An HPLC method to determine tryptophan and kynurenine in serum simultaneously

A method was established to measure tryptophan and kynurenine in serum simultaneously. Tryptophan is converted to kynurenine by the action of the enzyme indoleamine 2,3-dioxygenase induced by interferon-gamma (IFN-gamma). Since IFN-gamma is a Th1-cell derived cytokine, an increased tryptophan degradation rate via the kynurenine pathway can be found when the cellular immune system is activated as it is, e.g., in viral infections or in autoimmune diseases. Thus, the ratio kynurenine per tryptophan provides a possibility to estimate IFN-gamma activity in vivo and furthermore reflects the degree of immune activation. The HPLC method requires 100 microL serum. Protein is removed by trichloroacetic acid. An external albumin-based calibrator is applied, and analysis is referred to an internal standard, 3-nitro-L-tyrosine. Kynurenine and nitrotyrosine are detected via UV absorbance at 360 nm wavelength, and tryptophan is detected via its natural fluorescence at 285 nm extinction and 365 nm emission. Representative normal values of kynurenine and tryptophan were measured in the sera of 49 healthy blood donors.