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Fluorescence reveals early cataract growth

28 Apr 2011

Edinburgh Instruments believes it can objectively measure the protein changes causing the condition.

Assessing the growth of cataracts in human eyes in a notoriously subjective business, usually relying on the use of a conventional slit lamp and Rayleigh light scattering to allow direct observation of lens opacity by an ophthalmologist. A better way would be to quantitatively assess the structural changes underway in proteins within the lens which cause the opacity in the first place, and Edinburgh Instruments has developed a technique based on tryptophan fluorescence (TF) which could allow just that.

"Tryptophan is one of the standard amino acids and a component of the eye lens, and its fluorescence behavior varies depending on its microenvironment and structure," said Des Smith of Edinburgh Instruments. "When a tryptophan side chain is located in a non-polar microenvironment, its emission spectrum has a maximum at 308 nm. In folded multi-tryptophan proteins, spectra usually exhibit maxima between 325 and 335 nm, while a denatured protein exhibits a spectral shift to 355-360 nm."

It so happens that the cornea and aqueous humor in the eye is transparent in the 310 to 320 nm range, where tryptophan has a spectral feature that can be used for quantitative measurements. "If you move out to 317 nm there is a tail of the tryptophan excitation spectrum, where the absorption coefficient drops by a couple of orders of magnitude but is still sufficient for excitation," said Smith. "So it's possible to excite tryptophan fluorescence at the 'red edge' of the absorption band."

To test the correlation between lens transparency and changes in the tryptophan protein, the team used TF to examine complete eyeballs from pigs, along with human lenses supplied by the Bristol Eye Bank, UK. The lenses were deliberately UV-irradiated for periods of between 20 minutes and 12 hours, an irradiation model known to mimic the way that ambient UV light can bring about the formation of cortical cataracts in humans.

The results showed that the TF spectra of the UV-irradiated lenses were red-shifted, and that the intensity decreased with the radiation dose. At the same time, the intensity of a non-tryptophan emission with a maximum at 435 nm increased, which the team attributed to a photochemical conversion of the tryptophan population to 435 nm-emitting molecules as lens opacity develops.

"The ratio of intensity of the non-tryptophan and tryptophan emission bands was a parameter that turned out to be very sensitive to structural changes in the porcine lenses induced by UV irradiation," said Smith. "We could confidently detect changes after 20 minutes of irradiation. This parameter doubled within the first 30 minutes and approached saturation after 240 minutes, which was the first point at which structural defects could be also detected by the slit lamp method."

Towards a point-of-care instrument
The ability to diagnose cataract development at a molecular level through tryptophan changes could be of great benefit in surgical lens replacement decisions and pharmacological research, although transferring the technique into a robust analytical technique for human patients is currently at an early stage, not least because the complete human eye is a much more complicated system.

"For human eyes we will need an optical system that can handle the excitation and emission of the fluorescence, but also has an imaging ability on board," noted Smith. "Effectively we have to combine the fluorescence excitation and emission optical train with an existing hospital slit lamp. We are working on a method using a series of interference filters, which can pass more light through and have a bigger optical grasp. Being able to scan the spectrum using interference filters without having to build a grating spectrometer is the route forward to clinical trials of a point-of-care instrument for this technique."

Global research into cataracts is currently yielding some tantalizing results. "Russian studies suggest that treatment with N-acetylcarnosine may halt or possibly even reverse cataracts, while work carried out in Denmark has shown that treatment with femtosecond lasers at 800 nm wavelength may actually improve the transparency of human lenses where a cataract has already formed," said Smith. "So there are routes to both a possible pharmacological intervention and a possible laser intervention in cataract treatment. But because the testing and assessment of cataracts is so subjective, not all ophthalmologists agree on the validity of such findings. If TF can give quantitative information about lens transparency on a molecular scale, that will be very valuable."

*The work on tryptophan fluorescence was carried out by Edinburgh Instruments together with NHS Princess Alexandra Eye Pavilion and Heriot-Watt University.

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