Background: Fecal water provides information on the state of the gut microbiota and host
physiology and is a common biofluid to study, particularly for metabonomic purposes. Despite the interest
in fecal water, a method of preparation has yet to be standardized. Current methods of preparing
fecal water usually involve an extraction protocol combined with ultracentrifugation and filtration
steps. If metabonomic analysis is the final means of examining fecal water samples, consideration
should be given to the potential impacts of these preparation steps on the metabolites observed.
Objective: This work examines metabonomic effects of ultracentrifugation during fecal water preparation,
and provides an easily adaptable method based on considerations to metabonomic impacts of the
Methods: Both fecal water (prepared from human stool samples) as well as a controlled surrogate for
fecal water samples (prepared from in vitro fecal microbial communities) were used to perform systematic
evaluations of various preparation steps, and used metabonomic profiles to assess the protocols’
impact on the sample.
Results: 52 metabolites were observed and found that ultracentrifugation speed decreased metabolite
concentrations by approximately -2% per 10000 rpm increase, with one exception. P-cresol increased
by approximately +50% per 10000 rpm. Upon investigation of the potential sources of p-cresol, we
found evidence that these were tyrosine-rich cell proteins which broke down upon ultracentrifugation.
Conclusion: Based on these findings, we suggest that ultracentrifugation is an effective means of preparing
fecal water samples, with negligible impact on metabonomic analyses, though measurement of
p-cresol concentrations should be treated with discretion.