Plasma total calcium concentration is associated with blood pressure and systemic vascular resistance in normotensive and never-treated hypertensive subjects

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iblo20 Blood Pressure ISSN: 0803-7051 (Print) 1651-1999 (Online) Journal homepage: https://www.tandfonline.com/loi/iblo20 Plasma total calcium concentration is associated with blood pressure and systemic vascular resistance in normotensive and never-treated hypertensive subjects Lasse Oinonen, Jenni Koskela, Arttu Eräranta, Antti Tikkakoski, Mika Kähönen, Onni Niemelä, Jukka Mustonen & Ilkka Pörsti To cite this article: Lasse Oinonen, Jenni Koskela, Arttu Eräranta, Antti Tikkakoski, Mika Kähönen, Onni Niemelä, Jukka Mustonen & Ilkka Pörsti (2019): Plasma total calcium concentration is associated with blood pressure and systemic vascular resistance in normotensive and nevertreated hypertensive subjects, Blood Pressure, DOI: 10.1080/08037051.2019.1696180 To link to this article: https://doi.org/10.1080/08037051.2019.1696180 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group View supplementary material Published online: 02 Dec 2019. Submit your article to this journal Article views: 265 View related articles View Crossmark data ORIGINAL ARTICLE Plasma total calcium concentration is associated with blood pressure and systemic vascular resistance in normotensive and never-treated hypertensive subjects Lasse Oinonen a , Jenni Koskela a,b , Arttu Er€ aranta a , Antti Tikkakoski a,c , Mika K€ ah€ onen a,c , Onni Niemel€ a d , Jukka Mustonen a,b and Ilkka P€ orsti a,b a Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland; b Department of Internal Medicine, Tampere University Hospital, Tampere, Finland; c Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland; d Department of Laboratory Medicine and Medical Research Unit, Sein€ ajoki Central Hospital, Sein€ ajoki, Finland ABSTRACT Purpose: The underlying causes of primary hypertension are not fully understood. Evidence on the relation of plasma calcium concentration with blood pressure (BP) is inconsistent and relies largely on studies utilizing office BP measurements in populations using cardiovascular drugs. In many studies adjustment for confounders was not optimal. In this cross-sectional study we examined the association of plasma total calcium concentration with the haemodynamic determinants of blood pressure. Subjects and methods: Supine haemodynamics were recorded using pulse wave analysis, whole-body impedance cardiography, and heart rate variability analysis in 618 normotensive or never-treated hypertensive subjects (aged 19–72 years) without diabetes, cardiovascular or renal disease, or cardiovascular medications. Linear regression analysis was used to investigate factors associated with haemodynamic variables. Results: Mean age was 45.0 years, body mass index 26.8 kg/m 2 , seated office BP 141/89 mmHg, and 307 subjects (49.7%) were male. Mean values of routine blood and plasma chemistry analyses were within the reference limits of the tests except for low-density lipoprotein cholesterol (3.05 mmol/l). In the laboratory, mean supine radial BP was 131/75 mmHg, and both systolic and diastolic BP correlated directly with plasma total calcium concentration (r¼0.25 and r¼0.22, respectively, p<0.001 for both). In regression analysis plasma total calcium concentration was an independent explanatory variable for radial and aortic systolic and diastolic BP, and systemic vascular resistance, but not for cardiac output, pulse wave velocity, or any of the heart rate variability parameters. Conclusion: Plasma total calcium concentration was directly associated with systolic and diastolic BP and systemic vascular resistance in normotensive or never-treated hypertensive subjects without comorbidities and cardiovascular medications. Higher plasma calcium concentration potentially plays a role in primary hypertension via an effect on vascular resistance. ARTICLE HISTORY Received 12 September 2019 Revised 17 October 2019 Accepted 11 November 2019 KEYWORDS Plasma calcium; blood pressure; systemic vascular resistance; haemodynamics Introduction Despite the high global prevalence of hypertension and its known clinical consequences, the underlying causes of hypertension are still not fully understood. In a vast majority of hypertensive subjects (90%) the aetiology cannot be defined, and hence the term primary hypertension is used [1,2]. The identification of novel risk factors for hypertension is an