Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals.
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 degrees C), pH (7.4), and concentration of NO (less than 1 microM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC(50) values were calculated for rutin (1.30 +/- 0.02 microM, positive control), cysteine (321 +/- 8 microM), reduced glutathione (1106 +/- 93 microM), uric acid (134 +/- 12 microM), dipyrone (1.36 +/- 0.06 microM), and captopril (363 +/- 28 microM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC(50) 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.
Ribeiro
Joana P N
JP
REQUIMTE, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4099-030 Porto, Portugal.
Magalhães
Luís M
LM
Segundo
Marcela A
MA
Reis
Salette
S
Lima
José L F C
JL
D001331:Automation; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods*; D016166:Free Radical Scavengers / Q000032:analysis* / Q000097:blood; D006801:Humans; D009569:Nitric Oxide / Q000032:analysis*
D016166:Free Radical Scavengers; D009569:Nitric Oxide