Tryptophan and the kynurenine pathway in chronic renal failure patients on dialysis

Abstract

Tryptophan is metabolised along the kynurenine pathway under the influence of tryptophan 2,3 dioxygenase and indoleamine 2,3 dioxygenase. Quinolinic acid and kynurenine, two neuroactive metabolites of the kynurenine pathway are, in chronic renal failure patients, considered as uraemic toxins. Related research is generally hampered by the non-availability of relevant analytical techniques. The primary aim of this study was, therefore, to develop and validate suitable methods for the determination of tryptophan, kynurenine and quinolinic acid. The second aim was to quantify the levels of these substances in the blood of chronic renal failure patients on renal replacement therapies and to compare the levels of haemodialysis patients to those on peritoneal dialysis. Patients’ quality of life was investigated relative to disturbances in tryptophan metabolism. Gas chromatography coupled to mass spectrometry (GC-MS) gave the best results for the analysis of tryptophan, kynurenine and quinolinic acid. A Hewlett Packard HP GC 6890 series gas chromatographer was coupled to a MS 5973 series mass spectrometer. Analytes were separated on a DB-5MS column with a nominal length of 30 metres, a diameter of 250.0 µm and film thickness of 0.10 µm. Helium was used as carrier gas, and the chromatographic analysis run time 12.5 minutes. The validation results were within the acceptance criteria for newly developed methods. The linear calibration curves constructed for all of the analytes gave r2 correlation coefficients >0.99. Other validation data such as precision, bias, accuracy and stability all fell within acceptable validation limits. In the study on chronic renal failure patients significant differences were seen between patients and controls. Tryptophan levels were 5.34 SD 5.04 µM for the haemodialysis group, 6.73 SD 3.18 µM for the peritoneal dialysis group and 28.4 SD 4.31 µM for the control group. Kynurenine levels were 4.7 SD 1.9 µM for the haemodialysis group, 2.9 SD 2.0 µM for the peritoneal dialysis group and 2.1 SD 0.6 µM for the control group. Quinolinic acid levels were 4.9 SD 2.0 µM for the haemodialysis group, 2.8 SD 2.0 µM for the peritoneal dialysis group and 0.3 SD 0.1 µM for the control group. Tryptophan was lower for the total patient group than for controls with significantly lower levels for haemodialysis versus control (p<0.05) and peritoneal dialysis versus control (p<0.05). Kynurenine levels were higher in the total patient group with significantly higher levels for the haemodialysis versus control group (p=0.0001). The patient groups had higher quinolinic acid levels with significantly higher levels for the haemodialysis versus control (p<0.05) and peritoneal dialysis versus control (p<0.05) groups. This study was the first to determine the three substances simultaneously in both haemodialysis and peritoneal dialysis patients. The study showed significant tryptophan depletion, as well as kynurenine and quinolinic acid accumulation for both groups. No significant differences were found between the patient groups other than higher kynurenine levels in the haemodialysis group. The quality of life (SF-36) was largely similar in the two patient groups. This decrease in the quality of life strongly correlated with the degree of tryptophan depletion.