Effects of Supplemental Vitamin D and Calcium on Biomarkers of Inflammation in Colorectal Adenoma Patients: A Randomized, Controlled Clinical Trial

Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States and is consistently inversely associated with calcium intake and serum vitamin D levels (1–9). Inflammation is intricately linked to the etiology of CRC and may also be a key in understanding the mechanisms linking calcium and vitamin D to CRC risk reduction. Inflammatory conditions such as Crohn's disease and ulcerative colitis are established risk factors for CRC, nonsteroidal anti-inflammatory drug (NSAID) use reduced both polyposis in patients with familial adenomatous polyposis and sporadic colorectal adenoma recurrence in clinical trials, and specific proinflammatory markers such as C-reactive protein (CRP), TNF-α, and interleukin (IL)-6 are elevated in inflammatory bowel disease patients (10–14). These inflammatory markers are also associated with neoplastic growth, higher tumor grade, and increased risk of mortality in CRC patients (11, 12, 15–19). In addition, in a case–control study, risk factors for colorectal adenomas, such as old age, smoking, and adiposity, were found to be associated with higher levels of these inflammatory markers (10).

The mechanisms by which calcium is proposed to reduce risk for developing CRC are closely related to inflammation. Calcium binds to free fatty acids and bile acids, precipitating them from solution in the colon, which is hypothesized to reduce oxidative stress and inflammation in the colon (20). Calcium also activates the calcium sensing receptor, which is involved in cell-cycle events and differentiation, and promotes cell–cell and cell–matrix adhesion (21, 22). Vitamin D, along with increasing the absorption of calcium and regulating calcium homeostasis, also regulates more than 200 genes through the vitamin D receptor (VDR). Activation of the VDR is involved in bile acid degradation, direct transcriptional regulation of several inflammatory cytokines, cell-cycle regulation, DNA repair, differentiation, and apoptosis (22, 23).

Despite the basic science evidence, there are no published trials of the effects of vitamin D and/or calcium supplementation on blood markers of inflammation in patients at risk for developing CRC. To address this, we conducted a pilot, randomized, double-blind, placebo-controlled 2 × 2 factorial chemoprevention trial of calcium and vitamin D₃ supplementation, alone or in combination, versus placebo over 6 months to estimate their effects on a panel of circulating pro- and anti-inflammatory markers in patients with a history of sporadic colorectal adenoma. We hypothesized that vitamin D₃ and calcium, alone or in combination, would decrease tumor-promoting proinflammatory markers and increase tumor-inhibiting, anti-inflammatory markers.

This study was approved by the Emory University Institutional Review Board. Written informed consent was obtained from each study participant.

The detailed protocol of study recruitment and procedures was published previously (24). Briefly, study participants were recruited from the patient population attending the Digestive Diseases Clinic of Emory University. Eligibility included age 30 to 75 years, in good general health, capable of informed consent, and at least 1 pathology-confirmed sporadic colon or rectal adenoma in the past 36 months. Exclusions included contraindications to calcium or vitamin D₃ supplementation or rectal biopsy procedures, and medical conditions, habits, or medication usage that would otherwise interfere with the study (24).

Between April 2005 and January 2006, potential participants attended an eligibility visit during which they were interviewed, signed a consent form, completed questionnaires, provided a blood sample, and were entered into a 30-day placebo run-in trial (24). Diet was assessed with a semiquantitative food frequency questionnaire (25). After the 30-day placebo run-in trial, 92 participants without significant perceived side effects who had taken at least 80% of their capsules during the run-in trial were eligible for randomization. Eligible participants then underwent a baseline blood draw and rectal biopsy and were randomly assigned (stratified by sex and NSAID use) to the following 4 treatment groups: a placebo control group (n = 23), a 2.0 g elemental calcium (1 g twice daily as calcium carbonate) group (n = 23), an 800 IU vitamin D₃ (400 IU twice daily) group (n = 23), and a 2.0 g elemental calcium plus 800 IU vitamin D₃ group (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. Additional details and rationale for the doses and forms of calcium and vitamin D supplementation were described previously (24).

Over the 6-month treatment period, participants attended follow-up visits at 2 and 6 months after randomization and were contacted by telephone at monthly intervals between the second and final follow-up visits. 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, interim telephone call, and questionnaires and graded according to NIH Common Toxicity Criteria 2.0 and the 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 (but not nonaspirin NSAIDs) use for 7 days before each biopsy/blood draw 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 inflammatory cytokines (e.g., diet and NSAID use) were assessed at baseline, several were reassessed at the first follow-up visit, and all were reassessed at the final follow-up visit.

