Accepted Manuscript Effects of magnesium supplements on blood pressure, endothelial function and metabolic parameters in healthy young men with a family history of metabolic syndrome; A randomized crossover trial Elisa Cosaro , Sara Bonafini , Martina Montagnana , Elisa Danese , Maria S. Trettene , Pietro Minuz , Pietro Delva , Cristiano Fava , MD, PhD PII:

S0939-4753(14)00189-6

DOI:

10.1016/j.numecd.2014.05.010

Reference:

NUMECD 1304

To appear in:

Nutrition, Metabolism and Cardiovascular Diseases

Received Date: 13 December 2013 Revised Date:

13 May 2014

Accepted Date: 19 May 2014

Please cite this article as: Cosaro E, Bonafini S, Montagnana M, Danese E, Trettene MS, Minuz P, Delva P, Fava C, Effects of magnesium supplements on blood pressure, endothelial function and metabolic parameters in healthy young men with a family history of metabolic syndrome; A randomized crossover trial, Nutrition, Metabolism and Cardiovascular Diseases (2014), doi: 10.1016/ j.numecd.2014.05.010. 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 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.

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ACCEPTED MANUSCRIPT EFFECTS OF MAGNESIUM SUPPLEMENTS ON BLOOD PRESSURE, ENDOTHELIAL FUNCTION AND METABOLIC PARAMETERS IN HEALTHY YOUNG MEN WITH A FAMILY HISTORY OF METABOLIC SYNDROME; A RANDOMIZED CROSSOVER TRIAL. Short title: Magnesium supplements and blood pressure

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Elisa Cosaroa*, Sara Bonafinia *, Martina Montagnanab, Elisa Daneseb, Maria S Trettenea, Pietro Minuza, Pietro Delvaa, Cristiano Favaa. Department of Medicine and

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Department of Life and Reproduction Sciences, Azienda Ospedaliera Universitaria Integrata of

Verona, University of Verona, Italy.

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*These authors contributed equally to the manuscript. Correspondence and requests for reprints to: Cristiano Fava, MD, PhD Department of Medicine,

Piazzale LA Scuro 10, 37134 Verona, Italy.

fax: +39 45 8027465;

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Tel: +39 45 8124414;

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Division of Internal Medicine C,

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a

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e-mail: [email protected] Total word count: 4533 Number of tables: 1

Number of figures: 2 + 1 flow diagram

Conflicts of Interest and Source of Funding: none

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ABSTRACT Background and Aims: Magnesium plays an important role in the modulation of vascular tone and endothelial function and can regulate glucose and lipid metabolism. Patients with hypertension,

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metabolic syndrome (MetS) and diabetes mellitus (T2DM) have low body magnesium content; indeed, magnesium supplementation has been shown to have a positive effect on blood pressure (BP) and gluco-metabolic parameters. The aim of our study was to evaluate the effect of

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magnesium supplements on hemodynamic and metabolic parameters in healthy men with a positive family history of MetS or T2DM.

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Methods and Results: In a randomized, double-blind, placebo-controlled 8-week crossover trial with a 4 week wash-out period, oral supplements of 8.1 mmol of magnesium-pidolate or placebo were administered twice a day to 14 healthy normomagnesemic participants, aged 23 to 33 years. The primary endpoint was office BP, measured with a semiautomatic oscillometric device.

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Secondary endpoints included characteristics of the MetS, namely endothelial function, arterial stiffness and inflammation. Plasma and urinary magnesium were measured in all participants while free intracellular magnesium was measured only in a subsample.

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There was no significant difference in either systolic and diastolic BP in participants postmagnesium supplementation and post-placebo treatment when compared to baseline BP

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measurements. Further, the metabolic, inflammatory and hemodynamic parameters did not vary significantly during the study.

Conclusions: Our study showed no beneficial effect of magnesium supplements on BP, vascular function and glycolipid profile in young men with a family history of MetS/T2DM (trial registration at clinicaltrial.gov ID: NCT01181830; 12th of Aug 2010). Key words Magnesium, metabolic syndrome, diabetes, positive family history, blood pressure, flow mediated dilatation, arterial stiffness.

