Journal of the American College of Nutrition, Vol. 23, No. 6, 732S-737S (2004)
Published by the American College of Nutrition
Development of Cellular Magnesium Nano-Analysis in Treatment of Clinical Magnesium Deficiency
Burton B. Silver, PhD
University of Louisville, School of Medicine, Department of Physiology & Biophysics, Louisville, Kentucky (Ret), and IntraCellular Diagnostics, Inc., Foster City, California
Address reprint requests to: Burton B. Silver, Ph.D., IntraCellular Diagnostics, Inc. 553 Pilgrim Dr (B), Foster City, CA. E-mail: DocBurt{at}aol.com
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ABSTRACT
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A novel technique, using energy dispersive X-ray microanalysis (EXA tm), for noninvasive intracellular (i.c.) measurement of magnesium [Mg 2+] i has now been accomplished and proven to be a valuable tool in multiple aspects of normal as well as pathological magnesium metabolism. Since only 1% of total body Mg 2+ is found in the intravascular space, serum levels of Mg 2+ give little information about a patient’s overall Mg 2+ status with respect to this essential mineral. Using the EXA tm analysis it has shown been determined that Mg 2+ levels are significantly reduced in many physiological states which may lead to serious pathological conditions [15]. Description of the methodology and examples of data as well as potential applications will focus on intracellular (i.c.) [Mg 2+] i determinations obtained in cells from subjects with cardiovascular disease (CVD) syndromes related to Mg 2+ deficiency. Examples of the application of EXA tm evaluation include examination of intracellular magnesium and other minerals in a wide spectrum of conditions which include cardiovascular conditions, arrhythmias, heart failure, myocardial infarction, and bypass surgery. Standardization of control values were performed at NASA [12].
Key words: x-ray microanalysis, nano-analysis, cellular magnesium, non-invasive measurement
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INTRODUCTION
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Magnesium plays a protean role in intracellular (i.c.) metabolism, and is second only to potassium (K) as the most common i.c. cation in human physiology [1]. It participates in normal functions as well as in pathological syndromes that are associated with subnormal cellular levels and availability of the Mg stores within tissues [2]. Measuring cellular Mg 2+, accurately and easily within tissues is important in understanding and treating many human diseases. Cytosolic free Mg 2+, acts as a cofactor for ATPases and regulates calcium (Ca) and K transport into tissues. Because of its many effects, Mg 2+, has been called the "chronic regulator" of cell metabolism. Murphy et al. point out, though, that Mg 2+ i can only modulate intracellular functions if [Mg 2+] i changes in response to physiological stress [3]. Cellular Mg 2+, activates many enzyme systems including those vital reactions which govern cardiac function. Regulated by Mg 2+, levels, Na K-ATPase maintains the normal i.c. concentrations of Na + and K + vital to normal cardiac function. Given the net effect of a reduction i.c. Mg 2+, on the action potential and the multiple channels and enzymes influenced by a membrane depolarization would be expected due to cellular K + loss, Na + gain and Ca 2+ gain [4].
Methodology
The measurement of i.c. Mg [Mg 2+] i was performed by x-ray dispersive microanalysis utilizing a specially configured electron microscope which images and irradiates cells with a focused electron beam. Sublingual epithelial cells were chosen as markers since they are easily accessible, are non-cornified, are aerobic, have a high cytoplasm to nucleus ratio, turnover in less than 3 days, have long shelf life, exhibit 99% viability, and show significant correlations with cardiac and muscle biopsies taken during bypass surgery. Excitation of cellular atoms displaces inner orbital electrons which are replaced by electrons from higher energy cells releasing fluoresced x-rays which allows quantitation of i.c. elements. EXA tm units equals X-ray intensity (peak divided by background) divided by unit cell volume. Cardiac and muscle tissue biopsies were quick frozen in liquid nitrogen cooled with Freon and freeze dried tissue sections were then carbon coated for x-ray analysis [5].
EXA tm units are converted to mEq/L by a conversion constant for each element derived from reference standard of a highly stable matrix synthetic glass containing known amounts of the elements to be analyzed. The standard was certified by the National Bureau of Standards and Corning Diagnostics. The coefficient of variance of 40 determinations of X-ray emission energies for with the reference standard was 0.82%. Applications of i.c. Mg 2+ analysis have been reported in investigations incorporating this unique method [11,13,14] (Fig. A).
