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) have been used to obtain greater sensitivity.
Increased sensitivity of GC/MS has increased substantially in the past few
decades.
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 and NGS
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.
Next Generation Sequencing (NGS) is
another category of methods in genomics. It is reputed to provide higher
discovery power to detect novel genes and higher sensitivity to quantify rare
variants and transcripts (according to Illumina's website). The four steps
include nucleic acid isolation, library preparation, clonal amplification and
sequencing, and data analysis. The recent development by Illumina is a
departure from the classic Sanger chain-termination method.
Transcriptomics is another field that
is expanding significantly along with new methodologies and instruments.
The focus is on detecting the segments of messenger RNA (mRNA)
that code for various proteins under particular circumstances.
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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Revised:
February 19, 2024
