Quantification of diacylglycerol and triacylglycerol species in human fecal samples by flow injection Fourier transform mass spectrometry

Verena M Ertl,Louise Kjølbæk,Gerhard Liebisch,Marcus Höring,Hans-Frieder Schött,Ralph Burkhardt,Arne Astrup,Christina Blücher

doi:10.1007/s00216-020-02416-y

Verena M Ertl, Louise Kjølbæk + Show 6 more

Open Access

https://doi.org/10.1007/s00216-020-02416-y

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Journal: Analusis	Publication Date: Mar 21, 2020
Citations: 6	License type: open-access

Affiliation: University Hospital Regensburg, University of Copenhagen

Abstract

The intestinal microbiome plays an important role in human health and disease and fecal materials reflect the microbial activity. Thus, analysis of fecal metabolites provides insight in metabolic interactions between gut microbiota and host organism. In this work, we applied flow injection analysis coupled to Fourier transform mass spectrometry (FIA-FTMS) to identify and quantify lipid species in human fecal samples. Fecal homogenates were subjected to lipid extraction and analyzed by FIA-FTMS. The analysis of different subjects revealed a vast heterogeneity of lipid species abundance. The majority of samples displayed prominent signals of triacylglycerol (TG) and diacylglycerol (DG) species that could be verified by MS2 spectra. Therefore, we focused on the quantification of TG and DG. Method validation included limit of quantification, linearity, evaluation of matrix effects, recovery, and reproducibility. The validation experiments demonstrated the suitability of the method, with exception for approximately 10% of samples, where we observed coefficients of variation higher than 15%. Impaired reproducibility was related to sample inhomogeneity and could not be improved by additional sample preparation steps. Additionally, these experiments demonstrated that compared with aqueous samples, samples containing isopropanol showed higher amounts of DG, presumably due to lysis of bacteria and increased TG lipolysis. These effects were sample-specific and substantiate the high heterogeneity of fecal materials as well as the need for further evaluation of pre-analytic conditions. In summary, FIA-FTMS offers a fast and accurate tool to quantify DG and TG species and is suitable to provide insight into the fecal lipidome and its role in health and disease.

Highlights

It is generally accepted that the gastrointestinal system in particular the intestinal microbiome plays an important role in human health and disease [1]
Crude lipid extracts prepared by chloroform extraction according to the protocol by Bligh and Dyer [23] were analyzed in positive ion mode
None of the analyzed samples contained signals representing a relevant interference with the selected internal standards (IS) DG 40:0, TG 51:0, and TG 57:0 (ESM Fig. S1)

Summary

Introduction

It is generally accepted that the gastrointestinal system in particular the intestinal microbiome plays an important role in human health and disease [1]. Feces are composed of water, proteins, bacterial biomass, fat, and indigestible food components, e.g., fibers. Fat contained in feces is a heterogeneous mixture of different lipids and constitutes 8–16% of the dry weight of feces [3,4,5] and 2–8% of wet weight [6,7,8,9,10]. Fat found within feces comes from bacteria as well as from the undigested remains of dietary lipids [11]. Human feces contain, depending on diet and metabolism, different amounts of triacylglycerol (TG) and diacylglycerol (DG), which has been frequently studied in the context of steatorrhea [13] and colon cancer [14]

Methods

Results

Conclusion