4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic-acid-o-glucoside and 3-hydroxykynurenine-glucoside

To determine whether there are UVA light-responsive sensitizers in young human lenses capable of initiating the oxidation of ascorbic acid in the absence of oxygen.. Lens homogenates were fractionated, and low-molecular-weight (LMW) components were separated from the proteins by filtration through a 3000-MWt cutoff filter. Aliquots of each fraction were assayed for sensitizer activity by UVA irradiation (337-nm cutoff filter) with 0.1 mM ascorbic acid, measuring ascorbate oxidation by loss of absorbance at 265 nm. Two major peaks were isolated from a human lens water-soluble (WS)-LMW fraction on a reversed-phase column and were identified by mass spectrometry.. All human lens fractions oxidized ascorbate when irradiated by UVA light. Most of the sensitizer activity in young human lenses was in the LMW fractions. An action spectrum for the WS-LMW fraction from human lens showed activity throughout the UVA region. Assays with and without oxygen showed little or no difference in ascorbate oxidized, arguing for a direct transfer of an electron in a so-called type 1 reaction. A human lens WS-LMW fraction contained two major peaks of activity. The greater peak was identified as 3-hydroxykynurenine glucoside (3OHKG) by mass spectrometry and its absorption spectrum, whereas the lesser peak was identified as 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG). The activities were 1.1 and 2.8 nmol of ascorbate oxidized in 30 minutes/nmol 3OHKG and AHBG, respectively.. The filter compounds present in human lenses can absorb UVA light and cause the oxidation of ascorbic acid in the presence and absence of oxygen, possibly initiating the glycation of lens proteins.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Ascorbic Acid; Chromatography, High Pressure Liquid; Crystallins; Glucosides; Glutathione; Humans; Lens, Crystalline; Mass Spectrometry; Middle Aged; Oxidation-Reduction; Phenylbutyrates; Radiation-Sensitizing Agents; Subcellular Fractions; Tryptophan; Ultraviolet Rays

Age-dependent human lens coloration may be explained by the binding of UV filters to crystallins. It has been proposed that glutathione may compete for reaction with UV filter degradation products and therefore protect crystallins from modification. To understand this process, UV filters were quantified together with oxidized and reduced glutathione in human lenses of varying age.. Lens tissues were homogenized in ethanol to extract the UV filters. Metabolites were quantified by HPLC and correlations between them in the nuclear and cortical regions of the lens were examined.. The concentrations of the UV filters 3-hydroxykynurenine, kynurenine, and 3-hydroxykynurenine glucoside decreased linearly with age, with slightly lower levels in the nucleus than the cortex. 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside was found in higher levels in the nucleus than the cortex and decreased slowly in both regions with age. Glutathionyl-3-hydroxykynurenine glucoside was present in higher concentrations in the nucleus, barely detectable in young lenses, but increased significantly after age 50. Reduced glutathione levels were lower in the nucleus and decreased in both regions with age, yet oxidized glutathione increased in the nucleus but remained constant in the cortex.. Results are consistent with a predominantly nuclear origin for both 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside and glutathionyl-3-hydroxykynurenine glucoside. This is in accord with their proposed mechanism of formation, which involves an initial deamination of 3-hydroxykynurenine glucoside. This process is more pronounced in older lenses, possibly because of the barrier to diffusion. The barrier may also explain the increase in nuclear oxidized glutathione that is observed with age.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Chromatography, High Pressure Liquid; Glucosides; Glutathione; Humans; Kynurenine; Lens, Crystalline; Middle Aged; Phenylbutyrates; Radiation Injuries; Ultraviolet Rays