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Br. J. Surg. 1992, Vol. 79, February.
129-1 32
Influence of dietary calcium
supplements on ileoanal pouch
function and cytokinetics
A double-blind cross-over study was undertaken in 16 patients after
panproctocolectomy and ileoanal pouch reconstruction to compare
supplementary calcium (1.5 g J d a y ) with placebo over 2 months with a
2-week washout period. Stool frequency was recorded and the eflects
on pouch mucosal crypt cellular proliferation were determined using an
in vitro stathmokinetic technique which measures the crypt cell
production rate ( C C P R ) and an immunohistochemical method using
the Ki67 monoclonal antibody f o r proliferating nuclei. The median
(interquartile range) diurnal stool frequency was reduced by calcium
( 4 ( 3 - 5 ) per day) compared with values obtained before treatment ( 7
(5-10) per day, P < 0.002) and with placebo ( 7 ( 6 - 9 ) per day,
P = 0.002). Similarly, calcium reduced nocturnal stool frequency ( I
(0-1)per night) compared with pretreatment andplacebo (both 2 ( 1 - 3 )
per night, P < 0.05) values. Calcium reduced the mean(s.e.m.) C C P R to
1.88(0.41) cells per crypt per hour compared with pretreatment
(3.63(0.53), P = 0.01) and placebo (3.24(0.43), P = 0.002) values.
Median ( interquartile range) Ki67 activity was also reduced by calcium
(13.2 (9.7-16.7) per cent), compared with values obtained before
treatment (27.3 (14.3-30.2) per cent, P = 0.001) and with placebo
(26.0 (17.2-32.0) per cent, P = 0.001). Stool frequency was
significantly correlated with the C C P R (diurnal: r = 0.37; nocturnal:
r = 0.31, both P < 0.05). Nine patients used antidiarrhoeal medication
while receiving placebo compared with four patients receiving calcium
( P = 0.032). This study has shown that supplementary oral calcium
significantly reduced stool frequency in patients with pouches, u
reduction that was associated with reduced cell prolferation. The
mechanisms f o r this eflect are not known.
G. H. Barsoum,
M. Winslet, D. Youngs,
J. P. Neoptolemos and
M. R . B. Keighley
Academic Department of Surgery,
Queen Elizabeth Medical Centre,
Edgbaston, Birmingham, UK
Correspondence to:
Mr J. P. Neoptolemos, Department
of Surgery, Dudley Road Hospital,
Birmingham BIB 7QH, UK
Until recently, proctocolectomy and end-ileostomy was the
procedure of choice for surgical treatment of ulcerative
proctocolitis and familial adenomatous polyposis. However,
despite the remarkable improvements in stoma management,
there is still a high psychological and social price to pay for an
ileostomy'.*. The introduction of the ileoanal pouch procedure
(restorative proctocolectomy has changed the outlook of
patients requiring operation for these disorders. More than
1000 of these procedures have now been carried out3. Ileal
adaptation occurs after ileoanal pouch construction. The
changes in ileal morphology after pouch formation include a
chronic inflammatory cell infiltrate accompanied by intense
villous atrophy, increased crypt depth and an increase in
mucosal cell proliferation4-'. Frequency of defaecation after
ileoanal pouch construction is directly correlated with the ileal
crypt cell production rate (CCPR)', and hence factors that
influence pouch ileal mucosal cell proliferation could modify
its function.
Dietary calcium supplements have been shown to reduce
crypt cell proliferation and to abolish the toxic effects of topical
bile and fatty acids on colonic mucosa, either by sequestration
or by a direct action on the colonic m u ~ o s a * - ' ~Hence,
.
dietary
calcium supplements could exert a similar effect on ileal pouch
mucosa.
We sought to test this hypothesis by undertaking a
double-blind cross-over comparison of calcium with placebo.
We recorded the effects on stool frequency and on crypt cell
proliferation using monoclonal antibody Ki67 activityI4.
Paper bused on un ahstruct presented t o the Junuury 1991 meeting of
/lie Surgicul Research Socieij
_
_
0007-1323/92/020129-04
_
~
~
_
C 1992 Butterworth-Heinemann Ltd
~
Patients and methods
Trial design
Twenty patients who had undergone restorative panproctocolectomy
with a pelvic ileal pouch at least 12 months previously were recruited.
