Comparative phospholipid profiles of control and glaucomatous human trabecular meshwork

Abstract

Purpose: We compared phospholipid (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol) profiles of control and glaucomatous trabecular meshwork (TM) derived from human donors.

Methods: Control TM and most primary open angle glaucoma (POAG) TM were collected from cadaver donors. A select subset of POAG surgical TM samples also were collected for analyses. Lipid extraction was performed using a modification of the Bligh and Dyer method, protein concentrations were determined using the Bradford method, and for select samples confirmed with densitometry of PHAST gels. Lipids were identified and subjected to ratiometric quantification using a TSQ quantum Access Max triple quadrupole mass spectrometer with precursor ion scan (PIS) or neutral ion loss scan (NLS), using appropriate class specific lipid standards.

Results: The comparative profiles of phosphatidylcholine, phosphatidylserine, phosphoethanolamine, and phosphatidylinositol between control and glaucomatous TM showed several species common between them. A number of unique lipids in all four phospholipid classes also were identified in control TM that were absent in glaucoma TM and vice versa.

Conclusions: A number of phospholipids were found to be uniquely present in control but absent in glaucomatous TM and vice versa. Compared to a previous study of control and POAG blood, a number of these phospholipids are absent locally (TM), as well as systemically (in blood).

Figure 1.

Representative electrospray ionization tandem mass spectrometric analysis of PC class of lipids extracted from human TM in positive-ion mode. (A) Representative PIS scanning of m/z 184.0 corresponding to PC class. (B) Representative PIS as above with internal standard addition (arrowhead; m/z ratio of 650.6) enabling ratiometric quantification of all identified lipids in PC class. The experimental conditions for PIS are listed in Table 1. Parent ion scan for m/z 600 to 900 has been shown.

Figure 2.

A schematic diagram of potential conversion cycle of phospholipids in the TM. The conversion enzymes have been italicized. CK, choline kinase; CCTCTP, phosphocholine cytidylyltransferase; CPT, cholinephosphottransferase; PSS1&2, PS synthase 1 and 2; PEMT, phosphatidylethanolamine N-methyltransferase; EPT, ethanolamine-phosphotransferase; PSD, phosphoserine decarboxylase; ECTCTP, phosphoethanolamine cytidylyltransferase; EK, ethanolamine kinase.