Phospholipid profiles of control and glaucomatous human aqueous humor

Abstract

To compare phospholipid (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidylinositol) profiles of human control and glaucomatous aqueous humor (AQH). AQH samples were procured during surgery from human POAG and control subjects (n = 15 each). Samples were used following institutional review board approved protocols and adhering to the tenets of the Declaration of Helsinki. Lipid extraction was performed using a modification of the Bligh and Dyer method, protein concentrations were determined using the Bradford's method, and select samples were confirmed with Densitometry of PHAST gels. Lipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan (PIS) or neutral ion loss scan (NLS) using appropriate class specific lipid standards in a two step quantification process. The comparative profiles of phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols between control and glaucomatous AQH showed several species common between them. A number of unique lipids in all four phospholipid classes were also identified in control eyes that were absent in glaucomatous eyes and vice versa. A number of phospholipids were found to be uniquely present in control, but absent in glaucomatous AQH and vice versa. Compared with a previous study of control and POAG red blood cells, a number of these phospholipids are absent locally (AQH).

Keywords: Aqueous humor; Glaucoma; Lipidomics; Mass spectrometry; Phospholipids.

Figure 1

Representative electrospray ionization mass spectrometric analysis of the phosphatidylcholine (PC) class of lipids in human aqueous humor (AQH) analyzed in positive-ion mode. (A) Representative spectra of human control and (B) glaucomatous AQH as indicated with internal standard (arrowhead; m/z ratio of 650.6) that has been used for ratiometric quantification. Precursor ion scan for m/z 600–900 has been shown. Arrow shows presence of a unique species in control AQH.