Implications of Ocular Confounding Factors for Aqueous Humor Proteomic and Metabolomic Analyses in Retinal Diseases

To assess the impact of ocular confounding factors on aqueous humor (AH) proteomic and metabolomic analyses for retinal disease characterization.This study recruited 138 subjects (eyes): 102 with neovascular age-related macular degeneration (nAMD), 18 with diabetic macular edema (DME), and 18 with cataract (control group). AH samples underwent analysis using Olink Target 96 proteomics and Metabolon’s metabolomics platform Data analysis included correlation, differential abundance, and gene-set analysis.In total, 756 proteins and 408 metabolites were quantified in AH. Total AH protein concentration was notably higher in nAMD (3.2-fold) and DME (4.1-fold) compared to controls. Pseudophakic eyes showed higher total AH protein concentrations than phakic eyes (e.g., 1.6-fold in nAMD) and a specific protein signature indicative of matrix remodeling. Unexpectedly, pupil-dilating drugs containing phenylephrine/tropicamide increased several AH proteins, notably interleukin-6 (5.4-fold in nAMD). Correcting for these factors revealed functionally relevant protein correlation clusters and disease-relevant, differentially abundant proteins across the groups. Metabolomics analysis, for which the relevance of confounder adjustment was less apparent, suggested insufficiently controlled diabetes and chronic hyperglycemia in the DME group.AH protein concentration, pseudophakia, and pupil dilation with phenylephrine/ tropicamide are important confounding factors for AH protein analyses. When these factors are considered, AH analyses can more clearly reveal disease-relevant factors.Considering AH protein concentration, lens status, and phenylephrine/tropicamide administration as confounders is crucial for accurate interpretation of AH protein data.