67 I
Clinical Science (1998) 94, 671676 (Printed in Great Britain)
Effect of topical butyrate on rectal epithelial kinetics and
mucosal enzyme activities
Peter R. GIBSON, Diana KILIAS, Ourania ROSELLA, Jenny M. DAY, Margaret ABBOTT,
Caroline F. FINCH and Graeme P. Y O U N G
University of Melbourne Department of Medicine, The Royal Melbourne Hospital, Victoria 3050, Australia
1. This study aimed to determine the effect of luminal
butyrate on proliferative kinetics, a differentiation
marker (alkaline phosphatase), and a molecule that
controls cell-substratum adhesion (urokinase) in histologically normal human rectal mucosa.
2. Ten subjects with a colonoscopically normal colon
(seven had previous adenomas) were given either butyrate or saline enemas for 4 days in a double-blind
CroSS-OVer manner. Rectal biopsies were taken before
and after each course of enemas. Epithelial proliferative
kinetics were measured immunohistochemically using
antibodies to proliferating cell nuclear antigen. Urokinase and alkaline phosphatase activities were
measured spectrophotometrically in biopsy homogenates.
3. Both saline and butyrate enemas were well tolerated
and induced no histological change except for a significant increase in crypt length (P< 0.05). The number
of proliferating cells per crypt also increased significantly after butyrate (P= 0.018).
4. Compared with saline enemas, butyrate did not affect
kinetic indices nor alkaline phosphatase activities. However, mucosal urokinase activities were significantly
lower in butyrate-treated patients (9.5 & 2.0 i.u./g) than
in saline-treated patients (12.8 f2.0 i.u./g; P = 0.045).
5. Delivering of extra butyrate to the distal colon in
healthy subjects may stabilize cell-substratum adhesion
in surface epithelium and therefore offer a potential
mechanism by which elevating distal colonic luminal
butyrate concentrations might be beneficial in patients
with colitis or hyperproliferative large bowel epithelium.
INTRODUCTION
Fermentation of carbohydrates in the large bowel
lumen yields large amounts of the short-chain fatty
acid, butyrate [l]. Butyrate plays essential roles in
providing energy for colonic epithelium [2] and in
promoting sodium and water absorption [3]. Butyrate
has other powerful effects on cell lines in vitro,
including inhibition of cell growth, induction of
differentiated phenotype and apoptosis, and the stimulation or inhibition of the synthesis of a large variety of
proteins [4,5]. Isolated colonic crypt cells also respond
to butyrate but the response often does not parallel
that found in colon cancer cell lines. For example,
butyrate has no effect on the rate of DNA synthesis
and does not promote differentiation in isolated
normal cells [6] but suppresses it in cells from cancerbearing colons [7]. Butyrate also inhibits the production of the neutral protease, urokinase (u-PA) [8],
its receptor (u-PAR) [9], and interleukin-8 [lo]. However, it is uncertain whether the effects of butyrate on
normal cells in vitro also occur in the normal colonic
epithelium when exposed to butyrate in viva Extrapolation from the isolated cell system must be made
cautiously since normal epithelial-mesenchymal relationships are disrupted and the trauma of their
isolation appears to markedly induce the synthesis and
secretion of factors such as u-PA [8], u-PAR [9] and
interleukin-8 [lo].
In several studies, butyrate has been instilled luminally in rat large bowel which has been starved of
carbohydrate fermentation products by unphysiological manoeuvres such as the use of an elemental diet
or diversion of the faecal stream away from that
segment. These have consistently shown butyrate’s
trophic effect on colonic epithelium [11,121 although
the effects on mucosal hydrolase or u-PA activities
have not been reported. Under physiological conditions, however, greater degrees of luminal fermentation do occur and mucosal atrophy is not seen.
Modulating luminal butyrate concentrations more
than 10-fold by varying the amount and/or type of
dietary fibre ingested had, in rats, only small effects, if
any, on colonic epithelial proliferative kinetics and
hydrolase expression, but significantlyaltered mucosal
u-PA activity; the lowest activities were found in the
rats with the highest faecal butyrate concentrations
(the rats fed unprocessed wheat bran) [13]. However,
these effects do not necessarily specifically reflect
differences in butyrate concentration since multiple
other luminal characteristics, such as concentrations
of other short-chain fatty acids, pH and the spectrum
of the bacterial flora, are also affected by the diets.
