Global gas chromatography/time-of-flight mass spectrometry (GC/TOFMS)-based metabonomic profiling of lyophilized human feces

Abstract

Gas chromatography mass spectrometry (GC/MS)-based fecal metabonomics represents a powerful systems biology approach for elucidating metabolic biomarkers of lower gastrointestinal tract (GIT) diseases. Unlike metabolic profiling of fecal water, the profiling of complete fecal material remains under-explored. Here, a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) method was developed and validated for the global metabonomic profiling of human feces. Fecal and fecal water metabotypes were also profiled and compared. Additionally, the unclear influence of blood in stool on the fecal metabotype was investigated unprecedentedly. Eighty milligram of lyophilized feces was ultrasonicated with 1mL of methanol:water (8:2) for 30min, followed by centrifugation, drying of supernatant, oximation and trimethylsilylation for 45min. Lyophilized feces demonstrated a more comprehensive metabolic coverage than fecal water, based on the number of chromatographic peaks. Principal component analysis (PCA) indicated occult blood (1mgHb/g feces) exerted a negligible effect on the fecal metabotype. Conversely, a unique metabotype related to feces spiked with gross blood (100mgHb/g feces) was revealed (PCA, R(2)X=0.837, Q(2)=0.794), confirming the potential confounding effect of gross GIT bleeding on the fecal metabotype. This pertinent finding highlights the importance of prudent interpretation of fecal metabonomic data, particularly in GIT diseases where bleeding is prevalent.

Keywords: Colorectal cancer; Feces; GC/MS; GC/TOFMS; GIT; Gas chromatography mass spectrometry; LC/MS; MOX; MSTFA; Metabolic profiling; Metabolomics; Metabonomics; N-methyl-N-(trimethylsilyl) trifluoroacetamide; NIST; National Institute of Standards and Technology; PCA; PLSDA; R; RI; S/N; TMCS; TMS; correlation coefficient; gas chromatography/mass spectrometry; gas chromatography/time-of-flight mass spectrometry; gastrointestinal tract; liquid chromatography/mass spectrometry; methoxyamine; partial least square discriminant analysis; principal component analysis; retention index; signal-to-noise; trimethylchlorosilane; trimethylsilyl.