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Analysis of Tryptophan and Its Metabolites by High-Performance Liquid Chromatography

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Amino Acid Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2030))

Abstract

Tryptophan is a nutritionally essential amino acid for both humans and animals. Besides acting as a building block for protein synthesis, tryptophan (Trp) and its metabolites are crucial for maintaining neurological function, immunity, and homeostasis in the body. To uncover the regulatory role of Trp and its metabolites in cell nutrition, metabolism and physiology, various analytical methods, including high-performance liquid chromatography (HPLC), have been developed to determine key Trp metabolites. Here we describe a rapid and sensitive method for the simultaneous analysis of Trp and its metabolites along with other amino acids by HPLC involving in-line pre-column derivatization with o-phthaldialdehyde (OPA) and dual-channel fluorescence detection. OPA reacts very rapidly (within 1 min) with Trp, 5-hydroxytryptophan, 5-hydroxytryptamine, and tryptamine at room temperature (e.g., 20–25 °C) in an autosampler. Their derivatives are immediately injected into the HPLC column without the need for extraction. Trp metabolites that cannot react with OPA but are fluorescent can be detected by setting the excitation and emission wavelengths of the fluorescence detector in another detection channel. The autosampler is programmed to mix Trp and its metabolites with OPA for 1 min to generate highly fluorescent derivatives for HPLC separation and detection (Channel A, excitation = 270 nm and emission = 350 nm; Channel B, excitation = 340 nm and emission = 450 nm). The detection limit for Trp and its metabolites is 30 pmol/mL or 150 fmol/injection. The total time for chromatographic separation (including column regeneration) is 55 min for each sample. Our HPLC method can be used for the analysis of amino acids (including Trp) in alkaline protein hydrolysates and of Trp and its metabolites in biological samples.

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References

  1. Wu G (2010) Functional amino acids in growth, reproduction, and health. Adv Nutr 1(1):31–37

    Article  CAS  Google Scholar 

  2. Berger M, Gray JA, Roth BL (2009) The expanded biology of serotonin. Annu Rev Med 60:355–366

    Article  CAS  Google Scholar 

  3. Fernstrom JD (2016) A perspective on the safety of supplemental tryptophan based on its metabolic fates. J Nutr 146:2601S–2608S

    Article  CAS  Google Scholar 

  4. Deslandes B, Gariépy C, Houde A (2001) Review of microbiological and biochemical effects of skatole on animal production. Livest Prod Sci 71:193–200

    Article  Google Scholar 

  5. Presits P, Molnar-Perl I (2003) HPLC of tryptophan and its metabolites as OPA derivatives and on the basis of their UV and fluorescence spectra, simultaneously. Adv Exp Med Biol 527:695–704

    Article  CAS  Google Scholar 

  6. Vignau J, Jacquemont MC, Lefort A, Imbenotte M, Lhermitte M (2004) Simultaneous determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection. Biomed Chromatogr 18:872–874

    Article  CAS  Google Scholar 

  7. Yoshitake T, Fujino K, Kehr J, Ishida J, Nohta H, Yamaguchi M (2003) Simultaneous determination of norepinephrine, serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine. Anal Biochem 312:125–133

    Article  CAS  Google Scholar 

  8. Thorré K, Pravda M, Sarre S, Ebinger G, Michotte Y (1997) New antioxidant mixture for long term stability of serotonin, dopamine and their metabolites in automated microbore liquid chromatography with dual electrochemical detection. J Chromatogr B Biomed Sci Appl 694:297–303

    Article  Google Scholar 

  9. Rahman MS, Thomas P (2014) Restoration of tryptophan hydroxylase functions and serotonin content in the Atlantic croaker hypothalamus by antioxidant treatment during hypoxic stress. Front Neurosci 8:130

    Article  Google Scholar 

  10. Bourcier S, Benoist JF, Clerc F, Rigal O, Taghi M, Hoppilliard Y (2006) Detection of 28 neurotransmitters and related compounds in biological fluids by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 20:1405–1421

    Article  CAS  Google Scholar 

  11. Fürst P, Pollack L, Graser TA, Godel H, Stehle P (1990) Appraisal of four pre-column derivatization methods for the high-performance liquid chromatographic determination of free amino acids in biological materials. J Chromatogr 499:557–569

