Elsevier

Metabolism

Volume 60, Issue 3, March 2011, Pages 404-413
Metabolism

Plasma metabolomic profile in nonalcoholic fatty liver disease

https://doi.org/10.1016/j.metabol.2010.03.006Get rights and content

Abstract

The plasma profile of subjects with nonalcoholic fatty liver disease (NAFLD), steatosis, and steatohepatitis (NASH) was examined using an untargeted global metabolomic analysis to identify specific disease-related patterns and to identify potential noninvasive biomarkers. Plasma samples were obtained after an overnight fast from histologically confirmed nondiabetic subjects with hepatic steatosis (n = 11) or NASH (n = 24) and were compared with healthy, age- and sex-matched controls (n = 25). Subjects with NAFLD were obese, were insulin resistant, and had higher plasma concentrations of homocysteine and total cysteine and lower plasma concentrations of total glutathione. Metabolomic analysis showed markedly higher levels of glycocholate, taurocholate, and glycochenodeoxycholate in subjects with NAFLD. Plasma concentrations of long-chain fatty acids were lower and concentrations of free carnitine, butyrylcarnitine, and methylbutyrylcarnitine were higher in NASH. Several glutamyl dipeptides were higher whereas cysteine-glutathione levels were lower in NASH and steatosis. Other changes included higher branched-chain amino acids, phosphocholine, carbohydrates (glucose, mannose), lactate, pyruvate, and several unknown metabolites. Random forest analysis and recursive partitioning of the metabolomic data could separate healthy subjects from NAFLD with an error rate of approximately 8% and separate NASH from healthy controls with an error rate of 4%. Hepatic steatosis and steatohepatitis could not be separated using the metabolomic profile. Plasma metabolomic analysis revealed marked changes in bile salts and in biochemicals related to glutathione in subjects with NAFLD. Statistical analysis identified a panel of biomarkers that could effectively separate healthy controls from NAFLD and healthy controls from NASH. These biomarkers can potentially be used to follow response to therapeutic interventions.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from a simple accumulation of triglycerides in the hepatocytes (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis), fibrosis, and cirrhosis [1], [2]. Although hepatic steatosis is related to a number of clinical disorders and has been studied in several different animal models, NAFLD in humans is characterized by obesity, insulin resistance, and associated metabolic perturbations [1], [2]. Data from studies in humans and animal models have suggested a paradigm for the pathogenesis of nonalcoholic steatohepatitis (NASH) involving alterations in hepatic lipid metabolism, increased generation of reactive oxygen species and consequently oxidative stress, changes in mitochondrial function, DNA damage, and release of various cytokines resulting in progression of disease [3], [4]. Although such mechanisms have been inferred from data in animals and from indirect evidence in humans, no single biomarker or a cluster of metabolites in the plasma has been related to the progression of hepatic insult. Several diagnostic techniques have been developed to document impaired mitochondrial function, evidence of inflammation, or indices of hepatic fibrosis [3].

Metabolomics, the quantification of small molecules in plasma and tissue fluids, allows for a comprehensive view of the changes in several metabolic and signaling pathways and their interactions [5], [6], [7], [8]. Recent developments in high-throughput analysis, curation, and robust statistical analysis have allowed investigators to understand the changes in the cellular and tissue metabolism based upon the metabolomic data. In the present study, we have examined the plasma metabolome of a biopsy-proven group of nondiabetic subjects with steatosis and steatohepatitis and compared it with a group of healthy controls. Studies were done after a defined period of fasting.

The goals of the present study were (1) to describe the changes in plasma metabolome in subjects with hepatic steatosis and in those with NASH as compared with the controls; (2) to identify biomarkers, if any, associated with steatosis and NASH; and (3) by using appropriate statistical methods, to identify the metabolomic patterns that could discriminate steatosis and NASH from healthy controls.

Section snippets

Materials and methods

Metabolomic analyses were performed on 35 nondiabetic subjects with histologically confirmed hepatic steatosis (n = 11) and steatohepatitis (NASH; n = 24) and on 25 nondiabetic healthy controls. Subjects with NAFLD were recruited from the metabolic clinics of the Cleveland Clinic and MetroHealth Medical Center in Cleveland, OH. Hepatic ultrasonography was performed on all control subjects by the same investigator (SD) to confirm absence of steatosis. Patient demographics and clinical

Results

The demographics and clinical laboratory data are displayed in Table 1. The subjects were matched for age and sex. As anticipated, the subjects with NAFLD were obese and had significantly higher body mass index; plasma alanine aminotransferase, aspartate aminotransferase, and insulin levels; and insulin resistance (HOMA). The plasma levels of triglycerides and low-density lipoprotein cholesterol were higher, whereas the concentration of high-density lipoprotein cholesterol was lower in subjects

Discussion

In the present study, we performed metabolomic analysis of the plasma to obtain a comprehensive view of changes in several metabolic pathways in patients with NAFLD to identify disease-related patterns and to identify biochemical perturbations. The data revealed significant changes in certain key pathways, specifically bile acids, glutathione metabolism, and lipid and amino acid metabolism. The changes in the plasma metabolome were more evident in subjects with NASH than in individuals with

Acknowledgment

Supported by start-up funds from Cleveland Clinic Foundation and by National Institutes of Health grants DK079937 to SCK and CTSA 1UL1 RR024989 to Case Western Reserve University. Metabolomic analysis was done at Metabolon.

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    Conflict of interest: SCK and RWH are members of the Biochemistry Advisory Board of Metabolon Inc and have received honoraria and stock options from Metabolon.

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