concept of intestinal flora

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1 Hieronymous Bosch (ca ) - Garden of earthly delights painted in ; on display in Museo del Prado in Madrid concept of intestinal flora

2 Use of 13 C-labeled substrates to decipher fermentation of prebiotics in the human colon in vitro and in vivo Koen Venema TNO Healthy Living, Zeist, The Netherlands

3 Carbohydrate fermentation in the colon Who does what? What is made? What is the effect on health? proximal colon: primarily saccharolytic fermentation transverse colon: combination of saccharolytic and proteolytic fermentation distal colon: primarily proteolytic fermentation distal colon: colon cancer and IBD

4 Limited accessibility of the colon

5 Principle: Use of stable isotopes 13 C- labeled substrate Incubation 13 C labelled model substrates lactose inulin starch 13 C ends up in microbial metabolites 13 C ends up in microbial biomass (incl. DNA/RNA) De Graaf and Venema, AMP 2008

6 In vitro model of the colon: TIM-2 the in vitro model the inoculum

7 Analyses 13 C isotopomer analysis: LC-MS more sensitive lactate H-2 B6L Lactate C-2 extracted from HSQC butyrate H-2 B5 + butyrate H-3 butyrate H-4 92 % 61 % 23 % 64 % 25 % 64 % 26 % NMR positional data 6 % 2 % 2 % 14 % 11 % ppm ppm ppm ppm

8 Bacterial metabolism glucose 2 pyruvate CO 2 + CO 2 + acetate acetate butyrate + intracellular formate extracellular 97% 1% 1% 1% formate CO 2 lactate acetate butyrate Wood-Ljungdahl pathway

9 Propionate starch experiment NMR 13 C propionate isotopomers mm Propionate C2 1,8 1,6 1,4 1,2 1 PA2-s 0,8 PA2-d- PA2-d+ 0,6 PA2-dd 0,4 0, time (min) measurements = NMR NMR NMR NMR MS MS MS m+2 MS m+1 NMR NMR mm Propionate C3 3 2,5 2 1,5 PA3-s PA3-d 1 0, time (min) = + = + MS => all propionate isotopomers determinable mmol in lumen C3 in starch fermentation 3 M+0 M+1 2 M+2 M time (h)

10 Propionate pathways Acrylate and succinate-decarboxylating pathways (1-x).y x.y (1-x).(1-y) x ~= 0.33 CO 2 y x.(1-y) y ~= 0.40 acrylate pathway succinate decarboxylating pathway 50% 1-x x z ~= % CO 2 starch, t=4h acrylate 0 % succinate 100 %

11 Propionate lactose experiment Lactose t=15 min Lactose t=1h Lactose t=4h PROPIONATE ALA EtOH LACTATE C5 & C6

12 Propionate pathways 2,0 propionate production T[U-13C]inulin 2 propionate production T[U-13C]lactose 2,0 propionate production T[U-13C]starch mmol 1,6 1,2 0,8 0,4 Propionate M+0 Propionate M+1 Propionate M+2 Propionate M mmol 1 Propionate M+0 Propionate M+1 Propionate M+2 Propionate M+3 mmol 1,6 1,2 0,8 0,4 Propionate M+0 Propionate M+1 Propionate M+2 Propionate M+3 0, time (minutes) time (minutes) 0, time (minutes) Table Flux distribution over the propionate synthesis pathways on different 13 C-labeled substrates estimated from the isotopomeric labeling data. substrate acrylate pathway contribution (%) succinate decarboxylation pathway contribution (%) starch inulin lactose These results seem to indicate that a faster fermentation goes along with an increased contribution of the acrylate pathway, i.e. the pathway that has lactate as a precursor.

13 TIM-2 [U- 13 C]-starch experiment Computer model: Flux within intestinal microbiota V EMP 89.1 µmol/min [U- 13 C]Glc 13 C labeled substrate intracellular extracellular Unlabeled substrates Protein & Fatty acids V PYR V ACCOA V PDH Pyr 97.2 CO 2 AcCoA V LDH V PFL V FHL H2 CO 2 CO AcAcetylCoA V WLP V ACK Lactate 130 Formate Acetate V LACt Acetate V FORt V ACt 82.7 Lactate Formate Acetate V LACex V FORex V ACex > 90% reversibility 99.3 V BUK Butyrate 99.3 V BUt 35.7 Butyrate V BUex

