We prospectively enrolled 60 pediatric patients with NAFLD (age <19 years). The initial screening for the study enrollment included patients who visited the out-patient clinic with the chief complaint of elevated alanine aminotransferase (ALT) levels and the body mass index (BMI) >23.0 (Figure 1). They were also screened with questions, such as previous history of inborn errors of metabolism, other liver diseases and whether or not the patients had been administered drugs metabolized by the liver, and showed no signs of infection when they underwent laboratory testing. The patients who met the criteria underwent laboratory tests to exclude other causes of hepatitis and metabolic disease leading to hepatic steatosis as follows: Hepatitis A, B, and C virus, Epstein–Barr virus, levels of creatinine kinase, ceruloplasmin, fasting glucose, ammonia, and fluorescent antinuclear antibody. They also underwent ultrasonography to check for the presence of focal lesion or anatomical abnormalities in the hepatobiliary system and simultaneously check for the presence of fatty liver. Among patients for whom other causes of hepatitis and fatty liver were excluded, ultrasonography screening was used for confirmation; patients with a chest circumference of 75 cm or more were enrolled in the study as a condition for fibroscan examination. Of the 67 patients who were initially recruited, 7 patients dropped out because their chest circumference was <75 cm. Finally, total of 60 patients were included in the study. These 60 patients underwent additional blood tests including biomarkers after fasting and fibroscan to evaluate the stage of NAFLD (Figure 1).

The purpose of this study was to evaluate the diagnostic value of the two measured biomarkers: IL-6 (inflammatory cytokine) and P1NP (ECM product) to assess each stage of NAFLD. The significant differences were compared in patients with steatosis by classifying them into three groups according to their grade of fatty liver on ultrasonography. To evaluate the significant difference in patients with steatohepatitis (in the process of fibrogenesis), we divided the positive and the negative group with the fibroscan-AST (FAST) score and compared them. Advanced fibrosis was compared by dividing the groups based on the liver stiffness measurement (LSM) value obtained through fibroscan.

The second objective of this study was to find out their cut-off values to find meaningful biomarkers. The third objective of this study was to evaluate whether M2BPGi has the diagnostic capability to assess NAFLD with advanced fibrosis in children. All the methods of this study were performed in accordance with the relevant guidelines and regulations and were approved by the Clinical Research Ethics Committee of Samsung Medical Center. We have undergone appropriate written informed consent procedures for analysis of clinical data (IRB File No: SMC 2021-04-064).

Clinical data, such as age, sex, and weight (kg) were recorded. BMI was recorded as z-score because the patients were children. Venous blood samples were obtained after fasting over 8 h. The biochemical variables were measured using automated analyzer at the laboratory in the hospital.

We recorded the serum levels for the following: total bilirubin, aspartate aminotransferase (AST), ALT, triglyceride, cholesterol, low density lipoprotein, albumin, platelets, fasting plasma glucose, insulin, c-reactive protein, IL- 6, P1NP, osteocalcin, and M2BPGi.

The values of serum total procollagen type 1 amino-terminal propeptide (P1NP), osteocalcin, and IL-6 were measured using chemiluminescence enzyme immunoassay with the products of Roche Elecsys^® (Roche, Basel, Switzerland). P1NP is a representative bone formation marker and is used in the treatment of osteoporosis patients (16). In children and adolescents with rapid bone formation, the standard of normal values is different and higher than that of adults. As the bone formation is rapid, the normal level of P1NP is higher in children and adolescents than in adults. Data regarding osteocalcin, another bone formation marker, were also collected to correct the P1NP values (17). Therefore, in this paper, the value obtained by dividing P1NP by osteocalcin was used as a variable in analysis. In addition, since ALP is a test that can obtain values more easily than osteocalcin, P1NP corrected by ALP was also evaluated. The serum M2BPGi level was measured using chemiluminescence enzyme immunoassay with the HISCL™ M2BPGi™ reagent kit (Sysmex, Kobe, Japan).

