Vitreous Fatty Amides and Acyl Carnitines Are Altered in Intermediate Age-Related Macular Degeneration

**Background:** Age-related macular degeneration (AMD) is a leading cause of visual impairment, with intermediate AMD representing a high-risk stage for progression to late AMD. The retinal pigment epithelium (RPE) has high metabolic activity, and metabolic dysfunction is implicated in AMD pathogenesis. While previous metabolomics studies have used blood samples, the blood-retinal barrier limits direct inference to ocular metabolism. This study aimed to investigate vitreous humor metabolite profiles in intermediate AMD using untargeted metabolomics to better understand local metabolic alterations.

**Methods:** This prospective cross-sectional study recruited 31 patients with intermediate AMD and 30 controls (all undergoing vitrectomy for epiretinal membrane) from March 2017 to April 2018. AMD was defined using a modified AREDS grading system. Undiluted vitreous humor samples (≥1 mL) were collected during initial dry vitrectomy. High-resolution liquid chromatography-mass spectrometry (LC-MS) was performed using a Vanquish/Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Metabolite identification used Compound Discoverer software with databases including Human Metabolome Database and KEGG. Univariate analyses with false discovery rate (FDR) correction (Benjamini-Hochberg) were applied. Principal component analysis (PCA) and volcano plots were used for visualization. Metabolic pathway analysis was conducted using MetaboAnalyst 5.0. Adjustments were made for age and AREDS2 formula use.

**Key Results:** Of 858 metabolites identified, 258 significantly distinguished intermediate AMD from controls (P < 0.05 after FDR correction). PCA showed separation between groups. Key findings included: (1) Increased fatty amides: anandamide (0.90-fold), docosanamide (0.67-fold), oleoyl ethanolamide (0.80-fold), stearoyl ethanolamide (0.49-fold), and others. (2) Increased acyl carnitines: acetyl L-carnitine (1.37-fold), L-palmitoylcarnitine (1.36-fold), and 2-methylbutyrylcarnitine (2.31-fold). (3) Increased ascorbic acid (14.09-fold) and uric acid (8.94-fold). (4) Decreased metabolites: succinic acid (−1.69-fold), nicotinamide (−0.55-fold), and N-arachidonoyl-L-serine (−0.49-fold). Enrichment analysis revealed pathways including oxidation of branched chain fatty acids, carnitine synthesis, and mitochondrial electron transport chain. Baseline characteristics were similar between groups except for higher triglyceride levels in controls (P = 0.007).

**Clinical Implications:** This study provides direct evidence of altered lipid and energy metabolism in the vitreous humor of intermediate AMD patients. The increase in fatty amides (including endocannabinoid-like metabolites) and acyl carnitines suggests dysregulated lipid metabolism and mitochondrial fatty acid oxidation, which may contribute to RPE dysfunction and AMD progression. The decrease in succinic acid (TCA cycle intermediate) and nicotinamide (NAD precursor) points to impaired energy metabolism, with nicotinamide previously proposed as a potential therapeutic target. These findings highlight the importance of local ocular metabolism in AMD and suggest that vitreous humor metabolomics can identify early biomarkers. Limitations include the cross-sectional design, untargeted approach, lack of genetic data, and potential confounding by AREDS2 supplement use. Future targeted validation and multi-omics studies are needed to establish causality and clinical utility.