Multiplex analysis of age-related protein and lipid modifications in human Bruch's membrane

Abstract

Aging of the human retina is characterized by progressive pathology, which can lead to vision loss. This progression is believed to involve reactive metabolic intermediates reacting with constituents of Bruch's membrane, significantly altering its physiochemical nature and function. We aimed to replace a myriad of techniques following these changes with one, Raman spectroscopy. We used multiplexed Raman spectroscopy to analyze the age-related changes in 7 proteins, 3 lipids, and 8 advanced glycation/lipoxidation endproducts (AGEs/ALEs) in 63 postmortem human donors. We provided an important database for Raman spectra from a broad range of AGEs and ALEs, each with a characteristic fingerprint. Many of these adducts were shown for the first time in human Bruch's membrane and are significantly associated with aging. The study also introduced the previously unreported up-regulation of heme during aging of Bruch's membrane, which is associated with AGE/ALE formation. Selection of donors ranged from ages 32 to 92 yr. We demonstrated that Raman spectroscopy can identify and quantify age-related changes in a single nondestructive measurement, with potential to measure age-related changes in vivo. We present the first directly recorded evidence of the key role of heme in AGE/ALE formation.

Figure 1.

Raman spectral database of selected intermediates and final protein modifications detected in human tissues. DHPL, dihydropyridine lysine; MOLD, methylglyoxal-lysine dimer; GOLD, glyoxal-lysine dimer (GOLD); pento, pentosidine.

Figure 2.

Comparing Raman spectra of biochemical constituent 7 (A), G-H1 (B), AGE (C), CEP (D), and 2-AAA (E). Constituent 7 can be wholly accounted for by a combination of signals from AGEs/ALEs, with some slight shifts in band position (due to matrix interactions). Biggest band shift is observed in the band most sensitive to hydrogen bonding (carbonyl stretch), suggesting strong hydrogen bonding of G-H1 in Bruch's membrane.

Figure 3.

Column plots showing the changes in Raman signal levels of a range of proteins and lipids with chronological age in human Bruch's membrane. A) Collagen I (R²_l=0.58; P<0.05). B) Collagen III (R²_q=0.81; P<0.05). C) Collagen IV (R²_q=0.82; P<0.05). D) Elastin (R²_q=0.73; P<0.05). *Significant change between bracketed groups (P<0.05). E) α-Crystallin (R²_q=0.73; P<0.05). F) Heme (R²_l=0.54; P<0.05).

Figure 4.

A) Quantity of MUFAs (R²_q=0.00; NS; i), cholesterol (R²_q=0.74; P<0.05; ii), oxidized PUFAs (R²_q=0.73; P<0.05; iii), and G-H1/CEP/AGE/2-AAA (R²_q=0.44; NS; iv) in each decade, as predicted from PCA of Raman data. B) Raman maps showing the distribution of CEP (green) and oxidized PUFA (red) in donor eyes from 55-, 76-, and 83-yr-old females (i) and overlaid merge of the CEP and oxidized PUFAs (ii; yellow results from overlap of red and green). Scale bars = 5 μm. Intensity of images is related to magnitude of PC score (arbitrary units). *Significant change between bracketed groups (P<0.05).

Figure 5.

A–G) Raman distribution maps in human donor Bruch's membranes of heme (A), CML/CEL (B), G-H1/CEP (C), DHP-Lys (D), and the overlaid merge of these channels (yellow results from overlap of red and green); heme and CML/CEL (E), heme and G-H1 (F), and heme and DHP-Lys (G). Heme is colocalized with G-H1/CEP, excluded by CML/CEL, and has no consistent spatial relationship with DHP-Lys. H) Mean area of heme deposits against age; significant increase between 70s and 80s. *Significant change between bracketed groups (P<0.05). Scale bars = 5 μm. Intensity of images is related to magnitude of PC score (arbitrary units).

Figure 6.

Raman-PCA predicted AGE/ALE content of human Bruch's membrane against chronological age. A) CML. B) CEL (R²_q=0.88; P<0.01). C) HHE (R²_q=0.57; NS). D) DHP-Lys (R²_q=0.81; P<0.05). E) GO (R²_q=0.69; NS). F) Aggregate Raman-PCA predicted AGE/ALE content (R²_q=0.88; P<0.01). *Significant change between bracketed groups (P<0.05).

Figure 7.

Scatterplot of Raman-predicted pentosidine level vs. chronological age for human donor Bruch's membranes (R²_l = 0.87; P < 0.05). *Significant change between bracketed groups (P<0.05).