Gastrointestinal Simulation Model TWIN-SHIME Shows Differences

Subscriber access provided by UNIV OF NEWCASTLE

Article

The Gastrointestinal Simulation Model TWIN-SHIME® Shows Differences Between Human Urolithin-Metabotypes in Gut Microbiota Composition, Pomegranate Polyphenol Metabolism, and Transport Along the Intestinal Tract Rocio Garcia-Villalba, Hanne Vissenaekens, Judit Pitart, Maria Romo-Vaquero, Juan Carlos Espín, Charlotte Grootaert, María V. Selma, Katleen Raes, Guy Smagghe, Sam Possemiers, John Van Camp, and Francisco A. Tomas-Barberan J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Journal of Agricultural and Food Chemistry

The Gastrointestinal Simulation Model TWIN-SHIME® Shows Differences Between Human Urolithin-Metabotypes in Gut Microbiota Composition, Pomegranate Polyphenol Metabolism, and Transport Along the Intestinal Tract

Rocío García-Villalba,†* Hanne Vissenaekens, δde* Judit Pitart,‡ María Romo-Vaquero,† Juan C. Espín,† Charlotte Grootaert,δ María V. Selma,† Katleen Raes,d Guy Smagghe,e Sam Possemiers,‡ John Van Camp,δ Francisco A. Tomas-Barberan†# †

Research Group on Quality, Safety, and Bioactivity of Plant Foods, Laboratory of Food &

Health; Dep. Food Science and Technology, CEBAS-CSIC, 30100 Campus de Espinardo, Murcia, Spain. E-mail: [email protected]; Phone: +34-968396200. ‡

δ

ProDigest BVBA, Ghent, Belgium

Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent

University, Ghent, Belgium d

Department of Industrial Biological Sciences, Faculty of Bioscience Engineering, Ghent

University, Kortrijk, Belgium e

Department of Crop Protection, Ghent University, Faculty of Bioscience Engineering, Ghent,

Belgium *Contributed equally #Corresponding author 1 2

Title running header: Urolithin metabotypes and gastrointestinal simulation.

1 ACS Paragon Plus Environment


®

3

ABSTRACT: A TWIN-SHIME

system was used to compare the metabolism of pomegranate

4

polyphenols by the gut microbiota from two individuals with different urolithin metabotypes.

5

Gut microbiota, ellagitannin metabolism, short-chain fatty acids (SCFA), transport of

6

metabolites and phase II metabolism using Caco-2 cells were explored. The simulation

7

reproduced the in vivo metabolic profiles for each metabotype. The study shows for the first

8

time that microbial composition, metabolism of ellagitannins and SCFA differ between

9

metabotypes and along the large intestine. The assay also showed that pomegranate phenolics

10

preserved intestinal cell integrity. Pomegranate polyphenols enhanced urolithin and propionate

11

production, as well as Akkermansia and Gordonibacter prevalence with the highest effect in the

12

descending colon. The system provides an insight into the mechanisms of pomegranate

13

polyphenol gut microbiota metabolism and absorption through intestinal cells. The results

14

obtained by the combined SHIME®/Caco-2 cell system are consistent with previous human and

15

animal studies and show that, although urolithin metabolites are present along the

16

gastrointestinal tract due to enterohepatic circulation, they are predominantly produced in the

17

distal colon region.

18

19

KEYWORDS: ellagic acid, urolithin, phenotypes, gut microbiota, intestinal cells

20


Page 2 of 40

Page 3 of 40


21

INTRODUCTION

22

Dietary ellagitannins (ETs) and ellagic acid (EA) have been associated with important

23

health effects and benefits in diseases including cardiovascular disease.1,2 They are

24

present in dietary sources in larger amounts than previously estimated.3 In humans, ETs

25

are not absorbed as such, and the absorption of EA is rather low.4 Both ETs and EA are

26

catabolized by the gut microbiota leading to urolithin metabolites.5 The final metabolites

27

in this catabolic conversion are urolithin A (Uro-A), urolithin B (Uro-B), and

28

isourolithin A (Isouro-A) (Figure 1). Not all individuals have the appropriate gut

29

microbiota to produce the final urolithin metabolites and three different urolithin

30

metabotypes (UMs), UM-A, UM-B, and UM-0, have been reported.6 Species of the

31

genus Gordonibacter have been identified as gut microbiota constituents that are

32

involved in the conversion of EA into intermediary urolithins. 7,8 Fecal Gordonibacter

33

concentrations correlate positively with urolithin-A content in feces and urine,9 although

34

other unknown bacterial species are needed to produce the final urolithin metabolites.

