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Pseudoexfoliation aqueous humor lipidome suggests enrichment of specific pathways

ABSTRACT: Pseudoexfoliation syndrome (PEX) is systemic disorder that manifests as white, fluffy, proteinaceous fibrillar material throughout the body. In the eye such deposits result in Pseudoexfoliation glaucoma (PEXG), due to impeding aqueous humor outflow. Serum lipid alterations and increased lipid peroxidation have been reported in PEX. We report first ever comprehensive lipid profiling of the aqueous humor (AH) of PEXG. Our untargeted lipidomic analysis of 23 non-glaucomatous control, 19 primary open angle glaucoma, 9 PEX, and 14 PEXG AH with 13 deuterated lipid internal standards for normalization among the lipid classes resulted in the combined identification of 489 lipid species within 26 lipid classes across PEX, PEXG, POAG, and control AH. The mean total lipid content in the AH across samples showed that control AH (mean peak area 13.54 ± 56.1) had, on average, greater total lipid content than PEX (4.21 ± 10.90), PEXG (9.08 ± 25.97), and POAG (5.66 ± 15.75) samples. Multiple cholesterol esters (ChE), phosphatidylcholines (PC), triglycerides (TG), and ceramides (Cer) were present in higher concentrations for the PEXG AH samples. Some of the lipids found in high concentrations in the PEXG samples are ChE(16:0), ChE(20:3), ChE(18:1), ChE(18:3), ChE(22:6), ChE(18:2), ChE(20:4), PC(16:0/16:0), PC(16:0/18:2), TG(18:1/18:1/20:4), and Cer(t18:0/24:0). The CerG2GNAc1(d34:1) was enriched in control samples and depleted both in PEX and PEXG samples. The PC (18:0/18:2), PC (36:2), and PC (34:1e) are in low concentrations for PEX but highly concentrated in PEXG, despite both having similar material deposits, suggesting they are fundamentally different in composition. Elevations in Apolipoprotein A-I (APOA1) correlated to increase abundance of PC lipid species in the AH of patients with PEXG. Machine learning prediction with three supervised logistic regression binary classification tasks showed 1) POAG vs control, with 86% accuracy 2) PEXG vs control, with 71% accuracy and 3) PEX vs control, with 86% accuracy, respectively.

ORGANISM(S): Human Homo Sapiens

TISSUE(S): Eye Tissue

DISEASE(S): Pseudoexfoliation Syndrome

SUBMITTER: Sanjoy K. Bhattacharya

PROVIDER: ST001936 | MetabolomicsWorkbench | Thu Sep 16 00:00:00 BST 2021

REPOSITORIES: MetabolomicsWorkbench

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Project description:Serum samples were randomized and extracted using a modified version of the previously-published Folch procedure, as applied recently [20]. The maternal samples were analysed as one batch and the cord blood samples as a second batch. In short, 10 µL of 0.9% NaCl and, 120 µL of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was added to 10 µL of each serum sample. The standard solution contained the following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower layer of each sample was then transferred to a glass vial with an insert and 60 µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored at -80 °C until analysis. Calibration curves using 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(16:0e/18:1(9Z))), 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol (DG(18:0/18:2)), 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used in this work was a 1290 Infinity II system from Agilent Technologies (Santa Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 °C), a quaternary solvent manager and a column thermostat (maintained at 50 °C). Injection volume was 1 µL and the separations were performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 QTOF from Agilent Technologies interfaced with a dual jet stream electrospray (Ddual ESI) ion source. All analyses were performed in positive ion mode and MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. Quality control was performed throughout the dataset by including blanks, pure standard samples, extracted standard samples and control serum samples, including in-house serum and a pooled QC with an aliquot of each sample was collected and pooled and used as quality control sample. Relative standard deviations (% RSDs) for identified lipids in the control serum samples (n = 13) and in the pooled serum samples (n = 54) were on average 22.4% and 17.5%, respectively.

