Cecal Succinate Elevated by Some Dietary Polyphenols May Inhibit

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Cecal Succinate Elevated by Some Dietary Polyphenols May Inhibit Colon Cancer Cell Proliferation and Angiogenesis Tomoaki Haraguchi, Tomoko Kayashima, Yukako Okazaki, Junji Inoue, Shigeru Mineo, Kiminori Matsubara, Ei Sakaguchi, Noriyuki Yanaka, and Norihisa Kato J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501142k • Publication Date (Web): 23 May 2014 Downloaded from http://pubs.acs.org on June 4, 2014

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Journal of Agricultural and Food Chemistry

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Title:

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Cecal Succinate Elevated by Some Dietary Polyphenols May Inhibit Colon

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Cancer Cell Proliferation and Angiogenesis

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Author names:

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Tomoaki Haraguchi1, Tomoko Kayashima2*, Yukako Okazaki3, Junji Inoue4, Shigeru

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Mineo5, Kiminori Matsubara6, Ei Sakaguchi7, Noriyuki Yanaka1 and Norihisa Kato1

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Author address:

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1

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Higashi-Hiroshima 739-8528, Japan,

Graduate

School

of

Biosphere

Science,

Hiroshima

University,

1-4-4,

10

2

Faculty of Culture and Education, Saga University, 1, Honjou, Saga 840-8502, Japan

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3

Faculty of Human Life Sciences, Fuji Women’s University, 4-5 Hanakawa Minami,

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Ishikari 061-3204, Japan

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4

Ahjikan Co. Ltd., Hiroshima 733-0833, Japan

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5

Institutes of Health, BOURBON Corporation, Kashiwazaki 945-0114, Japan

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6

Graduate School of Education, Hiroshima University, 1-1-1, Higashi-Hiroshima,

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739-8524, Japan

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7

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Tsushimanaka, Kitaku, Okayama 700-8530, Japan

Graduate School of Natural Science and Technology, Okayama University, 1-1-1,

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Author responsible for correspondence:

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Tomoko Kayashima

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Faculty of Culture and Education, Saga University, 1, Honjou, Saga 840-8502, Japan

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Tel: +81-952-28-8380

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Fax: +81-952-28-8380

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E-mail: [email protected]

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Title running-header:

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Elevated succinate by polyphenols, and cancer

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Author Email address:

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Tomoaki Haraguchi ([email protected])

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Tomoko Kayashima*, Ph.D. ([email protected])

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Yukako Okazaki, Ph.D. ([email protected])

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Junji Inoue ([email protected])

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Shigeru Mineo ([email protected])

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Kiminori Matsubara, Ph.D. ([email protected])

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Ei Sakaguchi, Ph.D. ([email protected])

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Noriyuki Yanaka, Ph.D. ([email protected])

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Norihisa Kato, Ph.D. ([email protected])

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ABSTRACT

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This study demonstrated 0.5% dietary rutin, ellagic acid, or curcumin markedly

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increased cecal succinate levels in rats fed a high-fat diet, while catechin, caffeic acid,

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and quercetin did not. Other organic acids were modestly or hardly affected by

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polyphenols. To clarify the effects of succinate levels increased by polyphenols, we

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examined the effects of succinate on the growth and proliferation of colon cancer cells

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and angiogenesis. The growth and proliferation of HT29 human colon cancer cells and

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angiogenesis in an ex vivo model were significantly inhibited by succinate at a dose

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close to that in the cecum of rats fed polyphenols. Furthermore, succinate inhibited the

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migration of human umbilical vein endothelial cells. These findings suggest that

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consumption of some polyphenols affect the health and diseases of the large intestine

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by elevating succinate.