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ACCEPTED MANUSCRIPT INTRODUCTION

Metabolic syndrome (MetS), the clustering of central obesity with other features of insulin resistance, such as elevated glucose level or overt diabetes, dyslipidemia, and elevated BP, has been regarded as a worldwide pandemic.[1] Nevertheless, the precise pathogenesis of the syndrome is

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controversial, and the search for a unifying factor has been essentially fruitless. It has been proposed that several micro- and macro-nutrients play a role in the development of the metabolic and haemodynamic alterations in MetS.

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Many studies have focused on magnesium, which is involved in glucose and lipid metabolism, modulates vascular resistance and plays a fundamental role in many vital cellular pathways.[2-5]

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Magnesium is the second most abundant intracellular cation and the fourth most abundant cation in the body.[6] Most of the total storage of magnesium is in tissues (99%) with only 1% located extracellularly. Thus, the assessment of serum magnesium cannot adequately detect the presence of ion depletion, and a deficiency of ion stores can exist even in the presence of normal serum

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values.[6] Magnesium is mainly consumed through the diet, and it has been estimated that daily magnesium intake in a normal Western diet (approximately 300-360 mg/day) is often inadequate for the body's needs.[6]

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Intracellular magnesium is involved in essential functions for cell survival. It acts as a cofactor in more than 300 chemical reactions, including glycolysis, the process of phosphorylation, the

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response to growth factors, cell proliferation and mitochondrial energy production and promotes cell glucose uptake to optimize the action of insulin.[6] Magnesium acts also as a calcium antagonist by competing with calcium ions for many cation binding sites in membranes and proteins. Furthermore, magnesium is involved in the regulation of myocardial cell contractility, vascular tone and the basic response to pressor agents towards vasodilatation.[2-4;6] Numerous clinical studies have linked several components of the MetS with a low magnesium dietary intake.[7;8] This association has become increasingly important in Western countries where the burden of MetS and its long-term consequences is rising and constitutes a public health

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emergency.[1] It has been proposed that serum magnesium deficiency is an independent risk factor for all of the individual components of MetS.[7;9] Hypomagnesemia has been documented in patients diagnosed with impaired glucose tolerance, type 2 diabetes mellitus T2DM and MetS. [10;11]

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Some clinical trials in targeted populations, such as diabetics and obese or hypertensive participants, have reported a positive effect of magnesium supplementation on BP and glycometabolic parameters.[12-14] It has also been postulated that the effect of magnesium along

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with other nutrients and vitamins could be enhanced if the intake precedes the clinical onset of the disease.[15]

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Finally, because the offspring of patients with diabetes or MetS are at an increased risk of developing these metabolic disorders due to genetic and environmental factors, they represent a target population in whom preventive intervention could be addressed. Thus, we conducted a cross-over double blind placebo-controlled trial in young, healthy

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normomagnesemic men with a positive family history of MetS and/or T2DM to assess the effect of

hemodynamics.

METHODS

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magnesium supplementation for 8 weeks on several components of MetS and vascular

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Sixteen male patients, aged between 23 and 33 years, were recruited between offspring of patients who were followed in the “Hypertension outpatient clinic” in Verona University Hospital. Inclusion criteria were: good health and a positive family history of MetS and/or diabetes mellitus type 2 in at least one first-degree relative (AHA/NHLBI criteria).[1] The exclusion criteria were: a clinical diagnosis of hypertension (BP140>90 mmHg on several occasions and/or the use of antihypertensive treatment), diabetes mellitus (ADA criteria) or obesity (BMI >30 Kg/m2), use of lipid-lowering drugs or continuous therapy with nonsteroidal inflammatory drugs, use of vitamins or micro-nutrients supplements; hypermagnesemia at randomization; previous cardiovascular events

