Stability Studies of Kynurenine Pathway Metabolites in Blood Components Define Optimal Blood Processing Conditions.

The kynurenine pathway (KP) is the main pathway of tryptophan (TRP) metabolism that generates energy for multiple cellular processes. The activity of this pathway has been shown to be dysregulated in multiple human diseases. The resultant modulation of metabolites has been suggested to comprise biomarkers to track disease progression or could identify new therapeutic targets. While metabolite changes can be measured readily in blood, there is limited knowledge on the effect of blood matrices and sample processing time may have on the stability of KP metabolites. Understanding the stability of KP metabolites in blood is integral to obtaining accurate KP data to correlate with clinical pathology. Hence, the aim of this study was to assess the concentration of KP metabolites in matched whole blood, plasma and serum. The impact of pre-analytical sample processing time in the various blood matrices was also analysed. Serum and plasma had the higher concentration of KP metabolites compared to whole blood. Furthermore, concentrations of KP metabolites declined when the collected blood was processed after 24 hours storage at 4°C. Our study shows that that type of blood matrix and the time to processing have an impact on the stability of the KP metabolites. Serum or plasma are the preferred choice of matrix and the isolation of these matrices from whole blood is best performed immediately after collection for optimal analytical KP data.

Temporal Profile of Kynurenine Pathway Metabolites in a Rodent Model of Autosomal Recessive Polycystic Kidney Disease.

Autosomal recessive polycystic kidney disease (ARPKD) is an early onset genetic disorder characterized by numerous renal cysts resulting in end stage renal disease. Our study aimed to determine if metabolic reprogramming and tryptophan (Trp) metabolism via the kynurenine pathway (KP) is a critical dysregulated pathway in PKD. Using the Lewis polycystic kidney (LPK) rat model of PKD and Lewis controls, we profiled temporal trends for KP metabolites in plasma, urine, and kidney tissues from 6- and 12-week-old mixed sex animals using liquid and gas chromatography, minimum n = 5 per cohort. A greater kynurenine (KYN) concentration was observed in LPK kidney and plasma of 12-week rats compared to age matched Lewis controls (P ⩽ .05). LPK kidneys also showed an age effect (P ⩽ .05) with KYN being greater in 12-week versus 6-week LPK. The metabolites xanthurenic acid (XA), 3-hydroxykynurenine (3-HK), and 3-hydroxyanthranilic acid (3-HAA) were significantly greater in the plasma of 12-week LPK rats compared to age matched Lewis controls (P ⩽ .05). Plasma XA and 3-HK also showed an age effect (P ⩽ .05) being greater in 12-week versus 6-week LPK. We further describe a decrease in Trp levels in LPK plasma and kidney (strain effect P ⩽ .05). There were no differences in KP metabolites in urine between cohorts. Using the ratio of product and substrates in the KP, a significant age-strain effect (P ⩽ .05) was observed in the activity of the KYN/Trp ratio (tryptophan-2,3-dioxygenase [TDO] or indoleamine-2,3-dioxygenase [IDO] activity), kynurenine 3-monooxygenase (KMO), KAT A (kynurenine aminotransferase A), KAT B, total KAT, total KYNU (kynureninase), KYNU A, KYNU B, and total KYNU within LPK kidneys, supporting an activated KP. Confirmation of the activation of these enzymes will require verification through orthogonal techniques. In conclusion, we have demonstrated an up-regulation of the KP in alignment with progression of renal impairment in the LPK rat model, suggesting that KP activation may be a critical contributor to the pathobiology of PKD.

Recent advances in clinical trials targeting the kynurenine pathway.

The kynurenine pathway (KP) is the major catabolic pathway for the essential amino acid tryptophan leading to he production of nicotinamide adenine dinucleotide. In inflammatory conditions, the activation of the KP leads to the production of several bioactive metabolites including kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, kynurenic acid and quinolinic acid. These metabolites can have redox and immune suppressive activity, be neurotoxic or neuroprotective. While the activity of the pathway is tightly regulated under normal physiological condition, it can be upregulated by immunological activation and inflammation. The dysregulation of the KP has been implicated in wide range of neurological diseases and psychiatric disorders. In this review, we discuss the mechanisms involved in KP-mediated neurotoxicity and immune suppression, and its role in diseases of our expertise including cancer, chronic pain and multiple sclerosis. We also provide updates on the clinical trials evaluating the efficacy of KP inhibitors and/or analogues in each respective disease.

Effects of Sleep Deprivation on the Tryptophan Metabolism.

Sleep has a regulatory role in maintaining metabolic homeostasis and cellular functions. Inadequate sleep time and sleep disorders have become more prevalent in the modern lifestyle. Fragmentation of sleep pattern alters critical intracellular second messengers and neurotransmitters which have key functions in brain development and behavioral functions. Tryptophan metabolism has also been found to get altered in SD and it is linked to various neurodegenerative diseases. The kynurenine pathway is a major regulator of the immune response. Adequate sleep alleviates neuroinflammation and facilitates the cellular clearance of metabolic toxins produced within the brain, while sleep deprivation activates the enzymatic degradation of tryptophan via the kynurenine pathway, which results in an increased accumulation of neurotoxic metabolites. SD causes increased production and accumulation of kynurenic acid in various regions of the brain. Higher levels of kynurenic acid have been found to trigger apoptosis, leads to cognitive decline, and inhibit neurogenesis. This review aims to link the impact of sleep deprivation on tryptophan metabolism and associated complication in the brain.

Novel immune biomarkers in complex regional pain syndrome.

We investigated serum levels of 29 cytokines and immune-activated kynurenine and tetrahydrobiopterin pathway metabolites in 15 complex regional pain syndrome (CRPS) subjects and 14 healthy controls. Significant reductions in interleukin-37 and tryptophan were found in CRPS subjects, along with positive correlations between kynurenine/tryptophan ratio and TNF-α levels with kinesiophobia, tetrahydrobiopterin levels with McGill pain score, sRAGE, and xanthurenic acid and neopterin levels with depression, anxiety and stress scores. Using machine learning, we identified a set of binary variables, including IL-37 and GM-CSF, capable of distinguishing controls from established CRPS subjects. These results suggest possible involvement of various inflammatory markers in CRPS pathogenesis.