1. Lipidomic analyses of the Aqueous Humor and Trabecular Meshwork ABSTRACT CONCLUSION REFERENCES Katyayini Aribindi, Bogdan G. Gugiu, Richard K. Lee, Sanjoy K. Bhattacharya Bascom Palmer Eye Institute, University of Miami, Miami, Florida INTRODUCTION ACKNOWLEDGMENTS Known lipids were identified in normal and glaucomatous TM and AH. Glaucomatous TM and AH show distinct differences in phospholipid profiles compared to the phospholipid profiles of normal TM and AH. 1.Faulkner, R. et al. Aqueous humor concentrations of bimatoprost free acid, bimatoprost and travoprost free acid in cataract surgical patients administered multiple topical ocular doses of LUMIGAN or TRAVATAN. J Ocul Pharmacol Ther 26, 147-56 (2010). 2.Ansari, H. R., Davis, A. M., Kaddour-Djebbar, I. & Abdel-Latif, A. A. Effects of prostaglandin F2alpha and latanoprost on phosphoinositide turnover, myosin light chain phosphorylation and contraction in cat iris sphincter. J Ocul Pharmacol Ther 19, 217-31 (2003). 3. Yang, K., Cheng, H., Gross, R. W., & Han, X. (2009). Automated lipid identification and quantification by multidimensional mass spectrometry-based shotgun lipidomics Anal Chem, 81(11), 4356-4368. Supported by RPB Career award (SKB), an unrestricted grant from RPB to University of Miami, NIH grants R01EY016112 and P30EYEY014801. No Commercial Relationship Purpose: To identify the endogenous phospholipids of the aqueous humor (AQH) and trabecular meshdwork (TM) by mass spectrometric shotgun lipidomics. Methods: The human TM and AQH samples were collected adhering to tenets of declaration of Helsinki under IRB approved protocols. The TM and AQH were obtained from Caucasian donors with mean age of 55 ±8 years (n=10 each). Control TM and AQH were from cadavers and cataract surgery patients respectively. Lipids were extracted using the Bligh and Dyer method, and re-suspended in Isopropanol/Acetonitrile solution. For lipid analyses we have used recently developed shotgun lipidomic methods. Briefly, positive mode precursor ion scan (PIS) for phosphocholines (PC; product m/z of 184) and negative mode PIS for phosphoinositol (PI; product m/z 241) and phosphoethanolamine (PE; product m/z of 196) was used with a TSQ Quantum Access Max instrument. The samples were infused and scanned for one minute between 200 m/z to 1000 m/z. Ratiometric quantification was performed using quantitative standards for each lipid class. Results: All classes of phospholipids (PC, PI and PE) and corresponding lysophospholipids were found in both the AQH and TM. We found variation in lipid profile between AQH and TM. All phospholipid classes showed differences with respect to levels and species between glaucomatous and control groups. Unique phosphopholipids in controls that are absent in glaucomatous AQH and TM are being subjected to further characterization using high resolution mass spectrometry. Conclusion. We found unique phospholipids and lysophospholipids in control AQH and TM that are either absent or present in very low levels in glaucomatous samples. We identified different phospholipid species in AQH and TM using shotgun lipidomics. RESULTS The trabecular meshwork cells make up the Schlemm’s canal, dealing with the regulation of intraocular pressure. Mutations in the trabecular meshwork tissue create obstructions in the flow of aqueous humor, thus leading to the increase in IOP and subsequent development of peripheral blindness in the form of glaucoma. Topical prostaglandin analogs of the FP class have shown to be most affective in reducing IOP and open angle-glaucoma (Ansari, Davis, Kaddour-Djebbar, & Abdel-Latif, 2003). However, the actual physiological mechanism has yet to be understood. (Ansari et al., 2003).Ansari, Davis, Kaddour-Djebbar, & Abdel-Latif, 2003Ansari et al., 2003 One possible mechanism of IOP lowering caused by prostaglandin F(2alpha) or PGF(2alpha) is its involvement with myosin light kinase (MLC kinase). However, the end result showed the increase in inositol phosphate production, or more commonly known as phosphatidylinositol (PI), therefore prostaglandin analogs have a very high potential to change the amount of phospholipid of all types: phosphatidycholine (PC), phosphatidyserine (PS), and phosphatidyethanolamine (PE). A lipid profile of TM tissue will enable a plethora of other testable experiments and which may even lead to the reversal of the metabolic changes, not a simple prolongation. Changes in the lipid profile or glaucomatous TM tissue versus normal tissue can be quantified to see which lipids are in excess and which have been reduced. Prostaglandin analogs of the FP class are now able to be studied closer as