RICERCA ITALIANA     

Research program

ADVANCED TOOLS OF MOLECULAR IMAGING AND GENETIC ENGINEERING TO STUDY DISTRIBUTION AND DYNAMICS OF CELL MAGNESIUM: NOVEL APPROACHES TO LINK MAGNESIUM HOMEOSTASIS AND CELL FUNCTIONS.

University Co-ordinator

Università degli Studi di BOLOGNA - MEDICINA CLINICA E BIOTECNOLOGIA APPLICATA - ()

Research Unit Leader

Stefano Iotti

Description

The aim of this research unit is to study a new class of fluorescent molecules based on diaza-18-crown-6 appended with two 8-hydroxyquinoline groups (DCHQ) bearing different substituents in position 5. In particular, two molecules showed high affinity and specificity for magnesium, bearing in position 5 a H (DCHQ1) or a Cl (DCHQ2). This sensors for magnesium showed no interference with calcium, as displayed by commercial probes such as Mag-Fura and Mag-fluo. DCHQ1 has been previously characterized: it is well tolerated by cells, allows to evaluate intracellular total magnesium and monitor fast intracellular magnesium transients after mitochondrial stimuli. This molecule has the necessary requirements for filling the gap of the knowledge on the magnesium homeostasis, and could be a promising tool in the study of this ion in different physiological and pathological conditions, and an alternative to AAS in total intracellular magnesium evaluation, especially when only biological specimens of reduced dimensions are available.
The objectives of the first part of this project (first and second semester) are the photochemical characterization of new derivatives, such as DCHQ2 and new derivatives which can be entrapped within the cells. One of these (DCHQ3) is being synthesized by the group of Prof. Paul Savage, Brigham Young University, Provo, Utah, who collaborate with the participants of different U.O. of this project. These molecules will be evaluated for their applicability to spectrophotometric, microscopic and cytofluorimetric assays of magnesium concentration and its intracellular fluctuations in normal and transformed cells.
In the second part of the project (third semester) we plan to extend the application of the dye DCHQ1 to the evaluation of the total intracellular magnesium to blood cells, comparing the measures obtained with this dye in erythrocytes, platelets and lymphocytes of healthy donors with those obtained by AAS. Similar determinations will be performed with other members of DCHQ family if, in the first phase of the project, they showed suitable characteristics similar to DCHQ1.
Our group is currently involved in a study on the tissue distribution of magnesium performed by different spectroscopic techniques, as 31P-MRS and standard techniques (AAS, colorimetry and Mag-Fura fluorimetry) in patients affected by the chronic intestinal failure (CIF) in home parenteral nutrition. A deficit in plasma magnesium levels has been reported as a condition frequently occurring in patients affected by CIF. However, it is not yet clarified how this alteration influences the intracellular levels of total and free intracellular magnesium in different tissues. Furthermore, in this study an important analytical limits has been represented by the reduced size of blood samples. This constrain, due to the clinical characteristics of these patients and to obvious ethical issues, made impracticable the use of AAS to assess total magnesium measurements in blood cells of these samples. Therefore, DCHQ1 probe and possibly the other members of DCHQ family, can represent a valid alternative to overcome this problem.
Then, in CIF patients, the magnesium concentration will be measured, in different tissues, with two different spectroscopic techniques (fourth semester). In particular, free cytosolic Mg2+ will be measured in brain and in skeletal muscle by 31P-MRS, and in blood cells (lymphocytes, erythrocytes and platelets) by fluorescence spectroscopy and flow cytometry by commercial probes (Mag-fura and/or Mag-fluo). In addition, total magnesium will be measured by DCHQ probes in blood samples. Therefore, 20 CIF patients will be enrolled by the “Dipartimento di Medicina Interna e Gastroenterologia, Centro Regionale di Riferimento per Insufficienza Intestinale Cronica Benigna - Università di Bologna” together with 20 age- and sex-matched healthy subjects as controls enrolled by our research unit.
In each subject the blood sample, for the fluorimetric assays, will be taken after a fasted overnight, and muscle and brain 31P MRS examination performed the same day.

Experimental protocols

Atomic absorption spectroscopy.
Magnesium will be assayed on cellular acid extracts by a spectrometer mod. S11, Instrumentation Laboratory, USA equipped with an air/acetylene flame.

Fluorimetric assays.
Static spectrofluorimetry and time resolved spectrofluorimetry will be performed on a PTI Quanta Master C60/2000 and on a IBH system 5000 U respectively. To evaluate the membrane contribution to DCHQ fluorescence, vesicles (obtained by extrusion or sonication) with different phospholipid compositions will be used as model membranes.
Intracellular magnesium concentration will be determined by: i) the commercial probes Mag-fura-2 Mag-fluo-4 (Molecular Probes) for free Mg2 (1); ii) DCHQ sensors for total magnesium (2).

