Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

Summary

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Abstract

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.

Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.

This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method generates reliable results and can be further applied to other in vitro cellular models.

Introduction

Kynurenine pathway (KP) is the major route of tryptophan (Trp) catabolism in human cells. Indoleamine-2,3-dioxygenase (IDO-1) in extrahepatic cells is the first and limiting enzyme of KP and converts Trp into N-formylkynurenine¹. Further steps within KP generate other secondary metabolites, namely kynurenines that exhibit various biological properties. Kynurenine (Kyn) is the first stable Trp catabolite showing toxic properties and regulating cellular events after binding to the aryl hydrocarbon receptor (AhR)². Subsequently, Kyn is transformed into several molecules either spontaneously or in the enzyme-mediated process....

Protocol

1. Preparation of standard 3NT, Kyn, 3HKyn, 3HAA, XA standard stock solutions

1. Weigh the reagents in a vial with the highest accuracy (0.3 mg each). For better accuracy, scale up the reagents, adjusting the volume of the solvent according to step 1.2.
2. Dissolve the reagents in 300 µL of the solvent to obtain a stock solution of 1 g/L. Dissolve 3NT in 1% (v/v) formic acid (FA) in water; Kyn, 3HAA, and XA in dimethyl sulfoxide (DMSO); 3HKyn in water acidified to pH 2.5 with hydrochloric acid (HCl).
   CAUTION: 3HAA irritates eyes, nose, throat and lungs. It is harmful when inhaled, in contact with skin, and if swallowed. Wear appropriate pro....

Results

LC-MS presents indisputable advantages in the quantification of biologically active molecules, even though some components of complex specimens cause so-called matrix effects and compromise ionization of analytes. It leads to ion suppression or ion enhancement, strongly decreasing accuracy and affecting a limit of detection/quantification of LC-MS, which is considered as a ''weak” point of the method. In our protocol, the ions are generated by electrospray ionization (ESI) in the positive mode, which was also employed in.......

Discussion

This paper presents a detailed LC-SQ protocol for the simultaneous quantification of four major Trp metabolites (3HKyn, Kyn, 3HAA, XA) that are measured in a medium from in vitro cultured human cancer cells. Special attention is paid to the sample preparation, chromatographic/mass spectrometry procedure, and data interpretation, the most important points within the analysis.

In general, LC-MS analysis, due to high sensitivity, requires the highest standards of a protocol strictness an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-hydroksy-DL-kynurenina	Sigma-Aldrich	H1771
3-hydroxyanthranilic acid	Sigma-Aldrich	148776	97%
3-nitro-L-tyrosine	Sigma-Aldrich	N7389
Acetonitrile	Supelco	1.00029	hypergrade for LC-MS LiChrosolv
Activated charcoal	Supelco	05105	powder
Analytical balance	Ohaus
Analytical column	Agilent Technologies	959764-902	Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 µm)
Ammonium formate	Supelco	7022	eluent additive for LC-MS, LiChropur, ≥99.0%
Bovine Serum Albumin	Sigma	1001887398
Caps for chromatographic vials	Agilent Technologies	5185-5820	blue screw caps,PTFE/red sil sepa
Cell culture dish	Nest	704004	polystyrene, non-pyrogenic, sterile
Cell culture plate	Biologix	07-6012	12-well, non-pyrogenic, non-cytoxic, sterille
Cell incubator	Thermo Fisher Scientific		HERAcell 150i Cu
Centrifuge	Eppendorf		model 5415R
Centrifuge	Eppendorf		model 5428
Centrifuge tubes	Bionovo	B-2278	Eppendorf type, 1.5 mL
Centrifuge tubes	Bionovo	B-3693	Falcon type, 50 mL, PP
Chromatographic data acqusition and analysis software	Agilent Technologies		LC/MSD ChemStation (B.04.03-S92)
Chromatographic insert vials	Agilent Technologies	9301-1387	100 µL
Chromatographic vials	Agilent Technologies	5182-0714	2 mL, clear glass
Dimethyl sulfoxide	Supelco	1.02950	Uvasol
Dubelcco’s Modified Eagle’s Medium (DMEM)	PAN Biotech	P04-41450
Dual meter pH/conductivity	Mettler Toledo		SevenMulti
Evaporator	Genevac		model EZ-2.3 Elite
Fetal bovine serum	Sigma-Aldrich	F9665
Formic acid	Supelco	5.33002	for LC-MS LiChropur
Glass bottle for reagents storage	Bionovo	S-2070	50 mL, clear glass
Guard column	Agilent Technologies	959757-902	Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 µm)
Hydrochloric acid	Merck	1.00317.1000
L-kynurenine	Sigma-Aldrich	K8625	≥98% (HPLC)
Magnetic stirrer	Wigo		ES 21 H
Microbalance	Mettler Toledo		model XP6
Milli-Q system	Millipore	ZRQSVPO30	Direct-Q 3 UV with Pump
Quadrupole mass spectrometer	Agilent Technologies	G1948B	model 6120
Penicillin-Streptomycin	Sigma-Adrich	048M4874V
Plate reader	Bio Tek		Synergy 2 operated by Gen5
Potassium chloride	Merck	1.04936.1000
Potassium dihydrogen phosphate	Merck	1.05108.0500
Protein Assay Dye Reagent Concentrate	BioRad	500-0006
See-saw rocker	Stuart	SSL4
Serological pipette	Nest	326001	5 mL, polystyrene, non-pyrogenic, sterile
Sodium chloride	Sigma-Aldrich	7647-14-5
Sodium phosphate dibasic	Merck	1.06346.1000
Solvent inlet filter	Agilent Technologies	5041-2168	glass filter, 20 μm pore size
Solvent reservoir (for LC-MS)	Agilent Technologies	9301-6524	1 L, clear glass
Solvent reservoir (for LC-MS)	Agilent Technologies	9301-6526	1 L, amber glass
Spreadsheet program	Microsoft Office		Microsoft Office Excel
Stir bar	Bionovo	6-2003	teflon coated
Syringe filters for culture medium filtration	Bionovo	7-8803	regenerated cellulose, Ø 30 mm, 0,45 µm
Syringe filters for mobile phase components filtration	Bionovo	6-0018	nylon, Ø 30 mm, 0,22 µm
Tissue culture plates	VWR	10062-900	96-wells, sterille
Trypsin-EDTA Solution	Sigma-Aldrih	T4049-100 mL
Ultra-High Performance Liquid Chromatography system	Agilent Technologies	G1367D, G1379B, G1312B, G1316C	1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C)
Ultrasound bath	Polsonic	104533	model 6D
Vortex	Biosan		model V-1 plus
Xanthurenic acid	Sigma-Aldrich	D120804	96%