Fluorometric Determination of L-Kynurenine with Glycolaldehyde by High Performance Liquid Chromatography
L-Kynurenine (KYN) is one of the metabolites of L-tryptophan(Trp) on the kynurenine pathway. The first step of the kynurenine pathway is an oxidative cleavage of the indole ring of Trp, which is catalyzed by tryptophan 2, 3-dioxygenase (EC 188.8.131.52, TDO) to form N-formyl-L-kynurenine. In mammalian system, TDO is found only in liver and N-formyl-L-kynurenine is hydrolyzed instantly to KYN by an excess of formamidase present in liver. Therefore, in many cases assay of the TDO activity is based on the determination of KYN formed by TDO. We have developed a new fluorometric method for the determination of KYN by a single-pump high performance liquid chromatography (HPLC) using glycolaldehyde(GLA) as fluorogenic reagent. The method was successfully applied to the assay of the TDO activity.
KeywordsHigh Performance Liquid Chromatography Anthranilic Acid Kynurenic Acid Kynurenine Pathway Ammonium Sulfate Fraction
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- Cho-Chung Y.S. and Pitot H.C., 1967, Feedback control of rat liver tryptophan pyrrolase. I. End products inhibition of tryptophan pyrrolase activity, J. Biol Chem., 242: 192.Google Scholar
- Feigelson P. and Brady F.O., 1974, “Molecular Mechanisms of Oxygen Activation,” ed. by O.Hayaishi, Academic Press, New YorkGoogle Scholar
- Frieden E., Westmark G.W. and Schor J.M., 1961, Inhibition of tryptophan pyrrolase by serotonin, epinephrine, and tryptophan analogs, Arch. Biochem. Biophys., 92: 176. 2Google Scholar
- Knox W.E., Yip A. and Reshef L., 1970, L-Tryptophan 2,3-dioxygenase (tryptophan pyrrolase) (rat liver), in: “ Methods in Enzymology,” Vol. 17A, ed. by H. Tabor, C.W. Tabor, Academic Press, New York, pp. 415–428.Google Scholar
- Schimke R.T., Sweeney E.W,. and Berlin C.M., 1965, The roles of synthesis and degradation in the control of rat liver tryptophan pyrrolase, J. Biol. Chem., 240: 322.Google Scholar
- Seglen P.O. and Jervell K.F., 1969, Tryptophan oxygenase activation and ascorbate oxidation in whole homogenates from rat liver, Biochim. Biophys. Acta., 171: 47.Google Scholar