The presence of impaired fasting glycemia or diabetes was evaluated by a homeostatic model assessment for insulin resistance, which was calculated as follows: fasting insulin (microU/L) ^* fasting glucose (nmol/L)/22.5. A homeostatic model assessment for insulin resistance value of 3.8 or higher was considered as insulin resistance (18, 19). The FAST score, which is the only non-invasive scoring system to evaluate steatohepatitis status, was calculated as follows: e^{−1.65+1.07 × In(LSM)+2.66*10−8 × CAP3−63.3 × AST−1}/1+e^{−1.65+1.07 × In(LSM)+2.66*}10⁻8 × CAP³−63.3 × AST⁻¹.

The FAST score of 0.35 or higher is considered as steatohepatitis (20). The fibrosis-4 index is a representative scoring system evaluating advanced fibrosis in NAFLD and is calculated as follows: age (years)^*AST (U/L)/platelet count (^*10⁹/L)*ALT (U/L) (21).

The three trained radiology professors evaluated the degree of fatty liver according to the following four grades: (1) grade 0 (absence of steatosis, with normal liver echogenicity); (2) grade 1 (mild steatosis, the liver had a higher echogenicity than the right renal cortex; however, the echogenic wall of the main portal vein was preserved); (3) grade 2 (moderate steatosis, impaired echogenicity of the main portal vein wall); and (4) grade 3 (severe steatosis, impaired visualization of the posterior hepatic parenchyma or the diaphragm) (22, 23). The presence of hepatomegaly and splenomegaly was also evaluated.

Fibroscan (Echosens, Paris, France) was performed by trained nurses. The machine model of fibroscan in the center was 502 TOUCH, with M and XL probes. An automatic probe selection tool was embedded in the software; however, most patients (54/60, 90.0%) were tested using the M probe because most patients were aged under 18 years. The LSM and controlled attenuation parameter (CAP) were evaluated using vibration controlled transient elastography technology. LSM measures the speed of shear wave and expresses the degree of liver fibrosis numerically (unit: kPa). CAP measures the amount of attenuation of ultrasound transmitted into the liver and expresses the degree of fat distribution in the liver numerically (unit: dB/m). Eddewes et al. (24) presented the cut-off values of LSM and CAP in patients with NAFLD through a comparison with biopsy. For LSM, more than 8.2 kPa was evaluated as fibrosis stage 2 or higher (advanced fibrosis), and for CAP, more than 302 kPa was evaluated as steatosis stage 1 or higher (24).

For descriptive statistics, the continuous variables were expressed as mean (standard deviation) and categorical variables were expressed as an absolute number with percentages. In Table 1, one-way analysis of variance test was performed to analyze the mean value comparison between the three groups classified according to the fatty liver grades of ultrasonography. In Table 2, the t-test and the Mann–Whitney test were performed to analyze the mean value comparison between the two groups classified as positive and negative for the FAST score. In Table 3, the t-test and the Mann–Whitney test were performed to analyze the mean value comparison between the two groups classified as positive and negative for LSM (kPa) values. Univariable and multivariable analyses of the associations between steatohepatitis or fibrosis and other factors were performed using a logistic regression model (Table 3). Multivariable analysis was performed by selecting variables with p < 0.1 in univariate analysis. The diagnostic capabilities were evaluated using the receiver operating characteristic (ROC) curve using the items evaluated based on the results of the logistic regression analysis (Figures 2, 3). The diagnostic performances of the biomarkers were expressed as the diagnostic sensitivity, specificity, and area under the ROC (AUROC) curve. Youden's J statistic was computed to identify the cut-off values. In the analysis to evaluate the correlation between LSM and M2BPGi, the Pearson correlation analysis was used (Figure 4).

All of the above statistical analysis were conducted using SPSS version 27 (IBM Corporation, Armonk, NY, USA). A p-value <0.05 was considered as statistically significant.