to how each topical medication changes the lipid profile of the TM tissue, perhaps leading to improvements in treatment of glaucoma. On the same trend, the TM tissue is bathed in aqueous humor (AH), a clear gel like solution laying in between the lens and the cornea. It is the obstruction of AH outflow that leads to an increase in IOP. The same prostaglandin analogs have been shown to change the lipid concentration in AH exposed to these PGF2(alpha) drugs. The administration of these drugs led to a decrease in IOP, as was known, along with a change in the lipid profile (Faulkner et al., 2010). A phospholipid analysis of both TM and AH in glaucomatous and normal tissue will allow for a baseline to be established for future studies.Faulkner et al., 2010 Table 1. Summary of tandem mass spectrometric conditions for lipid analyses Name of the lipid standardParent MassLipid ClassScan TypeIon Mode Daughter Mass (m/z) CE (V) 1,2-ditridecanoyl-sn-glycero-3-phosphocholine649.89Phosphocholine (PC)PIS+18435 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine744.04Phosphoethanolamine (PE)PIS-19650 1-heptadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn- glycero-3-phospho-(1'-myo-inositol) 871.6Phosphoinositol (PI)PIS-24145 1,2-dioleoyl-sn-glycero-3-phospho-L-serine810.03Phosphoserine (PS)NLS-87.124 MATERIALS AND METHODS Table 1 Class of lipid species along with corresponding shotgun lipid scan type previously established for phospholipid analysis(Yang, Cheng, Gross, & Han, 2009)Yang, Cheng, Gross, & Han, 2009 Figure 1 TSQ Quantum Access Max triple quadrupole mass spectrometric instrument. Electrospray ionization (ESI) analysis used for phospholipid analysis with direct syringe infusion injection as preferred method of analysis. Device shown with sheath gas nitrogen and auxiliary gas Argon. Samples are suspended in 750 ul of Acetonitrile: Isopropanol (1:1) and scanned for one minute from range of 200m/z – 1000 m/z. A B NTM - PC GTM - PC Figure 2. Electrospray ionization mass spectrometric analysis of phosphocholines (PC) class of lipids in human trabecular meshwork (TM) in positive-ion mode. (A) and (B) are representative analysis of human glaucomatous and normal TM as indicated with internal standard was used for ratiometric quantification. Parent ion scan (see Table 1) was performed for m/z 200-1000. Arrow shows standard at 650.5 m/z PS NTM PS GTM A B Figure 3. Electrospray ionization mass spectrometric analysis of phosphoserines (PS) class of lipids in human trabecular meshwork (TM) in negative-ion mode. (A) and (B) are representative analysis of human glaucomatous and normal TM as indicated with internal standard was used for ratiometric quantification. Neutral loss scan (see Table 1) was performed for m/z 200-1000. Arrow shows standard at 787.0 m/z PC GAH A B PC NAH Figure 4. Electrospray ionization mass spectrometric analysis of phosphocholines (PC) class of lipids in human aqueous humor (AH) in positive-ion mode. (A) and (B) are representative analysis of human glaucomatous and normal AH as indicated with internal standard was used for ratiometric quantification. Parent ion scan (see Table 1) was performed for m/z 600-900. Arrow shows standard at 650.5 m/z Table 1: Unique lipid species present only in the control Trabecular meshwork Lipid Species* m/z** PC(12:0/13:0) 635.45 PC(13:0/20:4(5Z,8Z,11Z,14Z)) 739.52 PC(15:0/18:2(9Z,12Z)) 743.55 PC(16:0/20:5(5Z,8Z,11Z,14Z,17Z)) 779.55 PC(16:1(9Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 803.55 PC(17:0/18:1(9Z)) 773.59 PC(17:1(10Z)/0:0) 507.33 PC(18:1(11Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 831.58 PC(18:3(9Z,12Z,15Z)/0:0) 517.32 PC(18:4(9E,11E,13E,15E)/0:0) 515.3 PC(20:3(8Z,11Z,14Z)/0:0) 545.35 PC(21:4(6Z,9Z,12Z,15Z)/0:0) 557.35 PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 877.56 PC(25:0/18:0) 887.73 PC(O-12:0/O-12:0) 593.48 PC(P-15:0/0:0) 465.32 *The lipid species identification is based on Lipidmaps database, used as a *.csv file for bioinformatic analyses with MZmine 2.2 program. ** A representative mass/charge ratio is presented (variations in m/z was reconciled by MZmine 2.2) Table 2: Unique lipid species present only in the Glaucomatous Trabecular meshwork Lipid Species* m/z** PC(10:0/18:2(9Z,12Z))673.47 PC(O-1:0/16:0)509.35 PC(O-14:0/16:0)691.55 PC(O-14:0/18:2(9Z,12Z))715.55 PC(O-16:0/15:1(9Z))703.55 PC(O-16:0/22:5(7Z,10Z,13Z,16Z,19Z))793.6 Unknown1882.6 Unknown2600.0 *The lipid species identification is based on Lipidmaps database, used as a *.csv file for bioinformatic analyses with MZmine 2.2 program. ** A representative mass/charge ratio is presented (variations in m/z was reconciled by MZmine 2.2)