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Keywords:

  • OSTEOPOROSIS;
  • AUGMENTATION INDEX;
  • PULSE WAVE VELOCITY;
  • PERIPHERAL ARTERY TONOMETRY;
  • SUBENDOCARDIAL VIABILITY RATIO

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Calcium supplements have been associated with an increased risk of cardiovascular events. However, the validity of these findings has been questioned. A major concern is that the mechanism underlying an increase in cardiovascular events has not been demonstrated. Calcium initiates cardiac and vascular contraction following influx of calcium into cardiac and smooth muscle from extracellular fluid. We have investigated whether the acute rise in serum calcium following calcium supplement administration is associated with adverse changes in cardiovascular function. In an open interventional study, we recruited 25 volunteers (16 female, age 60.3 ± 6.5 years, body mass index 25.7 ± 2.7 kg/m2) from the community who were not taking calcium supplements. Participants were studied before and 3 hours after a single oral dose of 1000 mg calcium citrate. We assessed well-validated markers of arterial stiffness (pulse wave velocity [PWV]), arterial wave reflection (augmentation index [AIx]), and myocardial perfusion (subendocardial viability ratio [SEVR]) by pulse wave analysis and endothelial function (reactive hyperemia index [RHI]) by peripheral arterial tonometry. Total and ionized serum calcium were acutely increased by 0.10 ± 0.07 and 0.06 ± 0.03 mmol/L, respectively, 3 hours after calcium citrate administration (p < 0.0001 for both comparisons). Following administration of calcium citrate there was a fall in AIx from a median of 29.7% (23.8% to 34.0%) to 26.4% (22.7% to 34.0%, p = 0.03) and an increase in SEVR from 163% (148% to 174%) to 170% (149% to 185%, p = 0.007). PWV and RHI were not significantly altered. The change in total calcium was negatively correlated with the change in AIx (r = –0.48, p = 0.02). In summary, the acute increase in serum calcium following calcium supplement administration is associated with reduced arterial wave reflection and a marker of increased myocardial perfusion. If maintained long-term, these changes would be expected to reduce cardiovascular risk. Acute serum calcium–mediated changes in these parameters of cardiovascular function are unlikely to underlie an association between calcium supplementation and cardiovascular events. © 2013 American Society for Bone and Mineral Research


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Calcium supplementation is frequently prescribed to subjects with, or at risk of, osteoporosis. Calcium supplements increase bone mineral density by 1% to 2% in postmenopausal women1 and older men.2 However, the fracture risk reduction associated with calcium supplementation is substantially less than that achieved by other therapies for osteoporosis.3 Therefore, the role of calcium supplementation in fracture prevention is not fully established.4 Despite this, until recently the adverse effects of calcium supplementation were considered minimal and it is recommended that adult women and men receive a total daily calcium intake of 1000 to 1200 mg to prevent or treat osteoporosis.5 This daily calcium intake is not achieved by many patients without calcium supplementation.

A recent study raised questions about the safety of calcium supplementation.6 In a secondary analysis of a randomized-controlled trial with the primary aim of assessing the effect of calcium supplements on bone density in healthy postmenopausal women,1 Bolland and colleagues6 reported that subjects receiving calcium citrate 1000 mg/d in two divided doses had an increased rate of cardiovascular events. Subsequent meta-analyses of randomized-controlled trials by the same group have reported that the risk of myocardial infarction in patients randomized to receive calcium supplementation is increased by 20% to 30%.7, 8 However, the strength of the conclusions able to be drawn from these meta-analyses has been questioned.9 A particular concern has been the absence of a proven physiologic mechanism underlying the increase in cardiovascular risk.10

Acute administration of calcium supplements increases serum total and ionized calcium for several hours.11, 12 A consequent increase in vascular calcification with calcium supplementation has been proposed as a possible mechanism that could increase cardiovascular risk.13 However, vascular calcification develops slowly over time and the reported increase in cardiovascular risk occurred relatively early after starting calcium supplementation.7, 8 Calcium initiates cardiac and vascular contraction, with influx of calcium into cardiac and smooth muscle from extracellular fluid. Although influx of calcium into muscle is regulated in a voltage-dependent and ligand-dependent manner, there is evidence that an acute change in serum calcium could affect cardiovascular function. In a previous study, an intravenous calcium infusion that increased ionized calcium by 0.32 mmol/L increased systolic blood pressure and impaired endothelial-independent vasodilatation after 60 minutes.14

The primary aim of this study was to determine the acute effect of calcium supplementation on arterial stiffness and vasodilatory function. We hypothesized that the increase in serum calcium following supplement administration would adversely affect cardiovascular function and represent a mechanism that could underlie the relationship between cardiovascular disease and calcium supplementation.

