Tryptophan and Kynurenine Quantification

After separation from whole blood, plasma samples (200 uL) were treated with 8% Perchloric acid, to precipitate proteins and extract soluble metabolites. After extraction, the aqueous phase was analyzed by High Performance Liquid Chromatography (HPLC) as previously described (Mallmann et al., 2018). For the standard curve, a serial dilution was performed with TRP and KYN (TRP/KYN μM/L): 100/10; 50/5; 25/2.5; 12.5/1.25; 6.25/0.625; 3.125/0.325. HPLC flow rate was 1.0 mL min^–1 and 20 μL of clear sample was then injected into the HPLC system using an autosampler. TRP was identified at 278 nm and KYN at 360 nm by UV detection (Zhang et al., 2009). Retention time (Rt) was used to identify metabolites in the chromatogram and standard curve was constructed by plotting the ratio of peak area (computed by LCsolution software, Shimadzu) of TRP or KYN (y) against known TRP or KYN concentration (x), respectively. The linearity of the standard curves was confirmed using regression variance analysis and significance of correlation co-efficient (r) checked using Student’s t-test. KYN/TRP ratio was calculated using the formula KYN concentration (μM/L)/TRP concentration (μM/L).