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Title: Non-absorbable apple procyanidins prevent obesity associated with gut ... ACQUITY UPLC® BEH C18 (Waters Co., Milford, MA, USA) column (i.d. 2.1 × 50 ...
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SUPPORTING INFORMATION

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Title: Non-absorbable apple procyanidins prevent obesity associated with gut microbial

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and metabolomic changes

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Authors: Saeko Masumoto,1 Akari Terao,2 Yuji Yamamoto,2 Takao Mukai,2 Tomisato Miura,3

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Toshihiko Shoji1

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Afflications:

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National Institute of Fruit Tree Science, National Agriculture & Food Research, Tsukuba,

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Ibaraki, Japan

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2

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Aomori, Japan

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Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada,

Hirosaki University Graduate School of Health Sciences, Hirosaki, Aomori, Japan

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Corresponding author:

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Toshihiko Shoji, PhD

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NARO Institute of Fruit Tree Science, The National Agriculture & Food Research, 2-1 Fujimoto,

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Tsukuba, Ibaraki 305-8605, Japan. Tel: +81-298-38-6470; Fax: +81-298-38-6437; e-mail:

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[email protected]

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SUPPLEMENTARY METHODS

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Procyanidin analysis

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Apple procyanidins were filtered through a 0.45-µm PTFE syringe filter prior to injection into a

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Prominence HPLC system (Shimazu Corporation, Kyoto, Japan) equipped with a RF-20AXS

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fluorescence detector (Shimazu) and an Inertsil WP300 Diol (GL Science Inc., Tokyo, Japan)

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column (i.d. 4.6 × 250 mm; 5 µm). Mixtures of acetonitrile and acetic acid (mobile phase A,

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CH3CN:HOAc = 98:2) and methanol, H2O, and acetic acid (mobile phase B, MeOH:H2O:HOAc

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= 95:3:2) were used as mobile phases. Elution was performed using a linear gradient of 0–7% B

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for 0–3.0 min, followed by a linear gradient of 7–30% B for 57.0 min. Subsequently, mobile

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phase B content was increased from 30% to 100% over 60.0–70.0 min. The mobile phase was

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subsequently returned to the initial conditions (0% B) to re-equilibrate for 10.0 min. The sample

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injection volume was 5 µL, the flow rate was set at 1.0 mL/min, and fluorescence detection of

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procyanidins was performed with excitation and emission wavelengths of 230 and 321 nm,

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respectively.

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Metabolomic analysis by HPLC-QTOF/MS

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Urine samples were analyzed by high performance liquid chromatography–quadrupole time-of-

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fright/mass spectrometry (HPLC-QTOF/MS) coupled with a hybrid Q-TOF TripleTOF® 5600

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system (AB SCIEX) with a DuoSpray electrospray ionization (ESI) ion source and a Dionex

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UltiMate 3000 HPLC system (Dionex Corporation, Idstein, Germany). The column was an

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ACQUITY UPLC® BEH C18 (Waters Co., Milford, MA, USA) column (i.d. 2.1 × 50 mm; 1.7

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µm) at 40 °C. A mixture of 0.1% formic acid in distilled water (mobile phase A) and 0.1%

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formic acid in acetonitrile (mobile phase B) was used as the mobile phase. The initial eluent was

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0% B for 0–2.0 min, followed by a linear gradient from 0 to 50% B for 8.0 min. Subsequently,

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mobile phase B was increased from 50% to 95% over 10.0–11.5 min. The mobile phase was

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returned to the initial conditions (0% B) to re-equilibrate for 3.5 min. The injection volume was

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0.5 µL of the sample solution. The flow rate was set at 0.3 mL/min.

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Data were acquired using an ion spray voltage of -4500 mV. The declustering potential was -80

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V. The temperature was 650 °C. N2 was used as the curtain (25 arbitrary units) and nebulizer (50

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arbitrary units) gases. The instrument was set to perform one TOF/MS survey scan (150 ms) and

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10 MS/MS scans (30 ms). MS acquisition was performed in negative ionization mode with a

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mass-to-charge ratio (m/z) of 120–1400 from 0 to 12 min. Mass accuracy was maintained by the

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use of an automated calibrant delivery system (AB SCIEX) interfaced to the second inlet of the

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DuoSpray source. The TOF was calibrated with Irganox 1010 (m/z 1175.7768; Sigma-Aldrich,

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St. Louis, MO, USA). The MS/MS analyses were performed with collision energy (CE) of -30 ±

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15 V.

