Elsevier

Journal of Chromatography B

Volume 1054, 1 June 2017, Pages 36-43
Journal of Chromatography B

Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry

https://doi.org/10.1016/j.jchromb.2017.04.010Get rights and content

Highlights

  • A quantitative method to simultaneously assay tryptophan and 8 metabolites in plasma.

  • The method is quick (15 min per chromatographic run) and simple.

  • The method has been validated according to NF EN ISO 15189 criteria.

Abstract

Tryptophan (Trp) is an essential amino-acid and the precursor of many biologically active substances such as kynurenine (KYN) and serotonin (5HT). Its metabolism is involved in different physiopathological states, such as cardiovascular diseases, cancer, immunomodulation or depression. Hence, the quantification of Trp catabolites, from both KYN and 5HT pathways, might be usefulfor the discovery of novel diagnostic and follow-up biomarkers.

We have developed a simple method for quantification of Trp and 8 of its metabolites,involved in both KYN and 5HT pathways, using liquid chromatography coupled to tandem mass spectrometry. We also validated the methodin human plasma samples, according to NF EN ISO 15189 criteria.

Our method shows acceptable intra- and inter-day coefficients of variation (CV) (<12% and <16% respectively). The linearity entirelycovers the human plasma range. Stabilities of whole blood and of residues weredetermined, as well as the use of 2 different types of collectiontube, enabling us to adapt our process. Matrix effects and reference values showed good agreement compared to the literature.

We propose here a method allowing the simultaneous quantification of a panel of Trp catabolites, never used before to our knowledge. This method, witha quickchromatographic runtime (15 min) and simple sample preparation, has beenvalidated according to NF EN ISO 15189 criteria. The method enables the detailed analysis of these metabolic pathways, which are thought to be involved in a number of pathological conditions.

Introduction

L-Tryptophan (Trp) is an essential amino acid that is mostly involved in protein synthesis. It is also the precursor of many biologically active substances, such as kynurenine (KYN) and serotonin (5HT). The kynurenine pathway (95% of Trp catabolism) leads to nicotinamide adenosine dinucleotide (NAD) synthesis. The first reaction, is catalysed by two haem-dependent enzymes: the ubiquitous indoleamine-2,3-dioxygenase (IDO; EC 1.13.11.17) and tryptophan-2,3-dioxygenase (TDO; EC 1.13.11.11), localized mainly in the liver. In the last decade there has been considerable progress in research into the KYN pathway and cardiovascular diseases [1], [2], [3], [4]. Other studies show a strong link between IDO and immune tolerance [5], [6], and between the KYN pathway and cancerogenesis via activation of the aryl hydrocarbon receptor [7]. Our group has recently suggested a link between tryptophan metabolism, cardiovascular comorbidities and cancer progression in patients with sleep apnea [8]. Moreover, both the serotonin pathway and the KYN pathway have been implicated in neurological diseases [9], [10], [11]. While quantitatively the serotonin pathway consumes a small proportion of Trp (5% of Trp catabolism) its deregulation is responsible for depression and neurodegenerative diseases [12].1

Hence, the quantification of tryptophan catabolites, from both the KYN and 5HT pathways, presents an opportunity for the discovery of novel diagnostic and follow-up biomarkers. Several authors have described methods to determine the concentration of some metabolites of the KYN pathway [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], mainly using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), but to our knowledge no one has proposed the multiplex detection of all 9 metabolites presented in Fig. 1. Here, we introduce a reliable method using LC–MS/MS to quantitate Trp, KYN, anthranilic acid (AA), kynurenic acid (KA), 3-hydroxykynurenine (3HK), xanthurenic acid (XA), 3-hydroxyanthranilic acid (3HA), 5-hydroxytryptophan (5HTP) and 5HT. This simple method, without derivatization, has been validated for the analysis of human plasma according to NF EN ISO 15189 (range B) standard criteria [27].

Section snippets

Chemicals and reagents

Unlabelled Trp, KYN, AA, KA, 3HK, XA, 3HA, 5HTP and 5HT were purchased from Sigma-Aldrich (Lyon, France). 2H5-Trp was obtained from C/D/N Isotopes (Pointe-Claire, Canada) and 13C6-KYN, 13C6-AA, 2H5-KA, 13C6-3HK, 13C6-XA, 13C6-3HA and 2H4-5HT were purchased from Alsachim (Illkirch, France). Solvents for sample preparation and LC–MS/MS analysis were HPLC grade and purchased from Merck (Darmstadt, Germany), Carlo Erba Reagents (Val de Reuil, France) and VWR (Radnor, USA). Ascorbic acid (ASC), zinc

Results

A chromatogram of a typical plasma sample is shown in Fig. 3, representing the total ion current searched in each period.

Discussion

Variations in Trp metabolism have been shown to be implicated in many patho-physiological conditions [9]. In the present study we propose the simultaneous measurement of 9 Trp metabolite concentrations (i.e. Trp, KYN, 3HK, 5HT, 5HTP, 3HA, XA, KA and AA) using a simple protein precipitation coupled to HPLC with MS/MS detection. Other authors have proposed methods to measure the concentration of Trp and some of its metabolites, using different techniques, but to our knowledge no one has measured

Conclusion

We propose a cost and time efficient HPLC–MS/MS method for quantification of Trp and 8 metabolites involved in the KYN and 5HT pathways, using less than 200 μL of fasting plasma collected in EDTA-coated tubes. These analytes can be measured accurately, precisely and with high reproducibility. The potential clinical use of this method is wide-ranging, providing an opportunity for the routine detailed analysis of Trp metabolism in a number of pathological conditions.

Conflict of interest

The authors have no conflict of interest to declare.

Competing interests

The funding organizations played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Funding

This work was supported by an award from the French society of inborn error metabolism (SFEIM) and sponsored by SCIEX chromatography equipment suppliers.

Author contributions

All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Acknowledgement

We thank Dr Alison Foote (Grenoble Alpes University Hospital) for critical editing of the manuscript.

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