Development of a method for selective detection and quantification of S-nitrosothiols and DNICs in complex matrices.

• Grant number: 24/01221-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2024
• End date: February 28, 2025
• Field of knowledge: Physical Sciences and Mathematics - Chemistry - Inorganic Chemistry
• Principal Investigator: Daniela Ramos Truzzi
• Grantee: Fernanda Saito Matsuhira
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 19/17483-0 - Dinitrosyl iron complexes (DNICs): formation, reactivity, and implications for the physiopathology of nitric oxide, AP.JP

Abstract

Currently, all studies of biological DNICs use Electron Paramagnetic Resonance (EPR) for their detection and quantification. However, the EPR technique has limitations for DNICs studies such as the high limit of detection and quantification and the lack of detection of the binuclear form of DNICs. Additionally, Saville's method, which is widely used for quantification of NO-metabolites (nitrite, R-SNO, and NO-heme), was not developed considering the presence of DNICs in biological samples. This method takes advantage of the R-SNO species degradation promoted by HgCl2 which also exhibits an effect over DNICs degradation resulting in the overestimation of S-nitrosothiols levels. Therefore, we aim to develop a method for selective detection and quantification of S-nitrosothiols and total DNICs (mono and binuclear) in complex matrices (containing nitrite, R-SNO, NO-heme, and DNICs). Therefore, we will take advantage of the principles of Saville's spectroscopic method, in which HgCl2 is used to cleave the RS-NO bond and form nitrite (Saville reaction), which is then quantified by the classical Griess reaction (formation of the diazo compound with absorption at 540 nm). However, we will add an additional step of a sample treatment using chelating agents to promote selective degradation of DNICs to nitrite then, after this treatment, the sample will be treated with HgCl2 for exclusive degradation of S-nitrosothiols. Experimental conditions such as the excess of a chelating agent and pH will be tested in order to obtain optimal conditions for the complete degradation of DNICs. The chelating agents selected for the study are desferral (hydrophilic) and salicylaldehyde isonicotinoyl hydrazone (SIH) (hydrophobic), which may allow a selective quantification of extra and intracellular DNICs, respectively. Solutions containing known concentrations of DNIC-GS will be used in the initial experiments. After establishing the experimental conditions for the Saville and Griess's spectroscopic method, we aim to adapt it for detection by gas phase chemiluminescence using the Nitric Oxide Analyzer because it is a more sensitive method of detection and quantification of NO-derived species.