Abstracts

ARTICLE

mrh.2011.0284

Selected abstracts of European Magnesium Meeting EUROMAG June 8-10, 2011 Bologna, Italy Organized by the University of Bologna and by the International Society for the Development of Research on Magnesium

Plenary lectures

Magnesium in learning and memory

G. Liu

School of Medicine, Tsinghua University, Beijing, China; Center for learning and memory, University of Texas, Austin, USA

The human brain has roughly 10 billion neurons that communicate with one another through synaptic connections. Each neuron is capable of making 5,000-10,000 synapses its targets, leading to 50-100 trillion synaptic connections in the brain. The connections between neurons give rise to functional neural networks that provide the cellular substrate for higher cognitive functions such as learning, memory and, ultimately, consciousness. How these complex connections are established during the early phases of nervous system development and optimized in lifespan remains largely unknown. Continuing our inquiries into the regulation of the relationships among neurons and among synapses within a single neuron, we are increasing our understanding of the conditions necessary to maximize the efficacy of these relationships, and thus to optimize the performance of neural networks. Recently, we explored the molecular profiles, synaptic function, and network configuration necessary for circuitry plasticity and identified several endogenous factors, which serve as key regulators of synaptic plasticity and memory. One of molecules is Magnesium. Along the way, we also demonstrated that the memory capacity of synaptic network is controlled not only by the proteins critical for synaptic plasticity, but also by the functional organization of synapses on dendrites. In intact animal, increase in brain magnesium is effective to enhance the memory function of young animal, prevent age-associated memory decline in aging animal, and alleviate the cognitive impairment of the transgenic mice with Alzheimer diseases. Human clinical trials based our discovery are undergoing to translate the knowledge from our research to the new therapy for the treatment of neurological disease with decline of memory function.

Magnesium in aging

M. Barbagallo, L.J. Dominguez

University of Palermo, Italy

Aging is frequently associated with magnesium (Mg) deficit, which besides having a negative impact on the mitochondrial energy production pathways necessary to generate ATP also reduces the threshold antioxidant capacity of the aging organism. Mg itself acts as an antioxidant against oxidative mitochondria damage. Low-grade chronic inflammation and oxidative stress are intertwined mechanisms identified as pathogenic factors in the aging process per se and in various age-related diseases. Chronic Mg deficiency results in excessive production of oxygen-derived free radicals and low-grade inflammation. The total plasma concentrations of Mg are remarkably constant in healthy subjects at any age, however, total body Mg and intracellular Mg tend to decrease with age. The most common cause of Mg deficit in older persons is a low Mg intake, although secondary Mg deficit in aging may also results from diverse mechanisms, such as reduced Mg intestinal absorption, reduced Mg bone stores, and excessive urinary loss. Secondary Mg deficit may be linked to diverse disorders often present in older adults (i.e. insulin resistance and/or type 2 diabetes mellitus), and drugs (i.e. use of hypermagnesuric diuretics). Chronic Mg deficit has been associated with an increased risk of developing numerous preclinical and clinical age-related conditions, including hypertension, stroke, atherosclerosis, ischemic heart disease, cardiac arrhythmias, glucose intolerance, insulin resistance, type 2 diabetes mellitus, endothelial dysfunction, abnormal vascular remodeling, altered lipid metabolism, platelet aggregation/thrombosis, asthma, chronic fatigue, sarcopenia, as well as depression and other neuropsychiatric disorders. Mg deficit associated with aging may be at least one of the pathophysiological links helping to explain the interplay among inflammation/oxidative stress, the aging process and the age-related diseases.

References

Barbagallo M, et al. Magnes Res 2010; 23: 131-7.

Barbagallo M, Dominguez LJ. Curr Pharmaceutical Design 2010; 16: 832-9.

Barbagallo M, et al. Magnes Res 2009 ; 22: 235-46.

Magnesium and cancer: Dr Jekyll or Mr Hyde?

J.A.M. Maier

Università degli Studi di Milano, Italy

The relation between magnesium (Mg) and cancer is still a hank to disentangle. The knowledge derived from animals on a Mg restricted diet reveals a complex scenario in which Mg deficiency has both anti- and pro tumor effects. At the cellular level, Mg deficiency increases oxidative stress, which causes DNA damage, and impairs DNA repair mechanisms, thus leading to mutations that activate ptoto-oncogenes and inactivate tumor suppressor genes. Upon neoplastic transformation, Mg accumulates in the cell also when its extracellular availability is low, its distribution is altered and is not subjected to coordinated fluctuations in response to various stimuli. It is difficult to translate the lesson learnt from experimental models to humans. Based on epidemiological studies, Mg deficiency seems to be linked to increased risk of some types of cancers. The evidence that hypomagnesemia promotes inflammation and the demonstration of an impairment of Mg homeostasis in oncologic patients further complicate the field. We need more translational and clinical data to draw firm conclusions about the contribute of Mg to tumors.

References

Maier JAM, et al. Nutr Cancer 2007; 59: 192-8.

Wolf F, et al. Cancer Treat Rev 2009; 35: 378-82.

Wolf F, et al. Magnes Res 2009; 22: 5-9.

Insight into renal Mg²⁺ transporters

R.J.M. Bindels

Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

This presentation will describe the recent findings concerning novel Mg²⁺ transporters as putative interesting players in renal transepithelial Mg²⁺ transport. So far, the best characterized Mg²⁺ transport proteins are found in prokaryotes and yeast cells. In recent years, phylogenetic analysis and differential gene expression studies lead to the identification of numerous genes associated with Mg²⁺ transport in eukaryotes. Besides the well-known transient receptor potential channel melastatin, members 6 and 7, and the mitochondrial transporter Mrs2, additional Mg²⁺-transporting protein families can be acknowledged including the magnesium (Mag) transporters, solute carrier (SLC) family 41 members, ancient conserved domain proteins (ACDP), non-imprinted in Prader-Willi/Angelman syndrome (NIPA) proteins, membrane Mg²⁺ transporters (MMgT) and huntingtin-interacting protein 14 (HIP14). The identification of several mammalian proteins involved in Mg²⁺ transport highlights the physiological importance of this cation and its tight regulation in numerous tissues. Further investigation of these transporters might represent a key tool to complement our current knowledge about renal Mg²⁺ handling.

TRPM6 – physiological roles beyond magnesium?

T. Gudermann¹, V. Chubanov¹, U. Storch¹, M. Mederos y Schnitzler¹, T. Hofmann²

¹ University of Munich, Walther-Straub-Institute of Pharmacology and Toxicology; ² University of Marburg, Institute of Pharmacology, Germany

Divalent cation-selective outwardly rectifying currents, which are induced upon removal of intracellular Mg^2+, have been described in all mammalian cells examined so far. Accordingly, these currents were referred to as magnesium-inhibited currents (MIC) or magnesium nucleotide-regulated metal ion currents (MagNuM). TRPM6 and TRPM7 (melastatin-related members of the transient receptor potential gene family) were identified as molecular candidates mediating MIC/MagNuM. Recent studies revealed that TRPM7 is essential for the cell viability, embryonic development and Mg²⁺ homeostasis. Loss-of-function mutations in the human TRPM6 gene result in a hereditary human disease, i.e. hypomagnesemia with secondary hypocalcemia (HSH). HSH is an autosomal recessive disorder characterized by low serum Mg²⁺ and Ca²⁺ levels. TRPM6 is specifically expressed in the intestinal epithelium and the distal convoluted tubule in the kidney. Collectively, these findings support the concept that TRPM6 can directly participate in Mg²⁺ uptake by renal and intestinal epithelial cells. In order to investigate the role of TRPM6 in the pathomechanism of HSH, we initiated a phenotypic analysis of TRPM6 gene deficient mice carrying a LacZ reporter sequence and of additional mouse strains with TRPM6 genes conditionally inactivated in different organs. In stark contrast to what we know about the clinical picture of HSH, homozygous TRPM6-deficient mice die at a mid-gestational stage. Tracking LacZ expression and using TRPM6-specific antibodies, we observed an expression profile of TRPM6 much broader than that a previously assumed, supporting the notion that the phenotype of HSH patients only partially depicts the physiological relevance of TRPM6.

TRPM7: still the leader of magnesium transporters?

A. Fleig¹, A. Ryazanov²

¹ The Queen's Medical Center, University of Hawaii, Honolulu, USA; ² R. W. Johnson Medical School, UMDNJ, New Jersey, USA

TRPM7 is a member of the melastatin-like transient receptor potential (TRPM) subfamily. It has a unique protein combining an ion channel with a functional α-kinase domain in its cytosolic carboxy-terminal tail. Currently, the ubiquitously expressed TRPM7 is the only known functional channel conducting Ca²⁺, Mg²⁺ and trace metals into cells, and it is the only channel known to be essential for cellular viability. Most recent findings demonstrate that the channel is essential for normal Mg²⁺ absorption in the intestine, thereby regulating systemic Mg²⁺ in mammalian organisms in addition to maintenance of Mg²⁺ concentrations on a cellular level.

Reference

Ryazanova LV, et al. Nat Commun 2010; 1: 109.

Two-photon probes for magnesium ion

K. Chang Woo, C. Bong Rae

Department of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, Korea

Recently, two-photon microscopy (TPM) has emerged as an indispensable tool in biology due to the capability of imaging the intact tissue for a long period of time with minimum interference from the tissue preparation artifacts, self-absorption, auto-fluorescence, photobleaching, and photodamage. For this to become a common tool in biology, a variety of two photon probes for specific applications is needed. To address this need, we have developed a series of two-photon probes for intracellular free cations and lipid rafts by considering following requirements: significant TP cross section for the bright image, appreciable water solubility to stain the cells and tissues, cell permeability, sensitivity to the polarity of the environment for the selective detection of cytosolic and membrane-bound probes, high photostability for a long term imaging, and receptors that trigger the emission of the two photon excited fluorescence (TPEF) upon binding with the ions or membrane in the living system [1-4]. In this seminar, I will describe the photophysical properties and in-vivo applications of a few two photon probes for magnesium ions.

References

1. Kim HM, Cho BR. Acc Chem Res 2009; 42: 863.

2. Kim HM, Cho BR. Chem Asian J 2011; 6: 58.

3. Kim HM, et al. J Org Chem 2007; 72: 2088.

4. Kim HM, et al. Angew Chem Int Ed 2007; 46: 3460.

Advanced X-ray micro and nanoprobes techniques to assess Mg distribution and concentration in whole cells

S. Lagomarsino

CNR-IPCF–UOS Roma- c/o Dip. Fisica, Univ. Sapienza P.le A. Moro, Rome, Italy

The technological and methodological advancements in x-ray micro and nano-probes of the last 10-15 years constitute an invaluable potential for unraveling complex problems in biomedical sciences. X-ray microscopy and tomography, phase contrast imaging, x-ray fluorescence microscopy and spectromicroscopy are examples of advanced tools now ready for applications. Among the diverse microscopy techniques, those based on x-rays have the unique capability to give morphological, compositional and chemical information at the nanoscale on whole hydrated, non sectioned and unstained samples. In this lecture I will make a short review of the main x-ray micro and nano probes techniques, mostly based on third generation synchrotron radiation sources, I will then present a specific application aimed at measuring the distribution and the concentration of Mg in whole cells.

