A successful methodology for the sectioning and imaging of pre- and postcataractous ICR/f rat lenses has been established. Data collected from these analyses indicate that there are multiple αA-crystallin truncation products present in both pre- and postcataractous rats. Furthermore, these species have defined lenticular localizations and unique solubilities that may be a consequence of lens development and protein function within the lens environment.

Mass spectrometry with or without pre-analysis peptide fractionation can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen and electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighboring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4-hydroxynonenal (4HNE) of human bile acid CoA: amino acid N-acyltransferase (hBAT) and the cytosolic brain isoform of creatine kinase (CKBB) is associated with site-specific modifications. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) using nanoLC-electrospray ionization-mass spectrometry (ESI-MS) or direct infusion-ESI-MS with gas phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB, respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

Cataract-related loss of vision affects large numbers of people in today’s aging populations and presents a healthcare burden to many nations. The role of dietary supplements within the lens is largely unknown, although benefits from dietary antioxidants are expected. In this study, the effects of genistein as its aglycone, a genistein-containing dietary supplement (Novasoy®200), and a genistein-containing food (soy protein isolate, PRO-FAM 932) on the development of lens opacity were examined in the hereditary cataractous ICR/f rat. These studies were carried out in a background diet of semi-purified, isoflavone-free AIN-76A with casein as its protein source. The amount of genistein for the experimental diets was standardized to its concentration (as genistein aglycone as well as simple and complex β-glucoside conjugates) in the soy protein isolate supplement. Also tested was a high-dose genistein diet containing an 11-fold higher amount of genistein aglycone. The composition of each diet was verified by reverse-phase HPLC and blood plasma isoflavone concentrations were determined by LC-tandem mass spectrometry. The development of opacity in each lens was monitored and digitally recorded using slit-lamp examination over the course of the study. Each of the genistein-containing diets caused a significantly more rapid development of fibrous opacification in the anterior cortical region and development of apparent water clefts or vacuoles in the posterior subcapsular region than the AIN-76A control diet; however, the establishment of dense lens opacification was not significantly different between each of the diets. There was also no significant difference observed between the low-dose and high-dose genistein aglycone groups. These data suggest that genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat, with no dose-dependent effects.