Magnesium Modulation of Calcium Accumulation in Cardiac Mitochondria
Respiration-supported Ca ++ uptake in isolated heart mitochondria is decreased by the presence of Mg ++. Electron microscopic evidence also indicates that Mg ++ modulates the type of Ca ++ crystals formed in mitochondria with and without Mg ++ present during Ca ++ loading. In the presence of Mg ++, dynamic changes of intramitochondrial crystalization tends to produce spherical, amorphous crystals. Mitochondria lacking Mg ++ showed needle-like and apparently destructive dendritic Ca ++ accumulation during the same time period. Differential responses of mitochondrial respiration, as indicated by cytochrome b redox states indicate that the presence of Mg ++ "protects" the ability of heart mitochondria to phosphorylate ADP after Ca ++ uptake. Modulation of Ca ++ uptake by Mg ++ may, therefore, play an important role in protecting intact mitochondrion structural-functional relationships during ischemic episodes. These data thus indicate a role of Mg ++ as a modulator of both uptake of Ca ++ and the form it takes in cardiac mitochondria in response to ischemia and during normal metabolism, through its stimulation. Magnesium also produced marked stimulation of mitochondrial respiratory activity. Mg ++ may compete for mitochondrial Ca ++ binding and/or transport sites. This may alter the rate of Ca ++ entry into the mitochondria and subsequent effects of Ca ++ on stimulating respiratory activity thus playing a physiological role in heart muscle, considering the importance of calcium and ATP for normal cardiac function [7,18] (Fig. 1).
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Fig. 1. Destructive calcium crystals after infarct; no magnesium given (left panel). No destructive crystals after infarct; magnesium given prior to infarct (right panel).
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IA. Correlation of Mg levels between Sublingual cells and Atrial Tissues Taken at Surgical Bypass
Results.
We have found that energy dispersive x-ray analysis (EXA tm) of sublingual epithelial cells correlates well with atrial specimens obtained at cardiopulmonary bypass. Seventeen subjects had sublingual smears taken prior to bypass surgery, (average age 65.7 years), at which time atrial biopsies were taken at the time of surgery and frozen for later elemental x-ray analysis. Serum vs. Intracellular [Mg 2+] i in Surgical Patients: The mean serum Mg ++ levels for the cardiac surgery patients were within the normal range, (1.87 ± 0.06 mEq/L). Despite normal serum levels, intracellular [Mg 2] levels from the sublingual epithelial cells from the bypass patients were reduced when compared to healthy subjects. Linear regression comparing the individual values is shown in Fig. 2. A strong correlation exits between Mg ++ contents of sublingual and cardiac cells. (R = 0.68, p < 0.002) [5,10] (Fig. 2).
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Fig. 2. Correlation of atrial and sublingual [Mg]i. Atrial biopsy tissues frozen at surgery. Sublingual tissues taken within 8 hours of surgery or at surgery.
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IB. Effect of IV Mg ++ in Patients with ST elevation and Acute Myocardial Infarction
Using the EXA tm sublingual cell evaluation, magnesium intravenous intervention studies were done on 22 myocardial infarct patients who were compared to healthy controls and noncardiac patients. Mean Mg ++ in infarct patients was 30.7 ± 0.4 compared to 15 control subjects whose cellular Mg ++ levels were 35.0 ± 0.5, p < .0001. Infarct patients received a mean dose of 36 ± 6 mmol/24 hrs of intravenous Mg 2SO 4. No Mg ++ was given after the second 24 hours of the study. Intracellular [Mg 2+] i rose significantly in the infarct patients over the first 24 hours and the magnitude of the increase was greater in those who received higher doses of intravenous magnesium sulfate. Despite the fact that Mg 2SO 4 was not given after the first 24 hours, mean sublingual magnesium continued to rise for 48 hours after the first dose of magnesium sulfate (from 30.7 ± 0.4 to 35.2 ± 0.6 mEq/L) suggesting that magnesium may be moving from the vascular space to the tissues over 48 hours [16] (Fig. 3).
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Fig. 3. Increase of intracellular Mg in MI patients following Mg infusion from admission through 48 hours post-treatment.
