Justin Gray, Baochuan Guo, Rebecca Bearden
The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acid; μg/g stool) in stool. Therefore, a GC-MS method meeting this criterion was developed. Stool samples were homogenized, diluted with 80:20 water:methanol (v/v) and adjusted to a pH of 1.5 - 2.5. Samples were vortexed, centrifuged and directly injected into the GC-MS using pulsed splitless injection offering two-to-three-fold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses or retention time shifts after 1476 stool injections while other columns failed before 361 injections. A case-control study was conducted using 53 remnant raw stool samples with a positive diagnosis of either ulcerative colitis (UC) or Crohn’s Disease (CD) which comprised the Inflammatory Bowel Disease (IBD) group and were compared to a control group of 21 samples for SCFA and BCFA concentrations. Strong statistical differences were observed between groups whereas the IBD group contained less propionic, butyric and valeric acid (p < 0.05). A receiver operator curve was plotted using the sum of significantly different SCFAs normalized against acetic acid resulting in 96% AUC (95% CI: 0.89 – 0.98) demonstrating potential diagnostical application.
