Phospholipid composition and their interconversion enzymes differences in glaucomatous human trabecular meshwork compared to controls.

Abstract

Purpose : To determine differences in phospholipid profiles, their interconversion enzymes and steady state precursors between human glaucomatous trabecular meshwork and normal controls. To establish whether phospholipid containing vesicles are different in glaucomatous aqueous humor (AH) compared to controls.

Methods : Trabecular meshwork (TM) and AH samples were obtained from human cadaveric donors [control, primary open angle glaucoma (POAG) (each n=24, equal distribution of gender, all Caucasian age 68±5.0 and 69.0± 7.5, respectively)]. The samples were analyzed using a Q-Exactive orbitrap mass spectrometer after chromatography on an Acela 600 HPLC for Lipids after extraction using Bligh and Dyer method. All enzymes in the phospholipid conversion pathway were determined for protein level using Western blot and ELISA, and their activities at 15 minutes interval using established assays. The cadaveric TM and AH were collected under NIH category 4, IRB exempted provisions without identifiers. Extracellular vesicles (EVs) were prepared using established centrifugation and 101Bio P100L isolation kit. The EVs were characterized using nanotracker, electron microscopy (EM), atomic force microscopy (AFM) imaging and dynamic light scattering methods. Steady state pre-phospholipid metabolites entering and exiting interconversion cycle were measured using a stop flow apparatus as well as individual metabolite chase methods. All measurements were performed with samples identities masked to the estimators. Data were subjected to statistical analysis using MetaboAnalyst 4.0/STATA14.2 with recommended settings.

Results : Significant differences in phospholipid composition and their interconversion enzymes were found between control and POAG groups. POAG TM and AH was characterized by significant lower levels of phosphatidylserine (PS) and higher phosphatidylethanolamine (PE) levels. We also found significant up-regulation of phosphatidylserine decarboxylase in POAG TM. POAG AH demonstrated smaller lipid containing EVs compared to normal controls using nanotracker and EM, which was corroborated by other methods.

Conclusions : We found significant differences in phospholipid levels and their interconversion enzymes in glaucoma compared to control eyes. Consistent with these lipid changes, we found decreased size of lipid containing vesicles in the POAG AH.

This is a 2021 ARVO Annual Meeting abstract.