Effects of Vitamin D and Calcium Supplementation on Markers of Apoptosis in Normal Colon Mucosa: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Participant population

Participants were recruited from the patient population attending the Digestive Diseases Clinic of Emory University. Eligibility included age 30 to 75 y, in general good health, capable of informed consent, and a history of at least one pathology-confirmed sporadic colon or rectal adenoma within the past 36 mo. Exclusions included contraindications to calcium or vitamin D supplementation or rectal biopsy procedures, and medical conditions, habits, or medication usage that would otherwise interfere with the study. Specific exclusions are listed in Supplementary Appendix 1.

Clinical trial protocol

Participant recruitment and flow is depicted in Fig. 1. All age-eligible practice patients diagnosed with at least one pathology-confirmed adenomatous colonic or rectal polyp within the previous 36 mo were identified as potential study participants. Medical charts were screened, and potentially eligible patients were sent an introductory letter followed by a telephone interview during which willingness to participate and further eligibility was assessed, and, if appropriate, an in-person eligibility visit was scheduled. During the eligibility visit, potential participants were interviewed and signed a consent form, their medication and nutritional supplement bottles were reviewed, and they completed questionnaires (on sociodemographics, medical history, medication and nutrition supplement use, lifestyle, family history, and others) and provided a blood sample. Diet was assessed with a semiquantitative food frequency questionnaire (28). Medical and pathology records were reviewed. Those still eligible and willing to participate then entered a 30-d placebo run-in trial. Only participants without significant perceived side effects and took at least 80% of their tablets were randomized. Adherence for the run-in trial was assessed by questionnaire, interview, and pill count. Eligible participants then underwent a baseline rectal biopsy and were randomly assigned (stratified by sex and nonsteroidal anti-inflammatory drug use) over 9 mo to treatment group. Of those who passed initial chart eligibility, 42% were contacted and 20% were eligible and consented to participate.

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Fig. 1

Flow diagram of a trial of supplemental calcium and vitamin D₃, alone and in combination, versus placebo over 6 mo on markers of apoptosis in the normal colorectal mucosa.

Participants (n = 92) were randomly assigned to the following four treatments: placebo (n = 23), 2.0 g elemental calcium supplementation (as calcium carbonate in equal doses twice daily; n = 23), 800 IU vitamin D₃ supplementation (400 IU twice daily; n = 23), and 2.0 g elemental calcium plus 800 IU vitamin D₃ (n = 23).

Study tablets were custom manufactured by Tishcon Corp. The corresponding supplement and placebo pills were identical in size, appearance, and taste. The placebo was free of calcium, magnesium, vitamin D, and chelating agents.

Calcium carbonate was used for elemental calcium delivery in this trial because it was also successfully used in the Calcium Polyp Prevention adenoma recurrence (29) and Calcium and Colorectal Epithelial Cell Proliferation trials (30), it was used in most large studies using calcium long term for other reasons and therefore had the most established safety record, it is inexpensive, and it delivers more elemental calcium per tablet than other forms; thus, fewer tablets are required, enhancing adherence.

Vitamin D₃ was chosen as our form of vitamin D for several reasons, including avoidance of the toxicity risks associated with 1,25-(OH)₂-vitamin D or 25-OH-vitamin D. Supplementation with vitamin D₃ (a prohormone) takes advantage of natural metabolism to generate the most active moiety. Supplementation with even large doses of vitamin D₃ does not increase total 1,25-(OH)₂-vitamin D levels in individuals who are not vitamin D deficient (31). Multivitamins and calcium/vitamin D supplements typically provide 400 IU of vitamin D₃ daily, but numerous intervention studies (reviewed in refs. 32, 33) show that this dose will not suppress parathyroid hormone in most North American adults; however, 800 IU daily raises 25-OH-vitamin D levels toward the desired range and leaves a substantial margin of safety even when combined with dietary intake.