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Data processing

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The MS data were analyzed using MarkerView® Software (version 1.2.1, AB SCIEX). Peaks

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were identified for each sample, whereas alignment was performed using m/z values and

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retention times for the peaks for multivariate analysis. Peak finding was performed using a

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minimum spectral peak width of 1 ppm, a noise threshold of 5, and a subtraction multiple factor

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of 1.2. Alignment and filtering were used to ensure mass tolerance of 0.04 Da and retention time

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tolerance of 0.25 min.

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Multivariate analysis

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Several statistical analyses, including principal component analysis (PCA), t-tests, and PCA-

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discriminant analysis (PCA-DA) were performed using MarkerView® Software. The data were

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visualized by constructing principal component scores and loading plots. Each point on the

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scores plot represented an individual urine sample, whereas each point on the loadings plot

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represented a single mass spectrometry data point.

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Metabolite identification

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Markers contributing to the discrimination of the different groups of rats were identified on the

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basis of exact mass, which was compared to the theoretical mass of expected metabolites. The

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data for each urine sample was subjected to isotope pattern-matched peak mining using the

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Extracted Ion Chromatogram Manager add-on for PeakView® Software (version 1.1.1, AB

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SCIEX). Assignment of the spectral peaks was confirmed using appropriate standards and mass

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fragmentation (MS/MS analyses). The Human Metabolome Database (HMDB; www.hmdb.ca)

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and Phenol-Explorer (http://www.phenol-explorer.eu/) database were queried by molecular

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formula to identify chemical structures.

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Monoisotopic Catechin conjugates

L o a d in g s fo r D 2 (3 3 .3 %) v e rsu s D 3 (3 3 .3 %), P a re to (D A ) 117 0 .1 4

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PB Dimer conjugates

5 6 7 .2 / 5 .7 (2 0 4 4 0 ) 0 .1 3 163 0 .1 2

0 .1 1

Microbiota conjugates

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0.10

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0 .1 0

Bile acid deriva:ves

3 0 3 .0 / 4 .8 (7 0 8 9 )

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95

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23 0 .0 9

50 2 1 5 .0 / 5 .0 (2 6 5 4 )

0 .0 8

6 7 8 .1 / 4 .7 (2 4 3 9 6 ) 2 0 3 .0 / 5 .1 (2 1 6 0 )

2 8 4 .1 / 5 .7 (6 1 0 2 )

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3 7 0 .0 / 5 .3 (1 0 6 0 7 ) 12

0.05

4 7 5 .1 / 4 .8 (1 6 1 2 2 )

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0 .0 5

3 7 1 .0 / 5 .3 (1 0 6 5 0 )

3 0 4 .0 / 4 .8 (7 1 5 0 ) 161

1 8 8 .0 / 5 .8 (1 6 0 6 ) 3 6 1 .2 / 8 .1 (1 0 1 1 8 ) 3 2 4 .1 / 4 .4 (8 1 6 9 )

2 1 9 .0 / 4 .3 (2 8 4 6 )

5 5 9 .1 / 4 .8 (2 0 0 9 8 )

1 9 5 .1 / 0 .5 (1 8 8 5 )

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3 4 8 .1 / 5 .5 (9 4 2 3 )

2 4 4 .0 / 4 .8 (3 9 4 0 )

5 3 7 .1 / 4 .7 (1 9 0 9 5 ) 3 5 1 .1 / 0 .6 (9 5 7 9 )

2 4 7 .0 / 4 .9 (4 1 3 5 )

0 .0 2

D 3 L o a d in g

3 4 7 .1 / 6 .2 (9 3 8 1 )

5 6 9 .2 / 5 .7 (2 0 5 3 5 )

2 6 0 .0 / 4 .9 (4 8 3 9 ) 0 .0 3

2 2 8 .0 / 4 .7 (3 1 7 1 )

5 5 9 .1 / 4 .5 (2 0 0 9 7 )

0 .0 4

3 6 9 .2 / 9 .1 (1 0 5 7 7 )

3 8 0 .1 / 4 .2 (1 1 1 3 2 )

