Abstract
Purpose
To estimate the effects of calcium or/and vitamin D supplementation on blood pressure and serum lipid and carotenoid levels.
Methods
92 colorectal adenoma patients were randomized in a pilot, double-blind, placebo-controlled clinical trial of supplemental vitamin D3 800 IU and elemental calcium 2.0 g (as calcium carbonate) alone or in combination in divided doses twice daily with meals over six months.
Results
Relative to placebo, mean serum triglycerides decreased 30% (P=0.10) and 32% (P=0.10) in the calcium and calcium plus vitamin D3 treatment groups, respectively. When the two calcium intervention groups were pooled and compared to the pooled non-calcium groups, the estimated supplemental calcium treatment effects were statistically significant for triglycerides (P=0.04). Similar, but non-statistically significant decreases (5–7%) were observed for serum total cholesterol levels. Mean systolic blood pressure increased 6% (P=0.08) in the calcium group; otherwise, there were no appreciable changes in systolic or diastolic blood pressures in any active treatment group. Mean serum total carotenoid levels decreased 14% (P=0.07) in the calcium and 9% (P=0.10) in the calcium plus vitamin D3 groups.
Conclusions
Our results suggest that supplemental calcium alone or combined with vitamin D3 but not vitamin D3 alone may reduce serum lipids and lipophilic micronutrients.
Keywords: Calcium, vitamin D, lipids, blood pressure, carotenoids
INTRODUCTION
Hyperlipidemia and hypertension are considered causal risk factors for cardiovascular disease (CVD) [1–3], a leading cause of death in the United States [3, 4]. It is suggested that calcium may have a beneficial effect on CVD risk through its influences on serum lipid concentrations [5, 6] and blood pressure [7]. However, there is also evidence that calcium supplementation or high intakes of calcium may increase risk for myocardial infarction [8] and mortality [9] despite possible beneficial effects on serum lipid levels. Several [5, 10–17], but not all [18–21], clinical trials of calcium and serum lipids reported that supplemental calcium lowered serum total and LDL-cholesterol and increased HDL-cholesterol levels. Findings from human studies of calcium and circulating triglycerides have been inconsistent, with most studies finding no beneficial effects of calcium supplementation [5, 10, 11, 13, 16–18, 20, 21]. In addition, calcium treatment was found to reduce blood pressure in rats [22], and a previous meta-analysis of randomized, placebo-controlled clinical trials suggested that calcium supplementation may lead to a small reduction in systolic blood pressure in humans [23, 24].
Vitamin D is closely associated with calcium metabolism [25] and may also affect lipid concentrations due to its effects on insulin sensitivity [26]. Previous small-scale studies of the effects of supplemental vitamin D on circulating lipids produced inconsistent results [27–29]. Recently, in a large (n=1,259), randomized clinical trial [21], Raipahak et al. reported ≤ 5% changes in levels of circulating lipids, including cholesterol and triglycerides, after daily supplementation of 1.0 g elemental calcium (as carbonate) plus 400 IU vitamin D3 for 5 years. However, only the combined effects of calcium and vitamin D were assessed in the above study.
Fat intake may be important with respect to uptake of carotenoids, and previous research found an inverse association of carotenoids with CVD and cancer risk [30, 31]. Due to their lipophilic nature, the bioavailability of carotenoids is partially dependent on the presence of fat in the intestine [32], therefore, reduction of available fat for transport in the gut, caused by its interaction with calcium to form calcium-lipid complexes [6, 21], may lead to decreased carotenoid absorption and correspondingly lower serum concentrations.
Using data and stored blood samples from a pilot clinical trial [33], we examined the effects of calcium or/and vitamin D3 on blood pressure (systolic and diastolic) and serum levels of lipids (total cholesterol and triglycerides) and carotenoids.
METHODS
Participant Population and Protocol
We used data and stored serum samples from a previous pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial design, 6-month clinical trial to test the effects of calcium and/or vitamin D on biomarkers of risk for colorectal cancer [33]. In brief, 92 participants (30 to 75 years of age) in general good health; capable of informed consent; with at least one pathology-confirmed adenomatous colorectal polyp within the past 36 months; no contraindications to calcium or vitamin D supplementation or rectal biopsy procedures; no habits, medical conditions, or medication usage that would otherwise interfere with study interpretation; and had taken at least 80% of their study pills during the 30-day placebo run-in trial, were recruited from the patient population attending the Digestive Diseases Clinic of Emory University. Individuals were eligible for the study regardless of history of hypertension, hypercholesterolemia, or the use of anti-hypertensive or cholesterol-lowing medications. The Institutional Review Boards of Emory University and the University of Hawaii approved the current study protocol.
At baseline, eligible participants were randomly assigned to the following four treatment groups: a placebo control group (placebo), a 2.0 g elemental calcium (1.0 g twice daily as calcium carbonate) group (calcium), an 800 IU vitamin D3 (400 IU twice daily) group (vitamin D3), and a 2.0 g elemental calcium plus 800 IU vitamin D3 group (calcium plus vitamin D3). 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. All study pills were taken twice daily with meals. The treatment period was six months, and participants attended follow-up visits at two and six months after randomization and were contacted by telephone between the second and final follow-up visits. Pill-taking adherence was assessed by interview, questionnaire, and pill count. Participants were instructed to maintain their usual diet and not take any nutritional supplements (except for those provided by the study) upon study enrollment. Blood was collected at baseline (randomization) and at study completion (6-month follow-up) and diet was assessed with a semi-quantitative food frequency questionnaire [34]. Participants were not required to fast for their visits.