Subjects and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

This open interventional study was undertaken within the Endocrine Research Unit, Repatriation General Hospital, Adelaide, Australia. The study was approved by the Southern Adelaide Clinical Human Research Ethics Committee, Flinders Medical Centre, and all subjects provided written informed consent in accordance with the Declaration of Helsinki.

Subjects

We recruited 26 subjects aged ≥ 50 years by advertisement from the general community. Subjects were excluded if they had taken calcium supplements at any dose, vitamin D ( > 300 IU/d), proton pump inhibitors, or calcium channel blockers in the last 3 months, if they had primary hyperparathyroidism, diabetes mellitus, atrial fibrillation, or Raynaud's phenomenon, or if they were unable to provide written informed consent. One subject was not included in all analyses because his/her baseline blood results revealed previously undiagnosed primary hyperparathyroidism, leaving a final cohort of 25 subjects.

Study design

Subjects were asked to attend the Endocrine Research Unit at 9:00 a.m. after an overnight fast. Following resting for 30 minutes at 21°C to 24°C in a supine position, pulse wave analysis, pulse wave velocity (PWV), and peripheral artery tonometry were performed as outlined in the following sections. An intravenous cannula was then inserted into an antecubital vein and used to collect baseline blood samples. Following oral administration of 1000 mg calcium citrate (Citracal; Bayer Healthcare, Leverkusen, Germany), further hourly blood samples were collected for 3 hours. Pulse wave analysis, PWV, and peripheral artery tonometry were repeated in a standardized order between 120 and 180 minutes after calcium citrate administration (Fig. 1).

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Figure 1. Study protocol. Cardiovascular function was assessed before and 2 to 3 hours after 1000 mg calcium citrate administered orally. Rest = period of acclimatization before vascular studies; Ca = calcium; beta CTx = serum beta carboxyterminal peptide; PTH = parathyroid hormone.

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Pulse wave analysis

Pulse wave analysis was performed using a SphygmoCor device (AtCor Medical, NSW, Australia) and a high-fidelity micromanometer (SPC-301; Millar Instruments, TX, USA).15 A single investigator (BLM) performed all measurements. The radial pulse waveform was recorded and central aortic pressure was derived using an automated generalized transfer function. The augmentation index (AIx) was calculated as the increment in pressure from the first shoulder in the ascending aortic pressure wave to the peak of this wave. The day-to-day intraclass correlation for AIx for this operator is 0.82. To correct for changes in pulse rate, AIx results were normalized for a heart rate of 75 beats/min. One subject's pulse was < 40 beats/min and we were unable to correct the AIx. This subject was not included in the analysis of change in AIx. We used the quality control indices within the SphygmoCor device software to verify the quality of the recorded waveforms. This includes assessment of average pulse height, pulse height variation, diastolic variation, and shape deviation. If any of these indices were outside acceptable limits or if the overall quality index was < 80%, the waveform was discarded and the measurement was repeated.

Ventricular ejection duration and subendocardial viability ratio (SEVR), the ratio of the area under the aortic pressure curve in diastole to the area under the aortic pressure curve in systole, were also calculated from the radial pulse wave analysis. Reported results for AIx, ejection duration, and SEVR are an average of six consecutive recordings.

PWV

PWV was then calculated by combining pulse wave analysis at the carotid and femoral arteries with simultaneous electrocardiograph recording. PWV is calculated by measuring the difference in time the pulse waveform takes to reach the carotid and femoral arteries. Reported results are the average of six consecutive recordings. The average day-to-day coefficient of variation (CV) for PWV for this operator is 7.6%.

Peripheral arterial tonometry

Peripheral arterial tonometry (PAT) was performed using an Endo-PAT 2000 (Itamar Medical, Caesarea, Israel). The PAT signal is measured from a fingertip in both hands by recording finger arterial pulsatile volume changes using a pair of plethysmography-based biosensors. After a baseline pulse amplitude measurement was obtained from both hands, local ischemia was induced by inflating a blood pressure cuff to suprasystolic pressures for 5 minutes, causing occlusion of blood flow to the arm. The pulse amplitude response to hyperemia was then recorded electronically in both fingers (the nonoccluded arm serves as control) and analyzed by an automated computer algorithm for 5 minutes after cuff deflation. The time period between 90 and 150 seconds after deflation was used to calculate the reactive hyperemia index (RHI), as described.16 One subject declined a second RHI reading and was not included in the analysis of change in RHI. The average day-to-day CV for RHI at our institution is 11.4%.

