ARTICLE pubs.acs.org/jpr
A Comprehensive Untargeted Metabonomic Analysis of Human Steatotic Liver Tissue by RP and HILIC Chromatography Coupled to Mass Spectrometry Reveals Important Metabolic Alterations Juan C. García-Ca~naveras,†,‡ M. Teresa Donato,†,‡,# Jose V. Castell,†,‡,# and Agustín Lahoz*,† †
Unidad de Hepatología Experimental, Instituto de Investigacion Sanitaria Fundacion Hospital La Fe, Valencia, Spain Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Spain # CIBERehd, Centro de Investigaciones Biomedicas en Red de Enfermedades Hepaticas y Digestivas, FIS, Spain ‡
bS Supporting Information ABSTRACT: Steatosis, or excessive accumulation of lipids in the liver, is a generally accepted previous step to the development of more severe conditions like nonalcoholic steatohepatitis, fibrosis, and cirrhosis. We aimed to characterize the metabolic profile that defines simple steatosis in human tissue and to identify potential disturbances in the hepatic metabolism that could favor the switch to progressive liver damage. A total of 46 samples, 23 from steatotic and 23 from nonsteatotic human livers, were analyzed following a holistic LC MS-based metabonomic analysis that combines RP and HILIC chromatographic separations. Multivariate statistical data analysis satisfactorily classified samples and revealed steatosisassociated biomarkers. Increased levels of bile acids and phospholipid degradation products, and decreased levels of antioxidant species, were found in steatotic livers, indicating disturbances in lipid and bile acid homeostasis and mitochondrial dysfunction. Changes in hypoxanthine, creatinine, glutamate, glutamine, or γglutamyl-dipeptides concentrations, suggestive of alterations in energy metabolism and amino acid metabolism and transport, were also found. The results show that the proposed analytical strategy is suitable to achieve a comprehensive metabolic profile of steatotic human liver tissue and provide new insights into the metabolic alterations occurring in fatty liver that could contribute to its predisposition to damage evolution. KEYWORDS: metabonomics, mass spectrometry, steatosis, NAFLD, HILIC, ULPC-MS
’ INTRODUCTION Steatosis, or fatty liver, is an increased deposition of lipids, mainly triglycerides, in hepatocytes above 5% in weight.1 Lipid metabolism is tightly regulated in the liver. However, excessive fat accumulation occurs when fatty acid “input” (uptake from diet or adipose tissue or synthesis) exceeds its “output” (catabolism and VLDL export).2 Increased fatty acid influx from adipose tissue and the enhanced de novo synthesis of lipids in the liver are considered major mechanisms leading to hepatic fat accumulation.3,4 Although simple steatosis is considered a benign condition which does not apparently impair liver function, it is a generally accepted previous step of the development to more severe conditions such as nonalcoholic steatohepatitis (NASH, characterized by inflammation and hepatocellular infiltration and damage), fibrosis, and cirrhosis.1,5 All these severe conditions are different clinic-pathological situations encompassed in the term nonalcoholic fatty acid disease (NAFLD), a worldwide phenomenon currently considered the commonest cause of chronic liver disorder in western populations that affects up to one individual in three.6,7 NAFLD development has been related to multiple r 2011 American Chemical Society
factors (drugs, nutritional factors, viral infections, genetic disorders), and is closely associated with obesity, metabolic syndrome, and diabetes.8,9 In spite of its widespread and increasing prevalence, the pathophysiology of NAFLD remains poorly understood, particularly the events contributing to progressive hepatocellular damage after lipid accumulation. When conducting a holistic study, metabonomics provides global information on small molecules (MW < 1.5 kDa), such as metabolic substrates, products, cofactors, small peptides, or lipids, and is closely related with other “omics” (genomics, transcriptomics, proteomics). Nowadays, metabonomics has emerged as a promising tool for use in different areas related to human health, such as pharmacology, toxicology, oncology, and organ transplantation.10 13 Metabolites are the end product of cellular regulatory and metabolic processes and their levels can reflect tissue physiology as a measure of biochemical status. Therefore, a global comparison of different metabolomic profiles (i.e., control, Received: July 5, 2011 Published: August 11, 2011 4825
