Human Faecal ¹H NMR Metabolomics: Evaluation of Solvent and Sample Processing on Coverage and Reproducibility of Signature Metabolites

Abstract

The human faecal metabolome is complex, but rich in information and allows investigation of the host metabolism as a function of diet and health. The faecal metabolome is still much less explored than the plasma and urine metabolome, and in order to generate comparable data across laboratories and cohorts, standard operating procedures are required. This study evaluates 10 protocols, using different extraction solvents and sample processing methods for measuring the human faecal metabolome using proton nuclear magnetic resonance (1H NMR) spectroscopy. Three solvents: water, methanol, and dimethyl sulfoxide (DMSO) were investigated at varying concentrations for their ability to extract metabolites directly from faecal slurry or after freeze-drying. The protocols were evaluated on four different pools of human feces. The study also demonstrates a novel signature mapping (SigMa) method for rapid and unbiased processing of complex NMR spectra applied for the first time to human faecal metabolomics. The method is provided with a library containing the chemical shift ranges of 81 common faecal metabolites for future unambiguous and rapid faecal metabolite annotations. The result from the 10 faecal extraction protocols were investigated in terms of reproducibility, coverage, and ability to extract low concentration metabolites. The solvent type was shown to induce the highest variation in the data (45.7%) and the water based extractions allowed detection of the greatest number of metabolites and resulted in the highest reproducibility. Direct extraction of faecal slurry was proved to be more reproducible than freeze-drying. In addition, freeze-drying caused a relative loss of short chain fatty acids (SCFA). DMSO was used for the first time to extract faecal metabolites and enabled the detection of certain bile acids. Some derivatives of SCFA were only detected using methanol as solvent.

Human Fecal Metabolome Reflects Differences in Body Mass Index, Physical Fitness, and Blood Lipoproteins in Healthy Older Adults

“…The human fecal metabolome was investigated by employing two analytical platforms, namely 1 H NMR spectroscopy and GC-MS. Absolute concentrations of 36 human fecal metabolites and the relative concentrations of 76 unknown spin systems were quantified from 1 H NMR spectra, as described previously [16]. The mean absolute concentrations of the 36 human fecal metabolites are reported in Cui et al [26].…”

“…At least one control sample was included in the NMR analysis between 10 real samples. Phosphate buffer was employed as the extraction solvent; details of the fecal sample preparation protocol and phosphate buffer preparation have been described in Cui et al [16].…”

“…The receiver gain was set to 90.5. A more detailed description of NMR analysis has been reported in a previous paper [16].…”

“…This paper reports a fecal metabolomics study performed on healthy older adults using proton ( 1 H) nuclear magnetic resonance (NMR) spectroscopy [16] and untargeted gas chromatography-mass spectrometry (GC-MS) [17]. In addition, lipoprotein profiling of plasma samples was performed using 1 H NMR spectroscopy [18,19].…”

Human blood lipoprotein predictions from1H NMR spectra: protocol, model performances and cage of covariance

“…However, the SR of subfractions belonging to the same main fraction and the same molecular type were similar, though they possessed clear trends of chemical shift changes in cholesterol methyl, LP methyl and methylene signals according to the particle size. complex 1 H NMR metabolomics datasets 24,35 . The software is based on the latest developments in the NMR spectral data processing methods and through the regression models developed in this study, and able to predict LP concentrations from human blood plasma using 1 H NMR NOESY spectra.…”