important step to better understand its aetiology, find novel treatment targets, and reduce the associated burden of morbidity and mortality. Calcium and hormones regulating calcium balance have been shown to be linked to the regulation of BP [3–7] and risk of incident hypertension [8–10]. However, a comprehensive understanding of the relation between calcium metabolism and BP remains to be established. This is probably due to the complex nature of calcium ion as an intracellular and extracellular messenger [11,12], the complexity of calcium metabolism with many interdependent hormones [13], the bidirectional relationship between calcium metabolism and the renin-angiotensin-aldosterone-system [14–17], and the CONTACT Lasse Oinonen [email protected] Faculty of Medicine and Health Technology, Tampere University, Tampere, FI-33014, Finland Supplemental data for this article can be accessed here. ß2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/bync-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. BLOOD PRESSURE https://doi.org/10.1080/08037051.2019.1696180 putative direct functional role of the calcium-sensing receptor (CaSR) in the vasculature [18–20]. Alterations in extracellular calcium levels may influence calcium-mediated intracellular signalling and potentially play a role in the pathogenesis of primary hypertension in humans [21]. However, serum calcium concentration is tightly regulated by extracellular CaSR in the parathyroid glands, and a concerted interplay between the calcium regulating hormones parathyroid hormone (PTH), vitamin D 3 and to a lesser extent calcitonin [12]. Studies that have examined the association between serum total calcium concentration and BP have yielded conflicting results (Supplemental Table 1). Some studies reported a direct association, while others found no association. Some of these studies were characterized by relatively small sample sizes, and some by suboptimal adjustment for confounders like smoking habits, alcohol use, renal function, and plasma concentrations of vitamin D metabolites and PTH. In addition, all except two of the previous studies utilized office BP measurements without detailed haemodynamic analyses, and several investigations included subjects using antihypertensive medications or other drugs with direct cardiovascular influences (Supplemental Table 1). The inconsistent findings in the previous observations as well as the methodological questions presented above warrant further investigations of the associations between calcium metabolism and haemodynamics. We examined in a cross-sectional study whether plasma total calcium concentration is associated with the haemodynamic determinants of BP in 618 normotensive or never-treated hypertensive subjects without diabetes, cardiovascular or cerebrovascular disease, renal impairment, or medications with direct cardiovascular influences. Haemodynamics were characterized using pulse wave analysis, wholebody impedance cardiography and heart rate variability analysis. Subjects and methods Study population This study is part of an ongoing research on haemodynamics in primary and secondary hypertension (the DYNAMIC-study, clinical trial registration NCT01742702). The study complies with the Declaration of Helsinki, and was approved by the ethics committee of the Tampere University Hospital (code R06086M) and the Finnish Medicines Agency (Eudra-CT number 2006-002065-39). All methods were carried out in accordance with the relevant guidelines and regulations. The present study subjects were screened from 1349 participants who were recruited via announcements in local newspapers and in facilities of Tampere University Hospital, University of Tampere, Varala Sports Institute, and local organizations that provide occupational health care. Signed informed consent was obtained from all participants, and they were interviewed and examined by a physician. Smoking status was recorded and cigarette consumption calculated as pack-years. Alcohol consumption was recorded as standard drinks (12 grams of absolute alcohol) per week. The use of medicines, dietary supplements, and other substances not registered as drugs was documented. Dairy product consumption was recorded as standard daily doses. Seated office BP was measured by a physician according to ESH/ESC Guideline [22] using a manual sphygmomanometer (Heine Gamma G7, Herrsching, Germany). The phase I and V Korotkoff sounds were used to identify systolic and diastolic BP, respectively. The exclusion criteria