A list of the antibodies used in this experiment is listed in the supplementary materials. Pyfidoxal-5'-phosphate, 3-hydroxyanthranilic acid and 3-hydroxykynurenine were purchased from Sigma-Aldrich Co. LLC.
3 hydroxykynurenine
Manufactured by Merck Group
Sourced in United States
3-hydroxykynurenine is a biochemical compound that serves as a laboratory reagent. It is used in research applications to study metabolic pathways and biochemical processes.
Automatically generated - may contain errors
Lab products found in correlation
3 hydroxyanthranilic acid, by Merck Group (6 mentions)
Xanthurenic acid, by Merck Group (5 mentions)
Kynurenic acid, by Merck Group (4 mentions)
Tryptophan, by Merck Group (3 mentions)
Quinolinic acid, by Merck Group (3 mentions)
Kynurenine, by Merck Group (3 mentions)
L tryptophan, by Merck Group (2 mentions)
Picolinic acid, by Merck Group (2 mentions)
L kynurenine, by Merck Group (2 mentions)
Anthranilic acid, by Merck Group (2 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Rf 10axl fluorescence detector, by Shimadzu (1 mentions)
Uv 1601 pc spectrophotometer, by Shimadzu (1 mentions)
Rf 5301 pc fluorimeter, by Shimadzu (1 mentions)
D5 tryptophan, by Merck Group (1 mentions)
Serotonin, by Merck Group (1 mentions)
Melatonin, by Merck Group (1 mentions)
Tryptamine, by Merck Group (1 mentions)
Indole 3 lactic acid, by Merck Group (1 mentions)
Indole 3 acetamide, by Merck Group (1 mentions)
10 protocols using 3 hydroxykynurenine
1
Antibody Staining Protocol Optimization
2
Quantitative Chromatographic Analysis of Metabolites
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For chromatographic analysis, the samples (SGNc and SGC) were filtered through a 0.22-µm membrane, precipitated in ice pure methanol and centrifuged at 11 000g for 15 minutes, with 80 µL of the supernatant being analyzed. The secretome was analyzed in the High-Performance Liquid Chromatography (HPLC) system from Agilent (model infinity II 1260) coupled to a Diode array Detector (DAD). In order to elute the samples, a run took place in a reversed phase (300SB-C18) (150 mm × 4.6 mm i.d., 5 µm). The analysis was performed by gradients (methanol/acetonitrile acidified with 0.1% of formic acid) with its gradient increased linearly from 0% to 50% of acetonitrile in 8 minutes, going to 80% at 10 minutes and to 100% at 11 minutes, remaining in this condition for 12 minutes with a flow of 400 µL/minutes. The detection of the eluates by the photodiode was made between 280 and 360 nm. The samples were compared with chromatographic standard quinolinic acid, kynurenic acid, 3-hydroxykynurenine and tryptophan (Sigma–Aldrich, St. Louis, MO, USA), all diluted in water at a concentration of 500 µM.
3
HPLC Analysis of Squid Ommochromes
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Isolated ommochromes from squid were analyzed by high performance liquid chromatography (HPLC) using a Knauer chromatograph (Berlin, Germany) on a Diasphere 120 C18 column (4 × 250 mm; particle size, 5 μm). Solvent A was 10% aqueous acetonitrile containing 0.5% formic acid; solvent B was 100% acetonitrile containing 0.5% formic acid. The pigments were fractionated in a linear gradient (0–40%) of solution B in solution A for 60 min at a flow rate of 0.4 mL/min at 24 °C. Eluted pigments were registered with a Knauer K-2501 UV/Vis detector and an RF-10A-xl fluorescence detector (Shimadzu, Japan).
The ommochromes or standard compounds were dissolved in 100 μL of methanol containing 0.5% HCl. Tryptophan, kynurenine, 3-hydroxykynurenine, and xanthurenic acid (Sigma-Aldrich, Saint Louis, MO, USA) were used as standards. Xanthommatin was synthesized by autooxidation of 3-hydroxykynurenine. Absorption spectra were recorded with a Shimadzu UV–1601PC spectrophotometer. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorimeter. The obtained data were processed with the RFPC version 2.0 software (Shimadzu, Kyoto, Japan).
The ommochromes or standard compounds were dissolved in 100 μL of methanol containing 0.5% HCl. Tryptophan, kynurenine, 3-hydroxykynurenine, and xanthurenic acid (Sigma-Aldrich, Saint Louis, MO, USA) were used as standards. Xanthommatin was synthesized by autooxidation of 3-hydroxykynurenine. Absorption spectra were recorded with a Shimadzu UV–1601PC spectrophotometer. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorimeter. The obtained data were processed with the RFPC version 2.0 software (Shimadzu, Kyoto, Japan).