Peripheral venous blood samples were taken after the subject sat upright with his or her legs uncrossed for 5 minutes. Blood was drawn into red-coated, prechilled vacutainer tubes for whole blood, plasma, and serum and then immediately placed on ice and shielded from light. Blood fractions were aliquotted into amber-colored cryopreservation tubes, the air was displaced with argon gas, and then the aliquots were immediately placed in a −80°C freezer until analysis.

All samples were blinded to treatment group and treated identically. A single ELISA (R&D Systems) was used to measure CRP, in duplicate, according to the manufacturer's protocol. The average intra-assay coefficient of variation (CV) for CRP was 6.6%. A high-sensitivity multiplex ELISA (R&D Systems) was used to measure TNF-α, IL-6, IL-1β, IL-8, IL-5, IL-4, VEGF, IL-2, IL-10, IL-12, granulocyte macrophage colony-stimulating factor (GM-CSF), and IFN-γ, in duplicate, according to the manufacturer's protocol. The average intra-assay CV for TNF-α was 11.5%, for IL-6 11.7%, for IL-1β 10.6%, for IL-8 7.9%, for IL-5 34.1%, for IL-4 39.4%, for VEGF 21.0%, for IL-2 45.0%, for IL-10 11.5%, for IL-12 24.5%, and for GM-CSF 38.5%. Low plasma cytokine concentrations create very high variability, and the results for cytokines with CVs above 15% were considered too variable and inaccurate to be reported.

Treatment groups were assessed for comparability of characteristics at baseline and final follow-up by the Fisher exact test for categorical variables and ANOVA for continuous variables. ELISA reliability was assessed using CVs.

Primary analyses were based on assigned treatment at the time of randomization regardless of adherence (intent-to-treat analysis). Biomarker levels below the limits of detection were assigned a value equal to the lower limit of detection for that biomarker. Variables not normally distributed were transformed, as appropriate, before statistical testing. Mean biomarker concentrations were calculated for each treatment group for the baseline and 6-month follow-up visits. Treatment effects were evaluated by assessing the differences in biomarker concentrations from baseline to 6-month follow-up between each active treatment group and the placebo group by a repeated-measures linear mixed-effects model, as implemented using the Proc MIXED procedure of the Statistical Analysis System (SAS, version 9.2; SAS Institute Inc.). The model included the intercept, indicators for treatment group and visit (baseline and follow-up), and a treatment by visit interaction term. Study participant was treated as a random effect, and absolute treatment effects were calculated and reported. A cutoff level of P ≤ 0.05 (2-sided) was used for assessing statistical significance. Because concentrations of the measured biomarkers in plasma are not widely familiar, to provide perspective on the magnitude of treatment effects, relative effects were also calculated, defined as [(treatment group follow-up/treatment group baseline)/(placebo follow-up/placebo baseline); refs. (24, 26)]. The relative effect provides a conservative estimate of the average proportional change in the treatment group relative to that in the placebo group. The interpretation of the relative effect is somewhat analogous to that of an OR (e.g., a relative effect of 2.0 means that the relative proportional change in the treatment group was twice as great as that in the placebo group). Stratified analyses were conducted to investigate potential differential treatment effects by sex, age, body mass index (BMI), and NSAID use.

To assess the effects of vitamin D₃ and/or calcium supplementation on a summary score of all the pro- and anti-inflammatory markers combined, a summary inflammation z-score was calculated. This score was calculated as follows: first, a normalized z-score for each individual biomarker value, with a mean of zero and SD of 1.0, was calculated as z = (x − μ)/σ, where x is a participant's biomarker value at a given visit, and μ and σ are the study population mean and SD, respectively, at baseline; and then the combined inflammation z-score for each participant at each trial visit was created by summing the z-scores of each inflammatory marker [IL-10 was included with a negative sign, because it has been shown to protect against colonic inflammation (27)]. This inflammation z-score was then analyzed as that for the individual biomarkers.

Treatment groups were quite similar on characteristics measured at baseline (Table 1) or at final follow-up (data not shown; in particular, there was no change in NSAID use by treatment group over the course of the trial). The mean age of participants was 61 years, 70% were men, 71% were white, and 20% had a family history of CRC in a first-degree relative. Adherence to visit attendance averaged 92% and did not differ significantly among the 4 treatment groups. On average, at least 80% of pills were taken by 93% of participants at the first follow-up visit and 84% at the final follow-up visit. There were no complications attributed to study procedures or treatments. Seven participants (8%) were lost to follow-up due to perceived drug intolerance (n = 2), unwillingness to continue participation (n = 3), physician's advice (n = 1), and death attributed to cardiovascular disease (n = 1). Participant dropouts from the trial included 1 person from the vitamin D₃ supplementation group and 2 persons from each of other 3 groups.

