Abstract
Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.
Keywords: Tryptophan, Kynurenine, Immune surveillance, HPLC
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. HPLC chromatogram showing separation of tryptophan, kynurenine, kynurenic acid and nitrotyrosine standards. Quinolinate is also present in the standard mix but below the limit of quantification. The upper panel shows absorbance at 360 nm, and the lower panel shows absorbance at 286 nm. The tryptophan metabolite kynurenic acid and 3-nitro-L-tyrosine (nitrotyrosine), which can be used as an internal standard, are also separated. All analytes are present at a concentration of 12.5 µM. The standard solution was prepared as described in the Recipes section.
Notes
This method can be modified for compatibility with liquid chromatography-mass spectrometry by using a mobile phase consisting of 10 mM ammonium formate (pH 6.4), containing 2.7% acetonitrile. Other chromatographic conditions remain unchanged. If desired, a known quantity of 3-nitro-L-tyrosine can be added to samples prior to extraction for use as an internal standard.
Recipes
Acknowledgments
This work was supported by NIH grants 1R01CA135401-01A1, and 1R01DK082690-01A1, the Medical Service of the US Department of Veterans’ Affairs, and Dialysis Clinics, Inc. (DCI) (all to R. H. W.). This work was performed in part at the Research Resource for Biomedical Accelerator Mass Spectrometry, which is operated at LLNL under the auspices of the U. S. Department of Energy under contract DEAC52-07NA27344. The Research Resource is supported by the National Institutes of Health, National Institute of General Medical Sciences under Grant P40 RR13461. The extraction protocol was adapted from Villas-Boas et al. (2005). The HPLC method was adapted from Laich et al. (2002).
References
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