Over the 6-mo treatment period, participants attended follow-up visits at 2 and 6 mo after randomization and were contacted by telephone at monthly intervals between the second and final follow-up visits (Fig. 1). At follow-up visits, pill-taking adherence was assessed by questionnaire, interview, and pill count. Adverse events were monitored by interview at each study visit and interim telephone call and 2 wk after the last visit, questionnaire (included questions about hospitalizations, medical visits and diagnoses, medication changes, and symptoms) at each study visit, and by participant-initiated telephone calls, and graded according to NIH Common Toxicity Criteria and likelihood that they were study related. Participants were instructed to remain on their usual diet and not take any nutritional supplements not in use on entry into the study. At each follow-up visit, participants were interviewed and completed questionnaires. At the first and last visits, all participants underwent venipuncture and a rectal biopsy procedure. Participants were asked to abstain from aspirin use for 7 d before each biopsy visit. All visits for a given participant were scheduled at the same time of day to control for possible circadian variability in the outcome measures. Factors hypothesized to be related to the expression of apoptosis markers in normal colon mucosa (e.g., diet and medications) were assessed at baseline, several were reassessed at the first follow-up visit, and all were reassessed at the final follow-up visit. Participants did not have to be fasting for their visits and did not take a bowel cleansing preparation or enema.

Six 1-mm-thick biopsy specimens were taken from the rectal mucosa 10 cm proximal to the external anal aperture through a rigid sigmoidoscope with a jumbo cup flexible endoscopic forceps mounted on a semiflexible rod, teased off the forceps with and onto a strip of bibulous paper, then immediately placed in PBS and oriented under a dissecting microscope to ensure that they were not twisted or curled on the bibulous paper, and then immediately placed in 10% normal buffered formalin.

Immunohistochemistry protocol

The biopsies in formalin were left undisturbed for at least 6 h, transferred to 70% ethanol 24 h after being placed in formalin, embedded in paraffin blocks within 2 wk of the biopsy procedure, cut and stained within another 4 wk, and analyzed within another 4 wk. Five slides with four section levels each taken 40 μm apart were prepared for each biomarker, yielding a total of 20 levels per biomarker. Heat-mediated antigen retrieval was accomplished by steaming the slides for 40 min using a Pretreatment Module (Lab Vision Corp.) with 100× Citrate Buffer (pH 6.0; DAKO Corp.). Next, immunohistochemical processing by a labeled streptavidin-biotin method was accomplished using a DAKO Automated Stainer. The following reagents were used: antibody (Bcl-2 antibody, 1:100 dilution, Santa Cruz Biotechnology, Inc.; or Bax antibody, 1:200 dilution, DAKO) diluted with Antibody Diluent (DAKO), LSAB2 Detection System (DAKO), DAB (DAKO), and TBS buffer (DAKO). The slides, which were not counterstained, were coverslipped automatically with a Leica CV5000 Coverslipper (Leica Microsystems, Inc.) and placed in opaque slide folders. In each staining batch of biopsy slides, positive and negative control slides were included. Tonsil, used as a control tissue for both apoptosis biomarkers, was processed in the same manner as the patient's tissue except that antibody diluent was used rather than primary antibody on the negative control slide.

Protocol for quantifying staining density of immunohistochemically detected biomarkers in normal colon crypts (“scoring”)

The method (scoring) used to describe and quantify various characteristics of the labeled antigens in the colon crypts was a quantitative image analysis procedure for antigens that are labeled with a wide range of intensities in gradient distributions along the crypt axis—something that cannot be done manually. The unit of analysis was the “hemicrypt,” defined as one half of a crypt bisected from crypt base to colon lumen. A “scorable” hemicrypt was defined as an intact hemicrypt that extended from the muscularis mucosa to the colon lumen.

The major equipment and software for the image analysis procedures (scoring) were the following: personal computer, light microscope with appropriate filters and attached digital light microscope camera, digital drawing board, ImagePro Plus image analysis software (Media Cybernetics, Inc.), our custom-developed plug-in software for colorectal crypt analysis, and Microsoft Access (Microsoft Corp.). Equipment and imaging software settings were standardized. Slides were oriented in a standard manner and the section levels on the slides were viewed in sequence using light microscopy at ×200 magnification. The reader created a slide background correction image for the slide to be analyzed and, focusing on the first hemicrypt, captured and transferred the image as a 16-bit per pixel grayscale image from the camera to the image analysis program. Next, the hemicrypt was analyzed by precisely tracing the borders of the hemicrypt using a digital drawing board. The program then created a crypt length line midway along the hemicrypt axis, and then drew equally spaced perpendicular lines to the crypt length line at intervals to yield segments with the average widths of normal colonocytes. Finally, the program adjusted for any background levels on the slide, measured the absorbance of the labeling across the entire hemicrypt as well as within each segment, and entered the resulting data into the database automatically. Then, the reader moved to the next hemicrypt on the same or next image, section level, biopsy, and/or slide and repeated all the previously described analysis steps. The goal was to analyze a minimum of 16 hemicrypts on each of two biopsies, for a total of 32 hemicrypts.

