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Sreekumari Pushpoth, Martin Fitzpatrick, Stephen Young, Yit Yang, James Talks, Graham Wallace; Metabolomic analysis in patients with age related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3662.
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Age related macular degeneration is a complex disease where multiple factors show associations but do not explain the full nature of the disease. We hypothesized that a systems approach based on metabolomic analysis would be able to segregate diseases types and provide insights into the pathology. Metabolomics assesses the broad range of low molecular weight metabolites in biofluids and, since these are influenced by important AMD-related factors including diet, age and smoking, this approach may provide a useful novel window into the AMD-disease process.
Serum and urine samples were collected from 8 patients with dry and 35 with wet macular degeneration. Serum samples were centrifuged to remove cells, and 0.5ml aliquots stored at -80 degree C. After thawing, serum was filtered through 3kD MW cutoff filter to remove proteins. The filtrate was made with 10% in D2O, 100mM phosphate 0.5mM TMSP and pH 7.00.One-dimensional 1H spectra were acquired using a standard spin-echo pulse sequence on a Bruker DRX 600MHz NMR spectrometer equipped with a 1.7mm cryoprobe. 2D JRes spectra were also acquired to aid metabolite identification. Spectra were be segmented into 0.005-ppm (2.5 Hz) chemical shift ‘bins’ between 0.2 and 10.0 ppm, and the spectral area within each bin integrated. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the processed data was conducted using PLS Toolbox (Eigenvector Research) within MATLAB.
The samples from dry AMD patients cluster together reasonably well based on their metabolomics profile. With regards to samples from patients with wet AMD the clustering is far more complex. Several samples from wet AMD locate within the dry AMD cluster. Samples from patients with dry AMD show an increase in arginine, and decreased glucose, lactate, glutamine and reduced glutathione
Metabolomic analysis showed clear separation between body fluid samples from patients with wet and dry AMD. Investigation of the profiles produced identified arginine which may indicate increased nitric oxide production and a decrease in metabolites involved in the oxidative pathway that would support previous findings in AMD. That several samples from patients with wet AMD cluster with samples from dry AMD strongly indicates that common pathways are involved in both types of disease and that dry AMD can develop into the wet form.
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