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and/or cerebrovascular disease; chronic renal insufficiency, chronic inflammatory liver disease, kidney disease, malignancies, gastrointestinal dysfunction with hypo-mobility; smoking more than 5 cigarettes per day. Eligibility criteria were verified by the physicians involved in the study. STUDY DESIGN. The study was conducted according to a double-blind placebo controlled cross-

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over design lasting 8 weeks (see flow diagram). Between the first and the second phase of the study, at least 4 weeks of wash-out was instituted. The primary endpoint of the study was the effect of magnesium supplementation on “office” blood pressure. Secondary endpoints were significant

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changes in the other parameters associated with MetS according to the AHA/NHLBI criteria including the HOMA index, endothelial function, arterial stiffness and high sensitivity C reactive

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protein (HS-CRP). The study was approved by the Ethical Committee of the University Hospital of Verona, and written informed consent was obtained from each participant. The trial was registered in the ClinicalTrials.gov database with the identification code NCTO1181830 (12th of Aug 2010). RANDOMIZATION AND MAGNESIUM ALLOCATION. The randomization sequence was

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generated by a computer-generated random-number list without blocks. A pharmacist who was not otherwise involved in the study kept the randomization list until the trial was completed. The randomization numbers and the assigned treatment were contained in serially numbered envelopes

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and kept by staff who were not involved in the outcome assessment. Plastic bags containing 8.1 mmol of Magnesium pidolate powder or placebo (lactose) were

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provided by the Hospital Pharmacy and taken by the participants twice a day, in the morning and evening before meals (16.2 mmol of Magnesium pidolate per day equivalent to 368 mg of elementary Mg) for 8 weeks. Both drugs were prepared by the pharmacy in indistinguishable packs so that it was impossible for either the patients and the physician to know which medication was being assigned to each participant. Patient compliance was assessed both by interviews and by counting the number of empty bags. ASSESSMENTS. Each participant was evaluated 4 times as shown in the flow diagram. Before the first visit, a questionnaire was administered dealing with medical history, family history,

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physiological and pathological information, use of drugs and smoking. Then, the participants underwent a physical examination. They were advised not to engage in strenuous exercise and to avoid consuming caffeine containing beverages within 12 hours preceding the vascular studies. During each visit, blood pressure was measured with a semiautomatic oscillometric device (TM-

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2501, A&D instruments Ltd,, Abingdon Oxford, UK) 5 times and 5 minutes apart with the patient lying supine for at least 10 minutes before the first measurement in a room with controlled temperature (22-24°C). Body weight, height, and waist circumference were measured with the

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patient wearing light clothes.

Carotid intima-media thickness (IMT) measurements were performed with echo-Doppler (LogiQ P5

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pro, GE Healthcare, Indianapolis, USA) analysis using high-resolution probe 5-13 MHz with an axial resolution of 0.01 mm. IMT was measured at the level of the distal common carotid artery.[16]

Endothelial function was assessed by ultrasound of the brachial artery using the Flow Mediated

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Dilatation (FMD) technique according to international guidelines and with the aid of a dedicated hardware (Multimedia Video Engine II (MVE2) DSP Lab., Pisa CNR, Italy).[16;17] Common carotid artery distensibility (DC) was calculated as: DC = ∆A /( A* ∆P) where A is the diastolic

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lumen area, ∆A is the stroke change in lumen area, and ∆P is pulse pressure (PP). Changes in diameters were detected using ultrasound B-mode image sequences of the right and left common

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carotid arteries acquired at different steps and analyzed by the above mentioned automatic system.[18]

Stiffness Index (SI) and Reflection Index (RI) were estimated by the Digital Volume Pulse (DVP) method and were obtained with the digital photoplethysmography PulseTracePT1000 (MicroMedical Ltd, Gillingham, Kent, UK).[19] Laboratory tests including measurement of plasma magnesium, calcium, glucose, glycated hemoglobin, insulin, total cholesterol, HDL and LDL cholesterol, triglycerides, HS-CRP, creatinine, urinary magnesium in a 12-h urine collection were measured using standard methods.