Magnesium in clinical medicine – focus on sex hormones

R. Touyz

Ottawa Hospital Research Institute, Canada

Until recently the importance of magnesium in clinical medicine was essentially ignored. However with an increased understanding of the biology of magnesium, improved methods to assess magnesium in the clinic and the laboratory and with the recently identified magnesium-sensitive transporters that control cellular magnesium homeostasis, there has been a renewed interest in the role of this divalent cation in pathological processes. Magnesium deficiency has been linked to many pro-inflammatory diseases, including hypertension, diabetes, cardiac disease and neurological disorders. In addition in women, altered magnesium homeostasis has been implicated in conditions associated with changes in estrogen status, such as in postmenopausal osteoporosis, pre-eclampsia and menstrual migraines. Moreover cyclical menstrual changes in estrogen levels are associated with cyclical changes in plasma magnesium concentration. These conditions may respond favourably to magnesium therapy. Molecular mechanisms linking estrogen and magnesium are unknown but estrogen has been shown to regulate the magnesium transporter, transient receptor potential melastatin cation channel 6 (TRPM6). In fact estrogen has been described as a magnesiotropic hormone. This presentation will discuss the role of magnesium in clinical medicine, focusing on women's health and disease and will discuss putative mechanisms whereby sex hormones regulate magnesium homeostasis

Magnesium and preeclampsia

J. Vormann

Institute for Prevention and Nutrition, Ismaning/Munich, Germany

Preeclampsia (PE) is defined as pregnancy-induced hypertension and proteinuria, which can lead to eclampsia. PE is estimated to affect more than 8 million pregnant women worldwide every year and is a major cause of maternal, fetal and neonatal morbidity and mortality. It would be an advantage to be able to preselect pregnant women at risk for PE for close monitoring. A main cause of PE seems to be endothelial dysfunction in maternal blood vessels induced by substances from the placenta. Mg infusion is treatment of choice for eclampsia but is also effectively used in prevention of eclampsia in PE women. Low Mg intake is a risk factor for development of PE. Compared to uncomplicated pregnancies plasma Mg-concentrations in PE were reported to be unchanged, decreased or even increased. Intracellular total and ionized Mg however was found to be reduced as was membranous Mg in PE. Oral Mg supplementation is probably effective in reducing the incidence of PE. However, well designed large clinical trials are lacking. Pregnancy induced hypertension as a risk factor for PE can partly be prevented by oral Mg. Recently several Mg sensitive genes have been described which are also expressed in placenta. Only the gene SLC41A1 was found to be significantly over expressed in term placenta from PE women compared to placenta from women with uncomplicated pregnancy. SLC41A1 encodes for the Na⁺/Mg²⁺ exchanger. Na⁺/Mg²⁺ antiport seems to contribute significantly to maternal/fetal Mg transport and an increased activity could induce local Na⁺ derangements secondarily contributing to hypertension in PE. It is currently being investigated whether monitoring the expression of SLC41A1 in pregnant women could be used as a diagnostic tool for PE.

Magnesium in gynecological practice

F. Facchinetti, G. Pedrielli

Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Modena, Italy

Magnesium is the second most abundant intracellular divalent cation and is a cofactor for more than 300 metabolic reactions in the body. The importance of magnesium in obstetrical and gynecological disorders is now definitely established, and there is the need of study of its metabolism and body requirements. Even outside of the state of pregnancy and in the absence of gynecological disease, increased intake of magnesium in the diet appears to be desiderable in female subjects. It's known that increased production of estrogen in women may result in a lower availability of magnesium and the menstrual flow would favor the depletion of the ion. In gynecology there are various pathological conditions in which magnesium may play a role in etiopathogenesis or otherwise ion supplementation appears to be an important therapeutic significance. In particular, there are numerous experimental trials and clinical evidences for the effectiveness of magnesium in primary dysmenorrhoea and premenstrual syndrome (PMS). Dysmenorrhoea refers to the occurrence of painful menstrual cramps of uterine origin. Common treatment for dysmenorrhoea is medical therapy such as nonsteroidal anti-inflammatories or oral contraceptive pills, the efficacy of this therapies is considerable. However the failure rate is still often 20-25%. Many consumers are now seeking alternatives to conventional medicine. Magnesium could be one of them. A Cochrane review in 2009 found some trials comparing magnesium with placebo for relief of pain in primary dysmenorrhoea. Magnesium, given as pidolate, was more effective than placebo but the dose and regime were widely variable. Another study, magnesium significantly reduced menstrual fluid PGF2α to 45% of its pretreatment levels, which provides a mechanistic rationale for this therapy. Reduced magnesium (Mg) levels have been reported also in women affected by PMS. Three major studies evaluated the effect of magnesium in PMS: two have used magnesium oxide while the remainder took the magnesium pidolate. No significant differences emerged from studies with magnesium oxide, while the study conducted by Facchinetti et al. using a high dose and a different pharmaceutical preparation (350 mg/die) was obtained in a significant reduction of psychological symptoms.

References

Elin RJ. Am J Clin Pathol 1994; 102: 616-22.

Takaya J, et al. Magnes Res 2004; 17: 126-36.

Facchinetti F, et al. Obstet Gynecol 1991; 78:177-81.

Walker AF, et al. Med Hypothesis 2002; 58: 213-20.

Posters

TRPM7 downregulation mediates microvascular endothelial cell response to magnesium deficiency

E. Baldoli, J.A.M. Maier

Dipartimento di Scienze Cliniche, Università degli Studi di Milano, Italy

Evidence has accumulated to suggest that magnesium (Mg) plays a role in controlling angiogenesis. Because microvascular endothelial cells (MEC) are protagonists in angiogenesis, we asked whether different concentrations of Mg could affect the behavior of human MEC. In particular, we evaluated some crucial steps of the angiogenic process, i.e. proliferation, migration, protease production and organization in tridimensional structures of MEC cultured in low extracellular Mg. While Mg deficiency does not impact on MMP activity and cell differentiation, it inhibits MEC growth and migration. Since in MEC i) low Mg markedly downregulates TRPM7, which is essential for Mg homeostasis, and ii) silencing TRPM7 leads to the inhibition of cell proliferation and migration, we conclude that TRPM7 downregulation mediates low Mg-induced inhibition of cell growth and migration in these cells. On the contrary, in macrovascular endothelial cells low Mg induces TRPM7 and silencing TRPM7 results in increased cell growth and migration. Our results demonstrate a role of TRPM7 in modulating the angiogenic phenotype of MEC cultured in low Mg.

References

Carmeliet P. Nature 2005; 438: 932-6.

Bernardini D, et al. Front Biosci 2005; 10: 1177-82.

Ryazanova LV, et al. Nat Commun 2010; 1: 109.

The effect of UV-B radiation on Mg and Se concentrations and glutathione level in the HepG2 hepatic cells

E. Brucka-Jastrzębska, D. Kawczuga

University of Szczecin, Department of Physiology, Felczaka 3c street, Szczecin, Poland

We studied biological mechanisms of the UV-B influence (300 J cm-2) on the in vitro culture of the HepG2 hepatic cells. The tests were conducted on the HepG2 cell line, hepatocellular carcinoma (ATCC No. HB-8065), stimulated with UV-B generated by Philips TL12 fluorescent tubes (emission peak at 313 nm). The cells were examined at 0, 6, 24 and 48 hour post-exposure. Antioxidant status was measured fluorometrically. We examined the number of cells, the percentage of apoptotic and necrotic cells, and changes in the cell concentrations of glutathione antioxidants, Mg and Se. In the control group, no significant temporal changes were observed in the antioxidant, Mg and Se concentrations. In the UV-B-exposed cells, concentrations of total glutathione, Mg and Se were increasing significantly till the 24th hour (0.995-1.151 mg/mg-1 protein, 3.81-4.03 mg/g-1 wet weight and 0.092-0.125 mg/g-1 wet weight, respectively) and decreased at the 48th hour (0.855 mg/mg-1 protein, 3.67 mg/g-1 wet weight and 0.085 mg/g-1 wet weight, respectively). Not numerous apoptotic or necrotic cells were observed both in the study and control groups throughout the study. We concluded that the hepatic HepG2 cells are sensitive to UV-B radiation that increased the examined cell parameters till the 24th hour after exposure.

References

Shu B, et al. Chin J Traumatol 2002; 5:246-9.

Gohto Y, et al. Ophthalmic Surg Laser 2000; 31: 55-60.

Nomura K, et al. Lasers Med Sci 2001; 16: 218-23.

Pereira AN, et al. Lasers Med Sci 2002; 31: 263-7.

A new imidazothiazolylmethylen-oxindole induces apoptosis and decline in intracellular free and total magnesium in HT29 colon cancer cells

C. Cappadone¹, G. Farruggia¹, C. Marraccini², M. Sgarzi³, M. Lombardo³, S. Iotti²

¹ Dipartimento di Biochimica “G. Moruzzi” , Universita’ di Bologna; ² Dipartimento di Medicina Interna dell’Invecchiamento e delle Malattie Nefrologiche, Università di Bologna; ³ Dipartimento di Chimica “G. Ciamician”, Università di Bologna, Italy

Recent studies demonstrated that 2MM-67, a new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinone, analog of recently published compounds [1] is able to induce apoptotic cell death in HT-29, a colon carcinoma cell line. The cells treated with the proapoptotic agent (500 nM) showed arrest in G2/M phase and increased expression of p21 protein. Moreover it was observed a significant reduction of mitochondrial potential (Δψm), enhanced expression of Bax and, finally, a marked induction of effector caspases [2]. The aim of this work was to assess cellular magnesium levels during 2MM-67-induced apoptosis and if they correlate to Δψm decrease and to an increase in ROS production, characteristic feature of mitochondrion-mediated apoptosis. Intracellular free and total magnesium were evaluated in flow cytometry, respectively by the commercial probe MagFluo4-AM (Kd=4.7 mM), and the new synthesized DCHQ7 (Kd=8.3 μM) [3, 4], while ROS production was assessed by DiClFDA and Dihydrohetidine. Our results indicate that 2MM-67 treatment in HT29 cells can result in rapid declines in intracellular free and total magnesium concomitant with the decrease in mitochondrial membrane potential and the increase of ROS production.

References

1. Andreani A, et al. J Med Chem 2008; 51: 7508-13.

2. Andreani A, et al. (submitted).

3. Lee S, et al. Anal Chem 2009; 81: 538-42.

4. Farruggia, et al. J Org Chem 2010; 75: 6275-8.

Magnesium sulfate supplementation in exercise performance: the dynamic changes of energy metabolites in the peripheral and central systems

H.Y. Chen, Y.J. Chen, M.F. Wang, F.C. Cheng

Taichung Veterans General Hospital, 160, 3^rd Sect., Taichung Harbor Rd., Taichung, 40705 Taiwan

The effect of magnesium supplementation on exercise performance was still contentious. In our present study, magnesium sulfate on exercise performance and the dynamic changes of energy metabolism in the central and peripheral system were explored. Microdialysis technique was employed to explore continuously brain and muscle energy metabolites, and obtained the blood sample repeatedly via an auto-blood sampling system. Each Sprague-Dawley rat was pretreated with saline or magnesium sulfate (90 mg/kg, i.p.) 30 min before treadmill exercise. The treadmill speed was set at the speed 20 m/min for 60 min. All dialysates and blood samples were collected every 15 min before, during and after exercise. The results indicated that the blood, muscle and brain glucose levels immediately increased during exercise, and it were significantly higher (p<0.05) in the magnesium sulfate group compared with control group. Then, glucose levels were gradually decreased to approach to basal level in the recovery periods in both groups. Glucose levels in the blood, muscle, and brain were rapidly rose during exercise periods. After exercise, glucose levels in the blood and muscle were decreased to about basal levels in both groups, but brain glucose levels in the magnesium sulfate group was significantly higher (p<0.05) than control group in the recovery periods. Lactate levels in the blood, muscle, and brain rapidly and significantly increased during exercise and then blood lactate were declined to basal levels in both groups in the recovery periods. Nevertheless, muscle lactate in the magnesium sulfate group was delayed to reach the peak for 45 min during exercise. However, muscle lactate levels in the magnesium sulfate group were rapidly decreased to basal levels, but the phenomenon was not observed in the control group in the recovery periods. Moreover, brain lactate levels in the magnesium sulfate group were significantly higher than control group during exercise and recovery periods. In our present study, the effect of magnesium supplementation on the dynamic changes of glucose and its metabolites in the blood, muscle, and brain were investigated. However, the correlations between glucose, pyruvate, and lactate concentrations and exercise performance were still undetermined. Further study is required to determine the underlying mechanisms involved.

