Analyses of Cholesterol Metabolites of Optic Nerve Using GC-MS Methods

Sarohia, Gurkaran S.; Garza, Erik D; Bhattacharya, Sanjoy K.

doi:10.1007/978-1-4939-9488-5_5

Cited by 4 publications

(2 citation statements)

References 8 publications

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“…A modified Bligh and Dyer method was used to extract the lipids, which was described in our prior reports. 28 Methanol and chloroform were added in a 2:1 ratio to each sample. Samples were vortexed for 2 minutes followed by 2 minutes of sonication in an ultrasonic bath.…”

Section: Lipid and Protein Extractionmentioning

confidence: 99%

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves

Milbeck

Bhattacharya

2020

Invest. Ophthalmol. Vis. Sci.

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To determine whether lysophospholipid (LPL) profiles and corresponding conversion enzymes in the LPL pathways are altered in the optic nerve (ON) between human control and glaucoma samples. METHODS. Lipids extracted from control (n = 11) and glaucomatous (n = 12) ON samples using the Bligh and Dyer method were subjected to high-resolution mass spectrometry on a Q-exactive mass spectrometer coupled with a high-performance liquid chromatography (Accela 600) system. Analysis was performed for LPLs (lysophosphatidylcholines, lysophosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylinositols, and lysosphingomyelines) using LipidSearch v.4.1, MZmine v.2.0, and MetaboAnalyst v.4.0. LPL synthesis and degradation pathway maps, utilizing UniProt and BRENDA database entries as needed, were created using Kyoto Encyclopedia of Genes and Genomes (KEGG)-based tools. The mRNA expression level in normal and glaucomatous human ON were analyzed using Gene Expression Omnibus (GEO) entry GSE45570. Protein amounts were determined using PHAST gel and dot blot and were used for normalization of protein amounts across samples. Western blot, ELISA, and protein quantification were performed using established protocols. RESULTS. Principal component analysis of ON LPL profile placed control and glaucomatous ONs in two distinct separate groups. Mass spectrometric analysis of ON revealed decrease in lysophosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylcholine, and significant increase in diacylglycerol in glaucomatous ON. Statistical analysis of LPL conversion enzymes revealed significant overexpression of phosphatidate phosphatase LPIN2, phospholipid phosphatase 3, phosphatidylcholine-sterol acyltransferase, and calcium-dependent phospholipase 2, and significant downregulation of glycerol-3-phosphate acyltransferase 4 at mRNA level in glaucomatous ON. Western blot and ELISA confirmed proteomic differences between normal and diseased ON. CONCLUSIONS. Our analysis revealed alterations in specific LPL levels and corresponding select enzyme-level changes in glaucomatous ON.

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Section: Lipid and Protein Extractionmentioning

confidence: 99%

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves

Milbeck

Bhattacharya

2020

Invest. Ophthalmol. Vis. Sci.

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“…Briefly, twenty white prepupal-stage animals were collected and total cholesterol was extracted with 200 μL of chloroform: isopropanol: IGEPAL CA-630 (7:11:0.1) using a microhomogenizer. Then, total cholesterol was measured via GC-MS according to a previously published GC-MS method for the quantification of free cholesterol [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

Sterol Regulation of Development and 20-Hydroxyecdysone Biosynthetic and Signaling Genes in Drosophila melanogaster

Wen

Chen

Wen

et al. 2023

Cells

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Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit fly Drosophila melanogaster, both C27 and C28 ecdysteroids have been identified. While the biosynthetic pathway of the C27 ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C28 ecdysteroids from C28 or C29 dietary sterols remains unknown. In this study, we found that different dietary sterols (including the C27 sterols cholesterol and 7-dehydrocholesterol, the C28 sterols brassicasterol, campesterol, and ergosterol, and the C29 sterols β-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression in D. melanogaster Kc cells. We also found that a single dietary sterol was sufficient to support D. melanogaster growth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, our study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C27 ecdysteroid 20E biosynthetic pathway also participated in the conversion of C28 and C29 dietary sterols into C28 ecdysteroids.

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Advances in Chromatographic and Mass Spectrometric Techniques for Analyzing Reducing Monosaccharides and Their Phosphates in Biological Samples

Chen,

Lou,

Liu

et al. 2024

Critical Reviews in Analytical Chemistry

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Analyses of Cholesterol Metabolites of Optic Nerve Using GC-MS Methods

Cited by 4 publications

References 8 publications

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves

Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves

Sterol Regulation of Development and 20-Hydroxyecdysone Biosynthetic and Signaling Genes in Drosophila melanogaster

Advances in Chromatographic and Mass Spectrometric Techniques for Analyzing Reducing Monosaccharides and Their Phosphates in Biological Samples

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