and number of subjects excluded due to each reason were: 1) use of antihypertensives or other medications with direct haemodynamic influences (like a 1 -adrenoceptor blockers for prostate hyperplasia, and b 2 -adrenoreceptor agonists for asthma) (n¼619); 2) history of cardiovascular or cerebrovascular disease (n¼176); 3) heart rhythm other than sinus (n¼14); 4) diabetes mellitus (n¼108); 5) chronic kidney disease [estimated glomerular filtration rate (GFR) <60 ml/min/1.73 m 2 ,or proteinuria] (n¼73); 6) chronic liver disease (n¼0); 7) secondary hypertension [22](n¼221); 8) psychiatric illness other than mild depression or anxiety (n¼4); 9) alcohol or substance abuse (n¼23). No recordings were performed for subjects with any acute illness. The study group consisted of 618 subjects (307 men and 311 women) aged 19–72 years. Based on the office BP measurements, 248 (40.1%) of the participants were normotensive (95 men and 153 women) and 370 (59.9%) were hypertensive (212 men and 158 women) [22]. In total 240 (38.9%) of the participants used some medications. Seventy-six women used hormones for contraception or hormone replacement therapy, while 104 women were post-menopausal. Thirteen subjects used statins, 1 took ezetimibe, 22 used thyroxin for hypothyroidism, 15 took inhaled corticosteroids for asthma, 39 used antidepressants (mainly selective serotonin reuptake inhibitors, while 5 subjects used amitriptyline, 4 subjects venlafaxine, 2 2 L. OINONEN ET AL. subjects duloxetine, and 2 subjects mirtazapine), 6 were taking anxiolytics (benzodiazepines), and 2 subjects used antiepileptics. Four subjects used COX-2 inhibitors and 5 subjects non-selective NSAIDs. Laboratory analyses Blood and urine samples were collected after 12 h fasting. Plasma total calcium and other electrolytes, cystatin C, creatinine, C-reactive protein (CRP), alanine aminotransferase (ALT), alkaline phosphatase (ALP), glucose, uric acid, and lipid determinations were carried out using Cobas Integra 700/800 (F. Hoffmann-LaRoche Ltd., Basel, Switzerland) or Cobas 6000, module c501 (Roche Diagnostics, Basel, Switzerland). PTH and insulin were determined using electrochemiluminescence immunoassay (Cobas e411, Roche Diagnostics), and insulin sensitivity was evaluated by calculating quantitative insulin sensitivity check index (QUICKI) [23]. Blood cell counts were analysed using ADVIA 120 or 2120 (Bayer Health Care, Tarrytown, NY, USA). Plasma renin activity was measured using GammaCoat Plasma Renin Activity assay (Diasorin S.p.A., Saluggia, Italy), and aldosterone concentrations using active aldosterone radioimmunoassay (Diagnostics Systems Laboratories, Beckman Coulter, Webster, TX, USA). Plasma concentrations of calcidiol (25(OH)D 3 ) and calcitriol (1,25(OH) 2 D 3 ) were analysed using enzyme immunoassay (Immunodiagnostic Systems, Boldon, UK). Kidney disease was excluded by estimating GFR using CKD-EPI cystatin C formula [24], and by urine dipstick analysis using an automated refractometer test (Siemens Clinitec Atlas or Advantus, Siemens Healthcare GmbH, Erlangen, Germany). Protocol for haemodynamic measurements Prior to the recordings the participants were instructed to abstain from caffeine-containing products, smoking, and heavy meals for 4 h, and from alcohol for 24 h. Haemodynamics were recorded in a quiet temperature-controlled laboratory by a trained nurse. The subjects rested supine for 10 min, and then the nurse measured BP twice by a manual sphygmomanometer (Heine Gamma G7). Thereafter beat-to-beat supine haemodynamic data was recorded for 5 min. The mean values of the variables during the last 3 min were used in the analyses, as this was the most stable period. On average the data from each subject represented 180 cardiac cycles. For the heart rate variability analyses the mean values of the whole 5-minute recording period were used. Pulse wave analysis Radial BP and pulse wave form were continuously recorded using a tonometric sensor (Colin BP-508T, Colin Medical Instruments Corp., San Antonio, TX, USA) fixed on the left radial pulse. Radial BP measurement was calibrated twice during the 5-minute recording by contralateral brachial BP measurement using an oschillometric cuff at the upper arm. Aortic BP and pulse wave reflections were derived from the tonometric signal with the SphygmoCor V R system (SphygmoCor V R PWMx, AtCor Medical, Australia) utilizing a validated transfer function [25]. Heart rate, aortic pulse pressure, aortic augmentation pressure, and augmentation index (AIx, aortic augmentation pressure =aortic pulse pressure 100) were also determined. AIx adjusted for heart rate of 75 beats/ min (AIx@75) was determined with the SphygmoCor V R software. Whole-body impedance cardiography Whole-body impedance cardiography (CircMon V R device, JR Medical Ltd., Estonia), which detects changes in body’s electrical impedance during cardiac cycles, was utilized to determine beat-to-beat heart rate, stroke volume, cardiac output, aortic-to-popliteal pulse wave velocity (PWV), extracellular water volume (ECW), and ECW balance. The method and electrode configuration have been described previously [26]. Finger BP was recorded using a plethysmographic cuff (Finapres, Enschede, the Netherlands) in the left middle finger. Systemic vascular resistance (SVR) was calculated from tonometric BP and cardiac output so that the assumed normal central venous pressure (3mmHg) was subtracted from mean arterial pressure and the value was divided by cardiac output [26]. Cardiac output, stroke volume and SVR were normalized for body surface area to obtain cardiac index, stroke index and SVR index. ECW was evaluated with the CircMon device by the formula ECW¼k(height 2 /Z). The coefficient k (Xcm) is derived from blood resistivity and the relation between the distance of voltage electrodes and body height in centimeters, and Z is the recorded impedance of the body. The ECW balance was calculated as ECW/ECW predicted .TheformulaforECW predicted was 2.4 (0.0236height 0.725 weight 0.423 1.229) in males and 2.6(0.0248 height 0.725 weight 0.423 1.9549) BLOOD PRESSURE 3 in females [27–29]. For easier interpretation, the results of the ECW balance were adjusted so that the mean value in the lowest calcium tertile was 1.0. Stroke volume measured using CircMon V R correlates well with values obtained with 3-dimensional echocardiography [30]. Cardiac output measured with CircMon V R correlates well with values measured with the thermodilution and the direct oxygen Fick methods [26,31]. As the whole-body impedance cardiography slightly overestimates PWV, a validated equation (PWV¼PWV impedance 0.696 þ0.864) was used to calculate values corresponding to Doppler ultrasound method [32]. PWV values obtained using this equation correlate well with values measured with Doppler ultrasound or applanation tonometry [32,33]. Whole-body bioimpedance-derived ECW correlates well with 51 Cr-EDTA dilution-based measurement of ECW (r¼0.74, bias 0.2 ± 1.1 litres), as well as with perioperative weight changes of patients [34]. The repeatability and reproducibility of this measurement protocol has been previously established [32,35,36]. Frequency domain analysis of heart rate variability The electrocardiograms were recorded by the CircMon V R device at sampling rate of 200 Hz, and analysed using Matlab software (MathWorks Inc., Natick, MA, USA). Normal R-R intervals were recognized, and if the interval differed over 20% from the previous values the beat was considered as ectopic. The artefacts were processed using the cubic spline interpolation method, and the frequency domain variables were calculated using the Fast Fourier Transformation method [37], consisting of 1) power in low frequency (LF) range (0.04–0.15Hz), 2) power in high frequency (HF) range (0.15–0.40 Hz), and 3) LF/HF ratio. Both LF and HF power were transformed to the natural logarithm before analyses to yield normal non-skewed distributions. Statistical analyses The subjects were divided into tertiles of plasma total calcium concentration separately within each sex, and the sex-specific tertiles were applied in the subsequent analyses. The participants were also grouped according to the use of calcium and/or vitamin D supplements. One-way analysis of variance (ANOVA) was applied to assess the differences in the clinical characteristics, blood and plasma chemistry test values, haemodynamic variables, and consumption of supplements and dairy products. The Bonferroni correction was applied in the post-hoc analyses and Levene’s test was used to test homogeneity of variances. Variables for linear regression analysis were selected by utilizing Spearman’s nonparametric correlation analysis for radial systolic and diastolic BP, and p<0.05 was used as the inclusion criterion. The normality of the distributions of the variables was assessed using skewness and kurtosis data, and with normal Q-Q plot, and with the Shapiro–Wilk test. Due to skewed distributions, ECW balance, PTH, 1,25(OH) 2 D 3 , uric acid, CRP, triglycerides, highdensity