The urinary phenolic acid profile varies between younger and older adults after a polyphenol-rich meal despite limited differences in in vitro colonic catabolism
European Journal of Nutrition
https://doi.org/10.1007/s00394
The urinary phenolic acid profile varies between younger and older adults after a polyphenol-rich meal despite limited differences in in vitro colonic catabolism
Areej Alkhaldy 0 1
Christine A. Edwards 0 1
Emilie Combet 0 1
Emilie Combet 0 1
0 Clinical Nutrition Department, Faculty of Applied Medical Sciences, King Abdulaziz University , Jeddah , Saudi Arabia
1 Human Nutrition, School of Medicine Dentistry and Nursing, College of Medical Veterinary and Life Sciences, University of Glasgow , New Lister Building , Glasgow Royal Infirmary , Alexandra Parade, Glasgow G31 2ER , UK
Purpose To investigate whether age influences colonic polyphenol metabolism. Methods Healthy participants, younger (n = 8; 23-43 years) and older (n = 13; 51-76 years), followed a 3-day low-polyphenol diet (LPD) and a 3-day high-polyphenol diet (HPD). Urinary phenolic acids (PA), short chain fatty acids (SCFA), pH and gas were monitored, alongside selected colonic bacteria. Human faecal in vitro fermentations of rutin with or without raftiline were used to evaluate the gut microbiota capacity in a subset of both groups. Results Total urinary PA were higher in the older group after HPD compared to the younger group (1.5-fold; p = 0.04), with no difference between groups in terms of a change between diets (Δ high-low diet). While 17 PA were detected in all younger participants after HPD, a narrower range (n = 8 to 16 PA) was detected in most (n = 9/13) older participants, with lower level of benzoic acid (19-fold; p = 0.03), vanillic acid (4.5-fold; p = 0.04) but higher hippuric acid (2.7-fold; p = 0.03). Faecal SCFA concentration did not change after HPD within group, with similar differential excretion (Δ high-low diet) between groups. There were no differences between groups for faecal pH, total, faecal bacteria including Flavonifractor plautii, bifidobacteria, and bacteroides. In human in vitro faecal fermentations, seven PAs were detected in both groups after 24 h of rutin fermentation, with no quantitative and modest qualitative differences between groups. Total SCFA in faecal fermentation did not differ between groups, except for butyric acid (twofold higher in the older group; p= 0.009) when rutin was fermented with raftiline over 24 h. Conclusions Urinary phenolic acids were less diverse in older participants despite limited difference in functional capacity of in vitro faecal fermentations.
Ageing; Colon; Metabolism; Microbiota; Polyphenols; Inter-individual variability; Gut; Fermentation; Phenolic acid; Age
Introduction
The number of adults aged 60 years and over will double
between 2000 and 2050, from 11 to 22% [
1
] globally. The
increase in lifespan has a direct effect on the incidence of
age-related diseases, including colorectal cancer (CRC),
which can put a tremendous strain on health services [
2
].
The importance of the role of the gut microbiota in health
has been increasingly demonstrated. The composition of the
colonic microbiota is altered in the elderly with potentially
beneficial species, such as Bifidobacteria, Bacteroides, and
Lactobacilli declining, and potentially harmful bacteria,
such as Escherichia coli, Enterobacteriaceae and
Clostridium perfringens, increasing [
3
].
Changes in the colonic microbiota and its metabolic
products including short chain fatty acids (SCFA), butyrate,
acetate and propionate, could directly affect the proliferation,
differentiation, and gene expression of the colonic
epithelium [
4–6
]. Butyrate is the primary energy source for
colonocytes, and may play a key role in maintaining homeostasis
of the colonic mucosa inhibiting proliferation and inducing
apoptosis and differentiation in colorectal cancer cell lines
[
7, 8
].
Plant foods contain a large range of bioactive molecules,
including (poly)phenolics. The conversion of the parent
(poly)phenolic compound (poorly bioavailable) to smaller
phenolic acids (with increased bioavailability) by the colonic
microbiota is likely to be an important contributor to the
beneficial effect ascribed to the compounds.
There is an emerging body of work on the potential
impact of (poly)phenolic metabolites on age-related factors
(inflammation, oxidative stress, glycation, dysbiosis)
contributing to chronic diseases such as CRC [
9–14
]. Recently,
the International Scientific Association for Probiotics
and Prebiotic expanded the definition of the prebiotics to
include (poly)phenolics as they are selectively metabolised
by gut microbiota and release microbial bioactive molecules
(including phenolic acids) that may confer local and
systematic health benefits [15]. The interactions between the gut
microbiota and (poly)phenols have been described in two
ways: (1) some (poly)phenols metabolites have the
capability to promote and/or inhibit the growth certain bacteria
[
16
] and (2) the gut microbiota contributes to the production
of small potentially bioactive molecules including phenolic
acids [
17
]. (Poly)phenolics may have protective effects in the
gastrointestinal tract by: (1) inhibiting the growth of
pathogenic species, e.g. Clostridium spp, Staphylococcus aureus,
and Bacteroides spp. [
18
]; (2) suppressing the adhesion of
gut pathogens to human gut cells [
19
]; (3) enhancing
natural killer cell activity and cytokine secretion [
20
]. In older
adults (40–50 years old), a higher urinary concentration of
phenolic metabolites of anthocyanin such as syringic acid,
p-coumaric acid, 4-hydroxybenzoic acid, and homovanillic
acid was associated with higher bifidobacteria level (>4.47
vs. < 1.18 log10copies per g faeces) [
17
].