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Intracellular free magnesium concentration in circulating lymphocytes was measured by a spectrofluorimeter using the ratio technique.[20] Insulin resistance was estimated using the HOMA index and was calculated by Matthews’ equation (fasting insulin (µU/ml) × fasting glucose (mmol/l)/22.5).

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STATISTICS.

Data are presented as the mean ± standard deviation. The statistical analysis was performed using the software Statistical Package for Social Sciences software (SPSS / PC for Windows version

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21.0). Differences in the measured parameters were analyzed by both parametric (paired T-student test) and nonparametric tests (Wilcoxon test for paired data), but only parametric results are

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reported. We always used a two-tailed test, and p0.05 for DBP) as well as a test for carry-over effect (t=-1.64 with 12 d.f. p>0.05 for SBP; t=-2.165 with 12 d.f. p>0.05 for DBP). After adjustment for the period effect, we did not observe substantial modifications with respect to the above results that showed no effect of magnesium

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supplementation (data not shown).

EFFECT OF INTERVENTION ON COMPONENTS OF THE METABOLIC SYNDROME AND

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OTHER GLYCOMETABOLIC VARIABLES (SECONDARY ENDPOINTS) No significant differences were found in triglycerides, glucose, total cholesterol, HDL cholesterol, and LDL cholesterol (Table 1). HOMA Index, glycated hemoglobin and the HS-CRP did not vary significantly after administration of magnesium (Table 1). EFFECT OF INTERVENTION ON VASCULAR EXAMS FMD did not vary significantly after the administration of magnesium when compared to baseline and to post-placebo treatment, nor was there any difference in the DC of the carotid arteries assessed by ultrasounds, SI and RI by digital photoplethysmography (Table 1).

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DISCUSSION Patients affected by T2DM and or MetS have reduced levels of both plasma and intracellular

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magnesium.[10;11;20] Indeed some, but not all clinical trials, have demonstrated a positive effect of supplementation with magnesium, especially in hypertensive patients.[21;22] In fact, most of the evidence linking micro-nutrients and vitamins including magnesium to cardiovascular outcomes

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come from observational studies [8-11], whereas the results of clinical trials are often discordant. We hypothesized that normomagnesemic participants with a genetic background and an increased

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their susceptibility to metabolic diseases such as T2DM/MetS could be at risk of early haemodynamic and metabolic impairment and that supplementation with magnesium could improve their haemodynamics and the other features of the MetS. Different types of magnesium supplements are highly advertised in the media and their consumption is increasing, even in healthy populations.

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Nevertheless, preventive medicine guidelines do not recommend their use for primary or secondary prevention of cardiovascular disorders.[23]

In support of these guidelines, we found that the administration of magnesium pidolate for 8 weeks

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did not change any of the investigated parameters in healthy men who were offspring of patients with MetS and/or T2DM.

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Before concluding that magnesium is not beneficial, we should consider other reasons that could have led to treatment failure, perhaps due to magnesium itself or to the target population. For one, it can be argued that the dosage of magnesium was not sufficient, the 8 week duration of the trial was too short or that the type of magnesium supplements were not optimal, specifically in terms of bioavailability.[24] The dosage and timing of administration were chosen based on previous studies and two systematic reviews with meta-analyses.[21] In particular, the Cochrane meta-analysis reported that treating hypertensive patients with magnesium for periods ranging from 8 to 26 weeks

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is associated with a significant although modest reduction in DBP.[21] Still, we cannot exclude the possibility that a more prolonged intervention could have been beneficial. More recently, a study in which mild hypertensive patients were treated with our formulation (magnesium pidolate) for 12 weeks significantly decreased BP as measured by ambulatory blood

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pressure monitoring (ABPM).[25] Contrary to our study, serum and urinary magnesium levels increased significantly in that sample. [25] Previous studies have shown that oral magnesium bioavailability varies between different formulations.[24]

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It is also possible that magnesium alone is not sufficient to modify the hemodynamic and metabolic parameters and that a deficiency of other factors, such as fibers and micro-nutrients, such as zinc

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and antioxidants vitamins, are of equal or even greater important.[26]

