Nontargeted LC-MSn Profiling of Compounds in Ileal Fluids That

Sep 19, 2016 - Ileostomy studies provide a unique insight into the digestion of foods, allowing identification of physiologically relevant dietary phy...
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Nontargeted LC-MSn Profiling of Compounds in Ileal Fluids That Decrease after Raspberry Intake Identifies Consistent Alterations in Bile Acid Composition Gordon J. McDougall,*,† J. William Allwood,† Gema Pereira-Caro,‡ Emma M. Brown,§ Nigel Ternan,∥ Susan Verrall,† Derek Stewart,†,⊥ Roger Lawther,∥ Gloria O’Connor,∥ Ian Rowland,# Alan Crozier,○ and Chris I. R. Gill§ †

Environmental and Biochemical Sciences Group, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, Scotland Postharvest, Technology and Agrifood Industry Area, IFAPA, Córdoba, Spain § Centre for Molecular Biosciences, University of Ulster, Coleraine, BT52 1SA, Northern Ireland ⊥ School of Life Sciences, Heriot-Watt University, Edinburgh, EH14 4AS, Scotland ∥ Altnagelvin Area Hospital, Western Health and Social Care Trust, Londonderry, BT47 6SB, Northern Ireland # Department of Food and Nutritional Sciences, University of Reading, Reading, RG6 6AP, England ○ Department of Nutrition, University of California, Davis, California 95616, United States ‡

S Supporting Information *

ABSTRACT: Ileostomy studies provide a unique insight into the digestion of foods, allowing identification of physiologically relevant dietary phytochemicals and their metabolites that are important to gut health. We previously reported an increase of components, including novel triterpenoids, in ileal fluids of 11 ileostomates following consumption of raspberries using nontargeted LC-MSn techniques in combination with data deconvolution software. The current study focused on components that consistently decreased postsupplementation. After data deconvolution, 32 components were identified that met exclusion parameters of m/z signals and which decreased significantly in ileal fluids from eight of 11 participants post-raspberry supplementation. Two-thirds of these components were identified putatively from their MS properties. Consistent decreases were observed in components that possibly reflected “washing out” of presupplementation intake of common foods/drinks including (poly)phenol metabolites. Metabolites associated with fat metabolism such as hydroxylated fatty acids and cholate-type bile acids were specifically reduced. However, more directed re-examination of the data revealed that although some cholates were consistently reduced, the more polar glyco- and tauro-linked bile acid derivatives increased consistently, by as much as 100-fold over presupplementation levels. The possible reasons for these substantial alterations in bile acid composition in ileal fluids in response to raspberry intake are discussed.

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the metabolic fate of berry constituents are crucial to determining the components that function in vivo. If (poly)phenols and their derivatives are to be effective in vivo, they must first survive the effects of the digestive system. Studies that simulate digestive processes in vitro17,18 have provided evidence that berry (poly)phenols have different stabilities in the gastrointestinal tract (GIT) and that some components survive and could therefore influence events in the colon.19 However, such procedures can give only “broad brushstroke pictures” of relative stability in the gut, as they cannot mimic the active processes of digestion,20 and in vivo evidence is, therefore, more informative. Studies with ileostomy

erry consumption has been shown to have beneficial effects on a range of chronic diseases,1−4 and their underlying pathophysiologies5−7 are thought to be mediated through the activity of various (poly)phenols.8 However, many (poly)phenol classes exhibit low bioavailability. For example, urinary recoveries of anthocyanins, which reflect passage through the circulatory system, are, at best, 140 putative metabolites was extracted (Figure S1A−D; Table S1, Supporting Information) that most influenced the separation toward the presupplementation or “before” state. It should be noted that use of the loadings plot from the OPLS-DA analysis gave the same list. After removing possible adducts (such as formate), multiply charged variants, and in-source fragments, exclusion parameters were set to ensure that components that were consistently reduced after raspberry supplementation were selected. The parameters were that the components had to be reduced in eight out the 11 volunteers with no significant difference in the other three volunteers. This took into account the considerable interindividual variation already noted in these samples (Table S1, Supporting Information) and decreased the list to 32 putative metabolites. Lastly, pairwise metabolite−metabolite correlations (using Genstat for Windows, 16th ed., VSN International Ltd., Hemel Hempstead, UK) was used to confirm components that were generally reduced in abundance after supplementation. This used Pearson’s correlation coefficient (r) test using two sample sets, all samples, and then a subset consisting of the “before feeding” samples. The abundance graphs produced in the process allowed the data set to be interrogated for specific components of known m/z values (such as other putative bile acid components) to assess their relative abundance in the before and after samples and provide the data shown in the figures.

