Analytical method for determination of nitric oxide in zebrafish larvae: toxicological and pharmacological applications.
Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coli lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae.
Instituto de Toxicologia e Farmacologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, CEP 90619-900, Brazil. carlos.leite@pucrs.br
Carlos Eduardo
CE
Leite
Ariane da Cruz
Ada C
Teixeira
Fernanda Fernandes
FF
Cruz
Simone Cervieri
SC
Concatto
Jefferson Henrich
JH
Amaral
Carla Denise
CD
Bonan
Maria Martha
MM
Campos
Fernanda Bueno
FB
Morrone
Ana Maria Oliveira
AM
Battastini
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D007814:Larva / Q000737:chemistry; D057230:Limit of Detection; D009569:Nitric Oxide / Q000032:analysis* / Q000494:pharmacology / Q000633:toxicity; D015203:Reproducibility of Results; D015027:Zebrafish / Q000254:growth & development*
D009569:Nitric Oxide