At baseline, there were no significant differences between the 4 study groups in serum 25-hydroxy vitamin D (25-OH-vitamin D). By study end, serum 25-OH-vitamin D levels statistically significantly (P < 0.0001) increased by 60% to 29.5 ng/mL in the vitamin D₃ group and by 56% to 28.5 ng/mL in the calcium plus vitamin D₃ group relative to placebo (24).

Changes in CRP, TNF-α, IL-6, IL-1β, IL-8, and IL-10 plasma concentrations relative to placebo in the calcium, vitamin D₃, or combined supplementation groups are shown in Table 2. After 6 months of treatment, in the vitamin D₃ supplementation group, CRP decreased by 32%, TNF-α by 13%, IL-6 by 32%, IL-1β by 50%, and IL-8 by 15%, relative to placebo, although these changes were not statistically significant. In the calcium supplementation group, relative to placebo, CRP decreased 8%, IL-6 decreased 37%, IL-8 by 11%, and IL-1β by 27%, although these changes were also not statistically significant. In the vitamin D₃ plus calcium supplementation group, IL-6 decreased by 8%, IL-8 by 13%, and IL-1β by 35%, relative to placebo, although these changes were not statistically significant. IL-10 decreased by a minor and nonsignificant amount in all active treatment groups.

The effects of vitamin D₃ and/or calcium on the combined “inflammation z-score” of all reported inflammatory markers (CRP, TNF-α, IL-6, IL-8, IL-1β, and IL-10) are summarized in Table 3. An individual's inflammation z-score allows for the calculation of an aggregate score of all biomarkers by converting them to a comparable score, a z-score, and then totaling the values for each individual. The overall inflammation z-score significantly dropped 77% (P = 0.003) in the vitamin D₃ treatment group, 48% (P = 0.18) in the calcium treatment group, and 33% (P = 0.40) in the combined treatment group relative to placebo.

Men and women have differences in their biochemical makeup (such as estrogen levels) that could lead to differences in response to vitamin D and calcium supplementation; therefore, we investigated potential differences in response by sex (Table 4). In men, CRP decreased 37% (P = 0.05) in the vitamin D₃ treatment group relative to placebo but did not change substantially in women. Similar to the results for CRP, the inflammation z-score statistically significantly dropped in men (83%, P = 0.01) but not in women in the vitamin D₃ treatment group relative to placebo. Changes in TNF-α, IL-6, IL-8, IL-1β, and IL-10 did not differ substantially by sex (data not shown).

Because NSAID use may overwhelmingly affect inflammation pathways, we investigated the effects of vitamin D₃ and calcium among study participants who were not currently taking NSAIDs (Table 4). In non-NSAID users, the decrease in CRP (41%; P = 0.05) was slightly stronger than in all participants combined (32%; P = 0.11) in the vitamin D₃ treatment group relative to placebo. The inflammation z-score also decreased significantly by 58% (P = 0.01) among non-NSAID users in the vitamin D₃ treatment group. Changes in TNF-α, IL-6, IL-8, IL-1β, and IL-10 among non-NSAID users did not differ substantially from changes among all participants combined (data not shown).

The results from this pilot, randomized, controlled clinical trial suggest that supplementation with vitamin D₃ or calcium alone may decrease tumor-promoting proinflammatory markers in the plasma of sporadic colorectal adenoma patients. These findings are consistent with the hypothesis that vitamin D₃ or calcium may decrease inflammation in the colon and thus reduce risk for colorectal neoplasms. Consistent with previous findings in this same study on oxidative DNA damage in the normal colorectal mucosa (28), our findings also suggest that vitamin D₃ combined with calcium may have a lesser treatment effect on proinflammatory markers than do vitamin D₃ or calcium alone.