One slide reader analyzed all of the Bax-stained and Bcl-2–stained slides throughout the study. Blinded subsets of previously analyzed slides were resubmitted to the reader during the study to assess intrareader reliability, which was found to range from 0.95 to 0.98 throughout.

Protocol for measuring serum 25-OH-vitamin D and 1,25-(OH)₂-vitamin D levels

Laboratory assays for plasma 25-OH-vitamin D and 1,25-(OH)₂-vitamin D were done by Dr. Bruce W. Hollis at the Medical University of South Carolina using a RIA method as previously described (34, 35). Plasma samples for baseline and follow-up visits for all subjects were assayed together, ordered randomly, and labeled to mask treatment group, follow-up visit, and quality control replicates. The average intra-assay coefficient of variation for plasma 25-OH-vitamin D was 2.3% and 6.2% for 1,25-(OH)₂-vitamin D.

Statistical analysis

Treatment groups were assessed for comparability of characteristics at baseline and final follow-up by the Fisher's exact test for categorical variables and ANOVA for continuous variables. Slide scoring reliability was analyzed using intraclass correlation coefficients.

The mean density of staining for Bax, Bcl-2, and the Bax/Bcl-2 ratio in normal colon crypts was calculated for each patient at baseline and 6-mo follow-up by summing all the densities from all analyzed crypts from the biopsy specimens and dividing by the number of crypts analyzed. Measures of the within-crypt distributions of the apoptotic markers (e.g., the ratio of expression in the upper 40% to the lower 60% of the crypts) were calculated for each patient by taking the mean of the biomarker densities in the upper 40% of crypts, or in the lower 60% of crypts, and constructing ratios of expression in the upper 40% to the lower 60% of crypts. We decided a priori to use as measures of the within-crypt distributions of the apoptotic markers the ratio of expression in the upper 40% (differentiation zone) to the lower 60% (proliferation zone) of the crypts, and the ratio of expression in the upper 20% (closest to colon lumen contents) to the lower 20% (furthest colon lumen contents) of the crypts because they represent the ratios of well-recognized functional or exposure zones. We transformed biomarker expression density data by dividing each individual's measurement by the mean density of the staining batch to adjust for possible batch effects, and then mean transformed biomarker densities were calculated for each treatment group for the baseline and 6-mo follow-up visits.

Treatment effects were evaluated by assessing the differences in the transformed densities from baseline to the 6-mo follow-up visit between patients in each active treatment group and the placebo group. The differences in the transformed densities from baseline to 6 mo between each active treatment group and controls were tested with two-sided Wilcoxon exact nonparametric tests. The magnitude of the treatment effects on the biomarker staining densities and distributions was expressed as relative effects, defined as (treatment group follow-up mean/treatment group baseline mean)/(placebo follow-up mean/placebo baseline mean). The interpretation of the relative effect is somewhat analogous to that of an odds ratio (e.g., a relative effect of 2.0 would mean that the relative proportional change in the treatment group was twice as great as that in the placebo group). Primary analyses were based on randomization treatment assignment regardless of adherence status (intent-to-treat analysis).

The distributions of Bax and Bcl-2 batch-standardized staining densities were plotted along the colorectal crypts by normalizing each crypt to 50 sections, averaging within each section across all crypts separately for each patient, and then for each treatment group.

In sensitivity analyses, we also analyzed data without batch standardization by including batch as a covariate and using different transformations; the results from these analyses did not differ materially from those reported.

Statistical analyses were done using Statistical Analysis System software (version 9.1; SAS Institute, Inc.). A cutoff level of P ≤ 0.05 (two sided) was used for assessing statistical significance.