References

Cheng SM, et al. Eur J Appl Physiol 2010; 108: 363-9.

Chen YJ, et al. Appl Physiol Nutr Metabol 2009; 34: 1040-7.

Deep sea water mineral concentrate normalizes magnesium transporter genes expression in magnesium deficiency mice

F.C. Cheng¹, C.C. Chu², H.E. Chen², C.Y. Chen², S.H. Chen¹

¹ Stem Cell Center, Taichung Veterans General Hospital, Taichung; ² Taiwan Yes Deep Ocean Water Co., Ltd., Taipei, Taiwan

Magnesium (Mg) is involved in the regulation of numerous physiological functions. It may play a significant role in the genesis of various diseases such as DM, osteoporosis, essential hypertension, myocardial ischemia, stroke and depression. Mg deficiency at both the cell and organism levels has been demonstrated to be involved in the development and/or symptoms of the above diseases. The present study is to validate a newly developed test for intracellular Mg status, based on the expression levels of Mg transporter genes in mice lymphocytes. C57BL/6 mice were randomly divided into the Mg-deficiency group and control group. Nine Mg transporter gene expressions were determined by quantitative real-time PCR. Among them, SLC41A3, CNNM2, MAGT1, TRPM6/7, and N33 genes were hypersensitive in Mg-deficiency mice when compared to the control. These hypersensitive gene expressions were normalized except the SLC41A3 after four weeks supplementation by Nigari, concentrated deep ocean water in the magnesium-deficiency mice. Overall, the use of the newly developed test will deepen our knowledge of the Mg genetic variability in animal models. As Nigari is a safe, natural, and inexpensive Mg supplement, normalization of these above gene expressions by Nigari may have therapeutic effects on Mg-deficiency related diseases.

References

Kolisek M, et al. J Biol Chem 2008; 283: 16235-47.

Sponder G, et al. Magnes Res 2010; 23: 105-14.

Ferre S, et al. Curr Opin Nephrol Hypertens 2011; 20: 169-76.

Mg²⁺ dependent activity of TRPM7 channel as site target for natural and synthetic pharmacological compounds

V. Chubanov¹, M. Mederos y Schnitzler¹, M. Meißner¹, S. Schlsquäfer¹, K. Abstiens¹, T Hofmann², T. Gudermann¹

¹ Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians University of Munich; ² Institute for Pharmacology and Toxicology, Philipps-University of Marburg, Marburg, Germany

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bi-functional protein comprising a TRP ion channel segment linked to an α-type serine/threonine protein kinase domain. TRPM7 is a ubiquitously expressed Ca²⁺ and Mg²⁺ permeable cation channel with expanding roles in essential cellular processes such as Mg²⁺ homeostasis, cell motility, proliferation and mechanotransduction. Using an Aequorin bioluminescence-based assay of TRPM7 channel activity, we performed a hypothesis-driven screen for modulators of the channel. The candidates identified were further evaluated electrophysiologically. We found that TRPM7 currents are inhibited by common modulators of SKca channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31, UCL1684 and the scorpion peptide tamapin. The most potent compound, NS8593, specifically targets TRPM7 (IC50 1.6 μM) as compared to other TRPM channels, interferes with Mg²⁺ dependent activity of TRPM7 channel, and inhibits the motility of cultured cells. These results suggest that TRPM7 and SKCa channels display an unexpectedly similar design of their pore-forming segments. Our findings shed new light on the therapeutic potential of drugs targeting SKca channels, because they may prove instrumental in pathological conditions associated with TRPM7.

Overview of the NIH research portfolio on magnesium

R. Costello, C. Lentino, K. Regan

National Institutes of Health-Office of Dietary Supplements, USA

The National Institutes of Health (NIH) accomplishes its mission to improve the health of the population by funding research in the origin, diagnosis, prevention, and cure of human diseases. As part of its mission, NIH has funded many magnesium research projects. The following portfolio analysis is based on search results from the NIH RePORTER and the Human Nutrition Research Information Management databases. The search included the years 2000-2010 and was limited to nutrition and magnesium-related research, where magnesium was included in a stated research objective. A total of 63 unique projects were funded during this time period. In 2000, a total of 9 projects (4 basic, 1 RCTs, 4 epidemiological) were funded by 3 NIH Institutes. In 2010, a total of 23 projects (4 basic, 2 clinical, 7 RCTs, 10 epidemiological) were funded by 12 Institutes and Centers. For years 2000 to 2010, a bimodal distribution is seen in magnesium-related research. In 2001-2004, there was an increase in funding for RCTs sponsored by the National Institute of Child Health and Human Development due to the BEAM Study, which evaluated the beneficial effects of antenatal magnesium sulfate and fetal outcomes. In 2008-2010, the American Recovery and Reinvestment Act led to an increase funding by the National Institute of Diabetes and Digestive and Kidney Diseases principally for RCTs and epidemiological studies. In 2010, NIH invested 1.6 billion dollars in nutrition-related research with approximately 7.2 million dollars supporting magnesium-related studies.

References

Human Nutrition Research Information Management (HNRIM) (cited March 3, 2011) http://hnrim.nih.gov/

NIH RePORTER (cited March 3, 2011) http://projectreporter.nih.gov/reporter.cfm

Mg Acetyltaurinate as a photic inhibitor in photosensitive magnesium depletion: a physiological pathway in headache with photophobia treatment

P. Danhier¹, J. Durlach², N. Pages³, P. Maurois⁴, P. Bac³, J. Vamecq⁴, M. German Fattal⁴, V. Durlach⁵

¹ Biosciences_shift; ² Tri-Inov; ³ Laboratoire de Neuropharmacologie, Faculté de Pharmacie, Paris XI; ⁴ Faculté de pharmacie, Paris XI; ⁵ Faculté de Médecine, Reims, France

We previously described an actimetry-based test of photosensitization in mice, which can be used for a rapid and efficient screening of drugs of interest for photic cephalalgia and other photosensitive diseases. In summary, in absence of photostimulation, control mice had a basic motor activity whereas magnesium-deficient mice suffered nervous hyperactivity (NHE) with increased motor activity. While in each group, control and magnesium-deficient mice were submitted to photostimulation using a stroboscope, control mice developed the phenomenon of habituation whereas magnesium-deficient mice presented both sensitization with higher NHE and generalization, involving hypersensitivity to other type of stimuli. In the present study, we studied the efficiency of a new magnesium salt, which was not yet commercially available, magnesium N-acetyltaurinate (ATA Mg) on senzitisation suppression suggesting a possible therapeutic use in photosensitive diseases. In the magnesium-deficient, ATA-Mg-treated at dose of 100 mg/kg and photostimulated group, the phenomenon of sensitization disappeared. In the same conditions, neither magnesium ion under the form of MgCl2, nor taurine or acetyltaurine were efficient. Within our experimental conditions, ATA Mg among other commercial magnesium salts was the most efficient in restoring habituation capacity. Inhibiting properties of ATA Mg on the kainic acid receptor have been demonstrated in a model of magnesium depletion. Kainic acid is a neurotransmitter involved in NHE which is present in photosensitive diseases (migraine, convulsion, epilepsy). ATA Mg should be a therapeutic approach of interest to the treatment of headaches, migraine with reactional photophobia particularly. Clinical studies should confirm the results observed in vivo.

References

Bac P, et al. Meth Find Exp Clin Pharmacol 2005; 27: 681-4.

Durlach J, et al. Magnes Res 2004; 17: 314-26.

Bac P, et al. Magnes Res 1996; 9: 281-91.

Baran H. Amino Acids 2006; 31: 303-7.

Bac P, et al. Magnes Res 1993; 6: 11-19.

Role of magnesium in osteoblast-like cells proliferation: possible implications in the pathogenesis of osteoporosis

F. Dellera, J.A.M. Maier

Università degli Studi di Milano, Dipartimento di Scienze Cliniche, Laboratorio di Patologia Generale, Italy

Patients affected by osteoporosis show a decrease in osteoblasts number and this reduction is detected in animal models fed with a magnesium (Mg)-poor diet and in rare diseases with impaired magnesium homeostasis. We therefore investigated the influence of different concentrations of extracellular magnesium on osteoblast-like SaOS-2 cells behaviour. We found that low magnesium inhibited SaOS-2 cell proliferation by increasing the release of nitric oxide through the upregulation of inducible nitric oxide synthase (iNOS). Indeed, both pharmacological inhibition with the iNOS inhibitor L-NIL and genetic silencing of iNOS by siRNA restored the normal proliferation rate of SaOS-2. Interestingly, an addictive effect was observed when SaOS-2 cells cultured in low Mg were silenced for Transient Receptor Potential Melastatin (TRPM)7, which plays a prominent role in intracellular Mg homeostasis. It is noteworthy that Mg deficiency did not impact on SaOS-2 cell differentiation as evaluated by measuring alkaline phosphatase activity and evaluating the deposition of mineral matrix by Alizarin red staining. Because a moderate induction of nitric oxide is sufficient to potentiate bone resorption and a relative deficiency in osteoblast proliferation can result in their inadequate activity, we conclude that maintaining magnesium homeostasis is relevant to ensure osteoblast function and, therefore, to prevent osteoporosis.

References

Rude RK, Gruber HE. J Nutr Biochem 2004; 15: 710-6.

Wimalawansa SJ. Ann NY Acad Sci 2010; 1192: 391-403.

Dietary magnesium intake alters age-related changes in rat adipose tissue

S. Devaux¹, A. Markus ¹, P. Laurant¹, A. Berthelot¹, A. Quignard-Boulangé²

¹ EA 4267, IFR 133, Faculté de Médecine et Pharmacie, 25030 Besançon; ² INRA, UMR914, 75005 Paris, France

Obesity and related metabolic diseases are associated with increased risk of cardiovascular disease. We have previously showed some beneficial effects of Mg supplementation on cardiovascular disease. The aim of this study was to examine the effect of dietary Mg intake on adiposity in adult and old rats in relation to blood pressure (BP). Rats were fed for 1 (Young) or 22 months (Old) with deficient (Def-)(150 mg/kg), standard (800 mg/kg) or supplemented (Sup-) (3,200 mg/kg) Mg diets. Adipose tissue development and cellularity, BP and leptinemia were measured. In young rats, Mg supplementation resulted in a shift of frequency distribution of adipocytes toward greater sizes and increased adipose cell weight by 58%. Mg deficiency did not modify these parameters but adipocyte numbers was 30 % higher than in standard or Sup-diet. In old rats, Def-diet led to a relative adipocyte hypotrophy which was counterbalanced by an increased adipocyte number. By contrast, adipocyte size and number were similar in Sup-fed and standard rats. BP was modified in old rats according to the dietary Mg whereas it was unchanged whatever the diet in young rats. In conclusion, this study suggests that the level of magnesium intake may affect the age-related changes in rat adipose tissue cellularity.

