Study towards the quantitative definition of the kynurenine pathway
Murray, Toby Benjamin James
The kynurenine pathway is the prime pathway for the metabolism of the essential amino acid, tryptophan (TRP), and also the de novo pathway for nicotinamide adenine dinucleotide (NAD+) production. The kynurenine pathway is important in the pathogenesis of multi-organ dysfunction syndrome following severe acute pancreatitis (AP-MODS) due to the metabolism of kynurenine (KYN) into cytotoxic 3- hydroxykynurenine (3HK) by the enzyme kynurenine 3-monooxygenase (KMO). Mice with absent Kmo gene expression (KMOnull) have marked reductions in extrapancreatic organ injury post AP. This dissertation describes the pharmacokinetics (PK) of the kynurenine pathway after intravenous infusion of deuterated or heavy carbon stable isotopes (tracers) of four kynurenine pathway compounds (D5-TRP, 13C6-KYN, D5-kynurenic acid (KYNA) and 13C6-3HK) into rats (n=13). Liquid chromatography-electrospray ionisation tandem mass spectrometry (LCMS/ MS) is the most frequently used method to monitor kynurenine pathway compound levels in plasma. This dissertation reports a new method of extraction of plasma samples, using solid phase extraction (SPE), alongside improvement and optimisation of existing LC-MS/MS protocols using a reverse phase ultra-high performance C18-pentafluorophenyl column. This has enabled the analysis of each main metabolite in the kynurenine pathway in a single assay. Mass spectra of compounds were detected using electrospray ionisation (ESI) in both positive and negative polarity employing multiple reaction monitoring (MRM) modes over a 9 min total run time with an injection volume of 10 μL and flow rate of 0.4 mL/min. The method for each compound was shown to be reproducible and accurate (RSD < 25%) and each corresponding standard curve demonstrated linearity (R2 >0.99). Single compartment temporal PK analysis of tracers in rat plasma, during the elimination phase, reveals short mean half-lives for each compound, suggesting that metabolism through the kynurenine pathway is rapid (t1/2 12.14 – 29.12 mins). There was a marked difference in the volume of distribution of each analyte (D5 KYNA 0.12; 13C6-3HK 0.21; 13C6-KYN 0.96 and D5-TRP 0.92 (μg/kg)/(μg/L)). Enzyme rates of formation of each analyte were also identified (KYN 20.73; KYNA 2.21; 3HK 3.68 (μg/L)/min) In conclusion, a new accurate and reliable LC-MS/MS method for the analysis of kynurenine pathway metabolites has been developed. PK analysis has identified important and significant differences in the apparent volumes of distribution of each metabolite. Thus, it can be suggested that TRP and KYN are readily distributed to tissue whilst KYNA and 3HK are largely confined to the plasma compartment.