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KEYWORDS: rats, polyphenols, succinate, colon cancer cells, angiogenesis



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INTRODUCTION

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Polyphenols, which are abundant in plant foods, exhibit various beneficial

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activities such as anti-oxidative, anti-atherogenic, anti-diabetic, anti-cancer, anti-viral,

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anti-inflammatory, and anti-angiogenic activities1-3. We previously reported that the

18

dietary addition of some polyphenols including curcumin, caffeic acid, catechin, rutin,

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and ellagic acid suppress fecal levels of secondary bile acids, namely deoxycholic acid

20

and/or lithocholic acid (which are risk factors for colon diseases), in rats fed a high-fat

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diet4. A high-fat diet increases fecal secondary bile acids5, 6, causing compensatory

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proliferation of colonic epithelium cells7, 8. This suggests such polyphenols might have

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beneficial effects on colon health by reducing secondary bile acids in animals fed a

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high-fat diet4. High-fat diets not only increase fecal secondary bile acid levels, but also

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lower cecal organic acid production6. Mounting evidence indicates the increased

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production of intestinal organic acids including butyrate and propionate is associated

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with a decreased risk of colon cancer and colitis9, 10. Meanwhile, indigestible or limited

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digestible food constituents such as resistant starch11 and inulin12 increase cecal

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organic acid levels in both rats and humans. However, there is limited information

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concerning the effects of dietary polyphenols on cecal organic acids. Therefore, this

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study examined the effects of dietary polyphenols on cecal levels of organic acids in

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rats fed a high-fat diet.

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Succinate is an important metabolic molecule as an intermediate in the citric 4 ACS Paragon Plus Environment

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acid cycle. It is known as an umami-tasting substance and is found in shellfish, refined

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sake, and soy sauce. Recent studies suggest organic acids such as butyrate and

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propionate may contribute to intestinal immune and barrier function13-15. However, the

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role of succinate remains incompletely understood. In this study, we provided evidence

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that consumption of some polyphenols markedly elevated cecal succinate in rats fed a

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high-fat diet. Therefore, this study also examined the effect of succinate on the growth

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of colon carcinoma cells and angiogenesis.

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MATERIALS AND METHODS

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Materials

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Rutin, curcumin, catechin, caffeic acid, quercetin, succinate disodium salt, and

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), were purchased

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from Nacalai Tesque (Kyoto, Japan). Ellagic acid was obtained from Wako Pure

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Chemical Industry (Osaka, Japan). Human recombinant vascular endothelial growth

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factor (VEGF) was obtained from R&D Systems (MN, USA). Other reagents were

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special grade as commercially available.

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Animals and Diets

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Male Sprague-Dawley rats (Charles River Laboratories, Kanagawa, Japan)

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weighing 40–50 g (3 weeks old) were used. The rats were individually housed in metal

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separate cages in a temperature-controlled (24°C) room with a 12-h light–dark cycle 5 ACS Paragon Plus Environment


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(lights on from 08:00–20:00). The rats were allowed a 1-week acclimation period

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before the experiments. The rats were maintained according to the “Guide for the Care

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and Use of Laboratory Animals” established by Hiroshima University. The protocol

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was approved by “the Committee on the Ethics of Animal Experiments of the

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Hiroshima University” (Permit Number: D08-16). The control group received a

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high-fat diet that contained 30% fat, without polyphenols. The basal diet comprised

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30% beef tallow, 20% casein, 20.3% corn starch, 20% sucrose, 5% cellulose, 3.5% salt

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mixture (AIN-93G) 16, 1% vitamin mixture (AIN-93) 16, and 0.2% L-cystine (made up

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to 100% with corn starch). Rats allocated to the 0.5% polyphenol groups received the

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high-fat diet supplemented with 0.5% polyphenols including rutin, ellagic acid,

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curcumin, catechin, caffeic acid, or quercetin. All rats were fed the same amount of

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experimental diet (9, 10, 12, 14, and 15 g for days 1, 2–4, 5–7, 8–13, and 14–21,

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respectively) to suppress any variation in food intake due to ad libitum feeding. All

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diets were provided daily at 19:00 in food cups in the cages, and all rats had

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completely consumed the diets by the next morning. All rats had ad libitum access to

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deionized water. After 3 weeks of diet feeding, the rats were euthanized between

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13:00–15:00. The cecal contents were removed, weighed, and stored at -80°C for later

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analysis.

