Determination of serum kynurenine and hepatic tryptophan dioxygenase activity by high-performance liquid chromatography.
The status of the oxidative metabolism of L-tryptophan is usually evaluated by the determination of tryptophan metabolites in serum or urine and/or the activities of various oxidative enzymes in tissues. I have developed assays for serum kynurenine and hepatic tryptophan dioxygenase (TDO) activity based on the determination of kynurenine (KYN) by isocratic, reverse phase HPLC with spectrophotometric detection at 365 nm. Sample pretreatment prior to HPLC requires little more than perchloric acid precipitation of serum or a TDO incubation mixture. The analytical recovery for the serum assay was 101 +/- 2%, while the run-to-run coefficient of variation at normal KYN levels was approximately 8%. Serum KYN levels in 40 apparently healthy fasting humans were normally distributed and ranged from 0.27 to 0.69 microgram/ml (mean +/- SD: 0.47 +/- 0.1). Serum KYN in predialysis specimens from a group of 20 patients with chronic renal failure demonstrated a highly significant increase (mean +/- SD: 0.83 +/- 0.35 microgram/ml; P less than 0.001) as compared to the reference population. It is possible that such an increase might contribute to the pathophysiology of the uremic state. The analytical recovery of KYN from TDO incubation mixtures was approximately 90%. There was no evidence for the onward metabolism of KYN during the assay of whole liver homogenates. The mean (+/- SD) TDO activity of rat liver homogenates preincubated with ascorbate and hematin was 2.3 +/- 0.8 mumol/h/g wet wt (30 degrees C). The sensitivity, specificity, and convenience of these two methods suggest that they are suitable for routine use in the investigation of the biology and pathology of oxidative tryptophan metabolism.
Holmes
E W
EW
Department of Pathology, Stritch School of Medicine, Loyola University Medical Center, Maywood, Illinois 60153.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007676:Kidney Failure, Chronic / Q000201:enzymology / Q000378:metabolism; D007737:Kynurenine / Q000097:blood*; D008099:Liver / Q000032:analysis / Q000201:enzymology*; D008297:Male; D051381:Rats; D014366:Tryptophan Oxygenase / Q000378:metabolism*
D007737:Kynurenine; D014366:Tryptophan Oxygenase