Protein fluorescence explored for cataract diagnosis

Fluorescence of lens proteins could help understand, diagnose, and treat cataracts, researchers say.

“Rather than waiting for the condition to appear, it could be possible to diagnose and monitor cataract before it forms, allowing preventative measures to be taken where possible,” said Rory Duncan of Heriot-Watt University in Edinburh, United Kingdom in a press release.

Duncan and colleagues published their findings in Nature Scientific Reports.

The research focuses on post-translational modifications (PTMs) of Trp and Arg amino acid residues that accumulate as lenses age.

Courtesy of Dr Rory Duncan

Current cataract diagnosis depends on light scattering methods which can detect defects in the lens structure when they reach at least 0.5 µm, the optical wavelength. But the formation of PTMs may precede the micron-sized defects that make these light-scattering methods possible.

Matured eye lens fibre cells do not contain organelles apart from the proteasome, Duncan and colleagues point out. This gives the eye lens optical homogeneity and minimizes Rayleigh scattering. But it also precludes the synthesis of new proteins.

Courtesy of Dr Rory DuncanAs a result, proteins synthesised in new cells remain throughout the lifespan of mature cells. Without protein turnover, PTMS accumulate in crystallins, resulting in protein misfolding and aggregation and causing light scattering.

Also, some PTMS absorb light in the visible spectral range, reducing lens transparency and giving rise to coloration.

Modifications of Trp side chains can change the position of the Trp emission spectrum and fluorescence. So the researchers hypothesized that formation PTMs in crystallins endows the eye lens a unique fluorescence “signature” that could be measurable.

Exploring PTM fluorescence

To explore this proposition, Duncan and colleagues examined the fluorescence of PTMs in lenses from pigs and in donated human lenses.

They found that due to the spectral selectivity on the red edge of the Trp absorption spectrum, they could excite a fraction of Trp residues situated in hydrophilic pockets with a red-shifted absorption spectrum. A 317 nm light-emitting diode (LED) was effective for this excitation. They also measured fluorescence from Trp photo-degradation products and ArgP.

Courtesy of Dr Rory Duncan

They compared pig lenses irradiated with ultraviolet light to untreated lenses. And they compared human lenses from an eye bank, post-operational phaco-emulsified lens samples and clear emulsified lenses of 40 and 46 year old patients who underwent cosmetic surgery.

“The technology identifies how much oxidative damage lens proteins have accumulated through lifestyle or environmental factors,” said Duncan in the press release. “This accumulated damage may be important in determining risk factors for a number of age-related conditions.”

In summarizing, they wrote that they had developed a novel non-invasive method of semi-quantitative determination of the concentrations of fluorescent PTMs in the lens based on simultaneous measurements of fluorescence emission.

“We show that the fluorescence spectra of the individual fluorescent components normalised against Trp intensity enable determination of their relative concentration with an accuracy of 5–10%,” they wrote. “This makes this method useful for cataract grading and for monitoring cataractogenesis over a period of time.”

By monitoring changes in the lens structure at the molecular level, the hoped to also facilitate the development of medications that might slow down the development of cataracts.