All patients gave written consent to the study. Patients with pouchitis
and those taking metronidazole or any antibiotic were excluded. The
design was a double-blind cross-over study. Treatment was dispensed
by the hospital pharmacy in coded form with precise instructions to
each patient. Both the calcium and placebo were in powder form. The
dose of calcium was 1.5 g/day, given as calcium gluconate (BDH
Pharmaceuticals, Atherstone, U K ) . The placebo was lactose powder.
a substance known not to affect bowel habit in the absence of lactose
intolerance, which was excluded by history: no Afro-Caribbean patients
were included. The treatment period was 2 months o n the first powder
followed by a 2-week washout period before commencing the second
powder for a further 2 months. The patients were specifically instructed
to keep to their normal diet. Nocturnal and diurnal stool frequency
was recorded before the first treatment, after 2 months, and again after
the second 2-month course. The use of antidiarrhoeal medication was
recorded and pouch biopsies were also obtained from the posterior
wall of the pouch, l 0 c m from the anal verge, after each treatment.
Crypt cell production rute
Measurement of the CCPR was based on a previously described
technique". Biopsies were placed immediately into a circular Perspex
dish (Falcon Plastics. Los Angeles, California, USA) containing RPMl
1640 (Gibco Laboratories, Paisley, U K ) organ culture medium with
added gentamicin (40 mgiml), penicillin (100 units/ml) and fetal calf
serum (1.9, vol/vol). The medium was equilibrated with 95 per cent
oxygen and 5 per cent carbon dioxide at 37°C 1.h before use. The dish
was placed into a sealed gas jar and kept at 37°C. The same gas mixture
was introduced into the sealed jar via the inlet valve for 10 min. After
16 h, the medium was replaced with identical medium containing
~
~
129
Calcium supplements and ileoanal pouch function: G. H. Barsoum et al.
0.4 g/ml vincristine (Eli Lilly, Basingstoke, UK). Biopsies were
removed 60, 120 and 180 min later, fixed in Carnoy's solution for 2-6 h
and stored in 70 per cent ethanol.
Following rehydration the specimen was hydrolysed in 1 M
hydrochloric acid for 6 min at 6WC, stained with Schiffs reagent, and
the slide was rinsed with 45 per cent acetic acid. The mucosa was
microdissected from each specimen and the number of arrested
metaphase figures in ten crypts per specimen was counted. This was
undertaken by one person (D.Y.) on coded samples. Linear regression
analysis was used to determine the slope of metaphase accumulation,
which was expressed as cells per crypt per hour.
Biopsies were immediately frozen in liquid nitrogen and stored at
-40°C. Frozen specimens were cut 5 p n thick and fixed with
acetate:ether (60:40, vol/vol) on albumin-coated slides. A three-stage
peroxidase procedure was used ", involving sequential 30-min
incubations with the murine monoclonal antibody Ki67 and rabbit
antimouse and swine antirabbit immunoglobulins (Dako, Copenhagen,
Denmark ) with intermediate washings of Tris-buffered saline with
40 per cent human serum. The slides were developed with
diaminobenzidine and hydrogen peroxide for 8 min and counterstained
with haematoxylin. Negative controls were incubated with Tris-buffered
saline before development. Activity was expressed as the percentage of
positive cells to the total crypt cell population. This assay was performed
by one individual (G.H.B.) on coded slides.
Ethical approval
This was obtained from the ethical committee of the Central
Birmingham Health Authority.
Table 1 Summary data on stool frequency, CCPR and Ki67 activity
in patients before treatment and whilst receiving either calcium or placebo
Before
treatment
Calcium
7 (5-10)
4 (3-5)
2 (1-3)
l(0-1)
3.63
1.88
(0.53)
(0.41)
27.3
13.2
(14.3-30.2)
(9.7-16.7)
~~
Patients
Fifteen patients complied fully with the trial protocol. Four
patients did not comply with the study and were excluded.
