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Confocal Raman microscopy can quantify advanced glycation end product (AGE) modifications in Bruch’s membrane leading to accurate, nondestructive prediction of ocular aging

Josephine V. Glenn^*,1, J. Renwick Beattie^,,1, Lindsay Barrett^,, Norma Frizzell, Suzanne R. Thorpe, Mike E Boulton^||, John J. McGarvey^, and Alan W. Stitt^*,2

^* Centre for Vision Science, School of Biomedical Science, Faculty of Medicine and Health Sciences, Queen’s University of Belfast, Belfast, Northern Ireland;

School of Chemistry and Chemical Engineering,

Centre for Clinical Raman Microscopy, Queen’s University Belfast, Belfast, Northern Ireland;

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina, USA; and

^|| Department of Ophthalmology and Vision Sciences, the University of Texas Medical Branch, Galveston, Texas, USA

²Correspondence: Centre for Vision Science, School of Biomedical Science, Queen’s University Belfast, Royal Victoria Hospital, Belfast BT12 6BA, Northern Ireland. E-mail: a.stitt{at}qub.ac.uk

The modification of proteins by nonenzymatic glycation leading to accumulation of advanced glycation end products (AGEs) is a well-established phenomenon of aging. In the eyes of elderly patients, these adducts have been observed in retinal pigment epithelium (RPE), particularly within the underlying pentalaminar substrate known as Bruch’s membrane. AGEs have also been localized to age-related subcellular deposits (drusen and basal laminar deposits) and are thought to play a pathogenic role in progression of the major sight-threatening condition known as age-related macular degeneration (AMD). The current study has quantified AGEs in Bruch’s membrane from postmortem eyes and established age-related correlations. In particular, we investigated the potential of confocal Raman microscopy to identify and quantify AGEs in Bruch’s membrane in a nondestructive, analytical fashion. Bruch’s membrane and the innermost layers of the underlying choroid (BM-Ch) were dissected from fresh postmortem eye-cups (n=56). AGE adducts were quantified from homogenized tissue using reverse-phase HPLC and GC/MS in combination with immunohistochemistry. For parallel Raman analysis, BM-Ch was flat-mounted on slides and evaluated using a Raman confocal microscope and spectra analyzed by a range of statistical approaches. Quantitative analysis showed that the AGEs pentosidine, carboxymethyllysine (CML), and carboxyethyllysine (CEL) occurred at significantly higher levels in BM-Ch with age (P<0.05–0.01). Defined Raman spectral "fingerprints" were identified for various AGEs and these were observed in the clinical samples using confocal Raman microscopy. The Raman data set successfully modeled AGEs and not only provided quantitative data that compared with conventional analytical approaches, but also provided new and complementary information via a nondestructive approach with high spatial resolution. It was shown that the Raman approach could be used to predict chronological age of the clinical samples (P<0.001) and a difference in the Raman spectra between genders was highly significant (P<0.000001). With further development, this Raman-based approach has the potential for noninvasive examination of AGE adducts in living eyes and ultimately to assess their precise pathogenic role in age-related diseases.—Glenn, J. V., Beattie, J. R., Barrett, L., Frizzell, N., Thorpe, S. R., Boulton, M. E., McGarvey, J. J., Stitt, A. W. Confocal Raman microscopy can quantify advanced glycation end product (AGE) modifications in Bruch’s membrane leading to accurate, nondestructive prediction of ocular aging.

Key Words: RPE • Raman spectroscopy