RICERCA ITALIANA     

Magnesium Homeostasis in pathologies with primary and secondary defects of ion metabolism

Università degli Studi di Bologna

Abstract

Mechanisms which regulate magnesium homeostasis in different tissues, are still not completely understood. Most studies report data about the total serum magnesium concentration, while is well known the intracellular free fraction to be that functionally important. This research project will contribute to the knowledge of the regulatory mechanism of magnesium homeostasis by assessing its distribution in several tissues in humans, by measuring the intracellular free concentration in skeletal muscle, brain, blood cells, and the total and free concentration in plasma. We will use analytical methods specific for each cell type investigated: phosphorus magnetic resonance spectroscopy (31P MRS) to measure in vivo intracellular free magnesium concentration in skeletal muscle and brain, fluorimetry and cytofluorimetry to measure intracellular free magnesium concentration in blood cells, atomic absorption and Mg-selective electrode to measure in plasma total and free concentration respectively. It is well known that the intracellular Mg2+ content changes in many pathologies. These can be divided in two wide groups: a) pathologies with primary defect of ion metabolism, in which Mg transport is directly affected, as canalopathies; b) pathologies with secondary defect of ion metabolism involving alteration in hormonal regulation or absorption defects . This research project will study three pathologies representing an adequate in vivo experimental models for studying magnesium homeostasis, for which there is also a clinical interest in the functional evaluation of magnesium deficit.
The pathologies selected for this study are:
a) LQT syndrome, where an imbalance in cardiac re-polarization is caused by genetic defects associated to HERG gene mutations, which alter K+ channel functions. In this pathology, low levels of serum magnesium and high magnesium retention level are recurrent;
b) chronic intestinal failure, where magnesium deficit is mainly due to a reduced alimentary supplementation and reduced
absorption;
c) primary and secondary hyperaldosteronism, where magnesium deficit is correlated to stimulatory effects of aldosterone on membrane Na+/Mg2+ transporter.
The outcome of the present project will allow to obtain a detailed figure of magnesium tissue distribution in both healthy subjects and patients, and this will give information on the regulatory mechanism of the homeostasis of this ion.
Furthermore, the evaluation of tissue distribution of magnesium in the different pathologies will give pivotal indications on the opportunity of magnesium supplementation as therapeutic support for patient treatment putting the basis for the project of adequate clinical trials. <<<

Principal Investigator

Bruno BARBIROLI Università degli Studi di BOLOGNA

Research Objectives

This research project will contribute to the knowledge of the regulatory mechanism of magnesium homeostasis by assessing its distribution in several tissues in humans, by measuring the intracellular free concentration in skeletal muscle, brain, blood cells, and the total and free concentration in plasma. We will use analytical methods specific for each cell type investigated: phosphorus magnetic resonance spectroscopy (31P MRS) to measure in vivo intracellular free magnesium concentration in skeletal muscle and brain, fluorimetry and cytofluorimetry to measure intracellular free magnesium concentration in blood cells, atomic absorption and Mg-selective electrode to measure in plasma total and free concentration respectively. We will examine three pathologies representing an adequate in vivo experimental models for studying magnesium homeostasis, for which there is also a clinical interest in the functional evaluation of magnesium deficit. We choose to examine 14 patients affected by LQT syndrome enrolled by the research unit of "Dipartimento di Scienze Biochimiche, Fisiologiche e della Nutrizione-Università di Messina", 23 affected by primary or secondary aldosteronism enrolled by the research unit of "Dipartimento di Biomedica e Scienze Chirurgiche -Università di Verona", 12 affected by chronic intestinal failure enrolled by the research unit of "Medicina Interna, Centro Regionale di Riferimento per Insufficienza Intestinale Cronica Benigna -Università di Bologna" and 50 age- and sex-matched healthy subjects enrolled as controls by the research unit of "Dipartimento di Medicina Clinica e Biotecnologia Applicata-Università di Bologna".
The specific goals of this program are:
1) to define magnesium concentration in different tissues of patients affected by LQT syndrome and assess whether Mg2+ deficit is a critical cofactor in triggering ventricular arrhythmias and syncope, typical clinical features in this pathology;
2) to define magnesium concentration in different tissues of patients affected by primary or secondary aldosteronism and verify if the reported magnesium intra-lymphocyte deficiency in primary aldosteronism pertains also to secondary aldosteronism and if the deficiency affects other cell types;
3) to define magnesium concentration in different tissues of patients affected by chronic intestinal failure to understand i) the relationship between magnesium status and the pathogenethic mechanism of Mg deficiency in this pathology and ii) the potential physiopathologic consequences of Mg deficiency on the potassium nutritional status and on the parathyroid hormone function;
4) to obtain a detailed figure of Mg2+ distribution in different tissues correlating the Mg2+ of brain, skeletal muscle, and blood cells obtained in the healthy subjects examined;
5) to acquire significant information on the homeostasis of Mg2+ comparing the tissue distribution of healthy subjects to that of patients.
6) to set up new flow cytometric protocols, comparing the results obtained by classic fluorescence spectroscopy using routine protocols, to those obtained by flow cytometric assays.
7) to validate the 31P MRS as a clinical tool for the in vivo assessment of the intracellular free magnesium concentration. <<<