Theoretical and Raman spectroscopic study on the interaction of cimetidine (cim) with Mg⁺⁺ and Ca⁺⁺ ions

M. Di Foggia¹, A. Maris², E. Benassi³, S. Bonora¹

¹ Dipartimento di Biochimica “G. Moruzzi”, Università degli Studi di Bologna; ² Dipartimento di Chimica “G. Ciamician”, Università degli Studi di Bologna, Bologna; ³ Centro S3, CNR Istituto di Nanoscienze, Modena, Italy

Cimetidine (cim), a widely used drug for peptic ulcer disease, was found to affect serum levels of calcium and magnesium [1]. The complexes of cim with Mg⁺⁺ ions have been previously studied by nuclear magnetic resonance [2] and we deepened this study by using a combined theoretical (computational analysis) and experimental (spectroscopic techniques) approach. The theoretical analysis of complexes between cim and Mg⁺⁺ or Ca⁺⁺ ions showed that cim is a flexible molecule, acting as a bidentate ligand toward divalent ions, using nitrogen atoms. Moreover, it suggested that Mg(Cim)2⁺⁺ complex is the most stable; its structure is probably a distorted octahedron in which the two S atoms complete the coordination shell. Raman study on the solid 2:1 cim/Mg⁺⁺ and cim/Ca⁺⁺ complexes supported the theoretically provided interaction sites, as suggested by the noticeable wavenumbers shift observed in νC-H and νC-N vibrations. The study was extended in the very diluted solutions (ppm) range by using the Surface Enhanced Raman Spectroscopy (SERS), mimicking the physiological active concentrations of cim [3]. SERS spectra suggested, upon the binding of cim to silver colloids (with a mechanism that mimics the binding to a protein), the formation of a 1:1 adduct, as cim acts as a monodentate ligand. Thus, Mg⁺⁺ can bridge the substrate with other bioactive structures.

References

1. Ghishan FK, et al. J Nutr 1981; 111: 2157-61.

2. Teixeira F, et al. Gastroenterol Clin Biol 1984; 8: 879-80.

3. Bonora S, et al. J Raman Spectroscopy 2011; DOI: 10.1002/jrs.2775.

Expression of specific magnesium transporters in some epithelial tissues of rats under magnesium-restricted or magnesium-enriched diets

M. Di Nino¹, L. Mastrototaro¹, V. Trapani¹, H. Martin², S. Devaux², A. Berthelot², F.I. Wolf¹

¹ Istituto di Patologia Generale, Facoltà di Medicina e Chirurgia “A. Gemelli”, Università Cattolica del Sacro Cuore, Roma, Italy; ² EA 4267, Faculté de Médecine et Pharmacie, 25030 Besançon, France

Magnesium is essential for many biochemical processes such as signalling, proliferation and transport. In the last years, phylogenetic analysis and gene expression studies have identified and in part characterized numerous genes associated with Mg transport in eukaryotes. Of all these magnesium transporters, TRPM6 (Transient Receptor Potential Melastatin 6) is mostly involved in the regulation of systemic magnesium, it is expressed especially in colon mucosa and kidney tissues where it regulates magnesium absorption and re-absorption, respectively. TRPM6 expression is regulated by multiple factors, including dietary magnesium, magnesiotropic hormones and drugs. MRS2, analogous to prokaryotes CorA, is the first magnesium-specific channel found in eukaryotes, it represents the major mitochondrial Mg2+ uptake system in yeast, plants and mammals. We previously showed that mammary epithelial cells (HC11) adapt to decreased magnesium availability by upregulating TRPM6 and that also MRS2 expression is modulated by extracellular magnesium, namely higher MRS2 expression in high-Mg medium. Because of the importance of validating in vitro results with in vivo data, we studied the expression of these two Mg channels in epithelial tissues of rats under Mg-deficient or Mg-enriched diets. The expression of these channels were investigated by immunohistochemistry and western blot analyses, in mammary glands of female rats fed for 6 weeks with a diet containing lower (150 mg/kg) and higher amounts of Mg (4,000 mg/kg) (control diet 1,000 mg/kg). Kidney tissue was used as a positive control for TRPM6 expression. Our results show that in mammary glands dietary Mg affects TRPM6 expression consistent with that found in mammary HC11 cells, namely tissues of rats under low-Mg diet show higher level of TRPM6 and lower level of MRS2 compared to tissues of rats under normal or high-Mg diet. These data confirm that our in vitro observations are consistent with the in vivo conditions. Further investigations are needed in order to elucidate the role of these transporters in regulating magnesium content of mammary tissue.

Disclosure. Work supported in part by PRIN 2007ZT39FN.

References

Wolf FI, et al. J Cell Physiol 2010; 222: 374-81.

Modifications of plasma and intraerythrocyte magnesium concentration in NIDDM patients treated with biguanides and sulfonylureas

M.D. Doşa¹, T.L. Hangan¹, C. Galeş², M. Nechifor²

¹ University Ovidius, Faculty of Medicine, Constanţa; ² University of Medicine and Pharmacy Gr T Popa, Iaşi, Romania

The study was performed on a group of 30 subjects, diagnosed with type 2 diabetes mellitus that were treated with metformin for 3 months. After this period 8 patients had received treatment with sulfonylurea agents. Determination of plasma and intraerythrocyte magnesium concentration was done in advanced, after 3 months and after 6 months of therapy. Treatment with metformin evidenced that there were not significant differences compared with the initial moment for plasma magnesium (M=1.96, SD=0.10 vs M=1.95, SD=0.19, =0.735) while compared with control group there were significant differences (M=1.96, SD=0.105 vs M=2.21, SD=0.193, p<0.001), and there were significant differences for intraerythrocyte magnesium after 3 months of metformin therapy (M=5.75 SD=0.61 mg/dL vs M=5.09, SD=0.63, p<0.001) and no significant differences for intraerythrocyte magnesium (M=5.82 SD=0.74 mg/dL vs M=5.75, SD=0.56, p=0.786). Treatment with metformin for 3 months did not modify significant the plasmatic concentration of this cation but did modify significant the intraerythrocyte magnesium. Treatment with sulfonylureas did modify significant the plasmatic concentration of plasma magnesium and did not modify significant the intraerythrocyte magnesium.

References

Schnack C, et al. Diabetologia 1992; 34: 77-9.

Takaya J, et al. Magnes Res 2004; 171: 126-36.

Guerrero-Romero F, et al. Diabetes Metab 2004; 30: 253-8.

Sales CH, et al. Clin Nutr 2006; 25: 554-62.

Barbagallo M, et al. Arch Biochem Biophys 2007; 458: 40-7.

Molecular mechanism of hepatocyte nuclear factor 1b (Hnf1b)-related hypomagnesemia

S. Ferrè¹, G.J. Veenstra², R. Bouwmeester¹, J.G.J. Hoenderop¹, R.J.M. Bindels¹

¹ Department of Physiology, Radboud University Nijmegen Medical Centre; ² Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, The Netherlands

Background. Hepatocyte nuclear factor-1B (HNF-1B) is a transcription factor involved in embryonic development and tissue-specific gene expression in several organs, including the kidney. Hypomagnesemia is often reported in patients carrying HNF-1B defects. Interestingly, in silico analysis revealed HNF-1B binding sites in the FXYD2 gene, encoding two main variants of the γ-subunit of the Na+-K+-ATPase, namely γα and γβ. Previous studies described the γ-subunit to be involved in the Mg²⁺ reabsorption in the distal convoluted tubule (DCT) of the kidney. Aim of this study was to characterize the regulation of the alternative transcription of γα- and γβ- subunits by HNF-1B. Results. We demonstrated via two different reporter gene assays that HNF-1B specifically acts as an activator of the γα-subunit, whereas the γβ-subunit expression was not affected. Moreover, the HNF-1B mutations H69fsdelAC, H324S325fsdelCA, Y352finsA and K156E, previously identified in patients with hypomagnesemia, prevented transcription activation of γα-subunit via a dominant negative effect on wild type HNF1-B. By immunohistochemistry, it was shown that the γα- and γβ-subunits colocalize at the basolateral membrane of the DCT segment of mouse kidney. Conclusion. Our results appoint γα-subunit of the Na+-K+-ATPase as a new putative player in active Mg²⁺ reabsorption in the DCT.

References

Meij IC, et al. Nat Genetics 2000; 26: 265-6.

Nakayama M, et al. Pediatr Nephrol 2010; 25: 1073-9.

Adalat S, et al. J Am Soc Nephrol 2009; 20: 1123-31.

Degradation of magnesium alloys by “physiological” corrosion – setups and influencing factors

F. Feyerabend, D. Tie, L. Yang, N. Hort, K.U. Kainer, A. Schreyer, C. Vogt, H. Drücker, R. Willumeit

Department for Structural Research on Macromolecules, Helmholtz-Zentrum Geesthacht, Institute of Materials Research, Max-Planck-Str 1, D-21502 Geesthacht, Germany

Biodegradable metals like magnesium have a high potential as materials for non-permanent implants. However, there still is a lack of understanding of the degradation mechanisms, as well as a lack of correlation between the results of in vivo and in vitro corrosion measurements. This is most likely due to the choice of corrosion media not comparable to physiological fluids and/or the environmental conditions. The aim of this study was to compare different physiological solutions, the influence of proteins and oxygen and the introduction of defined environmental parameters (cell culture conditions, i.e. 37̊C, 5 % CO₂, 95% relative humidity). Cell culture conditions exhibited a huge influence on the formed corrosion products. Lower amount of oxygen in the gaseous phase is reducing the corrosion rates. Moreover, corrosion of magnesium samples is reducing the amount of available oxygen in solution nearly by half. The addition of proteins increased the amount of released magnesium in short term, but reduced the overall corrosion rate in the long term experiments. These results indicate that proteins may change the corrosion mechanism and that their presence also is one of the key factors to deal with in establishing in vitro systems for corrosion testing.

Association between magnesium, iron and zinc in critical care patient

D. Florea¹, J. Molina¹, E. Millan¹, L. Saez¹, A. Perez De La Cruz², E.M. Rodriguez², E. Planells¹

¹ Department of Physiology. Institute of Nutrition and Food Technology. University of Granada; ² Virgen De La Nieves Hospital, Granada, Spain

Introduction. Minerals like Zn, Fe, and Mg are essential nutrients for the cellular integrity. The lack of one or more of them may determine the big disorder on cell metabolism all this inducing dysfunction of multiples organs which is common in hypercatabolic critically ill patient. Objective. To assess the status of Zn, Fe and Mg in a group of ICU patients. Methodology. Multicentric observational study of 40 subjects, with mean age 58 from Granada area, Spain. Zn, Fe and Mg were analyzed by AAS in blood cells samples which were previously wet mineralized. Results. Zinc intake is 7.8±4.9, Mg 172.5±110.2, Fe 7.74±6.3 (mg/d). Mean of Zn in whole blood was 0.43±0.17 mg/dL in final of ICU period. In case of cellular Mg and Fe after 7 days of ICU period the mean values is 2.5±2.1 mg/dL, 59.7±30.3 mg/dL respectively. The results show statistical significance according whit Mg levels in blood cells at the end of the period monitored in critically ill patients (p=0.033), showing that patients with Mg deficiency, have 5.5 times more risk to suffer zinc deficiency. Conclusion. According to the results, the patients have no sufficient intake of Mg being necessary a nutritional assessment during ICU stay to know the status of minerals such as Zn, Fe and Mg which are key for critical ill patients avoiding gaps that may worsen their evolution.

References

Nahar Z, et al. Biol Trace Elem Res 2010; 133: 284-90.

Behara AS, et al. J Parenter Enteral Nutr 2008; 32: 113-9.