The effect of direct instillation of butyrate into large
bowel lumen on epithelial and mucosal characteristics
in humans has only been studied in patients with active
ulcerative colitis [14]. Butyrate enemas used twice daily
changed the distribution of proliferating cells and
favoured reduced proliferative activity. Since inflam-
~
Kay words: alkaline phosphatase, butyrate. cell proliferation, colonic epithelium, urokinase.
Abbreviations: PCNA. proliferatingcell nuclear antigen; U-PA. urokinase; U-PAR, urokinase receptor.
Correspondence: D r Peter R. Gibson.
672
P. R. Gibson and others
matory activity was also reduced, it is uncertain
whether the effects were secondary to butyrate itself or
to butyrate's effect on reducing inflammation. The aim
of the present study then was to use a similar regimen
of butyrate enemas in human subjects in whom rectal
histology was normal and to measure their effects on
rectal epithelial proliferative kinetics, a differentiation
marker (alkaline phosphatase), and the activity of uPA, a molecule that controls cell-substratum adhesion.
ME T H0 DS
Subjects studied
Ten subjects were studied. Their ages ranged from
42 to 70 (mean 56) years and six were female. The
subjects had had a colonoscopy within the previous 12
months which had revealed the large bowel free of
adenomas or other lesions. Seven of the subjects had
previous adenomatous polyps removed, one had previous hyperplastic polyps, and two had diverticular
disease alone. They all gave written informed consent
before commencement of the study and the protocol
was approved by the Board of Medical Research and
Ethics Committee of The Royal Melbourne Hospital.
Protocol
The subjects were instructed to ingest a diet low in
fibre and resistant starch for the duration of the study.
Three days after starting the diet, rectal biopsies were
taken 5-7 cm from the anal verge without sigmoidoscopy, using standard colonoscopic biopsy forceps.
The patients were then randomized to receive an
enema of either normal saline or sodium butyrate (for
composition, see below) twice daily for 4 days. Neither
the patient nor the investigator was aware of the
nature of the enema. The enemas were self-administered. At the end of that time a further set of rectal
biopsies were taken in the morning approximately 2 h
after the last enema. The subjects then crossed over to
the other enema (saline or butyrate) which was also
self-administered twice daily for 4 days. Biopsies were
taken at a similar time of the day about 2 h after the
last enema. The subjects also filled out a diary card
stating the times of day the enemas were administered
and when the bowels opened as well as any adverse
events or other effects experienced during the treatment. Six patients received the saline enema first and
four the butyrate enema first.
Four biopsies were taken on each occasion. Two
were placed immediately in Methacarn fixative and,
after 1 h, transferred to 100% ethanol and stored at
- 20 "C. The other two biopsies were placed in 1 ml of
ice-cold Tris-mannitol buffer (50 mM D-mannitol,
2 mM Trizma base in dH,O, pH 7.4) and stored at
-20 "C. The biopsies were coded in order to blind the
investigator to the timing of the biopsy.
corrected to 290 mOsmol/l using normal saline and
water for irrigation. Saline enemas comprised isotonic
sodium chloride at pH 7.0. The total volume of each
enema was 60 ml.
Epithelial proliferative kinetics
Thin sections of paraffin-mounted, Methacam-fixed
biopsies were cut (2 pm thick) and stained using an
immunoperoxidase technique with antibodies to the
proliferating cell nuclear antigen (PCNA, Sigma,
Castle Hill, NSW, Australia) as recently described and
validated by us [15]. Briefly, longitudinally cut crypts
were identified and the number of cells in each crypt
column counted. The number of crypt columns examined ranged from 28 to 58 (mean = 40). Cells
staining strongly positive for PCNA were identified
and their position in the crypt column recorded. From
these data, the following indices were determined :
crypt column height, the number of PCNA-positive
cells per crypt, the labelling index calculated by
dividing the number of positive cells by the total
number of cells in the crypt columns (mean 3480 cells
counted), and the distribution of positive cells according to five equal quintiles, quintile 1 being the base
of the crypt, and quintile 5 at the surface end of the
crypt, expressed as percentage of positive cells in that
quintile. All sections were evaluated by one experienced scientist who was blinded to the timing of the
biopsy. The performance of the latter in terms of
variances of component errors in cell counting has
recently been described in detail [15].