Project description:INTRODUCTION: Differences in the metabolite profiles between serum and plasma are incompletely understood. OBJECTIVES: To evaluate metabolic profile differences between serum and plasma and among plasma sample subtypes. METHODS: We analyzed serum, platelet rich plasma (PRP), platelet poor plasma (PPP), and platelet free plasma (PFP), collected from 8 non-fasting apparently healthy women, using untargeted standard 1D and CPMG 1H NMR and reverse phase and hydrophilic (HILIC) UPLC-MS. Differences between metabolic profiles were evaluated using validated principal component and orthogonal partial least squares discriminant analysis. RESULTS: Explorative analysis showed the main source of variation among samples was due to inter-individual differences with no grouping by sample type. After correcting for inter-individual differences, lipoproteins, lipids in VLDL/LDL, lactate, glutamine, and glucose were found to discriminate serum from plasma in NMR analyses. In UPLC-MS analyses, lysophosphatidylethanolamine (lysoPE)(18:0) and lysophosphatidic acid(20:0) were higher in serum, and phosphatidylcholines (PC)(16:1/18:2, 20:3/18:0, O-20:0/22:4), lysoPC(16:0), PE(O-18:2/20:4), sphingomyelin(18:0/22:0), and linoleic acid were lower. In plasma subtype analyses, isoleucine, leucine, valine, phenylalanine, glutamate, and pyruvate were higher among PRP samples compared with PPP and PFP by NMR while lipids in VLDL/LDL, citrate, and glutamine were lower. By UPLC-MS, PE(18:0/18:2) and PC(P-16:0/20:4) were higher in PRP compared with PFP samples. CONCLUSIONS: Correction for inter-individual variation was required to detect metabolite differences between serum and plasma. Our results suggest the potential importance of inter-individual effects and sample type on the results from serum and plasma metabolic phenotyping studies. NMR protocols and data are reported in the current study MTBLS470. UPLC-MS protocols and data associated to this study are reported in MTBLS465. Linked Studies: <a href='https://www.ebi.ac.uk/metabolights/MTBLS465' target='_blank'>MTBLS465</a>

Project description:INTRODUCTION: Differences in the metabolite profiles between serum and plasma are incompletely understood. OBJECTIVES: To evaluate metabolic profile differences between serum and plasma and among plasma sample subtypes. METHODS: We analyzed serum, platelet rich plasma (PRP), platelet poor plasma (PPP), and platelet free plasma (PFP), collected from 8 non-fasting apparently healthy women, using untargeted standard 1D and CPMG 1H NMR and reverse phase and hydrophilic (HILIC) UPLC-MS. Differences between metabolic profiles were evaluated using validated principal component and orthogonal partial least squares discriminant analysis. RESULTS: Explorative analysis showed the main source of variation among samples was due to inter-individual differences with no grouping by sample type. After correcting for inter-individual differences, lipoproteins, lipids in VLDL/LDL, lactate, glutamine, and glucose were found to discriminate serum from plasma in NMR analyses. In UPLC-MS analyses, lysophosphatidylethanolamine (lysoPE)(18:0) and lysophosphatidic acid(20:0) were higher in serum, and phosphatidylcholines (PC)(16:1/18:2, 20:3/18:0, O-20:0/22:4), lysoPC(16:0), PE(O-18:2/20:4), sphingomyelin(18:0/22:0), and linoleic acid were lower. In plasma subtype analyses, isoleucine, leucine, valine, phenylalanine, glutamate, and pyruvate were higher among PRP samples compared with PPP and PFP by NMR while lipids in VLDL/LDL, citrate, and glutamine were lower. By UPLC-MS, PE(18:0/18:2) and PC(P-16:0/20:4) were higher in PRP compared with PFP samples. CONCLUSIONS: Correction for inter-individual variation was required to detect metabolite differences between serum and plasma. Our results suggest the potential importance of inter-individual effects and sample type on the results from serum and plasma metabolic phenotyping studies. UPLC-MS protocols and data are reported in the current study MTBLS465. NMR protocols and data associated to this study are reported in MTBLS470. Linked Studies: <a href='https://www.ebi.ac.uk/metabolights/MTBLS470' target='_blank'>MTBLS470</a>

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Pseudoexfoliation aqueous humor lipidome suggests enrichment of specific pathways

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