These patients stopped taking the powder for various reasons
(holiday, inconvenient, forgot, changed mind) unrelated to any
change in bowel habit. One patient wished to stay on the initial
treatment (calcium gluconate) ; only pretreatment and
postcalcium data are therefore shown for this patient. The
median (range) age of the 16 patients who fully or partly
complied with the study was 39 (17-64) years; nine
(56 per cent) patients were female. The indications for surgery
were ulcerative colitis ( n = lo), chronic idiopathic constipation
( n = 3 ), megacolon ( n = 2) and familial adenomatous
polyposis (n = 1). The median (range) time since the formation
of the pouch was 22 (12-30) months.
Presentation of data
For simplicity, the data for each treatment period were grouped
together and are summarized statistically in Table I ;individual
values are given in Figures 1-3.
Placebo
7 (6-9)
2 (1-3)
3.24
(0-43)
26.0
(17.2-32.0)
~~
Values are *median (interquartile range); tmean(s.e.m.). CCPR, crypt
cell production rate
13
l4
12
Any differences in the CCPR were determined by multiple slope
analyses. Multiple stool frequency and Ki67 activity data were
compared by the Kruskal-Wallis test; if this was significant ( P < 0.05)
paired analysis was performed with the two-tailed Mann-Whitney U
test. Categorical data were analysed by Fisher's exact probability test
(two-tailed). Correlations were performed using Pearson's formula
(continuous data) and Spearman's rank correlation (categorical data ).
For correlations involving Ki67 and CCPR, normalization was
undertaken by log transformation.
Results
Ki67 immunohistochemistry
Stool frequency* ( n )
Day
Night
CCPRt
(cells per crypt per hour)
Ki67* ( % )
Statistical analysis
Stool frequency (Figure 1). Calcium treatment significantly
reduced diurnal stool frequency compared with values obtained
before treatment or with placebo (both P = 0.002). Similarly,
calcium reduced nocturnal stool frequency compared with
pretreatment ( P = 0.005) and placebo ( P = 0.031 ) values.
There was no difference in either instance when placebo and
pretreatment values were compared. Nine patients used
antidiarrhoeal medication while taking placebo, compared with
only four receiving calcium treatment ( P = 0.032).
10
1
9
-
8
11 10
z
U
C
%
0W
-
7
9 -
1
U
C
8 -
z
m
J
L
L
-0
c
VI
-m
L
C
.-J
6
c
0
7 -
5
I-'
VI
6 -
-m
5 -
c
C
4
L
J
U
0
z
4 -
3 -
3
2
2 I
1
l t
0 '
a
Before
treatment
Calcium
Placebo
0
b
Before
treatment
Calcium
Placebo
Figure 1 a Diurnal and b nocturnal stool frequency before treatment and during calcium and placebo treatment. Numbers within theJlgures indicate the
number of individuals with each stool frequency
130
Br. J. Surg., Vol. 79, No. 2, February1992
Calcium supplements and ileoanal pouch function: G. H. Barsoum et al.
L
3
1
0
L
W
a
c
a
?.
L
t
gl
lo
8
0
7 -
W
a
v)
6 -
U
W
v
W
c
m
L
.-0C
c
U
3
5 4 -
3 -
U
L
a
-a,
U
2 1 -
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a
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- 0
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Before treatment
Calcium
Placebo
Figure 2 Crypt cell production rate before treatment and during calcium
or placebo treatment
50
40
-
””
>.
..-c>
4-
30
m
U
.r
0
with an ileal pouch. In agreement with previously published
1 11 17.1 8 , this study also showed that supplementary
calcium in double the normal daily intake reduced the ileal
crypt cell production rate. The exact mechanism by which
calcium exerts its effect on mucosal cellular proliferation has
yet to be established. Newmark et a1.I’ have proposed that the
toxic (and also potentially tumour-promoting ) effects of fatty
acids and free bile acids on the colon may be inhibited by
ionized calcium. Calcium supplements using several grams per
day result in minimal variation of serum calcium in several
long-term human studies20-22,and an intake of 1.5 g/day has
been recommended to prevent osteoporosis in postmenopausal
womenz3.
