title: Assessment of the human faecal microbiota: I. Measurement and reproducibility of selected enzymatic activities.
abstract: BACKGROUND
  The intestinal microbial community has major effects on human health, but optimal research methods are unsettled. To facilitate epidemiologic and clinical research, we sought to optimize conditions and to assess reproducibility of selected core functions of the distal gut microbiota, β-glucuronidase and β-glucosidase bioactivities.
  
  
  RESULTS
  A colorimetric kinetic method was optimized and used to quantify activities of β-glucuronidase and β-glucosidase in human faeces. Enzyme detection was optimal with neutral pH, snap freezing in liquid nitrogen and rapid thawing to 37 °C before protein extraction. Enzymatic stability was assessed by delayed freezing for 2-48 h to mimic field settings. Activities decayed approximately 20% within 2 h and 40% within 4 h at room temperature. To formally assess reproducibility, 51 volunteers (25 men; mean age 39) used two devices to self-collect and rapidly chill four replicates of a stool. Devices were compared for mean enzymatic activities and intraclass correlation coefficients (ICC) in paired replicates of the self-collected specimens. Reproducibility was excellent with both devices for β-glucuronidase (ICC 0·92). The larger collection device had significantly higher reproducibility for β-glucosidase (ICC 0·92 vs. 0·76, P < 0·0001) and higher mean activities for both enzymes (P < 0·0001).
  
  
  CONCLUSIONS
  Optimal measurement of these core activities of the microbiota required a sufficient quantity of rapidly chilled or frozen specimens collected in phosphate buffered saline at pH7·0. Application of these methods to clinical and epidemiologic research could provide insights on how the intestinal microbiota affects human health.
authors.affiliation: Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD 20892, USA. floresr2@mail.nih.gov
authors.forename: Roberto
authors.initials: R
authors.lastname: Flores
authors.forename: Jianxin
authors.initials: J
authors.lastname: Shi
authors.forename: Mitchell H
authors.initials: MH
authors.lastname: Gail
authors.forename: Jacques
authors.initials: J
authors.lastname: Ravel
authors.forename: James J
authors.initials: JJ
authors.lastname: Goedert
mesh_terms: D000328:Adult; D004795:Enzyme Stability; D005243:Feces / Q000201:enzymology / Q000382:microbiology*; D005260:Female; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D054892:Metagenome; D015203:Reproducibility of Results; D013048:Specimen Handling; D013223:Statistics as Topic; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase; D005966:Glucuronidase