Targeted metabolomics for the analysis of p-Cresol in mouse brain by HPLC-ESI-MS/MS

p-Cresol, an environmental contaminant and endogenous metabolite primarily derived from the conversion of tyrosine by intestinal microflora, is gaining increasing attention due to its potential health impact. Once produced, the compound is converted into p-Cresyl-glucuronide and p-Cresyl-sulfate, then eliminated through urine via organic anion transporters (OAT), also expressed in the brain for efflux across the blood-brain barrier (BBB). p-Cresol and its metabolites, known as uremic toxins, are linked to various pathologies, causing central nervous system (CNS), immune system, and cardiovascular issues in chronic renal failure (CKD) patients. Elevated urinary levels of p-Cresol and p-Cresyl-sulfate are found in autistic children with altered intestinal microbiota, suggesting a link to increased autism severity and gut dysfunction [1,2]. Moreover, p-Cresol also interferes with dopamine metabolism, implicating it in PTSD and Parkinson's disease (PD) [3]. However, the evidence on the presence and concentration of p-Cresol in the CNS is virtually unknown, highlighting the need to develop an analytical method capable of quantifying this compound at a very low concentration. To address this, a new HPLC-MS/MS method was optimized [4] and validated for targeted metabolomics of this phenolic compound in brain areas, allowing detailed distribution within the CNS. Analysis in brain homogenates used a reversed-phase HPLC method under gradient elution coupled with electrospray ionization-mass spectrometry (ESI-MS/MS) detection, employing a triple quadrupole in multiple reaction monitoring (MRM) mode. The method was applied to quantify p-Cresol in brain tissues from adult male and female C57BL/6 mice, thus determining its distribution in seven different brain areas. Additionally, p-Cresol levels have been determined in the cortex of three different mouse strains, CD1 IGS, C57BL/6, and the model of idiopathic autism BTBR +tf/J, and correlated with the levels of noradrenaline, dopamine, and their metabolites to explore potential interactions and differences among the three genotypes. Furthermore, given the role of OAT in uremic toxin transport and BBB permeability, molecular docking was performed to explore the interaction of p-Cresol and its metabolites with the transporter. The determination of basal p-Cresol levels in brain lays groundwork for studying its role in neurodevelopmental and neurodegenerative diseases. Future research will explore whether targeting transporters can reduce p-Cresol accumulation, offering new therapeutic strategies for autism, PTSD, and PD.