177
ClinicalScience(1991) 80,177-182
The contribution of the large intestine to blood acetate in man
W. SCHEPPACH*, E. W. POMARE?, M. E m $
AND
J. H. CUMMINGSS
*Department of Medicine, University of Wuerzburg, Wuerzburg, F.R.G., ?Department of Medicine, Wellington School of Clinical
Medicine, Wellington Hospital, Wellington, New Zealand, and SMRC Dunn Clinical Nutrition Centre, Cambridge, U.K.
(Received 18 May/lS August 1990; accepted 14 September 1990)
SUMMARY
1. To test the hypothesis that the colon contributes
significantly to venous plasma acetate concentrations,
experiments were carried out in healthy volunteers and
ileostomy patients.
2. Fasting plasma acetate levels were measured in 10
ileostomy patients and compared with those in 21 control
subjects. Values in ileostomy patients (21.3 k 0.8 pmol/l)
were significantly lower than in control subjects
(48.0 f4.2 pmol/l).
3. Plasma acetate concentration was estimated in eight
healthy volunteers during 108 h of continuous fasting.
Acetate concentrations rose significantly from 12 h
(43.9 k4.4 ,umol/l) to 108 h of starvation (114.0 f 15.6
pmol/l) and fell back to normal fasting values on refeeding and another 12 h fast (44.3 k 4.7 pmol/l).
4. When colonic fermentation was stimulated after
oral ingestion of 10 g of lactulose, the plasma acetate concentration increased significantly (from 44.0 k 7.4 to
114.4 & 16.2 pmol/l) in seven healthy control subjects.
This rise was not affected by concomitant dosage of
metronidazole.
5. These data suggest that there are at least two major
sources of acetate in man, an endogenous source and the
colon which probably becomes more important when fermentation of carbohydrate is occurring.
energy requirements [2,3].In these animals the hind gut is
also a site for fermentation and may supply a further 10%
of energy requirement as SCFA [4, 51. Fermentation also
occurs in the caecum and colon of non-ruminant species
such as the horse, rabbit, pig and rat, where again it contributes to energy needs through the production of SCFA
[6-81.
In man the importance of the gut as a source of SCFA
which arise from fermentation is unknown [9]. Acetate is
rapidly absorbed from the human colon [lo] and is
usually present in human peripheral and portal blood
[ 11- 171. Acetate is used as a fuel by many tissues [ 12- 14,
18-20], especially cardiac muscle [ 11, 18, 211. In species
such as the rat, when portal blood levels fall below
200-250 pmol/l, net acetate synthesis occurs in the liver
[19,22-241.
In order to assess the contribution of the large intestine
to blood acetate in man we have measured plasma acetate
concentrations in healthy subjects and in ileostomy
patients after an overnight fast and in a further group of
healthy subjects during 5 days of complete fasting. We
have also measured peripheral venous acetate levels in
healthy subjects after a dose of water, the fermentable
carbohydrate lactulose, or the same dose of lactulose with
the addition of the antimicrobial agent metronidazole in
an attempt to suppress fermentation.
Key words: acetate, colon, dietary fibre, fermentation,
short-chain fatty acids.
METHODS
Abbreviation: SCFA, short-chain fatty acids.
Study 1 (healthy control subjects and ileostomy
patients). Blood samples were taken from 10 ileostomy
patients and 2 1 healthy volunteers after an overnight fast
of 12 h. The ileostomy patients (five male, five female),
mean age 67.1 years (range 56-80 years), had all undergone colectomy for ulcerative colitis at least 2 years previously and were in good health at the time of the study.
The average ileal resection in these patients was 10 cm
with a maximum of 21 cm. None had taken antibiotics in
the 3 months before the study. Subjects were asked to fast
from 20.00 hours the day before giving blood and were
visited at home between 08.00 and 10.00 hours when a
INTRODUCTION
Short-chain fatty acids (SCFA= acetate, propionate and
butyrate) arise principally from the fermentation of carbohydrates (starch and dietary fibre). In the rumen [l]SCFA
are important metabolic fuels providing 50-60% of
Correspondence: Dr Wolfgang Scheppach, Department of
Medicine, University of Wuerzburg, Josef-Schneider-Strasse 2,
D-8700 Wuerzburg, F.R.G.
Subjects and protocols
178
W. Scheppach et al.
10 ml blood sample was taken from an anticubital vein.
Twenty-one healthy volunteers ( 12 male, nine female),
mean age 39.0 years (range 19-73 years), served as controls. All were asked to fast for 12 h before having blood
taken and either came into the laboratory or were visited
at home. Blood was analysed (SCFA) on the same day as
it was collected.