lipoprotein (HDL) cholesterol, ALP, ALT, and renin were corrected by Lg 10 -transformation. Smoking status was categorized using two discrete variables that enabled classification to current smokers, previous smokers or never smokers (category for smoking or previous smoking was either 0 or 1, thus a never smoker received a category 0 for both). Alcohol consumption was categorized using three discrete variables (category either 0 or 1); cut-points for women 0 (abstinence), 1–7 (low consumption), 8–14 (moderate consumption), and 15 doses per week (high consumption); for men 0, 1–14, 15–24, and 25 doses per week, respectively, according to the Finnish Guidelines [38]. Linear regression analyses with backward elimination were used to investigate explanatory variables associated with the haemodynamic variables of interest. The final regression model included age, sex, body mass index (BMI), categorized alcohol consumption, smoking status; use of dairy products, calcium supplements, vitamin D supplements; QUICKI; plasma concentrations of calcium, phosphate, sodium, cystatin C, low-density lipoprotein (LDL) cholesterol; and Lg 10 of plasma concentrations of CRP , PTH, 1,25(OH) 2 D 3 , uric acid, ALP, ALT, triglycerides, HDL cholesterol, renin; and Lg 10 of ECW balance. In additional analyses the subjects were divided into tertiles according to age, and each tertile analysed separately. SPSS version 25.0 for Windows software (IBM SPSS, Armonk, NY, USA) was used for statistics. The results are presented as mean with standard deviation (SD) of the mean or with 95% confidence interval (CI) of the mean, and p<0.05 was considered statistically significant. Results Study population The total study population comprised of 618 subjects and 49.7% (307) were men. Mean age was 45.0 years 4 L. OINONEN ET AL. (range 19–72 years), mean BMI 26.8kg/m 2 , and mean seated office BP 141/89 mmHg. Mean plasma total calcium concentration was 2.28mmol/l (CI 2.27–2.30) in women, and 2.32mmol/l (CI 2.31–2.33) in men (p<0.001). Therefore, the results below concerning the tertiles of plasma calcium concentration were adjusted for sex. The demographics and clinical characteristics in each tertile of plasma total calcium concentration are presented in Table 1. Mean age, BMI, ECW, percentage of current smokers, dairy product consumption, and percentage of calcium or vitamin D supplement users did not differ between the calcium tertiles. Mean ECW balance was lower in the highest calcium tertile versus the lowest calcium tertile (0.97 vs. 1.00, respectively; p<0.01). Mean alcohol consumption as standard drinks per week was somewhat higher in the highest calcium tertile versus the lowest calcium tertile (5.4 vs. 3.7, respectively; p<0.05). Mean values of blood and plasma chemistry test results were within the reference limits of the tests in all calcium tertiles, except for plasma total and LDL cholesterol concentrations that were slightly above the upper limits of the reference values in the middle and highest tertiles (Supplemental Table 2). In addition to the obvious deviations in mean plasma total calcium concentration, there were small but statistically significant (p<0.05) differences in the mean plasma chemistry test results between the calcium tertiles: Plasma phosphate was higher in the highest calcium tertile versus the other tertiles, while plasma 25(OH)D 3 was higher in the highest calcium tertile versus the lowest calcium tertile. Plasma cystatin C, total and LDL cholesterol were higher, while estimated GFR was lower, in the highest and the middle calcium tertiles versus the lowest calcium tertile (Supplemental Table 2). In total 511 subjects used no calcium or vitamin D supplements, while 6 subjects took calcium supplements, 88 subjects took vitamin D supplements, and 13 subjects used both calcium and vitamin D supplements (Supplemental Table 3). There were no differences between the groups in mean plasma total calcium, phosphate or PTH concentrations, or in dairy product consumption. However, mean 25(OH)D 3 concentration was higher in the two groups using vitamin D supplements when compared with the group using neither calcium nor vitamin D supplements (p<0.05 for both). The plasma concentration of 1,25(OH) 2 D 3 was higher in the small group (n¼13) taking both calcium and vitamin D supplements when compared with the groups not taking the supplements or using only vitamin D supplement (p<0.05 for both) (Supplemental Table 3). BP and haemodynamic variables in