Bioinformatics Pertinent to Lipid Analysis in Biological Samples.

Electrospray ionization mass spectrometry has revolutionized the way lipids are studied. In this work, we present a tutorial for analyzing class-specific lipid spectra obtained from a triple quadrupole mass spectrometer. The open-source software MZmine 2.21 is used, coupled with LIPID MAPS databases. Here, we describe the steps for lipid identification, ratiometric quantification, and briefly address the differences to the analyses when using direct infusion versus tandem liquid chromatography-mass spectrometry (LC-MS). We also provide a tutorial and equations for quantification of lipid amounts using synthetic lipid standards and normalization to a protein amount.

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.

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 Bradford's 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.

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 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).

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.

Sphingolipids and ceramides in human aqueous humor.

PURPOSE: To determine the differential profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate and ceramide lipid species and their quantitative differences between control and glaucomatous aqueous humor (AQH) derived from human donors. METHODS: AQH from control and primary open-angle glaucoma donors was collected and subjected to lipid extraction using suitable modifications of the Bligh and Dyer method. Proteins were estimated using Bradford's method. Lipids were identified and ratiometrically quantified in a two-step process using precursor ion scan or neutral loss scan (NLS) with appropriate class-specific lipid standards on a TSQ Quantum Access Max mass spectrometer following established procedures. Primary human trabecular meshwork cells and video microscopic imaging were used to assess changes in cell shape and motility upon exposure to 20 pmol of Cer(d18:0/18:1(9Z)) in 10% dimethyl sulfoxide (vehicle). RESULTS: We identified several species of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramides that were common between control and glaucomatous AQH. Some unique lipid species in these classes were also identified in controls but not in glaucoma and vice versa. We found exposure to 20 pmol of Cer(d18:0/18:1(9Z)) resulted in changes in the trabecular meshwork cell shape and observed motility changes compared to vehicle-only control. CONCLUSIONS: Most lipids belonging to the sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide species were common between control and primary open-angle glaucoma donors. However, some sphingolipids and ceramides were found to be uniquely present in control but absent in the glaucomatous AQH and vice versa. Identification of unique lipid species present or absent in the pathophysiological context may contribute further insight into glaucoma pathology.

Cholesterol and glycosphingolipids of human trabecular meshwork and aqueous humor: comparative profiles from control and glaucomatous donors.

PURPOSE: To determine the differential profiles of cholesterol and glycosphingolipid species and their quantitative differences between control and glaucomatous aqueous humor (AQH) and the trabecular meshwork (TM) derived from human donors. METHODS: Control TM and selected primary open angle glaucoma (POAG) TM samples were collected from cadaveric donors. Other TM samples, glaucomatous AQH and control AQH were procured during intraocular surgery. Lipid extraction was performed using modifications of the Bligh and Dyer method. Protein concentration was estimated using the Bradford colorimetric assay. Cholesterol and glycosphingolipids were identified and subjected to ratiometric quantification utilizing precursor ion scan and neutral ion loss scan in positive ion mode using appropriate class specific lipid standards (Cholesterol and Psychosine) on a TSQ Quantum Access Max mass spectrometer. RESULTS: Control and glaucomatous AQH demonstrated 7 and 4 unique cholesterol species, whereas the TM demonstrated 7 and 12 unique species, respectively. The control and POAG AQH showed 6 and 0 whereas TM samples showed 5 and 1 unique glycosphingolipids, respectively. A total of 65 and 62 common cholesterol species and 59 and 58 common glycosphingolipids were found in AQH and TM, respectively. Increased zymosterol and glucopyranosyl cholesterol levels were found in glaucomatous AQH. Significantly decreased levels of galactosylceramide, glucosylceramide in glaucomatous TM were found compared to control TM. CONCLUSION: A high percentage of cholesterol and glycosphingolipid species was found to be common between control and POAG AQH and TM. Several cholesterol and glycosphingolipid species was found to be unique in a subset of POAG or controls. Glaucomatous aqueous humor and TM showed relatively higher levels of zymosterol (an intermediate precursor of cholesterol) and decreased glycoceramide levels, respectively.

Comparative phospholipid profiles of control and glaucomatous human trabecular meshwork.

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).