Regarding the target population, treatment failure could be because our sample population, which was composed of healthy men, was at low cardiovascular risk. In addition, subjects may not have been susceptible to the effects of magnesium supplements because they are already had an adequate

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body storage of magnesium. The study by Guerrero-Romero in patients with T2DM, hypertension and reduced plasma concentrations of magnesium showed that the administration of 2.5 g/day of MgCl2 for 4 months led to a decrease of the values of both SBP and DBP.[3] However, our results

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are consistent with other studies in healthy participants where the administration of magnesium did not significantly alter BP and insulin resistance.[13] The range of values of intra-lymphocyte

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magnesium in our subsample of participants is higher than those measured in previous work carried out in our laboratory in hypertensive people, supporting the hypothesis that our participants had adequate magnesium body storage.[20] No beneficial effects were observed in blood pressure or vascular parameters, particularly endothelial function and arterial stiffness, after magnesium treatment. Previous studies in populations with overt cardiovascular risk factors have shown a positive vascular effect of magnesium supplements. For example, Shechter et al. found that a six month administration of magnesium in patients with coronary artery disease led to a significant improvement of FMD

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validated for the noninvasive measurement of endothelial function. It is worth emphasizing that the coefficient of intra-subject variation for this measure can be as high as 15-20% in our laboratory. To minimize the problem of reproducibility, all vascular measurements were performed by the same

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operator, and attention was paid to finding the same point of measurement in the same participant during the different phases of the study. We also measured markers of localized (carotid artery DC) and systemic arterial stiffness (SI, a completely operator-independent measurement that can be

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assimilated to “Pulse wave velocity” [PWV] estimated with other devices), but no differences were detectable. In previous studies, lower serum magnesium was independently associated with PWV in

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renal transplant recipients[28] and Augmentation Index in hypertensives [5], whereas intracellular magnesium was significantly and positively related to aortic distensibility measured by magnetic resonance imaging (MRI) techniques in hypertensive patients.[29] Finally, after a MgSO4 bolus in preeclamptic patients, central arterial compliance was increased suggesting a positive effect of

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magnesium on arterial stiffness, at least in hypertensive patients.[30]

We did not find significant differences in any of the other parameters of MetS. Previous studies have shown a positive effect of magnesium on the HOMA-IR index and glycated hemoglobin in

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diabetic and non-diabetic patients.[12;14] Beyond the different dose and type of magnesium, our study differs from these other studies in that they were conducted in diabetic or insulin resistant

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participants.

In conclusion, our study showed that a dose of magnesium pidolate of 16.2 mmol/day for 8 weeks in healthy normomagnesemic men with a family history of MetS did not improve either hemodynamic and glycometabolic parameters. Possible limitations regarding magnesium formulation, duration of therapy, and the chosen target population (at a low cardiovascular risk), along with the limited sample size prompt us to be cautious before drawing a firm conclusion on this topic. Furthermore, lack of compliance with the assigned treatment suggested by the absence of a rise in plasma, intracellular and urine magnesium, could have contributed to the problem.

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Further studies are required to test if different target populations (i.e., people with MetS and/or T2DM) or different types of magnesium could be useful as supplements to prevent cardiovascular disease.

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2. Barbagallo M, Dominguez LJ. Magnesium metabolism in type 2 diabetes mellitus, metabolic syndrome and insulin resistance. Arch Biochem Biophys 2007; 458(1):40-47.

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3. Guerrero-Romero F, Rodrguez-Morn M. The effect of lowering blood pressure by magnesium supplementation in diabetic hypertensive adults with low serum magnesium levels: a randomized, double-blind, placebo-controlled clinical trial. J Hum Hypertens 2009; 23(4). 4. Sontia B, Touyz RM. Magnesium transport in hypertension. Pathophysiology 2007; 14(34):205-211.

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5. Afsar B, Elsurer R. The relationship between magnesium and ambulatory blood pressure, augmentation index, pulse wave velocity, total peripheral resistance, and cardiac output in essential hypertensive patients. J Am Soc Hypertens 2014; 8(1):28-35. 6. Swaminathan R. Magnesium metabolism and its disorders. Clin Biochem Rev 2003; 24(2):4766.