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Cecal Contents Analysis

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The levels of organic acids in the cecum were measured by high-performance 6 ACS Paragon Plus Environment

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liquid chromatography (HPLC) as described previously17. Cecal contents (300 mg)

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were homogenized by ultrasonication in 2 mL 10 mM sodium hydroxide aqueous

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solution containing 0.5 g/L crotonic acid and subsequently centrifuged at 10,000 × g

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for 15 min. Fat-soluble substances in the supernatant were removed by extraction with

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chloroform. The aqueous phase was filtered through a membrane filter, and the

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samples were subjected to HPLC. Organic acids were separated with an ion exclusion

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column and detected using an H-type cation exchanger column with a column

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temperature of 45°C, mobile phase of 5 mM p-toluene sulfonic acid aqueous solution,

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positive polarity electroconductivity detector at 45°C, and detection reagent of 20 mM

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bis-Tris aqueous solution containing 5 mM p-toluene sulfonic acid and 100 µM EDTA.

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Cell Lines and Culture Conditions

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HT29, human colon cancer cells were cultured routinely in Dulbecco’s

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modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum,

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penicillin (100 units/mL), and streptomycin (100 units/mL). Subcultures of HT29 were

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obtained by treating cell cultures with 2% trypsin/EDTA buffer. Human umbilical vein

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endothelial cells (HUVECs) were purchased from Kurabo Industries (Osaka, Japan)

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and grown in HuMedia EG 2 medium (Kurabo), which is modified MCDB 131

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medium containing 2% fetal bovine serum, 10 ng/mL recombinant human epidermal

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growth factor, 1 µg/mL hydrocortisone, 50 µg/mL gentamicin, 50 ng/mL amphotericin

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B, 5 ng/mL recombinant human basic fibroblast growth factor, and 10 µg/mL heparin. 7 ACS Paragon Plus Environment


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Subcultures of HUVECs were obtained by treating the HUVEC culture with

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Hanks’-based enzyme-free cell dissociation buffer (Gibco, New York, USA).

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HUVECs at passages 3−7 were used in the experiment. All cells were maintained at

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37°C in a humidified incubator with 5% CO2.

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MTT Assay

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For the growth assay of HT29 cells, cells were suspended in DMEM and

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plated onto 96-well culture plates (3.0 × 103 cells/100 µL). For the growth assay of

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HUVECs, cells were suspended in HuMedia EG2 Medium and plated onto 96-well

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culture plates (1.5 × 103 cells/100 µL). Cells were incubated for 24 h at 37°C in a

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humidified incubator with 5% CO2. The media were replaced with fresh media

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containing sodium succinate (1−30 mM), or vehicle (phosphate-buffered saline [PBS]).

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After exposing the cells for 24 h or 72 h, MTT solution (final concentration, 5 mg/mL

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MTT in PBS) was added to each well, and the cells were incubated for 3 h. The media

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were aspirated, and the cells were lysed in dimethyl sulfoxide. Absorbance was

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measured at 550 nm in an ELNX96 microplate reader (TFB Inc., Tokyo, Japan).

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Reported values represent the averages for 6 wells.

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BrdU Incorporation Assay

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Experiments were conducted in 6-well plates (1 × 106 cells/well) with 2

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replicates. After exposing the cells with or without the media containing sodium

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succinate (30 mM) for 24 h, HT29 cells were incubated with 100 µM 8 ACS Paragon Plus Environment

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bromo-deoxyuridine (BrdU) for 2 h at 37°C in a humidified incubator with 5% CO2.

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To detect BrdU immunoreactivity, cells were washed with PBS and fixed with 4%

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paraformaldehyde at room temperature for 10 min. After washing twice with PBS, cell

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samples were incubated with PBS with 1% Triton X-100 at room temperature for 10

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min. Cells were again washed twice with PBS and incubated with 2 N HCl. After

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removing the HCl, cells were washed 3 times with 0.1 mM borate buffer (pH 8.5). For

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blocking, 4% skim milk with PBS was added, and the cells were slowly shaken at

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room temperature. After 1 h incubation, the blocking buffer was discarded, and BrdU

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antibody (1:1000; 1 mL/plate) and DAPI (40 mg/mL, 1:1000) were added. The cells

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were incubated at room temperature for 1.5 h and then washed 3 times with PBS.

