SCFA Measurement: Advances And Current State of SCFA Analysis

Transcription

1 SCFA Measurement: Advances And Current State of SCFA Analysis Douglas Morrison, SUERC ILSI SCFA Workshop, Belgium Nov 2018

2 Overview 1. Why are SCFA important? 2. Methods for SCFA measurement 3. Challenges in interpretation 4. What can stable isotopes tell us? 5. Challenges and opportunities

3 Why are SCFA important?

4 Fermentable carbohydrates

5 Hierarchy of fermentation - CHO gases ph SCFA Carbohydrate Bile acids X Biomass α-diversity ~ β-diversity ~ Amines BCFA Indoles Protein Amino acids Phenols H 2 S Ammonia Fat

6 Fermentable CHOs - more than just bulk? SCFAs are metabolically active and act as signalling molecules Diet Morrison & Preston 2016

7 Regions matter! Korpela et al, 2018; Van der Beek et al, 2016

8 Which fibres - which SCFA in vitro? The top and bottom five ranked producers of acetate, propionate, butyrate, and total production at 24 hours of fermentation (mmol/g carbohydrate/ day) Top 5 Ranked on Acetate Ranked on Propionate Ranked on Butyrate Ranked on Total 1 Galactose 11.3 (7.2) 2 Rhamnose 4.7 (1.1) GOS 2.4 (2.5) GOS 13.7 (16.2) 2 GOS 8.5 (12.6) Arabinose 3.2 (2.5) Pectic oligosaccharide 1.5 (0.1) Galactose 12.3 (6.6) 3 Lactose 7.8 (8.5) Guar gum 2.9 (0.7) Raw starch 1.3 (1.3) Rhamnose 11.1 (5.6) 4 Pectic oligosaccharide 7.5 (1.3) Xylose 2.7 (2.8) Lactose 1.2 (1.4) Pectic oligosaccharide 9.9 (1.5) 5 Lactulose 7.0 (16.9) GOS 2.4 (1.8) Guar gum 1.0 (0.6) Arabinose 9.8 (7.2) Bottom 5 25 Pea fibre 1.5 (2.0) Modified pectin 0.4 (1.3) Pea fibre 0.3 (0.6) Pea fibre 2.5 (3.3) 26 Oat bran 0.6 (3.7) Cellulose 0.3 (0.6) Xylose 0.2 (0.9) Oat bran 1.0 (6.2) 27 Kiwi fibre 0.4 (0.2) Oat bran 0.2 (1.2) Oat bran 0.2 (1.2) Cellulose 0.9 (2.4) 28 Polydextrose 0.4 (4.0) Kiwi fibre 0.1 (0.1) Polydextrose 0.1 (0.9) Kiwi fibre 0.7 (0.3) 29 Cellulose 0.3 (1.4) Polydextrose 0.1 (1.7) Kiwi fibre 0.1 (0.1) Polydextrose 0.6 (6.5) Harris et al, Unpublished

9 Can we predict SCFA production? Harris et al, 2017

10 Complexity of fibre fermentation Reichardt et al, 2018

11 Function vs. diversity

12 SCFA Interconversion Morrison et al, 2006 den Besten et al, 2014

13 Which fibres in animals Body weight Body 8wks Fat / lean mass Cumulative food intake Liver fat content Caecal content Drew et al, 2018

14 Not explained by microbial changes Drew et al, 2018

15 Animal studies and human fibre intake Morrison & Preston, 2016

16 Which fibres in humans? Effect of type of fibre intervention on faecal bacteria Bifidobacteria Accepted prebiotic fibres 0.68 [0.38, 0.98] Candidate prebiotic fibres 0.77 [0.30, 1.24] General fibres 0.25 [-0.16, 0.65] Lactobacilli Accepted prebiotic fibres 0.34 [0.13, 0.55] Candidate prebiotic fibres -0.06[-0.29, 0.16] General fibres 0.22 [-0.31, 0.75] F. prausnitzii 0.14 [-0.12, 0.39] Roseburia spp [-0.14, 0.80] E. rectale -0.26[-1.20, 0.67] R. bromii 0.15 [-0.15, 0.45] Effect of type of fibre intervention on faecal SCFA Total SCFA 0.11 [-0.05, 0.27] Acetate 0.28 [-0.08, 0.63] Propionate -0.01[-0.20, 0.22] Butyrate 0.24 [0.00, 0.47]

17 Methods for SCFA measurement

18 SCFA - deceptively simple molecules! Acetate Propionate Butyrate O C CH 3 O - O CH 3 CH 2 C CH CH 2 O - 3 CH 2 O C O - Formate Lactate Succinate O O O H C O - CH 3 CH C O - HO C CH 2 CH 2 C O - OH O