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7. Larsson SC, Wolk A. Magnesium intake and risk of type 2 diabetes: a meta-analysis. J Intern Med 2007; 262(2):208-214.

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8. Song Y, Ridker PM, Manson JE, Cook NR, Buring JE, Liu S. Magnesium intake, C-reactive protein, and the prevalence of metabolic syndrome in middle-aged and older U.S. women. Diabetes Care 2005; 28(6):1438-1444. 9. Bayturan O, Tuzcu EM, Lavoie A, Hu T, Wolski K, Schoenhagen P et al. The metabolic syndrome, its component risk factors, and progression of coronary atherosclerosis. Arch Intern Med 2010; 170(5):478-484. 10. Pham PC, Pham PM, Pham SV, Miller JM, Pham PT. Hypomagnesemia in patients with type 2 diabetes. Clin J Am Soc Nephrol 2007; 2(2):366-373. 11. Lima Mde L, Cruz T, Rodrigues LE, Bomfim O, Melo J, Correia R et al. Serum and intracellular magnesium deficiency in patients with metabolic syndrome--evidences for its relation to insulin resistance. Diabetes Res Clin Pract 2009; 83(2). 12. Rodriguez-Moran M, Guerrero-Romero F. Oral magnesium supplementation improves insulin sensitivity and metabolic control in type 2 diabetic subjects: a randomized double-blind controlled trial. Diabetes Care 2003; 26(4):1147-1152.

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13. Lee S, Park HK, Son SP, Lee CW, Kim IJ, Kim HJ. Effects of oral magnesium supplementation on insulin sensitivity and blood pressure in normo-magnesemic nondiabetic overweight Korean adults. Nutr Metab Cardiovasc Dis 2009.

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14. Guerrero-Romero F, Tamez-Perez HE, Gonzalez-Gonzalez G, Salinas-Martinez AM, MontesVillarreal J, Trevino-Ortiz JH et al. Oral magnesium supplementation improves insulin sensitivity in non-diabetic subjects with insulin resistance. A double-blind placebo-controlled randomized trial. Diabetes Metab 2004; 30(3):253-258. 15. Minuz P, Fava C, Cominacini L. Oxidative stress, antioxidants, and vascular damage. Br J Clin Pharmacol 2006; 61(6):774-777.

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16. Bianchini E, Bozec E, Gemignani V, Faita F, Giannarelli C, Ghiadoni L et al. Assessment of carotid stiffness and intima-media thickness from ultrasound data: comparison between two methods. J Ultrasound Med 2010; 29(8):1169-1175.

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17. Gemignani V, Bianchini E, Faita F, Giannarelli C, Plantinga Y, Ghiadoni L et al. Ultrasound measurement of the brachial artery flow-mediated dilation without ECG gating. Ultrasound Med Biol 2008; 34(3):385-391. 18. Bianchini E, Bruno RM, Corciu AI, Faita F, Gemignani V, Ghiadoni L et al. Assessment of carotid elasticity during exercise: a reproducibility study. Ultrasound Med Biol 2012; 38(2):223-230.

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19. Millasseau SC, Kelly RP, Ritter JM, Chowienczyk PJ. The vascular impact of aging and vasoactive drugs: comparison of two digital volume pulse measurements. Am J Hypertens 2003; 16(6). 20. Delva P, Pastori C, Montesi G, Degan M, Micciolo R, Paluani F et al. Intralymphocyte free magnesium and calcium and insulin tolerance test in a group of essential hypertensive patients. Life Sci 1998; 63(16):1405-1415.

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21. Dickinson HO, Nicolson DJ, Campbell F, Cook JV, Beyer FR, Ford GA et al. Magnesium supplementation for the management of essential hypertension in adults. Cochrane Database Syst Rev 2006; 3.