Protocol for Measuring Blood Pressure
Blood pressures were measured at baseline and 6 months of follow-up by certified technicians following a standard protocol before blood draws or any other procedures. Briefly, arm circumference was measured, the appropriate size cuff was chosen, and then the participant remained quietly seated with both feet on the floor for 5 minutes. The correct size cuff was wrapped on the arm with the center of the bladder over the brachial artery. The time and then the pulse at the radial artery for 30 seconds were recorded. Using a calibrated standard aneroid sphygmomanometer, the cuff was then inflated to determine the pressure at which the radial pulse was obliterated, then deflated. The peak inflation level was determined as the pulse obliteration pressure plus 30 mm Hg. After the participant raised his or her arm for 5 seconds followed by a 30-second rest, the cuff was rapidly inflated to the peak inflation level, held constant for 5 seconds with the bell of the stethoscope placed on the brachial artery, then the cuff was slowly deflated at 2 mm Hg per second and the first and the fifth blood pressure phases were recorded. The cuff was completely deflated, then, after the participant raised his or her arm for five seconds and after another 30 seconds at rest, the blood pressure measurement was repeated. The average of the two recordings was taken as the blood pressure of the person on that visit.
Sample Analyses
Serum levels of total cholesterol and triglycerides were measured based on a latex particle enhanced immunoturbidimetric method using a Cobas MiraPlus clinical autoanalyser (Roche Diagnostics, Ltd., Rotkreutz, Switzerland) and an assay kit from Point Scientific, Inc. (Lincoln Park, MI).
Serum carotenoid (α-carotene, β-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein/zeaxanthin, lycopene, and total carotenoids) levels were analyzed by HPLC with pre-column electrochemical oxidation and post-column UV detection, as previously described in detail [35]. Assays were regularly validated for carotenoids through inclusion of external standards in each analysis batch and by participation in quality assurance programs organized by the US National Institute of Standards and Technology (NIST, Gaithersburg, MD) every year since 1994 [36].
Statistical Analysis
Treatment groups (placebo, calcium, vitamin D3, calcium plus D3) were assessed for comparability of characteristics at baseline and at final follow-up by ANOVA for continuous variables and Fisher’s exact test for categorical variables.
Primary analyses were based on assigned treatment at the time of randomization regardless of adherence status (intent-to-treat analysis). Outcome variable means were calculated for each treatment group for the baseline and six-month follow-up visits. A repeated-measures linear mixed-effects model was used to evaluate the absolute treatment effects by assessing the differences in the outcome variables from baseline to the 6-month follow-up visit between participants in each active treatment group and the placebo group. The model included the intercept, time effect (baseline and 6-month follow-up), and interactions between treatment groups and time (the absolute treatment effect). The model for total cholesterol also included a treatment group * cholesterol-lowering medication use (yes/no) interaction term. Analyses for total cholesterol were also stratified on cholesterol-lowing medication use.
Since the treatment groups were balanced on all other measured potentially influential factors at baseline, no adjustment was made for these covariates in the primary intent-to-treat analyses. However, the analyses for blood pressure were repeated adjusting for a history of having hypertension (yes/no) and taking anti-hypertension medication (yes/no) and there were no material changes in the results. To provide perspective on the magnitude of the treatment effects, we also calculated proportional treatment effects, defined as: absolute treatment effect/treatment group baseline × 100% (e.g., a proportional effect of −30% would mean an estimated 30% decrease in the active treatment group relative to the placebo group). SAS software (SAS Institute, Cary, NC) was used for analyses. All tests were 2-sided, and P < 0.05 was considered statistically significant.
RESULTS
The four treatment groups did not differ significantly on participant characteristics and dietary intakes measured at baseline (Table 1) or at the end of the study (data not shown) except for the higher proportion of individuals taking cholesterol-lowing medications in the calcium plus vitamin D3 intervention group compared to the placebo group. The mean age of participants was 60.5 ± 7.9 (SD) years and 64% of participants were men, 71% were white, 12% were current smokers, 82% were overweight or obese, 48% had history of hypertension, and 45% had a history of hypercholesterolemia.
Table 1.
Selected baseline characteristics and dietary intakes of study participants (n = 92)*
| Treatment Group | |||||
|---|---|---|---|---|---|
| Characteristics | Placebo (n = 23) |
Calcium (n = 23) |
Vitamin D3 (n = 23) |
Calcium + Vitamin D3 (n = 23) |
P† |
| Demographics | |||||
| Age (y) | 58.5 ± 8.2 | 61.9 ± 8.2 | 60.2 ± 8.1 | 62.1 ± 7.5 | 0.39 |
| Men (%) | 70 | 70 | 70 | 70 | 1.00 |
| White (%) | 74 | 83 | 65 | 61 | 0.39 |
| College graduate (%) | 65 | 61 | 57 | 44 | 0.53 |
| Medical history | |||||
| History of hypertension (%) | 57 | 35 | 39 | 61 | 0.21 |
| History of hypercholesterolemia (%) | 30 | 48 | 39 | 61 | 0.19 |
| Taking anti-hypertensive Medication (%) | 57 | 32 | 35 | 55 | 0.20 |
| Taking cholesterol-lowing medication (%) | 18 | 36 | 30 | 59 | 0.03 |
| Habits | |||||
| Current smoke (%) | 13 | 9 | 13 | 13 | 0.96 |
| Taking multivitamin (%) | 30 | 30 | 26 | 39 | 0.86 |
| Anthropometrics | |||||
| Body mass index (kg/m2) | 30.6 ± 7.2 | 29.4 ± 5.5 | 28.9 ± 5.6 | 31.6 ± 6.0 | 0.44 |
| Waist to hip ratio | 0.93 ± 0.08 | 0.92 ± 0.09 | 0.92 ± 0.10 | 0.98 ± 0.11 | 0.17 |
| Mean dietary intakes | |||||
| Total calcium (mg/d)‡ | 562 ± 273 | 681 ± 268 | 693 ± 426 | 560 ± 247 | 0.31 |
| Total vitamin D (IU/d)‡ | 132 ± 49 | 193 ± 99 | 177 ± 123 | 150 ± 88 | 0.14 |
| Total energy (kcal/d) | 1596 ± 528 | 1788 ± 691 | 1848 ± 821 | 1845 ± 752 | 0.59 |
| Total fat (g/d) | 67 ± 32 | 72 ± 35 | 70 ± 32 | 74 ± 28 | 0.59 |
| Saturated fat (g/d) | 20 ± 9 | 22 ± 10 | 22 ± 12 | 22 ± 8 | 0.89 |
| Cholesterol (mg/d) | 302 ± 174 | 302 ± 191 | 261 ± 161 | 272 ± 113 | 0.76 |
| Fiber (g/d) | 15 ± 7 | 17 ± 9 | 18 ± 9 | 17 ± 11 | 0.56 |
| Alcohol (g/d) | 9 ± 14 | 11 ± 15 | 14 ±18 | 10 ± 20 | 0.84 |
Data are given as means ± SD unless otherwise specified.
