Raman Spectroscopic Evaluation of Advanced Glycation & Lipoxidation Adducts in RPE

Abstract

Purpose: : Advanced glycation end products (AGEs) and advanced lipooxidation products (ALEs) accumulate with age in RPE and Bruch’s membrane and may have an important role in age-related macular degeneration (AMD). Raman confocal microscopy (RCM) is a non-invasive method that we have used to quantify AGEs in Bruch’s membrane (Glenn et al. FASEB J. 2007 21:3542-52). This study has extended the range of adducts that can be quantified in human ocular tissue. We have also used RCM to evaluate RPE monolayers that have been exposed to oxidised photoreceptor outer segments (POS) or defined AGEs/ALEs.

Methods: : Raman spectra were acquired by RCM to establish a database of a range of available free and protein-bound AGEs/ALEs. POS were isolated from bovine retinas and pre-oxidised by irradiation with white light or incubation with free radicals generators at 37oC. Human ARPE-19 were exposed to POS, pre-oxidised POS and AGE/ALE-modified proteins (1-14 days). RCM was used to monitor the accumulation of adducts in the RPE.

Results: : Raman spectra of over 20 defined AGE/ALE adducts have been collected, many of which have so far not been investigated in the eye. The probed adducts demonstrated distinctive spectral profiles, confirming the ability of Raman spectroscopy to discriminate and identify individual modifications. RPE exposure to oxidised POS resulted in RCM detection of several AGE/ALE adducts within cytoplasmic inclusions.

Conclusions: : We have completed an extensive Raman spectra database of AGE/ALE adducts. RCM can be used to identify diverse AGEs/ALEs in ocular tissue, many of which have proven pathogenic properties. This work is an important step towards Raman assessment of these adducts in the living eye

Keywords: retinal pigment epithelium • aging • image processing