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VOL. 73 (2), 441-452, 2007 GLUTAMATE DETERMINATIONS USING AMPLEX RED...

by diphosphohydrolases and nucleotidases and measuring the
amount of inorganic phosphate released with amplex red based-Pi
Per Phosphate Assay Kit. However, they incubated together BzATP
and HRP to know the phosphate concentration. Thus, in agreement
with the results obtained in this work it could be possible that
resorfurin fluorescence resulted affected by BzATP and not
corresponded, therefore, completely to BzATP catabolism. Similarly,
Parvathenani et al. (11) studied the role played by P2X7 receptor in
superoxide production in primary microglia. Thus, superoxide (O–2)
was measured indirectly through the detection of hydrogen peroxide
(H2O2) by using an Amplex Red based assay. Thus, microglia cells
were stimulated with BzATP in a 96-well plate containing 0.2 units/
mL horseradish peroxidase and 50 µM Amplex Red. It is relevant to
indicate that Amplex Red and BzATP were incubated together.
Accordingly to the results reported in this work where HRP-mediated
Amplex Red oxidation, in presence of BzATP, is not proportional to
H2O2 formed, it seems logical to hypothesize that resorufin
fluorescence detected by Parvathenani and co-workers are not
completely due to superoxide production but BzATP-mediated
resorufin production could also be involved.

In summary, the measurement of glutamate concentration by
Amplex® Red Glutamic Acid/Glutamic Oxidase Assay Kit results
altered when BzATP is present in the medium. Thus, Amplex Red
oxidation catalyzed by HRP is significantly enhanced by BzATP, even
in absence of H2O2. Taking into account that Amplex Red oxidation
by HRP is a shared reaction in Amplex Red-based assays, extra
caution is needed to take when these assays are used with BzATP in
order to avoid misinterpretations of the results.

ACKNOWLEDGMENTS

This work was supported by research grants from the Spanish
Ministry of Education and Science BFU2005-02079, CAM (SAL/
0551/2004), Fundación La Caixa n.º BM05-114-0 and Fundación
Marcelino Botín. David León is a postdoctoral fellow of the Junta
de Comunidades de Castilla-La Mancha and Fondo Social Europeo.

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