Research into the bioavailability and metabolism of
(poly)phenols, has so far been mostly limited to young
adults [
21–26
], and inter-individual factors likely to impact
on the outcome measures have rarely been studied in depth
[
27–33
]. One study, in particular, looked at the effect of
ageing on the absorption, metabolism, and excretion of
epicatechin in healthy younger and older subjects after the
consumption of cocoa flavanols, with little difference observed
between subjects; the study did not, however, consider the
colonic metabolism of epicatechin, or phenolic acids
production [34]. As a result, very little is currently known
about the impact of ageing on the metabolic fate of (poly)
phenolics in the human gastrointestinal tract, despite the fact
that age is a key risk factor for a non-communicable disease
development.
As the body ages, several changes may influence the
bioavailability and colonic bacterial metabolism of plant (poly)
phenolics: (1) longer transit times [
35
]; (2) increase in
conditions such as irritable bowel syndrome, diverticulosis, and
colon cancers which are linked with changes in gut
microbiota [
36–39
]; (3) reduced chewing strength, leading to
different food choices and lower fibre intake [40]; (4) reduced
physical activity which may affect gut function including
frequency of bowel movements [
41
], (5) changes in the
composition/diversity of the microbiota [
3
]. As the majority of
(poly)phenolics are metabolised by bacterial enzymes in the
colon, this may influence their colonic metabolism.
As there are very limited data on the effect of ageing on
the bacterial metabolism of (poly)phenols, this study aimed
to test whether age (≥ 50 years) affects the colonic
metabolism of dietary (poly)phenolics, with a focus on flavonols,
which are ubiquitous in the Western diet. Rutin was used
as a “model” flavonol as it is a well-characterised molecule
with published evidence related to its breakdown. Rutin is
a quercetin glycoside, which resists hydrolysis and is not
deglycosylated within the human small intestine by
cytosolic β-glucosidase and/or the lactase–phlorizin hydrolase
enzymes, and thus pass intact to the large intestine. Rutin is
degraded in the colon by the microbiota to low molecular
weight, phenolic acids, and is a well-characterised molecule
[
22, 23
]. We hypothesized that the colonic metabolism of
(poly)phenols would be less efficient in older adults.
Methods
Study design
Two designs were utilised to test the study hypothesis. The
first employed a human dietary intervention to compare
the colonic metabolism of (poly)phenols between healthy
younger (< 45 years) and older (> 50 years) adults, focusing
on urinary phenolic acid excretion and gut bacterial
composition (focusing on known polyphenol-degrading
bacteria). The age cut-off, while arbitrary, was designed to obtain
two groups distinct in age. The National Health Service in
Scotland uses the age of 50 years as the cut-off for their
bowel screening programme, justified by the increased risk
of colonic diseases in this age group [
42
]. The second design
employed an in vitro fermentation model, using faecal
samples collected during the dietary intervention, to study the
metabolic (functional) capacity of the faecal microbiota
according to age when a specific flavonol, rutin
(quercetin-3-O-rutinoside), was fermented.
Subjects and recruitment
Older (≥ 50 years) and younger (< 45 years) adults were
recruited using local advertisements, printed poster
displays, and online social networking sites. Exclusion
criteria included consuming alcohol (> 4 units/day), obesity
(BMI > 30 kg/m2), taking dietary supplements, pregnancy or
risk of pregnancy, smoking, taking any medication, or
having any conditions known to affect bowel function. Approval
for this study was obtained from the Ethics committee of the
University of Glasgow, College of Medical; Veterinary &
Life Sciences (ref FM03110). All participants gave informed
written consent.
Sample size and power calculation
The sample size was calculated with GPower version 3.1
(Dusseldorf university) using urinary phenolic acid
excretion as a primary outcome. In younger adults [
43
], selected
urinary phenolic acid excretion increased from 20.6 ± 7.6 to
62.7 ± 44.3 µmol/day after a high-polyphenol diet (∆
highlow diet 42.2 ± 41.0). Pilot analyses showed that excretion
varied between ethnic subgroups, with a difference for the
∆ high-low diet of 59 µmol/day between groups
(Caucasian ∆ high-low 60.5 ± 36.0 µmol/day; Asian ∆ high-low
diet 1.5 ± 10.2 µmol/day; groups with unmatched numbers
d = 1.6). Assuming a similar effect size of d= 1.6, a total of
n = 16 participants is sufficient to detect (or not) a similar
difference (β= 80%, two tails, α = 0.05). Based on previous
experience with similar trials, we allowed for a 40%
dropout rate to recruit a sample of n = 13 individuals per group.
While recruitment in the older group reached n = 13, this
was not the case for the younger group (n = 8)—as such,
the a priori power of the study to detect an effect size of
1.33 is 80% (and 92% for d = 1.6) given the sample size and
allocation.
Measurements and sample collection
Anthropometric measurements height, weight, body mass
index (BMI), and waist circumference (WC), and blood
pressure (BP) were collected at baseline and all subsequent
appointments using standard techniques [
44
]. All
participants followed two 3-day diets separated by a 1 week
washout: a low-polyphenol diet (LPD, avoiding all fruits,
vegetables, onions, coffee, tea, chocolate, vanilla and similar
flavourings, whole meal products, alcohol, spices, and all
dietary supplements) and a high-polyphenol diet (HPD,
including flavonoid-rich foods including tomatoes, plums,
provided along with cooking guidance and recipes)
(Supplementary Table 1). The phenolic composition and fibre
content of the HPD are available in Supplementary Table 2.
Urine (24 h) was collected on the last day of each diet, and a
morning stool sample was collected at the end of the 3-day
diet.