The mean age of the patients (n = 60) was 13.84 years, and the majority of the patients were male (81.7%) (Table 4). The mean value of BMI evaluated by the Z score was 3.37, indicating obesity (25). Six patients with insulin resistance assessed by a homeostatic model assessment for insulin resistance value, accounted for 10.0% of all patients. Two patients were diagnosed with type 2 diabetes. No patient had bilirubin level higher than normal; additionally, ALT elevation (mean value: 70.78 U/L) was more prominent than AST elevation (mean value: 115.90 U/L). Although the test was performed after fasting, the triglyceride level was 145.07 mg/dL, which was high when compared to the normal values considering the age. The cholesterol and low-density lipoprotein levels were 171.43 and 112.05 mg/dL, respectively.

The degree of fatty liver confirmed by ultrasonography was 31/60 (51.7%) in the moderate stage, accounting for more than half the cases. The quantified steatosis, CAP obtained through fibroscan was confirmed with the mean value of 297.87 dB/m. The FAST score for evaluating steatohepatitis had the mean value of 0.47. LSM, which evaluates fibrosis, showed the mean value of 6.57 kPa.

The clinical characteristics, laboratory results, and biomarker values were compared by dividing the patients into three groups according to their grade (mild, moderate, and severe) of fatty liver on ultrasonography (Table 1). Among 60 patients, 10 patients (16.7%) were identified as having mild fatty liver, 31 patients (51.7%) were identified as having moderate fatty liver, and 19 patients (31.7%) were identified as having severe fatty liver on ultrasonography. No statistically significant differences were observed between the three groups in clinical characteristics, laboratory results, scores, and biomarkers. However, when looking at the mean value of each group, there are items worth interpreting, although there is no statistical significance. In the laboratory results, the mean levels of AST, ALT, triglyceride, and insulin were observed to increase as the fatty liver grade increases. It was expected that the CAP test result would show a statistically significant difference between groups as it had a linear relationship with the degree of fatty liver evaluated by ultrasonography; however, it did not show a significant difference (p-value: 0.225).

The clinical characteristics, laboratory results, and biomarker values were compared by dividing the patients into two groups: FAST-positive group (FAST score of 0.35 or higher) and FAST-negative group (FAST score of <0.35) (Table 2). Among 60 patients, 47 patients (76.7%) were identified as the FAST-positive group and 14 patients (23.3%) were identified as the FAST-negative group. The mean value of the FAST score of FAST-positive group was 0.52 and that of FAST-negative group was 0.27. No significant differences were observed between the two groups in clinical characteristics, such as age, sex, and BMI z score. In the laboratory results, the AST, ALT, and insulin levels showed a significant difference (p-value 0.006, <0.001, and 0.018, respectively) between the two groups, and all three values showed significantly higher values in the FAST-positive group (AST: 80.96 vs. 37.36, ALT: 133.78 vs. 57.14, and insulin: 34.48 vs. 21.03).

There was also a statistically significant difference in the ratio of mild, moderate, and severe fatty liver confirmed by ultrasonography. In the FAST-positive group, the percentage of patients in the severe status was 37% and was significantly higher than 14.3% in the negative group. CAP and LSM showed a significant difference between the two groups with p-values of 0.033 and 0.005, respectively; additionally, the FAST-positive group showed a higher mean value (CAP: 303.78 vs. 278.43, LSM: 6.84 vs. 5.41). Among the biomarkers (IL-6, P1NP, osteocalcin, and M2BPGi), P1NP/osteocalcin ratio and P1NP/ALP ratio (corrected ECM product) showed significant differences with p-values of 0.008 and 0.003, respectively. Pearson's correlation between osteocalcin and ALP showed a significant correlation with a p-value < 0.001.

