TY - JOUR
T1 - Profiling human gut bacterial metabolism and its kinetics using [U-C-13]glucose and NMR
AU - de Graaf, Albert A.
AU - Maathuis, Annet J. H.
AU - de Waard, Pieter
AU - Deutz, Nicolaas E. P.
AU - Dijkema, Cor
AU - de Vos, Willem M.
AU - Venema, Koen
PY - 2010/1
Y1 - 2010/1
N2 - This study introduces a stable-isotope metabolic approach employing [U-C-13]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-C-13]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a C-13 recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed C-13 incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the C-12 contents and C-13 labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.
AB - This study introduces a stable-isotope metabolic approach employing [U-C-13]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-C-13]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a C-13 recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed C-13 incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the C-12 contents and C-13 labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.
KW - bacterial metabolism
KW - metabolic flux analysis
KW - metabolomics
KW - NMR
KW - stable isotopes
U2 - 10.1002/nbm.1418
DO - 10.1002/nbm.1418
M3 - Article
SN - 0952-3480
VL - 23
SP - 2
EP - 12
JO - NMR in Biomedicine
JF - NMR in Biomedicine
IS - 1
ER -