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 tyrosine conversion by intestinal microflora, is gaining increasing attention for its potential health impact. Once produced, the compound is converted into p-cresyl-glucuronide and p-cresyl-sulfate and excreted via organic anion transporters (OAT), which are also expressed in the brain, mediating efflux across the blood-brain barrier (BBB). While the impact of p-cresol and its metabolites as uremic toxins in chronic kidney disease (CKD) is well-known, affecting the central nervous, immune, and cardiovascular systems, their role in neurodegenerative and neurodevelopmental disorders is still under investigation. Elevated urinary levels of p-cresol and p-cresyl-sulfate have been found in autistic children with altered intestinal microbiota, suggesting a link to increased autism severity and gut dysfunction [1,2]. Moreover, p-cresol’s effects on dopamine metabolism suggest possible roles in post-traumatic stress disorder (PTSD) and Parkinson’s disease (PD) [3]. However, the evidence on the presence and concentration of p-cresol in the central nervous system (CNS) is virtually unknown, highlighting the need to develop an analytical method capable of quantifying this compound at very low concentrations. To address this gap, we optimized and validated a new HPLC-ESI-MS/MS method for targeted metabolomics of p-cresol in brain areas. Using reversed-phase HPLC with gradient elution coupled with electrospray ionization-mass spectrometry (ESI-MS/MS) detection in multiple reaction monitoring (MRM) mode, we analyzed brain tissue from male and female C57BL/6 mice, revealing p-cresol distribution across seven brain regions. Additional measurements in the cortex of three mouse strains - CD1, C57BL/6 and the model of idiopathic autism BTBR +tf/J - showed that p- cresol levels were influenced by both sex and genotype. This effect was also observed in experiments on wild-type (WT) and CX3CR1 knockout (KO) mice: while sex and genotype affected p-cresol distribution in the prefrontal cortex, treatment with lipopolysaccharide (LPS) did not produce any significant changes in p-cresol levels. Additional targeted metabolomic analyses were performed to further explore potential correlations between this compound, neurotransmitters and their metabolites, particularly in dopaminergic and noradrenergic pathways. Preliminary analyses on human cortex samples also confirmed the presence of p-cresol, underscoring its potential relevance to brain health. Finally, molecular docking studies on OAT provided insights into potential BBB transport mechanisms for p-cresol and its derivatives. The determination of basal p-cresol levels in the brain lays the groundwork for studying its role in neurodevelopmental and neurodegenerative diseases. Future research will explore whether targeting transporters may interfere with its accumulation, offering new therapeutic strategies for autism, PTSD and PD.