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Title

AN IMPROVED VALIDATED METHOD FOR THE DETERMINATION OF SHORT-CHAIN FATTY ACIDS IN HUMAN FECAL SAMPLES BY GC-FID

Creator

Freeman, Morganne M

Date

2022

Description

Short-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates....
Show moreShort-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota through the fermentation of non-digestible carbohydrates. Recent studies suggest that gut microbiota composition, diet and metabolic status play an important role in the production of SCFAs. Current methods for the analysis of SCFAs are complex and inconsistent between research studies. The primary objective of this study was to develop a simplified method for standardized SCFA analysis in human fecal samples by gas chromatography with flame ionization detection (GC-FID). A secondary objective was to apply the method to fecal samples from a previous randomized, crossover clinical trial comparing participants with pre-diabetes mellitus and insulin resistance (IR-group, n=20) to a metabolically healthy reference group (R-group, n=9) after daily consumption of a red raspberry smoothie (RRB, 1 cup fresh-weight equivalent) with or without fructo-oligosaccharide (RRB + FOS, 1 cup RRB + 8g FOS) over a 4-week intervention period. Extraction parameters, including solvent selection and water content of the sample, were investigated before finalizing the method. Freeze-dried fecal samples (0.5 g) were suspended in 5 mL of milli-Q water, vortexed and centrifuged at 3,214 x g for 10 minutes. The supernatant was transferred to a clean tube, acidified with 5.0 M HCl and centrifuged again at 12,857 x g for 5 minutes. The resulting supernatant was transferred to a GC vial for analysis by GC-FID. Linear regression data for standards at concentrations 5-2000 ppm ranged from 0.99994-0.99998. Limit of detection (LOD) ranged from 0.02-0.23 µg/mL. Limit of quantification (LOQ) ranged from 0.08-0.78 µg/mL. The validated method was then applied to fecal samples collected from a previously conducted study. Nine SCFAs were identified and quantified (acetic, propionic, iso-butyric, butyric, iso-valeric, valeric, 4-methyl valeric, hexanoic and heptanoic acids). Statistical analysis (Student’s t-test, ANCOVA) was performed on PC-SAS 9.4 (SAS Institute). Acetic acid was significantly lower in the IR-group compared to the R-group before starting intervention (baseline, Week 0, IR v R-group, p=0.014). Intervention analysis comparing RRB to RRB + FOS at 4 weeks (WK4) showed a significant difference in 4-methyl valeric acid (p = 0.040) in the R-group. Trends of decreased SCFA content after 4-weeks of RRB and RRB + FOS compared to baseline were observed in both groups, though changes were not significantly different between dietary interventions at 4 weeks (p>0.05). Metabolic status and dietary intervention are discussed in relation to their impact on SCFA content in fecal samples and mechanisms of biological use as a metabolite. Limitations of the study include sample size and using only feces and not other biological samples for SCFAs analysis, which may be considered for future research.
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