Serum metabolomics reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

Background & Aims

We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases.

Methods

Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls.

Results

We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis.

Conclusions

γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease.

Introduction

Acute or chronic viral hepatitis affects populations around the world, and the disease often progresses from chronic hepatitis and cirrhosis to hepatocellular carcinoma (HCC) [1]. Accurate diagnosis at earlier stages is necessary for improved therapeutic outcome. However, the diagnostic procedures are laborious and not risk-free. Patients with suspected liver damage are initially subjected to liver function tests that include the assessment of aspartate transaminase (AST), alanine transaminase (ALT), and γ-glutamyl transpeptidase (γ-GTP) serum levels. If these levels are abnormal, patients are then subjected to diagnostic imaging, such as ultrasound and computed tomography (CT), and assays to determine the presence of antibodies against hepatitis virus. Finally, a liver biopsy may be recommended to evaluate the severity of inflammation or fibrosis and to confirm the indications for antiviral therapy.

Recently, nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease in western countries. It encompasses a wide spectrum of conditions associated with over-accumulation of fat in the liver, ranging from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH), and cirrhosis [2]. Although SS typically follows a benign non-progressive clinical course, NASH may eventually develop into cirrhosis and HCC. To date, a liver biopsy remains the gold standard for the diagnosis of NASH [3]. However, since the biopsy procedures carry the risk of mortality [4], [5], the noninvasive identification of biomarkers, that can provide reliable differential diagnoses for the characterization of liver diseases, is desirable.

Metabolomics, which can be defined as measurement of the levels of all cellular metabolites, has emerged as a powerful new tool for discovering new low molecular weight biomarkers. Its utility has been demonstrated by the identification of new biomarkers for prostate cancer [6], Parkinson’s disease [7], type 2 diabetes mellitus [8], acute myocardial ischemia [9], and preeclampsia [10].

Recently, we developed new metabolomic profiling approaches based on capillary electrophoresis mass spectrometry [11] and capillary electrophoresis–time-of-flight mass spectrometry (CE–TOFMS) [12], [13], [14]. The efficacy of CE–TOFMS was demonstrated by the discovery of ophthalmate (γ-glutamyl-2-aminobutyrylglycine) as a biomarker; in mice, reduced glutathione (GSH) depletion produced acetaminophen-induced hepatotoxicity [12], [14]. In this study, to discover new noninvasive biomarkers for human liver diseases, we comprehensively analyzed the serum metabolites in a total of 248 samples from patients with nine types of liver disease or gastric cancer (GC) and from normal individuals using our metabolomic approaches, and found increased levels of γ-glutamyl dipeptides in the majority of the liver diseases. Moreover, we found that γ-glutamyl dipeptides were synthesized via the ligation of glutamate with various amino acids and amines by the γ-glutamylcysteine synthetase (GCS), an enzyme that is feedback-inhibited by GSH, and that the levels of γ-glutamyl dipeptides were indicative of the amount of GSH production. The concentrations of serum γ-glutamyl dipeptides varied with the stage and type of liver disease and can, therefore, act as new biomarkers for liver diseases. Here, we report that a highly specific set of γ-glutamyl dipeptides, alone or in combination with transaminases and methionine sulfoxide, can effectively distinguish specific liver diseases from other hepatic injuries and healthy control samples.