Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

Abstract

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver–operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.

Introduction

Hepatocellular carcinoma (HCC) is the sixth most prevalent malignant tumour worldwide and ranks the third as a cancer killer, causing more than half a million deaths annually in the world [1], [2]. The prognosis of HCC largely depends on the stage of the tumor. Early diagnosis can be obtained with surveillance of patients at risk using diagnostic modalities such as ultrasound (US) [3]. The commonly used serology tests for screening such as alphafetoprotein (AFP) are no longer considered as powerful screening tools in patients with chronic liver disease due to high false positive and false negative rates [4], [5]. The ideal biomarker should be the one that can be detected with good sensitivity and specificity in biological samples from the patient in a minimally invasive manner (e.g., blood, saliva, or urine). Recently, metabolomic profiling approaches have been increasingly used to elucidate significant changes in tumor metabolism and to explore candidate “biomarkers” from such variance within a huge number of endogenous metabolites.

Blood and urine are the most frequently used samples for exploring the systematic alteration in human metabolome. Compared with blood sample, utilization of urine samples is preferred as it enables non-invasive monitoring of metabolomic changes.

Metabolomics, defined as the quantitative measurement of all low-molecular-weight metabolites in an organism at a specified time under specific environmental conditions [6], has been shown to be an effective tool for disease diagnosis [7], [8], biomarker screening [9], [10], and characterization of biological pathways [11]. Metabolomic studies generally employ such techniques as nuclear magnetic resonance (NMR), high-performance liquid chromatography/mass spectrometry (HPLC/MS, and LC/MS/MS), Fourier transform infrared (FT/IR) spectroscopy, and gas chromatography/mass spectrometry (GC/MS) [12]. Among them, GC/MS has been described as a sensitive and reproducible method, which have been proposed as an ideal tool for metabolomic profiling of urine samples[13]. Metabolic profiling of urine has been successfully performed by GC/MS using bis-(trimethylsilyl)–trifluoroacetamide (BSTFA) as the derivatization agent [13], [14].

The aims of this study were to compare metabolite profiling from urine samples between HCC patients and healthy subjects using GC/MS and chemical derivatization and to establish a diagnotic model from these metabolic biomarkers to distinguish HCC from the normal subjects, using principal components analysis (PCA).