By Fisher’s exact χ2 test for categorical variables or ANOVA for continues variables.
Diet only (intakes exclusive of supplements).
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. No treatment related complications were observed. Seven people (8%) were lost to follow-up due to perceived drug intolerance (n=2), unwillingness to continue participation (n=3), physician’s advice (n=1), or death (n=1). Dropouts included one participant from the vitamin D3 intervention group and two from each of the other three treatment groups. The average adherence to visit was 92% and did not significantly differ across the four treatment groups.
Serum total cholesterol, triglycerides, and blood pressure (systolic and diastolic) levels are summarized in Table 2. There were no substantial or statistically significant differences in these parameters among the four treatment groups at baseline. Relative to placebo, serum levels of triglycerides decreased by 30.1% (P=0.10) and 32.1% (P=0.10) in the calcium and calcium plus vitamin D3 groups, respectively. Both calcium intervention groups also had small, non-statistically significant decreases in serum total cholesterol levels relative to placebo (calcium, 5.3%, P=0.31; calcium plus vitamin D3, 6.8%, P=0.19). Supplemental vitamin D3 alone did not appear to affect circulating lipid concentrations.
Table 2.
Serum levels of total cholesterol and triglycerides, and systolic and diastolic blood pressures (BP) at baseline and 6-month follow up
| Baseline |
6-mo follow-up |
Absolute Rx effect* |
Proportional Rx effect† |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Treatment Group | N | Mean ± SE | P‡ | N | Mean ± SE | P‡ | N | Mean ± SE | P‡ | (%) |
| Cholesterol (mg/dL) | ||||||||||
| Placebo | 23 | 225.5 ± 9.5 | 21 | 217.7 ± 9.9 | 21 | 0 | - | |||
| Calcium | 23 | 235.5 ± 9.8 | 0.46 | 21 | 215.3 ± 10.5 | 0.94 | 21 | −12.5 ± 12.2 | 0.31 | −5.3 |
| Vitamin D3 | 23 | 227.7 ± 9.5 | 0.87 | 22 | 219.6 ± 9.9 | 0.94 | 22 | −0.4 ± 11.7 | 0.97 | −0.2 |
| Calcium + Vitamin D3 | 23 | 229.3 ± 9.8 | 0.78 | 21 | 206.1 ± 9.9 | 0.45 | 21 | −15.5 ± 12.2 | 0.19 | −6.8 |
| Triglycerides (mg/dL) | ||||||||||
| Placebo | 23 | 112.9 ± 17.5 | 21 | 120.0 ± 18.3 | 21 | 0 | - | |||
| Calcium | 23 | 104.5 ± 17.9 | 0.82 | 21 | 80.0 ± 19.5 | 0.15 | 21 | −31.5 ± 24.4 | 0.10 | −30.1 |
| Vitamin D3 | 23 | 93.3 ± 17.5 | 0.27 | 22 | 93.9 ± 18.3 | 0.17 | 22 | −6.5 ± 23.5 | 0.68 | −7.0 |
| Calcium + Vitamin D3 | 23 | 98.0 ± 17.9 | 0.69 | 21 | 73.6 ± 18.2 | 0.11 | 21 | −31.5 ± 23.4 | 0.10 | −32.1 |
| Systolic BP (mmHg) | ||||||||||
| Placebo | 23 | 123.3 ± 3.2 | 22 | 118.9 ± 3.2 | 21 | 0 | - | |||
| Calcium | 23 | 125.1 ± 3.3 | 0.69 | 23 | 128.3 ± 3.2 | 0.05 | 20 | 7.6 ± 4.3 | 0.08 | 6.1 |
| Vitamin D3 | 22 | 125.9 ± 3.3 | 0.58 | 23 | 125.2 ± 3.2 | 0.19 | 22 | 3.7 ± 4.3 | 0.39 | 2.9 |
| Calcium + Vitamin D3 | 23 | 131.5 ± 3.3 | 0.07 | 22 | 128.1 ± 3.2 | 0.05 | 21 | 1.0 ± 4.3 | 0.81 | 0.8 |
| Diastolic BP (mmHg) | ||||||||||
| Placebo | 23 | 80.5 ± 2.2 | 22 | 80.7 ± 2.3 | 21 | 0 | - | |||
| Calcium | 23 | 79.1 ± 2.2 | 0.66 | 23 | 78.9 ± 2.3 | 0.58 | 20 | −0.4 ± 2.5 | 0.86 | −0.5 |
| Vitamin D3 | 22 | 76.5 ± 2.3 | 0.21 | 23 | 76.5 ± 2.3 | 0.19 | 22 | −0.2 ± 2.5 | 0.93 | −0.3 |
| Calcium + Vitamin D3 | 23 | 81.3 ± 2.2 | 0.82 | 22 | 78.5 ± 2.3 | 0.49 | 21 | −3.0 ± 2.5 | 0.23 | −3.7 |
Absolute treatment effect = [(treatment group follow-up) – (treatment group baseline)] – [(placebo group follow-up) – (placebo group baseline)].
Proportional treatment effect = [(absolute treatment effect / treatment group baseline) x 100%].
P value for difference between each active treatment group and placebo group from repeated measures MIXED model. P values for triglycerides were calculated based on log-transformed values.
There was no evidence that either vitamin D or calcium treatment had any beneficial effect on systolic blood pressure; indeed, there was a non-statistically significant increase in systolic blood pressure (6.1%, P=0.08) among individuals who received calcium supplements compared to placebo. Likewise, no estimated overall treatment effects were observed for diastolic blood pressure.