interference in recovery does explain why these components were increased, only that their particular enhancement would not be noted in individuals with an intact ileum and colon. In conclusion, a nontargeted LC-MSn-based examination of constituents that consistently decreased in ileal fluids following dietary raspberry supplementation revealed three main classes of components. First, there were compounds that could not be identified from the MS data available. Second, there were those that may decline (e.g., phenolics, Maillard products, lipids) as previously ingested common foods are being “cleared” from the GIT. Finally, the levels of certain endogenously produced components appeared to be modulated during digestion, including a leukotriene derivative, a putative neurotransmitter metabolite, and certain bile acids. Further examination indicated that the more polar glyco- and tauro-conjugated bile acid derivatives were consistently and substantially elevated in the postraspberry supplementation samples. Although it is not possible to be certain whether these alterations in bile acid profiles were due to the intake of raspberries or part of a general response to food intake, their consistent and substantial modulation has implications for digestion and gut health.

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EXPERIMENTAL SECTION

Chemicals. All chemicals were sourced as described previously.25 Bile acids (cholic acid, sodium taurocholate, and sodium glycocholate) were obtained from Sigma-Aldrich Chemical Co. Ltd., Poole, UK. Plant Material and Processing. Raspberries (30 kg, Rubus idaeus variety Glen Ample) were purchased locally and transported to the James Hutton Institute on the day of picking, where they were pureed as described before.25 These were then frozen, transported to the University of Ulster, and stored at −20 °C prior to use in the ileostomy feeding studies. Ileostomy Feeding Study. The ileal fluid samples were collected from a raspberry puree ileostomy feeding study25 (Reg. No. 11/NI/ 0112) conducted with the prior approval of the Office for Research Ethics Committees Northern Ireland (ORECNI) and the Ulster University Ethical Committee and with the informed consent of participants who were recruited from Clinics at Altnagelvin Area Hospital, with the assistance of Colorectal Consultant (Dr. R. Lawther) and Nurse Specialist (Dr. G. O’Connor). In brief, following a diet low in (poly)phenolic compounds, 11 ileostomates provided a baseline ileal fluid sample (T = 0 h), then consumed 300 g of pureed raspberries, and a second ileal fluid sample was collected at T = 8 h.25 The ileal fluid samples were collected, processed within 30 min, and stored as aliquots at −80 °C. Nontargeted Analysis of Metabolites in Ileal Fluids. Frozen ileal samples were thawed and vortexed, and duplicate 2.0 ± 0.1 g samples were weighed into 15 mL centrifuge tubes. These were extracted using 3 mL of ultrapure water containing aqueous 1% formic acid and 20 mM diethyldiothiocarbamate (DDC). The tubes were vortex-mixed for 3 × 30 s, then sonicated in a water bath for 1 min. All procedures were carried out at 5 °C. After centrifugation (2500g, 10 min, 5 °C), the supernatants were transferred to new tubes. The pellets were extracted twice using 3 mL of 1% formic acid in methanol containing 20 mM DDC, and the supernatants combined and vortexmixed. A subsample of 4 mL was removed and dried in a Speed-Vac. The dried samples were resuspended in 1 mL of 10% acetonitrile containing 0.2% formic acid and prepared in filter vials (0.45 μM PTFE filters, Bioprocess Engineering Services Ltd., Ashford, Kent, UK). Nontargeted analysis of the ileal fluids was performed on an HPLC system consisting of a quaternary pump (Thermo Fisher Scientific, Accella 600) and a PDA detector (Thermo Fisher Scientific, Accella) coupled to an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Duplicate 10 μL samples were injected onto a 2 mm × 150 mm (4 μm) Synergy Hydro-RP 80 fitted with a C18 4 × 2 mm Security Guard cartridge (Phenomenex Ltd.,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00532. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (G. J. McDougall): +44 (0) 1382 568782. Fax: +44 (0) 844 928 5429. E-mail: [email protected]. Present Address □

Environmental and Biochemical Sciences Group, The James Hutton Institute, Invergowrie, Dundee, Scotland.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the volunteers for their participation. C.G., R.L., and A.C. acknowledge funding from the National Processed Raspberry Council and Western Health and Social Care Trust. D.S., J.W.A., S.V., and G.McD. acknowledge funding from the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS). G.P.C. was supported by a postdoctoral fellowship from IFAPA (Programa Operativo del Fondo Social Europeo 2007−2013 de Andalucıa).

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