Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals |
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Authors: | Joana P N Ribeiro Luís M Magalhães Marcela A Segundo Salette Reis José L F C Lima |
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Institution: | 1. REQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4099-030, Porto, Portugal
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Abstract: | In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for
in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective
fluorogenic probe. The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of
putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37°C), pH
(7.4), and concentration of NO (less than 1 μM). This approach allowed the evaluation of scavenging capacity against NO by
endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC50 values were calculated for rutin (1.30 ± 0.02 μM, positive control), cysteine (321 ± 8 μM), reduced glutathione (1106 ± 93 μM),
uric acid (134 ± 12 μM), dipyrone (1.36 ± 0.06 μM), and captopril (363 ± 28 μM). A high degree of automation was attained
as the successive dilution of antioxidant standard solutions required for IC50 assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h-1 and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed
method to routine/screening analysis of scavenging capacity against NO. |
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