4
Tryptophan Metabolism Metabolite Analysis
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Methanol (CH3OH), ammonium acetate (CH3COONH4), acetonitrile (CH3CN), picolinic acid, 3-hydroxykynurenine, quinolinic acid, serotonin, 5-hydroxytryptophan, kynurenine, 3-hydroxyanthranilic acid, tryptamine, L-tryptophan, 5-hydroxyindole, acetic acid, indoxyl sulfate, N-Acetylserotonin, xanthurenic acid, indole-3-acetamide, kynurenic acid, DL-indole-3-lactic acid, indole-3-carboxaldehyde, indole-3-acetic acid, tryptophol, melatonin, 5-hydroxyindole acetic acid-D5, serotonin-D4, indole-3-lactic acid-D4, kynurenic acid-D5, melatonin-D4, picolinic acid-D3, tryptamine-D4, xanthurenic acid-D4, 3-hydroxyanthranilic acid-D3, 3-hydroxykynurenine-13C2-15N, 5-hydroxytryptophan-D4, indole-3-acetamide-D5, kynurenine-D4, L-tryptophan-13C11,15N, and tryptophan-D5 were purchased from Sigma Aldrich, Merck KGaA (Darmstadt, Germany) and other reagents and chemicals used in the study were purchased from local suppliers. The reagents and chemicals were of analytical grade or higher purity. Additionally, the yeast strain S. cerevisiae STG S101 was purchased from Fermentis (Marcq-En-Baroeul, France).
5
Comprehensive Metabolic Analysis Protocol
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Formic acid (HPLC grade) was purchased from Dikma Technologies Inc. (Lake Forest, CA, USA). Ultra-pure water was obtained from Hangzhou Wahaha Group Co., Ltd. (Zhejiang, China). Methanol (HPLC grade), acetonitrile (HPLC grade), chloroform (HPLC grade), isopropanol (HPLC grade), Tween, pCPA, serotonin hydrochloride, 5-hydroxyindoleacetate, kynurenine, kynurenate, 3-hydroxykynurenine, phenylalanine, tyrosine, sodium hippurate hydrate, creatinine, guanosine, hypoxanthine, 3-indolepropionic acid, citrate, oxoglutarate, succinate, xanthine, uric acid and indoxyl sulfate potassium salt were purchased from Sigma-Aldrich (St. Louis, MO, USA). T-AOC assay kit with Ferric Reducing Ability of Plasma (FRAP) method, SOD assay kit, total GPx assay Kit and lipid peroxidantion MDA assay kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). CAT assay kit and reactive oxygen species assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China).
6
HPLC Analysis of Tryptophan Metabolites
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HPLC-grade acetonitrile and formic acid were purchased from Tedia (Fairfield, OH, United States). 3-Hydroxykynurenine and 3-hydroxyanthranilic acid were obtained from Sigma-Aldrich (St. Louis, MO). Tyrosine and xanthurenic acid were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD.
7
Ferroptosis Pathway Inhibitors and Metabolites
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Erastin (S7242), RSL3 (S8155), ferrostatin‐1 (S7243), liproxstatin‐1 (S7699), 4‐HPPA (S2995) were pursued from Selleck. SYTOX Green (S34860) and BODIPY‐C11 581/591 (D3861) were pursued from Invitrogen. L‐kynurenine (K8625), 3‐hydroxy‐kynurenine (H1771), 3‐hydroxyanthranilic acid (148776), 4‐HPLA (H3253), 4‐HB (144088), 4‐HBz (H5501), L‐Carnosine (C9625), Ornithine (O6503), phenylpropionic acid (W288918), L‐pipecolate (P2519) were pursued from Sigma‐Aldrich. α‐ketoisocaproate (T5071), Agmatine (T4808L), 2‐ketobutyrate (T5060), 3‐hydroxyisobutyric acid (T4947), hippuric acid (T4815), Benzoic acid (T0833), cinnamoic acid (T2740), β‐hydroxy‐β‐methylbutyrate (T5550), guanidinoacetic acid (T4238), Histamine (T0965), L‐Tryptophan (T0439), 5‐HTP (T1379), 5‐HT (T2209), 5‐HIAA (T4744), melatonin (T1659), quinolinic acid (T4049), and tyrosine (T0493) were pursued from TOPSCIENCE. Trans‐urocanate (I0002) were pursued from J&K Scientific.