the tertiles of plasma total calcium concentration Mean seated office BP did not differ between the calcium tertiles (Table 1). However, mean supine systolic BP was higher in the highest and the middle calcium tertile versus the lowest calcium tertile when measured 1) at the brachial level indirectly by the research nurse using the auscultatory method, 2) at the radial Table 1. Demographics, clinical characteristics and laboratory results per tertile of plasma total calcium concentration. Demographics and clinical characteristics Tertile 1 (n¼201) Tertile 2 (n¼218) Tertile 3 (n¼199) Males (%) 49.3 53.2 48.2 Age (years) 44.9 (10.7) 45.8 (12.1) 44.4 (12.5) Body mass index (kg/m 2 ) 26.7 (4.4) 26.9 (4.3) 26.8 (4.3) Extracellular water volume (l) 13.02 (1.75) 12.87 (1.90) 12.84 (1.90) Extracellular water balance 1.00 (0.11) 0.98 (0.09) 0.97 (0.11) Current smokers (%) 10.9 11.0 16.1 Alcohol consumption (standard drinks/week) 3.7 (5.2) 4.4 (6.0) 5.4 (6.1) Dairy product consumption (standard dose/day) 3.0 (1.8) 3.0 (1.9) 3.4 (2.6) Calcium supplement users (%) 1.5 2.3 5.5 Vitamin D supplement users (%) 11.9 17.0 20.1 Seated brachial office blood pressure measured by physician (mmHg) 139 (22)/88 (13) 142 (21)/90 (12) 142 (20)/90 (12) Supine brachial blood pressure measured by nurse (mmHg) 127 (17)/78 (12) 134 (20)/81 (12) 135 (18)/83 (11) Supine radial laboratory blood pressure (mmHg) 127 (19)/72 (12) 133 (20)/76 (13) 135 (19)/77 (13) Supine finger laboratory blood pressure (mmHg) 118 (17)/68 (12) 124 (19)/69 (13) 124 (20)/70 (13) Results shown as mean (standard deviation). p<0.05 vs. Tertile 1; p<0.01 vs. Tertile 1. BLOOD PRESSURE 5 level using applanation tonometry, and 3) at the finger level using a plethysmographic cuff (p<0.01 for all, except for systolic finger BP p<0.05) (Table 1). Mean supine diastolic BP was also higher in the highest tertile versus the lowest tertile at the brachial level, and in the highest and the middle calcium tertile versus the lowest calcium tertile at the radial level (p<0.01 for all) (Table 1). Mean radial systolic and diastolic BPs were higher in the middle and the highest calcium tertiles when compared with the lowest calcium tertile (Figure 1(a,b)). Similarly, mean aortic systolic and diastolic BPs were higher in the middle and the highest calcium tertiles when compared with the lowest calcium tertile (Supplemental Figure 1(a,b)). Plasma total calcium concentration correlated positively with radial systolic and diastolic BP (r¼0.228, and r¼0.195, respectively; p<0.001 for both), aortic systolic and diastolic BP (r¼0.199, and r¼0.194, respectively; p<0.001 for both), and finger systolic and diastolic BP (r¼0.147, and r¼0.098; p<0.001, and p¼0.015, respectively), as well as with seated office systolic and diastolic BP (r¼0.141, and r¼0.129; p<0.001, and p¼0.001, respectively). Mean aortic pulse pressure was higher in the highest calcium tertile versus the lowest calcium tertile (Figure 1(c)), while PVW was not different between the calcium tertiles (Figure 1(d)). AIx, AIx@75, and aortic augmentation pressure were not different between the calcium tertiles (Supplemental Figure 1(c,d); AIx@75 not shown). Plasma total calcium concentration correlated with aortic pulse pressure (r¼0.144, p<0.001), but not with PVW, AIx, AIx@75, or aortic augmentation pressure (r<0.048, p>0.237 for all). There were no statistically significant (p<0.05) differences between the calcium tertiles in mean heart rate, stroke index, cardiac index, or SVR index (Figure 2(a–d)). Plasma total calcium concentration correlated with SVR index (r¼0.108, p¼0.007), but not with heart rate, cardiac index, or stroke index (r<0.064, p>0.113 for all). Also, no differences were observed between calcium tertiles in the low-frequency (LF) power, high-frequency (HF) power, and LF/HF ratio of heart rate variability (Supplemental Figure 2(a–c)). Plasma total calcium concentration did not correlate with any of the heart rate variability parameters (r<0.069, p>0.098 for all). BP and haemodynamic variables in linear regression analyses Linear regression analyses showed that plasma total calcium concentration was an independent explanatory variable for radial systolic and diastolic BP (standardized coefficient Beta ¼0.138, and 0.119; p¼0.001, and p¼0.003, respectively) (Table 2). In additional analyses plasma total calcium concentration was also an independent explanatory variable for aortic systolic and diastolic BP (Beta ¼0.149, and Beta ¼0.122; p<0.001, and p¼0.002, respectively), aortic pulse pressure (Beta ¼0.120, p¼0.002), and for finger systolic BP (Beta ¼0.137, p¼0.001), but not for seated Figure 1. Radial systolic blood pressure (a), radial diastolic blood pressure (b), aortic pulse pressure (c), and pulse wave velocity (d) in tertiles of plasma total calcium concentration adjusted for sex; mean (circle) with 95% confidence intervals of the mean (whiskers), one-way ANOVA with the Bonferroni correction in post-hoc analyses. 