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Cy3-labeled anti-mouse IgG was added as a secondary antibody (1:1000; 1 mL/plate),

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and the cells were incubated for 30 min at room temperature. The cells were then

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washed 3 times with PBS. The cells were visualized by fluorescence microscopy, and

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BrdU-positive cells were counted on 10 image fields. The average number of

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BrdU-positive cells in each sample was subsequently calculated.

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Ex vivo Angiogenesis Assay

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Male Wistar rats (6 weeks old, Charles River Laboratories) were maintained

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according to the guide established by Hiroshima University Animal Research

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Committee as mentioned above. The ex vivo angiogenesis assay was carried out in the

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rat aortic model18, 19 with slight modifications20. Briefly, a rat aortic segment coved 9 ACS Paragon Plus Environment


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with the collagen gel was overlaid with 2 mL culture medium (RPMI 1640 medium)

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containing sodium succinate (10 or 30 mM) or vehicle (PBS) and incubated for 10

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days in a fully humidified system of 5% CO2 at 37°C. The culture medium with

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sample was changed on 7th day. After 10 days incubation, microscopic fields were

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photographed with a digital camera (DSE330-A system, OLYMPUS, Tokyo, Japan),

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and the length of the capillary was measured using Adobe Photoshop software CS3.

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Each reported value represents the average of 3 or 4 culture samples.

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Endothelial Cell Tube Formation and Chemotaxis Assays

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The HUVEC tube formation assay was performed using BD Matrigel™ (BD

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Biosciences, New Jersey, USA) as described previously20. Reported values represent

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the averages of 3 samples. The HUVEC chemotaxis assay was performed in a

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modified Boyden chamber20,

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membrane at 200× magnification, and the average number of cells in each field was

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calculated. The experiment was performed in triplicate.

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Statistical Analysis

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. Migrated cells were counted in 3 fields of each

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Statistical analysis was conducted by one-way analysis of variation (ANOVA)

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and Dunnet’s multiple-range test (Excel Statistics 2006 for Windows, Social Survey

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Research Information Co. Ltd., Tokyo, Japan). Statistical significance was estimated at

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p < 0.05.

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RESULTS

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Effect of Polyphenols on Cecal Organic Acids in Rats Fed a High-Fat Diet

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Dietary addition of polyphenols did not affect the food intake, final body

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weight, or liver weight of the rats (data not shown). The wet weight of cecal contents

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was higher in the curcumin and ellagic acid groups than the control group (p < 0.05,

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Table 1). The cecal concentration (µmol/g wet digesta) of succinate was markedly

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higher in the rutin (6.4-fold), ellagic acid (5.3-fold), and curcumin (4.6-fold) groups

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than the control group (p < 0.05). Meanwhile, dietary catechin and caffeic acid tended

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to increase cecal concentrations of succinate (p < 0.1). Acetate and butyrate

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concentrations

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significantly lower in the quercetin group compared to the control group (p < 0.05).

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The concentrations of other organic acids including formate, propionate, isobutyrate,

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and valerate were unaffected by dietary polyphenols. Dietary rutin and ellagic acid

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significantly increased the concentrations of total organic acids compared to the

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control (1.9-fold and 1.6-fold, respectively, p < 0.05). The pH of digesta was

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significantly lower in the rutin group than the control group (p < 0.05).

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Effects of Succinate on HT29 Cell Growth and Proliferation

were

significantly higher and

isovalerate concentration

was

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Succinate significantly inhibited the growth of the HT29 cells at both 10 and

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30 mM (Figure 1, p < 0.05). Analysis of cell proliferation by BrdU incorporation into

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the cells indicated 30 mM succinate significantly reduced (−30%) the proportion of 11 ACS Paragon Plus Environment


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proliferative cells (control vs. succinate: 32.6 ± 2.0% and 22.8 ± 1.8%, respectively, n

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= 6, p < 0.05).

