Use of Gas Chromatography to Quantify Short Chain Fatty Acids in the Serum, Colonic Luminal Content and Feces of Mice

Background

The microbiota is a complex, heterogeneous and dynamic group of microorganisms (bacteria, viruses and fungi) that colonizes the tissues in direct contact with the external environment including the skin and the genitourinary, intestinal and respiratory tracts. Several studies have demonstrated recently that these microorganisms are important for maintaining host homeostasis. Indeed, changes in their composition can lead to dysbiosis, as observed in specific pathological conditions, and have been associated with their development, as shown for inflammatory bowel disease, asthma, obesity and other chronic inflammatory conditions (Ferreira et al., 2014; Nishida et al., 2018; Sokolowska et al., 2018).

Short chain fatty acids are bacterial metabolites produced by components of the microbiota. The most abundant and studied molecules of this class are acetic, propionic and butyric acids, which are normally found in their deprotonated forms (e.g., acetate, propionate and butyrate). SCFAs modify important processes including metabolism, immune system development and activation, and intestinal barrier function strategies (den Besten et al., 2013; Correa-Fiz et al., 2016; Koh et al., 2016) have been used for changing SCFAs bioavailability in the gut including administration of probiotics (Andrade-Oliveira et al., 2015; Mendes et al., 2017), antibiotics (Fellows et al., 2018), diets with different fiber contents (Vieira et al., 2017), SCFAs in the drinking water or the butyric acid pro-drug (tributyrin) (Vinolo et al., 2012; Vieira et al., 2017). Taken together, these strategies allow us to investigate the role of these metabolites in vivo in different disease models. In this context, it is essential to have a robust method for measuring the levels of these molecules in biological samples such as feces, luminal content or serum.

Gas chromatography (GC) is most commonly used for SCFA analysis due to compatibility with the chemical properties of SCFAs, such as volatility, and the suitability of the detectors that can be coupled to this equipment, such as flame ionization detector (FID). FID is the most widely used for analysis of SCFAs. Due to the large amount of compounds present in the matrix, the sample should be pretreated using extraction and derivatization procedures. Recently, several techniques of derivatization have been described to obtain more stable compounds and provide greater compatibility between the stationary phase and the analytes (Karlsson et al., 2010; Walton et al., 2012; Zhang et al., 2013). On the other hand, the use of derivation techniques has critical disadvantages, including the points that it is time-consuming, there can be losses of SCFAs, it is costly due to the use of large quantities of reagents, and the risk of occupational exposure to allergenic and toxic reagents. Some authors have described filtration and ultracentrifugation techniques to avoid the disadvantages of derivatization and obtain a fast sample preparation (Cuervo et al., 2013; Salazar et al., 2015). In spite of this, the run time is usually increased due to low purification of the samples, which increases the quantities and variety of compounds needed for separation in the column. We have chosen the ultracentrifugation technique in this protocol. Here, we describe a method that we have used to measure SCFAs in biological samples using gas chromatography equipped with FID and a liquid-liquid extraction technique. The column used in this method is composed of a high polarity stationary phase, which makes analysis of the SCFAs possible. The liquid-liquid extraction offers some advantages such as greater sample recuperation, higher sample purification, time optimization and greater separation and peak resolution in chromatograms.