The troublesome diarrhoea seen in patients suffering from
the short bowel syndrome and after jejunoileal bypass or
ileostomy may be reduced by increased dietary calcium24. The
8-week duration of treatment with calcium or placebo in this
study was chosen to ensure that exposure was long enough to
exert an effect on intestinal cell kinetics, although the normal
turnover time of ileal epithelial cells is only a few days and the
proliferation pattern can change within 1 week of altering
dietary intake25.Patients who had had a pouch reconstruction
for at least 12 months were chosen because the CCPR increases
for up to 6 months after surgery’. Patients with pouchitis were
also excluded because of changes in the CCPRz6. Lactose
powder was chosen as a placebo because of the similar
appearance, consistency and taste compared with calcium
gluconate. Lactose does not affect bowel transit except in the
presence of lactose intolerance. Although lactose intolerance
was excluded by history, this may not have been absolutely
reliable. The choice of placebo does seem appropriate, however,
since the stool frequencies, CCPR and Ki67 activity were all
very similar to the pretreatment values during treatment with
placebo.
To date, only one human studyz7 has failed to show any
effect of calcium on cell renewal in the bowel. We have shown
that supplementary oral calcium reduces the frequency of
nocturnal and diurnal defaecation and that this is significantly
associated with changes in cell proliferation using two different
techniques, measurement of the CCPR” and Ki67 activity“.
It remains to be determined whether the effects on cell
proliferation in patients with pouches were secondary to
reduced stool frequency or whether such effects were responsible
for the beneficial clinical effects.
reports8.9,
20
Y
10
0
Before treatment
Calcium
Placebo
Figure 3 xi67 cell proliferative activity before treatment and during
calcium and placebo treaiment
Crypt cell production rate (Figure 2 ) . The CCPR was
significantly reduced by calcium compared with pretreatment
( P = 0.01) and placebo ( P = 0.002) values. There was no
difference between placebo and pretreatment values.
Ki67 activity (Figure 3). Ki67 activity was significantly
reduced by calcium treatment compared with pretreatment and
placebo values (both P = 0.001 ). There was no difference
between placebo and pretreatment levels of Ki67.
Correlations
Diurnal stool frequency was significantly correlated with CCPR
( r = 0.37, P < 0.05) and Ki67 activity ( r = 0.52, P < 0.001).
Similarly, nocturnal stool frequency was correlated with CCPR
( r = 0.31, P < 0.05) and Ki67 activity ( r = 0.43, P < 0.01).
There were also significant correlations between diurnal and
nocturnal stool frequency ( r = 0.7, P < 0.001) and between
CCPR and Ki67 activity ( r = 0.58, P < 0.001 ).
Discussion
Dietary calcium supplements in the dose used significantly
reduced stool frequency in patients after ileoanal anastomosis
Br. J. Surg., Vol. 79, No. 2, February 1992
Acknowledgements
We are grateful to the Medical Photography Department, Dudley Road
Hospital for the diagrams and to Fay Cox for typing the manuscript.
References
1.
2.
3.
4.
5.
Life with an ileostomy. Lancet 1982; i i : 1079-80 (Editorial).
Whates PD, Irving M . Return to work following ileostomy. Br
J Surg 1984; 71 : 619-22.
Mortensen N. Progress with the pouch -restorative proctocolectomy for ulcerative colitis. Gut 1988; 2 9 : 561-5.
Shepherd NA, Jass JR, Duval I, Moskowitz RL, Nicholls RJ,
Morson BC. Restorative proctocolectomy with ileal reservoir:
pathological and histochemical study of mucosal biopsy
specimens. J Clin Pathol 1987; 40: 6 0 - 7 .
Nasmyth DG, Codwin PGR, Dixon MED, Williams NS,
Johnson D. Ileal ecology after pouch anal anastomosis or
ileostomy. A study of mucosal morphology, faecal bacteriology,
faecal acids and their interrelationship. Gastroentero/ogy 1989;
96: 817-24.
6.
7.
8.
Moskowitz RL, Shepherd NA, Nicholls RJ. An assessment of
inflammation in the reservoir after restorative proctocolectomy
with ileoanal ileal reservoir. Int J Color Dis 1986; 1 : 167-74.
Kmiot WA, Youngs D, Winslet M, Curran FT, Keighley MRB.
Ileal adaptation following restorative proctocolectomy. Br J Surg
1989; 76: 625 (Abstract).
Wilson RG, Brydon WG, Smith AN. Antitropic effect of dietary
calcium in subjects at increased risk for colon cancer. Br J Sury
1990; 77: 691 (Abstract).
