Challenges in Developing Analytical Methods to Measure Tryptophan and Kynurenine in Human Blood Serum
Challenge: Diteba was contracted to develop and validate an appropriate analytical method capable of quantifying the levels of L-tryptophan (0.06 μmol/L to 222 μmol/L) and kynurenine (0.09 μmol/L to 9.84 μmol/L) in human serum. Several HPLC methods have been developed over time to determine serum tryptophan and kynurenine. However, none of this research has met desirable levels of sensitivity or reproducibility. The methods described in the literature were complicated by the interference of other amino acids with the kynurenine UV absorption in HPLC chromatography. In addition to the analytical method development and validation exercise, it was necessary to develop a simple and accurate method of extraction for tryptophan and kynurenine from patients' blood serum. The extraction procedures were complicated because of the nature of the samples. The patients' samples were infected with a chronic hepatitis C which required extreme precaution in drawing and handling these samples.
Degradation of the essential amino acid tryptophan leads to reduced availability and is considered as an immune defense mechanism, which suppresses the growth of intracellular bacteria, viruses and parasites like toxoplasma, malignant tumour, or chronic hepatitis C. In an alternative reaction series, the so-called kynurenine pathway, tryptophan is catabolized forming the intermediate kynurenine with nicotine amides and the vitamin niacin as end products. Decrease of serum tryptophan and an increase of kynurenine indicate an enhanced cytokine-induced degradation of tryptophan, when the cellular immune system is activated.
Solution: Degradation of the essential amino acid tryptophan leads to reduced availability and is considered as an immune defense mechanism, which suppresses the growth of intracellular bacteria, viruses and parasites like toxoplasma, malignant tumour, or chronic hepatitis C. In an alternative reaction series, the so-called kynurenine pathway, tryptophan is catabolized forming the intermediate kynurenine with nicotine amides and the vitamin niacin as end products. Decrease of serum tryptophan and an increase of kynurenine indicate an enhanced cytokine-induced degradation of tryptophan, when the cellular immune system is activated.
Diteba developed and validated a new specific HPLC-UV-FL method for the simultaneous quantification of tryptophan and its metabolite kynurenine in human blood serum. This method allows the sponsor to calculate the ratio of tryptophan/kynurenine, enabling indirect examination of patients with chronic hepatitis C and monitoring interferon-based immunotherapy.
Result: Not only was Diteba able to efficiently develop and validate an analytical method capable of quantifying the levels of L-tryptophan and kynurenine in human serum, Diteba's scientists also were able to provide a simple extraction method that was safe and easy to administer.