First Results

At the end of the first phase of the project, a first set of results obtained by 31P MRS and by the other analytical techniques will be available, and the study of patients affected by primary aldosteronism will be completed. A database will be made containing data coming from the exams and analytical assays of the subjects examined which will allow to get preliminary information about magnesium tissue distribution. The assessment of the skeletal muscle intracellular free magnesium concentration obtained in different metabolic condition by 31P MRS together with the measure of the post exercise phosphocreatine kinetics, both in healthy subjects and patients, will give information on the influence of magnesium on muscle mitochondrial functionality. Useful indication for the functional characterisation of the examined samples and for flow cytometric protocols setting up will be provided by comparing the results obtained by flow cytometry and spectrofluorimetryThe database will be completed containing all data coming from all exams and analytical assays of all subjects which will allow to obtain a detailed figure of magnesium tissue distribution and information on the regulatory mechanism of this ion.
Furthermore, the evaluation of tissue distribution of magnesium in the different pathologies will give pivotal indications on the opportunity of magnesium supplementation as therapeutic support for patient treatment putting the basis for the project of adequate clinical trials. Finally, on the basis of results obtained by flow cytometric assays and by classic fluorescence spectroscopy, the reliability of the analytical protocols for the different cell types and pathologies examined will be tested. <<<

Timescale

24 months

National and international background

Magnesium is one of the most abundant cations in the cells of soft tissues, its cellular concentration ranging, in most of them, between 14 and 20 mM (1). Magnesium is known to be involved in many cellular process, such as transport systems, receptor, signal transduction, enzyme activities, energetic metabolism, DNA and protein synthesis and membrane functionality (1,2). Its relatively high intra-cellular concentration accompanied to limited fluctuations have suggested that this ion could act as a long term regulator for enzymes and cellular function (1,2). Its concentration depends both on the positive gradient between the intra- and the extra-cellular compartment which regulates the influx, and on transport mechanisms regulating the efflux mainly represented by the exchanger Na+/Mg2+ (1). Recently, new transepithelial magnesium transporters TRPM6 and TRPM7 have been identified which seems to be involved in Mg2+ homeostasis (3,4). However, the mechanisms which regulate magnesium homeostasis in different tissues, are not yet completely understood. Studies report data about the total serum magnesium concentration, while there are growing evidence that the intracellular free fraction is that functionally important (5-8). Magnesium was defined as the "forgotten ion" as, despite of being the second intracellular ion, it is rarely considered in medical practice (9). Furthermore not many data regarding its role in cellular homeostasis and its regulation mechanisms are available. This is mainly due to the analytical limitations in measuring the concentration of freeMg2+ which is the biologically active form. Therefore, so far the great majority of data refer mainly to plasma magnesium which represents less than 1% of total body magnesium thus preventing any extrapolation to magnesium homeostasis. Nevertheless, a number of techniques are available for the assay of cytosolic free Mg2+ (Mg2+), as ion-selective microelectrodes (8), phosphorus magnetic resonance spectroscopy (31P MRS) (11-13), fluorescence spectroscopy (12). All these techniques have different fields of application, depending on the different cells studied; but they all represents a demanding analytical approach requiring competence, instrumental resources and adequate experimental protocols.
Most interest has been recently focused on the study of the cellular mechanisms involved in maintaining the intracellular magnesium concentration constant despite the inwardly-directed magnesium gradient. Furthermore, a great deal of interest has been appointed on the hormonal mechanisms involved in such a regulation. The intracellular free magnesium concentration is mainly regulated by catecholamines (15-17), insulin (16), aldosterone (16,19) and by extracellular glucose concentration (20). In fact Romani and Scarpa (15,17) have shown that norepinephrine and isoproterenol are capable of stimulating adelinate cyclase, increasing AMPc concentrations and increasing the activity of a membrane Na+ Mg2+ exchanger. The magnesium efflux thus obtained is capable of decreasing intracellular magnesium concentration. The same authors have demonstrated that insulin is capable of inhibit the catecholamine-induced magnesium efflux.
It is well known that the intracellular Mg2+ content changes in many pathologies (5, 8, 18-32). These can be divided in two wide groups: a) pathologies with primary defect of ion metabolism, in which Mg transport is directly affected, as canalopathies; b) pathologies with secondary defect of ion metabolism involving alteration in hormonal regulation or absorption defects. The pathologies selected for this study are:
a)Congenital or aquired LQT syndrome, where an imbalance in cardiac re-polarization is caused by genetic defects associated to mutations in ion channel genes. In particular, HERG gene mutation alters K+ channel functions, causing the low levels of serum magnesium and high magnesium retention level, recurrent in this pathology (21-24);
b) chronic intestinal failure, where magnesium deficit is mainly due to a reduced alimentary supplementation and reduced
absorption (25-27);
c) primary and secondary hyperaldosteronism, where magnesium deficit is correlated to stimulatory effects of aldosterone on membrane Na+/Mg2+ transporter (28-31).