Bowel movements
The usual frequency of bowel movements (twice daily or
more, daily, every 2–3 days or less than twice a week) was
self-reported retrospectively at the beginning of the study.
Dietary assessment of (poly)phenol intake
Prospective, weighed dietary records were kept by
participants throughout each study period. Diaries were analysed
using the WinDiets Nutritional Analysis Software (Robert
Gordon University, Aberdeen, UK) [
45
]. The mean
flavonoid content of foods was sourced from the Phenol-Explorer
database (http://www.phenol-explorer.eu/contents) [
46
]. The
flavonoid content of low-polyphenol foods such as pasta,
bread, biscuits, cakes and pastry was estimated from their
wheat flour content [
47
].
In vitro fermentation (faecal incubation)
The faecal incubation was prepared as previously described
[
48
]. The substrates used were (a) control (no substrate),
(b) 28 µM rutin, and (c) 28 µM rutin (Sigma–Aldrich
Company Ltd; Dorset, UK) with 1 g raftiline HP (Orafti, Tienen,
Belgium). Raftiline (fibre) was added to the fermentation
medium as a source of energy (carbon) to help mimic in vivo
conditions (compared to fermentations without a source of
carbon or a fast-fermented source such as glucose).
Freshly voided human faecal samples were homogenised
with sodium phosphate buffer (0.1 M, pH 7.0) in a blender
(Braun™) to make a 32% faecal slurry (16 g faecal
sample with 50 ml sodium phosphate buffer). The faecal slurry
(5 ml) was added to 44 ml of fermentation medium in 100 ml
glass sterilised bottles. The substrate (rutin, 28 µmols) was
added to the faecal slurry with or without 1 g of a highly
fermentable fibre (raftiline). Control cultures containing
no substrates were incubated at the same time. Participants
were asked to provide a whole bowel movement, and the
same sample was used to produce the faecal slurry added to
the four fermentation bottles (a single bottle for each
condition). The anaerobic conditions were established by using
anaerobic reagents in the media (reducing solution checked
with resazurin) and by flushing the media and bottles with
oxygen-free nitrogen. The fermentation bottles were sealed
with a gas-tight septum, which was fitted with a gas
collection syringe monitoring gas production. Fermentation
bottles were kept upright in a shaking water bath at 37 °C,
60 strokes/min for 24 h to simulate the colonic lumen
conditions. Two samples (3 ml) of fermentation fluid were
collected after 0, 2, 4, 6 and 24 h. One sample for each time
point was immediately stored at − 80 °C for phenolic acid
analysis. The other was mixed with 1M NaOH (1 ml) and
stored at − 20 °C for SCFA production measurements.
Colonic fermentation markers: pH and gas production
A 50-ml disposable syringe and a three-way tap were used to
measure gas production in each fermentation bottle at
different time points (0, 2, 4, 6, 24 h). An auto-calibrated portable
digital pH meter model (Hanna pH20 instruments, USA)
was used to measured faecal pH by preparing a suspension
of ~ 1 g faecal sample from each participant and diluted in
3 mL of distilled water. The pH of fermentation fluid was
determined at 0, 2, 4, 6, and 24 h with universal pH indicator
paper from 1 to 14 (Fisher Brand, UK).
Phenolic acid extraction, derivatization and analysis by gas chromatography mass spectrometry (GC–MS)
Phenolic acids were measured in urine and fermentation
fluid. Extraction and derivatisation were carried out as
previously described [
43
]. Derivatized phenolic acids were
analysed on a Trace GC interfaced to a DSQ mass
spectrometer equipped with a split/splitless injector and an AI3000
autosampler (Thermo Fisher, Hemel Hempstead, UK) as
previously described [
42
]. Identification of phenolic acids
was based on retention time (tR) and target ions [
49
].
Quantification was based on 2.5 to 15 µg calibration curves of
derivatised and analysed phenolic acid standards. The area
ratio of each standard was averaged and the coefficient of
variance calculated (R2 > 0.98).
SCFA analysis by gas chromatography with flame ionization detection (GC‑FID)
Short chain fatty acids were measured in dry faeces and
fermentation fluid. Extraction and analysis were carried out
according to Laurentin and Edwards [
50
]. SCFAs were
estimated using a TRACE™ 2000 gas chromatograph (Thermo
Quest Ltd, Manchester, UK) equipped with a flame
ionization detector (250 °C) and a Zebron ZB-Wax capillary
column (15 m × 0.53 mm id × 1 µm film thickness, catalogue
No.7 EK-G007 22, Phenomenex, Cheshire, UK). EK-G007
22, Phenomenex, Cheshire, UK).
Extraction, concentration, and purity of bacterial DNA
Bacterial DNA was isolated and purified as previously
described [
51
]. DNA concentration and purity were
determined by measuring 1.5 µl of undiluted DNA extract with
a NanoDrop ND-1000 (software version 3.7.4; Fisher
Scientific, UK). An absorbance ratio (A260/A280) greater
than 1.8 was used to assess high purity, and the absorbance
ratio at 230/260 nm was used to assess guanidinium salt
carried. Furthermore, DNA was assessed for shearing by
electrophoresis.
Determination of bacterial diversity and composition
Flavonifractor plautii was selected for quantification in
faecal samples because of its previous reported contribution to
the colonic metabolism of flavonoids [
52–54
].
Bifidobacteria, bacteroides, and total bacteria were also measured.
Realtime PCR with species-specific probes was used to quantify
individual species, bacterial populations, and total bacteria
(Supplementary Methods) on a 7500 Real-Time PCR System
(Applied Biosystems, Carlsbad, CA) using TaqMan.
