To protect against oxidative-stress induced protein damage, the lens contains high levels of antioxidants, such as glutathione and ascorbic acid. The development of age-related cataract is thought to be related to changes in lens antioxidants and other metabolites in specific lens regions, and leads to the protein damage, insolubilisation and opacification that characterises lens cataract. In this study, lens small molecules and metabolites have been mapped in the aging human lens to define lens metabolome changes associated with normal lens aging, and a method to quantitatively map the distribution of predominant lens antioxidants developed using glutathione as a model.
Cryosections from human lenses ranging in age from 29 to 82, and lens homogenates spiked with different concentrations of heavy GSH, were coated with NEDC matrix and analysed simultaneously by a MALDI FTICR mass spectrometer (Bruker Solarix-XR 7T), in negative ion mode with MALDI IMS spatial resolution of 100μm. Data were imported into SCiLS Lab 2015b for analysis, while standard curves were plotted using Microsoft Excel.
Signal for glutathione was detected in lenses of all ages, in addition to human lens-specific UV filters. For example, 3-hydroxykynurenine glucoside (3-OHKG) was more abundant in the lens nucleus than the cortex, and signal intensity decreased with lens age. In contrast, a signal for putative lens fluorophore 3-OHKG-GSH was detected only in the old human lens nucleus. With age, glutathione signal declined, first in the nucleus, then in the cortex. For glutathione quantitation, a standard curve using heavy GSH was used to quantitate GSH signal levels in the human lens sections, which ranged from 5μmol/g in the lens cortex to 0.7μmol/g in the nucleus. This study highlights the metabolic changes that take place in the aging human lens, providing a baseline measure for age-related changes to be compared with metabolic changes associated with cataract formation.