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  The Magnesium Project - Methods

Measurement of Magnesium
There are a number of methods available for measuring levels of Magnesium in samples.  One challenge is to determine levels without interference from other ions, since normally samples are mixtures of many ions.  The second and even greater challenge is to measure the amount of Mg that is free and available to participate in biochemical reactions.  Most methods give total Mg.  It has been only recently that an Ion Selective Electrode (ISE) has become available that will measure the free Mg++ (from Nova Biomedical in Waltham, MA).  This method however, while fine for body fluids that can be extracted (like blood or urine), it cannot be used on the cellular level.

Some scientists have developed colorimetric tests.  The following material is extracted from a draft document on cation selectivity and measurements.

A number of compounds are known to have a high affinity for various cations including for calcium, magnesium, manganese and zinc.  There are a variety of proteins that have special binding sites for metal ions but they have their own unique set of characteristics.  The present study is directed to compounds other than proteins, compounds that typically are of much smaller size than metal-binding proteins.  Among these are EDTA and EGTA that have acidic carboxyl groups and negatively polarized nitrogen atoms to which cations may be attracted.  Others have oxygen from hydroxyl or phosphate groups (for example pyrophosphate, orthophosphate, ATP/ADP, etc.).  Still others are compounds not prevalent in biological systems as indicated in the following list and elaborated upon in the text and tables below:

* 4-oxo-4H-quinolizine-3-carboxylates reported by Otten et al.

* 1,5-bis(2-hydroxyphenyl)-3-cyanoformazans cited in Kodak patents 4753890 and 5215925

* ortho-cresolphthalein complexones

* Arsenazo III-based methods

* Calmagite (3-hydroxy-4-[(2-hydroxy-5-methylphenyl)azo]-1-naphthalenesulfonic acid)

* Eriochrome Black T (3-hydroxy-4-[(1-hydroxy-2-naphthalenyl)azo]-7-nitro-1-naphthalene
                   sulfonic acid monosodium salt) (Denney US Pat. 4383043)

These latter compounds have various functional groups to which metal ions bind (including sulfonic).  In some instances the binding brings about a change in light absorption or a change in fluorescence that can be measured.

Other techniques include use of radioactive isotopes.  Radioactive isotopes are available for other ions - Ca, Na, K for membrane flux studies and binding.  However the radioactive isotope for Mg is fairly short lived and requires special neutron activation in a nuclear reactor.

 

Electrolyte Determination Methods

Type

Interferences

Ion Plasma

Optical

 Ca++

Atomic Absorption

Optical

 Mg++

UV-Visible spectrophotometry

Optical

Ca++, Zn?, Mn

EDTA Titration

Titration

Ca++, Zn?, Mn

Ion Selective Electrodes

electrochemical

 Ca++, pH

   

 

Measurements of Other Cations
Ion Selective Electrodes are available for H+, Na+, K+, and Ca++ and many years of research has been carried out using such techniques.  An advantage of these techniques is that they measure the actual chemical activity level (ionized forms).

Spectrophotometric procedures to measure cation concentrations are well developed and used frequently in laboratory and clinical practice.  However, most techniques determine the total amount present, not just the ionized forms.

Measurements of Enzymes
Spectrophotometric procedures to measure enzymes are also well developed and used frequently in laboratory and clinical practice.  The challenge is to obtain sufficient levels and sufficient specificity.  The more carefully one isolates an enzyme from a cellular milieu of thousands of enzymes, the less the quantity of sample obtained.

Measurements of Metabolites
The problem of multi-factor screening is sensitivity.  Gas chromatography using a Mass Spectrometer detector (GC/MS) can measure metabolic intermediates for high output reactions but often cannot determine levels in the microgram or nanogram levels at which hormones and many enzymes occur.  Radioactive tagging or immunological techniques (using labelled antibodies) are used to obtain greater sensitivity.

Genomic Research Techniques
A variety of spectrophotometric and other procedures are  well developed and used frequently in laboratory and clinical practice including:  HPLC, GC/MS, immuno-fluorescence, Electrophoresis, Western blot, northern blot, etc.  Commercial vendors can provide reactants on micro-arrays for nearly simultaneous detection of hundreds of chemical species.

High Throughput Systems for GWAS et al.
High Throughput Systems (HTS) are a focus of attention as genetic and other biochemical analyses are being performed by more and more researchers and clinicians.  Accelyrs is one of many manufacturers of automated equipment who provide associated software and reference databases.  Genome Wide Association Studies (GWAS) rely on collecting massive quantities of data and looking for meaningful correlations between genetic anomalies and diseases.

Bio-informatics
With high throughput screen techniques a massive amount of data is produced.  Some of the top researchers talk in terms of terabytes of data.  This requires powerful computers if not super-computers and/or parallel distributed computing by networked computers.  Super-computer applications are also being used for analysis of protein folding, 3D structure prediction, and other computational challenges like comparing protein or gene sequences looking for homologies or defects.  See Software Tools for further details on these topics.

References
Various compilations of references and bibliographies have been made and are available to qualified researchers.

 


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