Serum carotenoid levels at baseline and at six-month follow-up are summarized in Table 3. Total carotenoid levels were observed to decrease 13.5% in the calcium group (P=0.07) and 9.1% in the calcium plus vitamin D group (P=0.10) relative to the placebo group. Serum concentrations of individual carotenoids decreased approximately 2 – 17% in both of the calcium intervention groups relative to placebo, and the finding for lycopene was borderline statistically significant (−17%, P=0.08) in the calcium alone group. Serum β-carotene and lycopene decreased 15.6% (P=0.40) and 19.6% (P=0.07), respectively, in the supplemental vitamin D3 alone group compared to placebo.
Table 3.
Serum levels of carotenoids at baseline and 6-month follow up
| Baseline |
6-mo follow-up |
Absolute Rx effect* |
Proportional Rx effect† |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Treatment Group | N | Mean ± SE | P‡ | N | Mean ± SE | P‡ | N | Mean ± SE | P‡ | (%) |
| α-Carotene (ng/mL) | ||||||||||
| Placebo | 23 | 40.1 ± 5.8 | 21 | 39.7 ± 6.0 | 21 | 0 | - | |||
| Calcium | 23 | 53.2 ± 6.0 | 0.12 | 21 | 45.5 ± 6.4 | 0.52 | 21 | −7.4 ± 6.8 | 0.27 | −13.9 |
| Vitamin D3 | 23 | 39.6 ± 5.8 | 0.95 | 22 | 39.3 ± 6.0 | 0.96 | 22 | 0.1 ± 6.4 | 0.98 | −0.2 |
| Calcium + Vitamin D3 | 23 | 43.7 ± 6.0 | 0.67 | 21 | 36.9 ± 6.0 | 0.74 | 21 | −6.4 ± 6.4 | 0.32 | −14.6 |
| β-Carotene (ng/mL) | ||||||||||
| Placebo | 23 | 176.3 ± 48.5 | 21 | 190.8 ± 49.3 | 21 | 0 | - | |||
| Calcium | 23 | 220.7 ± 50.0 | 0.52 | 21 | 202.4 ± 51.3 | 0.87 | 21 | −32.9 ± 38.7 | 0.40 | −14.9 |
| Vitamin D3 | 23 | 204.6 ± 48.7 | 0.68 | 22 | 187.3 ± 49.3 | 0.96 | 22 | −31.9 ± 37.6 | 0.40 | −15.6 |
| Calcium + Vitamin D3 | 23 | 302.9 ± 49.5 | 0.07 | 21 | 267.5 ± 49.8 | 0.28 | 21 | −49.9 ± 36.8 | 0.18 | −16.5 |
| α-Cryptoxanthin (ng/mL) | ||||||||||
| Placebo | 23 | 40.8 ± 3.4 | 22 | 39.3 ± 3.4 | 21 | 0 | - | |||
| Calcium | 23 | 47.3 ± 3.4 | 0.18 | 23 | 45.0 ± 3.6 | 0.25 | 20 | −0.7 ± 2.9 | 0.81 | −1.5 |
| Vitamin D3 | 22 | 46.8 ± 3.4 | 0.21 | 23 | 44.4 ± 3.4 | 0.30 | 22 | −0.9 ± 2.8 | 0.74 | −1.9 |
| Calcium + Vitamin D3 | 23 | 49.1 ± 3.4 | 0.09 | 22 | 46.2 ± 3.5 | 0.16 | 21 | −1.4 ± 2.7 | 0.62 | −2.9 |
| β-Cryptoxanthin (ng/mL) | ||||||||||
| Placebo | 23 | 156.5 ± 29.6 | 22 | 162.7 ± 30.3 | 21 | 0 | - | |||
| Calcium | 23 | 181.2 ± 30.0 | 0.56 | 23 | 158.7 ± 31.8 | 0.92 | 20 | −28.8 ± 31.8 | 0.37 | −15.9 |
| Vitamin D3 | 22 | 194.8 ± 29.4 | 0.36 | 23 | 222.8 ± 30.3 | 0.16 | 22 | 21.8 ± 30.6 | 0.48 | 11.2 |
| Calcium + Vitamin D3 | 23 | 194.9 ± 30.1 | 0.36 | 22 | 185.8 ± 30.3 | 0.59 | 21 | −15.3 ± 30.3 | 0.62 | −7.9 |
| Lutein/zeaxanthin (ng/mL) | ||||||||||
| Placebo | 23 | 474.2 ± 40.8 | 21 | 449.6 ± 42.1 | 21 | 0 | - | |||
| Calcium | 23 | 550.4 ± 41.7 | 0.20 | 21 | 496.3 ± 44.3 | 0.44 | 21 | −29.5 ± 44.9 | 0.51 | −5.4 |
| Vitamin D | 23 | 553.9 ± 40.8 | 0.17 | 22 | 541.8 ± 42.1 | 0.13 | 22 | 12.5 ± 43.2 | 0.77 | 2.3 |
| Calcium + Vitamin D3 | 23 | 548.0 ± 41.7 | 0.21 | 21 | 483.4 ± 42.1 | 0.57 | 21 | −40.8 ± 42.8 | 0.35 | −7.4 |
| Lycopene (ng/mL) | ||||||||||
| Placebo | 23 | 313.0 ± 27.9 | 21 | 321.8 ± 29.2 | 21 | 0 | - | |||
| Calcium | 23 | 395.8 ± 28.5 | 0.04 | 21 | 335.5 ± 31.2 | 0.75 | 21 | −69.0 ± 39.8 | 0.08 | −17.4 |
| Vitamin D3 | 23 | 357.2 ± 27.9 | 0.27 | 22 | 295.5 ± 29.2 | 0.53 | 22 | −70.0 ± 38.4 | 0.07 | −19.6 |
| Calcium + Vitamin D3 | 23 | 383.2 ± 28.5 | 0.08 | 21 | 351.5 ± 29.0 | 0.47 | 21 | −40.0 ± 38.1 | 0.29 | −10.4 |
| Total carotenoids (ng/mL) | ||||||||||
| Placebo | 23 | 1,296 ± 123 | 21 | 1,290 ± 125 | 21 | 0 | - | |||
| Calcium | 23 | 1,583 ± 126 | 0.11 | 21 | 1,402 ± 130 | 0.54 | 21 | −175 ± 95 | 0.07 | −13.5 |
| Vitamin D3 | 23 | 1,536 ± 123 | 0.17 | 22 | 1,461 ± 125 | 0.34 | 22 | −69 ± 92 | 0.45 | −4.5 |
| Calcium + Vitamin D3 | 23 | 1,646 ± 124 | 0.05 | 21 | 1,491 ± 127 | 0.26 | 21 | −149 ± 91 | 0.10 | −9.1 |
Absolute treatment effect = [(treatment group follow-up) – (treatment group baseline)] – [(placebo group follow-up) – (placebo group baseline)].
