Background

Short-chain fatty acids (SCFAs) are important molecules that regulate and control biological functions (Siri-Tarino et al., 2015). SCFAs typically refer to volatile organic fatty acids with less than six carbon atoms (Wong et al., 2006; Marette and Jobin, 2015). They mainly include acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid. SCFAs are mainly produced by the intestinal microbiota, which ferments undigested carbohydrates and dietary fiber in the colon. (Henningsson et al., 2001). Many studies have shown that branched fatty acids are derived from branched-chain amino acids and leucine (Koh et al., 2016). A small amount of branched-chain fatty acids are generated by fermenting undigested proteins (Cummings and Englyst, 1987), such as isobutyric acid and isovaleric acid. Numerous types of SCFAs are present in the colon, and have different functions, because bacteria are complex and fermentation substrates vary in the colon (Topping and Clifton, 2001). These metabolites are formed and absorbed in the colon and then transported through the hepatic vein to the liver. Only approximately 5% of SCFAs are excreted in feces and the remaining are absorbed by colon cells (Ruppin et al., 1980; Hu et al., 2010; Stumpff, 2018). SCFAs play an important role in maintaining the normal function of the large intestine and the morphology and function of colonic epithelial cells. Among other features, SCFAs contributes to the main role of the intestinal flora in the homeostasis of the host. Studies have shown that propionic acid, butyric acid and 3-hydroxybutyric acid, which are signaling molecules, play important roles in activating G protein-coupled receptors and inhibiting histone deacetylase activity (Johnstone, 2002; Xiong et al., 2004). The most abundant molecules in SCFAs are acetic acid, propionic acid, and butyric acid, which are usually present in deprotonated form. SCFAs are quickly absorbed by the hindgut and store energy. Acetic acid provides 10% of the body’s energy expenditure and is an important component of the collective energy source (Hu et al., 2010). The close relationship between SCFAs content in the human body and human health has always been the focus of research (O'Keefe, 2016). Therefore, a method for the rapid qualitative and quantitative analyses of SCFAs in complex biological matrices is needed to be developed (Fiorini et al., 2015).

Many detection techniques can be used for the qualitative and quantitative identification of SCFAs. Those include high-performance liquid chromatography (HPLC) (Torii et al., 2010; de Sá et al., 2011; Schiffels et al., 2011; De Baere et al., 2013), capillary electrophoresis (Saavedra et al., 2000), and ion exchange HILC (Horspool et al., 1991). However, these methods have several limitations in terms of poor sensitivity and low recovery (Saavedra et al., 2000; Destandau et al., 2005; Horspool et al., 1991). Gas chromatography (GC) is the most commonly used method for SCFA analysis because SCFAs are volatile. SCFA biological samples are prepared differently in terms of sample processing such as pretreatment, distillation, ultrafiltration, and extraction (Zhao et al., 2006; Fiorini et al., 2015; Hoving et al., 2018). However, the tedious and time-consuming pretreatment process may be one of the reasons for the decrease in detection sensitivity and recovery of SCFA.

Therefore, GC was chosen to detect SCFA, but a pretreatment method for processing biological samples needs to be developed. Such method must not only be simple and easy to implement, but also time-efficient. In this study, a simple and effective pretreatment method for the rapid qualitative and quantitative determination of SCFAs in mice feces via GC was developed. This approach can provide a basis for the analysis of SCFAs in other biological samples.