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Effects of Succinate on ex vivo Angiogenesis and Endothelial Cell Functions

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In the ex vivo angiogenesis model using a rat aortic ring, microvessels

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appeared from the ends of aortic rings and elongated in the absence of succinate

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(Figure 2A). Meanwhile, microvessel growth was significantly inhibited in the

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presence of 30 mM succinate (Figures 2C, D, p < 0.01).

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The effect of succinate on HUVEC functions including tube formation on a

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reconstituted basement membrane, chemotaxis, and growth are shown in Figure 3.

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HUVECs inoculated onto a reconstituted basement membrane migrated, attached to

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each other, and finally formed tube structures in the tube formation assay model. In

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this assay, succinate did not significantly suppress HUVEC tube formation (Figure 3A).

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However, succinate at both 10 and 30 mM significantly inhibited HUVEC migration

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stimulated with VEGF in a gelatin-coated Boyden chamber (Figure 3B, p < 0.01).

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However, succinate did not significantly affect the growth of HUVECs (Figure 3C).

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DISCUSSION

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We previously reported that some dietary polyphenols suppress fecal levels of

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secondary bile acids, namely deoxycholic acid and/or lithocholic acid, in rats fed a

193

high-fat diet4. This study further provides the novel evidence indicating dietary 12 ACS Paragon Plus Environment

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supplementation of rutin, ellagic acid, and curcumin to a high-fat diet markedly

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increase cecal succinate concentrations in rats. However, dietary catechin, caffeic acid,

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and quercetin hardly affected cecal succinate concentrations. Aprikian et al. report that

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adding a polyphenol-rich apple concentrate (~0.7 g polyphenol/kg diet) to a normal

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diet (with 5% corn oil) did not affect cecal succinate concentrations in rats22.

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Furthermore, in our preliminary study, dietary addition of 0.5% ellagic acid to a low

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fat-diet (5% beef tallow) failed to elevate cecal succinate levels in rats (Haraguchi et

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al., unpublished data), but the reason of no such effect is unknown. Thus, the effect of

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polyphenols on cecal succinate levels might be dependent on dietary fat intake as well

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as the kinds and levels of polyphenols in the diet. Intriguingly, the dietary addition of

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rutin significantly increased cecal succinate levels, whereas quercetin did not.

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Quercetin and its glycosidic form, rutin, are common flavonoids in edible foods.

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However, the absorption processes of these polyphenols are quite different. Crespy et

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al. report quercetin can be partly absorbed in the stomach whereas rutin cannot23.

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Dietary rutin likely escapes absorption in the stomach and small intestine to reach the

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large intestine, leading it to significantly affect cecal succinate production.

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Because succinate is the major metabolite of Bacteroides in the large

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intestine24, we examined the effect of the polyphenols on the cecal profile of the

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microflora by 16S rDNA-based terminal restriction fragment length polymorphism

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analysis. The results indicate dietary 0.5% curcumin, ellagic acid, and rutin did not 13 ACS Paragon Plus Environment


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significantly affect the proportion of cecal microflora including Bacteroides,

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Clostridium, Lactobacillales, and Bifidobacterium (unpublished data). Thus, the

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increased production of cecal succinate by such polyphenols cannot be accounted for

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by the microfloral profile. In our previous study, besides 0.5% dietary curcumin,

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ellagic acid, and rutin, 0.5% dietary caffeic acid and catechin also markedly reduced

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fecal toxic secondary bile acids such as deoxycholic and lithocholic acid (which are

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risk factors for colon diseases) in rats fed high-fat diet4. Therefore, the overall

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association between alterations in the cecal profile of organic acids and fecal

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secondary bile acids appears to be weak.

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To gain further insight into the polyphenol-induce increase in cecal succinate

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levels, we examined the effects of succinate on colon cancer cells and angiogenesis.

