PURPOSE. We characterized differences in biochemical composition of the vitreous of different animal species with respect to small molecule constituents. METHODS. Vitreous samples were extracted from sheep, pig, Dutch Belted rabbits, and New Zealand white rabbits. The vitreous samples were investigated for acetylcholinesterase (AChE) activity and, in addition, were subjected to metabolomics determination using mass spectrometry. RESULTS. AChE activity varied across the species investigated with

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... greater activity noted in larger animals. Principal component analysis demonstrated species differentiation in relation to metabolomic profile. Key peaks identified the importance of animal diet on small molecule composition of the vitreous. CONCLUSIONS. Our results highlighted principal and consistent differences in small molecule composition and enzymatic activity of the vitreous depending on species. Interesting differences were demonstrated, showing that diet potentially can impact on components of and metabolites contained within the vitreous. Material will be exchanged between vascular and retinal tissue with the vitreous compartment and as a nonvascular, slowly equilibrating "sink" might reflect changes in transporter activity. As a first step, understanding the differences in the metabolic profile of vitreous from different species may impact interpretation of such activity across different species. (Invest Ophthalmol Vis Sci. 2012; 53:4778-4786)