Br. J. Surg. 1992, Vol. 79, June, 581 -583

Reduction of mucosal crypt cell

proliferation in patients with colorectal adenomatous polyps by dietary calcium supplementation

G. H. Barsoum, C. Hendrickse*, M. C. Winslet, D. Youngs, I. A. Donovan*, J. P. Neoptolemos* and M. R. B. Keighley Academic Department of Surgery, Queen Elizabeth Hospital and *Department of Surgery, Dudley Road Hospital, Birmingham, UK Correspondence to: Mr J. P. Neoptolemos, Department

of Surgery, Dudley Road Hospital, Birmingham B18 7QH, U K

The crypt cell production rate was measured in 14 patients with adenomatous colorectal polyps, I7 patients with colorectal cancer and 12 control subjects. The median (interquartile range) rate (cells per crypt per hour) was found to be signijicantly higher ( P < 0.001) in the polyp (2.45 (1-94-3.20)) and cancer (3.01 (2.35-3.68)) groups compared with controls (1.25 (0.70-1.85)). A double-blind cross-over study was performed in patients with adenomatous polyps consisting of 2 months' treatment, 2 weeks' washout,followed by 2 months' treatment with dietary calcium supplementation (1.25 g day-') versus placebo. A signijicant reduction in the crypt cell production rate occurred with calcium treatment compared with the placebo (1.25 (06-2.25) versus 2.15 (1.58-3.08) cells per crypt per hour, P = 0.03.5). This study demonstrates a signijicant reduction in mucosal cell proliferation by dietary calcium supplementation in patients with adenomatous polyps. Such treatment may be worthy of further investigation in patients at high risk of developing colorectal polyps.

There is strong evidence that increases in mucosal cell proliferation precede the development of colonic cancer in humans and in animal models'.2. Expansion of the colonic crypt cell proliferative compartment has been demonstrated in patients with familial adenomatous p o l y p ~ s i s ~ in - ~ ,subjects with a history of familial or non-familial colonic cancer6-', and in patients with sporadic adenomasg-' or ulcerative colitis' 2-1 '. Wargovich et a1.I6 showed that dietary calcium significantly lowered the toxicity of bile acids, which are implicated in the pathogenesis of colorectal polyps and cancer. Free bile acids (representing 98 per cent of colonic bile acids) and fatty acids are precipitated in the colon as calcium phosphate complexes or soaps, provided there is sufficient calcium in the A number of experimental studies has suggested that this is the mechanism of action of dietary calcium in reducing colorectal carcinogenesisl6-' 8*20-23 . Alternatively, calcium may have a direct inhibitory effect on colonic cellular p r ~ l i f e r a t i o n ~ ~ . Epidemiological studies have suggested that calcium reduces the risk of colorectal c a n ~ e but r ~the~ data ~ ~are ~ ~onflicting~~. Of four dietary intervention studies ~ n d e r t a k e n ~ ' - ~only ' , one did not reveal a reduction in colonic crypt cell proliferation by dietary calcium30. There has been no detailed study, however, of calcium supplementation in patients with adenomatous polyps. The effects of supplementary dietary calcium on mucosal cell proliferation using a double-blind cross-over study were, therefore, investigated in a group of patients with adenomatous polyps. The colonic crypt cell production rate (CCPR) was examined in patients with colorectal cancer because the technique used is s t a t h m o k i n e t i ~and ~ ~ ,differs ~ ~ from that in other studies mentioned above, which used the tritiated thymidine labelling index.