Laboratory analysis

Serum total calcium was measured on a Roche Modular Analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan) with a CV of < 1% across the range of calcium concentrations measured. Ionized calcium was measured on a Radiometer blood gas analyzer (Radiometer Medical ApS, Bronshoj, Denmark) with a CV of 1.9% at 1.06 mmol/L and 1.2% at 1.65 mmol/L. Parathyroid hormone (PTH) and serum beta C-terminal telopeptide (beta CTx) were measured by electrochemiluminescent immunoassay (Roche Diagnostics, GMBH, Mannheim, Germany). The between-run CV for PTH is 4.3% at 2.1 pmol/L and 3.5% at 18.4 pmol/L. The CV for beta CTx is 6% at concentrations between 280 and 600 pg/mL. 25-hydroxy vitamin D was measured using an automated chemiluminescent assay (IDS-iSYS; IDS Ltd, Boldon, UK). The between-run CV is 7% to 11% at concentrations between 30 and 165 nmol/L.

Statistical analysis

Data were analyzed using SPSS 20.0 for Windows (SPSS Inc, Chicago, IL, USA) with p < 0.05 considered statistically significant. Results are presented as mean ± SD if normally distributed and median (interquartile range) if the distribution was not normal. Changes in serum total and ionized calcium were assessed by repeated measures ANOVA followed by post hoc testing using a Bonferroni correction. Changes in other variables were assessed using paired t tests for normally distributed data and a Wilcoxon signed rank test for data that were not normally distributed. Simple linear correlations were used to assess the relationship between changes in serum calcium and PTH and changes in vascular function. The primary endpoints were the change in PWV (primary marker of arterial stiffness) and RHI (primary marker of peripheral vasodilation) following calcium citrate administration. A sample size of 25 subjects has 85% power to detect a 1.5 m/s change in PWV (assuming a SD of 2.5 m/s) and 80% power to detect a 0.35 change in RHI (assuming a SD of 0.6) at the two-tailed 0.05 significance level.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Subject characteristics

Sixteen of the 25 subjects were female. The subjects' mean age was 60.3 ± 6.5 years, weight was 73.1 ± 11.1 kg, height was 1.69 ± 0.09 m, body mass index was 25.5 ± 2.7 kg/m2, and waist circumference was 88 ± 9 cm. Four subjects were treated for hypertension and 4 subjects for hypercholesterolemia. No subject had a history of ischemic heart disease, cerebrovascular disease, peripheral vascular disease, or renal impairment, and no subject was a current smoker.

Biochemistry

Total (p < 0.0001) and ionized (p < 0.0001) serum calcium were significantly increased following administration of 1000 mg calcium citrate (Fig. 2A, B). In post hoc testing total calcium was significantly greater than baseline at 120 and 180 minutes and ionized calcium was significantly greater than baseline at 60, 120, and 180 minutes after calcium citrate administration. PTH (4.7 ± 1.1 versus 2.8 ± 1.0 pmol/L, p < 0.0001) and beta CTx (378 ± 150 versus 305 ± 138 pg/mL, p < 0.0001) were significantly lower than baseline 180 minutes after administration of 1000 mg calcium citrate. Mean 25 hydroxy-vitamin D concentration was 75 ± 26 nmol/L.

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Figure 2. Total (A) and ionized (B) serum calcium concentration before and up to 3 hours after oral administration of 1000 mg calcium citrate. Values represent mean ± SE. Values of p were calculated using repeated measures ANOVA. #p < 0.05 versus baseline; *p < 0.0001 versus baseline. Ca = calcium.

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Cardiovascular function

Pulse rate was significantly lower than baseline after administration of 1000 mg calcium citrate ( < 0.0001). However, there were no significant changes in systolic or diastolic blood pressure. There was a significant fall in AIx (p = 0.03) following administration of 1000 mg calcium citrate, consistent with a reduction in arterial wave reflection. Ejection duration was lower (p = 0.005) and SEVR higher (p = 0.007) following administration of calcium citrate. There were no significant changes in PWV or RHI after administration of 1000 mg calcium citrate (Table 1).

Table 1. Measures of Cardiovascular Function Performed Before and Between 120 and 180 Minutes After Oral Administration of 1000 mg Calcium Citrate
 Before calcium citrateAfter calcium citratep
  1. Values represent mean ± SD or median (interquartile range).

