IV. RESULTADOS Y DISCUCIONES
4.2. DETERMINACIÓN DE LA INFLUENCIA DEL TIEMPO Y
Between January 1990 and June 1991, 28 patients with FAP were recruited into the
trial. There were 19 males and 9 females, and the median age was 38 years (range 16-65). As is routine practice at St M ark's for this disease, all patients had previously undergone total abdominal colectomy with ileorectal anastomosis and were attending the outpatient clinic for follow-up. The median period since colectomy and ileorectal anastomosis was
12.5 years (range 2-30, mean 12 years). Exclusion criteria included a past history of hypercalcaemia, renal failure or peptic ulceration. The trial was fully explained to each patient and, after acceptance, a questionnaire was completed including the following
history and recent gastrointestinal symptoms. At each subsequent visit the medical history was updated. Patients were randomised in a double-blind fashion to receive either 1500 mg/day of calcium carbonate for a six month period or an inert placebo (Inositol) both supplied by Dalgety Pilsbury Ltd, Birmingham, U.K; this involved taking eight tablets per day. Coding and dispensing of the tablets was performed at the pharmacy department at St
M ark's Hospital. The code was broken only after completion of the trial. Compliance was assessed at subsequent visits to the outpatients by using a simple questionnaire and by examining returned medication containers. Entry into the trial did not alter the treatment of the patients. At St M ark's, the managment of polyps within the retained rectum is usually simple observation, unless the size increases greatly. None of the patients recruited were admitted for fulguration of the polyps during the trial period. Fully informed, written consent was obtained from all the patients, and local and regional ethical committee approval was granted before commencement of the trial. A contact telephone number was given to all patients.
Procedure
Abdominal examination and rigid sigmoidoscopy were performed at each visit to the clinic. At sigmoidoscopy, the position of the ileorectal anastomosis was first visualised,
then the site, size and number of polyps were recorded systematically in all four quadrants of the rectum while slowly withdrawing the sigmoidoscope. The information was recorded
on a standard sheet completed at each visit. Two rectal biopsies were taken from adjacent macroscopically normal rectal mucosa. The first biopsy was transported in culture medium, cooled on ice, to allow the determination of CCPR by in-vitro organ culture. The second
histological examination (to exclude the presence of microadenomas). Blood samples were taken to determine serum calcium, phosphate and vitamin D metabolites.
Crvpt cell production rate (CCPR)
Rectal biopsies were divided into multiple small explants and were orientated mucosal surface uppermost on a metal grid within an organ cuture dish (Lux laboratories). The crypt cell production rate was determined as previously described ( Materials and
Methods). Breifly, after 15 hours, vincristine 0.6 jug/ml (Oncovin, Eli Lily, Basingstoke U.K.) was added to the culture medium to produce metaphase arrest within the rectal crypts. Explants were removed one, two and three hours later, fixed (for 2-4 hours) in Camoy's fluid and then stored in 70% alcohol. Later, biopsies were rehydrated and after acid hydrolysis in IM HCL at 60°C for six minutes, the tissue was stained with Schiff's reagent. Using a dissecting microscope at least 20 crypts were isolated in each specimen, and the number of metaphase figures was counted per crypt. The mean number of arrested metaphases was plotted against time from vincristine administration, the slope of this line (determined by least squares linear regresssion analysis ) giving a value for the crypt cell production rate in cells/crypt/hour.
Serum profiles
Serum levels of 1,25 OHg Dj and 25 OH D, were analysed at the Regional Assay
Centre, The Middlesex Hospital, London. Calcium, albumin phosphate liver function tests and electrolytes were measured at the Hammersmith Hospital, London.
Stastical analysis.
Pre-and post-treatment values were compared using the paired Student's t-test within the test groups and the Mann-Whitney U-test between groups. Polyp counts were compared using analysis of variance, Chi-squared test and the Mann Whitney U-test. Serum electrolytes were compared using the Spearman correlation test.
Results.
Twenty-five patients completed the trial. Three violated the protocol and were therefore
excluded. Pre-and post-treatment polyp counts were obtained in all 25 patients who completed the trial. Initial rectal CCPR values were obtained in 22 patients, but one of these values was later excluded owing to the presence of a microadenoma on histological examination. Paired CCPR values before and after treatment were obtained in 16 patients (nine placebo and seven calcium patients). The tablets were well tolerated in all subjects with no adverse side effects being reported.
CCPR
There was considerable variation in the 21 pre-treatment values obtained, the overall mean being 5.96+ 0.88 cells/crypt/hour (m ean+s.e.m .) with a range o f 1.14- 19.80
cells/crypt/hour (Tablel). The median control metaphase count was 11.72 cells/crypt (range 4.16-18.70) at 1 hour, 14.36 cells/crypt (range 5.11-33.46) at two hours and 17.42 cells/crypt (range 13.22-35.87) at three hours.
Calcium reduced CCPR by 49% from a pre-treatment value of 4 .72+ 0.48 to a post-treatment value of 2.42+0.48 cells/crypt/hour (p < 0 .0 5 ) There was no significant difference in the placebo group, CCPR being 5.46+1.21 before treatment and 5.08+ 1.17 cells/crypt/hour after treatment (Tables 2 and 3).
Table 1.
Pre-treatment CCPR values in 21 patients (cells/crypt/hour).
Patient IRA yrs/ago Age Sex CCPR
A. N. 13 38 M 5 .0 7 ± 0 .5 6 D. M. 3 16 M 19.80+1.2 T. A. 37 65 M 1.14+0.02 M. S. 8 29 M 11.4+1.14. R. H. 2 46 M 11.09+0.5 C. W. 18 39 F 3.77+ 0.47 C. S. 29 53 F 5.18+ 0.54 H. M. 6 22 F 3.70+0.75 M. J. 30 56 M 7 .30+ 1.10 E. F. 13 37 F 4.16+ 0.38 H. B. 12 29 M 6.11 + 1.88 R. S. 6 46 M 7 .36+ 1.10 R.H. 6 30 M 5.60+ 1.74 D. N. 12 36 M 5.20+ 0.94 J. M. 14 40 F 4.47+ 0.24 R. T. 6 43 M 1.74+1.87 S. M. 6 40 F 3.65+2.25 S. H. 20 40 M 3.90+ 1.77 M. B. 10 29 M 5.47+ 0.14 H. H. 13 40 M 3.60+ 1.87 P.T. 4 33 M 5.45+ 2.74 Mean 12.50-hl.52 36.70-f-2.87 5.96+0.88
IRA = Ileorectal anastomosis. CCPR = Crypt cell production rate.