Proportional treatment effect = [(absolute treatment effect / treatment group baseline) x 100%].
P value for difference between each active treatment group and placebo group from repeated measures MIXED model.
Since the estimated effects of supplemental vitamin D3 observed in the current study were minimal, we pooled the calcium and calcium plus vitamin D3 treatment groups (pooled calcium group) and compared them to the pooled placebo and vitamin D3 alone treatment groups (pooled non-calcium group) (Table 4). In the pooled calcium group relative to the pooled non-calcium group, serum triglyceride levels statistically significantly decreased by 28% (P=0.04), serum total cholesterol decreased 6% (P=0.09), and total carotenoids decreased 8% (P=0.06).
Table 4.
Serum levels of total cholesterol, triglycerides, and total carotenoids for pooled calcium and pooled non-calcium intervention groups*
| Baseline |
6-mo follow-up |
Absolute Rx effect† |
Proportional Rx effect‡ |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Treatment Group | N | Mean ± SE | P§ | N | Mean ± SE | P§ | N | Mean ± SE | P§ | (%) |
| Cholesterol (mg/dL) | ||||||||||
| Non-calcium | 46 | 226.6 ± 6.7 | 43 | 218.7 ± 7.0 | 43 | 0 | - | |||
| Calcium | 46 | 232.4 ± 6.8 | 0.55 | 42 | 210.4 ± 7.1 | 0.41 | 42 | −14.0 ± 8.3 | 0.09 | −6.0 |
| Triglycerides (mg/dL) | ||||||||||
| Non-calcium | 46 | 103.1 ± 12.3 | 43 | 107.0 ± 12.9 | 43 | 0 | - | |||
| Calcium | 46 | 101.3 ± 12.6 | 0.74 | 42 | 76.7 ± 13.2 | 0.22 | 42 | −28.4 ± 16.7 | 0.04 | −28.0 |
| Total carotenoids (ng/mL) | ||||||||||
| Non-calcium | 46 | 1,416 ± 87 | 43 | 1,375 ± 88 | 43 | 0 | - | |||
| Calcium | 46 | 1,614 ± 89 | 0.69 | 42 | 1,448 ± 90 | 0.05 | 42 | −125 ± 65 | 0.06 | −7.7 |
Pooled calcium supplementation group (calcium) included participants from calcium alone and calcium plus vitamin D groups. Pooled non-calcium supplementation group (non-calcium) included participants from placebo and vitamin D alone groups.
Absolute treatment effect = [(pooled calcium group follow-up) – (pooled calcium group baseline)] – [(pooled non-calcium group follow-up) - (pooled non-calcium group baseline)].
Proportional treatment effect = (absolute treatment effect / pooled calcium group baseline) x 100%.
P value for difference between pooled calcium group and pooled non-calcium group from repeated measures MIXED model. P values for triglycerides were calculated based on log-transformed values.
Estimated treatment effects on serum total cholesterol levels stratified by cholesterol-lowing medication use are presented in Table 5. Among participants who were not taking a cholesterol-lowering medication, there was a 7.7% decrease (P=0.09) in cholesterol concentrations in the pooled calcium supplementation group relative to the pooled non-calcium group, whereas no changes were observed among the medication users (0.3% decrease, P=0.96) (Pinteraction=0.10).
Table 5.
Serum levels of total cholesterol for pooled calcium and pooled non-calcium intervention groups, stratified by cholesterol-lowering medication use*
| Baseline |
6-mo follow-up |
Absolute Rx effect† |
Proportional Rx effect‡ |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Treatment Group | N | Mean ± SE | P§ | N | Mean ± SE | P§ | N | Mean ± SE | P§ | (%) |
| Taking cholesterol-lowering medications | ||||||||||
| Cholesterol (mg/dL) | ||||||||||
| Non-calcium | 11 | 231.6 ± 12.6 | 9 | 211.1 ± 13.4 | 9 | 0 | - | |||
| Calcium | 21 | 215.6 ± 9.1 | 0.31 | 17 | 194.5 ± 9.7 | 0.32 | 17 | −0.73 ± 13.9 | 0.96 | −0.3 |
| Not taking cholesterol-lowering medications | ||||||||||
| Cholesterol (mg/dL) | ||||||||||
| Non-calcium | 35 | 225.0 ± 7.7 | 31 | 220.7 ± 8.0 | 31 | 0 | - | |||
| Calcium | 23 | 247.7 ± 9.5 | 0.07 | 21 | 224.5 ± 9.8 | 0.77 | 21 | −19.0 ± 10.9 | 0.09 | −7.7 |
| P for interaction¶ = 0.10 | ||||||||||
Pooled calcium supplementation group (calcium) included participants from calcium alone and calcium plus vitamin D groups. Pooled non-calcium supplementation group (non-calcium) included participants from placebo and vitamin D alone groups.
Absolute treatment effect = [(pooled calcium group follow-up) – (pooled calcium group baseline)] – [(pooled non-calcium group follow-up) - (pooled non-calcium group baseline)].
Proportional treatment effect = (absolute treatment effect / pooled calcium group baseline) x 100%.
P value for difference between pooled calcium group and pooled non-calcium group from repeated measures MIXED model.
Interaction between treatment (calcium/non-calcium) and cholesterol-lowing medication use (yes/no).