Study 2 (fasting). Eight healthy students (seven male,
one female), aged 20.4 years (range 19-21 years), who
undertook to fast for charity, were studied. The fast lasted
4 days, during which time they carried on with their
normal lives but ate no food and drank only water. Blood
was taken after an overnight fast (12 h, day 1)and then in
the morning of days 3 and 5 (60 and 108 h). The students
were allowed to eat on day 5 after the morning blood
sample. A fiial sample was taken on the morning of day 6
after an overnight fast of 12 h (132 h from the beginning
of the study). Blood was analysed immediately (SCFA)or
stored frozen at -20°C until analysis (free fatty acids,
3-hydroxybutyrate).
Study 3 (lactulose and metronidazole). Eight healthy
male subjects who were university or laboratory staff or
students (average age 27.7 years, range 20-41 years)
participated in this study. The experiments were carried
out at the metabolic suite of the MRC DUM Clinical
Nutrition Centre. Subjects lived at the centre and their
diet was supplied from the metabolic kitchen. They were
encouraged to lead as normal a life as possible except for
the days of the tests. The day before each test subjects
were asked to minimize the amount of dietary fibre and
starch they ate. At 19.00 hours the evening before each
study day a polysaccharide-free meal was eaten consisting
of 50 g of prawns, 50 g of mayonnaise (made from olive
oil, egg yolk and salt), 50 g of chicken, two eggs ( = 100 g),
18 g of butter, 41 g of meringue, 53 g of double cream, 44
g of cheddar cheese and 330 ml of Coca Cola. The
subjects then fasted until 08.00 hours the next day when
the tests commenced.
In the initial experiment (six subjects) a 400 ml drink of
mineral water was given at 08.00 hours and venous blood
samples were obtained at 30 min intervals for 2 h and
then hourly for a further 2 h. The subjects rested in a
comfortable chair throughout.
Next, seven of the subjects followed a similar dietary
protocol but instead of 400 ml of water took 10 g of
lactulose as a syrup (15 ml; Duphalac; Duphar Ltd,
Southampton, U.K., containing 3.35 g of lactulose, 0.3 g
of lactose and 0.5 g of galactose/5 ml) with 385 ml of
water. Venous blood was sampled half-hourly for 6 h.
Thirdly, five subjects took metronidazole for 5 days
beforehand (400 mg three times daily for 4 days and 800
mg three times daily on the day before the test) and then
repeated the study which involved ingestion of 10 g of
lactulose. Metronidazole (800 mg) was also taken with the
dose of lactulose. Once the subjects had taken metronidazole they were not allowed to participate in any other
part of the study.
End-expiratory breath samples were collected at 10
min intervals throughout these studies for measurement
of hydrogen and methane.
For all these studies blood was taken from the forearm
without (or with minimal) compression. For the lactulose
and metronidazole study an indwelling 20G intravenous
catheter (Gelco Ltd, Tampa, FL, U.S.A.) was used, kept
open by flushing with 1 ml of dilute heparin (100 units/
ml) each time blood was collected. Blood was taken
directly into lithium-heparin tubes, centrifuged ( 1000 g
for 10 min) and plasma was taken immediately for SCFA
analysis.
The studies were approved by the Ethical Committee
of the MRC DUM Nutrition Unit, Cambridge.
Chemical methods
Plasma SCFA were measured in duplicate by gas
chromatography after freeze-transfer as previously
described [25]. Free fatty acids were estimated by the
colorimetric method of Duncombe [26], and 3-hydroxybutyrate by the 3-hydroxybuyrate dehydrogenase method
of Williamson & Mellanby described in [27]. Breath
hydrogen was measured with a selective electrochemical
cell (GMI Exhaled Hydrogen Monitor; GMI Medical Ltd,
Redrew, Paisley, Scotland, U.K.), calibrated with a 96
p.p.m. standard gas. Ail breath samples were taken in
duplicate and simultaneous samples of room air were
used on each occasion as a control. Methane was
measured by gas chromatography using a F'ye 104 gas
chromatograph fitted with a 2 ml gas sampling loop and a
flame ionization detector. Methane was separated on a
2 m x 4 mm glass column packed with Poropak-Q at 50°C
using nitrogen as a carrier gas. The instrument was calibrated with a 50 p.p.m. standard gas (British Oxygen
Company Special Gases).
Statistical analysis
Results are given as means fSEM. Statistical calculations were performed by Student's t-test throughout using
SPP (a statistical package for personal computers by
Patrick Royston, London School of Hygiene and Tropical
Medicine).
