Electrochemical Determination of Kynurenine Pathway Metabolites—Challenges and Perspectives

Abstract

In recent years, tryptophan metabolism via the kynurenine pathway has become one of the most active research areas thanks to its involvement in a variety of physiological processes, especially in conditions associated with immune dysfunction, central nervous system disorders, autoimmunity, infection, diabetes, and cancer. The kynurenine pathway generates several metabolites with immunosuppressive functions or neuroprotective, antioxidant, or toxic properties. An increasing body of work on this topic uncovers a need for reliable analytical methods to help identify and quantify tryptophan metabolites at physiological concentrations in biological samples of different origins. Recent methodological advances in the fabrication and application of electrochemical sensors promise a rise in the future generation of novel analytical systems. This work summarizes current knowledge and provides important suggestions with respect to direct electrochemical determinations of kynurenine pathway metabolites (kynurenines) in complex biological matrices. Measurement challenges, limitations, and future opportunities of electroanalytical methods to advance study of the implementation of kynurenines in disease conditions are discussed.

Establishing a quantitative fluorescence assay for the rapid detection of kynurenine in urine

“…33 Electroanalytical methods enhance the high-throughput capabilities but currently still require both chromatographic and pre-treatment steps for reducing interference from other species in complex media. 23 Comparing our fluorescencebased approach with currently available alternatives shows that our approach delivers selectivity and enough sensitivity (LOD 0.7 μM) to quantify kynurenine at physiological concentrations in urine samples, without the need for the time-consuming and costly sample preparation. The developed fluorescence-based approach is distinguished by its simplicity, low cost and speed and suitability for automation, enabling highthroughput measurements of large number of samples.…”

“…Currently, quantitative analysis for kynurenine in biological samples has been reported using HPLC, 15,16,19 GC-MS 7,8 and LC-MS. 7,8,17 An ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method has been established that can detect kynurenine in urine with a LOD < 11.5 nM. 23 Urinary concentrations of kynurenine are found at low micromolar levels (>1.4 μM). 8 Thus, LC-MS-based approaches offer specificity and sensitivity, which is well below the levels required, but it is not without limitations.…”

“…[20][21][22] However, whilst electroanalytical methods can produce sensitive measurements in point of care formats such as glucose sensing, there is still some challenges to address before mass clinical testing. 23 Ideally, a useful kynurenine sensor would have highthroughput capabilities allowing for rapid results from multiple patient samples. In clinical settings, fluorescence is routinely utilised in formats such as immunoassays, where existing high-throughput technology is available in routine clinical biochemistry laboratories.…”

“…20–22 However, whilst electroanalytical methods can produce sensitive measurements in point of care formats such as glucose sensing, there is still some challenges to address before mass clinical testing. 23…”

Electrochemistry meets mass spectrometry

“…LC-EC, relying on analytical standards, is able to detect and quantify compounds belonging to many different chemical classes, such as vitamins 121 , hormones 122 , carotenoids 123 and drugs of abuse 124 . Neurotransmitters can also be detected by LC-EC 125,126 and this analytical platform has been successfully implemented for the discovery of gut bacterial metabolites 34,35 . Furthermore, EC detection has been used for the determination of serotonin levels in feces from mice 127,128 and short chain fatty acids in rat and human feces 129 , as well as in mouse feces 130 , proving to be a suitable method for the detection of metabolites in complex biological samples.…”