Quantification was performed against serial dilution of bacterial
DNA standards obtained from pure cultures
(Supplementary Methods). DNA standards for F. plautii (DSM 4000;
6.3 ng/µl) were obtained from the German Collection of
Microorganisms and Cell Cultures (DSMZ). Standard DNA
for bifidobacteria (Bifidobacterium longum, DSM 20219T,
9.8 ng/µl), bacteroides [Bacteroides vulgatus (DSM 1447T,
27.2 ng/µl)], and total bacteria [Bacteroides vulgatus (DSM
1447T, 27.2 ng/µl)] were available in-house [
51
]. A set of
seven bacterial reference standards were prepared for each
target. The serial dilution to measure the Eubacterium
ramulus and F. plautii was 1:5, and 1:10 to measure
bifidobacteria, bacteroides, and total bacteria.
Statistical analysis
Data were analysed using Minitab 16. The
Anderson–Darling test was used for normality. Descriptive statistics are
presented as mean and standard deviation, or medians and
inter quartile range (IQR). Comparisons between groups
were carried out using Mann–Whitney test for
non-parametric data and paired test and two sample t tests for normally
distributed data. Chi-squared test was used to compare the
frequency of bowel movement between groups.
Results
Subject characteristics
13 older adults aged between 51 and 76 years old and eight
younger adults aged between 23 and 43 years old were
recruited, with no subsequent drop-out. The baseline data
for participants are presented in Table 1, with no significant
differences in anthropometric characteristics between the
two groups. Median BMI and WC were within the normal
*p value using Fisher’s exact test between age group
aBMI cut-off points of overweight [adult= 25, older adult (55–65 years old) = 28]; [
55, 56
]
bWC cut-off points of high risk (adult women= 80 cm, men = 94 cm; older women = 99 cm, men = 106 cm); [
55, 56
]
cut-off range for younger and older healthy adults. The male
to female ratio was similar between groups, with two males
and six females in the younger group and three males and ten
females in the older adult group (p = 0.99). Diastolic BP was
higher (by 17 mmHg) in older adults (p = 0.005).
Frequency of bowel movements was not different
between younger and older groups (p = 0.84) with 37.5%
in the younger group and 25% in the older group having
twice daily or more frequent bowel movement; 37.5% in
the younger group and 50% in the older group having daily
bowel movement; and 25% in the younger group and 25% in
the older group having a bowel movement every 2–3 days.
Flavonoid intake during low‑ and high‑polyphenol diets (LPD and HPD)
Flavonoid intake during the 3-day LPD was 6 mg/day in
the younger (IQR 2–10) and older groups (IQR 2–16).
During the 3-day HPD, flavonoid intake in younger adults was
510 mg/day (IQR 499–539) and 496 mg/day (IQR 438–540)
in older adults. Flavonoid intake increased from low to high
diet for both groups (p = 0.000), with no difference between
groups (Fig. 1). Moreover, there were no differences between
the groups in term of flavonols or phenolic acids intake after
the HPD (Supplementary Table 3). As urinary phenolic acid
excretion has been shown to be markedly increased between
8 and 24 h following ingestion [
31
], urinary phenolic acid
excretion was corrected for flavonoid intake on day 3 of the
diet, given that the 24 h urine collection was carried out
from the second urine of day 3, and including the first urine
of day 4.
There was no difference between groups for intake on
day 3, during the LPD [5 mg (IQR 2–10) versus 9 mg (IQR
5–21) for younger and older groups, respectively] or the
HPD [553 mg (IQR 474–666) versus 497 mg (IQR 305–643)
for younger and older groups, respectively].
Dietary fibre intake during low‑ and high‑polyphenol diets (LPD & HPD)
The fibre intake increased after the HPD in both groups
(younger: p = 0.0009; older: p = 0.002) with no difference
between groups in terms of change to dietary fibre intake
(p = 0.6). Dietary fibre intake during the 3-day LPD was
9 g/day (IQR 8–10) in the younger and 12 g/day in the older
group (IQR 8–14). During the 3-day HPD, dietary fibre
intake in younger adults was 27 g/day (IQR 26–29) and 27 g/
day (IQR 24–34) in older adults (Supplementary table 4).
Macronutrient/ micronutrient intake during low‑ and high‑polyphenol diets (LPD and HPD)
There was no difference between groups in terms of energy,
fat, protein, carbohydrate, total sugars, starch, or alcohol
intake during either low or HPD, or when considering the
difference in macronutrient intake between dietary periods
(Δ high-low diet) (Supplementary Table 4). The intake of
vitamins or dietary minerals did not differ either, except for
thiamine and copper intake during the HPD, with thiamine
intake higher (1.3-fold; p = 0.05) in the older group, and
copper intake higher (1.4-fold; p = 0.01) in the younger group
(Supplementary Table 5).
b
a
a
participant after low- and high-polyphenol diets. Median flavonoid
intake for each group is indicated by a red horizontal line. a, b
symbols indicate differences within group (LPD to HPD; p = 0.000)
Identification of phenolic acids in urine
A total of 18 urinary phenolic acids were identified and
quantified by GC-MS after LPD and HPD in the younger
group. However, 4-OHPAA was excluded from the sum of
urinary phenolic acid excreted for between-group
comparisons, as it is produced by pathways unrelated to the colonic
degradation of (poly)phenols [
31
] and did not increase in
young or older adults after the HPD. Subsequently, only
n = 17 phenolic acids are reported (Table 2).