Magnesium concentration in cisplatin-sensitive and -resistant human ovarian cancer cell lines: preliminary results

C. Frassineti¹, D. Guerrieri¹, G. Marverti¹, F. Chiavolelli¹, S. De Lucia¹, G. Farruggia², S. Iotti²

¹ Università di Modena e Reggio Emilia; ² Università di Bologna, Italy

Among the mechanisms responsible for cisplatin (cDDP)-resistance, the higher plasma and mitochondrial membrane potential is central for the resistant phenotype [1] in a cDDP-resistant human ovarian cancer cell line (C13*) in comparison to its -sensitive parental line (2008). Since mitochondrial membrane potential Dym has been shown to be also involved in the homeostasis of intracellular magnesium concentration [2], we hypothesized that modifications of Mg²⁺ concentrations ([Mg]) and of their sub-cellular distribution, might be involved in the occurrence of cDDP- resistance. ICP, AA and fluorescence studies revealed a higher total [Mg] in C13* than in 2008 cells with a ratio [Mg]C13/[Mg]2008=1.3. RH4 cells (reverted from C13* cells by exposure to the lipophilic cationic mitochondrial poison rhodamine 123) showed a [Mg] as well as Dym and sensitivity to cDDP intermediate between the two cell lines. On the contrary, another couple of cDDP-sensitive and –resistant human ovarian cancer cell lines (A2780 and A2780-CP) showed a ratio of 0.8 which might be the consequence of the lower mitochondrial mass in the resistant cells, compared to the sensitive ones [3]. In addition, we noticed a cDDP dose-dependent increase of [Mg] in 2008 cells but not in C13* cells after 72 h-treatment; whereas the exposure to compounds that inhibit cell growth also by affecting mitochondrial functionality caused a concentration-dependent decrease of [Mg] in both lines [4]. All together, these preliminary studies suggest a causal relationship between total intracellular [Mg] and mitochondrial alterations raised during the selection of cDDP-resistance phenotype.

References

1. Zinkewich-Péotti K, Adrews PA. Cancer Res 1992; 52: 1902-6.

2. Kubota T, et al. Biochim Biophys Acta 2005; 1744 : 19-28.

3. Hunakova L, et al. Neoplasma 2007; 54 : 541-8.

4. Marverti G, et al. Invest New Drugs 2011; 29: 73-86.

Therapeutic hypothermia (selective head cooling) enhanced by MgSO₄ for hypoxic-ischemic encephalopathy in the neonates - first polish experience

E. Gulczynska^1,2, B. Sobolewska³, T. Talar³, M. Nowiczewski³, B. Cyranowicz³, L. Zylinska⁴

¹ Department of Neonatology, Research Institute of Polish Mother Health Center; ² Department of Neonatology, Medical University of Lodz; ³ Research Institute of Polish Mother Health Center; ⁴ Department of Neurobiochemistry, Medical University of Lodz, Lodz, Poland

At present therapeutic hypothermia has become a standard care among newborn with HIE. Since planning new trials with a control group – without hypothermia is considered as unethical, further studies should be focused (focus) on the possibility of the enhancement of beneficial effect of hypothermia by an additional effect of some neuroprotective drugs. Materials and methods: At Research Institute of PMHC 15 newborn with HIE were enrolled to the therapeutic hypothermia program. The Apgar score was ≤5@ 10 min, mean BBW 3,230 g (2,420-4,070) and GA 38,8 weeks (36-41). A moderate or severe brain injury was confirmed by aEEG. The studied neonates were underwent the method of selective head cooling. According to previously applied protocol, MgSO4 infusion -250 mg/kg was also given. All the neonates were examined by NMR Results: In the pilot group 2 patients died because of extremely poor condition. 8/15 babies (60%) received inotropic agents due to moderate hypotension. Several patients required single doses of diuretics owing to moderate low urine output, transient oliguria was observed in one neonate born with signs of hypovolemic shock and severe anemia (21%) caused by placenta abruption. In the same newborn severe thrombocytopenia (connected with significant volume of used donor blood) was noticed. None of the patients suffered from PPHN and none required iNO therapy. Conclusions: In our pilot group we didn’t observe significant adverse effect of combined therapy (hypothermia and MgSO4). At present we have decided to start RCT to assess the safety and efficiency of hypothermia enhanced by magnesium.

Disclosure. Supported by the grant number NN407547538 form Polish Ministry of Education and Science.

References

1. Marks K, et al. Isr Med Assoc J 2010; 12: 494-500.

2. Cooper DJ. Neonatal Netw 2011; 30: 29-35.

3. Thoresen M. J Pediatr 2011; 158 (2 Suppl.): e45-9.

Magnesium and potassium and the metabolism of Ca in the muscle cells

R. Hunger

Private Practice, Lürlibadstrasse 80, CH-7000 Chur, Switzerland

Mg and K regulate together the metabolism of Ca in the cell. Mg competes Ca from the Ca-ATPase and activates the fast Na/Ca-exchange molecule. K activates the fast Na/K-exchange and reduces the slower pump mechanisms. In the retraction test the length of the blood clot correlates to the velocity of the Ca export out of the cell. The length of the blood clot after the retraction correlates to the ATP consumption of the export of Ca. Mg helps the cell to export Ca as fast as possible. Mg competes Ca from the slower Ca-ATPase and activates the faster Na/Ca-exchange which delivers Na ions to the Na/K-ATPase. The Na/Ca-exchange pumps with ATP and one molecule conformation 3 Na ions out of the cell. With K⁺, Mg²⁺ and PO₄^3- it may exchange 1 Na against 1 K. With increasing concentration of K the exchange increases and increases its part compared to the pump mechanism. This leads to a decrease of the MP and the Ca channels are opened. At a K concentration of more the 6 mmol/L cardiac arrhythmias may occur. The Na/Ca exchange is crucial for the velocity of the export of Ca. The Na/Ca-exchange may exchanges up to 4,000 Cai against 12’000 Na/s. As the affinity of the Ca ATPase is higher to Ca than to the Na/Ca-exchange, Mg is needed to shift the export of Ca to the faster Na/Ca-exchange. The Na/K-ATPase is overloaded with Na. At K deficiency there may be an accumulation of Na and Ca.

Magnesium therapy in borderline hypertension

K. Kisters¹, B. Gremmler², O. Micke³, M. Hausberg⁴, H. Liebscher⁵

¹ Mrd. Clinic I, St. Anna Hospital, Hospitalstr. 19, 44649 Herne; ² Med. Clinic I, Marienhospital Bottrop; ³ Inst for Radiation Oncology, St. Franziskus Hospital Bielefeld; ⁴ Med. Clinic I, Stlsquädt. Clinic, Karlsruhe; ⁵ SHO-Mineralimbalancen, Berlin, Germany

A magnesium deficiency can play a pathogenetic role in the development of primary hypertension. Therefore we have measured plasma and intracellular Mg⁺⁺ levels in erythrocytes of 18 untreated borderline hypertensive patients, and in 35 untreated normotensive healthy subjects as controls. In hypertensive patients intracellular Mg⁺⁺ content was significantly lower (1.61 +0.09 mmol/L, mean +SD), than in controls (1.84 +0.14 mmol/l, p<0.05). After 12 - 15 weeks of an oral supplementation with 240-500 mg Mg⁺⁺/day, the erythrocyte Mg⁺⁺ content had increased significantly in the borderline hypertensive group (1.78 +0.11 mmol/L) (p<0.05). There was no significant difference between the normotensive and borderline hypertensive group in plasma Mg⁺⁺ concentrations (0.87 +0.13 vs 0.83 +0.17 mmol/l). Systolic and diastolic blood pressure values of the borderline hypertensive group normalized after oral Mg⁺⁺ administration (before therapy: 147.6 +8.5/82.2 +4.4 mmHg; after therapy: 137.2 +4.6/83.7 +3.4 mmHg, p<0.05). We conclude that M⁺⁺ deficient borderline hypertensives can benefit from an oral magnesium supplementation with regard to high blood pressure and quality of life.

References

Kisters K, et al. Magnes Res 1993; 4: 355-60.

Kisters K, et al. Clin Sci 1998; 95: 583-7.

Kisters K, et al. Magnes Res 2005; 18: 207-13.

Kisters K, et al. Clin Nephrol 2007; 68: 130-1

Kisters K, et al. Trace Elem Electrolyt 2011 (in press).

Intracellular concentration map of light elements in whole cells by complementary nano-probe methods

S. Lagomarsino¹, S. Iotti^3,9, G. Farruggia⁴, A. Cedola², V. Trapani⁵, M. Fratini², I. Bukreeva^2,10, A. Notargiacomo², L. Mastrototaro⁵, C. Marraccini³, A. Sorrentino^2,11, I. McNulty⁶, S. Vogt⁶, D. Legnini⁶, S. Kim⁶, A. Gianoncelli⁷, J.M. Maier⁸, F.I. Wolf⁵

¹ IPCF-CNR –uos Roma c/o Dip Fisica Universita’ “Sapienza”, P.le A. Moro, 2, Rome, Italy; ² IFN-CNR - V. Cineto Romano, 42, 00156 Rome, Italy; ³ Dipartimento di Medicina Interna, dell’Invecchiamento e Malattie Nefrologiche Università di Bologna, Via Massarenti, 9, 40138 Bologna, Italy; ⁴ Dipartimento di Biochimica « G. Moruzzi » Università di Bologna, Via Irnerio, 48, 40126 Bologna, Italy; ⁵ Istituto di Patologia Generale - Università Cattolica del Sacro Cuore, Facoltà di Medicina “A. Gemelli” L.go F. Vito, 1, 00168 Rome, Italy; ⁶ Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, USA; ⁷ SINCROTRONE TRIESTE, S.S.14 km 163.5 in Area Science Park, I-34149 Basovizza, Trieste, Italia; ⁸ Dipartimento di Scienze Cliniche, Università di Milano, Via GB Grassi 74, 20157 Milan, Italy; ⁹ Istituto Nazionale Biostrutture e Biosistemi, Rome, Italy; ¹⁰ Shubnikov Institute of Crystallography, Leninskii prospekt 59, Moscow, 119333 Russia; ¹¹ Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, V. della Vasca Navale, 84, 00146 Rome, Italy

We present an innovative approach to couple elemental and morphological information from individual cells with high spatial resolution, and apply it to the problem of localization of magnesium and other light elements. The integration of Scanning fluorescence X-ray microscopy (SFXM), and Atomic Force Microscopy provided the relative concentration map of the elements in a single whole dehydrated cell. In this specific experiment spatial resolution of the order of 0.3 μm was obtained, but better resolution is in principle possible. The concentration map shows significant differences with respect to the distribution map, indicating higher Mg concentration in cell periphery.

Impact of extracellular and intracellular magnesium-calcium balance on reactive oxygen species generation by phagocytic cells

P. Libako¹, J. Galli¹, W. Nowacki¹, A. Mazur²

¹ Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Science, Wroclaw, Poland; ² Human Nutrition Unit, INRA, Theix, France

Disturbances in the Mg homeostasis lead to multiple pathophysiological events including an altered immune and inflammatory response. The relationship between low Mg status (Mg-deficiency) and inflammation is strongly supported by experimental animal studies. Rodents fed with Mg-deficient diet developed classical signs of inflammation including peripheral vasodilatation, leukocytosis and abnormal phagocyte activity. Several works from our and other laboratories show that during the progression of Mg deficiency leukocytes and macrophages become primed/activated what is directly linked with exacerbated production of a wide range of inflammatory molecules and mediators [cytokines, nitric oxide (NO) and reactive oxygen species (ROS)]. The purpose of our study was to determine the consequence of Mg/Ca ratio alterations with regard to non-specific immune response, this focused on respiratory burst response. In vitro studies were performed on the selected cell lines: primary mouse neutrophils (Balb/C strain) and macrophage-like continuous cell line J774.E. cells were cultured in medium with different concentrations of magnesium (in the range of 0.1 mmol/L-5.0 mmol/L). Extracellular (verapamil) and/or intracellular (TMB-8) calcium (Ca) channel inhibitors were additionally applied into the experimental system to asses the role of extra- and intracellular Ca. The measurement of oxidative burst was carried out using the chemiluminescence assay by monitoring the oxidation of luminol by phagocyte-generated reactive oxygen species. Cells activated with phorbol 12-myristate 13-acetate (PMA) showed an enhanced respiratory burst under severe Mg–restricted conditions. Pivotal role of calcium in the inflammatory issue of Mg-deficiency was confirmed.

