Seriously underestimating iron

Seriously underestimating iron

Does the calcein-AM method assay the total cellular 'labile iron pool'
- or only a fraction of it?
Tenopoulou M, Kurz T, Doulias PT, Galaris D, Brunk UT
Biochem J. 2007 Jan 18;

The calcein-AM (acetoxymethyl ester) method is a widely used technique
that is supposed to assay the intracellular 'labile iron pool' (LIP).
When cells in culture are exposed to this ester, it passes the plasma
membrane and reacts with cytosolic unspecific esterases. One of the
reaction products, calcein, is a fluorochrome and a hydrophilic alcohol
to which membranes are non-permeable and which, consequently, is
retained within the cytosol of cells. Calcein fluorescence is quenched
following chelation of low mass labile iron, and the degree of
quenching gives an estimate of the amounts of chelatable iron. However,
a requirement for the assay to be able to demonstrate cellular LIP in
total is that such iron be localized in the cytosol and not in a
membrane-limited compartment. For some time it has been known that a
major part of cellular redox-active labile, low mass iron is
temporarily localized in the lysosomal compartment as a result of the
autophagic degradation of ferruginous materials, such as mitochondrial
complexes and ferritin. Even if some calcein-AM may escape cytosolic
esterases and enter lysosomes to be cleaved by lysosomal acidic
esterases, resulting calcein does not significantly chelate iron at pH
<5. Here we show that the calcein-AM method does not capture lysosomal
low mass iron and, therefore, that the method seriously underestimates
total cellular labile iron.

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pbaux@lpart . fr : Seriously underestimating iron

pbaux@lpart . fr

<pbaux@lpart . fr > a écrit dans le message de news:
1169525387.860934.86390@m58g2000cwm.googlegroups . com ...
> Does the calcein-AM method assay the total cellular 'labile iron pool'
> - or only a fraction of it?
> Tenopoulou M, Kurz T, Doulias PT, Galaris D, Brunk UT
> Biochem J. 2007 Jan 18;
>
> The calcein-AM (acetoxymethyl ester) method is a widely used technique
> that is supposed to assay the intracellular 'labile iron pool' (LIP).
> When cells in culture are exposed to this ester, it passes the plasma
> membrane and reacts with cytosolic unspecific esterases. One of the
> reaction products, calcein, is a fluorochrome and a hydrophilic alcohol
> to which membranes are non-permeable and which, consequently, is
> retained within the cytosol of cells. Calcein fluorescence is quenched
> following chelation of low mass labile iron, and the degree of
> quenching gives an estimate of the amounts of chelatable iron. However,
> a requirement for the assay to be able to demonstrate cellular LIP in
> total is that such iron be localized in the cytosol and not in a
> membrane-limited compartment. For some time it has been known that a
> major part of cellular redox-active labile, low mass iron is