Patients and methods Study design The rectal CCPR was measured in a group of control patients, a group with adenomatous polyps and a group with colorectal cancer. Rectal biopsies (2 mm thick) were obtained by rigid sigmoidoscopy from the posterior wall, 10 cm from the anal verge or at least 5 cm from a rectal

0007-1323/92/0605Sl-03

0 1992 Butterworth-Heinemann

Ltd

tumour if present. Patients with histological evidence of proctitis were excluded, as was any patient with a recent change from normal bowel habit (other than bleeding). None of the control subjects or patients had a family history of colorectal cancer. All the individuals were white Caucasians, taking a free-range omnivorous diet. The effect of calcium supplementation on rectal CCPR was assessed in the adenomatous polyp group using a double-blind cross-over study. The treatment was dispensed by the pharmacy in coded form with precise instructions to each patient, and prescriptions randomized by the pharmacy before the start of the trial using random number generation. The calcium and placebo were in powder form. The dose of calcium was 1.25 gday-' given as calcium gluconate (BDH, Atherstone, UK). The placebo was lactose powder, a substance known not to affect bowel habit in the absence of lactose intolerance, which was excluded by history. The study period consisted of 2 months on the first treatment followed by a 2-week washout period before starting on the second powder for a further 2 months. Patients The control group consisted of six men and six women with a median (interquartile range (i.q,r.))age of 64 (52-71 ) years. The patients were included as controls only if the large bowel was shown to be free of neoplastic lesions by rigid sigmoidoscopy and a double contrast enema. The diagnoses were haemorrhoids ( n = 8), diverticular disease ( n = 2) and non-specific abdominal pain ( n = 2). Patients with irritable bowel syndrome were excluded. The adenomatous polyp group consisted of eight men and six women with a median (i.q.r.) age of 64 (52-73) years. All had villous adenomas, which were solitary in all but two. There were 17 patients with colorectal cancer, of whom ten were men. The median (i.q.r.) age was 72 (57-78) years. Eleven cancers were rectal, four were rectosigmoid, and two were in the sigmoid colon. Three cancers were classified as Dukes' A, nine as Dukes' B and five as Dukes' C. Crypt cell production rate

of CCPR was based on previously described technique^^^,'^. Biopsies were immediately placed into a circular

Measurement

Perspex dish (Falcon Plastics, Los Angeles, California, USA) containing RPMI 1640 (Gibco, Paisley, U K ) organ culture medium with added gentamicin (40 mg ml-I), 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 for 1 h before use. The dish was incubated at 37°C in the above gas mixture. After 16 h the medium was replaced with identical medium but containing 0.4 g ml- ' vincristine (Eli Lilley, Basingstoke, UK). Biopsies were

581

Mucosal cell proliferation in colorectal polyposis:

G. H. Barsoum e t al.

removed 60,120 and 180 min later, fixed in Carnoy's solution for 2-6 h and stored in 70 per cent ethanol. After rehydration the specimen was hydrolysed in 1 mmol 1 hydrochloric acid for 6 min at 60T, stained with Schiffs reagent and 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 by one person (D.Y.) on coded samples. Linear regression analysis was used to determine the slope of metaphase accumulation and was expressed as cells per crypt per hour. ~

Ethical approval

Approval to undertake the study was obtained from the ethical committees of the Central and West Birmingham Health Authorities. Statistical analysis

Comparisons between multiple groups or multiple time-points were undertaken using the Kruskall-Wallis test adjusted for ties. If this was significant, paired analysis was performed using the two-tailed Mann-Whitney U test. Significance was taken as P < 0.05.

Results There was no significant difference in the ages between any of the groups. The median (i.q.r.) CCPRs in the three groups were: control group 1.25 (0.70-1%5) cells per crypt per hour; polyp group 2.45 (1.94-3.20); and cancer group 3.01 (2.35-3.68). This was significant overall ( H = 21.8, 2 d.f., P < 0.001). Compared with controls, values were significantly higher for the polyp ( P < 0,001 ) and cancer ( P < 0.001 ) groups but there was no difference between the latter two. Individual data are shown in Figure 1 . In the cross-over part of the study, calcium supplementation significantly reduced the CCPR in the patients with polyps ( P = 0.034). For simplicity, data for patients on calcium supplementation or placebo were grouped (Table I ); individual data are shown in Figure 2 .