dx.doi.org/10.1021/pr200629p | J. Proteome Res. 2011, 10, 4825–4834
Journal of Proteome Research disease) seems relevant to understand the disease mechanism and its manifestations. Mass spectrometry (MS)-based metabonomics has been applied to study different aspects of fatty liver disease and its progression to steatohepatitis or hepatocarcinoma. Such studies have mainly focused on studying NAFLD metabolic alterations in serum14 16 or animal tissues,17,18 but very little attention has been paid to comprehensively study the metabolic alterations caused by progressive lipid accumulation in the human liver and its implication in NAFLD development. LC MS metabonomics studies have been usually performed by reversed-phase high-performance liquid chromatography (RP-HPLC), in which only nonpolar and medium polar analytes can be separated and resolved by the column to be detected properly. Recent advances in column technology, such as hydrophilic interaction chromatography (HILIC), allow the detection of polar/ionic analytes which, typically, are poorly retained by RP phases or indeed are not retained at all.10,19,20 In HILIC chromatography, retention of a metabolite is a combination of liquid liquid partitioning, adsorption, ionic interactions, and hydrophobic retention and is heavily dependent on the nature of the analyte and the composition of the mobile phase. Using HILIC chromatography coupled with MS may also offer higher sensitivity than conventional RP phases because of increased ionization efficiency resulting from the use of mobile phases containing a high proportion of organic solvents. No single separation technique is able to resolve and detect the complete range of metabolites that may be present in a complex biological sample such as liver tissue; therefore, achieving the most comprehensive metabonome coverage may require the use of several chromatographic column chemistries. Here, RP and HILIC ultra performance liquid chromatography (UPLC) were used as complementary metabolite separation techniques, while quadrupole time-of-flight mass spectrometry (Q-ToF) was employed for unbiased analyte detection. The intention of this metabonomic approach, together with careful liver tissue fractionation, was to detect the widest possible chemical coverage of metabolites, which is an especially important fact in terms of an unbiased metabonomic analysis of complex samples (i.e., tissue) when broad chemical diversity is involved. In the present study, we used the proposed analytical strategy to comprehensively analyze the human metabonome of steatotic human liver tissue to gain new insights into the metabolic alterations caused by lipid accumulation.
’ MATERIALS AND METHODS Chemicals
All the solvents were of LC MS grade and were purchased from Fisher Scientific (Loughborough, U.K.). All the additives and standards were purchased form Sigma-Aldrich Quimica SA (Madrid, Spain). The kits used for the determination of total lipids, triglycerides (TG), phospholipids, and cholesterol were purchased from SpinReact (Girona, Spain). Human Liver Samples
A total of 46 liver tissue samples obtained from the Liver Bank at the Hospital La Fe (UHE-LAFE/CIBERehd, Valencia, Spain) were analyzed. The study was approved by the Ethics Committee of the Hospital Universitario La Fe (Valencia, Spain). Liver tissues were classified as steatotic and nonsteatotic according to available histological information, and were confirmed by determining
ARTICLE