The role of Mg2+ in LQT syndrome
Numerous experimental and clinical data have suggested that Mg2+ deficiency can induce elevation of intracellular Ca2+
concentrations, formation of oxygen radicals, proinflammatory agents and growth factors and changes in membrane permeability and transport processes in cardiac cells. The opposing effects of Mg2+ and Ca2+ on myocardial contractility may be due to the competition between Mg2+ and Ca2+ for the same binding sites on key myocardial contractile proteins such as troponin C, myosin and actin (32). Stimulants, for example, catecholamines can evoke marked Mg2+ efflux which appears to be associated with a concomitant increase in the force of contraction of the heart. It has been suggested that Mg2+ efflux may be linked to the Ca2+ signalling pathway (33). Hypomagnesemia is commonly associated also with hypokalemia in patients with hypertension or myocardial infarction as well as in chronic alcoholism (32). Potassium depletion is associated with increased urinary excretion of magnesium, calcium and phosphatein hypertensive conditions. In fact, hypokalemia together with increased kaliuresis occur in a significant number of hypomagnesemic patients. Mg2+ supplemented K+ cardioplegia modulates Ca2+ accumulation and is directly involved in the mechanisms leading to enhanced post ischemic functional recovery in the aged myocardium following ischemia. In severe Mg2+ deficiency, some electrocardiographic (ECG) changes may occur, involving alterations of T-wave, QT segment, and PR and QRS intervals. The ECG changes may, therefore, be secondary to hypokaliemia which is commonly seen in Mg2+ deficiency (34). In this context, long QT syndrome consists of a long QT interval in ECG and episodes of ventricular arrhythmias following
emotional of physical stress. LQTS is of two types, i.e. congenital or acquired (35, 36). Congenital LQTS seems caused by modifications in proteins for the cardiac potassium and sodium channels as a consequence of mutations in genes coding for these ion channels. At time, four genes involved in LQTS have been successfully identified based on diagnostic criteria, i.e. K+ channel genes KVLQT1 on chromosome 11p15.5, HERG on 7q35-36 and minK on 21q22, and sodium channel gene SCN5A on chromosome 3p21-24 (35, 37). Recently, it has been reported that in patients with congenital LQTS magnesium deficiency may be associated with syncope, whereas magnesium supplementation may prevent recurrent syncope. Therefore, it is possible to hypothesize that Mg deficiency may be identified as a significant cofactor in some forms of LQT syndrome (2).