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II. Abnormal QT Dispersion Correlates with Tissue Magnesium
Abnormal QT dispersion (QTd) on the 12 lead electrocardiogram (ECG), and Mg ++ deficiency have both each been shown to predict a higher risk for sudden cardiac death, in patients with CHF, alcoholic liver disease, or arrhythmias requiring antiarrhythmic drug therapy. We tested the hypothesis that Mg ++ deficiency predisposes to abnormal repolarization in the (QTd). In 37 subjects with malignant arrhythmias, serum Mg ++ and i.c. Mg ++ in sublingual epithelial cells, were measured in a blinded fashion. Results were correlated with the ECG, (r = 0.56, p = 0.0001). Linear regression showed QTd correlated with depressed i.c. Mg measured using energy dispersive x-ray analysis, (EXA tm). Mean serum levels showed no such correlations. Repeat Mg ++ levels obtained in 12 treated patients 3–7 days later showed increase in tissue Mg ++ (1.5 ±/–0.6 mEq/l (p < 0.0001) and a decrease in QTd by 10 ± 12 msecs (p < 0.0001). Delta QTd [Mg ++] correlated at r = 0.61, p < 0.05. QT dispersion was shown to correlate with tissue Mg ++ levels suggesting Mg ++ deficiency may be a risk factor for sudden cardiac by its effect on cardiac repolarization as indexed by QTd [8,16] (Fig. 4).
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Fig. 4. QT dispersion and change in sublingual intracellular magnesium (EXAtest) in 12 patients having two measurements 3 to 7 days apart.
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III. Effect of Experimental Heart Failure on Sublingual Tissue Mg ++ and Cardiac Ionized Mg ++
To test the hypothesis that CHF results in a rapid loss of tissue Mg ++ in the absence of confounding drugs or diseases, sublingual smears were obtained from normal dogs (n = 4) which were exposed to 4 weeks of ventricular pacing at 250 bpm. All animals manifested bi-ventricular failure. Sublingual cell Mg ++ was measured by energy dispersive x-ray analysis (EXA tn). Cardiac myocytes were measured with the fluorescent probe Mg-Indo to measure [Mg 2+] i. Pacing the dogs resulted in significantly lower [Mg 2+] i in their sublingual cells demonstrated in paced dogs (34.8 mEq/L ± 0.8 vs. 38.7 mEq/l ± 0.5 2 p < 0.05 when they were unpaced). The fluorescence ratios indexing [Mg 2+] i were also significantly reduced in the paced animals compared to ratios before they were paced:. the normal dogs. (0.573 ± 0.007, n = 49) paced vs normal: (0.749 ± 0.029), (n = 18 cells, 2 p < 0.001). The reduction in total cellular Mg ++ detected in the sublingual cells was (8%) yet was associated with a 
40% reduction in [Mg 2+] i compared to controls. It seems that free Mg ++ is not well buffered but fluctuates with changes in changes in total Mg ++. A small reduction in total cellular Mg would cause a much large decrement in free Mg ++ which constitutes <5% of total stores. The data suggest that a reduction in tissue Mg ++ results in a relatively large fall in free cardiac Mg ++. Measures of sublingual Mg ++ (EXA tn) my be useful in future clinical investigation regarding the significance of Mg ++ in heart failure. More importantly, EXA tm testing will allow accurate serial measurement of Mg ++ to guide Mg ++ repletion therapy in a wide variety of syndromes in which Mg ++ deficiency is a factor [9] (Figs. 5, 6).
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Fig. 5. Comparison of [Mg2+] from cardiac myocytes and [Mg]i in sublingual cells from pacing-induced heart failure in dogs.
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Oral Magnesium Improves Endothelial Function in Coronary Artery Disease Patients
Mg ++ has biologic effects that parallel the pharmacologic effects of Ca ++ blocker drugs. To determine whether increased i.c. levels of Mg ++ [Mg ++] i enhance brachial artery (BA) vasoreactivity, prospectively flow-mediated endothelium-dependent (FMED) and endothelium-independent sublingual nitroglycerin (NTG) BA vaso-reactivity (BRT) were determined in 49 stable CAD patients (40 men, 9 women, mean age 68 ± 9 years) [6].
Method.
Energy-dispersive x-ray analysis (EXAtest) was used to measure [Mg ++] i in sublingual epithelial cells (normal value 34.5 ± 0.7 mEq/L.). BRT was measured using 10 MHz ultrasound at 1:30 minutes (min) following 3 min of blood pressure cuff arterial occlusion and after NTG administration.
Results.
Flow-mediated % diameter change at 1:30 min (%D 1:30) was significantly associated with [Mg ++] (r = 0.86, p < 0.001). Patients were then divided into two groups < and 
median [Mg ++] level of 32.3 mEq/L.