01

Before treatment

Calcium

Placebo

Figure 2 Crypt cell production rates of patients with adenomatou.\ polyps, before and after treatment w'ith calcium or placebo

Discussion The stathmokinetic method used in this study differs from the tritiated labelling index method but provides an excellent measure of mucosal cell proliferation because it takes into account the cell cycle time, growth fraction and the crypt cell p ~ p u l a t i o n Despite ~~. differences in technique these findings are consistent with those from other studies which have shown an increase in mucosal cell proliferation in patients with colorectal polyps or cancer1.4s35,36.The tendency to develop synchronous or mctachronous tumours may be related to the finding of a hyperproliferative state throughout the colonic mucosa' Reduced proliferative activity, however, has been found in Seventh-Day Adventist vegetarians, a group with a lower mortality rate from colonic cancer than the general population in the USA3'. In agreement with previously published s t ~ d i e s ' ~ - ~this '-~~, study suggests that supplementary calcium intake in double the normal daily amount reduces the CCPR in patients at risk of developing colorectal cancer. Lipkin and Newmark2' showed a reduction in the labelling index of ten individuals at high risk from non-polyposis cancer of the colon. Buset et ~ 1 showed . ~ ~ a reduction in cellular proliferation in six of nine individuals with a previously determined high labelling index (four with a family history, four with tumours and one with neither). Using a similar technique Rozen et ~ 1 . ~showed ' a reduction of cell proliferation in a group of 26 first-degree relatives with sporadic colorectal cancer and nine patients who had previously undergone adenoma resection. In the only previous doubleblind study (non-cross-over), Gregoire et a/.30 studied 15 patients after resection for cancer and 15 control subjects. The labelling index appeared to increase after calcium supplementation ( P = 0.06), perhaps due to instability of colonic crypts following resection. Calcium supplementation of several grams per day results in minimal variation of serum calcium and has been shown to be safe in several long-term human s t ~ d i e s ~An ~ - intake ~ ~ . of 1500 mg day has been recommended to prevent osteoporosis in postmenopausal women without causing ill effects43. The 8-week duration of treatment with calcium or placebo was chosen to ensure that exposure was long enough to exert an effect on intestinal cell kinetics. The normal turnover time of ileal epithelial cells is only a few days. Within 1 week of altering dietary intake the intestinal epithelium can change its proliferation pattern44. In conclusion, calcium supplementation caused a reduction in the mucosal cell turnover rate of patients with colorectal polyps. Whether such an approach can prevent or delay the development of colorectal polyps can be determined only by long-term studies.

'.

8

c

8

lr

d

t 0

Controls (n = 12)

Adenomatous Colorectal PO~YPS ( n = 14)

cancer

( n = 17)

Figure 1 Crypt cell production rates of individuals in the three groups. Bars are median values

Table 1 Summary data of cross-ouer study in the 14 patients H,ith adenomatous polyps

~

Median crypt cell production rate (cells per crypt per hour) Before treatment

Calcium

2.25 (1.78-3.1)*

(0.6-2.25)t

1.25

Kruskall- Wallis test (2 dS.)

Placebo

H

P

2.15

6.81

0.034

(1.58-3.08)$

Values in parentheses are interquartile ranges. * P = 0.022 (before treatment uersus calcium); t P = 0.035 (calcium uersus placebo); $ P not significant (before treatment uersus placebo); all two-tailed Mann-Whitney U test

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Br. J. Surg.. Vol. 79, No. 6. June 1992

Mucosal cell proliferation in colorectal polyposis: G. H. Barsoum et al.

Acknowledgements Mr C. Hendrickse is a Sheldon Research Fellow supported by the West Midlands Regional Health Authority. The authors are grateful to the Medical Photography Department and to Fay Cox and Dilys Thomas of Dudley Road Hospital for preparing the manuscript.

References 1.

2. 3. 4. 5. 6. 7.

8.

9. 10.

II.

12.

13. 14. 15. 16. 17. 18. 19.

20. 21.

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Paper accepted 16 January 1992

583

Reduction of mucosal crypt cell proliferation in patients with colorectal adenomatous polyps by dietary calcium supplementation.

The crypt cell production rate was measured in 14 patients with adenomatous colorectal polyps, 17 patients with colorectal cancer and 12 control subje...
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