The role of Mg2+ in chronic intestinal failure (CIF)
Chronic intestinal failure (CIF) can be due to four major pathophysiological conditions (each one due to more than one
gastrointestinal disease): short bowel syndrome, motility disorders of the small bowel, intestinal fistulas or small bowel parenchymal disease (38). In CIF, nutrient deficiencies can be due to decreased oral intake (motility disorders), malabsorption or increased gastrointestinal losses (other causes of CIF). Magnesium (Mg)and potassium (K) deficiency (26, 39) and metabolic bone disease are among the most frequent issues (33, 40). Low serum concentrations of Mg and K have been reported in 2-30% of patients receiving intravenous magnesium with home parentereral nutrition (HPN) (26, 39). Furthermore, low concentrations of magnesium in red blood cells have been observed in about 30% of the patients on HPN having normal serum magnesium levels (25). The presence of a bone disease, assessed by bone densitometry, was observed in 84% of adult patients on HPN, about one half of them having osteoporosis according with the World Health Organization diagnostic categories (40). Mg is normally absorbed by passive diffusion in the distal small bowel and colon (41). 1,25-dihydroxy-vitamin D may increase the intestinal absorption of Mg (42). However, in patients with CIF, Mg deficiency may cause a reduction of both secretion and peripherical function of parathyroid hormone, that, reduce the renal production of 1,25-dihydroxy-vitamin D (42). In patients with CIF, Mg deficiency may be due also to the presence of secondary hyperaldosteronism to losses of water and salt,because aldosterone increases renal excretion of Mg (43, 44).
In patients with CIF, Mg deficiency may play a role both in the maintenance of K deficiency and in the pathogenesis of bone disease (45).

The role of Mg2+ in primary and secondary aldosteronism
Primary aldosteronism represents an interesting in vivo model for studying magnesium homeostasis. Aldosterone is believed to influence renal magnesium handling causing magnesium wasting (43, 44). This effect would follow the volume expansion and hypertension associated with this disease. Horton and Biglieri described an increased magnesium clearance in patients with primary aldosteronism (43). Despite of this the great majority of data in literature report no variations in magnesium plasma levels in primary aldosteronism (43, 44), while the intralymphocyte free magnesium concentration is signiflcantly lower in patients with primary aldosteronism than in normotensive control subjects (19). A decrease in cellular magnesium may play a rolein the pathogenesis of arterial hypertension in primary aldosteronism in that it is well known that in vitro magnesium deficiency is followed by an increase in vascular tone and potentiates the pressor effects of angiotensin II. Furthermore, there are anecdotical data on an augmented incidence of idiopathic intracranial hypertension in patients with primary aldosteronism (46), but there are yet no data on magnesium concentration in the brain. These data may be provided by 31P MRS technique. In vitro studied on the effect of aldosterone on free intralymphocyte magnesium showed a dose-effect curve with an EC50 value of approximately 0.5-1 nmol/l aldosterone (19).This value is close to the physiological concentration of plasmatic aldosterone suggesting that the hormone may play a role as a physiological regulator of free intracellular magnesium. In fact, aldosterone is capable of stimulating the membrane Na+-Mg2+ exchanger and the decrease of intracellular magnesium produced by aldosterone is accounted for by an increased Mg2+ efflux (19). The secondary aldosteronism too seems to represent an interesting model for studying the role of magnesium in cardiovascular system. Patients with congestive heart failure are characterized by secondary aldosteronism mainly due to the adrenergic upregulation and increased renin concentration. The recent RALES study (47) on 1633 patients with congestive heart failure has shown that the by adding to conventional therapy spironolactone there is a 30% reduction in mortality. Among the different effects of spironolactone there is also a magnesium spering capacity which was not studied by the RALES investigators. Aldosterone is believed to play a central role in modulating cardiac fibrosis through a direct action on cardiac fibroblasts. An increase in plasma aldosterone is followed by biventricular fibrosis in rats the latter being prevented by spironolactone (48). Finally ACE-inhibitors even at non ipotensive doses prevent cardiac fibrosis in hearts subjected to a pressure-overload (49). Even if the link beteen fibrosis and magnesium homeostasis is hypothetical there a few data on this subject. Rats following a magnesium-deficient diet are characterized by an increased fibrosis in the heart and in the aorta (50, 51). It has been recently shown that human fibroblasts incubated in the absence of extracellular magnesium showed an increase in mRNA collagen I and III gene expression as compared to control cells incubated with magnesium (52). It is possible to speculate that in congestive heart failure secondary aldosteronism by inducing a negative magnesium balance is capable to increse myocardial fibrosis leading to a progressive deterioration of cardiac function (31). <<<