Newly developed method for quantification of tryptophan and its metabolites : clinical application

Tryptophan, an essential amino acid, follows important metabolic pathways for protein, niacin and serotonin synthesis. The kynurenine pathway is a major pathway for tryptophan degradation. Indications are that tryptophan metabolism is disturbed in chronic renal failure patients (CRF), with tryptophan depletion and accumulation of kynurenine metabolites. Assessment of tryptophan metabolism is generally hampered by the non availability of analytical techniques for these substances. Aim: to develop and validate a suitable method for the quantification of tryptophan, kynurenine and quinolinic acid in the blood. The second aim was to demonstrate the clinical application of this method by the quantification of the said metabolites in the blood of chronic renal failure patients. The levels of the substances in the blood of peritoneal dialysis patients, haemodialysis patients and controls were compared. Method: A sensitive, selective and repeatable method was developed that employed gas chromatography coupled to mass spectrometry (GC-MS). The gas chromatographer was a Hewlett Packard HP GC 6890 series instrument coupled to a MS 5973 series mass spectrometer. Separation of the analytes was achieved on a DB-5MS GC column with a nominal length of 30 metres, a diameter of 250.0 ìm and film thickness of 0.10 ìm. Results: Method validation results fell within the international acceptance criteria for a newly developed method. Tryptophan levels were 5.34 SD 5.04 ìM for the haemodialysis group, 6.73 SD 3.18 ìM for the peritoneal dialysis group and 28.4 SD 4.31 ìM for the control group. Kynurenine levels were 4.7 SD 1.9 ìM for the haemodialysis group, 2.9 SD 2.0 ìM for the peritoneal dialysis group and 2.1 SD 0.6 ìM for the control group. Quinolinic acid levels were 4.9 SD 2.0 ìM for the haemodialysis, 2.8 SD 2.0 ìM for the peritoneal dialysis group and 0.3 SD 0.1 ìM for the control group. Tryptophan was significantly lower (p<0.05) and kynurenine and quinolinic acid levels were higher (p<0.05) in the patient groups. Conclusions: GC-MS is suitable for simultaneous assessment of tryptophan, kynurenine and quinolinic acid. Significant tryptophan depletion and accumulation of the metabolites kynurenine and quinolinic acid occurs in CRF patients on both haemodialysis and peritoneal dialysis treatment.