Predictors of Nonalcoholic Steatohepatitis and Significant Fibrosis in Non-obese Nonalcoholic Fatty Liver Disease

Donghee Kim; Won Kim; Sae Kyung Joo; Jung Ho Kim; Stephen A. Harrison; Zobair M. Younossi; Aijaz Ahmed


Liver International. 2019;39(2):332-341. 

In This Article

Materials and Methods

Subjects and Study Design

In this study, we analysed data based on a previously described "Boramae NAFLD cohort" (NCT 02206841).[13,14] Briefly, we prospectively enrolled eligible Asian subjects from January 2013 to February 2018. The eligibility criteria for this cohort were as follows: (a) adults ≥18 years, (b) bright echogenic liver on ultrasound scanning (increased liver/kidney echogenicity and posterior attenuation) and (c) unexplained high alanine aminotransferase (ALT) levels above the reference range within the past 6 months.[15] We used the following exclusion criteria: (a) significant alcohol consumption (>30 g/d for men and >20 g/d for women),[16] (b) hepatitis B or C virus infection, (c) pre-existing chronic liver disease (Wilson's disease, alpha-1-antitrysin deficiency, hemochromatosis, primary sclerosing cholangitis, autoimmune hepatitis, primary biliary cholangitis and drug-induced liver injury) and (d) malignancy diagnosed within the past year. Out of the eligible study participants, those with at least two of the following risk factors underwent liver biopsy:[17] abdominal obesity (waist circumference ≥90 cm for male or ≥80 cm for female), high triglycerides level (≥150 mg/dL), low high-density lipoprotein (HDL) cholesterol level (<40 mg/dL for male or <50 mg/dL for female), presence of insulin resistance and/or diabetes mellitus, hypertension and "clinically suspected NASH or fibrosis" by non-invasive panels and transient elastography. Non-obese and obese controls included subjects with no suspicion of NAFLD who underwent liver biopsy (a) during evaluation for living donor liver transplantation or (b) during characterization of solid liver masses that were suspected to be hepatic adenoma or focal nodular hyperplasia based on abdominal imaging study. We obtained two different liver tissue samples at the time of liver biopsy for a solid liver mass; one sample was obtained from the hepatic lesion and the other from the surrounding normal liver parenchyma. Liver biopsy performed in these controls showed none or <5% macrovesicular steatosis without any evidence of histological features of NASH. This study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki for the participation of human subjects and approved by the Institutional Review Board of Boramae Medical Center (IRB No.16-2014-86). We obtained written informed consent from each subject.

Clinical and Laboratory Evaluations

We used a previously described method for this cohort.[13,14] In brief, each subject underwent an anthropometric assessment as well as a laboratory testing. A well-trained nurse recorded anthropometric measurements according to a standard protocol. The BMI was calculated with the following formula: BMI = weight (kg)/height squared (m2). A BMI <25 kg/m2 was used to define the non-obese population. The subjects with NAFLD and BMI <25 kg/m2 were defined as non-obese NAFLD. Waist circumference was assessed in accordance with a previously published protocol.[18] The methods used for clinical and laboratory evaluations are shown in Appendix S1.

Measurement of Abdominal Adipose Tissue Areas and Abdominal Diameter

The methods used for adipose tissue area measurement have been described in detail elsewhere.[19] Briefly, the subjects were examined for abdominal fat at umbilicus level with a 128-detector computed tomography (CT) scanner (Ingenuity CT; Philips Medical Systems, Cleveland, OH, USA) in the supine position. The area at the umbilicus level was measured with commercially available CT software (Rapidia 2.8; INFINITT, Seoul, Korea), which determined the adipose tissue area by setting the attenuation values for a region of interest within a range of −250 to −50 Hounsfield units.

The technique used for abdominal diameter measurements in umbilical CT images has been previously described.[20] Sagittal abdominal diameter (SAD) was measured electronically with the cursor extending from skin to skin through the centre of the abdomen in the anterior-posterior direction. We defined transverse abdominal diameter as the largest span width from skin to skin through the centre of the abdomen in both lateral directions.

Liver Histology

We used a previously described protocol for the liver biopsy.[13,14] In brief, liver specimens were required to be at least 20 mm in length. An experienced liver pathologist assessed and reviewed all liver biopsies. We defined NAFLD as the presence of ≥5% macrovesicular steatosis and controls as the absence or <5% of macrovesicular steatosis. We diagnosed NASH based on an overall pattern of histological hepatic injury consisting of macrovesicular steatosis, lobular inflammation or hepatocyte balloon degeneration according to Brunt's criteria.[21,22] NASH activity was also assessed using the NAFLD activity score (NAS), which ranges from 0 to 8 points composing of steatosis (on a scale of 0–3), hepatocyte balloon degeneration (on a scale of 0–2) and lobular inflammation (on a scale of 0–3), with higher scores representing increasing severity.[23] Fibrosis was assessed according to a 5-point scale proposed by Brunt and modified by Kleiner et al:[23] F0, absence of fibrosis; F1, perisinusoidal or periportal fibrosis; F2, perisinusoidal and portal/periportal fibrosis; F3, bridging fibrosis; and F4, cirrhosis. Significant fibrosis was defined as F2-F4.

Statistical Analysis

We conducted comprehensive analyses of collected data including demographic, anthropometric, clinical, laboratory and histological parameters. Continuous variables were presented as mean ± standard deviation (SD) and categorical variables as frequencies (%). The statistical significance of differences between groups was evaluated using the independent t test, the Mann-Whitney U test, analysis of variance (ANOVA) or the Kruskal-Wallis test for continuous variables and the chi-square test for categorical variables. To investigate the independent determining factors for the presence of NASH or significant fibrosis, analyses were performed using backward stepwise logistic regression. The candidate set for the multivariable-adjusted models was limited to determinants that were based on biological plausibility—including demographics (age and sex), diabetes, hypertension, components of metabolic syndrome (BMI, triglycerides, HDL cholesterol and insulin resistance [homeostasis model assessment, HOMA-IR]), biochemical liver injury biomarkers (ALT and aspartate aminotransferase [AST]) and VAT and subcutaneous adipose tissue (SAT) areas. The VAT and SAT areas were standardized to a mean of 0 and a SD of 1. The odds ratio (OR) per 1-SD was used to show the relative strength of the relationship between non-obese and obese populations. The diagnostic accuracy between anthropometric measures was assessed by the area under the receiver operating characteristic (AUROC) curve. The AUROC curves were compared according to DeLong et al[24] All analyses were performed using STATA 13.0 (StataCorp, College Station, TX, USA) and MedCalc 18.21 (MedCalc Software, Ostend, Belgium). Two-sided P values were used and were considered statistically significant if P ≤ 0.05.