Urinary phenolic acid excretion after low‑ and high‑polyphenol diets (LPD and HPD)
The sum of the 17 urinary phenolic acids excreted
increased ~ 5.6-folds in the younger group from 205 µmol/
day (IQR 158–525) after the LPD to 1197 µmol/day (IQR
730–1345) after the HPD (p = 0.0009), and ~ 5.5-fold in
the older group, from 336 µmol/day (IQR 190–469) to
1819 µmol/day (IQR 986–2831) respectively (p = 0.0002).
The change in urinary excretion (Δ high-low diet) was not
different between groups; however, the urinary phenolic
acid excretion was higher after the HPD in the older group
(p = 0.04) compared to the younger group (Fig. 2), with a
large variability in urinary PA excretion in the older group.
Correcting for flavonoid intake did not reveal further
difference in differential excretion between the groups. Based on
the key (statistical) differences seen between LPD and HPD,
the main phenolic acids linked to rutin metabolism that are
excreted in urine, by at least threefold, are 3-OHPAA, HVA,
3,4DOHPAA, 3,4 DIOHPPA, and 3-OHhippA.
Hippuric acid (HA) was always the most abundant acid in
urine samples in both groups (80 and 98% of total phenolic
acids for younger and older groups, respectively), and was
higher in the older group after the HPD (2.7-fold; p = 0.03)
compared to LPD. However, the change in excretion, Δ
highlow diet, was not different between groups.
The sum of urinary phenolic acids minus HA was
considered (as HA is most likely to be formed in the liver by
conjugation of benzoic acid and glycine, rather than from flavonoid
metabolism). The sum of PA excreted after the HPD increased
in both groups (compared to LPD), ~ twofold in the younger
group, from 104 µmol/day (IQR 91–129) to 211 µmol/day
(IQR 146–233) (p = 0.003) and in the older group, from
56 µmol/day (IQR 48–77) to 100 µmol/day (IQR 78–127)
(p = 0.007). The difference in urinary excretion (Δ high-low
diet) remained similar between groups. However, urinary
phenolic acid concentration was higher after the LPD (p = 0.03)
and HPD in the younger group (p = 0.02); Fig. 3.
Most notably, there were important qualitative and
quantitative differences between groups for the excretion of
individual phenolic acids (∆ high-low diet). While 17
phenolic acids were identified in all younger participants after
both diets, only eight phenolic acids were excreted by all
older participants (BA, MA, 3-OHPAA, HVA, 4-OHMA,
3,4diOHPAA, HA, 3-OHhippA) (Table 2). A narrower
range of urinary phenolic acids (n = 8–16) was detected in
most (n = 9/13) older participants. The older group excreted
19-fold less BA (p = 0.03), and 4.5-fold less VA (p = 0.04)
than the younger group (Table 2).
Faecal pH after low‑ and high‑polyphenol diets (LPD and HPD)
After the HPD, the faecal pH significantly decreased in the
older group from 7.7 (IQR 7.4–7.9) to 6.9 (IQR 6.7–7.4;
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Fig. 2 24-hour urinary phenolic acid profile excretion (µmol/day)
after low and high-polyphenol diets in younger (n = 8) and older
(n = 13) participants. Each circle indicates the measurement of the
urinary phenolic acids profile for each participant after low and
highpolyphenol diets. Median urinary phenolic acids for each group are
indicated by a red horizontal line. a, b symbols indicate differences
within group (LPD to HPD; younger: p = 0.0009; older: p = 0.0002).
§Symbol indicates differences between groups (HPD vs. HPD;
p = 0.04)
b §
a †
b §
b §
Fig. 3 24-hour urinary phenolic acid profile excretion without
hippuric acid (µmol/day) after low- and high-polyphenol diets in younger
(n = 8) and older (n = 13) participants. Each circle indicates the
measurement of the urinary phenolic acids profile without hippuric acid
for each participant after low- and high-polyphenol diets. Median
urinary phenolic acids profile without hippuric acid for each group
p = 0.006), but not in the younger group (7.2, IQR 6.9–7.5
to 6.7, IQR 6.4–7.1). Looking at pH changes with diet (Δ
high-low diet for pH), there was no significant difference
between the groups (Supplementary Fig. 1).
Faecal SCFA after the low‑ and high‑polyphenol diets (LPD and HPD)
Total faecal SCFA concentration (sum of all SCFA) did
not change significantly from low to HPD in either group
is indicated by a red horizontal line. a,b symbols indicate differences
within group (LPD to HPD; younger: p = 0.003; older: p = 0.007).
†Symbol indicates differences between groups (LPD vs. LPD;
p = 0.03). §Symbol indicates differences between groups (HPD vs.
HPD; p = 0.02)
despite a threefold increase in fibre intake [younger from
164 µmoles/g dwt (IQR 124–218) to 192 µmoles/g dwt (IQR
162–214); older from 258 µmoles/g dwt (IQR 168–280) to
265 µmoles/g dwt (IQR 208–301)]. Although there were
no significant differences (Δ high-low diet) between the
groups, the mean SCFA concentration was 1.4 fold higher
in the older group compared to the younger group after the
HPD (p = 0.01; Supplementary Fig. 2). There were no
differences in the changes (Δ high-low diet) in each specific
acid between groups. However, the absolute levels of acetic
acid were higher after both the LPD (1.5-fold; p = 0.01)
and HPD (1.4-fold; p = 0.02) in the older group, compared
to the younger group. Absolute levels of heptanoic acid
were higher after the HPD in the younger group (1.8-fold;
p = 0.03; Supplementary Table 6).