  2. BP = blood pressure.

Pulse (beats/min)63.6 ± 7.958.6 ± 5.6< 0.0001
Systolic BP (mm Hg)138 ± 18140 ± 170.55
Diastolic BP (mm Hg)83 ± 1080 ± 90.09
Augmentation index (%)29.7 (23.8–34.0)26.4 (22.7–34.0)0.03
Ejection duration (milliseconds)346 (338–356)340 (334–351)0.005
Subendocardial viability ratio (%)163 (148–174)170 (149–185)0.007
Pulse wave velocity (m/s)8.2 (7.7–9.4)8.9 (7.6–9.5)0.33
Reactive hyperemia index2.39 ± 0.522.35 ± 0.620.72

There was a significant negative correlation between the maximal change in total serum calcium after administration of 1000 mg calcium citrate and the change in AIx (Fig. 3). There were no significant correlations between the change in total serum calcium and other markers of vascular function (data not shown). Changes in ionized calcium and PTH were not significantly correlated with changes in cardiovascular function (data not shown).

thumbnail image

Figure 3. Correlation between the maximal change in total calcium and change in augmentation index after oral administration of 1000 mg calcium citrate.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

This study reports the acute changes in cardiovascular function in healthy volunteers following administration of 1000 mg calcium citrate. Total and ionized serum calcium were increased after 3 hours by an average of 0.10 and 0.06 mmol/L respectively, which was sufficient to reduce serum PTH and consequently bone resorption. These changes in serum calcium were associated with a reduction in pulse rate and AIx, an increase in SEVR and no significant change in PWV or RHI. These findings demonstrate that the increase in serum calcium following administration of calcium supplements does not acutely induce deleterious changes in these parameters of cardiovascular function. The reported increase in cardiovascular risk associated with calcium supplementation is thus unlikely to be secondary to acute alterations in arterial stiffness and vasodilatory function.

Recent meta-analyses7, 8 and epidemiologic studies17 reporting an increase in cardiovascular events in patients on calcium supplements have raised concerns regarding their safety. However, others have highlighted potential flaws in the study design and raised concerns regarding event ascertainment.9, 10 Furthermore, another meta-analysis reported that the combination of calcium and vitamin D supplementation was associated with a reduction in all-cause mortality whereas vitamin D supplementation alone was not.18 It is highly unlikely that there will ever be a randomized-controlled trial of sufficient size and duration to conclusively determine the effect of calcium supplementation on cardiovascular risk. Lacking these data, assessment of the effect of calcium supplements on plausible physiologic mechanisms that could underlie an increase in cardiovascular risk will greatly enhance the final interpretation of the above studies.7, 8, 17, 18

We found no significant change in PWV following calcium citrate administration. Carotid-femoral PWV is considered the gold standard measure of arterial stiffness because it is highly reproducible and directly reflects arterial stiffness in the aorta, which makes the largest contribution to arterial buffering following left ventricular ejection.19 PWV is not only a manifestation of structural changes in the aortic wall,20, 21 but can change within 90 minutes of administration of a single dose of a pharmacologic agent such as propranolol or captopril.22 PWV is a consistent predictor of fatal and nonfatal cardiovascular events in a range of subject populations, independent of other cardiovascular risk factors.23 An increase in PWV of 1 m/s is associated with a 14% risk factor–adjusted increase in relative risk of cardiovascular events.24 This study was powered to detect a 1.5 m/s change in PWV, which would equate to about a 20% increase in relative risk of cardiac events, similar to the increase in relative risk reported in recent calcium supplement meta-analyses.7, 8 The failure to find a significant change in PWV suggests an acute serum calcium–mediated increase in arterial stiffness does not explain the reported increase in cardiovascular events with calcium supplements.

Contrary to our hypothesis, we found a reduction in median AIx of about 10% following calcium citrate administration, with the change in serum total calcium negatively correlated with the change in AIx. Based on the findings of a recent meta-analysis, this reduction in AIx, if maintained long-term, would be associated with about a 30% reduction in relative risk of cardiovascular events.25 Unlike PWV, AIx is an indirect marker of arterial stiffness that is also influenced by the amplitude of the reflected wave, the reflection point, and the pattern and duration of left ventricular ejection.19 Given that PWV did not significantly change following administration of calcium citrate and because we controlled for the change in heart rate, our data suggest that the reduction in AIx is likely to be secondary to a reduction in left ventricular ejection volume.19 Consistent with this we found a significant reduction in left ventricular ejection duration after administration of calcium citrate. Further studies should directly explore the acute changes in cardiac function associated with calcium supplementation.

