Simultaneous Measurement of Serum Tryptophan and Kynurenine by HPLC,

Abstract

l-Tryptophan is an essential amino acid that is required for the biosynthesis of proteins and is the precursor for several important biological compounds, e.g., 5-hydroxytryptamine (serotonin). In an alternative reaction series, the so-called kynurenine pathway, tryptophan is catabolized to form nicotine amides and the vitamin niacin (1) as end products. A cytokine-inducible indoleamine-(2,3)-dioxygenase (IDO) catalyzes the first step of tryptophan degradation (2)(3)(4)(5), forming the intermediate kynurenine. Interferon-γ has been shown to be a potent stimulating cytokine for IDO in vitro (6)(7). Similarly in vivo, a decrease of serum tryptophan and an increase of kynurenine in parallel was described (8)(9)(10)(11)(12), indicating an enhanced cytokine-induced degradation of tryptophan, when the cellular immune system is activated. In particular, the ratio of kynurenine to tryptophan seems to be a sensitive indicator for interferon-γ-induced tryptophan degradation and therefore for an activated immune system (10)(13). Thus, simultaneous measurements of kynurenine and tryptophan, allowing calculation of the ratio, enables indirect examination of endogenous interferon-γ formation. We describe a new HPLC method to determine the amounts of kynurenine and tryptophan in serum simultaneously with use of an external albumin-based calibrator and an internal calibrator. The human subjects involved within the scope of this method development correspond to the ethical standards of our institutions’ responsible committee. l-Tryptophan was obtained from Serva, l-kynurenine and 3-nitro-l-tyrosine were from Sigma, and potassium phosphates and acetonitrile for the HPLC elution buffer were from Merck. All chemicals used were of analytical grade. The HPLC pump was a Model 9010 (Varian) controlled by a DS …