Cytofluorimetric assays.
Cytofluorimetric assays will be performed on a Bryte-HS (BioRad) flow cytometer of the “Centro Interdipartimentale Ricerche Biotecnologiche” Università di Bologna, equipped by a 100 W Xe-Hg lamp for UV and visible excitation. This technique will be used to evaluate DCHQ dyes uptake kinetics, their fluorescence stability in the intracellular milieu and their potential cytotoxicity.
Mag-Fluo-4 will be used to measure intracellular Mg2+ by flow cytometry. To define the correct experimental protocols, intracellular calibration curves of the dyes will be performed on normal and trasformed cells and on lymphocytes, erithrocytes and platelets isolated from blood of healthy donors. These curves are made by changing the Mg2+ cellular concentration, permeabilizing the cells by ionophores and leaving magnesium to equilibrate with specific magnesium calibration buffers (3). To obtain a correct evaluation of Mg2+ concentration only in viable cells, the samples will be counterstained by Propidium Iodide (PI), which penetrates only in cells with altered membrane permeability, allowing to exclude dead cells (4). To discriminate Mg2+ in the different cellular compartments, partial permeabilization will be performed with digitonin, evaluating the residual fluorescence due to the dye complexed to Mg2+ in the different organelles (5). We will compare data obtained by fluorescence spectroscopy, which evaluates an average fluorescence in the examined samples, with data obtained by flow cytometry, which evaluates the fluorescence of the single cells, giving statistical distribution of fluorescence intensity of the population examined.

MRS
Brain and muscle 31P-MRS studies will be performed in a 1.5 Tesla clinical body magnet (General Electric, Milwaukee, Wisconsin, USA) available at “Azienda Ospedaliera di Bologna Policlinico S.Orsola-Malpighi” under an agreement with the University of Bologna, using a 8 cm diameter double-tune surface coil, tuned at the resonance frequencies of 1H (63.89 MHz) and 31P (25.88 MHz). 31P-MRS pulse sequences used will be FID-CSI and SPIN-ECHO-CSI with a repetition time (TR) of 5 s. Data will be processed using an Octane work-station (Silicon Graphics) and the software package j-MRUI (6) for spectra deconvolution and quantification. Brain and muscle free Mg2+ concentration will be assessed from the chemical shift of beta-ATP signal using the calibration curves implemented in the software “Magic BMC” (7,8).

Muscle 31P-MRS
31P-MRS will be performed on gastrocnemius muscle at rest. The region of interest will be selected acquiring axial T1-weigthed Spin-Echo images (repetition time TR = 500ms; Echo Time TE = 12ms, thickness = 4mm; gap = 1mm). The duration of the MR imaging studies will be less than 2 minutes. Surface coil will be placed directly on the skin beneath the calf and fixed by a vacuum pillow, then a slice will be selected to acquire signal mainly from the medial and lateral head. 64 FIDs will be acquired at rest to obtain a signal to noise ratio (S/N), measured on the beta-ATP peak, above 30 (9). Whole exam will last about 10 minutes.

Brain 31P-MRS
In this exam the subject will be placed in a supine position with head at the centre of the magnet. Phosphorus spectra will be acquired from the medial parietal occipital area by placing the surface coil directly on the skull in the occipital region, and fixed by a vacuum pillow. The region of interest (ROI) will be selected acquiring three-plane scout images and axial T1-weigthed Spin-Echo
images ( repetition time TR = 500ms; Echo Time TE = 12ms, thickness = 4mm; gap = 1mm). The duration of the MR imaging studies will be less than 4 minutes. Then 31P MRS spectra will be acquired from ROI averaging a number of FIDs to achieve a S/N of the beta-ATP above 9 (10).
Whole exam will last about 30 minutes.


The research project will articulate in the following work plan:
First semester:
Photochemical characterization, by means of UV-visible absorption spectroscopy, static and dynamic fluorescence spectroscopy, of DCHQ2 and DCHQ3 and their complexes with Mg2+ in aqueous solutions and in presence of model membrane, such as micelles and vesicles with different phospholipid compositions. The same measurements will be repeated with other metallic ions of biological relevance (Ca2+, Mn2+, Zn2+), to evaluate the possible interference. Absorption spectroscopy and static fluorescence spectroscopy will allow to determine the association constant of the dyes with Mg2+ in solution and in model membranes. All these results of this first part will form the experimental basis for the application of these dyes to advanced system of molecular imaging as single and double photon confocal microscopy, performed by the U.O. of Prof. Wolf, which in turn will provide the background for the X-ray microscopy performed by the U.O. of Prof. Lagomarsino.
Time resolved fluorescence spectroscopy will allow to evaluate the fluorescence lifetimes distribution of these probes in aqueous and lipofilic milieu. This will allow to evaluate the potential applicability of these probes to “Fluorescence Lifetime Imaging Microscopy” (FLIM) which could represent another tool in mapping the intracellular magnesium distribution.

