Kynurenine Pathway Metabolite Quantification

All reagents and KP metabolites were analytical reagent grade and were purchased from Sigma-Aldrich (St Louis, MO), unless otherwise stated. Deuterated internal standards were purchased from Medical Isotopes, Inc (Pelham, NH). KP metabolites were extracted using 10% (w/v) trichloroacetic acid (TCA) with equal volume of serum samples in accordance with methods previously described15 . CSF samples were prepared similarly to serum samples except that deproteinization with TCA was not performed.
Concurrent analysis of tryptophan, kynurenine, 3-hydroxykynrenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), and anthranilic acid (AA) was performed with UHPLC as described by Jones et al.16 (link), using an injection volume of 20 μL of the prepared extract from each samples. KA detection was performed using a gradient mobile phase comprise of 50 mM sodium acetate buffer supplement with 25 mM zinc acetate (dihydrate) to enhance fluorescence intensity and 2.25% acetonitrile as organic modifier (Solvent A), and 10% acetonitrile (Solvent B). Each sample (10 μL) was injected into a Poroshell RRHT C-18, 1.8 μm 2.1 × 100 mm column (Agilent Technologies, Inc, Santa Clara, CA) maintained at 38 °C for 12 min run time at a unison flowrate of 0.75 mL/min. The gradient elution consisted of 100% solvent A for 3 min and then 50% solvent A and 50% solvent B for 2 min, followed by 100% B for 2 min and 100% solvent A (run time 10 min). This gradient ensures sufficient time for KA retention while minimizing potential build-up of pressure due to precipitation of the high salt buffer. Detection of KA used fluorescence (excitation and emission wavelengths of 344 and 388 nm, respectively with a retention time of 1.5 min). Agilent OpenLAB CDS ChemStation (Edition C.01.04) was used to analyze the chromatograms (Supplementary Figure S1A and B).
For GCMS, 50 μL of the prepared extract were derivatized. Concurrent analysis of PA and QA were carried out as described by Smythe et al.17 (link) with slight modification using an Agilent 7890 A GC system coupled with Agilent 5975 C mass spectrometry detector and Agilent 7693 A autosampler (Agilent Technologies, Inc, Santa Clara, CA) with one microliter of derivatized mixture. Separation of PA and QA were achieved with a DB-5MS column, 0.25 μm film thickness, 0.25 mm × 30 m capillary column (Agilent Technologies, Inc, Santa Clara, CA) within 7 min but the assay run time was set for 12 min to prevent sample carryover. Concentrations of PA and QA were analyzed using Agilent GC/MSD ChemStation software (Edition 02.02.1431) and interpolated from the established six-point calibration curves based on the abundance count ratio of the metabolites to their corresponding deuterated internal standards within each standards and samples (Supplementary Figure S1C and D).
The intra- and inter-assay CV was within the acceptable range of 4–8% for UHPLC assays and 7–10% for GCMS assays calculated from the repeated measures of the metabolites standards incorporated during the sequence run. Nicotinamide adenine dinucleotide (NAD⁺) was measured with 20 μL of neat serum using a previously described method18 (link).