Quantifying Hydrolytic Enzymes in the Vitreous Humor of Humans and Nonhuman Species Using Targeted Proteomics

The vitreous humor is a viscous ocular fluid that contains a complex proteome including hydrolytic enzymes. Differences between concentrations of these enzymes may contribute to the variable hydrolysis of some intravitreal drugs/implants. Here, we developed a targeted proteomics method to quantify six hydrolytic enzymes in the vitreous humor of eight human donors as well as in the vitreous humor of rabbits, minipigs, and monkeys. These enzymes include cathepsin D (CTSD), ectonucleotide pyrophosphatase 2 (ENPP2), B-hexosaminidase subunit alpha (HEXA), serum paraoxonase 1 (PON1), tripeptidyl peptidase 1 (TPP1), and ubiquitin carboxy-terminal hydrolase lysozyme 1 (UCHL1). The results showed that enzyme concentrations were different among human subjects and between nonhuman species. Within human samples, CTSD and ENPP2 showed the highest levels. A gender-based comparison also revealed that two enzymes, UCHL1 and TPP1, showed significantly higher levels in female subjects compared to those in male subjects. Comparisons between humans and other species revealed potentially significant inter- and intraspecies differences between the levels of hydrolytic enzymes. Although performed on a limited number of samples to draw a robust biological conclusion, these results can guide researchers in the design of <i>in vitro</i> models of vitreous humor for prodrug metabolism and implant polymer degradation studies.