Development of a Fluorophore-Bound l-Tryptophan Derivative for Evaluating Indoleamine 2,3-Dioxygenase Activity by HPLC with Fluorescence Detection: An In Vivo Microdialysis Study Using Rat Kidney

TL;DR

A new fluorescent tryptophan derivative was developed to measure IDO enzyme activity in rat kidneys using microdialysis and HPLC with fluorescence detection.

Contribution

A novel fluorophore-bound l-tryptophan derivative was created for in vivo IDO activity evaluation.

Findings

5-DBD-l-Trp was metabolized by renal IDO, producing detectable fluorescence peaks.

The main metabolite was identified as 5-DBD-kynurenine using mass spectrometry.

IDO activity was inhibited by 1-methyl-d-Trp, reducing fluorescence peak intensity.

Abstract

Evaluating the activity of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan (Trp) metabolism, is important because IDO is involved in immune tolerance and drives the production of Trp metabolites implicated in psychiatric disorders and cancer. This study aimed to design and develop a novel fluorescent l-Trp derivative to fluorometrically monitor Trp-catabolizing enzyme activity via IDO. To evaluate IDO activity in vivo, 7-N,N-dimethylamino-2,1,3-benzoxadiazole (DBD), a fluorophore, was covalently bound at the 5-position of the indole ring in Trp to produce 5-DBD-l-Trp. An in vivo microdialysis (MD) study was conducted using the kidneys of Sprague–Dawley rats. Specifically, 5.0 μM 5-DBD-l-Trp in phosphate-buffered Ringer’s solution was infused into the rats, and the MD sample was analyzed via high-performance liquid chromatography with fluorescence detection. In the MD sample, two fluorescence peaks other than 5-DBD-l-Trp were observed during the 5-DBD-l-Trp infusion, and the main metabolite peak was proposed to be 5-DBD-kynurenine, verified by liquid chromatography-tandem mass spectrometry. The intensity of the fluorescent peak was significantly attenuated by co-infusion with an IDO inhibitor, 1-methyl-d-Trp. These results suggest that 5-DBD-l-Trp may be metabolized by renal IDO and can be used to evaluate IDO activity in vivo.

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