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The results indicate 30 mM succinate, which corresponds to the cecal level in the rats

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fed various polyphenols, inhibits the growth and proliferation of HT29 colon cancer

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cells. An animal study by Inagaki et al. also demonstrates 100 mM succinate inhibits

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epithelial cell proliferation in the colon mucosa in rats25. Succinate accumulates in

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colon and stomach cancers26, 27, although it remains unclear if succinate itself is related

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to colon cancer. Succinate is known to inhibit the function of HIF prolyl hydroxylases

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(PHDs); a line of data indicates PHDs have essential functions in cell growth and

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proliferation28. Therefore, it would be of interest to determine if the inhibitory effect of

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succinate on the growth and proliferation of colon cancer cells is mediated by the 14 ACS Paragon Plus Environment

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inactivation of PHDs.

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Angiogenesis plays key role in the development of malignant tumors and

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inflammatory bowel disease29. The inhibition of angiogenesis prevents tumor growth

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and metastases, and thus inflammatory bowel disease. The results of the present ex

238

vivo angiogenesis model indicate succinate has anti-angiogenic effects at 30 mM,

239

which is the dose corresponding to cecal levels in rats fed various polyphenols.

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Furthermore, the addition of succinate inhibited HUVEC migration but not HUVEC

241

tube formation or growth. Thus, the anti-angiogenic effect of succinate might be at

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least in part mediated by suppression in VEGF-induced HUVEC migration. Succinate

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at the µM order is reported to enhance retinal angiogenesis by mediating

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G-protein-coupled receptor 91330. However, in our study, the concentrations of

245

succinate at 100-500 µM did not affect angiogenesis in an ex vivo model (unpublished

246

data). This discrepancy might be due to differences in experimental models. Although

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succinate inhibits PHDs, some conflicting evidence indicates PHDs either suppress or

248

activate angiogenesis28. Dietary rutin, ellagic acid, and curcumin are reported to

249

suppress colon carcinogenesis in rodents31, 32. This raises the question of whether the

250

suppression of colon cancer by polyphenols is related to the concentrations of

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intestinal succinate elevated by polyphenols.

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Interestingly, this study indicated that dietary supplementation of quercetin

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increases cecal concentrations of butyrate and acetate in rats. Butyrate produced by 15 ACS Paragon Plus Environment


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intestinal microbial fermentation exerts potentially useful effects on prevention and

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inhibition of colonic carcinogenesis and other intestinal and extraintestinal disoders33.

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Recent

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LPS-stimulated RAW264.7 cells34. It was reported that fermentation of apple extracts

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containing quercetin compounds resulted in an increase of butyrate and acetate in vitro

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with human fecal flora35. To our knowledge, our study provides the first in vivo

260

evidence for the elevations in these organic acids in the cecum of animals fed

261

quercetin.

study

indicated

moderate

anti-inflammatory

activity

of

acetate

in

262

In summary, this study provides the first evidence that the consumption of some

263

polyphenols remarkably increases cecal succinate levels in rats. Furthermore, succinate

264

at the levels close to those in the colonic ruminal environment suppressed the

265

proliferation of colon cancer cells and angiogenesis. These findings provide novel

266

insights into the roles of some polyphenols in the health and diseases of the large

267

intestine.

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ABBREVIATIONS USED

269

BrdU, bromo-deoxyuridine;

270

DMEM, Dulbecco’s modified Eagle medium;

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HPLC, high-performance liquid chromatography;

272

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;

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PBS, phosphate-buffered saline; 16 ACS Paragon Plus Environment

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VEGF, vascular endothelial growth factor;

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HUVEC, human umbilical vein endothelial cell;

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PHDs, prolyl hydroxylases

277 278

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Figure 1.

392

Effects of Succinate on the Growth of HT29 cells.

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Cultured cells (3.0 × 103 cells/well) were exposed to the medium for 24 h. Cell growth

394

was determined by the MTT assay. Values are means ± SEM (n = 6). Significantly

395

different from the control by Dunnet’s multiple-range test (*p < 0.05).