Tissue content of As in hypomagnesemic rats

J. Llopis¹, C. Lopez-Chaves¹, C. Bermudez-Peña², M. Montes-Bayon³, P. Aranda¹, A. Sanz-Medel³, C. Sanchez-Gonzalez¹

¹ Department of Physiology. Faculty of Pharmacy. University of Granada, Spain; ² Biomedical Research Unit, Mexican Social Security Institute, Durango, Mexico; ³ Physical and Analytical Chemistry, University of Oviedo, Spain

Purpose. There is a well known direct relation between nutritional Mg deficit, resistance to insulin [1]. On the other hand, there is accumulating evidence that the metabolism of several trace elements is altered in magnesium deficiency and that these nutrients might have specific roles in the pathogenesis and progress of this disease. Several studies had demonstrated that arsenic induces pancreatic β-cell apoptosis via the oxidative stress-regulated mitochondria-dependent and endoplasmic reticulum stress-triggered signaling pathways [2]. This study investigates the tissue distribution of As and its interactions with magnesium in healthy and in Mg-deficient rats, in order to determine the possible existence of metabolic interactions and its possible implication in the development of type 2 diabetes in hypomagnesemic rats. Methods. Two groups were used: Control and Mg deficient (164.4 mg Mg/kg food). The experiment had a duration of five weeks. We measured As levels in serum, skeletal muscle, kidney, liver, adipose tissue, heart and femur. Total metal content was analyzed by ICP-MS [3]. Results. No significant changes were observed in As serum levels. The Mg-deficient diet led to a fall in the Mg content in the kidney, liver and femur and increases in skeletal muscle, kidney, liver and femur content of As. Although no significant changes were observed in As concentration in heart and adipose tissue following consumption of the Mg-deficient diet, an upward tendency in these values was observed. Conclusion. The results show interactions between Mg and As in the tissues studied.

References

1. Guerrero-Romero F, et al. Eur J Clin Invest 2008; 38: 389-96.

2. Lu TH, et al. Toxicol Lett 2011; 201: 15-26.

3. Gracas Fernández K, et al. J Anal At Spectrom 2005; 20: 115-20.

Cytosolic [Mg²⁺] in three different muscle groups assessed in vivo by ³¹P-MRS in human healthy subjects

E. Malucelli¹, G. Layec², C. Testa¹, A. Tonson², D. Manners², D. Bendahan², S. Iotti¹

¹ Dipartimento di Medicina Interna, dell’Invecchiamento e Malattie Nefrologiche,Università di Bologna, Italy; ² Centre de Résonance Magnétique Biologique et Médicale, UMR CNRS 6612 - Faculté de Médecine de la Timone, 27, Bd J. Moulin, 13385 Marseille, France

Many relevant pathological conditions, such as cardiovascular diseases, essential hypertension, diabetes mellitus, metabolic syndrome, neurological disorders [1-3] are associated with reduced Mg availability and/or increased excretion either at a systemic level or in specific tissues. Although the clinical effects of variations in serum [Mg²⁺] have not been widely recognised, in routine clinical practice [Mg²⁺] is assayed in serum and not directly in tissues. ³¹P-MRS offers the unique opportunity to measure in vivo [Mg²⁺] in several tissues [4, 5]. Due to the chemical equilibrium between the major Mg fraction bound to ATP and the free Mg fraction, the chemical shift of ATP signals is sensitive to free [Mg²⁺]. On the basis of appropriate calibration the [Mg²⁺] has been measured in skeletal muscle during standardized rest-exercise-recovery protocols [5]. However, the variability among different exercising muscles has not been reported. The aim of this study was to assess and compare the [Mg²⁺] in calf, quadriceps and forearm muscles in 8 healthy control subjects using ³¹P-MRS. We measured significant differences in resting muscles (calf 0.37±0.03, quadriceps 0.15±0.01 and forearm 0.23±0.02 mM). Studies are in progress to investigate [Mg²⁺] changes during exercise in the different muscle groups and how they correlate with different fiber types.

References

1. Toyuz RM. Front Biosci 2004; 1: 1278.

2. Barbagallo M, Dominguez LJ. Arch Biochem Biophys 2007; 458: 40.

3. Barbiroli B, et al. J Cerebr Blood Flow Metab 1999; 19: 528.

4. Iotti S, et al. NMR Biomed 1996; 9: 24.

5. Iotti S, et al. Magnetic Resonance Imaging 2000; 18: 607.

The modification of blood magnesium and occupational exposure to lead

M. Margineanu, E. Danulescu, C. Croitoru, F. Gradinariu

National Institute of Public Health, Public Health Center Iasi, Romania

The impact of socioeconomic condition and occupational exposure to lead with the metabolism of Calcium (Ca) and Magnesium (Mg) is an issue that occurs with high frequency in the explanation of the toxic's innermost mechanism. The aim of our study was to determine the changes of Mg²⁺ serum levels during occupational exposure to lead in a porcelain painting factory. MATERIALS AND METHOD: A cross-sectional study was performed. The exposed group consisted of 150 persons with mean age of 32 years and mean length of service (LS) 14.5 years. We investigated: the phases of the technological processes; the level of lead in the air of workplaces. The estimation of lead exposure impact upon health status has been carried out by clinical and paraclinical investigations. Blood lead, urinary lead, urinary deltaaminolevulinic and serum magnesium were determined. The interpretation of the results was realized compared with an adequate control group. RESULTS AND CONCLUSIONS: The concentration of lead in the air of workplaces was 2 times higher than the Toxic Limit Value (TLV). Many people were over 10 year professional experience. Correlation between Pb-s (as indicator of exposure) >65 mg/ml and DALA-u (as indicator of biological effect) >20mg% confirms the suspicion of high absorption of lead in 80% of exposed persons. Concentration of serum Mg had levels between 1.3-2.3 mg% (67% of the subjects had low values). The statistical analysis points out a decrease of serum Mg levels with statistically significant from the control group. There was an inverse correlation between serum Mg²⁺ and the frequency of spasmophilia (r=-0.3819, p=0.000). Correlation between LS and serum Mg levels was significant at p=0.05. 95% of subjects were diagnosed with two or more syndromes, due to the action of lead on the body. The most frequent main syndromes were chronic digestive syndrome: 44% (persons that prefer to consume products reach in alimentary fibers present a low incidence of digestive disturbances); astenic vegetative syndrome (41%); sensorial-motor polineuropathies (15%). The high number of cases with low levels of serum Mg²⁺ allow us to conclude that exposure to lead interferes with Mg²⁺ metabolism. The study confirms the hypothesis of the competitive effect between the alimentary fibers from the diet and intestinal lead. This suggests that alimentary fibers have a protective role for persons chronically exposed to Pb. Monitoring the work environment and health status are efficient means of prevention.

References

Tefas L, et al. J Med Pharm 1997; 3 (Suppl. 1): 32-9.

Boyden JD, et al. J Nutr 1995; 125: 990-1002.

8-hydroxyquinoline derivatives as fluorescent sensors for total intracellular magnesium

C. Marraccini¹, G. Farruggia², M. Sgarzi³, M. Lombardo³, C. Trombini³, S. Iotti¹

¹ Dipartimento di Medicina Interna dell’Invecchiamento e Malattie Nefrologiche University of Bologna; ² Dipartimento di Biochimica University of Bologna; ³ Dipartimento di Chimica University of Bologna, Italy

Although magnesium is essential for a number of cellular processes such as proliferation and death, its distribution and intracellular compartmentalization have not yet been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular magnesium distribution. For this reason, particular interest has been recently demonstrated by a family of fluorescent molecules, the DCHQ series (Diaza-18-Crown-16 8-Hydroxyquinoline), that have shown an affinity and specificity for magnesium higher than all the other commercial probes, thus permitting the detection of the total intracellular magnesium [1]. The synthesis of the DCHQ compounds has been optimized by using microwave heating [2]: this approach allowed us to easily modify the basic structure with the introduction of various functional groups to obtain a number of derivatives with improved features of fluorescence, uptake and localization with respect to the original compound (DCHQ1). Fluorescence quantum yield has been enhanced via introduction of aromatic side groups (DCHQ7 and 13), that contemporary conferred improved features of uptake, since the probes are highly retained inside the cells after washings. Enhanced uptake has also been achieved with DCHQ12, a DCHQ-AM derivative that is recognized by the intracellular esterases. DCHQ11, with two long hydrophobic side chains, instead, allowed a better staining of the membranes due to its high affinity to the lipophilic environments. Results show the potentiality of these new fluorescent probes in providing novel insight in the study of intracellular magnesium homeostasis.

References

1. Farruggia G, et al. J Am Chem Soc 2006; 128: 344-50.

2. Farruggia G, et al. J Org Chem 2010; 75: 6275-8.

Effect of extracellular magnesium availability on the magnesium-specific mitochondrial channel MRS2 and cell growth in normal and tumor cells

L. Mastrototaro, V. Trapani, M. Di Nino, A. Boninsegna, A. Cittadini, F.I. Wolf

Universita’ Cattolica del Sacro Cuore, Roma, Italy

Magnesium is the most abundant intracellular divalent cation playing an important role in many processes, such as proliferation, cell division and apoptosis; in mitochondria, magnesium is a key factor of the ATP-synthesizing machinery. Proliferating cells require more magnesium than resting ones, and the lack of magnesium or defects in magnesium transport affect the proliferation rate, which can be resumed by magnesium supplementation. The MRS2 gene encodes a mitochondrial protein belonging to the CorA metal ion transporter family. The existing data support the notion that the MRS2 is the major transport protein for Mg (+) uptake into mitochondria and that its expression is essential for the maintenance of respiratory complex I and cell viability. A recent study shows an association between multidrug resistance (MDR) and MRS2 expression and in particular it seems that MRS2 can protect gastric cancer cells from adriamicin-induced apoptosis and promote their growth [1]. Using our model of mouse mammary epithelial cells HC11 grown in different Mg containing media (0.5 mM, 0.05 mM, 40 mM) [2], we investigated the modulation of MRS2 expression by Mg availability. We found that HC11 cells grown in high Mg, up-regulate MRS2 protein and show a stronger resistance to apoptosis after doxorubicin treatment compared to control cells. In addition, the MRS2 expression is rapidly up-modulated in control HC11 cells acutely supplemented with excess Mg (10mM) for 24h. Furthermore, by using three different strains of tumour cells sensitive and resistant to chemotherapeutic drugs, we found an over-expression of MRS2 protein in all the resistant ones. These results suggest that MRS2 expression: 1) is regulated by magnesium availability and 2) is correlated to MDR phenotype in cancer cells. The exact role of MRS2 in the regulation of cell growth and apoptosis by magnesium needs to be further investigated.

References

1. Chen Y, et al. Cancer Biol Ther 2009; 8: 607-14.

2. Wolf F, et al. J Cell Physiol 2010; 222: 374-81.

Influence of a magnesium supplementation in mineral and lipidic status in high performance athlete

J. Molina¹, J.M. Molina², L.J. Chirosa², D. Florea¹, L. Sáez¹, E. Millán¹, B. López-González¹, E. Planells¹

¹ Physiology Department, Institute of Nutrition and Food Technology, University of Granada; ² Physical Education Department, Faculty of Sport Sciences, University of Granada, Spain

Introduction. Magnesium is involved in more than 300 essentials metabolic reactions. Magnesium deficiency has been shown to decrease physical performance and worsening of cardiovascular disease. Magnesium levels are increased with high intensity exercises and short duration, but decrease with exhaustion in prolonged exercise. Objective. To study the effect of a magnesium supplementation in mineral a lipidic status in high performance athletes. Method. 14 professional handball players more than 18 years, were evaluated during 4 months of competition and daily supplemented for initial 2 months with 100 mg of Mg. Magnesium intake and plasma levels were known at initial (control 1), two months (control 2) and four months (control 3), by 72h recall and Nutriber^® software, and AAS, respectively. Results. 28.6% of subjects were initially below two thirds of the RDAs. This value reduced to 7.1% of subjects in controls 2 and 3. However, plasma levels were normal for all subjects in each control. In plasma levels, there were statistically significant differences (p<0.05) of magnesium between the control 1 and control 2. Magnesium was significantly negative correlated with cholesterol, LDL (p<0.05), and calcium (p<0.01). Conclusion. High performance athlete needs to control magnesium levels during the competition period to keep a correct nutritional balance, monitoring possible mineral alterations caused by magnesium deficiency4 that may compromise performance and health.

