Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain

Abstract

We describe here a GC/MS/MS method for the sensitive and concurrent determination of extracellular tryptophan and the kynurenine pathway metabolites kynurenine, 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) in rat brain. This metabolic cascade is increasingly linked to the pathophysiology of several neurological and psychiatric diseases. Methodological refinements, including optimization of MS conditions and addition of deuterated standards, resulted in assay linearity to the low nanomolar range. Measured in samples obtained by striatal microdialysis in vivo, basal levels of tryptophan, kynurenine and QUIN were 415, 89 and 8 nM, respectively, but 3-HK levels were below the limit of detection (<2 nM). Systemic injection of kynurenine (100 mg/kg, i.p.) did not affect extracellular tryptophan but produced detectable levels of extracellular 3-HK (peak after 2-3 h: ~50 nM) and raised extracellular QUIN levels (peak after 2 h: ~105 nM). The effect of this treatment on QUIN, but not on 3-HK, was potentiated in the NMDA-lesioned striatum. Our results indicate that the novel methodology, which allowed the measurement of extracellular kynurenine and 3-HK in the brain in vivo, will facilitate studies of brain kynurenines and of the interplay between peripheral and central kynurenine pathway function under physiological and pathological conditions.

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“…The proposed protective actions were related to the stimulation of glutathione S-transferase and superoxide dismutase activities and to the elevations in the protein contents of the transcription factor and antioxidant regulator nuclear factor (erythroidderived 2)-like 2 (Nrf2) and some of its related proteins [223]. It is important to mention that the basal extracellular concentrations of 3-OH-L-KYN in the rat brain (2 nM) are far below the above mentioned levels [224]. A recent study assessing the effects of 3-OH-L-KYN and 3-OH-ANA with regard to mitochondrial dysfunction demonstrated that both compounds decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction in a concentration-dependent manner in association with an impairment of mitochondrial membrane potential [225].…”

Reduced kynurenine pathway metabolism and cytokine expression in the prefrontal cortex of depressed individuals

“…We quantified tryptophan, kynurenine and QUIN levels in the brain with GC/MS using a 7890A GC coupled to a 7000 MS/ J Psychiatry Neurosci 2016;41(6) MS (Agilent Technologies), using electron capture negative chemical ionization as described previously 40 (Appendix 1).…”

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

“…One l was then injected into the GC. GC/MS analysis was carried out with a 7890A GC coupled to a 7000 MS/MS (Agilent Technologies, Santa Clara, CA), using electron capture negative chemical ionization (45).…”