Fecal 1H-NMR Metabolomics: A Comparison of Sample Preparation Methods for NMR and Novel in Silico Baseline Correction.

Catherine L J Brown,Michael P Opyr,Amy S Freund,Gerlinde A S Metz,Tony Montina,Hannah Scott,Crystal Mulik,G Douglas Inglis

doi:10.3390/metabo12020148

Catherine L J Brown, Michael P Opyr + Show 6 more

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https://doi.org/10.3390/metabo12020148

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Abstract

Analysis of enteric microbiota function indirectly through the fecal metabolome has the potential to be an informative diagnostic tool. However, metabolomic analysis of feces is hampered by high concentrations of macromolecules such as proteins, fats, and fiber in samples. Three methods—ultrafiltration (UF), Bligh–Dyer (BD), and no extraction (samples added directly to buffer, vortexed, and centrifuged)—were tested on multiple rat (n = 10) and chicken (n = 8) fecal samples to ascertain whether the methods worked equally well across species and individuals. An in silico baseline correction method was evaluated to determine if an algorithm could produce spectra similar to those obtained via UF. For both rat and chicken feces, UF removed all macromolecules and produced no baseline distortion among samples. By contrast, the BD and no extraction methods did not remove all the macromolecules and produced baseline distortions. The application of in silico baseline correction produced spectra comparable to UF spectra. In the case of no extraction, more intense peaks were produced. This suggests that baseline correction may be a cost-effective method for metabolomic analyses of fecal samples and an alternative to UF. UF was the most versatile and efficient extraction method; however, BD and no extraction followed by baseline correction can produce comparable results.

Highlights

The enteric microbiota plays a critical role in the health of mammals and avians, and the structure and function of the microbiota can be an indicator of the health status of the host
There are many different methods proposed for the preparation of fecal samples for 1 H-NMR; there is no universal consensus on the most cost-effective and reproducible method for water-soluble metabolite extraction
The aims of the current study were to: (i) examine the variance observed in the fecal metabolomic data obtained from NMR following the application of the most common small molecule water-soluble metabolite extraction methods; (ii) assess the utility of applying in silico baseline correction as a means to deal with incomplete removal of macromolecules from fecal samples; and (iii) provide a recommendation for the best extraction method, in terms of efficiency, reproducibility, and cost, to be utilized for NMR-based metabolomic studies of feces

Summary

Introduction

The enteric microbiota plays a critical role in the health of mammals and avians, and the structure and function of the microbiota can be an indicator of the health status of the host. Alterations to the normal structure of the microbiota (i.e., dysbiosis) are associated with a number of adverse acute and chronic health conditions such as pseudomembranous colitis [3,4], inflammatory bowel disease (IBD) [5], and irritable bowel syndrome (IBS) [6]. Autoimmune disorders such as multiple sclerosis (MS) [7], mood disorders such as major depressive disorder (MDD) [8], autism spectrum disorder (ASD) [9], and neurodegenerative diseases such as human motor neuron disease (MND) [10] have been linked to dysbioses in the enteric microbiota. This technique does not directly measure bacterial functions such as metabolic processes [12]

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