Concentration of bacterial DNA isolated from faecal samples after LPD and characteristics of the qPCR run condition
High quality and high yield DNA was obtained from all
faecal samples. The purity and yield of the extracted DNA was
high (1.7–2.0 absorbance ratio 280/260 nm; yield 463 ng/
µL (IQR 429–552) for older group samples, 519 ng/µL (IQR
480–576) for the younger group samples). The faecal DNA
appeared intact and compact as a high-molecular-weight
band when electrophoresed through a 1.5% agarose gel. The
qPCR amplification efficiency was within the normal range
(90–105%) for the total bacteria, Bacteroides–Prevotella,
and Flavonifractor plautii in both groups; however,
efficiency for Bifidobacterium spp. was just below the normal
range (87%) in both groups. The coefficient of determination
(R2) range was between 0.994 and 0.999 for all bacterial
groups and species in both groups. Absolute levels of
bifidobacteria, bacteroides, and Flavonifractor plautii did not
differ between younger and older groups (Table 3).
In vitro fermentation of rutin
Ten fermentations were carried out using the stool samples
of six younger (one male and five females) and four older
subjects (one male and three females), collected after the
LPD. Rutin was fermented for 24 h with or without fibre
(raftiline) to test the metabolic capacity of the gut faecal
contents, including the microbiota, in relation to ageing.
There was no change in the pH of the fermented faecal
fluids containing rutin alone, over time, in either group
(Supplementary Fig. 3). However, raftiline and the combination
of rutin + raftiline reduced pH from 7 to 5 by 24 h, in both
groups. There was no difference in total gas production
between groups after 24 h. In both younger and older groups,
the combination of rutin + raftiline increased gas
production more than rutin alone (p = 0.02; p = 0.03; respectively).
Moreover, the older group produced 1.9-fold more gas over
24 h fermentation than the younger group when raftiline was
fermented alone (p = 0.02; Supplementary Table 7).
Metabolism of rutin in faecal fluids and phenolic acids formation
Seven phenolic acids were detected in fermentations of
rutin with faecal samples from younger and older adults,
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over 24 h, with great variability within and between groups
(Table 4).
Only PAA and 3-OHPPA were detected in all
fermentations, while 3-OHPAA (younger: 5/6; older 3/4) and
4-OHPPA (younger: 5/6; older 4/4) were detected in most
fermentations. 3,4diOHPAA, a recognised rutin
metabolite, was seen in all younger donors and most older donors
(younger: 6/6; older 3/4). Finally, 4-OHBA (younger: 1/6;
older 2/4) and 3,4diOHPPA (younger: 2/6; older 2/4) were
detected in the fermentations of some donors only. Five
of these phenolic acids were also detected in the control
fermentation at 0 h: PAA was detected in all younger and
older donors; however, 4-OHPPA (younger: 2/6; older 3/4),
3-OHPPA (younger: 2/6; older 4/4), 4-OHBA (older only
2/4), and 3-OHPAA (younger only 1/6) were detected in the
fermentations of some donors (Table 4).
The sum of the seven phenolic acids increased over time
in all rutin fermentations, with or without raftiline in the
younger group (35-fold with rutin only, p = 0.000, sixfold
with rutin + raftiline, p = 0.001) and in the older group when
rutin was fermented alone (21-fold, p = 0.04), without
significant increase with the addition of raftiline (ninefold change,
p = 0.06). There was no difference in the sum of the seven
acids between groups after 24 h in all rutin fermentations,
with or without raftiline. The addition of raftiline to the
fermentation inhibited the formation of all seven phenolic acids
after 24 h, by sevenfold (p = 0.02) in the younger group and
2.8-fold (p = 0.33) in the older group with no detectable
difference between groups (Fig. 4).
Individual phenolic acids formed after the fermentation of rutin
PAA increased over time in the fermented faecal fluids
from the younger group with rutin (25-fold; p = 0.000) and
rutin + raftiline (2.1-fold; p = 0.03), and in the fermented
faecal fluids from the older group with rutin only (19-fold;
p = 0.04) but not with the addition of raftline. The older
group formed more PAA in the fermentations of rutin +
raftiline (threefold; p = 0.01) and raftiline only (fourfold;
p = 0.03) compared with the younger group but no
differences were detected between groups when rutin alone was
fermented. 3-OHPPA also increased in the fermented
faecal fluids containing rutin only in both younger (13-fold,
p = 0.02) and older (25-fold, p = 0.03) groups, with no
differences between the groups (Table 4).
Metabolism of rutin in and SCFA production faecal incubation
Total SCFA concentrations increased over time in the
fermentations of rutin + raftiline (38-fold for the younger
group, p = 0.005; 58-fold for the older group, p = 0.03) and
rutin only (13-fold for younger group, p = 0.005; 14-fold
for the older group, p = 0.03) with no differences between
groups. Acetic and propionic acid did not differ between the
groups but butyric acid concentration was higher in the older
group (two-fold; p = 0.009) at 24 h (Supplementary Table 8).
Discussion
This study tested the hypothesis that age influences the
metabolism of dietary (poly)phenols, which may be relevant
for gut health and the development of chronic diseases. To
our knowledge, this is the first dietary semi-controlled study
investigating the colonic metabolism of dietary polyphenols
in different age groups using human feeding and in vitro
faecal fermentation designs. 3 days on each diet was enough
for (poly)phenol-rich foods to be supplied to the colon and
be fermented over the course of several meals. The present
study showed:
1. Inter and intra variability in younger and older groups in
terms of urinary excretion of phenolic acids after LPD/
HPD.
2. Limited differences between groups in phenolic acid
formation after fermentation of rutin with in vitro batch
faecal incubations.