Enzymic activities
Before assay, mucosal samples in Tris-mannitol
buffer were thawed and mechanically homogenized at
4 "C. Triton X-100 was added to a final concentration
of 0.1 YO.Myeloperoxidase activity was measured
within 5 min of homogenization spectrophotometrically using guaiacol as substrate [16]. The results
were expressed as change in absorbance ' units'/min
per mg of protein of the homogenate. Alkaline
phosphatase activities were assayed in aliquots of the
homogenate spectrophotometrically using p-nitrophenol as substrate as previously described [17]. u-PA
activity was measured in mucosal homogenates by the
colorimetric method of Coleman and Green [18]. The
assay has previously been shown to be specific for uPA and the results not affected by the addition of
antibody specific for tissue plasminogen activator [191.
Because of the presence of plasminogen in the assay,
the activities of both pro-u-PA and u-PA are
measured. Enzyme activities were expressed relative to
the mucosal protein content which was measured
using bovine y-globulin as standard [20].
Statistical evaluation
Enema composition
Butyrate enemas comprised sodium n-butyrate at a
concentration of 80 mM at pH 7.0. Osmolality was
Results are expressed as means S.E.M. unless
otherwise stated. Paired data were compared using a
two-tailed paired t-test. Because of the study's cross-
673
Topical butyrate and the rectum
over design, the order in which the enemas were
applied was taken into consideration but found to
have no influence on the results. The number of bowel
actions over the 4-day period during saline or butyrate
enema treatment was compared using the Wilcoxon
signed rank test. A P value of 0.05 or less was
considered statistically significant.
Q
3 4
+
20
RESULTS
Compliance to and tolerability of butyrate and saline
enemas
All subjects were able to retain the enemas for at
least 1 h. Compliance, as assessed by daily diary cards,
was very good with only one subject failing to use the
final butyrate enema due to spillage. Compared with
their normal bowel habit, all but one subject experienced reduced frequency of bowel actions after
commencement of the low fibre diet. Over the 4-day
treatment period, the median (range) number of times
the subjects opened their bowels was 7 (1-1 1) during
use of saline enemas, which was significantly more
frequent than that during butyrate enema treatment [4
(2-7); P = 0.0431. Only one patient reported an
adverse event, comprising the sensation of being
‘bound up’ together with tiredness during butyrate
enema therapy only. The number of bowel actions
during butyrate enema therapy in this patient was 4
compared with 10 during saline enema therapy.
Effect on proliferative kinetics
Histology of all biopsies was normal and no features
of inflammationwere seen. As shown in Figure 1A, the
&
!j
10
OJ
Q3
QS
Figure 2 Distribution of epithelial cells staining w i t h
P C N A within the crypt in rectal mucosa M o r e (‘Pra’)
and after 4 days of enemas containing either saline or
butyrate
The crypt was divided into five equal parts, quintile I being at the
base of the crypt. The results are expressed as the mean of the
percentage of stained cells in that quintile. There were no
significant differences across the groups.
use of enemas per se (irrespective of whether they
contained saline or butyrate) resulted in significantly
longer crypts. Likewise, the number of PCNA-positive
cells per crypt tended to be greater after the use of
enemas (Figure lB), but this only reached statistical
significance in association with butyrate enemas.
However, the labelling index was unaffected (Figure
1C) and the distribution of PCNA-positive cells across
the 5 quintiles was similar (Figure 2). When the effect
of butyrate was compared with that of saline enemas,
B
A
C
2.5
120
T
T
n
s 100
%
W
0 80
8
3-
60
fi
8U
40
20
0
Pre
Lenemas --J
Saline Butyrate
Lenemas --J
Figure I Epithelial prolifentive kinetics In rectal mucosa before (‘Pro’)
Pre
SalineButyrate
enemas -I
and after 4 days of enemas containing either saline o r
butynta
Indices measured were (A) the crypt column height (CCH), (6) the number of PCNA-positive cells per crypt column, and (C) the labelling
index (number of PCNA-positive cells per 100 crypt cells). The results are expressed as means+S.E.M. and the P values shown derive from
paired t-tests.
674
P. R. Gibson and others
butyrate enemas compared with those after treatment
with saline enemas (P= 0.045). u-PA activities after
butyrate treatment also tended to be lower than those
before enema therapy but failed to reach statistical
significance (P= 0.074). Saline enemas had no effect
on u-PA activity compared with pretreatment levels (P
= 0.61) The order in which the enemas were given had
no influence upon the results.
1
DISCUSSION
PIX?
Saline Butyrate
I
enemas 1
PIX?
