A mass spectrometric method for quantification of tryptophan-derived uremic solutes in human serum

© 2017 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )

Download PDF

Cite

XML

HTML

Abstract

In addition to various physiologic roles, emerging evidence strongly points to pathogenic roles of tryptophan and of its metabolites, especially in diseases such as renal failure. Accurate estimation of levels of these metabolites in blood is important to mechanistically probe their contribution to disease pathogenesis, while clinically, such a panel can be used to risk stratify patients for a clinical phenotype. Herein, we describe a comprehensive liquid chromatography-mass spectrometry (LC/MS)-based method to determine the level of tryptophan and its metabolites (kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate and indoxyl acetate). Human sera samples were processed through a C18 column followed by application of a binary gradient and quantitation by MS/MS. The linearity, lower limit of detection, inter- and intraassay variabilities and recovery were determined, yielding a precise, reproducible method for all the metabolites. Unlike previous studies, we further validated these methods in a well-characterized set of human sera from end stage renal disease patients compared to age-, gender- and ethnic-background matched human controls. Overall, we report an optimized LC/MS-based estimation of a comprehensive panel of tryptophan-derived metabolites with quality features within FDA standards, underscoring their readiness for translational use.

Keywords

uremic solutes

mass spectrometry

indoxyl sulfate

kynurenine

References

1. Michael, A.F., et al., Tryptophan Metabolism in Man. J Clin Invest, 1964. 43: p. 1730-46.
2. Vanholder, R., et al., Uremic toxicity: present state of the art. Int J Artif Organs, 2001. 24(10): p. 695-725.
3. Yao, K., et al., Tryptophan metabolism in animals: important roles in nutrition and health. Front Biosci (Schol Ed), 2011. 3: p. 286-97.
4. Pawlak, K., et al., Kynurenine, quinolinic acid--the new factors linked to carotid atherosclerosis in patients with end-stage renal disease. Atherosclerosis, 2009. 204(2): p. 561-6.
5. Oxenkrug, G.F., Metabolic syndrome, age-associated neuroendocrine disorders, and dysregulation of tryptophan-kynurenine metabolism. Ann N Y Acad Sci, 2010. 1199: p. 1-14.
6. Nguyen, N.T., et al., Aryl hydrocarbon receptor and kynurenine: recent advances in autoimmune disease research. Front Immunol, 2014. 5: p. 551.
7. Kawasaki, H., et al., A tryptophan metabolite, kynurenine, promotes mast cell activation through aryl hydrocarbon receptor. Allergy, 2014. 69(4): p. 445-52.
8. Shivanna, S., et al., The Aryl Hydrocarbon Receptor is a Critical Regulator of Tissue Factor Stability and an Antithrombotic Target in Uremia. J Am Soc Nephrol, 2016. 27(1): p. 189-201.
9. Shashar, M., J. Francis, and V. Chitalia, Thrombosis in the uremic milieu--emerging role of "thrombolome". Semin Dial, 2015. 28(2): p. 198-205.
10. Pawlak, D., et al., Accumulation of toxic products degradation of kynurenine in hemodialyzed patients. Int Urol Nephrol, 2001. 33(2): p. 399-404.
11. Itoh, Y., et al., Protein-bound uremic toxins in hemodialysis patients measured by liquid chromatography/tandem mass spectrometry and their effects on endothelial ROS production. Anal Bioanal Chem, 2012. 403(7): p. 1841-50.
12. Chen, Y., et al., RRLC-MS/MS-based metabonomics combined with in-depth analysis of metabolic correlation network: finding potential biomarkers for breast cancer. Analyst, 2009. 134(10): p. 2003-11.
13. EMA. Guidance for industry: bioanalytical method validation. US Department of health and human service, US FDA, Rockville, MD USA. .
14. Zhu, W., et al., Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem, 2011. 401(10): p. 3249-61.
15. Fuertig, R., et al., LC-MS/MS-based quantification of kynurenine metabolites, tryptophan, monoamines and neopterin in plasma, cerebrospinal fluid and brain. Bioanalysis, 2016. 8(18): p. 1903-17.
16. Meyer, T.W., et al., The clearance of protein-bound solutes by hemofiltration and hemodiafiltration. Kidney Int, 2005. 68(2): p. 867-77.
17. Manni, A., et al., Bioavailability of albumin-bound testosterone. J Clin Endocrinol Metab, 1985. 61(4): p. 705-10.
18. Forker, E.L., et al., Effect of albumin binding on the hepatic transport of rose bengal: surface-mediated dissociation of limited capacity. J Pharmacol Exp Ther, 1982. 223(2): p. 342-7.
19. Bipath, P.a.M.V., Tryptophan and the kynurenine pathway in haemodialysis and peritoneal dialysis. Biomedical Research, 2010. 09(April).

Previous article in this issue

Next article in this issue

Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific

Publisher's Core Philosophy

We are committed to support the scientific community by publishing impactful research and enhancing communication among scientists. At POL Scientific, we are continuously looking for ways to accelerate scientific progress and to strive for transparency and open communication, making knowledge freely accessible without barrier.

171 Skyview St. San Francisco, CA 94131 U.S.A.

info@polscientific.com