19 Analytical approach

20 Summary of analytical techniques Technique Advantages Disadvantages GC-FID Inexpensive High resolution Free acid analysis GC-MS Inexpensive (?) High-resolution Sensitivity Derivatisation (EI) Isotope dilution LC-MS NMR Little / no sample processing Medium / high resolution Sensitivity Isotope dilution Positional information Flux (isotope) Metabolomic / metabonomic Poor sensitivity (plasma, urine) No molecular selectivity / confirmation Sample processing Derivatisation (EI, acetate) Expensive (?) Derivatisation (acetate) Poor sensitivity (plasma, urine)

21 Workflow Sample collection / storage Choice of collection tube Blank control IS or ID spike / purification / concentration GC-FID / GCMS / LCMS / IRMS Blank control Extraction / derivatisation GCMS / LCMS Blank control Data reduction

22 GC / GCMS Morrison et al, 2004 Hoving et al, 2018

23 LC / MS Underivatised, plasma O-benzylhydroxylamine derivatives van Eijk et al, 2009 Zeng & Cao, 2018

24 Sampling Site Plasma - µmol / L Urine - µmol / L Faeces - mmol / L Lumen sampling - mmol / L Portal sampling - µmol / L - mmol / L

25 Colonic SCFA production - the holy grail! Plasma concentrations reflect the balance of production and sequestration. Time-course Faecal concentrations cannot directly measure SCFA production because they are readily absorbed in colon In vitro and animal models provide useful mechanistic data but how do they (or can they be) compared with in vivo? Stable isotopes

26 Isotope techniques for measuring SCFA production 1. Use labelled CHO 2. Isotope dilution

27 Complex CHO utilisation is complicated Breath 13 CO2 (ppm 13 C excess) Breath 13C Breath H Breath H2 (ppm) Tracer and Test meal Meal One, No Tracer Meal Two, No Tracer Time (hours) Edwards et al, 2002

28 13 C CHO quantifying production 1. Need a second tracer to quantify production (flux) a) Known SCFA spike 2. Need a comparator / reference compound b) Two study days

29 Stable Isotope Dilution Endogenous production of tracee (Ra) Time Constant infusion of tracer Rate of disappearance (Rd) At isotopic equilibrium Ra = Rd Infusion rate Flux = Plateau enrichment

30 Acetate production from lactulose 9 hour tracer infusion protocol Site of tracer infusion: iv, oral or colonic delivery? Pouteau et al, 1998

31 Limitations 1. Long i.v. infusion times 2. Correction factors for splanchnic uptake 3. Underlying assumption that endogenous acetate production is constant 4. Underestimates true colonic production > interconversion in colon 5. All SCFA simultaneously?

32 Colonic 13 C SCFA infusion > colonic SCFA flux

33 GCMS and GC-C-IRMS Low isotope precision (0.5%) High sensitivity High isotope precision (0.0002%) Low sensitivity Choice of tracer, sampling site, cost

34 Colonic SCFA delivery 13 C SCFA colonic delivery capsules Boets et al, 2017

35 Colonic SCFA production Quantitation of SCFA contribution to metabolism + SCFA flux Boets et al, 2015; 2017

36 SCFA flux - challenges How to handle the SCFA blank (especially acetate, blank often > sample) Propionate/butyrate conc. for GC-C-IRMS Sample size (~ 10ml whole blood) >> urine 2 H tracers for GCMS 13 C tracers for GC-C-IRMS

37 Summary 1. SCFA may be important mediators of diet-microbiome-health axis 2. SCFA relatively easy to measure quantitatively 3. SCFA measurements more difficult to interpret 4. Stable isotope methods look promising

38 Challenges and opportunities 1. No satisfactory routine method to quantify colonic SCFA production a) Stable isotope dilution studies using colonic 13 C SCFA delivery are promising 2. The impact of NDC on specific SCFA remains opaque in vivo b) Phenotype, microbiome, diet 3. Direct delivery of colonic SCFA useful for RCTs c) Would benefit from direct colonic SCFA production method 4. A colonic production method would enhance our understanding of the role of SCFA in human health

39 Acknowledgements Acknowledgements Tom Preston Hannah Harris Christine Edwards Graeme Milligan Nicole Reichardt Catriona Tedford Kenneth MacDougall Emma Hamilton David Barn Robin Stewart Gary Frost Ed Chambers Claire Byrne Alexander Viardot Arianna Psichas Steve Bloom Waljit Dhillo Kevin Murphy Harry Flint Silvia Duncan Petra Louis Janice Drew Linda Williams