“…Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich Co. HPLC-grade solvents (acetonitrile, dichloromethane, hexanes, and 2-propanol) were purchased from Thermo Fisher Scientifi c, Inc. Syntheses of [25,26,26,26,27,27,27 -d 7 ]7-DHC, 4 ␣ -hydroxy-7-DHC, 4  -hydroxy-7-DHC, 7-ketocholesterol, and d 7 -7-ketocholesterol have been described elsewhere ( 13,17 ). …”
Section: Methodsmentioning
confidence: 99%
“…The hydrolyzed mixture was directly extracted with hexanes (3 ml × 3), and the combined organic layers were dried under nitrogen, reconstituted in methylene chloride (200 l), and stored at Ϫ 80°C until analysis. Oxysterols in all samples were analyzed by normal-phase HPLC-APCI-MS/MS following the previously reported method with a slightly modifi ed HPLC condition ( 13,17 ). HPLC conditions: silica 150 × 4.6 mm column (Phenomenex, Inc.), 3 m; 1.0 ml/min; elution solvent: 10% 2-propanol in hexanes.…”
Section: Analysis Of Oxysterols By Hplc-ms/msmentioning
“…Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich Co. HPLC-grade solvents (acetonitrile, dichloromethane, hexanes, and 2-propanol) were purchased from Thermo Fisher Scientifi c, Inc. Syntheses of [25,26,26,26,27,27,27 -d 7 ]7-DHC, 4 ␣ -hydroxy-7-DHC, 4  -hydroxy-7-DHC, 7-ketocholesterol, and d 7 -7-ketocholesterol have been described elsewhere ( 13,17 ). …”
Section: Methodsmentioning
confidence: 99%
“…The hydrolyzed mixture was directly extracted with hexanes (3 ml × 3), and the combined organic layers were dried under nitrogen, reconstituted in methylene chloride (200 l), and stored at Ϫ 80°C until analysis. Oxysterols in all samples were analyzed by normal-phase HPLC-APCI-MS/MS following the previously reported method with a slightly modifi ed HPLC condition ( 13,17 ). HPLC conditions: silica 150 × 4.6 mm column (Phenomenex, Inc.), 3 m; 1.0 ml/min; elution solvent: 10% 2-propanol in hexanes.…”
Section: Analysis Of Oxysterols By Hplc-ms/msmentioning
“…1), 7-ketocholesterol, 7-ketocholestanol, and 7␣-hydroxycholesterol were purchased from Steraloids Inc. (Newport, RI). [25,26,26,26,27,27,27-d 7 ]-7-Dehydrocholesterol was synthesized as described previously (13). [25,26,26,26,27,27,27-d 7 ]-7-Ketocholesterol was purchased from Toronto Research Chemicals (North York, Ontario, Canada).…”
Section: Methodsmentioning
confidence: 99%
“…Some humans are deficient in 7-dehydrocholesterol reductase (DHCR7) and exhibit Smith-Lemli-Opitz syndrome (SLOS), 2 with elevated levels of 7-dehydrocholesterol and attenuated levels of cholesterol (3,(7)(8)(9)(10). 7-Dehydrocholesterol is exceptionally susceptible to free radical oxidation (11), and more than one dozen biologically active oxysterol products have been characterized from 7-dehydrocholesterol oxidation in solution (12)(13)(14). In particular, we have been interested in the enzymatic conversion of 7-dehydrocholesterol to oxysterols.…”
“…In the context of SLOS, a mixture of 7-DHC-derived oxysterols induced changes in gene expression in control Neuro2a cells that are similar to those identifi ed in Dhcr7-defi cient Neuro2a cells ( 44 ). DHCEp is a major product of 7-DHC peroxidation and can metabolize to another oxysterol, DHCEO, a biomarker for 7-DHC oxidation that can affect neural development in a SLOS model mouse ( 16,45 ). Because some of the 7-DHCderived oxysterols are electrophilic, such as DHCEp, it seems reasonable to consider covalent modifi cation of proteins as a contributing factor in the pathogenesis of SLOS.…”
Section: Protein Adduction Increases In the Cellular Slos Modelmentioning
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