RESULTS
Study 1 (Fig. 1)
After an overnight fast the plasma acetate concentration in 21 healthy subjects was 48.0k4.2 pmol/l, which
was significantly greater than that found in the 10
ileostomy patients (21.3f0.8 pmol/l; t=4.3, PcO.01).
Fig. 1 shows that there was no overlap between the values
in the two groups, since the lowest value in healthy
controls was 27 pmol/l and the highest in ileostomy
patients was 24 pmol/l. Since the ileostomy patients were
on average an older group than the healthy volunteers
(67.1 versus 39.0 years), the fasting plasma acetate
concentrations for the five oldest healthy control subjects
(mean age 70.6f1.7 years) were compared with those
obtained in five ileostomy patients matched for age as
nearly as possible (71.4 k 2.6 years). The acetate values
Gut-derived blood acetate
.
t
i
-E3
179
Normal
Starvation oeriod, diet
70
*Ol
....
I
I
lleostomy
patients
Control
subjects
Fig. 1. Venous plasma acetate concentration after 12 h
fasting in 10 ileostomy patients (O),in five age-matched
older control subjects ( B ) and in 16 younger control subjects (.). For details, see the text.
were 34.0 f2.7 ,umol/l in healthy control subjects and
20.0 f 1.0 ,umol/l in ileostomy patients ( t =4.7, P < 0.01).
Study 2 (Fig. 2)
After an overnight fast the plasma acetate concentration was similar in the eight students (43.9 f4.4 pmol/l)
to that seen in the 21 healthy volunteers in study 1. However, after 60 h of continuous fasting, plasma acetate concentration had risen significantly to 104.6 f 13.2 ,umol/l
( t = 12.4, P<O.OOl), remaining elevated at 108 h
(114.0 f 15.6 ,umol/l) and finally falling back to normal
fasting levels on refeeding (44.3 k 4.7 ,umol/l). There was
no sigmficant difference between the initial and final
fasting values (r=0.34, P < 0 . 5 ) or between values at 60
and 108 h ( t= 0.45, P < 0.5).
Fig. 2 shows the changes in circulating free fatty acid
and 3-hydroxybutyrate concentrations. The free fatty acid
concentration rose significantly from 0.6 f0.1 mmol/l
after a 12 h fast to 1.8k0.1 mmol/l after 60 h of starvation ( t = 16.9, P < O . O O l ) . No further increase was seen
between 60 and 108 h of fasting (1.9k0.1 mmol/l at
108 h; r=0.36, P<O.5). After refeeding (108-120 h) and
another overnight fast (120-132 h) the free fatty acid
concentration decreased to 0.3 k 0.02 mmol/l, which was
not significantly different from that observed after the
initial overight fast (132 vkrsus 12 h; t = 1.95, P< 0.5).
The venous
3-hydroxybutyrate concentration
increased significantly from 0.2 t- 0.1 mmol/l (12 h) to
3.3f0.4 mmol/l (60 h) (t=8.32, P<O.OOl). A further
rise was observed from 60 to 108 h (3.8 f 0.5 mmol/l)
(108 h versus 60 h t=2.80, P<O.O25). After refeeding
and another overnight fast )132 h) the 3-hydroxybutyrate
concentration fell to 0.1 k-0.02 mmol/l (132 h versus
Time (h)
Fig. 2. Venous plasma acetate, free fatty acid (FFA) and
3-hydroxybutyrate concentrations in eight healthy subjects after fasting periods of 12, 60 and 108 h. The last
blood sample (132 h) was drawn after refeeding and
another 12 h fasting period. Vertical bars indicate fSEM.
108 h t=6.57, P<O.OOl; 132 h versus 60 h: t=7.61,
P < O . O O l ) . Values at 12 and 132 h were not significantly
different from each other ( t= 0.79, P< 0.5).
As a clinical sign of starvation the body weight of the
fasting subjects fell from 66.6 3.3.3 kg (12 h) to 63.2 k 3.0
kg(108 h) (P<0.05).
Study 3 (Figs. 3-5)
Fig. 3 shows the changes in the plasma acetate concentrations after an overnight fast and then after a drink
of water only, 10 g of lactulose, or lactulose plus metronidazole. Fasting values were again in the expected range,
except for the five subjects taking metronidazole who had
elevated plasma acetate concentrations at 70.2 f 13.6
,umol/l. No change was seen after drinking water ( t= 0.34,
P<0.5) but ingestion of 10 g of lactulose led to a rapid
rise in plasma acetate concentration which peaked at
114.4 f 16.2 ,umol/l after 3 h. Adding metronidazole to
the regimen did not alter the response of plasma acetate
concentration to lactulose. The area under the plasma
acetate concentration curve in the subjects who ingested
lactulose alone was similar to that in the subjects who
took lactulose and metronidazole ( f = - 0.1, P< 0.5).
