Doctor of Philosophy
Department of Chemistry
Wood, Andrew Martin, Tryptophan metabolism in the human lens, Doctor of Philosophy thesis, Department of Chemistry, University of Wollongong, 1993. http://ro.uow.edu.au/theses/1213
The aim of this thesis was to discover more about the pathway in the human lens which leads to the formation of 3-hydroxykynurenine glucoside, the major UV-filter substance. This biosynthetic pathway has received scant attention since the discovery of the glucoside by van Heyningen in 1971.
In order to further examine the unique metabolism of tryptophan in human lenses, an intact lens organ culture system was employed. Radiolabelled tryptophan was added to the artificial aqueous humor of organ cultured-lenses and its incorporation was followed by analysis of the low molecular weight lens fraction by high pressure liquid chromatography and liquid scintillation counting.
Significant conversion of tryptophan into metabolites was observed in 24 hours. The major proportion of tryptophan radiolabel was found in 3-hydroxykynurenine glucoside, with smaller quantities detected in both kynurenine and 3-hydroxykynurenine. It was found, using tritiated 3-hydroxykynurenine, that synthesis of the 3-hydroxykynurenine glucoside is a diversion from normal tryptophan oxidative metabolism, in which 3-hydroxykynurenine is glucosylated by an enzyme system(s) localised primarily in the equatorial region of the lens.
The concentration of 3-hydroxykynurenine glucoside in the lens was, on average, approximately six times as great as the concentration of free tryptophan. Kynurenine and 3-hydroxykynurenine were present at much lower levels. No Nx-formylkynurenine, the immediate product of tryptophan oxidation, was detected in lens extracts.
The concentration of 3-hydroxykynurenine glucoside was found to decline from the levels in very young lenses to those aged about 40 years, after which the level was relatively constant. A similar trend was observed for the much lower concentrations of kynurenine and 3-hydroxykynurenine. No satisfactory explanation for this age-related change in lenticular tryptophan metabolites could be found.
Tryptophan, kynurenine, 3-hydroxykynurenine and 3-hydroxykynurenine glucoside were detected in the cortex and nucleus of the lens. The concentration of these compounds along the visual axis was found to be approximately twice as high as in the equatorial region. Conversion of tryptophan to 3-hydroxykynurenine glucoside, however, appeared to take place only in the organehecontaining cells of the lens. It was shown that epithelial explants from human lenses could also convert tryptophan to 3-hydroxykynurenine glucoside.
3-Hydroxykynurenine glucoside was found to efflux from cultured lenses at a linear rate. No further metabolism of 3-hydroxykynurenine glucoside was, however, detected, and it was therefore proposed that efflux represents the major, if not the only, pathway for removal of 3-hydroxykynurenine glucoside from lenses. 3-Hydroxykynurenine glucoside was also detected in human vitreous humor.
No evidence was found in the lens for the further metabolism of 3-hydroxykynurenine via the normal tryptophan oxidative pathway found in other mammalian tissues.