Genea Edwards

Phospholipid profiles of control and glaucomatous human aqueous humor.

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 Bradfords 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).

Aqueous humor phospholipids of DBA/2J and DBA/2J-Gpnmb(+)/SjJ mice.

PURPOSE To compare phospholipid profiles [phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylinositol (PI)] of normotensive and hypertensive aqueous humor (AH) from DBA/2J and compare them with phospholipid profiles of DBA/2J-Gpnmb(+)/SjJ mice. METHODS AH was obtained from young-normotensive DBA/2J, old -hypertensive DBA/2J mice, young and old DBA/2J-Gpnmb(+)/SjJ mice (aging control). Lipids were extracted using modified Bligh and Dyer method and subjected to mass spectrometric identification using appropriate class-specific lipid standards and ratiometric quantification. Corresponding aqueous phase (of extraction) protein concentrations were measured using Bradford method. RESULTS The total amount of phospholipids showed a decrease in the hypertensive state compared to normotensive state. The total PE and total PS contributed over 50% of the total amount. Total PS showed a remarkable decrease in hypertensive compared to normotensive state. In contrast, total PE in the hypertensive stage presented an increase in amount. Unique lipid species were found encompassing all four phospholipid classes in normotensive as well as in the hypertensive state. Several phospholipid species were found common to both states but with remarkable differences in amount in individual states. The ratio of lysophospholipids to total phospholipids is significantly reduced in the hypertensive state. Commensurate with reduced level of lysophospholipids, we found an increased level of lysophospholipase D (Autotaxin) in the hypertensive state. The difference of total phospholipids between young and old was 35.4% in DBA/2J group but 10% in DBA/2J-Gpnmb(+)/SjJ mice. CONCLUSION The significant change of phospholipids including lysophospholipids was found commensurate with the elevated intraocular pressure (IOP).

Segmental outflow of aqueous humor in mouse and human.

ABSTRACT The main and only modifiable risk factor in glaucoma, the group of usually late onset progressive and irreversible blinding optic neuropathies, is elevated intraocular pressure (IOP). The increase in IOP is due to impeded aqueous humor (AH) outflow through the conventional pathway. The aberrant increased resistance at the trabecular meshwork (TM), the filter‐like region in the anterior eye chamber is the major contributory factor in causing the impeded outflow. In normal as well as in glaucoma eyes the regions of the TM are divided into areas of high and low flow. The collector channels and distal outflow regions are now increasingly being recognized as potential players in contributing to impede AH outflow. Structural and molecular make‐up contributing to the segmental blockage to outflow is likely to provide greater insight. Establishing segmental blockage to outflow in model systems of glaucoma such as the mouse in parallel to human eyes will expand our repertoire of tools for investigation. Further study into this area of interest has the potential to ultimately lead to the development of new therapeutics focused on lowering IOP by targeting the various components of segmental blockage of outflow in the TM and in the distal outflow region. HIGHLIGHTSElevated intraocular pressure is a characteristic of glaucoma.Segmental outflow consists of high and low outflow areas.There are several factors that contribute to segmental outflow.Segmental outflow has been found in the DBA/2J mouse model.

Sphingolipids and ceramides of mouse aqueous humor: Comparative profiles from normotensive and hypertensive DBA/2J mice

PURPOSE To identify the sphingolipid and ceramide species and their quantitative differences between normotensive and hypertensive intraocular pressure states in DBA/2J mouse aqueous humor (AH). METHODS Normotensive and hypertensive AH was sampled from mice by paracentesis. Lipid extraction was performed using modifications of the Bligh and Dyer method. Protein concentration was estimated using the Bradford colorimetric assay. Sphingolipids and ceramides were identified and subjected to ratiometric quantification using appropriate class specific lipid standards on a TSQ Quantum Access Max triple quadrupole mass spectrometer. RESULTS The comparative profiles of normotensive and hypertensive DBA/2J mouse AH showed several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate (S1P) and ceramides common between them. A number of unique lipids in each of the above lipid classes were also identified in normotensive AH that were absent in hypertensive AH and vice versa. CONCLUSION A number of sphingolipid and ceramide species were found to be uniquely present in normotensive, but absent in hypertensive AH and vice versa. Further pursuit of these findings is likely to contribute towards expanding our understanding of the molecular changes associated with increased intraocular pressure (IOP) and glaucoma.

