An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-flight mass spectrometry

Abstract

Abstract:In this paper, we present a tissue metabonomic method with an optimized extraction procedure followed by instrumental analysis with gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) and spectral data analysis with multivariate statistics. Metabolite extractions were carried out using three solvents: chloroform, methanol, and water, with design of experiment (DOE) theory and multivariate statistical analysis. A two-step metabolite extraction procedure was optimized using a mixed solvent of chloroform-methanol-water (1:2:1, v/v/v) and then followed by methanol alone. This approach was subsequently validated using standard compounds and liver tissues. Calibration curves were obtained in the range of 0.50-125.0 μg/mL for standards and 0.02-0.25 g/mL acceptable for liver tissue samples. For most of the metabolites investigated, relative standard deviations (RSD) were below 10% within a day (reproducibility) and below 15% within a week (stability). Rat liver tissues of carbon tetrachloride-induced acute liver injury models (n = 10) and healthy control rats (n = 10) were analyzed which demonstrated the applicability of the developed procedure for the tissue metabonomic study.

Metabolic profiling reveals the protective effect of diammonium glycyrrhizinate on acute hepatic injury induced by carbon tetrachloride

“…Each 100 mg liver tissue was extracted following the two-step extraction procedure described in our previous report (Pan et al 2010). Briefly, for each 100-mg liver tissue sample, 500 μl of each of the two solvents (the mixture of chloroform, methanol and water (1:2:1, v/v/v) and methanol alone) were used as the two extraction solvents.…”

“…Briefly, unprocessed GC/MS files were converted into NetCDF format via DataBridge (Perkin-Elmer Inc., U.S.A.) and directly processed by our custom scripts in MATLAB (The MathWorks, Inc., U.S.A.). This process performed data smoothing, filtering, de-noising, baseline correction, peak discrimination and alignment (for identification and extraction of the peaks of the chromatogram indicating the existence and intensities of potential metabolites), internal standard exclusion, and normalization to the total sum of the chromatogram (Bao et al 2009;Wang et al 2009;Pan et al 2010). The resulting threedimensional (retention time, M/Z and Intensity of peaks) matrix was introduced into the SIMCA-P 12.0 Software package (Umetrics, Umeå, Sweden) for multivariate statistical analysis.…”

“…Because the CCl 4 induced hepatic injury model can clearly reflect the function, metabolism and morphological variations of hepatic cells with high reproducibility, the model is commonly used to simulate acute/chronic hepatitis (Okamoto and Okabe 2000;Cherkashina and Petrenko 2006) and hepatic fibrosis (Abdel-Salam et al 2007). The metabolic impact of CCl 4 has been evaluated by profiling differential expressed metabolites of plasma, tissue and urine during poisoning process (Robertson et al 2000;Pan et al 2010;Lin et al 2009). In this study, we set up an acute hepatic injury animal model with CCl 4 to test the biochemical and metabolic mechanism of the anti-hepatotoxicity of DG.…”

“…Metabolic profiling of biological samples using GC/MS has been extensively used to evaluate the toxic/disease status and test the efficacy of drug treatment (Pan et al 2010;Qiu et al 2007;Wang et al 2007). This technology provides quantitative information on metabolite levels increase or decrease in response to xenobiotic interventions, especially in hepatotoxicity research (Chen et al 2009;Beger et al 2010;Sun et al 2009).…”

Method for liver tissue metabolic profiling study and its application in type 2 diabetic rats based on ultra performance liquid chromatography–mass spectrometry

“…Besides, various liver metabolic profiling determinations were carried out by gas chromatography/mass spectrometry (GC/MS) in current research. The disadvantages are the complexity of solid-phase extraction (SPE) pretreatment and derivatization [9,[15][16][17][18]. SPE are not only used in GC/MS research of liver tissue, but also in liquid chromatogram/mass spectrometry (LC/MS) study.…”