Tryptophan metabolites fluorescence

Griffiths, H.R, Lunec, J. and Blake, D.R. (1992). Oxygen radical-induced fluorescence in proteins identification of the fluorescent tryptophan metabolite N formyl kynurenine as a biological index of radical damage. Amino Acids 3, 183-194. 

In the same year paper chromatography was first attempted by Benassi (B4) for the simultaneous analysis of 8 tryptophan metabolites (kyn-urenine, 3-hydroxykynurenine, kynurenic acid, xanthurenic acid, anthra-nilic acid, 3-hydroxyanthranilic acid, 2-aminoacetophenone, and 2-amino-3-hydroxyacetophenone), separated by means of a mixtiue of methanol, n-butanol, benzene, and water and revealed through the fluorescence in ultraviolet light of 3655 A. Each compound elicits a different fluorescent color (cf. Table 1). 

Detailed analyses by HPLC revealed that there were more than 60 minor contaminants in the implicated L-tryptophan from Showa Denko.10 Of these contaminants, six were considered to be associated with EMS. The structures of the three contaminants are known and have been described previously, but the full identities of the other three contaminants are unknown. Simat et al.17 used single reversed-phase high-performance liquid chromatography run using UV and fluorescence detection on implicated L-tryptophan and observed 17 contaminants. Most of these contaminants were classified as tryptophan metabolites, nonphysiological oxidation, or carbonyl condensation compounds of tryptophan. 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Jain, S., Kumar, C.V., Kalonia, D.S. (1992). Protein-peptide interactions as probed by tryptophan fluorescence Serum albumins and enkephalin metabolites. Pharm. Res., 9, 990-992. 

Tryptophan and tyrosine are intrinsic fluorophores that are present in many peptides, which then can be identified with fluorescence detection. However, most peptides have no native fluorescence, thus making derivatization a prerequisite for fluorescence detection. Derivatization has been described with naphthalene-2,3-dicarboxaldehyde-(S-mercaptoethanol for determination of substance P and its metabolites, fluorescamine, and 5-carboxyfluorescein succinimidyl ester for luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, and 3-endorphin. Kostel and Lunte compared various postcolumn reactor designs, whereas Advis et al. employed precolumn derivatization, among others. In order to improve sensitivity, laser light is frequently employed for exciting the fluorescent molecules referred to as laserinduced fluorescence (LIE) and provides a 500-1000 times improved sensitivity compared to UV detection. 

Joseph, M. H., Baker, H. F., and Ridley, R. M., 1981, Analysis of CSF amine metabolites and precursors including tryptophan, 5-HIAA and HVA by HPLC using fluorescence and electrochemical detection in primates effect of probenecid, in Central Neurotransmitter Turnover C. Pycockand P. V. Taberner, eds.), pp. 162-167, Groom-Helm, London. 

Fig. 1. A generalized scheme showing the kinds of secondary products that arise from the aromatic amino acids in higher plants. Several similarities are found in fungi and bacteria some fungi produce alkaloids ftom tryptophan and lignin-like materials from phenylalanine. Plant pathogenic fungi produce cinnamate and para and meta hydroxy phenyl-acetate from phenylalanine. Certain bacteria produce antibiotics and fluorescent pigments from metabolites in the shikimate pathway. Microorganisms are not known to produce coumarin, substituted coumarins, flavonoids and isoflavonoids.

Presits, P. Molnfir-Perl, I. HPLC of tryptophan and its metabolites using simultaneously UV, native fluorescence and pre-column fluorescence derivatization, Chromatographia, 2003, 57, S87-S92. [UV detection fluorescence detection niacin niacinamide melatonin]... 

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