DISCUSSION
The results from this pilot, randomized, double-blind, placebo-controlled clinical trial suggest that daily supplementation with 2.0 g elemental calcium (as calcium carbonate) alone or in combination with 800 IU vitamin D3 for 6 months may decrease serum triglyceride levels by approximately 30%. Although only small, non-statistically significant decreases (5 – 7%) in circulating total cholesterol levels were observed in both calcium intervention groups, the findings were stronger (−7.7%, P=0.09) among persons who were not taking cholesterol-lowering medications, suggesting that calcium supplementation may lower total cholesterol levels among persons who are not taking cholesterol-lowering medications, but have no effect among those already taking such drugs. It has been suggested that a high calcium intake may reduce lipid absorption by forming indigestible calcium-lipid complexes [6]. Also, calcium binds bile acids in the gut and increases their excretion, further contributing to decreased fat absorption [6, 19]. Previous animal studies also suggest that calcium supplementation may have beneficial effects on circulating lipid profiles [37–41].
A few early, small, clinical trials suggested that supplemental calcium may reduce serum cholesterol; however, these studies had major design limitations [10–14]. Subsequently, two randomized, placebo-controlled trials with larger sample sizes (n=193 – 223) and longer follow-up (6 – 12 months) [5, 19] reported no substantial effects of calcium on serum total cholesterol levels; however, the study by Reid et al. [5] found statistically significant increases in HDL and the HDL/LDL ratio after calcium supplementation. Similarly, no substantial changes (≤ 5%) were observed in a large, recent, long-term (5-year follow-up) clinical trial (n=1,259) that assessed the joint effect of calcium and vitamin D3 on serum lipid levels. The 5 – 7% decreases in total cholesterol observed in our study were comparable to those found in most of the previously reported clinical trials [5, 13–15, 17, 19, 21].
Our results that suggested that calcium supplementation may moderately lower (≈30%) serum triglyceride levels are in contrast to the majority of published clinical trials that found no beneficial effects of calcium on triglyceride levels [5, 11, 13, 16–18, 21]. This could be attributable to factors such as calcium dose, length of follow-up, study population, and chance. In the current study, a 2.0 g elemental calcium/day dose was used, which was twice that used in the two large clinical trials [5, 21] in which no substantial changes in serum triglyceride levels were observed for supplemental calcium alone or in combination with vitamin D.
One of the steps in the absorption process of carotenoids that may affect their bioavailability involves the incorporation of carotenoids into micelles and the presence of intestinal fat, which is thought to be crucial to micelle formation [32]. As stated above, calcium and lipids bind to one another in the gut, each interfering with the other's absorption [6, 21]. Thus, the possible formation of intestinal calcium-lipid complexes may explain in part the observed decreases in circulating carotenoids in both calcium intervention groups since serum lipid levels also decreased after six months of calcium supplementation. While the reduction in fat absorption may be beneficial, a reduction in carotenoid level may offset certain health benefits associated with calcium. However, the relative importance of calcium’s potential lipid-and carotenoid-lowering effects is unknown, and there is other evidence suggesting that calcium supplements may increase risk of myocardial infarction [8] and mortality [9]. Therefore, caution is needed in deciding whether or not to take large daily doses of calcium supplements.
There are plausible mechanisms of action for a proposed effect of calcium on blood pressure in normal individuals as well as for individuals at high risk of developing hypertension [42–44]; however, none of the these mechanisms has been clearly established. Overall, in our intention-to-treat analyses there was no support for either vitamin D or calcium treatment having any beneficial effects on systolic and diastolic blood pressures. The 6% non-statistically significant increase in systolic blood pressure observed in the calcium group may have been due to chance since we did not observe a similar finding in the calcium plus vitamin D3 group; however, future studies are necessary to investigate this more fully.
The strengths of the current study included the randomized, double-blind, placebo-controlled trial design; high protocol adherence by the study participants; and that it was the first trial to evaluate both the independent and combined effects of calcium and vitamin D3 on blood pressure and serum lipids and lipophilic micronutrients. Limitations included the relatively small sample size and relatively short follow-up period. In addition, we did not measure circulating HDL- and LDL-cholesterol levels. However, the findings from this pilot study are novel, biologically plausible, and have potentially important clinical and public health implications.
In conclusion, the results from the current study suggest that daily supplementation with 2.0 g of elemental calcium alone or in combination with 800 IU of vitamin D3 for 6 months may reduce serum triglyceride and, possibly, total cholesterol levels, but that it may decrease serum carotenoid levels, and thus support further investigation in a larger trial to establish the value or risk of calcium supplementation. Our findings provide little or no support for the hypothesis that supplemental calcium or vitamin D, alone or combined, can lower blood pressure.