However, in both groups there were significant movements above baseline (lactulose: t = 4.4, P< 0.01; lactulose plus metronidazole: t = 2.39, P < 0.05).
Breath hydrogen responses were variable and did not
follow the same pattern as the plasma acetate concentration responses (Fig. 4). The area under the curve in
response to 10 g of lactulose was significantly different
180
W. Scheppach et al.
from the fasting area ( t= 2.9 1, P< 0.02). Adding metronidazole reduced the breath hydrogen response from
1631 k 349 p.p.m. x min (lactulose)to 910 k 230 p.p.m. x
min (lactulose plus metronidazole) but this difference was
not significant ( I = - 1.57, P>O.l). However, the breath
hydrogen area for lactulose plus metronidazole was not
significantly different from the fasting area either
(t=1.76,P>0.1).
In the two subjects who had detectable methane in
breath, this was almost totally suppressed by metronidazole (Fig. 5).
01
0
I
I
1
I
I
2
I
I
'
I
3
Time (h)
"
"
5
4
6
.,
Fig. 3. Venous plasma acetate concentration in healthy
volunteers after a 12 h fasting period and the following
interventions at 0 min: drink of 400 ml of water ( n= 6);
0 , 10 g of lactulose in 400 ml of water ( n = 7); A , 10 g of
lactulose in 400 ml of water plus metronidazole (for
dosage see the text) ( n = 5).Vertical bars indicate fSEM.
0
1
2
6
5
4
3
.,
Time (h)
Fig. 4. Breath hydrogen in healthy subjects after a 12 h
fast and the following interventions at 0 min: drink of
400 ml of water ( n = 6); 0 , 10 g of lactulose in 400 ml of
water ( n = 7); A , 10 g of lactulose in 400 ml of water plus
metronidazole ( n = 5).Vertical bars indicate fSEM.
6o
I
I J-
2
lo,l----l--- -l---L -
-
J-- -
1- -1
-
DISCUSSI 0N
In previous studies from this laboratory [25] we have
shown that ingestion of carbohydrates, such as lactulose
and pectin, which are fermented in the colon after escaping digestion in the small bowel, are associated with
increases in blood acetate which can be quantitatively
related to the amount of carbohydrate broken down.
Since the fermentation of carbohydrates in the colon is
brought about by the indigenous anaerobic flora, we
attempted to alter fermentation with metronidazole, an
antimicrobial agent which is known to be active against
selected gut anaerobes [28]. Its effects were to reduce
breath hydrogen and methane but no change in acetate
was seen. Metronidazole is active mainly against bacteria
or protozoa that have a low redox potential and high
thymine content in their DNA. Such organisms include
Trichornonas vaginalis, clostridial species and fusiform
bacteria. These microbes are predominantly hydrogen
producers, and metronidazole acts as an electron acceptor in the pyruvate dehydrogenase reaction, thus inhibiting
hydrogen production [29]. Methane production would be
expected to fall as a result of reduced hydrogen availability [30]. In the rumen inhibition of methanogenesis by
antibiotics leads to increased propionate production [311.
In man, propionate does not reach peripheral blood in
amounts which are measurable by present techniques
(less than 5 pmol/l) so any change would not be detectable. Studies of faecal SCFA excretion in man [32] failed
to show an effect of metronidazole on propionate excretion. This was ascribed to the poor penetration of metronidazole into bowel contents. However, the present study
has shown a clear effect on hydrogen and methane production and suggests a direct effect on the colonic
bacteria.
After an overnight fast, significant amounts (aveage 50
pnol/l) of acetate are still detectable in peripheral blood.
In previous studies we have shown that these levels persist
even when fermentable carbohydrate is withdrawn from
the diet for 24 h [25]. In these circumstances a small
amount of acetate may still arise from fermentation of
mucus and proteins [33,34] which gives rise to SCFA and
branched-chain fatty acids. Branched-chain fatty acids are
products of the fermentation of valine, leucine and isoleucine and are found in the human caecum [ 171. Protein
fermentation is therefore an additional gut source of
acetate and may contribute even during an overnight fast,
Gut-derived blood acetate
since considerable protein is secreted into the gut,
especially by the pancreas. However, in ileostomy patients
who have no colon, detectable acetate is still present in
peripheral blood, although at significantly reduced levels.
Furthermore, the pronounced rise in plasma acetate
during prolonged fasting (60-108 h) in healthy subjects is
unlikely to be from the colon, since substrates entering the
colon are reduced and cell turnover of the epithelium is
slowed down 135,361.
