More Rapid Method for Simultaneous Measurement of Tryptophan and Kynurenine by HPLC

Abstract

The essential amino acid l-tryptophan is important in nitrogen balance and the maintenance of muscle mass and body weight in humans (1). Moreover, tryptophan is the precursor for the biosynthesis of the neurotransmitter serotonin (5-hydroxytryptamine). Insufficient availability of tryptophan may increase susceptibility for mental depression (2). On activation of cellular immunity, the T-cell-derived cytokine interferon-γ stimulates the enzyme indoleamine-(2,3)-dioxygenase (IDO) in various cells (3)(4). IDO catalyzes the initial step of tryptophan catabolism within the biosynthetic pathway of nicotinamide dinucleotides, and N -formyl-kynurenine is formed as a first intermediate. The kynurenine-to-tryptophan ratio has been a sensitive estimate to monitor the activation status of IDO and of cellular immunity both in vivo and in vitro (3)(5). In patients, a decrease in serum tryptophan and a parallel increase of kynurenine attributable to IDO activation is found in various diseases associated with T-cell activation, such as viral infections, autoimmune disorders, and malignant diseases (3)(5)(6)(7)(8). More recently, activation of IDO in monocytes/macrophages was found to interfere with the proliferative capacity of T cells in response to antigenic stimulation by the withdrawal of tryptophan (9). This finding has attracted immunologic researchers especially to explore the possible involvement of IDO in tolerance induction and in diseases that are associated with acquired immunodeficiency. We have described a reverse-phase HPLC method to quantify serum concentrations of kynurenine and tryptophan in parallel with use of 3-nitro-l-tyrosine as an internal standard (10). Here we report an optimized protocol that uses a shorter HPLC column and a different elution buffer, allowing faster throughput of samples …