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22. He K, Liu K, Daviglus ML, Morris SJ, Loria CM, Van Horn L et al. Magnesium intake and incidence of metabolic syndrome among young adults. Circulation 2006; 113(13). 23. Perk J, De BG, Gohlke H, Graham I, Reiner Z, Verschuren M et al. European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts). Eur Heart J 2012; 33(13):1635-1701. 24. Walker AF, Marakis G, Christie S, Byng M. Mg citrate found more bioavailable than other Mg preparations in a randomised, double-blind study. Magnes Res 2003; 16(3):183-191. 25. Hatzistavri LS, Sarafidis PA, Georgianos PI, Tziolas IM, Aroditis CP, Zebekakis PE et al. Oral magnesium supplementation reduces ambulatory blood pressure in patients with mild hypertension. Am J Hypertens 2009; 22(10):1070-1075.

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26. Czernichow S, Vergnaud AC, Galan P, Arnaud J, Favier A, Faure H et al. Effects of long-term antioxidant supplementation and association of serum antioxidant concentrations with risk of metabolic syndrome in adults. Am J Clin Nutr 2009; 90(2):329-335. 27. Shechter M, Sharir M, Labrador MJ, Forrester J, Silver B, Bairey Merz CN. Oral magnesium therapy improves endothelial function in patients with coronary artery disease. Circulation 2000; 102(19):2353-2358.

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28. Van LS, Marechal C, Verbeke F, Peeters P, Van BW, Devuyst O et al. The relation between hypomagnesaemia and vascular stiffness in renal transplant recipients. Nephrol Dial Transplant 2011; 26(7):2362-2369.

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29. Resnick LM, Militianu D, Cunnings AJ, Pipe JG, Evelhoch JL, Soulen RL. Direct magnetic resonance determination of aortic distensibility in essential hypertension: relation to age, abdominal visceral fat, and in situ intracellular free magnesium. Hypertension 1997; 30(3 Pt 2):654-659.

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Figure legends

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30. Rogers DT, Colon M, Gambala C, Wilkins I, Hibbard JU. Effects of magnesium on central arterial compliance in preeclampsia. Am J Obstet Gynecol 2010; 202(5):448.

Flow diagram: Flow diagram of the progress through the phases of the cross-over randomized trial.

Figure 1: Effect of magnesium on systolic blood pressure compared to baseline (A), post-placebo treatment (B) and delta placebo (C).

Figure 2: Effect of magnesium on diastolic blood pressure compared to baseline (A), post-placebo treatment (B) and delta placebo (C).

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Table 1. Effects of 8 weeks of magnesium supplementation versus placebo on cardiovascular, inflammatory, and metabolic outcomes in healthy, young, normo-magnesemic men. Post Mag.

∆-Mag.

P value (Pre vs. post Mag.)

Pre plac.

SBP (mmHg) DBP (mmHg) Total cholesterol (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) Triglycerides (mmol/L) Glucose (mmol/L) Insulin (µU/mL) HOMA-IR Index HbA1c (%) HS-CRP (mg/L) P-Ca2+ (mmol/L) P-Mg2+ (mmol/L) U-Mg2+ (mmol/mmol creatinine) U-Mg2+ (mmol/12h) U-Na+ (mmol/12h)

126.0±13.1 71.4±5.9 4.49±0.77 1.32±0.27 2.75±0.74 0.89±0.51 5.04±0.37 6.0±3.2 1.4±0.8 5.5±0.16 0.46±0.46 2.30±0.09 0.81±0.05 0.32±0.13 2.55±1.32 68.2±32.2

123.7±13.2 71.4±4.5 4.68±0.78 1.43±0.27 2.81±0.84 0.93±0.52 5.19±0.37 6.2±5.1 1.5±1.2 5.5±0.21 0.48±0.43 2.33±0.08 0.81±0.04 0.40±0.13 2.94±1.16 61.6±32.5

-2.3±14.4 0.1±5.3 0.19±0.35 0.11±0.21 0.06±0.34 0.05±0.56 0.15±0.39 0.28±5.04 0.09±1.1 -0.14±0.16 0.03±0.61 0.03±0.09 -0.01±0.03 0.08±0.18 0.39±1.49 -6.71±42.9