35

Urolithins reach in human plasma concentrations within the µM range,10,11 and these

36

bioavailable metabolites, trigger different molecular and cell responses that may

37

account, at least partially, for the antioxidant, anti-inflammatory, anticancer, cardio-

38

metabolic, and neuroprotective effects attributed to ETs and (or) to ET-containing

39

foods.2,

40

identification of the specific regions of the intestine where they are formed, and the gut

41

microbiota involved are of special interest. After pomegranate polyphenols intake,

42

several urolithins have been detected in human feces, urine and also in biopsies taken

43

from prostate and different regions of the colon in cancer patients.14,

44

production of urolithins from EA by human fecal microbiota from both metabotype A

45

and B has also been described.16 However, the gastrointestinal tract site for urolithin

5, 12, 13

Therefore, the study of the mechanisms for urolithin production, the


15

In vitro


46

production, the stability and absorption of the metabolites in the gut are still unknown.

47

To date, Gordonibacter levels have only been quantified in human fecal samples, but its

48

distribution throughout the digestive tract and its role in urolithin production are still

49

unknown. Fecal Gordonibacter levels are higher in urolithin metabotype A (UM-A)

50

individuals than in those with urolithin metabotype B (UM-B) and urolithin metabotype

51

0 (UM-0).17 Modulation of some human fecal bacteria by consumption of ET- rich food

52

such as pomegranate has recently been described,18,19 and the increase of fecal

53

Gordonibacter levels was highlighted.19 However, modulation of Gordonibacter and

54

other bacterial groups by ET-rich foods along the digestive tract as well as their

55

differences between metabotypes have not been explored and require further research.

56

In the present study, a simulator of the human intestinal microbial ecosystem (TWIN-

57

SHIME®) was used to shed light on ET gut microbiota metabolism in the different

58

regions of the intestine. A pomegranate extract (PE) supplement was subjected to a

59

stomach and small intestine (SI) digestion to estimate the bioavailability of native

60

polyphenols and their catabolism in the upper part of the gastrointestinal tract. Long-

61

term microbial colon fermentation was also investigated in the TWIN-SHIME®, thus

62

determining the gut microbiota metabolism of ETs in the colon, the urolithin production

63

pathway, the sites of transformation and the metabolite profile of (poly)phenolics which

64

have potential to be absorbed . The production of specific SCFA was also evaluated, as

65

well as the modulation of gut microbiota. The intestinal transport and cell metabolism of

66

the (poly)phenolics were also evaluated through direct addition of diluted phenolics-

67

containing SHIME® matrix to Caco-2-cells. Overall, our results are of interest to

68

validate this system when comparing with the results previously obtained in vivo.

69 70

MATERIALS AND METHODS


Page 4 of 40

Page 5 of 40


71

Pomegranate Extract (PE) and Chemicals. A characterized PE was provided by

72

Laboratorios Admira S.L. (Alcantarilla, Murcia, Spain).19 EA, punicalagin and 6,7-

73

dihydroxycoumarin (DHC) were from Sigma-Aldrich (St. Louis, MO, USA). Urolithins

74

were obtained as previously described.3 Purity was higher than 95% for all tested

75

compounds. Organic solvents such as methanol, acetone, and acetonitrile were from

76

Merck (Darmstadt, Germany). All chemicals and reagents were of analytical grade.

77

Volunteer stratification and characterization. To select the fecal donors for UM-A

78

and UM-B, 13 individuals consumed 30 g walnuts/day for three days, and urine samples

79

were collected on the third day. Urolithin production and metabolic profiles were

80

evaluated using HPLC-DAD-MS,20 and eight individuals were stratified as UM-A, four

81

as UM-B and one as UM-0. This distribution is consistent with normal values as

82

previously reported.20,6 Representatives of UM-A and UM-B were selected as fecal

83

donors for the assay of ET metabolism in the gastrointestinal simulator.

