Methods to assess effects of prebiotics in humans Lieselotte Cloetens 1,2, Willem F. Broekaert 3,4, Björn Åkesson 1, Gunilla Önning 1, Jan A. Delcour 3, Kristin Verbeke 2 1 Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, Lund, Sweden 2 Laboratory of Digestion and Absorption, TARGID, Catholic University of Leuven, Leuven, Belgium 3 Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Center (LFoRCe), Catholic University of Leuven, Leuven, Belgium 4 Fugeia NV, Leuven, Belgium
Prebiotics non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improve host health (Gibson et al., Nutr Res Rev 2004;17:259-275) Health-promoting bacteria: bifidobacteria and lactobacilli Health-related effects: effects on gastrointestinal transit improvement of mineral absorption lipid-lowering effects reduced risk of colon cancer reduced protein fermentation
Prebiotics Fructo-oligosaccharides (FOS) and inulin: well-known prebiotics Arabinoxylan-oligosaccharides (AXOS): prebiotic potential? = degradation products of arabinoxylan flour bran germ outer layer of a cereal: 20-25% arabinoxylans
Methods Gastrointestinal parameters o Motility and digestion Prebiotic effects o Microbial composition o Metabolic activity of the microbiota
Motility and digestion dietary intake and digestive processes substrate availability microbial composition colonic transit time colonic fermentation metabolites Cloetens et al., e-spen 2008
Digestive processes In the proximal GI-tract: o gastric emptying rate (GE) o lipid digestion o protein digestion o oro-caecal transit time (OCTT) o (total gastrointestinal transit) o prebiotics can alter digestion and absorption of other nutrients: physicochemical properties (viscosity, water-holding capacity and osmolarity)
Breath tests
GI parameters parameter labelled substrate dose sample collection gastric emptying 1 oro-caecal transit time 2,3 protein digestion 4 [ 13 C]-octanoic acid or [ 14 C]-sodium octanoate lactose-[ 13 C]-ureide or inulin-[ 14 C]-carboxylic acid egg protein intrinsically labelled with [ 13 C]-leucine 91mg 74kBq 500mg 74kBq 200mg lipid digestion 5 [ 13 C]-mixed triglyceride 250mg 6h 4h 10h 6h 1 Ghoos et al. Gastroenterology 1993 2 Geypens et al. J Nucl Med 1999 3 Verbeke et al. Aliment Pharmacol Ther 2005 4 Evenepoel et al. J Nutr 1997 5 Vantrappen et al. Gastroenterology 1989
Methods Gastrointestinal parameters o Motility and digestion Prebiotic effects o Microbial composition o Metabolic activity of the microbiota
Metabolic activity of microbiota Lactose-[ 15 N, 15 N ]-ureide to measure colonic ammonia metabolism
Lactose-[ 15 N, 15 N ]-ureide 15 N-LU 15 NH 3 COLON + prebiotics blood liver bacterial metabolism faecal 15 N-excretion kidneys urinary 15 N-excretion
Urinary and faecal 15 N-excretion p<0.05 p<0.05 significant shift from urinary to faecal 15 N-excretion Cloetens et al., Am Coll Nutr 2008 Cloetens et al., Br J Nutr 2010
15 N-excretion in bacterial fraction p<0.05 significant increased uptake of nitrogen by bacteria due to stimulation of bacterial growth/activity Cloetens et al., Am Coll Nutr 2008 Cloetens et al., Br J Nutr 2010
Metabolic activity of microbiota Lactose-[ 15 N, 15 N ]-ureide to measure metabolic ammonia metabolism P-cresol and phenol to measure protein fermentation
Phenolic compounds tyrosine bacterial degradation p-cresol and phenol excretion via faeces absorption detoxification urinary excretion (GC-MS method) (De Loor et al., Clin Chem 2005;5:1535-1538) urinary excretion of p-cresol and phenol is a measure of the extent of colonic protein fermentation
Phenolic compounds Prebiotics: decreased proteolytic activity, less p- cresol and phenol excretion o o o o increased carbohydrate fermentation = saccharolytic activity increased uptake and assimilation of nitrogen decreased colonic ph reduced protease activity Results o significantly decreased amount of p-cresol after 10g AXOS Cloetens et al., Br J Nutr 2010
Metabolic activity of microbiota Lactose-[ 15 N, 15 N ]-ureide to measure metabolic ammonia metabolism P-cresol and phenol to measure protein fermentation Bacterial enzyme activities: β-glucuronidase and β-glucosidase
Bacterial enzymatic activities LIVER toxic or carcinogenic compounds detoxification glucuronide, glucoside COLON bacterial enzyme activities: ß-glucuronidase ß-glucosidase (Goldin and Gorbach, J Natl Cancer Inst 1976;57:371-375) toxic or carcinogenic compounds
Metabolic activity of microbiota Lactose-[ 15 N, 15 N ]-ureide to measure metabolic ammonia metabolism P-cresol and phenol to measure protein fermentation Bacterial enzyme activities: β-glucuronidase and β-glucosidase Metabolic profiles of volatile organic compounds
Volatile organic compounds 100 95 90 85 3.22 7.20 analysis with GC-MS NL: 6.08E6 TIC F: MS G3_F8_T 0_B 80 75 70 65 1.70 22.53 Relative Abundance 60 55 50 45 40 1.49 2.86 4.89 18.92 19.47 10.11 20.65 24.48 35 30 25 20 3.70 5.02 21.95 8.22 17.10 5.35 10.26 23.36 15 10 5 0 0.04 9.25 14.90 17.56 13.39 16.61 12.79 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Time (min) 24.87 28.67 28.21 25.20 28.04 29.64 30.40 ±60 volatile organic components per sample
Metabolic profiles multivariate data analysis score plot loading plot AXOS and wash-out placebo baseline discrimination was explained by differences in diet
Methods to assess effects of prebiotics in humans Lieselotte Cloetens 1,2, Willem F. Broekaert 3,4, Björn Åkesson 1, Gunilla Önning 1, Jan A. Delcour 3, Kristin Verbeke 2 1 Biomedical Nutrition, Pure and Applied Biochemistry, Lund University, Lund, Sweden 2 Laboratory of Digestion and Absorption, TARGID, Catholic University of Leuven, Leuven, Belgium 3 Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Center (LFoRCe), Catholic University of Leuven, Leuven, Belgium 4 Fugeia NV, Leuven, Belgium