0.55 0.96 0.06 0.07 0.51 0.77 0.17 0.85 0.78 0.74 0.88 0.19 0.50 0.11 0.35 0.56

122.7±6.5 72.1±5.0 4.70±0.96 1.33±0.31 2.97±0.89 0.83±0.45 5.13±0.37 4.1±3.0 1.0±0.8 5.5±0.2 1.89±3.98 2.34±0.05 0.83±0.06 0.33±0.12 2.68±0.91 76.8±50.6

FMD (%)

4.9±2.7

5.0±1.9

0.1±2.5

30.6±7.6

32.4±9.4

Left carotid DC (KPa 10 )

30.2±6.8

SI (m/sec) RI (%)

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Right carotid DC (KPa 10 ) -1

-3

117.2±7.5 69.7±4.1 4.63±0.88 1.35±0.19 2.82±0.80 0.97±0.48 5.01±0.33 4.7±4.1 1.1±1.0 5.5±0.2 0.36±0.38 2.34±0.08 0.82±0.05 0.32±0.08 2.66±1.07 66.7±31.6

M AN U

TE D

EP

∆- plac.

P value (Post Mag. vs. post Plac.) 0.06 0.15 0.75 0.27 0.96 0.81 0.04 0.23 0.19 0.54 0.48 0.97 0.27 0.11 0.49 0.64

P value ∆-Mag vs.∆-plac

-5.6±8.3 -2.4±8.7 -0.070±0.50 0.02±0.25 -0.15±0.37 0.14±0.36 -0.11±0.24 0.59±2.0 0.10±0.45 0.01±0.13 -1.5±3.7 -0.00±0.07 -0.01±0.04 -0.01±0.16 -0.02±1.40 -10.1±38.8

P value (Pre vs. post Plac.) 0.06 0.20 0.62 0.77 0.14 0.17 0.09 0.29 0.41 0.69 0.003 0.97 0.51 0.76 0.95 0.35

0.40 0.31 0.14 0.38 0.09 0.64 0.053 0.89 0.96 0.60 0.18 0.32 0.93 0.17 0.51 0.83

0.88

3.1±2.31

7.0±3.3

3.9±4.8

0.009

0.07

0.04

1.8±9.6

0.49

31.0±7.6

31.8±6.0

0.8±6.9

0.49

0.81

0.72

30.5±9.1

0.3±10.2

0.91

30.0±7.4

32.9±5.8

2.9±8.5

0.24

0.45

0.86

6.1±0.59

6.4±0.89

0.3±0.7

0.15

6.3±0.52

6.4±0.65

0.8±13.4

0.62

0.85

0.54

65.4±11.9

68.9±14.1

3.5±10.9

0.25

63.5±11.7

64.4±13.6

0.1±0.6

0.83

0.29

0.60

AC C

-1

Post Plac.

RI PT

Pre Mag.

SC

Variable

SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; P-, plasma; U-, Urine; IL, Intralymphocyte; HS-CRP, high sensitive C-reactive protein, ∆-Mag., delta magnesium, ∆-plac., delta placebo; FMD, Flow Mediated Dilatation; DC, distensibility; SI, Stiffness Index; RI, Reflection Index, ∆Mag., delta magnesium, ∆-plac., delta placebo.

ACCEPTED MANUSCRIPT

Enrollment

Assessed for eligibility (n=16)

RI PT

Excluded (n = 0)

SC

1 declined to continue for personal reason

Four weeks of wash out period (n=15)

Crossed over to placebo for 8 weeks (n=7) Received allocated intervention (n=6)

AC C

EP

Crossed over to placebo for 8 weeks (n=8) Received allocated intervention (n=8)

Analysis

Allocated to magnesium pidolate for 8 weeks (n=8) Received allocated intervention (n=8)

M AN U

Allocated to placebo for 8 weeks (n=8) Received allocated intervention (n=8)

TE D

Follow-up

Allocation

Randomized (n = 16)

1 declined to continue for personal reason Analyzed (n=14)