Date/Time Date(s) - 02/12/20211:30 pm - 2:30 pm
Title: The Analysis, Preservation and Quantificationof the Fecal Metabolome with Multi-Segment Injection Capillary ElectrophoresisMass Spectrometry
Date: Thursday, December 2, 2021
Time: 1:30-2:00
Zoom contact chemgrad@mcmaster.ca for link
Host: David Emslie
Abstract: Human stool samples are ofgreat interest to metabolomics analysis and have recently gained greaterpopularity as a specimen of choice for the monitoring and detection of keymarkers of human health. Fecal samples provide researchers a non-invasive processfor examining biomarkers for diseases affecting not only the gastrointestinal(GI) tract, but also the functioning and overall health of a patient.Currently, standardized and mutually supported sample preparation and storageguidelines for fecal samples is lacking. Herein, we introduce a high throughputmethod based on multisegmented injection capillary electrophoresis-massspectrometry (MSI-CE-MS) for the development of an optimized protocol for thesample preparation, extraction, and storage of human fecal sample. As part ofthe protocol optimization, we have looked at the differences in extractionsolvents on metabolite recoveries. Stool samples from a previous study onpediatric IBD patients underwent extraction with both the previously optimizedmodified Bligh Dyer (MeOH:H2O:CHCl3) and a more simple methanol-water(MeOH:H2O) extraction method recommended in literature. Results from this studyshowed increased extraction efficiencies using the methanol-water extraction(mean bias 32.2%) and provided us with a reputable extraction solvent forforthcoming studies. Lyophilization offers a new approach to limit inter-samplevariability by removing the majority (~70%) of the water from the samples,allowing for fecal sample metabolites levels to be used in a clinical setting,due to the ability to normalize the metabolite concentrations to dry weight. Inliterature It has been suggested that due to their highly volatile nature,important metabolites such as the short chain fatty acids (SCFAs) may be lostduring lyophilization. As demonstrated by this study, due to the complex natureof the human fecal sample matrix, the attraction to these SCFAs by the samplematrix has shown to prevent the evaporation of these metabolites duringlyophilization. Aside from allowing for normalization of metabolite levels todried stool weight, lyophilized stool samples also showed elevated metaboliterecoveries in certain metabolites (i.e. Amino acids). This suggests thelyophilization process may cause bacterial cell lysis allowing for analysis ofintracellular metabolites, or simply greater extraction efficiencies due to theremoval of water prior to extraction causing greater solvent penetration of thesample. Lyophilization also has the potential to provide better long-term stabilityof the fecal sample’s metabolite profile, as the removal of water may limit thebioactivity of samples post collection. Effects of delays to storage andfreeze-thaw cycling on the long-term (4 months) stability of the fecal extractsand lyophilized stool metabolite profiles is currently underway. Preliminaryfindings show both preparation methods offer up a robust fecal preparationprotocol. As the study is still underway conclusions and further analysis willbe assessed after completion of the study. Overall, MSI-CE-MS offers apropitious platform for comparing and contrasting two promising fecal samplehandling protocols, which will help build up the repository of information andassist in the development of a more standardized set of fecal preparationguidelines.