A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

Abstract

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography mass spectrometry (GC-MS) analysis. A one-step derivatization using 100 µL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations (RSDs) of all targeted compounds showed good intra- and inter-day (within 7 days) precision (< 10%), and good stability (< 20%) within 4 days at room temperature (23–25 °C), or 7 days when stored at −20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistics analysis of the concentrations of these target metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.

Effect of lactulose intervention on gut microbiota and short chain fatty acid composition of C57BL/6J mice

“…In addition, straightchain SCFAs are able to improve immune function, glucose regulation, and prevent obesity (Polyviou et al, 2016). In contrast, BCFAs are always derived from catabolism of branched-chain amino acids (Zheng et al, 2013), and are major markers of protein fermentation, which is likely to be detrimental to the host (Yang & Rose, 2015).…”

“…In addition, straightchain SCFAs are able to improve immune function, glucose regulation, and prevent obesity (Polyviou et al, 2016). In contrast, BCFAs are always derived from catabolism of branched-chain amino acids (Zheng et al, 2013), and are major markers of protein fermentation, which is likely to be detrimental to the host (Yang & Rose, 2015).Although some studies have assessed the effects of lactulose on gut microbiota, the gel-or PCR-based methods used limit our ability to evaluate the full extent of the impact of lactulose on the gut microbiotic community. In this study, 16S rRNA high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS) were used to evaluate effect of lactulose on gut microbiota and their metabolites in mice.…”

Quantification of Plasma or Serum Short-Chain Fatty Acids: Choosing the Correct Blood Tube

“…In particular for acetate and propionate, the extraction efficiency was low, which is evidenced from the relatively low octanol/water partitioning coefficients (log P acetate = 0.2; log P propionate = 0.3 and log P butyrate = 0.8), and therefore not sufficiently reproducible. Prior derivatization of the SCFA with reagents such as propyl chloroformate or 1-(tert-butyldimethylsilyl)imidazole increases the extraction efficiency but also complicates the sample preparation protocol and increases the risk for contamination [15,21]. In a recently published paper, the need for extraction of underivatized SCFA was circumvented by direct injection of an acidified water concentrate on a GC-FID system.…”

Crystal Structure and Substrate Specificity of Human Thioesterase 2

“…Triplicate reactions were quenched at different time points by the addition of 50% fresh KOH (60 l of sample: 300 l of KOH) and stored at Ϫ80°C. The amount of C8, C10, C12, C14, and C16 free fatty acids was determined by GC-MS using a modified protocol by Zheng et al (30). The protocol was modified to use C9 and C15 fatty acids (Nuchek Prep, Inc., Elysian, MN) as internal standards, which were doped into each sample before derivatization with propyl chloroformate, such that the final concentration was 800 pg/l at injection.…”