Background

Research

Cataract occurs in the aging population due to a variety of factors, leading to len protein (crystallin) damage. These changes include browning, the formation of fluorophores and protein aggregation and crosslinking. All of these effects can be mimicked by the incubation of lens proteins with sugars. Dr. Ortwerth's lab is pursuing the hypothesis that oxidative stress is normally prevented by ascorbic acid, but in the process ascorbic acid degradation products accumulate which can modify lens proteins. These compounds cause cataractous-like changes in vitro, and react 100-fold more rapidly than glucose in this process. Dr. Ortwerth and his associates are currently involved in the isolation of the damaged amino acids, and have recently shown the protein adducts can absorb UVA light and generate oxygen free radicals. Therefore, two mechanisms may be involved in protein damage.

Quantitation of the Singlet Oxygen Produced by the UVA Irradiation of Human Lens Proteins:

Ultraviolet light has long been implicated in cataract formation. The effect of UV light on lens proteins has been investigated in several model systems and in rats. Dr. Ortwerths'slaboratory proposed the major damage to lens proteins comes from the absorption of UVA, rather than UVB light. Further, this light is absorbed by the nuclear sclerotic compounds that accumulate with age and generate oxygen free radicals. This has been demonstrated by irradiating human lens proteins and measuring the amount of reactive oxygen species produced. With a UV dose 200 times sunlight, the major species is singlet oxygen, being produced at levels in excess of 2.0 mM. This largely occurs within the protein aggregates, thereby preventing the access of antioxidants (ascorbic acid and glutathione) and protective enzymes (superoxide dismutase and catalase) from reaching the site of active oxygen formation. This has recently been demonstrated in vitro with human lens proteins, by showing increased effectiveness of antioxidants when the aggregates are either proteolyzed enzymatically, or the protein is denatured with urea or guanidine hydrochoride.

Ascorbic Acid Glycation of Lens Proteins Produces UVA Sensitizers Similar to those Present in Human Lens:

The previous two projects have merged recently with the demonstrated that bovine lens proteins, after incubation with ascorbic acid under oxidative conditions, produces protein-bound compounds which generate reactive oxygen species when irradiated with UVA light. These proteins have the same absorption spectrum and fluorescence spectrum as the proteins isolated from aged human lenses (70-90 yr). Quantitation of the reactive oxygen species show that after four weeks of incubation of the bovine lens proteins with ascorbic acid, the distribution and level of these products are almost identical to those obtained by irradiated aged human lens proteins. These data support the idea that oxidized ascorbic acid may be responsible for the browning and UVA sensitivity of human lenses. This may occur only later in life when these oxidation products are allowed to accumulated, and the proteins become aggregated. Normally, ascorbic is essential to retain a reducing atmosphere in the lens, protecting against any possible light damage.

Media

Photos

Publications

Linetsky M, Ortwerth BJ. Quantitation of the Singlet Oxygen Produced by the UVA Irradiation of Human Lens Proteins. Photochemical Photobiology. 65:522-529, 1997.

Ortwerth BJ, Linetsky M, Olesen P. Ascorbic Acid Glycation of Lens Proteins Produces UVA Sensitizers Similar to those Present in Human Lens. Photochemical Photobiology. 62:454-462, 1995.