Saline Butyrate
enemas 1
Figure 3 Enzyme activities in rectal mucosa before
(‘Pre’) and after 4 days of enemas containing either saline
or butyrate
Indices measured were (A) the crypt alkaline phosphatase activities and (6) urokinase activities expressed relative to mucosal
protein content. The results are shown as means+S.E.M. and the
P value shown derives from a paired t-test.
there was no difference for any of the epithelial kinetic
indices.
Effect on enzyme activities
Myeloperoxidase activity was very low (<
0.8 units/min per mg of protein) in all biopsies which
corresponded to the lack of inflammation evident
histologically. Alkaline phosphatase activities were
similar across the groups (Figure 3). In contrast, u-PA
activities differed across the groups (Figure 3). u-PA
activities were significantly lower after treatment with
Previous studies of the direct effects of butyrate or a
combination of short-chain fatty acids on the colonic
mucosa in humans and experimental animals have
used one of three models: atrophic colon due to
luminal butyrate deficiency after faecal diversion or
elemental diet [1 1,121, hyperproliferating colon due to
bile salt injury [21], or inflamed colon [14]. In these
models, the control comparator comprises abnormal
mucosa and this creates difficulties in interpreting the
physiological effects. For example, in butyrate deficiency, the mucosa is atrophic mainly because of the
dependence of the epithelium on butyrate as an energy
source [2]. It is therefore difficult to distinguish effects
of enhanced energy supply per se from the effects
directly mediated by butyrate. Similarly, in colitis
models, distinguishingthe effects of the direct action of
butyrate from those indirectly mediated by reduction
in mucosal inflammation is not readily achieved. An
alternative approach is to identify the effects of
butyrate in vivo by delivering more butyrate to the
distal colon via the ingestion of slowly fermented fibres
such as wheat bran [13]. This dietary change is,
however, associated with multiple other effects on
luminal contents, such as changes in pH and in the
concentrations of other short-chain fatty acids. It
introduces, therefore, too many variables to be able to
confidently dissect the effects of butyrate alone. The
approach applied in the present study - to examine the
effect of supplementing butyrate delivery to a histologically normal rectum (albeit mostly in patients with
previous adenomas) - has not previously been reported. An attempt was made to reduce luminal
butyrate levels by the use of a low residue diet. This did
constipate most of the subjects but the rectal mucosa
showed no histological evidence of energy deficiency
since the crypts were of normal length. Thus the
experimental design addressed the issue of whether a
modest increase in delivery of butyrate to the normal
rectal mucosa induced changes.
Butyrate has been shown to cause a variety of effects
on epithelial proliferative kinetics. It suppresses cell
proliferation [4] and induces apoptosis [5] in colon
cancer cell lines. Treatment of normal colonic mucosa
in organ culture with butyrate results in shortening of
crypts with an increased relative number of proliferating cells [22]. In vivo, luminally instilled butyrate
increases epithelial proliferation and crypt length in
atrophic mucosa [l 11 but decreases proliferation in
inflamed mucosa [14]. Despite these varied but substantial effects, rectal butyrate supplementation under
Topical butyrate and the rectum
the conditions of the present study did not affect
epithelial population kinetics. These findings mimic
butyrate’s effect on isolated normal crypt cell populations in which no change in the rate of DNA
synthesis nor DNA content was observed [6].
The use of an enema per se did, however, induce
longer crypts, most probably by increasing proliferative activity to support the expanded population. The
underlying mechanisms are unclear but may involve a
‘cleansing action’ of either enema in washing away
toxic substances that might contribute to cell death in
the surface epithelium. Reduction of such injury may
lead to prolongation of the life span of cells and
subsequent lengthening of crypts. Alternatively, the
effect may represent the result of ‘physical stimulation’
of the colonic epithelium by luminal contents as
previously reported in mice [23].
Mucosal alkaline phosphatase activity was unaffected by butyrate supplementation. Alkaline phosphatase has previously been used as a marker of
differentiation [6,7] because of its gradient of expression from crypt to surface [24]. However, whether
measurement of mucosal alkaline phosphatase activity is a sensitive or even valid marker of differentiation remains uncertain (P. R. Gibson, R. Nov, M.
Fielding, A. McIntyre, C. F. Finch, 0. Rosella, J. M.