6 L. OINONEN ET AL. office systolic or diastolic BP (Beta ¼0.044, and Beta ¼0.052; p¼0.241, and p¼0.182, respectively). As the major determinants of BP, the regression analyses also examined cardiac output and SVR as the variables of interest. Plasma total calcium concentration was not an explanatory variable for cardiac output, but was an independent explanatory factor for SVR (Beta ¼0.098, p¼0.029) (Table 3). Finally, plasma total calcium concentration was not an independent explanatory variable for PWV, AIx or AIx@75 (Beta ¼0.007, Beta ¼0.047, and Beta ¼0.046; p¼0.823, p¼0.145, and p¼0.137, respectively). Additional analyses in tertiles of age Although age did not correlate with plasma total calcium concentration (r¼0.004, p¼0.923), additional analyses were performed so that the subjects were divided into tertiles of age. The age ranges in the Tertiles 1, 2 and 3 were: 19.0–40.0, 40.1–52.0, and 52.1–72.0years, with the mean (SD) ages of 31.1 (6.0), 46.8 (3.3), and 58.0 (4.1) years, respectively. When each tertile was analysed separately using linear regression analysis, plasma total calcium concentration was an independent explanatory variable for radial systolic BP in all tertiles (Tertile 1 Beta ¼ 0.203, p¼0.001; Tertile 2 Beta ¼0.151, p¼0.029; and Tertile 3 Beta ¼0.265, p<0.001), and for radial diastolic BP in the middle and the highest tertile (Tertile 1 Beta ¼0.117, p¼0.076; Tertile 2 Beta ¼ 0.158, p¼0.023; and Tertile 3 Beta ¼0.246, p¼0.001, respectively). Discussion In our study in 618 Caucasian normotensive or never-treated hypertensive subjects, plasma total calcium concentration was directly associated with radial Figure 2. Heart rate (a), stroke index (b), cardiac index (c), and systemic vascular resistance index (d) in tertiles of plasma total calcium concentration adjusted for sex; mean (circle) with 95% confidence intervals of the mean (whiskers), one-way ANOVA with the Bonferroni correction in post-hoc analyses. Table 2. Significant explanatory variables for radial systolic and diastolic blood pressure in linear regression analysis. Systolic blood pressure (mmHg) B Beta pDiastolic blood pressure (mmHg) B Beta p Model (R 2 ¼0.320) Model (R 2 ¼0.310) (constant) 66.325 (constant) 9.582 Calcium 27.414 0.138 0.001 Cystatin C 16.029 0.181 <0.001 Cystatin C 18.017 0.137 0.002 LDL cholesterol 1.898 0.140 0.001 Age 0.222 0.135 0.002 PTH 12.181 0.139 <0.001 Body mass index 0.566 0.128 0.004 Age 0.147 0.133 0.003 LDL cholesterol 2.408 0.119 0.006 QUICKI 37.719 0.124 0.001 Sex (male) 4.319 0.112 0.005 Calcium 16.023 0.119 0.003 QUICKI 48.678 0.108 0.008 Phosphate 7.887 0.101 0.014 PTH 12.566 0.096 0.013 Sex (male) 2.312 0.089 0.025 Smoking (current) 4.917 0.085 0.022 Alcohol consumption (high) 6.619 0.078 0.034 Calcium supplement use 8.808 0.080 0.030 1,25(OH) 2 D 3 7.212 0.076 0.047 LDL: low-density lipoprotein; QUICKI: quantitative insulin sensitivity check index; PTH: parathyroid hormone; 1,25(OH) 2 D 3 : 1,25-dihydroxyvitamin D 3 .Variables in Model: Age, sex, body mass index, alcohol consumption (moderate, high), smoking status (current, previous), calcium, phosphate, Lg 10 of PTH, Lg 10 of 25(OH)D 3 ,Lg 10 of 1,25(OH) 2 D 3 ,QUICKI,Lg 10 of triglycerides, HDL cholesterol, LDL cholesterol, uric acid, alkaline phosphatase, alanine aminotransferase, sodium, Lg 10 of renin, Lg 10 of C-reactive protein, cystatin C, calcium supplement use, vitamin D supplement use, and dairy products use (standard doses/day). BLOOD PRESSURE 7 and aortic systolic and diastolic BP, as well as systemic vascular resistance, but not with cardiac output, pulse wave velocity, or any of the heart rate variability parameters. All participants were without previously diagnosed diabetes, cardiovascular or cerebrovascular disease, renal insufficiency, and medications with direct cardiovascular influences. The association of plasma total calcium concentration with haemodynamics was investigated using whole-body impedance cardiography and analyses of pulse wave and heart rate variability, and a comprehensive adjustment for