14 Principle: Use of stable isotopes 13 C- labeled substrate Incubation 13 C ends up in microbial metabolites 13 C ends up in microbial biomass (incl. DNA/RNA) TIM-2 De Graaf and Venema, AMP 2008

15 16S rrna-sip 13 C-labeled substrate Incubation RNA extraction Density gradient ultracentrifugation (CsTFA) Gradient fractionation Molecular analyses heavy vs. light fractions 16S rrna - based molecular fingerprinting of microbial diversity Cloning and sequencing of 16S rrna

16 Which microbes enjoy the heavy meal? 16S rrna RT-PCR (Bacteria) MspI Fraction density Control 2h 4h 8h Fraction number R. bromii Control, fraction 5 4h, fraction g ml g ml bp T-RF length 1000 bp Fermentation products 8h after starch addition: acetate > butyrate > propionate Ruminococcus bromii > Bac./Prev.spp acetate Eubacterium rectale propionate butyrate Kovatcheva-Datchary et al. (2009), Environ Microbiol

17 Use of GOS by the intestinal microbiota - Implications for claim substantiation Bifidobacterium bifidum t0 t1 Lactobacillus gasseri t2 t4 50 t ratio(%) 25 ratio(%) t8 t4 t2 t1 time(h) t fraction t8 t4 t2 t1 time(h) t fraction Maathuis et al., (2010) in preparation

18 Determination of caloric value - Implication for obesity 1 gram of inulin has a caloric value of 2.05 kcal similarly, 1 gram of lactose has a caloric value of 2.41 kcal 1 gram of starch 1.69 kcal Therefore, depending on the fermentable substrate, the energy-harvest of the body is different ----> link of microbiota with obesity Venema (2010) Curr Opin Clin Nutr Metab Care

19 Conclusions Stable isotope-labeled substrates are excellent tools to study the processes occurring in the inaccessible colon - even in human individuals The label can be traced in metabolites and microbial biomass This allows to create the food-chain in the gut: substrate microbe metabolite Precise determination of the metabolites produced allows the exact calculation of energy harvested from fermentable substrates

20 Outlook human feeding Maastricht University hospital Substrate delivery via a nasal tube 13 C Colon sampling via a stoma in the colon transversus or back through catheter Plasma sampling LC-MS analyses

21 First experiments in humans!!

22 Use of the nasal catheter in 2 healthy volunteers delta plasma glucose delta breath CO 2 breath hydrogen (ppm) C-CO 2 H 2 13 C-glucose individual h 2 h 3 h 1 h 2 h 3 h - no 13 C-glucose in plasma - increase in 13 C-CO 2 in breath - in crease in breath H 2 Bifidobacterium Collinsella Propionibacterium Streptococcus bovis et rel. Streptococcus intermedius et rel. Streptococcus mitis et rel. Allistipes et rel. Bacteroides fragilis et rel. Bacteriodes ovatus et rel. Bacteriodes plebeius et rel. Bacteroides splachnicus et rel. Bacteroides stercoris et rel. Bacteroides uniformis et rel. Parabacteroides distasonis et rel. Prevotella tannerae et rel. Tannerella et rel. Sporobacter termitidis et rel. Butyrovivrio crossotus et rel. Clostridium symbiosum et rel. Eubacterium rectale et rel. Ruminococcus obeum et rel. Fusobacteria Alcaligenes faecalis et rel. Anaerobiospirillum Burkholderia Enterobacter aerogenes et rel. Escherichia coli et rel. Haemophilus Klebsiella pneumonia et rel. Moraxellaceae Oxalobacter formigenes et rel Proteus et rel. Pseudomonas Serratia Sutterella wadsworthia et rel. Vibrio Xanthomonadaceae Yersinia et rel. Uncultured Mollicutes Akkermanisa fermentation of 13 C-lactose in colon

23 Acknowledgements The team! Bart van Rossum Mick Deutz Roland Meesters Hans van Eijck Johanne Bloemen Daisy Jonkers Fred Troost Steven Vanhoutvin Henrike Hamer Andrea Kodde Annet Maathuis Albert de Graaf Markus Egert Petia Kovatcheva-Datchary Hauke Smidt Willem de Vos Tao He Saed Lahham Ad Sprenkels Ton Jenneboer Jan Sikkema