PURPOSE: Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over a lifetime and may be precursors of mature cataracts. The authors analyzed changes in proteins, phospholipids, and cholesterol in these opacities using in situ techniques: Raman microspectroscopy, filipin cytochemistry for cholesterol, and transmission electron microscopy (TEM). METHODS: Human lenses with verified focal opacities were fixed in 1% paraformaldehyde. Slabs with opacities were analyzed using confocal Raman spectroscopy, then filipin Raman analysis of cholesterol, and finally TEM. RESULTS: Compared with normal fibers, opacities consistently showed elevated levels of cholesterol and aliphatic chains, increased phospholipid acyl chain disorder, and changes in phospholipid lateral packing. Disulfide bridges of specific geometry (trans-gauche-trans) were found. Although protein content was unchanged, compared with normal fibers, aromatic amino acid content was significantly lower. The hydrophobicity of tyrosine residues showed a significant decrease, and a change in the tryptophan indole ring angle was found. The changes were abrupt and sharply delineated focal opacities. TEM confirmed this sharp boundary and showed that the opacities were densely packed with vesicles of varying size and electron density embedded in a homogenous matrix. CONCLUSIONS: The Raman and TEM analyses of opacities showed that early cataractogenic events led to disruption of fiber membranes, formation of vesicles from the membrane constituents, and protein changes. The aberrant morphology of the membranes enveloping the focal opacities may have segregated the affected fibers from the surrounding normal tissue, thus explaining the stationary or slowly progressing character of these opacities.
PURPOSE: Focal opacities are signs of early cataractogenesis in the human lens. They progress slowly over a lifetime and may be precursors of mature cataracts. The authors analyzed changes in proteins, phospholipids, and cholesterol in these opacities using in situ techniques: Raman microspectroscopy, filipin cytochemistry for cholesterol, and transmission electron microscopy (TEM). METHODS:Human lenses with verified focal opacities were fixed in 1% paraformaldehyde. Slabs with opacities were analyzed using confocal Raman spectroscopy, then filipin Raman analysis of cholesterol, and finally TEM. RESULTS: Compared with normal fibers, opacities consistently showed elevated levels of cholesterol and aliphatic chains, increased phospholipid acyl chain disorder, and changes in phospholipid lateral packing. Disulfide bridges of specific geometry (trans-gauche-trans) were found. Although protein content was unchanged, compared with normal fibers, aromatic amino acid content was significantly lower. The hydrophobicity of tyrosine residues showed a significant decrease, and a change in the tryptophan indole ring angle was found. The changes were abrupt and sharply delineated focal opacities. TEM confirmed this sharp boundary and showed that the opacities were densely packed with vesicles of varying size and electron density embedded in a homogenous matrix. CONCLUSIONS: The Raman and TEM analyses of opacities showed that early cataractogenic events led to disruption of fiber membranes, formation of vesicles from the membrane constituents, and protein changes. The aberrant morphology of the membranes enveloping the focal opacities may have segregated the affected fibers from the surrounding normal tissue, thus explaining the stationary or slowly progressing character of these opacities.
Authors: Shuhua Yue; Juan Manuel Cárdenas-Mora; Lesley S Chaboub; Sophie A Lelièvre; Ji-Xin Cheng Journal: Biophys J Date: 2012-03-06 Impact factor: 4.033