The Carnitine Shuttle Pathway is Altered in Patients With Neovascular Age-Related Macular Degeneration

Abstract

PurposeTo identify metabolites and metabolic pathways altered in neovascular age-related macular degeneration (NVAMD).MethodsWe performed metabolomics analysis using high-resolution C18 liquid chromatography-mass spectrometry on plasma samples from 100 NVAMD patients and 192 controls. Data for mass/charge ratio ranging from 85 to 850 were captured, and metabolic features were extracted using xMSanalyzer. Nested feature selection was used to identify metabolites that discriminated between NVAMD patients and controls. Pathway analysis was performed with Mummichog 2.0. Hierarchical clustering was used to examine the relationship between the discriminating metabolites and NVAMD patients and controls.ResultsOf the 10,917 metabolic features analyzed, a set of 159 was identified that distinguished NVAMD patients from controls (area under the curve of 0.83). Of these features, 39 were annotated with confidence and included multiple carnitine metabolites. Pathway analysis revealed that the carnitine shuttle pathway was significantly altered in NVAMD patients (P = 0.0001). Tandem mass spectrometry confirmed the molecular identity of five carnitine shuttle pathway acylcarnitine intermediates that were increased in NVAMD patients. Hierarchical cluster analysis revealed that 51% of the NVAMD patients had similar metabolic profiles, whereas the remaining 49% displayed greater variability in their metabolic profiles.ConclusionsMultiple long-chain acylcarnitines that are part of the carnitine shuttle pathway were significantly increased in NVAMD patients compared to controls, suggesting that fatty acid metabolism may be involved in NVAMD pathophysiology. Cluster analysis suggested that clinically indistinguishable NVAMD patients can be separated into distinct subgroups based on metabolic profiles.

A Plasma Metabolomic Profiling of Exudative Age-Related Macular Degeneration Showing Carnosine and Mitochondrial Deficiencies

“…Valerylcarnitine is a short-chain fatty acid (C5) associated with carnitine. The alteration of the carnitine shuttle was already identified in metabolomics studies as a key feature of neovascular AMD [19]. Our study shows that, in addition to the long-chain acylcarnitines already reported, short-chain acylcarnitines such as valerylcarnitine can also be involved, as well as the fatty acids carrier itself, carnitine.…”

Metabolomics and Age-Related Macular Degeneration

“…Recently, the limitations of the original paper by Osborn et al [126] have been improved on in a study, which investigated the metabolites and associated metabolic pathways of a larger cohort of NVAMD patients [131]. Plasma samples were collected from NVAMD patients, who exhibited extensive CNV, subretinal hemorrhaging or fibrosis, or photocoagulation scarring in one or both eyes.…”

Untargeted high-resolution plasma metabolomic profiling predicts outcomes in patients with coronary artery disease

“…Plasma aliquots (65 μL) were treated with 130 μL acetonitrile (2:1 volume/volume) containing 3.5 μL of an internal isotopic standard mix and placed on ice for 30 minutes. [ 14 – 17 ] The internal standard mix consisted of 14 stable isotopic chemicals, [ 14 ] which cover a broad range of chemical properties represented in small molecules: [ 13 C 6 ]-d-glucose, [ 15 N]-indole, [2- 15 N]-l-lysine dihydrochloride, [ 13 C 5 ]-l-glutamic acid, [ 13 C 7 ]-benzoic acid, [3,4- 13 C 2 ]-cholesterol, [ 15 N]-l-tyrosine, [trimethyl- 13 C 3 ]-caffeine, [ 15 N 2 ]-uracil, [3,3- 13 C 2 ]-cystine, [1,2- 13 C 2 ]-palmitic acid, [ 15 N, 13 C 5 ]-l-methionine, [ 15 N]-choline chloride, and 2’-deoxyguanosine- 15 N 2 , 13 C 10 -5’-monophosphate. Samples were analyzed using a Thermo LTQ Velos Orbitrap high-resolution (60,000 mass resolution) mass spectrometer (Thermo Fisher Scientific, San Diego, California) and C18 column chromatography (Higgins Analytical Inc., Targa, 2.1 × 10 cm) in positive ionization mode with a scanning m/z range of 85–2000 over 10 minutes.…”

“…All subjects underwent an overnight fast before blood collection and untargeted high-resolution metabolomic profiling using liquid-chromatography mass spectrometry (LC/MS) was performed using standardized techniques after thawing each subject's plasma stored at -80C elsius. [11][12][13][14][15][16][17] Plasma samples were run in a randomized order in batches of 20 and three technical replicates were analyzed for each sample in a sequential manner. Plasma aliquots (65 μL) were treated with 130 μL acetonitrile (2:1 volume/volume) containing 3.5 μL of an internal isotopic standard mix and placed on ice for 30 minutes.…”

“…Plasma aliquots (65 μL) were treated with 130 μL acetonitrile (2:1 volume/volume) containing 3.5 μL of an internal isotopic standard mix and placed on ice for 30 minutes. [14][15][16][17] The internal standard mix consisted of 14 stable isotopic chemicals, [14] which cover a broad range of chemical properties represented in small molecules: [ 13…”