Inflammation is intricately linked to the etiology of CRC, as evidenced by inflammatory conditions of the colon, such as Crohn's disease and ulcerative colitis, which are established risk factors for the disease (29). Several inflammatory molecules, including CRP, TNF-α, IL-6, and IL-8, were found to be higher in the blood of CRC patients than in controls (11, 12, 19) and have been associated with other risk factors for CRC, such as age, smoking, and high BMI (10). In addition, CRP, TNF-α, and IL-6 are associated with higher tumor grade and poorer prognosis (15, 19), and higher levels of CRP and IL-6 are associated with increased mortality among CRC patients (11). In a case–control study, polymorphisms in the genes for IL-6, TNF-α, IL-1β, and IL-8 that are linked to increased expression of their corresponding cytokines were associated with increased adenoma risk (18, 30). IL-1β is involved in COX-2 activation and activates the Wnt cell-cycle activation pathway, the primary pathway of colon cell proliferation (31). Vitamin D₃ inhibited this pathway in vitro by decreasing IL-1β production by macrophages, thus decreasing colon carcinoma cell proliferation (31).

Calcium and vitamin D have several mechanisms of action relevant to our hypothesis that they may decrease inflammatory markers and risk for developing CRC. Only about 30% of calcium is absorbed in the gastrointestinal tract, with the other 70% free to bind with and precipitate bile acids, which have been shown to cause damage to epithelial cell membranes and produce an inflammatory response in these cells (32, 33). This inflammatory response, in turn, may represent a large source of circulating cytokines. Vitamin D, acting through the vitamin D receptor, also reduces bile acids in the colon by increasing the bile acid–catabolizing enzyme CYP3A4 (21, 34). 1,25-Dihydroxycholecalciferol or 1,25-dihydroxyvitamin D₃ [1-25-(OH)₂-vitamin D₃] binding of the vitamin D receptor acts as a transcriptional regulator to enhance IL-10 transcription and represses several proinflammatory cytokines, including IL-6, IL-8, and TNF-α (26, 35). In addition, the vitamin D receptor, when activated by vitamin D, suppresses the transcription of RelB, a component of the global transcriptional regulator nuclear factor kappaB (NF-κB; ref. 36), a key regulator of inflammation and response to oxidative stress and a downstream target of TNF-α (37). NF-κB induces the transcription of inflammatory cytokines and antiapoptotic proteins that together promote cellular transformation and tumor formation (38). Mice lacking IL-10 quickly develop inflammatory bowel disease, but supplementation with vitamin D₃ ameliorated symptoms and blocked the progression of the disease (27). Combined with this biological evidence, the results of our study support vitamin D₃ and calcium as possible inflammation-reducing agents in humans.

Contrary to our original hypothesis, and the findings of some epidemiologic and clinical studies, we found no evidence for a greater than additive effect of combined supplementation of calcium and vitamin D₃ (28, 39–42). Our estimated treatment effects in the calcium plus vitamin D₃ group tended to be less than those for the individual agents. In this same population, we previously reported that combined calcium and vitamin D₃ supplementation may have lesser effects on colorectal epithelial apoptosis, differentiation, and oxidative DNA damage than do calcium or vitamin D₃ alone (24, 28, 43). These statistically nonsignificant findings of a smaller treatment effect in the combined treatment group may simply be due to chance because of our small sample size. However, given the consistency of this pattern, it is possible that calcium and vitamin D negatively regulate one another. 1,25-(OH)₂-vitamin D₃ regulates calcium absorption, and calcium suppresses 1,25-(OH)₂-vitamin D₃ synthesis by 1α-hydroxylase (44). One animal study found that high calcium supplementation led to lower circulating levels of 25-OH-vitamin D (34); however, in humans, risk of adenoma recurrence was only decreased by calcium supplementation in individuals with higher serum 25-OH-vitamin D levels (40). In human colon carcinoma cells, calcium and vitamin D synergistically enhanced the expression of E-cadherin; however, the enhanced expression of p21 and p27 by calcium and vitamin D separately was not changed with a combined treatment (45). Another possible explanation for the less than additive effects of calcium plus vitamin D₃ is that too little vitamin D₃ was given. Although 800 IU daily vitamin D₃ supplementation in this population statistically significantly raised serum 25-OH-vitamin D levels, the mean in all treatment and placebo groups was below 32 ng/mL, the suggested level to be considered sufficient for this vitamin (24, 46). Taken together, the combined effect of calcium and vitamin D₃ on biomarkers of colon carcinogenesis and inflammation in humans is unclear and requires further clarification through larger studies.

