Evaluation of 6-nitrohomovanillic acid as an endogenous metabolite of 6-nitrodopamine

• Grant number: 24/06033-2
• Support Opportunities: Scholarships in Brazil - Support Program for Fixating Young Doctors
• Start date: April 01, 2024
• End date: September 30, 2025
• Field of knowledge: Biological Sciences - Pharmacology - Autonomic Pharmacology
• Agreement: CNPq
• Principal Investigator: Roberto Zatz
• Grantee: Felipe Fernandes Jacintho
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 23/01376-6 - Evaluation of 6-Nitrodopamine effects on renal and urogenital function, AP.R

Abstract

The 6-nitrodopamine (6-ND), a product of nitric oxide (NO)-dependent "nitration" of dopamine (DA), is produced endogenously, and increasing evidence shows its role as the most potent mediator of in vivo and in vitro chronotropism in rats, as well as the spasmogenic activity of the vas deferens in rats and humans. Despite being the catecholamine released in greater quantities from the studied vascular tissues, circulating levels of 6-ND have not been identified thus far. Considering that dopamine is metabolized by the enzymes monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT), generating homovanillic acid found in plasma and urine, the objective of this project is to evaluate in vitro and subsequently in vivo release of 6-nitro-homovanillic acid as a possible endogenous metabolite of 6-ND. Quantification of 6-nitro-homovanillic acid in Krebs-Henseleit's solution, urine, and plasma, will be performed through high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In tissues where basal release of 6-nitro-homovanillic acid is observed, investigation of metabolic pathways will be conducted using selective MAO-A and MAO-B inhibitors, as well as non-selective MAO and COMT inhibitors. Once the methodology for urine and plasma is validated, the presence and establishment of levels of this new metabolite will be investigated in matrices obtained from laboratory animals, healthy volunteers, and patients, comparing them with the levels of homovanillic acid reported in the literature. It is important to note that the "gold standard" methodology for quantifying homovanillic acid used in clinical laboratories, which is high-performance liquid chromatography coupled with ultraviolet or fluorescence detectors, does not differentiate between homovanillic acid and 6-nitro-homovanillic acid. The identification, quantification, and establishment of plasma and urinary levels will provide a better understanding of the physio pathological relevance of this potent endogenous catecholamine, 6-ND.