SEVR is a reflection of the relative time in diastole and systole, with a lower SEVR associated with greater time in systole and a resultant reduction in coronary perfusion relative to cardiac workload.26 A recent study reported that a lower SEVR was associated with a reduction in coronary flow reserve, independent of other variables.27 As such, our finding of an increase in SEVR following administration of calcium citrate is indirectly indicative of an increase in coronary perfusion.

There was no significant change in RHI following calcium citrate administration. RHI, which is in part dependent on release of nitric oxide from the endothelium,28 is associated with risk factors for cardiovascular disease,29 coronary artery atherosclerosis on angiography,30 and cardiovascular events.16 Our findings differ from a previous study reporting that peripheral vasodilation was reduced following an intravenous calcium infusion.14 These contrasting results may reflect the different method of assessment of vasodilatory function in the study by Nilsson and colleagues14 or relate to the fivefold greater increase in ionized calcium following intravenous calcium infusion. However, the dose of calcium citrate used in our study is at the upper end of the dosing range for osteoporosis treatment and it is unlikely that most patients with osteoporosis will be exposed to greater changes in serum calcium.

Our results following acute calcium supplement administration contrast with studies in patients with primary hyperparathyroidism that reported an increase in PWV and AIx.31–33 A possible explanation for this difference is that changes in vascular stiffness in primary hyperparathyroidism could be PTH-mediated, and not calcium-mediated. A previous study reported that carotid stiffness was correlated with PTH, but not serum calcium.34 Another explanation for the discordant findings is that primary hyperparathyroidism is generally associated with a greater degree of hypercalcemia and the duration of hypercalcemia is far longer. Consequently, there are structural arterial changes in patients with primary hyperparathyroidism that will contribute to an increase in arterial stiffness.34

How should we interpret the studies of Bolland and colleagues7, 8 and Li and colleagues17 in light of our findings? Unfortunately, our study neither confirms nor refutes the safety of calcium supplementation. However, our findings do demonstrate that the rise in serum calcium following supplement administration does not acutely exert an adverse effect on these parameters of cardiovascular function. Future studies should focus on the effect of longer-term calcium supplementation on these parameters of cardiovascular function.

The strengths of this study include that it is a within-subject analysis on a single day and as such minimizes potential confounding from other variables. Furthermore, all investigations were performed by a single operator who has demonstrated good reproducibility in pulse wave analysis and PWV measurements. However, we acknowledge our study is subject to limitations. First, the study was not randomized or blinded. However, we report results that refute our original hypothesis, suggesting it is unlikely the lack of blinding introduced any bias. Second, the sample size was relatively small. Nevertheless, the study was adequately powered to detect the magnitude of change in PWV we would expect to be associated with a 20% to 30% increase in cardiovascular events if changes in arterial stiffness were the underlying mechanism. We do acknowledge, however, that a smaller increase in PWV than this study was powered to determine could be clinically significant. One subject was taking a beta blocker medication that was administered approximately 2 hours before the first measurement of AIx. It is possible that there could have been a small difference in atenolol concentration at the time of the second AIx measurement that might have a minor effect on results. Finally, the calcium dose in this proof of concept study was higher than that used by many patients. However, it is unlikely that lower calcium doses will exert an opposite effect and adversely affect cardiovascular function acutely.

In summary, the acute increase in serum calcium following administration of 1000 mg calcium citrate was associated with a reduction in arterial wave reflection and an indirectly measured increase in coronary perfusion. As such, acute serum calcium–mediated changes in cardiovascular function are unlikely to underlie the increase in cardiovascular events reported in previous studies of calcium supplementation. Future studies should examine the effect of longer-term calcium supplementation on structure and function of the cardiovascular system.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

This study was supported by a competitive research grant from Foundation Daw Park. Australian New Zealand Clinical Trials Registry number: ACTRN12612000232831. We thank the participants who volunteered for this study.

Authors' roles: Study design: MGB. Study conduct: MGB, BLM, DS, and CJP. Data collection: BLM and DS. Data analysis: MGB. Data interpretation: MGB, BLM, DS, and CJP. Drafting manuscript: MGB. Revising manuscript content: BLM, DS, and CJP. Approving final version of manuscript: MGB, BLM, DS, and CJP. MGB takes responsibility for the integrity of the data analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Subjects and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
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