D. P. BUDAC1, D. SONG1, M. CAJINA1, A. LEE1, B. M. CAMPBELL1, G. LI1, C. SÁNCHEZ1, C. FORRAY1, V. S. PALAMARCHOUK2, G. N. SMAGIN1,2
1Lundbeck USA, Paramus, NJ; 2PsychoGenics Inc, Tarrytown, NY
Degradation of the amino acid tryptophan along the kynurenine pathway (KP) yields several neuroactive intermediates regulated by enzymes localized in astrocytes and microglial cells. The limited ability of many KYN-derived metabolites to cross the blood brain barrier suggests that CNS concentrations of these metabolites is largely regulated by local enzyme activity. KYN itself is actively transported into the brain by large neutral amino acids transporter. The fate of KYN in the brain is poorly understood and was investigated using a stable labeled analogue of KYN ([13C6]L-KYN) and in vivo microdialysis A sensitive LC/MS/MS method was developed and utilized to provide a time course for stable isotope labeled metabolite formation. Mice and rats were implanted with guide cannula placed into the striatum. One week after surgery, a microdialysis probe was inserted for the microdialysis experiment. Microdialysis samples were collected for 6 hrs after i.p. administration of 5 mg/kg of [13C6]L-KYN and analyzed for labeled and unlabeled L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA) and quinolinic acid (QUIN), using LC/MS/MS method. In mice, [13C6]L-KYN metabolism showed a stronger kynurenine 3-monooxygenase (KMO) branch response with increased incorporation of 13C label in 3-HK, 3-HAA, and QUIN. [13C6]L-KYN metabolism in rats progressed predominantly down the kynurenine aminotransferase (KAT) pathway as suggested by the higher amounts of labeled KYNA. The results demonstrate the utility of LC/MS/MS analysis of stable isotope labeled KP metabolite from in vivo microdialysis samples since this type of technique can be utilized to delineate the role of central and peripheral KP metabolites in diseases involving dysregulation of kynurenines metabolites. Further studies of brain-penetrable modulators of KP may be beneficial for the treatment of these diseases.