- • Baseline 72% of all patients were Mg ++ deficient by sublingual i.c. analysis.
- • Mg ++ increased significantly post-intervention versus placebo.
Conclusion.
FMED BRT is enhanced in stable CAD patients with higher [Mg ++] suggesting a potential role for [Mg ++] in the treatment of CAD patients [6] (Figs. 7, 8).
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Fig. 7. Correlation of percent change in baseline brachial artery flow-mediated vasodilation (%FMD) and baseline intracellular lever, ([Mg]i), in 50 subjects showing a linear correlation.
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Fig. 8. Percent change in endothelium-dependent brachial artery flow-mediated vasodilation (%FMD) in placebo (horizontal line) (n = 25) and magnesium (diagonal line) (n = 25) at baseline and after 6 months.
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Exercise Tolerance, Chest Pain and Quality of Life
Analytical Electron Microprobe studies of sublingual cells, i.c. Mg ++ (EXAtest) correlating with clinical data, has demonstrated that Mg ++ supplementation improves endothelial function in patients with coronary artery disease (CAD). However, the impact on clinical outcomes, such as exercise-induced chest pain, exercise tolerance, and quality of life, was not established a study in 2003 [17].
In that multicenter, multinational, prospective, randomized, double-blind, placebo controlled trial, 187 CAD patients (151 men, 36 women; mean age 63–10 years, range 42 to 83) were randomized to receive either oral Mg ++ 15 mmol twice daily (Magnosolv-Granulat, total Mg ++ 365 mg provided as Mg-citrate) (n 94) or placebo (n 93) for 6 months. Symptom-limited exercise testing (Bruce protocol) and responses given on quality-of-life questionnaires were the outcomes measured.
Mg ++ therapy significantly increased i.c. Mg ++ levels ([Mg]i) in a substudy of 106 patients at 6 months compared with placebo (EXAtest) i.c. readings indicated increased i.c. Mg ++ (35.5 mEq/l vs 32.6 mEq/L, p = 0.0151).
Mg ++ treatment significantly increased exercise duration time compared with placebo (8.7 +/– 2.1 vs 7.8 +/– 2.9 minutes, p = 0.0075), and lessened exercise-induced chest pain (8% vs 21%, p = 0.0237).
Quality-of-life parameters significantly improved in the Mg ++ group. These findings suggest that oral Mg ++ supplementation in patients with CAD for 6 months results in a significant improvement in i.c. Mg ++ concentration, exercise tolerance, exercise-induced chest pain, and quality of life, suggesting a potential mechanism whereby Mg ++ therapy and use of the i.c. EXAtest could beneficially alter outcomes in patients with CAD [17] (Figs. 9, 10).
Summary
Energy dispersive X-ray microanalysis, (EXA tm), for non-invasive. Measurement of i.c. Mg ++ has proven to be a valuable tool in many aspects of normal as well as pathological Mg ++ metabolism. Over the last 35 years data have accumulated using this newly available unique method which is now available to non-invasively, and accurately evaluate tissue Mg ++ levels.
While serum levels of Mg give little information about a patients overall Mg ++ status with respect to this essential mineral, utilizing i.c. analysis has shown that Mg ++ levels are significantly reduced in tested patho-physiological states. Description of the methodology and experimental data will focus on i.c. [Mg 2+] i determinations obtained from subjects repleted with Mg ++ or with syndromes related to Mg ++ deficiency. I.c. analysis studies, (EXA tm), of tissue magnesium levels include:
- Effects of oral Mg ++ on tissue levels in CAD patients.
- Effects of i.c. Mg ++ on exercise and endothelial function.
- I.c. Mg’s role in dysrhythmias, heart failure, and infarction patients.
- Correlations of i.c. Mg ++ with cardiac tissue levels.
- Depletion studies of i.c. Mg ++ in human subjects and large animals.
Mg’s role in intracellular metabolism is second only to that of K+ as the most common i.c. cation in human physiology. Numerous normal functions, as well as pathological syndromes, are affected by the cellular levels and availability of magnesium stores. Logically, measuring Mg ++ accurately and easily within tissues is of immense value to understanding and treating many diseases. Applications of i.c. Mg ++ analysis are reported from studies utilizing this procedure.
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FOOTNOTES
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Intracellular magnesium analysis performed by B.B. Silver of IntraCellular Diagnostics, Inc.
Received August 5, 2004.
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ABSTRACT
INTRODUCTION