84

Metabolism of the ETs in the TWIN-SHIME® and sampling. A SHIME® setup

85

(registered name from Ghent University and ProDigest), simulating the entire human

86

gastrointestinal tract, was used as previously reported.21-23 To investigate two different

87

metabotypes (UM-A and UM-B) at the same time, a TWIN-SHIME® setup23 was used

88

by operating two systems in parallel. The first two reactors are of the fill-and-draw

89

principle to simulate different steps in food uptake and digestion, with peristaltic pumps

90

adding a defined amount of SHIME® nutritional medium (140 mL 3x/day) and

91

pancreatic and bile liquid (60 mL 3x/day), respectively, to the stomach and small

92

intestine (SI) compartments and emptying the respective reactors after specified

93

intervals. The last three compartments simulate the ascending (AC), transverse (TC) and

94

descending (DC) colon. Inoculum preparation, retention time, pH, temperature settings

95

and reactor feed composition were previously described.23 After inoculating the colon



96

reactors of the TWIN-SHIME® system with fresh fecal samples from UM-A and UM-B,

97

a two-week stabilization period allowed the microbial community to differentiate in the

98

reactors. After the stabilization period, the standard SHIME® nutrient matrix was further

99

dosed to the model for two weeks. Analysis of samples in this control period allowed to

100

determine the baseline microbial community composition and activity in the different

101

reactors, which were used as a control to compare with the results from the treatment

102

period where the basic diet was supplemented with 1.8 g/day of PE during three weeks.

103

TWIN-SHIME® samples obtained from the different reactors (SI, AC, TC, and DC)

104

were analyzed . Samples were taken for chemical (ET derived metabolites and SCFA

105

profile) and microbial (denaturing gradient gel electrophoresis (DGGE) and quantitative

106

PCR (qPCR)) analyses.

139

EA and urolithins LC-MS analysis. Samples (2 mL) from the different vessels of the

140

TWIN-SHIME® were extracted with 2 mL of ethyl acetate acidified with 1.5% formic

141

acid. The mixture was vortexed for 2 min and centrifuged at 3500g for 10 min. The

142

organic phase was separated and evaporated under reduced pressure to dryness. The dry

143

samples were then redissolved in 400 µL of methanol and filtered through a 0.22 µm

144

PVDF filter. Then 5 µL of 10 µg/mL of internal standard (6,7-dihydroxycoumarin) was

145

added to 50 µL of the sample before the injection onto a column for HPLC-DAD-single

146

Q analysis. Several samples were also analyzed after 1:10 dilution in methanol to

147

quantify compounds present at very high concentrations (saturated compounds).

148

Samples (0.5 mL) from the Caco-2 transport experiment were extracted with 0.5 mL

149

acetonitrile: formic acid (2%). After vortexing the samples for 2 minutes they were

150

centrifuged for 15 min at 14,000 g. The supernatants were collected, dried using

151

nitrogen and stored at -80 ºC until HPLC-MS analyses. Samples were analyzed as

152

described previously,3,20 using HPLC-DAD-MS with a reversed-phase column. All


Page 6 of 40

Page 7 of 40


153

metabolites were quantified with their standards at 305 nm, except for EA, punicalagin,

154

and Uro-M7 at 360 nm. These were quantified with EA calibration curve at 360 nm.

155

The method validation previously reported,3,20 was adapted regarding recovery and

156

limits of detection and quantification using this matrix (SHIME®

157

(Supplementary Table 1).

medium)

158

SCFA analysis. TWIN-SHIME® samples were analyzed as previously described.24

159

Briefly, SCFA were extracted from the samples with diethyl ether, after the addition of

160

2-methyl hexanoic acid as an internal standard. Extracts were analyzed using a GC-

161

2014 gas chromatograph (Shimadzu, Hertogenbosch, The Netherlands), equipped with a

162

capillary fatty acid-free EC-1000 Econo-Cap column (dimensions: 25mm0.53 mm, film

163

thickness 1.2 mM; Alltech, Laarne, Belgium), a flame ionization detector and a split

164

injector. The injection volume was 1 mL, and the temperature profile was set from 110

165

to 160 °C, with a temperature increase of 6 °C/min. The carrier gas was nitrogen, and

166

the temperature of the injector and detector were 100 and 220 °C, respectively.

