Figure 1. Human RPE utilizes nitrogen from proline to generate amino acids and exports them into the media.

(A) Human RPE cells were grown for 20 weeks and switched into DMEM media with or without 1 mM ¹⁵N proline. The spent media were collected at 0h, 24h and 48h and cells were collected at 48h for metabolite analysis with GC MS. (B) A schematic of proline nitrogen metabolism into amino acids (Colored in blue) through proline dehydrogenase (PRODH), pyrroline-5-carboxylic acid (P5C) dehydrogenase (P5CDH), ornithine transaminase (OAT), glutamate decarboxylase (GAD), glutamine synthetase (GS) and aminotransferases (AT). These amino acids in proline metabolism were selected for GC MS analysis. (C-D) ¹⁵N fraction of metabolites derived from ¹⁵N proline in RPE cells and the total abundance of all isotopologue relative to control RPE cells without ¹⁵N proline. *P<0.05, ***P<0.001, N=3. (E-F) ¹⁵N fraction of metabolites derived from ¹⁵N proline in RPE spent media at different time points and the total abundance of all isotopologue relative to control RPE culture media without ¹⁵N proline at 24h or 48h. *P<0.05, **P<0.01, ***P<0.001, N=3.