References

Cinar V, et al. Biol Trace Elem Res 2007; 119: 97-102.

Lukaski HC. Nutrition 2004; 20: 632-44.

Berryman CE, et al. Nutr Rev 2011; 69; 171-85.

Planells E, et al. Br J Clin Nutr 1994; 72: 315-23.

New proposition of ADHD treatment using magnesium in children

M. Mousain-Bosc, C. Siatka

Ecole de l’ADN, 19 Grand’rue 30000 Nîmes, France

ADHD, hyperactivity, impulsivity, attention deficit, is diagnosed in 3 to 5 % of the young population. It is a huge problem of public health in Europe. It has been shown that hyperactivity and learning disorder are linked with magnesium deficiency. A single magnesium supplementation, up to 6 mg /kg /day for a period of 4 to 12 weeks, allows to observe an improvement of all the ADHD symptoms. Actually, the most common medication is the use of psycho-stimulant as methylphenidate. This kind of treatment listed as stupefacient, shows strong side effects at short term but also at long term as, growth retardation, psychotic disorders, vascular heart diseases and risk of sudden death. European health regulations concerning children treatment disapprove the use of such drugs. In first intention, magnesium treatment should be strongly recommended in ADHD children.

Magnesium concentration in breast milk of women treated with oral steroidal contraceptives

M. Nechifor¹, D. Urzica², C. Gales³

¹ Department of Pharmacology “Gr.T.Popa” University of Medicine and Pharmacy, Iasi; ² “Cuza-Voda” Clinic Obstetrics-Gynecology Hospital, Iasi, Romania; ³ Department of Histology “ Gr.T.Popa” University of Medicine and Pharmacy, Iasi, Romania

The plasma and breast milk magnesium concentration was determined in mothers that were using oral steroidal contraceptives during lactation. The start of oral contraception was three months after birth. The study was performed on two groups of breast feeding mothers which received oral contraception and on a group of control 15 mothers (which don’t received oral contraception). The first group (17) received daily combination pill (levonorgestrel 0.15 mg + ethinylestradiol 0.03mg) and the second group received mini-pill norethindone 0.35mg .The total magnesium concentration was determined before the start of contraception and after 30days of contraception. During this time the mothers didn’t received other drugs. In all cases the children were healthy .The obtained data were statistic interpreted .The results showed that the contraceptive drugs do not modify the breast milk magnesium concentration (23.8±2.7 mg/L before treatment vs 24.7±3.1 mg/L after in first group NS) and 24.2±3.9 mg/L before contraception vs 27.5±2.9 mg/L after 30 days of contraception NS in the second group).There were no differences between control group breast milk t-magnesium concentration (28.21±3.5mg/L) and the groups which received steroidal contraception. The oral contraception used didn’t change the plasma t-magnesium concentration.

Reference

Schiappacasse V, et al. Contraception 2002; 66: 57-65.

The human “magnesome”: how to detect human proteins that can bind Mg ions

D. Piovesan, R. Casadio

Biocomputing Group, Department of Biology and Health Science and Technologies-CIRI, University of Bologna, Italy

How many human proteins bind Mg ions? We addressed this question computationally with our BAR-PLUS (BAR+), a non hierarchical clustering method that relies on the pair wise sequence comparison of about 14 millions proteins from over 300,000 species, of whom 998 are complete proteomes and include Homo sapiens [1]. All the sequences cluster in over 900,000 clusters with the constraints that their sequences identity is ≥40% on ≥90% of the alignment length. From this they can inherit from the cluster in a validated manner functional and structural annotation (PDB+/SCOP+/Pfam+/GOterms+/Ligands). With our procedure we find that 2,402 human proteins bind Mg ions in 88 clusters and another 1,140 in 161 clusters bind cofactors through Mg ions. Some 30% of all the sequences are annotated for the first time as endowed with putative Mg binding sites. A cell localisation of the 3,542 Mg binding proteins (the human Magnesome) is developed considering that the most populated Cell Components are: Intracellular part, Endomembrane system, Cell periphery, Protein complex. In turn the most populated Biological Processes are: Cellular metabolic process, Primary metabolic process, Multicellular organismal development, Macromolecule metabolic process, Nitrogen compound metabolic process, Small molecule metabolic process, Anatomical structure development, Cell cycle, Cell death. The most populated Molecular Functions are: Hydrolase activity, Transferase activity, Nucleotide binding, Ion Binding, Protein binding, Oxideraductase activity, Signal transducer activity, Isomerase activity. We also characterise typical Mg binding signatures useful in annotating Mg binding sites from protein sequences.

Reference

1. Piovesan et al. NAR 2011 (in press).

TRPM7 channel-kinase and Mg²⁺ homeostasis in stem cells.

A. Ryazanov, L. Ryazanova

Robert Wood Johnson Medical School, UMDNJ, New Jersey, USA

We have recently generated TRPM7 kinase-deficient mice and demonstrated that TRPM7 is essential for the control of cellular and whole body magnesium homeostasis [1]. We also generated mice with the deletion of both channel and kinase domain of TRPM7. Embryonic stem cells with disrupted TRPM7 gene were obtained. We found that embryonic stem cells lacking TRPM7 gene (TRPM7-/-) displayed a growth arrest phenotype that can be completely rescued by magnesium supplementation. Interestingly TRPM7-/- embryonic stem cells incubated at 1 mM extracellular Mg²⁺ can be maintained for several days in a quiescent state without loss of cell viability. Quiescent TRPM7-/- embryonic stem cells did not undergo spontaneous differentiation and can be induced to proliferate by additional magnesium supplementation. Our results suggest that TRPM7 plays an essential role in proliferation of stem cells most likely by mediating Mg²⁺ influx that is necessary for the progression through G1 phase of the cell cycle. A novel model describing the interrelationship between TRPM7, Mg²⁺ homeostasis and cell cycle control is discussed.

Reference

1. Ryazanova LV, et al. Nature Communications 2010; 1: 101.

Risk groups for magnesium deficiency among females

R. Rylander¹, J. Vormann²

¹ BioFact Environmental Health Research Center, Sweden; ² Institute for Prevention and Nutrition, Ismaning/Munich, Germany

Magnesium deficiency is related to health risks such as arrythmia, cardiovascular disease, and muscular contractions. Beneficial effects of intervention with magnesium have demonstrated in some investigations but not in others. One reason for an absence of an effect could be a poor representation of risk groups in the population studied. Characteristics of two such risk groups is reported. 1. The relation between acid-base balance and excretion of magnesium was investigated in a sample of females > 50 years. There was a relation between the secretion of urea and magnesium. In the group with the highest excretion of urea (2nd quartile, >390 mmol) there was an inverse relation between blood pressure and the secretion of magnesium. 2. The urinary excretion of magnesium was measured among pregnant females. Blood pressure was recorded throughout the pregnancy. In the group with a high excretion of magnesium (>3.5 mmol, 2^nd quartile) in week 12, an increase in blood pressure was found in weeks 36-39. These examples demonstrate that groups at risk can be defined by the excretion of magnesium and other characteristics. Magnesium intervention studies with the aim to assess causality should be confined to such risk groups.

References

Rylander R, et al. Magnes Res 2009; 22: 1-4.

Nielsen FH, Rylander R. Arch Gynecol Obstet 2011; 283: 443-7.

Rylander R, et al. The acid-base balance and mineral excretion relationship – effects of mineral water intervention. (submitted).

Bioavailability of vanadium in magnesium-deficient rats

C. Sanchez-Gonzalez¹, C. Lopez-Chaves¹, C. Bermudez-Peña², M. Montes-Bayon³, P. Aranda¹, A. Sanz-Medel³, J. Llopis¹

¹ Department of Physiology. Faculty of Pharmacy. University of Granada, Spain; ² Biomedical Research Unit, Mexican Social Security Institute, Durango, Mexico; ³ Physical and Analytical Chemistry, University of Oviedo, Spain

Purpose. Vanadium is an element whose role as a micronutrient has yet to be fully clarified. The present study was undertaken to investigate the bioavailability of vanadium and its interactions with magnesium in healthy and in Mg-deficient rats, in order to determine its role as a micronutrient. Methods. Two groups were used: Control (456.4 mg Mg and 0.06 mg V/kg food) and Mg deficient (164.4 mg Mg/kg food and 0.06 mg V/kg food). The experiment had a duration of five weeks. We measured V and Mg in excreta, serum, skeletal muscle, kidney, liver, adipose tissue and femur. Results. Consumption of the Mg-deficient diet produced a significant reduction in urinary losses of V, increases in retained V and in R/I %, together with higher serum V levels. However, the absorption of V in the Mg-deficient rats was similar to that observed in the control animals. Conclusion. In the present study the Mg deficient diet was associated with reduced V absorption. These results highlight the existence of interactions between Mg and V in the digestive and renal system.

References

Bogden JD, et al. J Nutr 1982; 112: 2279-85.

Nielsen FH. Present Knowledge in Nutrition. Washington, DC: ILSI Press, 1996: 364-70.

Thompson KH, et al. J Inorganic Biochem 2006: 1925-35.

Tissue distribution of vanadium in magnesium-deficient rats

C. Sanchez-Gonzalez¹, C. Lopez-Chaves¹, C. Bermudez-Peña², M. Montes-Bayon³, P. Aranda¹, A. Sanz-Medel³, J. Llopis¹

¹ Department of Physiology, Faculty of Pharmacy, University of Granada, Spain; ² Biomedical Research Unit, Mexican Social Security Institute, Durango, Mexico; ³ Physical and Analytical Chemistry, University of Oviedo, Spain

Purpose. The fact that Mg and V participate in glucose metabolism, intervening in the same processes, led us to consider that these two elements could be related and be acting jointly in the metabolism of carbohydrates. This study investigates the tissue distribution of vanadium and its interactions with magnesium in healthy and in Mg-deficient rats, in order to determine the possible existence of metabolic interactions. Methods. Two groups were used: Control and Mg deficient (164.4 mg Mg/kg food). The experiment had a duration of five weeks. We measured V and Mg in excreta, serum and tissues. Results. No significant changes were observed in Mg concentration in the skeletal muscle and adipose tissue in Mg-deficient rats. There was a significant reduction in Mg in the kidney, liver and femur. The Mg deficient diet raised V content in the liver, kidney and femur. Conclusion. The intake of a Mg-deficient diet led to a fall in the Mg content in the kidney, liver and femur, and an increase in the V content in the same tissues. It is interesting to observe that V deposits were only produced in the organs where Mg had been depleted, which indicates the existence of possible interactions between these elements in tissues.

References

Aranda P, et al. Ann Nutr Metab 1990; 34: 244-51.

Thompson KH, et al. J Inorganic Biochem 2006; 100: 1925-35.

Tubafart S, et al. Med Chem Res 2010; 19: 854-63.

