare to healthy control. M2BPGi had a positive correlation with estimated fibrosis formulae such as FIB-4 (R=0.293, p< 0.001) and NAFLD fibrosis score (R=0.248, p<0.001). While the prevalence of advanced fibrosis in total population was just 1.6% (FIB-4 >2.65), the prevalence of advanced fibrosis increased to 50% in high M2BPGi (> 1.0) group with diabetes. It was 31.25 times higher than total population group. Conclusions There was high possibility of advanced hepatic fibrosis in subjects with abnormal M2BPGi levels (> 1.0) in diabetes.

3 Background Non-alcoholic fatty liver (NAFLD) is seemed to be a major cause of chronic liver disease in worldwide. [1,2] It is important to select the advanced liver fibrosis in general population. The liver biopsy is a gold standard for assessing the fibrosis status, but it has several limitations, such as, invasiveness, sampling error and cost. Until now, various noninvasive approaches, which are transient elastography Glycoproteins are known to be synthesized, secreted and metabolized in the liver, and increased in liver disease.[9, 10] Among serum glycoproteins for liver fibrosis, Mac-2 binding protein glycosylation isomer (M2BPGi, Wisteria floribunda agglutinin-positive Mac-2 binding protein) has been recently studied as a useful serum marker for evaluating liver fibrosis in various chronic liver diseases such as chronic hepatitis C virus infection, autoimmune hepatitis, primary biliary cholangitis and NAFLD. [9][10][11] In biopsy proven cohort, M2BPGi discriminate from NASH at cut off 0.95 (AUROC 0.759) and significant fibrosis (>F2) at cut off 1.0 (AUROC 0.758). [11] However, most of the studies so far focused on role of M2BPGi for NASH diagnosis or prediction of advanced fibrosis in patients with high risk group. Now we are called to make a strategy for screening patients with advanced liver fibrosis in the general population. There is few study on clinical characteristics of M2BPGi in average risk group. Moreover, cut off value of M2BPGi to predict advanced fibrosis was quite different according to etiology of liver fibrosis. There is still little research on whether MGBPGi can apply to screen advanced liver fibrosis patients in the general population.
The aim of this study was to find clinical role of M2BPGi in health check-up clinic setting Methods 1.

Study design.
The medical records of Hanyang University Hospital were collected for analysis, prospectively. 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 (m 2 ). 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/height 2 ). 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

Measurement of M2BP
M2BPGi quantification was based on a lectin antibody sandwich immunoassay performed using a fully 6.

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] 7.

Statistical analysis
Participants were divided into 2 groups (Abnormal M2BPGi/Normal groups) by using cut off 1.0 (>2SD

Characteristics of study population
A thousand and seventy-three subjects were finally included for analysis, except for 952 subjects with risk factors for liver disease and insufficient data (Figure 1). The baseline characteristics, demographic data of the 1,073 study subjects according to the M2BPGi level and Pearson correlation between M2BPGi and other variables are presented in Table 1. The mean age of the subjects was 47.11 years, and the proportion of males was 58.3%. The mean value and standard deviation of serum M2BPGi were 0.511 and 0.264, respectively. Upper normal limit (> 2SD) was 1.04 (Figure 2A and B).

Clinical characteristics of M2BPGi
Serum M2BPGi level increased, as the subjects were older (p for trend < 0.001) ( Figure 2C) Table 2).

Correlation with hepatic steatosis and hepatic fibrosis burden
Serum M2BPGi level was higher in NAFLD than control group ( Figure 3A).

Clinical implication of M2BPGi in diabetes subjects
The Pearson coefficients between of M2BPGi and estimated fibrosis formulae (FIB-4 and NFS) higher in diabetes group than general population ( Figure 4A and B). But, Pearson coefficients between To the best of our knowledge, this is the first study to compare the hepatic fibrosis burden according to underlying conditions, such as NAFLD and diabetes and M2BPGi abnormality in average risk group.
Kamada et al also conducted a study in average risk group, but their study was focused only to predict the proportion of suspected advanced hepatic fibrosis patient in healthy cohort by cut-off value, resulted from biopsy proven cohort. However, our study was focused on clinical usage of M2BPGi in average risk group and further analyzed the hepatic fibrosis burden of subjects according to underlying condition and M2BPGi abnormality. As a result, we know patients identified as high M2BPGi group with diabetes in health checkup patients have a relatively high fibrosis burden and we recommend they should be evaluated immediately.
In the European NAFLD guidelines, the screening for insulin resistance, metabolic syndrome or T2DM subjects in the entire subject group. Large-scale studies will be needed for a more accurate analysis.
Third, further study is needed to compare the performance of M2BPGi with performance of FIB-4 and NFS, which are frequently used for the screening of liver fibrosis, or merged method.

Conclusions
In conclusion, M2BPGi could discriminate high-risk patients with advanced fibrosis from the general population by cut off 1.0 (>2SD). Moreover, when abnormal M2BPGi group was accompanied by diabetes, the prevalence of advanced fibrosis increased up to 50%. Therefore, patients identified as   Data are presented as number (percent). Chi-square test or ┼Fisher's Exact test was used for comparing each group. If there cell , which have expected count less than 5, Fisher's exact test was used. Abbreviation : body weight index (BMI); weight or height adjusted skeletal muscle index (w/h SMI); metabolic syndrome (MetS); Impaired fasting glucose (IFG); triglyceride (Tg high density lipoprotein (HDL); hypertension (HTN); diabetes mellitus (DM); non-alcoholic fatty liver disease (NAFLD) Figure 1 Study flow diagram.   were divided at the cut-off value of 1.0. T indicate total subjects. Chi-square test or ┼Fisher's Exact test was used for categorical variables to compare between groups.