A Comparison of Trabecular Meshwork Sphingolipids and Ceramides of Ocular Normotensive and Hypertensive States of DBA/2J Mice

PURPOSE To determine the differential profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide and their quantitative differences between trabecular meshwork (TM) derived from normotensive and hypertensive intraocular pressure states of DBA/2J mice. METHODS Normotensive and hypertensive state TM were collected from mice and analyzed. Lipid extraction was performed using the Bligh and Dyer method, and the protein concentrations were determined using the Bradford method. The lipids were identified and quantified using appropriate standards with a TSQ Quantum Access Max triple quadrupole mass spectrometer applying class-specific lipid identification settings. RESULTS The comparative profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide between normotensive and hypertensive TM showed several species unique to a phase and as well common between states. CONCLUSION The presence or absence of several sphingolipids and ceramides in the normotensive or hypertensive states may contribute to better understanding of the glaucomas.

Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism

PURPOSE We determined the profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide, and their quantitative differences between control and glaucomatous trabecular meshwork (TM) derived from human donors. METHODS Control and primary open angle glaucoma (POAG) TM samples were collected from cadaver donors. In addition, POAG TM surgical specimens also were procured. Lipid extraction was performed using suitable modifications of the Bligh and Dyer method. Protein concentrations were determined using Bradfords method. Lipids, identified using standardized class-specific lipid mass spectrometry, were quantified using a two-step ratiometric process. Gomori methenamine silver (GMS) staining was performed for detection of presence of Fusarium in the anterior eye tissue sections. PCR analyses were performed for detection of Fusarium species in the donor TM samples. RESULTS Several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide were common between control and POAG TM. Only a subset of species in some of these classes were identified uniquely either in controls or in POAG TM. Several lipid species of fungal origin (many from Fungi imperfecti, Fusarium species) were found to be common between control and POAG TM. The GMS staining and PCR analyses showed presence of DNA belonging to Fusarium species suggesting latent commensalism. CONCLUSIONS Most sphingolipids and ceramides (except a few unique to a specific donor TM group) were found to be common in the control and POAG TM. Latent commensalism by Fusarium was suggested by identification of Fusarium-specific lipids, which was supported further by PCR amplification and sequencing of DNA.

Aberrant glycosylation in the human trabecular meshwork.

To determine the difference in protein glycosylation and glycosylation enzyme levels between glaucomatous and control trabecular meshwork (TM).

Shotgun Sphingolipid Analysis of Human Aqueous Humor

This protocol provides a step-by-step guide to shotgun sphingolipid analysis of aqueous humor. We describe the Bligh and Dyer crude lipid extraction method and electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled with MZmine 2.21 data processing for identification and ratiometric quantitation of sphingosine, sphingosine-1-phosphate, sphingomyelin, and ceramide.

Sample preparation and analysis for imaging mass spectrometry

Imaging mass spectrometry (IMS) is a novel quantitative technique used to investigative diverse biomolecules in tissue sections. Specifically, IMS uses analytical separation of mass spectrometry to determine the spatial distribution of certain lipids and/or proteins located directly on biological sections from a single tissue sample. Typically, IMS is combined with histological analysis to reveal additional distribution details of characterized biomolecules including cell type and/or subcellular localization. In this chapter, we describe the use of Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight/Time-Of-Flight (TOF/TOF) to analyze various cholesterol and phosphatidylcholine species in atherosclerotic plaque of murine heart aortic valves. In particular, we detail animals used, tissue collection, preparation, matrix application, spectra acquisition for generating a color-coded image based on IMS spectral characteristics.