L kyn

KYNA, L-KYN, D-KYN, dimethylsulfoxide (DMSO), DL-penicillamine, diethylenetriaminepentaacetic acid (DTPA), H₂O₂, ethylenediaminetetraacetic acid (EDTA), 3-methylpyrazole-5-carboxylic acid (MPC), kojic acid and benzoic acid were obtained from Sigma Aldrich Company (St. Louis, MO, USA). All other chemicals were of the highest commercially available purity. Solutions were prepared using deionized water obtained from a Milli-RQ (Millipore) purifier system.

l-KYN and QUNA were purchased from Sigma-Aldrich (St. Louis, MO). l-TRP was purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). l-KYN sulfate (ring-D4, 3, 3-D2) (was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). Hippuric acid-d5 were purchased from Toronto Research Chemicals (Toronto, Canada). All other solvents and reagents used were commercially available LC/MS or HPLC grade.
Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma-Aldrich. The recombinant human cytokines used in this study and their providers were as follows: interferon (IFN)-α 2a and IFNα-2b, PBL Assay Science (Piscataway, NJ); IFNγ and interleukin (IL)-4, Thermo Fisher Scientific (Waltham, MA); IFNβ, IL-1β, and tumor necrosis factor α (TNFα), R&D systems (Minneapolis, MN); IL-6 and IL-10, BioLegend (San Diego, CA). All cytokines were prepared at 10 µg/mL in phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS) and stored at − 80 °C.

In an open‐label design, the participants received a continuous intravenous infusion of 50, 100, and 150 µg/kg LKYN (manufactured by Sigma‐Aldrich under conditions and practices required by the good manufacturing practice (GMP) regulations, reference number CSQ‐23526R2) over 20 minutes (min) on three days separated by at least 1 week (Figure 2B). LKYN was dissolved in 0.1 M NaOH and was further diluted with saline to its final concentration just prior to the experiment. The final pH was around 7.4.

To detect IDO/TDO activity decapsulated testes, TLN and ILN were cut with scissors. Samples were incubated with lysis buffer as described above and no protease inhibitors were added. Protein precipitation was performed by adding 70% perchloric acid to the supernatant. The suspension was left on ice for 15 min, and then centrifuged at 22,000 g for 10 min. The pellet was discarded and 100 μl of the supernatant was analyzed by HPLC. Kynurenine (Kyn) release and tryptophan (Trp) degradation were measured. Standard curves were generated with L-Kyn and L-Trp (Sigma-Aldrich) in the same solvent. The chromatographic system was equipped with a Phenomenex Luna 5 μm, C18, 250 mm × 4.60 mm column and an ultraviolet detector (UVIS 204, Linear Instruments, Reno, USA). The wavelength was set at 360 nm for Kyn and at 278 nm for Trp. The mobile phase composed of acetate buffer (15 mM, pH 4) (92%) and acetonitrile (8%) was pumped at a flow rate of 1.0 ml min⁻¹. Results were shown as the Kyn/Trp ratio.

2′,7′-dichlorofluorescein (DCF), DCF-diacetate (DCF-DA), L-KYN, 3-nitropropionic acid (3-NP), FeSO₄, xylenol orange, ammonium iron (II) sulfate hexahydrate, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), bovine serum albumin (BSA), nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), ethylenediaminetetraacetic acid (EDTA), nicotinamide adenine dinucleotide (NADH), diethylenetriaminepentaacetic acid (DTPA), sodium hypochlorite (NaOCl), H₂O₂ and N,N-dimethyl-4-nitrosoaniline (DMNA) were all obtained from Sigma Chemical Company (St. Louis, MO, USA). All other reagents were reactive grade and obtained from known commercial suppliers. Solutions were prepared using deionized water obtained from a Milli-RQ (Millipore, Burlington, MA, USA) purifier system.

CGRP (Tocris Bioscience, Bio‐Techne Ltd, UK) was dissolved in saline to a concentration of 0.5 mg/ml. LKYN (Sigma‐Aldrich, Denmark) was dissolved in 0.1 M NaOH. CGRP and LKYN were further diluted with saline or 0.1 M NaOH to their final concentration just prior to the experiment.