Previous work compared absorption, metabolism, and
excretion of epicatechin in healthy younger and older
subjects, with limited differences in flavonol metabolites
levels in plasma and 24-h urinary collection between the age
groups [
34
]. Our own results align with these findings,
showing limited quantitative differences between groups in
term of phenolic acid excretion, as catabolites of
(poly)phenolics or colonic metabolism capacity. These same findings,
however, highlight important qualitative differences between
the two age groups. While all younger participants excreted
all 17 phenolic acids in urine after the 3-day
high-polyphenols diet, not all older participants excreted the full panel.
This varied from one participant excreting 8 out of 17
phenolic acids to another excreting 17 out of 17 phenolic acids.
This is particularly relevant as inter-individual variability
in the response to (poly)phenolics (including flavonols) is
now being increasingly considered [
61
]. However, whether
inter-individual variability of (poly)phenols metabolism
suggests a biological difference between the age groups is still
unclear.
The phenolic acids consistently seen in urines from
both younger and old subjects were BA, MA, 3-OHPAA,
HVA, 4-OHMA, 3,4diOHPAA, HA, and 3-OHhippA
with 3-OHPAA, HVA, and 3,4diOHPAA, well described
rutin metabolites [
27, 48
]. Intermediates (3,4-diOHBA,
3,4-diOHPAA, 3,4-diOHPPA) were detected in variable
24 h
ND
ND
32.9 (n = 6/6)
7.1 (n = 6/6)
ND
3.2 (n = 1/4)
65.8 (n = 3/4)
52.0 (n = 3/4)
ND
ND
0.4 (n = 2/6)
ND
ND
ND
0.5 (n = 2/4)
0.6 (n = 1/4)
PAA phenylacetic acid; 3-OHPAA 3-hydroxyphenylacetic acid; 3-OHPPA 3-hydroxyphenylpropionic acid; 4-OHBA 4-hydroxybenzoic acid;
4-OHPPA 4-hydroxyphenylpropionic acid; 3,4diOHPAA 3, 4-dihydroxyphenylacetic acid; 3,4diOHPPA 4-hydroxy-3-methoxy-phenylpropionic
acid; ND not detected
aPhenolic acids previously described in the literature
are labelled “Y”. #There was a statistically significant increase in the
sum of PA in younger adults over time in the blank fermentations
(p < 0.001) and when rutin and raftiline were combined (p = 0.001). §
There was a statistically significant increase in the sum of PA in older
adults over time in the fermentations with rutin alone (p = 0.04)
amounts at different time points, but the absence of these
phenolic acids at any given point does not simply
illustrate capacity, but also the kinetics in f lavonol
catabolism. The evidence in this study and that of others is
clearest 3-OHPAA and 3,4-diOHPAA, as they are both
found in the in vivo and in vitro studies. Three more,
HVA, 3,4-diOHPPA, and 3-OHhippA were present in the
in vivo study after the HPD in both age groups, but not in
the in vitro fermentations—highlighting the likely role of
phase II metabolism in the liver in the synthesis of these
compounds. However, VA, GA, 4-OHMA, 3,4-diOHBA
which were present in all younger participants, but only
in some of the older participants, could be linked to
agerelated variations.
With their simpler structure and increased
bioavailability, phenolic acids are one of the major (poly)phenol
metabolites present in blood with a maximum plasma
concentration of 0–4 µmol/L with an intake of 50 mg
aglycone equivalents [
62
], and reported anti-inflammatory,
anti-carcinogenic, anti-proliferative, anti-glycative and
prebiotic properties in in vitro and/or in vivo studies [
16,
62–65
]. As the concentration in the colon is much higher
than in the circulation with concentration ranging from
46 to 479 µmol/L in human faecal water [
59, 66
], some
effects exerted at high doses may still be physiological
[67]. Moreover, while phenolic acid intake is usually in
the range of 50–900 mg per day, phenolic acids in urines
can reach levels close to 1 mmol/day—as urinary phenolic
acid largely come from the colonic metabolism of (poly)
phenolics, but also other compounds such as benzoic acid
and precursors (quinic acid, aromatic amino acid
tryptophan, tyrosine, and phenylalanine). Other sources of
benzoic acid are benzoates (E numbers 210–219) which are
commonly used in food, medications, and mouthwashes
[
68, 69
].
The bioactivity of specific phenolic acids is less well
defined, with examples including 3,4diOHPAA an
inhibitor of enzymes involved in detoxification (GSTT2),
inflammation (COX-2) and anti-proliferative activity in
HCT116 colon cancer cells [
67, 70
]; 3,4-diOHPPA (3 µM) and
3,4-diOHPAA (3 µM), inhibitors of the pro-inflammatory
cytokines such as TNF-a, IL-1b and IL-6 in
lipopolysaccharide-stimulated peripheral blood mononuclear cells [
70, 71
];
and gallic acid (882 µM) inhibitor of Clostridium
histolyticum in in vitro faecal fermentation [
65, 72
].
The intra-group variation we observed could be due to
the variation in gut microbiota, themselves influenced by
genetics, lifestyle, diet and ageing. Older adults excreted
a higher amount of hippuric acid in their urine. Hippuric
acid is formed in the liver by conjugation of colonic benzoic
acids with glycine; therefore, the high amount of HA could
come from sources other than (poly)phenol-rich food
metabolism, such as amino acids [
68, 69
]. Moreover, urinary
hippuric acid correlated with hypertension and obesity [
73, 74
],
which would be consistent with the characteristics of some
in our older group. The high intra-group variability within
the older group for the sum of the phenolic acids was mainly
attributable for hippuric acid (30-fold difference between
low and high excreters); however, the variability reduced to
only threefold when hippuric acid was not included in the
sum of phenolic acids.
