Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry
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.
References (43)
- et al.
Kynurenine pathway—a new link between endothelial dysfunction and carotid atherosclerosis in chronic kidney disease patients
Adv. Med. Sci.
(2010) - et al.
Is tryptophan metabolism involved in sleep apnea-related cardiovascular co-morbidities and cancer progression?
Med. Hypotheses
(2015) - et al.
Kynurenine pathway metabolites in humans: disease and healthy states
Int. J. Tryptophan Res.
(2009) - et al.
Development and validation of a single analytical method for the determination of tryptophan, and its kynurenine metabolites in rat plasma
J. Chromatogr. B
(2012) - et al.
Plasma tryptophan, kynurenine and 3-hydroxykynurenine measurement using automated on-line solid-phase extraction HPLC-tandem mass spectrometry
J. Chromatogr. B
(2009) - et al.
Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry
J. Chromatogr. B
(2008) - et al.
Stable isotope dilution ultra-high performance liquid chromatography–tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid
J. Chromatogr. A
(2016) - et al.
A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma
J. Pharm. Biomed. Anal.
(2015) - et al.
Quantitation of tryptophan, kynurenine and kynurenic acid in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2002) - et al.
High throughput analysis of tryptophan metabolites in a complex matrix using capillary electrophoresis coupled to time-of-flight mass spectrometry
J. Chromatogr. A
(2007)
Concurrent quantification of tryptophan and its major metabolites
Anal. Biochem.
Targeting tryptophan and tyrosine metabolism by liquid chromatography tandem mass spectrometry
J. Chromatogr. A
High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma
J. Chromatogr. B
Stability study of 81 analytes in human whole blood, in serum and in plasma
Clin. Biochem.
Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain
Anal. Biochem.
Selective and sensitive liquid chromatographic determination method of 5-hydroxyindoles with fluorous and fluorogenic derivatization
J. Pharm. Biomed. Anal.
Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection
J. Chromatogr. B
Simultaneous quantification of neuroactive dopamine serotonin and kynurenine pathway metabolites in gender-specific youth urine by ultra performance liquid chromatography tandem high resolution mass spectrometry
J. Pharm. Biomed. Anal.
Tryptophan catabolism and vitamin B-6 status are affected by gender and lifestyle factors in healthy young adults
J. Nutr.
Indoleamine 2,3-dioxygenase activity associates with cardiovascular risk factors: the health 2000 study
Scand. J. Clin. Lab. Invest.
Immune activation and degradation of tryptophan in coronary heart disease
Eur. J. Clin. Invest.
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