Mariadason, D. H. Barkla and G. P. Young, unpublished work). While butyrate potently stimulates cell
differentiationin cell lines [4], there is no evidence that
butyrate exerts this effect on normal colonic epithelial
cells. Normal crypt cells studied in vitro neither change
their expression of alkaline phosphatase nor change
the rate of glycoprotein synthesis on exposure to
butyrate [6]. Furthermore, in colonic mucosa in organ
culture, butyrate leads to a loss of mucosal alkaline
phosphatase activity, probably due to loss of mature
surface epithelial cells by the induction of apoptosis
[221*
In contrast to the lack of a butyrate-specificeffect on
epithelial kinetics or alkaline phosphatase activity,
butyrate enemas did significantly reduce mucosal
u-PA activity independently of an effect of the enema
itself. This effect was similar to that observed in rats
ingesting wheat bran where mucosal u-PA activity was
significantly reduced compared with that in rats fed
diets containing soluble or no fibre [13]. Wheat bran
leads to considerable elevation of distal colonic luminal butyrate concentrations [26]. Butyrate-mediated
reduction of mucosal u-PA activity also mimics the
effect we have previously observed of butyrate on
u-PA expression and secretion by isolated colonic
crypts PI.
The disparity of effects of butyrate on two different
end-points, epithelial proliferation and mucosal u-PA
activity,might reflect either or both of two possibilities.
Firstly, the mechanisms by which butyrate acts on
these might differ. Butyrate-mediated stimulation of
epithelial proliferation is best seen in butyrate-starved
cells. Thus, stimulation is seen only when mucosa is
atrophic due to nutrient depletion. Recent evidence
has shown that butyrate inhibits cell proliferation by
675
inhibition of histone deacetylase [27]. In cells not
dependent upon butyrate for its energy supply and not
usually exposed to butyrate, such as cell lines, this
effect is likely to predominate. In normal colonic
epithelium, histone deacetylase may be less sensitive to
butyrate-mediated suppression due to chronic exposure to butyrate. The mechanism by which butyrate
inhibits u-PA synthesis is not known but, like alkaline
phosphatase [27], may not be due to inhibition of
histone deacetylase. The disparity of findings, therefore, might reflect different mechanisms of action of
butyrate, each having different concentration curves.
Secondly, differences in butyrate delivery to the
target cells may be responsible. It is likely that
luminally instilled butyrate is being effectively delivered to the surface epithelium. Since this is a major
site of u-PA production in the mucosa (P. R. Gibson,
I. Birchall, 0. Rosella, V. Albert, C. F. Finch, D. H.
Barkla and G. P. Young, unpublished work), butyrate
will have the best chance of exerting its modulatory
effects on that enzyme. In contrast, proliferating cells
are present in the lower portion of the colonic crypt
which, by virtue of the unidirectional flow of mucus
towards the lumen, would be exposed to lower
concentrations of butyrate. In support of this view is
the observation that butyrate does inhibit abnormal
proliferation when the proliferative compartment is
expanded into the upper crypt, as observed in bileacid-injured colon [21] and in ulcerative colitis [14].
The biological significance of reduced mucosal uPA activity is uncertain. In the healthy subjects in the
present study, a change in mucosal u-PA activity did
not correlate with a change in epithelial kinetic indices.
However, reduction of mucosal u-PA activity might be
important in colitis or hyperproliferating epithelium
induced in rats by the ingestion of diets containing
resistant starch or soluble fibre, where mucosal
u-PA activity is elevated (P. R. Gibson, I. Birchall,
0. Rosella, V. Albert, C. F. Finch, D. H. Barkla and
G. P. Young, unpublished work and [29]). Its reduction in the surface epithelium might stabilize cellsubstratum adhesion and thereby reduce the rate of
epithelial death. This would, in turn, lead to a
reduction in the rate of epithelial proliferation. In
support of this contention, mucosal u-PA activity in
the distal colon of rats directly correlates with proliferative indices (P. R. Gibson, 1. Birchall, 0. Rosella,
V. Albert, C. F. Finch, D. H. Barkla and G. P.
Young, unpublished work). In colitis, the effect of
butyrate on u-PA activity might be one mechanism by
which it reduces inflammatory activity when given by
enema [141.
In conclusion, rectal supplementation of butyrate in
humans with a histologically normal rectum has no
discernible effect on rectal epithelial proliferative
kinetics beyond that induced by an enema itself nor on
differentiation, but inhibits mucosal u-PA activity.
The findings mimic those observed with isolated
normal colonic crypt cells. The results demonstrate a
potential mechanism - stabilizing cell-substratum adhesion in surface epithelium - by which elevating distal
676
P. R. Gibson and others
colonic luminal butyrate concentrations might be
beneficial in patients with colitis or hyperproliferative
large bowel epithelium.
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