Abstract
Abstract: :
Purpose: Tryptophan oxidation is believed to play a key role in the lens matrix damage that ultimately results in the development of cataracts. Previous studies have suggested that oxindolealanine (OIA), an oxidation product of tryptophan, is present in human cataractous lenses. The purpose of this study was to confirm the presence of OIA in cataracts and to examine whether oxindolealanine is elevated in human cataractous lenses using LC/PDA/MS. Methods: Human lenses were obtained from several sources, including surgically removed whole human cataractous lenses and donor globes. Lens protein samples were hydrolysed in 6N HCl and analyzed by HPLC. System I consisted of a HPLC/PDA (Waters) and employed isocratic conditions on a Brownlee Spheri–5 C18 column with 20mM KH2PO4/10% methanol (pH 7) at 0.5 ml/min. Identification of OIA was confirmed by identical retention times and characteristic absorbance spectra from chemically synthesized OIA. A ratio was then expressed with respect to the phenylalanine peak for standardization. System II was used for definitive confirmation of OIA, and consisted of a LC/MS (ThermoElectron) with a Surveyor HPLC/PDA and Synergi Max–RP C12 analytical column. A gradient of 10% – 100% CH3OH (0.1% TFA in water) with a flow rate of 0.2 mL/min was used. Mass spectra were recorded by a quadrupole ion trap mass analyzer (LCQ Advantage) equipped with an electrospray ionization source. Hydrolysates of calf lenses were used as a methods control. Results: OIA was detected by both chromatographic systems with Rt=19min (max=250nm and 280nm shoulder) for system I and Rt=79.5min for system II (m/z =221). The mean content (+/– S.D.) of OIA (expressed as a ratio to phenylalanine) was 1.47 (+/– 0.48) in 10 human cataracts and 0.35 (+/– 0.08) in 4 aged (average 60 years) human controls, corresponding to a 4.2X increase (p<.01). OIA levels were also higher in the cataract nucleus (3.57 +/– 2.31) versus cortex (1.58 +/– 0.86) in five cataractous lenses, corresponding to a 2.26X difference (p<.04). Using the LC/MS system, OIA was confirmed in human cataract samples with an m/z = 221 and max=250nm/280nm shoulder. OIA was not detected in calf lens hydrolysates, which served as a methods control. Conclusions: The results suggest that oxindolealanine is a useful biomarker for the determination of cataract development as well as being additional evidence for the role of tryptophan oxidation in the aging of the human lens.
Keywords: crystallins • oxidation/oxidative or free radical damage • cataract