ACKNOWLEDGMENTS
This research was supported in part by the National Institutes of Health grants R01CA104637 and R03CA132149, and Georgia Cancer Coalition Distinguished Scholar award (to RMB). WC was supported by a postdoctoral fellowship on grant R25CA90956.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
REFERENCES
- 1.van Dijk SB, Takken T, Prinsen EC, Wittink H. Different anthropometric adiposity measures and their association with cardiovascular disease risk factors: a meta-analysis. Neth Heart J. 2012;20:208–218. doi: 10.1007/s12471-011-0237-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Galassi A, Reynolds K, He J. Metabolic syndrome and risk of cardiovascular disease: a meta-analysis. Am J Med. 2006;119(10):812–819. doi: 10.1016/j.amjmed.2006.02.031. [DOI] [PubMed] [Google Scholar]
- 3.Bass KM, Newschaffer CJ, Klag MJ, Bush TL. Plasma lipoprotein levels as predictors of cardiovascular death in women. Arch Intern Med. 1993;153(19):2209–2216. [PubMed] [Google Scholar]
- 4.Klag MJ, Ford DE, Mead LA, He J, Whelton PK, Liang KY, et al. Serum cholesterol in young men and subsequent cardiovascular disease. N Engl J Med. 1993;328(5):313–318. doi: 10.1056/NEJM199302043280504. [DOI] [PubMed] [Google Scholar]
- 5.Reid IR, Mason B, Horne A, Ames R, Clearwater J, Bava U, et al. Effects of calcium supplementation on serum lipid concentrations in normal older women: a randomized controlled trial. Am J Med. 2002;112(5):343–347. doi: 10.1016/s0002-9343(01)01138-x. [DOI] [PubMed] [Google Scholar]
- 6.Reid IR. Effects of calcium supplementation on circulating lipids: potential pharmacoeconomic implications. Drugs Aging. 2004;21(1):7–17. doi: 10.2165/00002512-200421010-00002. [DOI] [PubMed] [Google Scholar]
- 7.Cappuccio FP, Elliott P, Allender PS, Pryer J, Follman DA, Cutler JA. Epidemiologic association between dietary calcium intake and blood pressure: a meta-analysis of published data. Am J Epidemiol. 1995;142(9):935–945. doi: 10.1093/oxfordjournals.aje.a117741. [DOI] [PubMed] [Google Scholar]
- 8.Li K, Kaaks R, Linseisen J, Rohrmann S. Associations of dietary calcium intake and calcium supplementation with myocardial infarction and stroke risk and overall cardiovascular mortality in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition study (EPIC-Heidelberg) Heart. 2012;98(12):920–925. doi: 10.1136/heartjnl-2011-301345. [DOI] [PubMed] [Google Scholar]
- 9.Mic1haelsson K, Melhus H, Warensjo Lemming E, Wolk A, Byberg L. Long term calcium intake and rates of all cause and cardiovascular mortality: community based prospective longitudinal cohort study. BMJ. 2013;346:f228. doi: 10.1136/bmj.f228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Yacowitz H, Fleischman AI, Bierenbaum ML. Effects of Oral Calcium Upon Serum Lipids in Man. Br Med J. 1965;1(5446):1352–1354. doi: 10.1136/bmj.1.5446.1352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Carlson LA, Olsson AG, Oro L, Rossner S. Effects of oral calcium upon serum cholesterol and triglycerides in patients with hyperlipidemia. Atherosclerosis. 1971;14(3):391–400. doi: 10.1016/0021-9150(71)90067-0. [DOI] [PubMed] [Google Scholar]
- 12.Bierenbaum ML, Fleischman AI, Raichelson RI. Long term human studies on the lipid effects of oral calcium. Lipids. 1972;7(3):202–206. doi: 10.1007/BF02533064. [DOI] [PubMed] [Google Scholar]
- 13.Bhattacharyya AK, Thera C, Anderson JT, Grande F, Keys A. Dietary calcium and fat. Effect on serum lipids and fecal excretion of cholesterol and its degradation products in man. Am J Clin Nutr. 1969;22(9):1161–1174. doi: 10.1093/ajcn/22.9.1161. [DOI] [PubMed] [Google Scholar]
- 14.Denke MA, Fox MM, Schulte MC. Short-term dietary calcium fortification increases fecal saturated fat content and reduces serum lipids in men. J Nutr. 1993;123(6):1047–1053. doi: 10.1093/jn/123.6.1047. [DOI] [PubMed] [Google Scholar]
- 15.Ditscheid B, Keller S, Jahreis G. Cholesterol metabolism is affected by calcium phosphate supplementation in humans. J Nutr. 2005;135(7):1678–1682. doi: 10.1093/jn/135.7.1678. [DOI] [PubMed] [Google Scholar]
- 16.Bell L, Halstenson CE, Halstenson CJ, Macres M, Keane WF. Cholesterol-lowering effects of calcium carbonate in patients with mild to moderate hypercholesterolemia. Arch Intern Med. 1992;152(12):2441–2444. [PubMed] [Google Scholar]
- 17.Major GC, Alarie F, Dore J, Phouttama S, Tremblay A. Supplementation with calcium + vitamin D enhances the beneficial effect of weight loss on plasma lipid and lipoprotein concentrations. Am J Clin Nutr. 2007;85(1):54–59. doi: 10.1093/ajcn/85.1.54. [DOI] [PubMed] [Google Scholar]
- 18.Mitchell WD, Fyfe T, Smith DA. The effect of oral calcium on cholesterol metabolism. J Atheroscler Res. 1968;8(6):913–922. doi: 10.1016/s0368-1319(68)80005-5. [DOI] [PubMed] [Google Scholar]
- 19.Bostick RM, Fosdick L, Grandits GA, Grambsch P, Gross M, Louis TA. Effect of calcium supplementation on serum cholesterol and blood pressure. A randomized, double-blind, placebo-controlled, clinical trial. Arch Fam Med. 2000;9(1):31–38. doi: 10.1001/archfami.9.1.31. [DOI] [PubMed] [Google Scholar]
- 20.Karandish M, Shockravi S, Jalali MT, Haghighizadeh MH. Effect of calcium supplementation on lipid profile in overweight or obese Iranian women: a double-blind randomized clinical trial. Eur J Clin Nutr. 2009;63(2):268–272. doi: 10.1038/sj.ejcn.1602921. [DOI] [PubMed] [Google Scholar]
- 21.Rajpathak SN, Xue X, Wassertheil-Smoller S, Van Horn L, Robinson JG, Liu S, et al. Effect of 5 y of calcium plus vitamin D supplementation on change in circulating lipids: results from the Women’s Health Initiative. Am J Clin Nutr. 2010;91(4):894–899. doi: 10.3945/ajcn.2009.28579. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Sallinen K, Arvola P, Wuorela H, Ruskoaho H, Vapaatalo H, Porsti I. High calcium diet reduces blood pressure in exercised and nonexercised hypertensive rats. Am J Hypertens. 1996;9(2):144–156. doi: 10.1016/0895-7061(95)00333-9. [DOI] [PubMed] [Google Scholar]
