Abstract
Purpose: :
To quantify plasma protein carboxymethyllysine (CML) and pentosidine (advanced glycation end products) as possible biomarkers for age-related macular degeneration (AMD).
Methods: :
Blood was collected from clinically documented AMD and age-matched normal, healthy donors at the Cole Eye Institute, Cleveland Clinic Foundation. Plasma proteins were precipitated with acetone and hydrolyzed in 6N HCl to determine amounts of protein, CML and pentosidine. Protein and furosine were quantified by amino acid analysis. CML was quantified by LC MS using a Sciex API 3000 triple quadrupole mass spectrometer. Pentosidine was measured by LC fluorescence monitoring (excitation = 335 nm; emission = 385 nm) using a Waters 474 scanning fluorometer. Carboxyethylpyrrole (CEP) adducts were quantified in plasma by ELISA. Logistic regression modeling for c-statistics, odds ratios and p values was performed with SAS 9.1. Sensitivity and specificity were calculated to maximize the sum of the two values using receiver operating characteristic (ROC) curves.
Results: :
Quantitative analyses of plasma from AMD (n = 60) and normal control (n = 30) donors showed that mean CML and pentosidine levels were elevated in AMD plasma by ~60% and ~80%, respectively, while furosine was largely unchanged. In these samples, CEP adducts were 2-fold higher in AMD (n = 56) than in controls (n = 30). The odds ratio for both CML and pentosidine elevated was 15 fold greater in AMD than in control patients. For these plasma samples, C-statistics and ROC curves indicate that CML discriminated between AMD and control donors with ~79% accuracy, CEP adducts with 81% and pentosidine with 92% accuracy. In combination with CEP adduct levels, CML provided 89% accuracy and pentosidine provided ~96% discrimination accuracy.
Conclusions: :
CML and pentosidine levels are elevated in AMD plasma and may prove useful for predicting AMD susceptibility and for monitoring therapeutic efficacy, especially in combination with CEP biomarkers.
Keywords: age-related macular degeneration • proteomics • protein modifications-post translational