8
Quantitative Analysis of Kynurenine Pathway Metabolites
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Tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, picolinic acid, and quinolinic acid were obtained from Sigma Aldrich (Poole, United Kingdom). The stable isotope–labeled internal standards were 2H5-Tryptophan (QMx Laboratories, Thaxted, United Kingdom), 2H6-kynurenine sulfate and 2H5-kynurenic acid (CK isotopes, Ibstock, United Kingdom), and 2H3-picolinic acid and 13C3,15N-quinolinic acid (LGC standards, Teddington, United Kingdom). Acetonitrile and methanol were obtained from Rathburn Chemicals (Walkerburn, United Kingdom) and Fisher Scientific UK (Loughborough, United Kingdom), respectively.
Standards of each analyte were used to automatically tune in multiple reaction monitoring (MRM) mode. Transition optimizations were performed manually. xanthurenic acid was significantly more sensitive in negative ion mode, but, with the chromatographic conditions used, there was massive matrix ion suppression. Consequently, xanthurenic acid was measured in positive ion mode.
Because analyte retention times on Chirobiotic-T columns tend to be matrix dependent, there is a critical requirement for the appropriate stable isotope internal standards. Consequently, when stable isotopes were unavailable, 2 separate transitions, when possible, were used to calculate and then confirm the result.
Standards of each analyte were used to automatically tune in multiple reaction monitoring (MRM) mode. Transition optimizations were performed manually. xanthurenic acid was significantly more sensitive in negative ion mode, but, with the chromatographic conditions used, there was massive matrix ion suppression. Consequently, xanthurenic acid was measured in positive ion mode.
Because analyte retention times on Chirobiotic-T columns tend to be matrix dependent, there is a critical requirement for the appropriate stable isotope internal standards. Consequently, when stable isotopes were unavailable, 2 separate transitions, when possible, were used to calculate and then confirm the result.
9
Kynurenine Pathway Metabolite Analysis
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L-tryptophan (Trp,#T0254), L-kynurenine (Kyn, #K8625), Kynurenic acid (KA, #K3375), Xanthurenic acid (XA, #D120804), Anthranilic acid (AA, #A89855), 3-hydroxyAnthranilic acid (HAA, #148776), 3-Hydroxykynurenine (HK, #H1771), Nicotinamide (NIC, #N9976), 2,3-pyridinedicarboxylic acid (QA, #P63204), Quinaldic acid (QAA, #160660), and Tetrachlorodibenzo-p-dioxin, (TCDD, #48599) were purchased from Sigma-Aldrich. 6-Formylindolo[3,2-b]carbazole (FICZ, #BML-DR206-0100) was purchased from Enzo Life Sciences. The IDO1 inhibitor Epacadostat was obtained from Synnovator, Inc. TDO inhibitor (PF06845102/EOS200809) is described by Schramme et al20 (link) and in the published patent application US 20150225367. The AHR antagonist CH-223191 (#182705) was purchased from Merck. LAT1 inhibitor BCH (#5027) was purchased from Tocris and Selleckchem, respectively. All metabolites were solubilized in HBSS medium except for HAA and KA, which required to be dissolved in DMSO. Each compound was aliquoted immediately after reconstitution and was stored at −80°C. Each metabolite was controlled by high performance liquid chromatography (HPLC) after dilution for degraded by-products. Salubrinal (#SML0951 ) and Isrib (#SML0843 ) were purchased from Sigma Aldrich. To inhibit the transcription, 5 µg/mL Actinomycin D (#A4262 from Sigma) prediluted in DMSO were added to the medium.
10
Targeted Metabolomics of Tryptophan Metabolites
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Reinforced Clostridium Medium (RCM) was purchased from Qingdao Hope Bio-Technology Co., Ltd. (Supplementary Table S1 ). Acetonitrile (CAS.75-05-8, ≥99.9% purity), formic acid (CAS.64-18-6, ≥99.0% purity), ammonium acetate (CAS 631-61-8, ≥99.0% purity), methanol (CAS.67-56-1, ≥99.0% purity), dimethyl sulfoxide (DMSO, CAS.67-68-5, ≥99.9% purity), TRP (CAS.73-22-3, 98% purity), 3-hydroxykynurenine (3-HK, CAS.484-78-6, ≥99.0% purity), xanthurenic acid (XA, CAS.59-00-7, 96% purity), and IBA (CAS.133-32-4, ≥99.0% purity) were obtained from Sigma-Aldrich (United States). 3-Hydroxyanthranilic acid (3-HAA, CAS.548-93-6, ≥97.0% purity), 5-HIAA (CAS.54-16-0, 98% purity), 5-hydroxytryptamine (5-HT, CAS.50-67-9, ≥98% purity), IAA (CAS.87-51-4, ≥99.0% purity), IPA (CAS.830-96-6, ≥98% purity), KYNA (CAS.492-27-3, 97% purity), KYN (CAS.13441-51-5, 98% purity), and 5-hydroxytryptamine-d4 (5-HT-d4, CAS.58264-95-2, 98% purity) were purchased from Aladdin (China).
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