    Article  Google Scholar 

  12. Wu G, Knabe DA (1994) Free and protein-bound amino acids in sow’s colostrum and milk. J Nutr 124:415–424

    Article  CAS  Google Scholar 

  13. Wu G, Meininger CJ (2008) Analysis of citrulline, arginine, and methylarginines using high-performance liquid chromatography. Methods Enzymol 440:177–189

    Article  CAS  Google Scholar 

  14. Dai Z, Wu Z, Jia S, Wu G (2014) Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection. J Chromatogr B 964:116–127

    Article  CAS  Google Scholar 

  15. Ji Y, Wu Z, Dai Z, Sun K, Zhang Q, Wu G (2016) Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells. Amino Acids 48:149–156

    Article  CAS  Google Scholar 

  16. Hou Y, Li X, Dai Z, Wu Z, Bazer FW, Wu G (2018) Analysis of glutathione in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde. Methods Mol Biol 1694:105–115

    Article  CAS  Google Scholar 

  17. Wu G, Flynn NE, Knabe DA, Jaeger LA (2000) A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning. Am J Physiol Regul Integr Comp Physiol 279:R554–R559

    Article  CAS  Google Scholar 

  18. Dai Z, Wu Z, Wang J, Wang X, Jia S, Bazer FW, Wu G (2014) Analysis of polyamines in biological samples by HPLC involving pre-column derivatization with o-phthalaldehyde and N-acetyl-l-cysteine. Amino Acids 46:1557–1564

    Article  CAS  Google Scholar 

  19. Li H, Meininger CJ, Bazer FW, Wu G (2016) Intracellular sources of ornithine for polyamine synthesis in endothelial cells. Amino Acids 48:2401–2410

    Article  CAS  Google Scholar 

  20. Hou Y, Jia S, Nawaratna G, Hu S, Dahanayaka S, Bazer FW, Wu G (2015) Analysis of L-homoarginine in biological samples by HPLC involving precolumn derivatization with o-phthalaldehyde and N-acetyl-l-cysteine. Amino Acids 47:2005–2014

    Article  CAS  Google Scholar 

  21. Downing SR, Klement GL (2012) Isolation and proteomic analysis of platelets by SELDI-TOF MS. Methods Mol Biol 818:153–170

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Work in our laboratories was supported by grants from the National Natural Science Foundation of China (31301979), National Key Basic Research Program of China (2013CB127303), National Key R&D Program of China (2017YFD0500501), the Zhengzhou 1125 Talent Program, Agriculture and Food Research Initiative Competitive Grants (2014-67015-21770, 2015-67015-23276, and 2016-67015-24958) from the USDA National Institute of Food and Agriculture, and Texas A&M AgriLife Research (H-8200).

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Authors and Affiliations

  1. State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China

    Zhaolai Dai, Shiqiang Sun, Hui Chen, Moyan Liu, Lianhua Zhang, Zhenlong Wu & Guoyao Wu

  2. Henan Yinfa Animal Husbandry Co., Xinzheng, Henan, China

    Ju Li

  3. Department of Animal Science, Texas A&M University, College Station, TX, USA

    Guoyao Wu

Authors
  1. Zhaolai Dai
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  2. Shiqiang Sun
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  3. Hui Chen
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  4. Moyan Liu
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  5. Lianhua Zhang
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  6. Zhenlong Wu
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  7. Ju Li
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  8. Guoyao Wu
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Corresponding author

Correspondence to Zhaolai Dai .

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Editors and Affiliations

  1. Office of Policy for Pharmaceutical Quality Office of Pharmaceutical Quality, Center for Drug Evaluation and Research US FDA, Silver Spring, MD, USA

    Michail A. Alterman

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Dai, Z. et al. (2019). Analysis of Tryptophan and Its Metabolites by High-Performance Liquid Chromatography. In: Alterman, M. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 2030. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9639-1_11

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  • DOI: https://doi.org/10.1007/978-1-4939-9639-1_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9638-4

  • Online ISBN: 978-1-4939-9639-1

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