Second semester:
1.Determination by static and dynamic fluorescence and flow cytometry of uptake kinetics and chemical stability of DCHQ probes in different cell types. The cellular models chosen in this phase will be mainly normal cells and stabilized cell lines (immortalized and tumour cell lines). In particular, the normal cells will be the endothelial cells supplied by the Prof. Maier U.O., while the stabilized lines will be leukemic cells HL60, epithelial cells (HC11) and connectival cells (Rat1). These cells are widely utilized in studies on the correlation between the intracellular magnesium content and cell proliferation, differentiation and death.
2.Determination of the analytical characteristics of DCHQ2 and DCHQ3 comparing the measures of magnesium obtained with these probes, to the evaluation of total intracellular magnesium obtained by AAS and of free cytosolic magnesium obtained by the commercial fluorescent dyes Mag-fluo-4 and Mag-fura-2.
3.Determination, by flow cytofluorimetric assays, of possible cytotoxycity of DCHQ probes and of their effects on cell functions.

Third semester:
Determination, by static spectrofluorimetry and by flow cytofluorimetry, in cells isolated from peripheral blood samples of healthy volunteers, of intracellular Mg2+, by commercial fluorescent dyes Mag-fluo 4 and Mag-fura 2, and of total intracellular magnesium, by AAS and DCHQ probes. These measures will be firstly performed on purified cell populations (lymphocytes, erythrocytes and platelets). Then, to limit blood sample manipulations, the applicability of these dyes to flow cytofluorimetric assays on whole blood samples will be evaluated, basing the indentification of cells subpopulation on light scattering signals and on the use of specific monoclonal antibodies.

Fourth semester:
Determination, by 31P-MRS, of cytosolic Mg2+ in skeletal muscle and in brain of healthy volunteers and in CIF patients in home parenteral nutrition. On the same subjects, in cells isolated from peripheral blood samples, both total and free intracellular magnesium will be determined by fluorescence spectroscopy and flow cytometry by using Mag-fluo 4, Mag-fura 2 and DCHQs. The same analysis will be repeated on blood cells at different purification steps.

References

1)The Handbook. A Guide to Fluorescent Probes and Labeling Technologies,10th ed,; Haugland, R. P., Ed.; Molecular Probes: Eugene, OR, 2005.
2)Farruggia G. et al. 8-Hydroxyquinoline derivatives as fluorescent sensors for magnesium in living cells, J. Am. Chem. Soc. 128 (2006) 344-350.
3)Watanabe M., et al. Intracellular calibration of the fluorescent Mg2+ indicator furaptra in rat ventricular myocytes. Pflugers Arch. 442:35-40, 2001
4)Rabinovitch P.S., et al. Studies of Cell Function in Current Protocols in Cytometry, JP Robinson managing editor, John Wyley and Son, 1997
5)Delva P. et al., Degan M, Pastori C, Faccini G, Lechi A. Glucose-induced alteration of intracellular ionized magnesium in human lymphocytes. Life Science 71:2119-2135, 2002.
6)http://carbon.uab.es/mrui/mruiHomePage.htm
7)Iotti S, et al. In vivo assessment of free magnesium concentration in human brain by 31P MRS. A new calibration curve based on a mathematical algorithm. NMR in Biomedicine 9:24-32, 1996.
8)Iotti S, et al. 'In Vivo 31P-MRS assessment of cytosolic [Mg2+] in the human skeletal muscle in different metabolic condition. Magnetic Resonance Imaging 18:607-614, 2000.
9)Iotti S. et al. In vivo assessment of mitochondrial functionality in human gastrocnemius muscle by 31P MRS. The role of pH in the evaluation of phosphocreatine and inorganic phosphate recoveries from exercise. NMR in Biomedicine 6:248-253, 1993
10)Barbiroli B., et al. Defective brain energy metabolism shown by in vivo 31P MR spectroscopy in 28 patients with mitochondrial cytopathies. Journal of Cerebral Blood Flow and Metabolism 13:469-474, 1993.