167

DNA extraction and microbial analysis. Powerfecal® DNA isolation kit (Mo-Bio

168

Laboratories, Carlsbad, CA, USA) was used to isolate total DNA from different

169

SHIME® samples. An additional step was done consisting on vigorous shake using a

170

FastPrep® Instrument and 2mL tubes containing special beads (MP Biomedicals, LLC,

171

Ohio, USA). After DNA extraction, DGGE was used to monitor the most prominent

172

shifts within the overall microbial community together with group-specific shifts within

173

the total bacteria25 and Clostridium cluster XIVa.26 After DNA extraction and PCR with

174

general or group-specific primers, DGGE was performed to separate PCR products.25, 26

175

Gels had a denaturizing gradient from 45% to 60% and were run using a DCodeTM

176

Universal Mutation Detection System (Bio-Rad). Data analysis was carried out using

177

GelCompar version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium). Pearson



178

correlation and UPGMA (Unweighted Pair Group Method using Arithmetic Mean)

179

clustering were used to calculate dendrograms of DGGE profiles.

180

qPCR for total bacteria, Firmicutes, Bacteroidetes, Bacteroides spp., Bifidobacterium

181

spp., Lactobacillus spp., Akkermansia spp and Gordonibacter spp. was performed as

182

previously reported.17,19 Real-time qPCR was run on the ABI 7500 system (Applied

183

Biosystems, ABI, Madrid, Spain) following manufacturer’s conditions.

184

Maintenance of intestinal cell culture. The human colon adenocarcinoma cell line

185

Caco-2 (HTB37™) was obtained and cultured as reported previously.27 Cells were

186

maintained in an incubator with a water saturated atmosphere of 10% CO2 at 37 ºC

187

(Memmert CO2 incubator, Memmert GmbH & Co., Nurnberg, Germany). The cell

188

culture medium was replaced 3 times a week, and when Caco-2 cells reached 80-90%

189

confluence, the cells were sub-cultured using 0.25% (v/v) trypsin-EDTA solution

190

(Sigma-Aldrich).

191

Cytotoxicity measurements. Using MTT and SRB assays, the cytotoxicity of (i) PE-

192

free intestinal matrix, (ii) digested PE and (iii) undigested PE on the intestinal Caco-2

193

cellswas investigated.27,28 Intestinal Caco-2 cells were maintained 21 days to obtain

194

confluent monolayers of differentiated intestinal cells as previously reported.27,28 21

195

Days post-seeding, the Caco-2 monolayers were loaded for 4 h with 1/5 (v/v) dilutions

196

of the intestinal matrix and the (un)digested PE in HBSS.29 Subsequently the MTT and

197

SRB assays were performed to monitor mitochondrial activity and protein content,

198

respectively.27,28

199

Intestinal transport. Caco-2 cells were seeded on the apical side of the Transwell®

200

filters of 6-well Transwell® plates (0.4 µm pore diameter, 24 mm insert, Corning Costar

201

Co., Elscolab, Kruibeke, Belgium) and after 21 days, a 100% confluent monolayer was

202

obtained. Intestinal monolayer integrity measurements were performed as previously 8 ACS Paragon Plus Environment

Page 8 of 40

Page 9 of 40


203

reported.27,28 To ensure that the intestinal Caco-2 cell monolayer (i) is intact during the

204

transport assays and (ii) is not permanently damaged by the treatment, the

205

transepithelial electrical resistance (TEER) of the monolayer was measured before,

206

immediately after, and 24 h after the transport assays. The paracellular transport was

207

also assessed 24 h after the transport assays using the fluorescent paracellular transport

208

marker Lucifer Yellow. Based on in-house experience,27,28 Hank’s balanced salt

209

solution (HBSS) (Gibco Life Technologies) was selected as a transport medium. Cells

210

were washed and pre-incubated with the transport medium (HBSS) for 1 h. Next, cells

211

were treated with dilutions of (i) PE-free intestinal matrix, (ii) digested PE and (iii)

212

undigested PE dissolved in HBSS (2mL, pH 6.5), while fresh HBSS (2.5 mL, pH 7.5)

213

was loaded in the basal compartment, and incubated for 4 h (37 °C and 10% CO2).

214

Samples (0.5 mL) from the apical and basal compartments were obtained after 2 and 4 h

215

of treatment.

216

Statistical analysis. All samples were analyzed in triplicate. All data are expressed as

217

mean value ± SD. Statistical analyses were performed using SPSS V.23 for Windows

218

(SPSS, Chicago, IL, USA). Two-way ANOVA was performed to evaluate differences

219

between groups. Bonferroni posthoc test was used to investigate differences between

220

stages. Statistical significance was accepted at P

Gastrointestinal Simulation Model TWIN-SHIME Shows Differences

Recommend Documents