Magnesium intake is associated with strength performance in elite basketball, handball and volleyball players

D.A. Santos, C. Matias, C.P. Monteiro, A.M. Silva, P.M. Rocha, L.B. Sardinha, M.J. Laires

Exercise and Health Laboratory, Faculty Human Kinetics, Technical University of Lisbon, Portugal; Physiology and Biochemistry Laboratory, Faculty Human Kinetics, Technical University of Lisbon, Portugal; CIPER - Centro Interdisciplinar de Performance Humana, Faculty Human Kinetics, Technical University of Lisbon, Portugal

We aim to understand the impact of magnesium intake on strength in a pre-season training period in elite male basketball (n=11), handball (n=7), and volleyball (n=8) players (age 20.1±4.9 yrs). Mg intake (Mg) was assessed from 7-day diet record analyzed with Food Processor SQL. Strength tests consisted in maximal isometric trunk flexion, extension, and rotation, maximal handgrip, squat (SJ) and countermovement abalakov (CMJA) jump, and maximal isokinetic knee extension and flexion peak torques: velocities, 60°/s(PT60), and 180°/s(PT180). Pearson and Spearman correlations were performed and significance was set at p<0.1. Data were analyzed with SPSS v19.0. Mg intake in these athletes was 244.7±78.8 mg/day, significantly lower than the RDA (p<0.001). Mg was directly associated with trunk flexion (r=0.44; p=0.025), trunk rotation (r=0.357; p=0.073), handgrip maximal strength (r=0.468; p=0.016); jumping performance tests: SJ (r=0.390; p=0.081) and CMJA (r=0.539; p=0.012), and all isokinetic strength variables: PT60(extension) (r=0.460; p=0.020), PT60(flexion) (r=0.641; p=0.001), PT180(extension) (r=0.741; p<0.001) and PT180(flexion) (r=0.797; p<0.001). These athletes consume diets with less magnesium than the recommended which may compromise the adequate cellular availability of magnesium. The observed direct correlations between Mg and muscle strength performance may result from the important role of magnesium in energetic metabolism, transmembrane transport and muscle contraction and relaxation. Whether magnesium supplementation will improve performance remains to be shown.

References

Matias C, et al. Magnes Res 2010; 23: 138-41.

New diaza-18-crown-6 hydroxyquinoline (DCHQ) derivatives as fluorescent chemosensors for magnesium ions in bio-medical applications

M. Sgarzi¹, L. Prodi¹, N. Zaccheroni¹, M. Montalti¹, C. Trombini¹, M. Lombardo¹, C. Marraccini²

¹ Università di Bologna - Dipartimento di Chimica “G.Ciamician”; ² Università di Bologna - Dipartimento di Medicina Interna, dell’Invecchiamento e Malattie Nefrologiche, Italy

Magnesium is the most important divalent cation in cells, not only for its abundance, but also because it is involved in the regulation of hundreds of enzymatic processes and different functions, in cell proliferation and death. Despite its key role, information about its intracellular regulation is still poor, due to the lack of appropriate detection methods. Some diaza-18-crown-6 hydroxyquinoline (DCHQ) derivatives were previously reported for total Mg²⁺ assessment and as effective fluorescent Mg²⁺ probes [1]. Here we present new DCHQ derivatives designed and synthesized, following an innovative strategy [2], in order to modulate cellular uptake, membrane selectivity and to optimize fluorescence quantum yield when they are injected in cells.

References

1. Farruggia G, et al. J Am Chem Soc 2006; 128: 344-50

2. Farruggia G, et al. J Org Chem 2010; 75: 6275-8

The influence of maternal magnesium serum concentration on anthropometric parameters and postpartum status of newborn babies

B. Sobolewska-Nowakowska

Department of Neonatology, Polish Mothers’ Research Institute, Lodz, Poland

Objective. This study investigated the influence of serum magnesium concentration in pregnant women on afterbirth status and anthropometric parameters of newborns. Methods. We investigated 500 healthy pregnant women and their newborns. Materials were analyzed by flame absorption atomic spectrometry. Serum magnesium were measured I: 8 - 12 GA, II: 30-34 GA, III: umbilical cord blood. Neonate status was assessed by Apgar score at 5’. Body weight, height, head and chest circumference were measured after birth. Results. Newborns: GA 34.5-42 weeks (mean 39.2), Apgar 6-10 points (mean 9.4), body birth weight 2,000-3,409 g (mean 3,409), height 45-64cm (mean 55), head circumference 30-39 cm (mean 34.5), chest circumference 26-39 cm (mean 34.3). Magnesium serum concentration I 0.75-2.7 (1.78), II 1.08-3.23 (1.73), III 1.06-3.26 (1.9) mg/dL. We found statistically significant correlation between serum magnesium and head circumference r=0.113 p.

A study of the effect of [Ca²⁺]/[Mg²⁺] ratio on the growth of osteoblastic cells

T. Theophanides, I. Anastassopoulou

National Technical University of Athens, Greece

The magnesium hydrated cation , [Mg(H₂O)₆]²⁺ is unique in biology because of its water coordinated hydration sphere's size and reactivity [1]. More than half of the total human body's content of magnesium is found in bones [2, 3]. It plays a significant role in biological systems and helps the bones to absorb calcium. On the other hand, Ca²⁺ cation occurs in all metabolic pathways in advanced biological systems. Calcium cation possesses a central role in cellular functions and is also known as a major constituent of the skeleton, in the form of hydroxyapatite. In this work human osteoblastic bone marrow cells were used to examine the influence of calcium signaling and its alterations to the bone. Human bone marrow was cultured in conditions that favour osteoblastic differentiation, however without using dexamethasone. The cells were cultured in a-MEM medium at the following [Ca²⁺]/[Mg²⁺] molecular ratios: 3/1, 3/2, 2/1, 1/1, 1/2 in unmodified medium and the cells were monitored for differences in their proliferation, viability and morphology. For different [Ca²⁺]/[Mg²⁺] culture medium ratios it was shown that each ratio provided a different proliferation pattern for osteoblastic cells. From the growth curves it was found that the [Ca²⁺]/[Mg²⁺] =3:2 ratio provides a growth pattern that resembles the typical growth curve with a plateau that maintains the same level for 15-25 days, while for the other ratios the growth curve was 10 days. It is interesting that this ratio of calcium to magnesium ratio of 3/2 happens to be also the natural ratio in cells as hydrated cations [4].

References

1. Theophanides T, et al. Μagnesium and water. In: Y. Rayssiguier, A. Mazur, J. Durlach, eds. Advances in Magnesium Research: Nutrition and Health. London: John Libbey & Co, Ltd: 219-25.

2. Anastassopoulou J, Theophanides T. The role of metal ions in biological systems and medicine. In: Kessissoglou DP, ed. Bioinorganic Chemistry. Kluwer Academic Publishers, 1995: 209-18.

3. Petra M, et al. Synchrotron micro-FT-IR spectroscopic evaluation of normal paediatric human bone. J Mol Structure 2005; 78: 101.

4. Theophanides T. Magnes Res 1996; 9: 259.

Mechanisms of doxorubicin resistance in mammary epithelial cells adapted to non-physiological extracellular magnesium availability

V. Trapani, L. Mastrototaro, M. Di Nino, A. Boninsegna, A. Cittadini, F.I. Wolf

Istituto di Patologia Generale, Facoltà di Medicina e Chirurgia “A. Gemelli”, Università Cattolica del Sacro Cuore, Rome, Italy

The development of multidrug resistance hampers the treatment of many human tumours. Multiple mechanisms may contribute to this phenomenon, but only few of them have been identified. Recent findings suggest that magnesium, a key component in cellular proliferation, might also play a role in the apoptotic process and in particular in the development of drug resistance. Indeed, expression of the mitochondrial magnesium channel mrs2 has been associated with doxorubicin resistance in a multidrug resistant gastric cancer cell line [1]. We sought to clarify the relationship between mrs2 expression and doxorubicin resistance in a model of mammary epithelial cells chronically adapted to grow in a low-magnesium (0.05 mM vs. 0.5 mM) or high-magnesium (30 mM) medium [2]. Work in our lab showed that high extracellular magnesium availability upregulates mrs2 expression and in parallel increases resistance to doxorubicin-induced apoptosis. We did not find any significant change in drug accumulation in high-magnesium cells in comparison to normal or low-magnesium cells, neither did we detect any difference in reactive oxygen species formation in the three cell lines. We are presently investigating whether adaptation to non-physiological magnesium availability and the consequent mrs2 modulation might affect the mitochondrial apoptotic pathway. The old concept of a possible network between drug sensitivity and mitochondrial function will be enriched by new findings clarifying whether mitochondrial magnesium has a coincidental role, or rather acts as a pivotal factor in the downstream signaling cascades leading to life versus death.

Disclosure. Work supported by PRIN 2007ZT39FN.

References

Chen Y, et al. Cancer Biol Ther 2009; 8: 607-14.

Wolf FI, et al. J Cell Physiol 2010; 222: 374-81.

Serum-magnesium paradox during repeated Mg-infusion in patients with Mg-depletion(MD)-a possible simple diagnostic tool in latent MD?

B. von Ehrlich¹, K. Kisters²

¹ Internal Medicine Practice; ² St. Anna Hospital Herne, Germany

Proof of Mg-depletion(MD) is crucial in patients with suspected MD-symptoms but borderline Mgs. This retrospective observational study reports 8 Mg-infusion(MI) courses/5 patients with MD-symptoms/codiagnosis, previous oral Mg-supplementation, ongoing symptoms, T0 Mgs below 0.85; 4/5>0.75mmol/L. We observed decrease of Mgs during first control (T1) after first series of MI compared to (T0)values in 7/8. This paradox was detected between 7-40 days, ranged Sigma 2,6-50.5 mmol Mg, single doses 3.15-9.45mmol Mg-glutamate in 50 ml NaCl 0.9% 30-40 min. No complications observed. 2 patients (Stress, Tinnitus) reported slight dizziness initially infused. No correlation between intensity of Mgs decrease and baseline Mgs. Discussion: We described case-reports of Mgs-decrease during oral Mg supplementation in diabetes 2004 and 2008. Song reported this phenomenon after 8 weeks oral supplementation of 12-20 mmol Mg/day [1]. Coincidence with clinical improvement (HR, Stress, Tinnitus, diabetic-control) gives rise to speculation that extra-to-intracellular shift (“sponge effect”) due to alternated compartmentalisation -intracellular depletion- is causal of decrease. To our knowledge the decrease of Mgs following parenteral supplementation has not jet been described. We suggest the quotient of two Mgs-determinations T1/T0 (“Sponge-effect quotient”).

References

1. Song, et al. Diab Med 2006; 23: 1050-6.

Inflammation pathways linked to impaired cardiac function during hypomagnesemia

W.B. Weglicki, I.T. Mak, J.J. Chmielinska, J.H. Kramer

Departments of Biochemistry and Molecular Biology, and Medicine, George Washington University Medical Center, Washington DC 20037, USA

In our studies of the complex systemic response of rodent models to dietary and drug-induced hypomagnesemia we found: oxidative and nitrosative stress, diminished antioxidant reserve, NMDA channel activation, elevated cytokines and neuropeptides, tissue infiltration of activated WBCs, loss of neutral endopeptidase activity, endotoxemia with increased CD14 expression, and, eventually, depressed cardiac contractility. In recent years using echocardiography we evaluated cardiac systolic and diastolic dysfunction with progressive hypomagnesemia. After only three weeks of severe dietary magnesium restriction, early significant diastolic dysfunction (p<0.05) appeared, and at five weeks reduced (p<0.05) ventricular fractional shortening occurred and this was partially reversed by NK1 receptor blockade. In other studies with normomagnesemic rats, EGFR blockade by a tyrosine kinase inhibitor, tyrphostin AG1478, moderately decreased circulating magnesium levels (35%) occurred at three weeks; after five weeks systolic dysfunction (p<0.05) became evident. All of the above cardiac responses were preceded by activation of the above inflammatory processes. We submit that these experimental animal findings may be particularly relevant, in those cardiac and cancer patients with preexisting hypomagnesemia who require prolonged therapy with agents that promote additional magnesium wasting. Also, since drug-related gastrointestinal side effects may preclude effective oral magnesium replacement, additional therapies to block some of the above key inflammatory pathways leading to cardiac dysfunction may be required.

Disclosure. Supported by NIH RO1-HL-62282, NIH 1R01 HL66226.