Holistic Lipidomics of the Human Gut Phenotype Using Validated Ultra-High-Performance Liquid Chromatography Coupled to Hybrid Orbitrap Mass Spectrometry

Abstract

As lipids are assigned a plethora of biological functions, it is evident that dysregulated lipid metabolism signifies a key element in many pathological conditions. With this rationale, this study presents a validated lipidomics platform to map the fecal lipidome, which integrates unique information about host-gut microbiome interactions, gastrointestinal functionality, and dietary patterns. This particular method accomplished coverage across all eight lipid categories: fatty acyls, glycerolipids, phosphoglycerolipids, polyketides, prenols, saccharolipids, sphingolipids, and sterols. Generic extraction of freeze-dried feces was achieved by solid-liquid extraction using methanol and methyl tert-butyl ether. Extracted components were separated by liquid chromatography, whereby the selected ethylene-bridged hybrid phenyl ultra-high-performance liquid chromatography stationary phase allowed fast separation of both individual lipid species and categories. Detection was achieved by high-resolution full-scan Q-Exactive Orbitrap mass spectrometry and covered a broad m/z scan range (67-2300 Da). Method validation was performed in a targeted fashion to evaluate the analytical performance across all lipid categories, revealing excellent linearity (R ≥ 0.9921), acceptable repeatability (coefficients of variance ≤15.6%), and stable recovery (coefficients of variance ≤11.9%). Method suitability for untargeted fingerprinting was verified, demonstrating adequate linearity (R ≥ 0.90) for 75.3% and acceptable repeatability (coefficients of variance ≤30%) for 84.5% of about 9000 endogenous fecal compounds. Eventually, the potential of fecal lipidomics was exemplified within a clinical context of type 2 diabetes, thereby revealing significant perturbations [orthogonal partial least-squares discriminant analysis Q(Y) of 0.728] in the fecal lipidome between participants with normal blood glucose levels (n = 26) and those with type 2 diabetes (n = 17).

Nutrimetabolomics: An Integrative Action for Metabolomic Analyses in Human Nutritional Studies

“…Similarly, Vanden Bussche et al . and Van Meulebroek et al . proposed protocols for untargeted polar metabolomic and lipidomic analysis of feces, where fecal samples were frozen at −80 °C (for 1 or 2 days) and lyophilized, prior to aliquoting and final storage at −80 °C.…”

“…However, freeze drying may also potentially decrease the content of volatile compounds such as SCFAs, which may be of interest as potential biomarkers . In this context, proper execution of the freeze‐drying process and the absence of any defrosting is crucial as freeze drying was shown to deliver the richest fecal metabolomes (over 9000 compounds) in recent untargeted studies …”

“…From the powder a collection of subsamples were prepared and stored at −80°C. [100] Similarly, Vanden Bussche et al [63] and Van Meulebroek et al [111] proposed protocols for untargeted polar metabolomic and lipidomic analysis of feces, where fecal samples were frozen at −80°C (for 1 or 2 days) and lyophilized, prior to aliquoting and final storage at −80°C. Freeze drying in sample preparation may remove the water from the sample, thus minimizing the effect of confounding factors for NMR spectroscopy.…”

“…[109] In this context, proper execution of the freezedrying process and the absence of any defrosting is crucial as freeze drying was shown to deliver the richest fecal metabolomes (over 9000 compounds) in recent untargeted studies. [63,111]…”

Describing the fecal metabolome in cryogenically collected samples from healthy participants

“…The characterization of lipids is an emerging field that is often studied for metabolic disorders as lipids can make up 15% of the feces composition. 15,16,17 Several research groups have developed state-of-the-art fecal fingerprinting methods, including chemical labeling with isotopic reagents and high-performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HRESMS) 18,19 20 , nuclear magnetic resonance (NMR) for compounds like SCFAs and BCAAs [21][22][23] and the use of LC-MS 24,25 and GC-MS 26,27 . In this study, we aim to measure molecules in feces with in-house analytical pipelines using cryogenically drilled samples 28,29,30 , focusing on quality control, sampling procedure and biochemical pathways that can describe feces from healthy volunteers.…”