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M.D. Linetsky, F. Hu; Dehydroalanine Crosslinks in the Human Normal Aged Lenses and in Brunescent Cataract . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2350.
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Purpose: The aim of this work was to elucidate the occurrence and to determine the levels of the dehydroalanine crosslinks (DHA), histidinoalanine (HAL), lanthionine (LAN) and lysinoalanine (LAL) in the aged human (AHL) and brunescent cataractous (BCL) lenses. Methods: Human brunescent cataractous lenses (stage I and stage II) and AHL (all of similar age) were separately homogenized in 1 ml of water. The extracted water-soluble (WS) and sonicated water-insoluble (WI) lens proteins were exhaustively dialyzed against water and hydrolyzed with 6.0 N HCL at t=110oC for 24 h.The crosslinks from the particular lens fractions were obtained by passing lens protein hydrolysates through Dowex 1x2-200 column (10 ml) in acetic acid cycle using pyridine: acetic acid buffers. The crosslinks were further derivatized with dansyl chloride and their concentrations were determined by an HPLC equipped with a Luna C18 4.6x250 mm column and a fluorescence detector set at Eex/Eem= 340/540 nm. Results: HAL was the most abundant crosslink amongst DHA crosslinks and the levels of DHA crosslinks in both WS and WI lens proteins were HAL>LAN>LAL. The levels of HAL in the WI proteins from the stage I BCL were 2.2-fold higher than in the WI proteins from AHL (1.33±2.32 vs 0.59±0.25 nmoles/mg) and 3.5-fold higher in the WI proteins from stage II BCL (2.72±2.36 nmoles/mg; t<0.05) compared to their counterparts from the normal donors. The same trend was observed for the WS proteins from both AHL and BCL. The concentration of HAL in the WS from BCL (stage I) was 3-fold higher compared to the concentration of HAL in the WS proteins from the AHL (1.04±0.61 vs 0.33±0.03 nmoles/mg; t<0.05) and 6-fold higher in the WS proteins from stage II BCL (2.03±0.08 nmoles/mg; t<0.001). In general, the concentrations of the LAL crosslink were in the sub-nanomolar range, approximately three to ten-fold lower than HAL in BC and AHL proteins and were found to be elevated in BCL compared to AHL (e.g. stage II WS 0.67±0.31 nmoles/mg vs normal WS 0.04±0.01nmoles/mg; t<0.02; stage II WI 0.09±0.01 vs normal WS 0.03±0.01 nmoles/mg; t<0.05). The levels of LAL in both WS and WI lens proteins were determined to be either absent or in the picomolar range and in the BCL only. Conclusions: Based on our results we conclude that histidinoalanine, similar to ε-(γ-glutamyl)-lysine should be considered as one of the major crosslink/modifications in brunescent cataractous lenses. Provided that it is the product of the reaction between dehydroalanine (which, in turn, is formed from protein's and/or glutathione's cystines residues) and the imidazole ring of histidine, it should also be viewed as an oxidation marker in cataractous and aged human lenses.
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