Calcium and vitamin D have several known and likely unknown downstream targets involved in inflammation regulation as discussed earlier and therefore biological effects of these agents may be best measured using a combined detection method. We developed an inflammation z-score to assess the inflammation status of an individual more comprehensively and then analyzed the effects of calcium and/or vitamin D₃ on this inflammation z-score. We hypothesized that vitamin D₃ and/or calcium would affect this inflammation z-score more substantially than any single measure of inflammation. Vitamin D₃, but not calcium or the 2 combined, significantly reduced the inflammation z-score in this study population by 77% (P = 0.003) relative to placebo. This finding suggests that vitamin D₃ may reduce inflammation in multifactorial ways. Inflammatory markers, including CRP, IL-6, and TNF-α, were found to be significantly higher in CRC patients than in controls (11, 12, 19); however, it is not known whether these individual markers are also elevated in colorectal adenoma patients. We propose the use of this inflammation z-score to measure subclinical inflammation or to detect small changes in multiple cytokines that combined may produce clinically important changes in inflammation and risk for developing disease. Further investigation is needed, however, and this score should be explored in cohort and case–control studies to investigate whether it is associated with risk for developing colorectal adenomas or cancer, as well as in larger chemoprevention trials to investigate its usefulness as an intervention response marker.

In our analysis stratified by sex, there was a significant reduction in CRP and the inflammation z-score with vitamin D₃ supplementation in men but not in women. There are several possible explanations for this, the most obvious one being chance related to the small sample size, especially in women. Another possible explanation is that most women in this study were postmenopausal and not taking hormone replacement therapy and therefore likely had low estrogen levels. Estrogen supplementation was found to increase 1-25-(OH)₂-vitamin D signaling and downregulate inflammation pathways in the rectal epithelium of postmenopausal women (47). The findings of our study support the hypothesis that low estrogen levels may interfere with response to vitamin D supplementation, VDR signaling, and inflammatory pathways; however, larger studies are needed to investigate these issues more definitively.

When only non-NSAID users were considered, CRP and the inflammation z-score were found to be statistically significantly reduced with vitamin D₃ supplementation. Other than chance due to the small sample size, a possible explanation is that NSAIDs have powerful effects on inflammation pathways that could mask effects of vitamin D₃ or calcium. NSAIDs largely reduce risk for developing CRC by blocking a major colon carcinogenesis and inflammatory pathway enzyme, COX-2 (48, 49). Vitamin D supplementation effects on inflammation may only be detectable and important in individuals not already using NSAIDs, although more investigation is needed to clarify this issue.

Our pilot study has several limitations and strengths. First, the sample size was small, limiting the statistical power for detecting treatment effects. A second potential limitation to the study is that all of the blood biomarker analyses except for CRP were done using a high-sensitivity multiplex ELISA. Although the low limit of detection allowed for a higher number of samples with detectable analytes, the measurements may have been less reliable and accurate than would have been found with nonmultiplex ELISA. We accounted for this lower accuracy of the ELISA by calculating CVs and did not report data for those cytokines with a CV of more than 15%. Third, with the relatively low dose of vitamin D₃ supplementation, although serum 25-OH-vitamin D levels increased significantly, the average serum levels did not reach the “sufficiency” range of above 32 ng/mL (24, 46). Therefore, higher doses of supplemental vitamin D₃ may produce more definitive changes in proinflammatory markers.

Strengths of this study included the randomized, double-blind, placebo-controlled clinical trial design; the high protocol adherence by the study participants; investigation of both the individual and combined effects of calcium and vitamin D₃; and the balance in the treatment groups on many potential confounding risk factors for CRC and inflammation.

In summary, our preliminary findings suggest vitamin D₃ or calcium alone may decrease tumor-promoting proinflammatory markers in the plasma of sporadic colorectal adenoma patients. Also, taken together with previous literature, this study supports further investigation of (a) vitamin D₃ or calcium supplementation for reducing inflammatory biomarkers in sporadic colorectal adenoma patients, (b) our investigated biomarkers of inflammation or a combined inflammation z-score as potential treatable biomarkers of risk for developing CRC, and (c) a larger trial with higher doses of vitamin D₃ on biomarkers of inflammation and risk for developing colorectal neoplasms.

The NIH, the Georgia Cancer Coalition, and the Franklin Foundation had no influence on the design of the study; the collection, analysis, and interpretation of the data; the decision to submit the manuscript for publication; or the writing of the manuscript.

We thank Bill Liang for equipment management and advice, Jill Woodward for managing the trial, and all study participants for their time and dedication to the study.

This study was funded in part by grants from the NIH, the Franklin Foundation, and a Georgia Cancer Coalition Distinguished Scholar Award (to R.M. Bostick); R01 CA104637/CA/NCI NIH HHS/United States; R03 CA136113/CA/NCI NIH HHS/United States; and The Franklin Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.