MSACL 2024 Abstract

The Investigation of Tryptophan Metabolites and Vitamin B in Myalgic Encephalomyelitis via High-Resolution Mass Spectrometry Sandy Abujrais, Kumari Ubhayasekera, Jonas Bergquist Analytical Chemistry and Neurochemistry, Department of Chemistry – BMC, Uppsala University, Uppsala, Sweden The ME/CFS Collaborative Research Centre at Uppsala University, Sweden Sandy Abujrais (Presenter) Uppsala University

Presenter Bio: Highly motivated and professional analytical chemist with more than 5 years of experience in performing chemical analysis of variety of clinical samples using specialized equipment and analytical techniques with experience of metabolomics and proteomics analysis using high resolution mass spectrometry. Possess teaching experience, researching, report-writing, and familiar with GLP and ISO 17025.

Introduction

Tryptophan is mainly metabolised by two biological pathways, the kynurenine pathway (KP) and the serotonin pathway. KP is important for producing both neuroprotective compounds, such as kynurenic acid and neurotoxic compounds like 3-hydroxy kynurenine, 3-hydroxy anthranilinic acid and quinolinic acid. Kynurenine has neurotoxic potential and has been linked to depression and anxiety. Enzymatic cofactors involved in the tryptophan kynurenine pathway include the B vitamins, particularly pyridoxal 5′-phosphate and riboflavin.
Myalgic encephalomyelitis (ME) is a chronic multisystem disease that affects the nervous, immune system and energy metabolism. The cause of the disease remains unknown with no diagnostic test available. The investigation of these metabolites in ME will help increase our understanding of the disease pathology.

Methods

Plasma samples from 38 ME patients and 24 healthy participants were extracted using a protein precipitation method. The isotope dilution method was used to quantify 12 analytes of tryptophan metabolites (quinolinic acid , 3-hydroxykynurenine , kynurenine , 3-hydroxyanthranilic acid , tryptophan , anthranilic acid , xanthurenic acid , kynurenic acid , nicotinamide , 5-hydroxytryptophan, melatonin and serotonin) and five metabolites of vitamin B (riboflavin, pantothenic acid, pyridoxine, biotin and pyridoxal 5′-phosphate). A parallel reaction monitoring (PRM) is employed in a positive electrospray mode. The analyses were performed using a Waters Acquity UHPLC (Waters™) coupled to a high-resolution Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific™).

Preliminary Data

Using direct infusion, mass spectrometric detection parameters, including MS/MS mass transitions were optimized for each analyte . Thereafter, the method was validated according to the European Medicines Agency (EMA) guidelines for bioanalytical method development. Precision, accuracy, freeze-thaw stability, recovery and matrix effect were evaluated by preparing quality control samples at four levels; lower limit of quantification, low, medium and high quality control. These validation parameters were found to be within the limits outline in the EMA guidelines.
There was a wide dynamic range for the analytes investigated ranging from 0.02 ng/ml for melatonin and 60000 ng/ml for tryptophan.

Discussion

This rapid and sensitive method allows for the simultaneous analysis and absolute quantification of 15 analytes related to tryptophan metabolism focusing on KP and its enzyme cofactors.
This method can be employed in ME to increase our understanding of the pathological changes associated of the disease.