Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals

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 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.

High-throughput Total Cupric Ion Reducing Antioxidant Capacity of Biological Samples Determined Using Flow Injection Analysis and Microplate-based Methods

“…Finally, the CUPRAC-FIA method provides measurement of total antioxidant capacity, which can be more useful for assessing biological oxidative status than the targets of previously reported automatic methods that aimed at assessing scavenging capacity against a given species, namely NO, 36,37 or H2O2, ClO -, O2 • -, and 1 O2. 37 Compared to previously described automatic flow systems for TAC, this CUPRAC-FIA system is not invasive, 38 requiring a minimum amount of sample (between 2.7 and 10 μL per analysis, considering dilution factors between 75 and 20 times, respectively).…”

Nitric oxide (NO) scavenging capacity of natural antioxidants

“…Although it is well-known that free radicals to be scavenged by antioxidants include nitric oxide (NO), NO has never been a subject for scavenging capacity measurement in ORAC-like methods (Al-Laith, 2010;Cook, Wink, Blount, Krishna, & Hanbauer, 1996;Nishibayashi, Asanuma, Kohno, Gomez-Vargas, & Ogawa, 1996;Poderoso et al, 1999;Rubbo et al, 2000;Sreejayan & Rao, 1997). Recently, using the chemiluminescence-based method with the multisyringe flow injection analysis (MSFIA), the NO scavenging capacity for some antioxidants was evaluated; however the MSFIA method has a few experimental disadvantages such as the low selectivity for NO and the requirement of careful choice of experimental conditions (Miyamoto et al, 2007;Ribeiro, Magalhases, Segundo, Reis, & Lima, 2010). In living biological systems, a high level of NO and its oxidised derivatives such as peroxynitrite are known to be toxic (Szabo & Thiemermann, 1994), but the ability of antioxidants to scavenge NO has been hardly evaluated.…”

High-sensitivity programmable flow method for assessment of total antioxidant capacity in biological samples

“…The general downscaling of the assay also provides reduced sample consumption, as illustrated by the semi-micro FIA microdialysis probe hyphenated system that was applied for evaluation of the ex vivo antioxidant effect of the microdialysate after ascorbic acid administration in rats for preclinical assays [20]. The strictly controlled timing in flow assays also provides the valuable possibility for in-line in vitro production of highly reactive radical species [21][22][23], additionally to testing of biological samples against various reactive species is also possible resorting to a single flow set up [24]. However, the analytical assays described do not measure the TAC of biological samples in concentrations similar to those found in cellular environment.…”