131
Calcium supplements and ileoanal pouch function: G. H. Barsoum et al.
9.
10.
11.
12.
13.
14.
15.
Lipkin M, Friedman E, Winawar S, Newmark H. Biomarker of
colonic cancer risk modified by calcium. Clin Res 1987 ; 35 : 589
(Abstract ).
Appleton GVN, Davies PW, Bristol JB, Williamson RCN.
Inhibition of intestinal carcinogenesis by dietary supplementation
with calcium. Br J Surg 1987; 74: 523-5.
Buset M, Lipkin M, Winawer S, Swaroop S, Friedman E.
Inhibition of human colonic epithelial cell proliferation in uivo
and in oitro by calcium. Cancer Res 1986; 46: 5426-30.
Wargovich MJ, Eng VWS, Bruce WR. Calcium ameliorates the
toxic effects of deoxycholic acid on colonic epithelium.
Carcinogenesis 1983; 4 : 1205-7.
Wargovich MJ, Eng VWS, Newmark HL. Calcium inhibits the
damaging and compensatory proliferative effects of fatty acids
on mouse colonic epithelium. Cancer Lett 1984; 23: 253-8.
Gerdes J, Lemke H, Baisch H et al. Cell cycle analysis of a cell
proliferation associated human nuclear antigen defined by the
monoclonal antibody Ki-67. J Immunol 1984; 133: 1710-15.
Goodlad RA, Wright NA. Quantitative studies on epithelial
replacement in the gut. I n : Tichen DA, ed. Techniques in the Life
Sciences Digestiue Physiology. Amsterdam : Elsevier Scientific
Publishers, 1982: 1-23.
Franklin WA, McDonald G P , Stein H O et al. Immunohistochemical demonstration of abnormal colonic crypt cell kinetics
in ulcerative colitis. Hum Pathol 1985; 16: 1129-32.
Rozen P, Fireman Z, Fine N , Wax Y, Ron E. Oral calcium
suppresses increased rectal epithelial proliferation of persons at
risk of colorectal cancer. Gut 1989: 30: 650-5.
Lipkin M , Newmark H. Effect of added dietary calcium on
colcnic epithelial cell proliferation in subjects at high risk for
19.
20.
21.
22.
23.
24.
25.
-
16.
17.
18.
132
26.
27.
familial colonic cancer. N EnglJ Med 1985: 313: 1381-4.
Newmark HL, Wargovich MJ, Bruce WR. Colon cancer and
dietary fat, phosphate, and calcium : a hypothesis. J Nut/ Canwr
Inst 1984; 72: 1323-5.
Carlson LA, Olsson AG, Oro I, Rossner S. Effects of oral calcium
upon serum cholesterol and triglycerides in patients with
hyperlipidemia. Atherosclerosis 1971 ; 41 : 391-400.
Bierenbaum ML, Fleishman AI, Raichelson RI. Longterm
human studies of the lipid effects of oral calcium. Lipids 1972;
14: 391-400.
Belizan J M , Villar J, Pineda 0 et PI. Reduction of blood pressure
with calcium supplements in young adults. J A M A 1983; 249:
1161-5.
Heaney RP, Recker RR, Savill PD. Menopausal changes in
calcium balance performance. J Lab Clin Med 1978; 92: 953-63.
Le-Veen HH, Borek B, Axelrod DR, Johnson A. Cause and
treatment of diarrhoea following resection of the small intestine.
Surg Gvnecol Obstei 1967; 124: 766-70.
Stadler J, Stern HS, Yeung KS et al. Effect of high fat
consumption on cell proliferation activity of colorectal mucosa
and on soluble fecal bile acids. Gut 1988; 29: 1326-31.
Kmiot WA, Youngs DJ, Keighley MRB. Acute reservoir ileitis
(pouchitis) : are the mucosal morphological changes due to
bacterial overgrowth? Br J Surg 1990; 77: 1417 (Abstract).
Gregoire RC, Stern HS, Yeung KS et ul. Effect of calcium
supplementation on mucosal cell proliferation in high risk
patients for colon cancer. Gui 1989; 30: 376-82.
Paper accepted 27 September 1991
Br. J. Surg.. Vol. 79, No. 2. February1992
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