Several potential mechanisms could explain the absence
of some of the phenolic acids in the urine of participants as
well as the low amount of phenolic acid excreted in the urine
of the older group.
The first mechanism involves a decrease in colonic
absorption. As the individual ages, colonic absorption is
reduced and the mucosal surface area is diminished [
75, 76
].
During in vitro faecal fermentation of rutin, a high amount
of PAA was detected in the faeces-only control at 0 h in the
older group, suggesting that bacterial phenolic metabolism
is not the sole source of increased PAA. The comparatively
greater formation of PAA in the fermentation fluid of the
older participants in the presence of rutin may also point
toward a higher capacity of faecal material from older
participants to effectively metabolise rutin with the low urinary
phenolic acid excretion due to poorer absorption of the
phenolic compounds in the colon. Variability in enterohepatic
(re)circulation is not well studied and could be an additional
contributing factor [
77
].
The second mechanism relates to the effect of
ageing on the colonic microbiota composition and associated
microbial and enzymatic activities. In the human colon,
bacterial enzymes (β-glucosidases, β-glucuronidases, and
α-rhamnosidase) hydrolyse rutin to release the
quercetin aglycone [
78
]. Insufficient or lower levels of bacterial
enzymes in the colon could be one reason for the absence
and low urinary phenolic acid in the older group. Although
these bacterial enzymes were not measured directly in this
present study, we could not detect a difference between
groups in the quantity of bifidobacteria, bacteroides, or
Flavonifractor plautii bacteria. The aim of the current protocol
focusing on targeted bacteria was not to study the impact of
dietary components, but instead to establish how pre-HPD
levels of these bacteria could factor in the differences in
phenolic acid excretion. Selected bacteria were measured
in representative faecal samples and the values were not
corrected for the total amount of daily faecal output, which
could affect the accuracy of the measurement. Indeed, as
the bacteria measurements were performed after the
lowpolyphenol diet which was lower (but not devoid of) in
fibre (younger: 9 g/day; older: 12 g/day), with a view to
describe the levels of the targeted bacteria before exposure
to the HPD. It is not clear how fibre intake impacts, short
term (here, 3 days) on bacterial diversity and total numbers.
Additionally, the selected bacteria measured in this study
may not be representative of all bacteria responsible for
(poly)phenol metabolism found in the colon such as
Enterococcus casseliflavus [
53, 79
] Butyrivibrio spp. [80], and
Bacteroides distasonis [
58
], and may have been influenced
by the nutrients in the low-polyphenolic diet (although the
impact of such a short-term intervention on bacterial
populations themselves is not clear). Based on this study’s finding,
the differences in colonic fermentation between groups, as
modeled in vitro, are unlikely to be linked to the differences
seen in the urinary phenolic acid excretion between groups.
Woodmansey et al. [
81
] reported that a high faecal pH in the
elderly (due to a low fibre intake) may lead to a reduction in
SCFA production. In the present study, faecal pH was close
to 8.0 in the older group after the LPD and above 7.0 after
the HPD. However, even though the SCFA were higher in
the older group after the HPD, there were no differences
between groups when considering the change from the low
to high-polyphenol diet. As mentioned above, the
measurement of the SCFA was performed in a representative faecal
sample and correction for the total daily faecal output was
not possible. A pH above 7.0 could be due to other
compounds such as ammonia, which have been reported in older
adults [
81
], either due to lower fibre intake and/or increased
activity of proteolytic bacterial species, such as
Fusobacteria, Propionibacteria, and Clostridia.
It is important when studying the bioavailability of
bioactive molecules to consider stratification of the population
into responders and non-responders. Age and other factors
such as gender, genetics, gut microbiota or physiological
status involved in creating inter-individual variability are
important to consider before developing such stratification
models. Improvement in the knowledge of factors such as
age, gender, genetic and gut microbiota composition, and
their influence on the metabolism of plant food bioactive
molecules, together with the development of methods to
identify responsiveness profiles will enhance the
development of effective and innovative products for prevention of
chronic disease [
82
]. The strength of this study includes the
combination of in vivo and in vitro designs to assess the
colonic bacterial metabolic ability of each group. However,
the full profile of (poly)phenol metabolites was not measured
in the urine and the phenolic acids were not measured in
faecal samples, which could have provided useful information
regarding the absorption and accumulation of the phenolic
compounds in the colon. Only selected bacterial species
were measured in the faecal samples, and other gut
bacteria such as, Clostridium scindens, Eubacterium desmolans,
Eubacterium ramulus [
52, 60, 79, 83
], Butyrivibrio sp [80],
and Bacteroides distasonis [
58
] could potentially contribute
to the colonic metabolism of dietary (poly)phenols.
The differential excretion of phenolic acids in older adults
may be linked to differences in the gut microbiota profile or
functionality, but may also be linked to other
gastrointestinal factors. Age was shown to impact on the phenolic acid
profiles excreted in urine between groups qualitatively, as
well as quantitatively (the younger group excreted higher
phenolic acid in urine after both low- and
high-polyphenolic diets). These differences were not explained by the in
vitro fermentations, as a similar range of phenolic acids was
formed from the substrates in both groups, with minimal
quantitative differences. Our study highlights the importance
of study participant selection (and description) in
experimental design aiming to explore the colonic metabolism of
plant bioactives. Age may be a factor influencing the
considerable variance in measurements seen in some studies.
The restricted urinary phenolic acid profile observed in
some participants in the older group may have relevance for
colonic health, and should be investigated further.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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