confounders was applied. Our data indicates a direct association between plasma total calcium concentration and central and peripheral BP. Some previous reports observed a direct relation between serum total calcium concentration and seated office BP (Supplemental Table 1), while in the present regression analyses plasma calcium concentration was not an explanatory factor for seated office systolic and diastolic BP. Probably, the powerful confounding by the white-coat effect interfered with the BP measurements in the office [22], as statistically significant (p<0.05) differences in BP between the present calcium tertiles were found in the laboratory measurements performed by the research nurses using the auscultatory, tonometric and plethysmographic techniques. The observed direct association between plasma total calcium concentration and BP was not driven by age, as a direct association was present when all age tertiles were analysed separately. These findings indicate that plasma total calcium concentration should be considered as a potential contributor to the pathophysiology of primary hypertension. The observed differences in the supine systolic BP (7mmHg) and diastolic BP (5 mmHg) between the highest and lowest calcium tertiles are also clinically significant, as such a difference is associated with an increased risk of cardiovascular disease [39,40]. Previously, Kim et al. [8] found that higher plasma total calcium concentration (2.37 vs. <2.37mmol/l) was associated with 1.24-fold increase in the incidence of hypertension during 6 years of follow-up in a Korean cohort of 5560 subjects. The present regression analyses indicate that the observed direct association between plasma total calcium concentration and BP was mediated via SVR. In most of the earlier studies, the haemodynamic components involved in BP regulation were not addressed (Supplemental Table 1). However, Schutte et al. [41] investigated the associations of serum total calcium concentration with 24-h ambulatory BP and Finometer V R measurements during a stress test in 99 African men. They found that in younger men (<43years, n¼50), but not in older men, serum total calcium concentration was directly associated with 24-h ambulatory BP and total peripheral resistance during the stress test [41]. Previous experimental studies have suggested that higher extracellular calcium level increases calcium influx into arterial smooth muscle, induces smooth muscle contraction, and thus elevates SVR [4,42]. Experimental studies have found that CaSR activation in vascular smooth muscle sensitizes the arterial wall to contractile stimuli [20] and potentially influences the regulation of SVR [43]. The CaSR is also expressed in the human arterial wall [18]. Furthermore, acute calcium infusion has been reported to impair endothelium-dependent relaxation in healthy subjects [44], whereby extracellular calcium concentration may induce changes in endothelium-mediated control of arterial tone that contributes to the increase in SVR. In the present study, plasma total calcium concentration was not associated with PWV, wave reflection (AIx), cardiac index, or ECW. Therefore, changes in large arterial stiffness, cardiac output, or volume status did not explain the higher BP associated with higher plasma total calcium concentration. ECW balance was actually lowest in the highest calcium tertile. Nevertheless, plasma total calcium concentration was directly associated with aortic pulse pressure. This finding can readily be attributed to higher vascular resistance, as SVR has been shown to be one of the major determinants of central pulse pressure in middle-aged subjects [45]. Recently Mateus-Hamdan et al. [46] reported a direct association between plasma total calcium concentration and supine brachial pulse Table 3. Significant explanatory variables for cardiac output and systemic vascular resistance in linear regression analysis. Cardiac output (ml/min) B Beta pSystemic vascular resistance (dynes s/cm 5 ) B Beta p Model (R 2 ¼0.234) Model (R 2 ¼0.146) (constant) 5.231 (constant) 390 Sex (male) 0.946 0.403 <0.001 Sex (male) 152 0.253 <0.001 Body mass index 0.033 0.124 0.003 Age 5 0.195 <0.001 QUICKI 2.695 0.098 0.019 Vitamin D supplement use 91 0.111 0.010 Smoking (current) 0.332 0.094 0.014 LDL cholesterol 32 0.100 0.041 Calcium 306 0.098 0.029 Smoking (current) 80 0.088 0.042 QUICKI: quantitative insulin sensitivity check index; LDL: low-density lipoprotein. Variables in Model as in Table 2. 8 L. OINONEN ET AL.