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145

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3 7 1 .2 / 1 0 .1 (1 0 6 9 0 )

2 5 5 .0 / 0 .7 (4 5 8 7 )

0 .0 0

2 9 1 .1 / 7 .2 (6 5 0 2 ) 3 5 0 .2 / 6 .2 (9 5 4 3 )

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2 8 2 .1 / 7 .7 (5 9 8 7 )

2 0 4 .0 / 4 .5 (2 2 1 2 ) 1 9 8 .1 / 7 .2 (1 9 8 3 )

4 5 3 .2 / 7 .3 (1 4 9 9 4 ) -0 .0 2

3 6 7 .1 / 8 .4 (1 0 4 5 6 )

3 3 1 .1 / 7 .9 (8 5 4 8 )

0

-0 .0 1

Tyrosine deriva:ves

2 9 9 .1 / 5 .5 (6 8 9 8 ) 2 1 5 .0 / 4 .6 (2 6 5 7 )

0 .0 6

D3 (33.3 %)

5 6 8 .2 / 5 .7 (2 0 4 8 5 )

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0 .0 7

0 .0 1

Tryptophan deriva:ves

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3 3 4 .2 / 7 .8 (8 7 0 9 )

2 8 1 .1 / 7 .8 (5 9 3 9 )

3 1 9 .1 / 5 .3 (7 9 4 1 ) 4 4 1 .2 / 8 .6 (1 4 4 0 6 )

2 2 2 .0 / 5 .1 (2 9 6 2 ) -0 .0 3

6 7 7 .3 / 7 .4 (2 4 3 8 5 )

2 2 7 .1 / 1 0 .0 (3 1 5 8 )

7 3 3 .3 / 7 .8 (2 5 8 0 2 ) -0 .0 4 4 0 4 .2 / 5 .6 (1 2 4 6 1 )

-0.05

3 2 7 .1 / 7 .0 (8 3 1 6 )

2 4 8 .1 / 4 .9 (4 2 2 8 )

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1 8 6 .1 / 6 .8 (1 5 3 4 )

2 3 2 .0 / 4 .4 (3 3 7 1 )

4 2 5 .2 / 6 .9 (1 3 5 8 5 )

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-0 .0 5

3 5 6 .1 / 7 .8 (9 8 2 7 )

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3 2 9 .1 / 7 .3 (8 4 2 7 )

6 1 5 .3 / 8 .3 (2 2 3 6 0 )

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2 1 6 .1 / 6 .4 (2 7 2 6 )

2 7 1 .0 / 5 .7 (5 3 9 7 )

4 5 3 .1 / 6 .2 (1 4 9 8 1 )

2 7 9 .1 / 7 .3 (5 8 3 9 ) 90

156 5 1 3 .0 / 5 .2 (1 7 9 8 7 )

7 1 1 .3 / 7 .8 (2 5 2 6 4 )

-0 .0 7

4 0 7 .1 / 5 .3 (1 2 5 8 4 ) 1 7 2 .1 / 5 .9 (1 1 6 5 )

-0 .0 8

133 3 4 1 .1 / 6 .6 (9 0 1 6 )

5 6 9 .3 / 8 .3 (2 0 5 5 3 )

93 4 1 5 .2 / 8 .0 (1 3 0 6 3 )

-0 .0 9

1 9 1 .0 / 0 .7 (1 7 4 8 )

-0.10

3 7 1 .1 / 8 .3 (1 0 6 7 1 )

-0 .1 0 4 8 8 .2 / 6 .9 (1 6 7 9 2 )

-0 .1 1

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115 -0 .0 8

-0 .0 6

-0.05

-0 .0 4

-0 .0 2

0 .0 0

0

0 .0 2

0 .0 4

0 .0 6

D 2 L o a d in g

0.05

0 .0 8

0 .1 0

0.10

0 .1 2

0 .1 4

0 .1 6

0.15

D2 (33.3 %)

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Supplementary figure 1. PP-treated mice urine metabolomes are distinct from those of HFHS diet-induced obesity mice. The loading plot obtained via principal component analysis-discriminant analysis (PCA-DA) of the urine metabolites of mice treated for 20 weeks are shown. Metabolomics by HPLC-QTOF/MS was performed by the method described in the online supplementary method.

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