Tryptophan Fluorescence of Ocular Lens Protein for Early Diagnosis of Cataracts
- Created: Saturday, 01 October 2011
Findings could spur the development of a clinically useful, non-invasive tool sensitive enough to detect, diagnose, and monitor lens change earlier than current methods.
Cataracts are the leading cause of blindness worldwide; they affect more than 20 million people and result in ~1.3 million operations annually in the United States. Current methods of cataract detection are based on subjective observation of lens opacity by Rayleigh light scattering using a slit lamp. These methods are not sensitive enough to reveal structural changes on a molecular level; they can only reveal defects once their size becomes comparable with the optical wavelength (400-600 nm). This occurs at a very late stage of cataract development.
Surgical treatment requires the eye lens to be destroyed by ultrasound and replaced by a plastic lens. Though it is effective, it requires very skilled surgeons, and, like all surgeries, presents risks. A detection method that provides protein-level detail would be valuable, facilitating the discovery of the very first changes in the structure of the lens, thus enabling research on preventative or pharmacological treatment of cataracts. Such a technique would be of great importance in identifying the role of risk factors. While factors like dehydration, exposure to naturally occurring UV, and high blood sugar in diabetes are believed to be important, only a very sensitive way of detecting the smallest changes in lens proteins can hope to clarify this.
Tryptophan fluorescence is a sensitive probe of protein structure. It has been widely used for monitoring protein changes in biophysical research. Any process that introduces changes in the emission spectrum e.g. protein folding, conformation and aggregation, can be detected. So far, it has not been exploited for cataract diagnostics. However, excitation is possible through the cornea and aqueous humour to access the ocular lens where there is extremely high tryptophan absorption. Close to total absorption of the excitation light in a narrow surface layer of the lens prevents penetration of the excitation light to the interior of the lens at ~300 nm. Yet, if the tryptophan is excited at 317 nm, where the absorbance is significantly lower, a sub-population of tryptophan is selectively excited. The subpopulation is a residue situated on the protein surface that is exposed to the aqueous environment. Therefore, protein changescanbedetected.
The concept has been validated by studying changes in porcine lenses after UVA irradiation to mimic a cataract. The measurements were carried out on fresh porcine lenses on the same day that they were obtained from an abattoir. The UVAmodel was chosen since UV is a known risk factor in humans. Some of these lenses were imaged at NHS Princess Alexandra Eye Pavilion (Livingston, UK) by a conventional slit lamp (Haag Streit BQ); after four hours, they showed visible signs of cataract damage. Fluorescence images of irradiated and normal lenses were taken with a FinePix S5600 digital camera, using excitation by an Edinburgh Instruments 320 nm LED at the same UV exposure time and numerical aperture. Spectral measurements were made on a series of irradiated samples with an Edinburgh Instruments FLS920 spectrometer. The tryptophan fluorescence was found to change significantly within 20 minutes.
It was shown that the UVA irradiation brings about a significant decrease in the intensity of tryptophan fluorescence, a red shift of its emission spectrum, and a creation of an additional non-tryptophan fluorescence band with maximum at 435 nm. The non-tryptophan emission increased with the duration of irradiation at the same time the tryptophan fluorescence intensity decreased. This suggests conversion of the tryptophan is sensitive to the changes in the lens structure. Hence, either could be used to quantify defects at a molecule level. In contrast, after four hours of UVA irradiation, when the structural changes in the lens were seen by the conventional slit-lamp method, the changes had reached their saturation level. The experiments offer a sensitive method for monitoring very early changes in the lens structure that cannot be detected by the standard slit-lamp method.
Red-edge measurements in a whole pig’s eye were made due to the transparency of cornea and aqueous humour at this wavelength of 317 nm; hence there is potential for in vivo applications. Importantly, application of a robust optical design and the light grasp of interference filters should enable these measurements to be conducted at irradiation levels well below the daily safety threshold for UVA exposure.
Exploiting this discovery could allow the development of a clinically useful, non-invasive tool sensitive enough to detect, diagnose, and monitor lens change before significant damage occurs. This method could help to establish the point at which the irreversible crystalline protein change occurs. It also has the potential to act as a screening method for potential pharmacological treatment. In addition, clinical applications of this method would help in diagnostics of early stages of metabolic disorders such as diabetes, preventative treatment of which could delay the development of chronic diseases.
This work was done by Edinburgh Instruments in conjunction with the NHS Princess Alexandra Eye Pavilion, Livingston, UK. For more information, visit http://info.hotims.com/34459-199.