- 23.Allender PS, Cutler JA, Follmann D, Cappuccio FP, Pryer J, Elliott P. Dietary calcium and blood pressure: a meta-analysis of randomized clinical trials. Ann Intern Med. 1996;124(9):825–831. doi: 10.7326/0003-4819-124-9-199605010-00007. [DOI] [PubMed] [Google Scholar]
- 24.Bucher HC, Cook RJ, Guyatt GH, Lang JD, Cook DJ, Hatala R, et al. Effects of dietary calcium supplementation on blood pressure. A meta-analysis of randomized controlled trials. JAMA. 1996;275(13):1016–1022. doi: 10.1001/jama.1996.03530370054031. [DOI] [PubMed] [Google Scholar]
- 25.Haynes RC. Agents affecting calcification: calcium ph, calcitonin, vitamin D, and other compounds. In: Gilman AG, Rall TW, Nies AS, et al., editors. The pharmacologic basis of therapeutics. 8th ed/ New York: Pergamon Press; 1990, p. pp. 1498–1522. [Google Scholar]
- 26.Lind L, Hanni A, Lithell H, Hvarfner A, Sorensen OH, Ljunghall S. Vitamin D is related to blood pressure and other cardiovascular risk factors in middle-aged men. Am J Hypertens. 1995;8(9):894–901. doi: 10.1016/0895-7061(95)00154-H. [DOI] [PubMed] [Google Scholar]
- 27.Heikkinen AM, Tuppurainen MT, Niskanen L, Komulainen M, Penttila I, Saarikoski S. Long-term vitamin D3 supplementation may have adverse effects on serum lipids during postmenopausal hormone replacement therapy. Eur J Endocrinol. 1997;137(5):495–502. doi: 10.1530/eje.0.1370495. [DOI] [PubMed] [Google Scholar]
- 28.Carlson LA, Derblom H, Lanner A. Effect of different doses of vitamin D on serum cholesterol and triglyceride levels in healthy men. Atherosclerosis. 1970;12(2):313–317. doi: 10.1016/0021-9150(70)90111-5. [DOI] [PubMed] [Google Scholar]
- 29.Lips P, Wiersinga A, van Ginkel FC, Jongen MJ, Netelenbos JC, Hackeng WH, et al. The effect of vitamin D supplementation on vitamin D status and parathyroid function in elderly subjects. J Clin Endocrinol Metab. 1988;67(4):644–650. doi: 10.1210/jcem-67-4-644. [DOI] [PubMed] [Google Scholar]
- 30.Rock CL, Natarajan L, Pu M, Thomson CA, Flatt SW, Caan BJ, et al. Longitudinal biological exposure to carotenoids is associated with breast cancer-free survival in the Women’s Healthy Eating and Living Study. Cancer Epidemiol Biomarkers Prev. 2009;18(2):486–494. doi: 10.1158/1055-9965.EPI-08-0809. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.van Poppel G. Epidemiological evidence for beta-carotene in prevention of cancer and cardiovascular disease. Eur J Clin Nutr. 1996;50(Suppl 3):S57–S61. [PubMed] [Google Scholar]
- 32.van Het Hof KH, West CE, Weststrate JA, Hautvast JG. Dietary factors that affect the bioavailability of carotenoids. J Nutr. 2000;130(3):503–506. doi: 10.1093/jn/130.3.503. [DOI] [PubMed] [Google Scholar]
- 33.Fedirko V, Bostick RM, Flanders WD, Long Q, Shaukat A, Rutherford RE, et al. Effects of vitamin D and calcium supplementation on markers of apoptosis in normal colon mucosa: a randomized, double-blind, placebo-controlled clinical trial. Cancer Prev Res (Phila) 2009;2(3):213–223. doi: 10.1158/1940-6207.CAPR-08-0157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Willett WC, Sampson L, Browne ML, Stampfer MJ, Rosner B, Hennekens CH, et al. The use of a self-administered questionnaire to assess diet four years in the past. Am J Epidemiol. 1988;127(1):188–199. doi: 10.1093/oxfordjournals.aje.a114780. [DOI] [PubMed] [Google Scholar]
- 35.Franke AA, Custer LJ, Cooney RV. Synthetic carotenoids as internal standards for plasma micronutrient analyses by high-performance liquid chromatography. Chromatogr. 1993;614(1):43–57. doi: 10.1016/0378-4347(93)80222-p. [DOI] [PubMed] [Google Scholar]
- 36.National Institute of Standards and Technology special publication no.874. Methods for analysis of cancer chemo-preventive agents in human serum. Washington (DC): Government Printing Office; 1994. [Google Scholar]
- 37.Fleischman AI, Yacowitz H, Hayton T, Bierenbaum ML. Effects of dietary calcium upon lipid metabolism in mature male rats fed beef tallow. J Nutr. 1966;88(3):255–260. doi: 10.1093/jn/88.3.255. [DOI] [PubMed] [Google Scholar]
- 38.Fleischman AI, Yacowitz H, Hayton T, Bierenbaum ML. Long-term studies on the hypolipemic effect of dietary calcium in mature male rats fed cocoa butter. J Nutr. 1967;91(2):151–158. doi: 10.1093/jn/91.2.151. [DOI] [PubMed] [Google Scholar]
- 39.Dougherty RM, Iacono JM. Effects of dietary calcium on blood and tissue lipids, tissue phospholipids, calcium and magnesium levels in rabbits fed diets containing beef tallow. J Nutr. 1979;109(11):1934–1945. doi: 10.1093/jn/109.11.1934. [DOI] [PubMed] [Google Scholar]
- 40.Diersen-Schade DA, Richard MJ, Jacobson NL. Effects of dietary calcium and fat on cholesterol in tissues and feces of young goats. J Nutr. 1984;114(12):2292–2300. doi: 10.1093/jn/114.12.2292. [DOI] [PubMed] [Google Scholar]
- 41.El-Merhie N, Sabry I, Balbaa M. Effect of calcium treatment on blood parameters, gonadal development and the structure of bone in immature female rats. J Physiol Biochem. 2012;68:219–227. doi: 10.1007/s13105-011-0133-z. [DOI] [PubMed] [Google Scholar]
- 42.Smith HT. Electrolytes in the epidemiology, pathophysiology, and treatment of hypertension. Prim Care. 1991;18(3):545–557. [PubMed] [Google Scholar]
- 43.Stein PP, Black HR. The role of diet in the genesis and treatment of hypertension. Med Clin North Am. 1993;77(4):831–847. doi: 10.1016/s0025-7125(16)30227-9. [DOI] [PubMed] [Google Scholar]
- 44.Reusser ME, McCarron DA. Micronutrient effects on blood pressure regulation. Nutr Rev. 1994;52(11):367–375. doi: 10.1111/j.1753-4887.1994.tb01367.x. [DOI] [PubMed] [Google Scholar]