396 397 398 399 400

Figure 2.

401

Effect of Succinate on ex vivo Angiogenesis with the Use of a Rat Aortic Ring.

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(A-C) Representative result as to the inhibitory effects of succinate. (D) Microvessel

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length was measured on day 10 of culture. Values are means ± SEM (n = 3, 4).

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Significantly different from the control by Dunnet’s multiple-range test (*p < 0.01).

405 406 407 408 409

Figure 3.

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Effect of Succinate on HUVEC Functions. 23 ACS Paragon Plus Environment


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(A) Effect of succinate on HUVEC tube formation on reconstituted basement

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membrane. Capillary length was measured. Values are means ± SEM (n = 3). (B)

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Effect of succinate on HUVEC chemotaxis. HUVECs that migrated to the lower

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415

filters. Means of a field of three filters ± SEM (n = 9) are shown. NC, negative control

416

(medium without VEGF and sample); PC, positive control (VEGF containing medium

417

without sample). (C) Effect of succinate on the growth of HUVEC. Cultured cells (1.5

418

× 103 cells/well) were exposed to the medium for 72 h. Cell growth was determined by

419

the MTT assay. Values are means ± SEM (n = 6). Significantly different from the

420

positive control (PC) by Dunnet’s multiple-range test (*p < 0.01).


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Table 1. Effects of dietary addition of 0.5% polyphenols on cecal organic acids in rats fed a high-fat diet

Control

Rutin

Ellagic acid

Curcumin

Catechin

Caffeic acid

Quercetin

1.44 ± 0.12

1.83 ± 0.21

2.47± 0.16*

2.50 ± 0.18*

1.81 ± 0.16

1.75 ± 0.15

1.69 ± 0.11

Wet weight of cecal digesta (g) Organic acids (µmol/g wet digesta) Succinate

7.0 ± 1.6

44.5 ± 9.8*

37.4 ± 6.4*

32.5 ± 9.0*

25.1 ± 4.8

23.8 ± 5.7

7.1 ± 3.0

Formate

1.40 ± 0.20

1.48 ± 0.73

0.54 ± 0.26

0.82 ± 0.27

0.99 ± 0.30

1.27 ± 0.31

1.88 ± 0.43

Acetate

24.1 ± 2.3

29.8 ± 2.8

26.6 ± 1.1

26.3 ± 2.1

29.0 ± 2.6

29.6 ± 4.4

35.4 ± 2.0*

Propionate

6.34 ± 0.77

6.55 ± 0.83

6.66 ± 0.52

4.82 ± 0.60

5.18 ± 0.62

6.44 ± 0.74

7.63 ± 0.46

Isobutyrate

1.24 ± 0.20

ND

ND

ND

0.80 ± 0.22

1.34 ± 0.41

0.67 ± 0.18

Butyrate

4.50 ± 0.38

3.60 ± 0.48

2.96 ± 0.36

2.99 ± 0.48

5.99 ± 0.45

5.12 ± 0.98

7.78 ± 0.72*

Isovalerate

1.58 ± 0.14

0.74 ± 0.31

0.78 ± 0.14

1.22 ± 0.34

1.24 ± 0.24

1.33 ± 0.28

0.58 ± 0.10*

Valerate

1.12 ± 0.12

ND

0.32 ± 0.12

0.56 ± 0.24

0.88 ± 0.26

1.01 ± 0.32

ND

Total

46.2 ± 5.0

86.9 ± 8.0*

75.6 ± 6.8*

69.8 ± 7.8

68.3 ± 3.2

70.2 ± 8.0

60.8 ± 5.8

7.15 ± 0.12

6.40 ± 0.27*

6.97 ± 0.12

6.77 ± 0.19

6.71 ± 0.09

6.55 ± 0.20

6.81 ± 0.13

pH

Mean ± SE (n = 7). Significantly different from the control group by Dunnett’s multiple-range test (*p

Cecal Succinate Elevated by Some Dietary Polyphenols May Inhibit

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