A logistic regression analysis was performed on the basis of the results of Table 2, the P1NP/osteocalcin ratio and ALT were evaluated as suitable factors in the regression formula with statistical significance (Table 5A). For the P1NP/osteocalcin ratio, the odds ratio was 1.850, with a p-value of 0.018; additionally, for ALT, the odds ratio was 1.078, with a p-value of 0.022. Based on these results, whether the P1NP/osteocalcin ratio had a diagnostic capability in predicting steatohepatitis (early fibrosis) or not was evaluated using the ROC curve (Figure 2). Even the P1NP/osteocalcin ratio alone, with AUROC of 0.782, p-value of 0.002, and cut-off value of 3.54 or higher, had a diagnostic capability to evaluate steatohepatitis (early fibrosis); however, the diagnostic capability for assessing the steatohepatitis was higher when the ratio was multiplied by ALT. The AUROC of P1NP/osteocalcin ratio^*ALT value was 0.939 and the p-value was <0.001. The cut-off value was 305.38. P1NP/ALP ratio evaluation through AUROC curve also showed a diagnostic capability, with AUROC of 0.788, p-value of 0.001, and cut-off value of 1.46 or higher. Similarly, the P1NP/ALP ratio showed better diagnostic capability when the ratio was multiplied by ALT, with AUROC of 0.894, p-value of <0.001, and cut-off value of 119.08 or higher.

The clinical characteristics, laboratory results, and biomarker values were compared by dividing the patients into two groups with LSM-positive group (LSM of 8.2 kPa or higher), and LSM-negative group (LSM of <8.2 kPa) (Table 3). Among 60 patients, 7 patients (11.7%) were identified as the LSM-positive group and 53 patients (88.3%) were identified as the LSM-negative group. The mean value of LSM of the LSM-positive group was 10.30 and that of the LSM-negative group was 6.00. No significant differences were observed between the two groups in clinical characteristics, such as age, sex, and BMI z score. In the laboratory results, the AST and ALT levels showed a significant difference (p-value <0.001 and 0.017, respectively) between the two groups, and the two values showed significantly higher values in the LSM-positive group (AST: 148.29 vs. 60.55 and ALT: 177.71 vs. 107.74).

The FAST score and fibrosis-4 score showed a significant difference between the two groups with both p-values of <0.001; additionally, the LSM-positive group showed higher mean value (FAST score: 0.71 vs. 0.43, fibrosis-4 score: 0.62 vs. 0.27). Among the biomarkers (IL-6, P1NP, osteocalcin, and M2BPGi), only IL-6 and M2BPGi showed significant differences with p-values of 0.005 and <0.001, respectively.

A logistic regression analysis was performed based on the results of Table 3; the IL6 and AST were evaluated as suitable factors in the regression formula with statistical significance (Table 5B). For the IL-6, the odds ratio was 1.844, with a p-value of 0.010, and for ALT, the odds ratio was 1.010, with a p-value of 0.045. Based on these results, whether the IL-6 has a diagnostic capability in predicting advanced fibrosis was evaluated using the ROC curve (Figure 3). The AUROC of IL-6 as 0.719, p-value of 0.037, and cut-off value of 5.25 or higher had a diagnostic capability to evaluate advanced fibrosis; however, the diagnostic capability for assessing advanced fibrosis was higher when the ratio was multiplied by AST. The AUROC of IL-6^* AST value was 0.821 (p = 0.002) and the cut-off value was 228.15.

M2BPGi, which has already proven its diagnostic value for advanced fibrosis or cirrhosis in chronic liver disease, was evaluated to check whether it can be utilized in pediatric NAFLD. LSM (kPa) and M2BPGi showed a significant linear relationship in the Pearson correlation analysis (Pearson correlation coefficient = 0.382 with a p-value of 0.003) (Figure 4A). The diagnostic capability evaluation of advanced fibrosis with AUROC also showed an acceptable result (AUROC = 0.742 with a p-value of 0.022) (Figure 4B).