Hydroxykynurenine isolation

Kynurenine and 3-hydroxykynurenine are the precursors or chromogens of the pigments. One of them, xanthommatin, was obtained from the eyes of insects 7800 heads of Calliphora erythrocephala gave 19 mg of the pigment (B32). Xanthommatin was also isolated from the molting secretion of Vanessa urticae 10,000 butterflies yielded 100 mg of the substance (B30). 

All 90 hemoblastotic patients, including 30 cases of myeloid leukemia, 30 of lymphoid leukemia, 2 of hemocytoblastotic and 1 of histiocytic leukemia, 21 of Hodgkin s disease, 3 of multiple myeloma, and 1 of lympho-sarcomatosis, were followed chromatographically for several days and the increased excretion of the two kynurenines was confirmed. Fever is not a determining factor since the remission of fever did not have a normalizing effect on the metabolite excretion. The investigation was completed with the isolation of kynurenine and 3-hydroxykynurenine in pure form. 

From 9 liters of urine from a subject with chronic myeloid leukemia 16.4 mg 3-hydroxykynurenine and 40 mg kynurenine, as the sulfate, were isolated through the mercuric salts, resolution with hydrogen sulfide, preparative paper chromatography of the concentrate, elution of the fluorescent bands, and repeated concentrations and crystallizations. From 5.5 liters of urine from a patient with Hodgkin s disease 34 mg kynurenine and 21.1 mg 3-hydroxykynurenine were obtained. A third patient also with Hodgkin s disease gave 23.8 mg kynurenine and 9 mg 3-hydroxykynurenine... 

The amounts of the two substances originally present in the urine were higher than those actually isolated. It must be emphasized, however, that tryptophan metabolites have rarely been extracted from urine when neither special diets nor supplementary tryptophan are used. Furthermore, urine of hemoblastotic patients can be considered, together with Calliphora erythrocephala pupae, as one of the rare natural sources of 3-hydroxykynurenine. 

M19. Musajo, L., Benassi, C. A., and Parpajola, A., Isolation of kynurenine and 3-hydroxykynurenine from human pathological urine. Nature 175, 855-856 (1955). 

Hydroxykynurenine was isolated from the pupae of Calliphora erythro-cephala (115, 623) and was identified as the cn-f—substance referred to above, and its constitution was proved by s5mthesis (128, 500, 623). It was also obtained from the larvae of the silkworm Bonibyx mori (392) and was shown to give nicotinic acid in a Neurospora mutant blocked after the kynurenine stage (970), which accumulated iV -acetylkynurenine (diagram 20) in the medium. 

No enzyme system which will oxidize kynurenine to hydroxykyiiurenitie has yet been isolated in a cell-free state from any species, and there is some evidence that direct conversion may not normally occur, at least in mammals. Riboflavin was suggested to be concerned in hydroxykynurenine formation at a comparatively early stage (387), and this has been supported by nutritional experiments. 

W -Acetylkynurenine (i.e., acetylated on the aliphatic amino group) was isolated from a Neurospora mutant culture (970) and was also found in the urine of pyridoxine-deficient rats together with the analogous iV -acetylhy-droxykynurenine (171). It is possible, though there is no direct evidence, that these Af -acetyl derivatives play some part in normal tryptophan metabolism in the rat (c/. 170, 173), or they may, as is probably the case in Neurospora, merely be products of side reactions occurring in the presence of abnormal amounts of kynurenine and hydroxykynurenine. Hydroxykynurenine is also excreted by the pyridoxine-deficient rat as its 0-sulfate... 

I) Kynurenine and Hydroxykynurenine. Initially kynurenine was suggested as a possible precursor of nicotinic acid because it was active in supporting the growth of a particular niacinless strain of Neurospora. a-A -Acetylkynurenine, isolated from the culture filtrate of another niacinless strain,was found to be Moo as active as niacin. When kynurenine is supplied in excess, nicotinic acid accumulates in the culture medium, just as it does when an excess of tryptophan is introduced. 

Interest in hydroxykynuienine in relation to niacin formation was aroused when it was observed that it yielded nicotinic acid in a Neurospora mutant with a genetic block after kynurenine (298). The compound was first isolated from the larvae of several insects (299, 300). Its importance in the kyniuenine pathway in the mammal was established when it was shown that it was converted to nicotinic acid and could replace the latter as a growth factor (301, 302). No similar conversion of anthranilic acid took place (503). Subsequently it was found that hydroxykynurenine was a constituent of the urine and that its excretion was increased in pyridoxine-deficient animals (304). 

This conversion has been found to be catalyzed by an enzyme isolated from the liver and kidney of several mammalian species (6Jir67), from tryptophan-adapted Pseudomonas (68, 69), and from Neurospora (60). The enzyme appears to be identical with the system which causes the hydrolysis of 3-kynurenine to anthranilic acid. The products of the hydrolysis of hydroxykynurenine are hydroxyanthranilic acid and alanine. Pyri-doxal phosphate is required for the reaction. 

Apart from the results reported by Jori et al. 211-214) who claimed quantitative conversion of tryptophan to formylkynurenine residues when proteins are photooxidized in aqueous buffered solution with proflavine as sensitizer, no other author has reported similar quantitative conversion. On the other hand it has been found that loss of tryptophan clearly does not match the formation of formylkynurenine 63, 304). Gomyo and Fujimaki 148) detected kynurenine and 3-hydroxykynurenine as products of the lumiflavine sensitized photooxidation of lysozyme in buffered aqueous solution. Kravchenko and Lapuk 229, 232), careful purified a photooxidized sample of lysozyme and isolated a group of monooxidized enzymes in which a single tryptophan entity was oxidized. Even this relatively homogeneous product could be separated into different species of enzyme with different degrees of residual activity and different ultraviolet spectra (229). 

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