In most mammals the plasma acetate concentration is
maintained as a result of simultaneous production and
utilization by a variety of tissues. In the rat the liver is a
site for net uptake of acetate when portal acetate concentrations are above 200-250 pmol/l [19, 22-24] and
net release when the portal acetate concentration is below
200-250 pmolll. In man, net output of acetate by liver
and leg muscle has been observed when the arterial
plasma acetate concentration falls below 80 pmol/l [13].
Liver cells contain both acetyl-CoA synthetase (acetate-CoA ligase; EC 6.2.1.1), which is present in the cytosolic fraction, and acetyl-CoA hydrolase (EC 3.1.2.1),
which is present in mitochondria [18, 23,37, 381. Starvation increases hepatic acetyl-CoA hydrolase activity in
rats and sheep [18], but leaves acetyl-CoA synthetase
activity unchanged. Acetate is formed from acetyl-CoA
under conditions where the citric acid cycle activity is
reduced. Elevated fasting plasma acetate concentrations
have been reported by Akanji et al. [39] in diabetic
patients compared with healthy control subjects. This
increase is probably due to a high availability of acetylCoA from enhanced /3-oxidation of fatty acids at a
reduced citric acid cycle activity. In addition, acetate turnover is slowed in diabetic patients [40] and depancreatized animals [411. Under these circumstances acetate
serves along with ketone bodies to distribute oxidizable
substrates throughout the body. Many tissues take up
acetate, but heart muscle [ll,18,211 is particularly active
in this respect, and acetate may account for up to 75% of
oxygen consumption during fasting by the rat [21].
In the light of the above, it is perhaps not surprising
that plasma acetate concentrations increased during starvation along with ketone bodies and free fatty acids. However, Skutches et af. [ 131 failed to find a change in blood
acetate in six obese subjects who fasted overnight (116
,umol/l)and then for 7 days (143 ,umol/l),whereas Seufert
et al. [16] did show a change in seven obese volunteers
who fasted for 10 days (overnightfast, 29 pmol/l; 10 days
fast, 92 ,umol/l). The much higher values obtained by
Skutches et al. [16] may.relate to differences in either the
patient group or the methodology. Neither of the studies
in obese subjects reported sequential measurements
during the starvation period or after refeeding. In this
study the pronounced rise of plasma acetate concentration during starvation (colonic fermentation presumably
decreased) and its rapid fall after refeeding (colonic fermentation presumably increased) suggests that there is an
endogenous source of acetate. This is in keeping with data
from animal studies, which have shown that acetate may
either be taken up or released by tissues depending on the
nutritional state of the animal [20,21].
181
As the ileostomy patients (study 1)were on average an
older group than the healthy volunteers (67 versus 39
years), a potential age dependence of plasma acetate concentration has to be considered. In another study (J. H.
Cummings, unpublished work) plasma acetate concentration was measured after an overnight fast (12 h) in 144
healthy subjects (mean age 38.4 years, range 18-84 years)
of various ethnic origins (Black, Indian, Caucasian). Fasting plasma acetate concentrations did not correlate with
age ( r =0.07), which is in agreement with the findings of
Akanji et al. [39].
It is clear from these and other studies that both the
colon and other tissues contribute to blood acetate in
man. In the fed state acetate is likely to be derived principally from the colon, but during starvation the liver probably regulates production ensuring, along with ketone
bodies, an adequate supply of oxidizable substrates to
tissues such as the myocardium and brain [ll, 18, 381.
These fuels may be preferred to long-chain fatty acids in
tissues such as the brain, partly because they are water
soluble and readily cross the blood-brain barrier, and
partly because appropriate enzymes exist for their oxidation.
The present work does not allow a quantitative estimate of the contribution of fermentation in the human
colon to energy metabolism in human tissues. To make
such a determination dynamic studies of acetate are
needed, although the relative contributions from different
tissues, and particularly the lack of access to the portal
vein, make such studies much more difficult in man than
in animals [42]. Some idea of the colonic contribution has
been made by estimating the amount of substrate available for fermentation [43].Depending on diet and the efficiency of fermentation, SCFA probably contribute
between 2 and 10% of energy supply to subjects living on
Western-style diets.
ACKNOWLEDGMENTS
We thank the volunteers for their willing and patient cooperation. E.W.P. was supported by the University of
Otago, Wellington, New Zealand, the Royal Australasian
College of Physicians and the Todd Foundation. W.S.
received a grant from Deutsche Forschungsgemeinschaft
(Sche 252/1-1).
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