their lipid and TG content. Subjects with other forms of liver disease or who evidenced alcohol consumption were excluded by reviewing the subject’s clinical history. Total lipids, TG, phospholipids, and cholesterol content were determined as previously described.21 Briefly, liver samples homogenates were obtained and an aliquot of the homogenates was used for the determination of total protein content using a 96-well adapted Lowry method.22 Another aliquot was extracted with a methanol/ chloroform mixture. The organic phase was separated, aliquoted, and evaporated to dryness. The lipid residue was resuspended and lipidic determinations were carried out following the manufacturer’s instructions. Safety considerations: Work in a fume hood while using chloroform. Sample Preparation for the UPLC-Q-ToF Analysis
Liver tissue extraction was performed following a slightly modified version of a previous procedure.23 Frozen tissue (∼100 mg) was placed in a 2 mL tube containing CK14 ceramic beads (Precellys, France), and 4 mL/g of methanol and 1.25 mL/g of an aqueous solution containing 2.4 μM Leucine Enkephalin and 1.2 μM Erythromycin (internal standards, IS) were added sequentially. Liver tissues were homogenized twice for 25 s at 6000 rpm in a Precellys 24 Dual system (Precellys, France). Tissue homogenates were transferred to a 2 mL clean Eppendorf tube. Then, 2 mL/g of the aqueous solution was used to improve homogenate recovery. Sample homogenates were extracted using 4 mL/g of chloroform stabilized with ethanol, vortexed three times for 15 s for extraction, and mixed for 20 min at 4 °C in a tube rotator. After mixing, samples were left on ice for 15 min and centrifuged for 15 min at 10 000g and 4 °C. The liquid phase, but not the solid interphase, was transferred to a clean Eppendorf tube and centrifuged for 7 min at 10 000g. The aqueous (upper) and organic (lower) phases were carefully withdrawn into separate clean Eppendorf tubes and stored at 80 °C until analyzed. Prior to the analysis, frozen aqueous liver extract samples were allowed to thaw on ice. Safety considerations: Work in a fume hood while using chloroform. UPLC-Q-ToF Mass Spectrometry
A Waters Acquity UPLC chromatograph coupled with a Q-ToF SYNAPT (Waters, U.K.) was used for the acquisition of tissue metabolic profiles. The proposed strategy comprises two complementary chromatographic approaches: RP chromatography for the aqueous and organic extract analyses, using C18 and C8 columns, respectively, and HILIC chromatography (amide column) for the aqueous extract analysis. For the RP metabonomic profiling analysis of the aqueous liver extracts, an 80 μL aliquot was placed in a clean Eppendorf tube and 9.6 μL of an aqueous solution containing 0.83% (v/v) FA and 0.42 μg/mL of reserpine as IS were added. The analysis was performed in an Acquity C18 UPLC HSS T3 (100 2.1 mm, 1.8 μm) column with a VanGuard precolumn (5 2.1 mm, 1.8 μm). A 15 min gradient from 100% A (0.1% v/v FA in water) to 95% B (0.1% v/v FA in methanol) was used. The RP analysis of the organic extracts was performed using an Acquity UPLC BEH C8 (100 2.1 mm, 1.8 μm) column with a VanGuard precolumn (5 2.1 mm, 1.8 μm). An 80 μL aliquot was placed in a clean Eppendorf tube and evaporated to dryness in a SpeedVac. Organic dry residue was dissolved in 240 μL of an acetonitrile/isopropanol/chloroform (49:49:0.2) solution containing 0.1% FA and 1 μg/mL of terfenadine as IS. A 30 min gradient from 100% A (0.1% v/v FA in water) to 100% B (0.1% v/v FA in acetonitrile) was used. 4826
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Journal of Proteome Research
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For the HILIC metabonomic profiling analysis, 100 μL of the aqueous liver extract was placed in a clean Eppendorf tube and evaporated to dryness in a SpeedVac. Subsequently, the dry residue was dissolved in 100 μL of an acetonitrile/water (70:30) Table 1. Study Cohort Demographic and Clinical Laboratory Analysisa nonsteatotic
steatotic
(n = 23)
(n = 23)
FC
p-value
Age, years Male, %
50 ( 20 43
48 ( 15 70
1.0
0.59
BMI, kg/m2
26 ( 5
30 ( 4
1.1
0.045
AST, IU/L
40 ( 40
50 ( 20
1.2
0.064
patient characteristics
ALT, IU/L
40 ( 30
40 ( 30
1.1
0.33
GGT, IU/L
40 ( 40
40 ( 30
1.1
0.93
Lipids, μg/mg prot
210 ( 80
1400 ( 700
6.5
