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

ABSTRACT: The kynurenine metabolite is associated with many diseases and disorders, ranging from diabetes and sepsis to more recently COVID-19. Here we report a fluorescence-based assay for the detection of kynurenine in urine using a specific chemosensor, 3-formyl-4-(ethylthio)-7-(diethylamino)-coumarin. The assay produces a linear response at clinically relevant ranges (1-20 μM), with a limit of detection of 0.7 μM. The average standard addition recoveries of kynurenine in synthetic urine samples are near to 100%, and the relative standard deviation values are less than 8%. The established fluorescence assay for quantitative analysis of kynurenine in urine is facile, sensitive and accurate and holds great potential for low-cost and high-throughput analysis of kynurenine in clinical laboratory settings.

SUBMITTER: Patel K

PROVIDER: S-EPMC9063859 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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

Patel Kamlesh K Fernandez-Villamarin Marcos M Ward Craig C Lord Janet M JM Tino Peter P Mendes Paula M PM

The Analyst 20220503 9

The kynurenine metabolite is associated with many diseases and disorders, ranging from diabetes and sepsis to more recently COVID-19. Here we report a fluorescence-based assay for the detection of kynurenine in urine using a specific chemosensor, 3-formyl-4-(ethylthio)-7-(diethylamino)-coumarin. The assay produces a linear response at clinically relevant ranges (1-20 μM), with a limit of detection of 0.7 μM. The average standard addition recoveries of kynurenine in synthetic urine samples are ne ...[more]

PMID: 35388832

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Project description:Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157-167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The highly metastatic rat mammary adenocarcinoma and murine lung carcinoma cell lines had four to ten times the heparanase activity of non-metastatic variants, confirming the correlation of heparanase activity with metastatic potential. Human cancer patients had twice the serum heparanase levels of normal healthy adults. The assay will be valuable for the determination of heparanase activity from a variety of tissue and cell sources, as a diagnostic tool for the determination of heparanase potential, and for the development of specific inhibitors of heparanase activity and metastasis.

Dataset Information

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

Publications

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

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