title: A highly sensitive LC-ESI-MS/MS method for the quantification of cholesterol ozonolysis products secosterol-A and secosterol-B after derivatization with 2-hydrazino-1-methylpyridine.
abstract: Cholesterol ozonolysis products, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) have been found in atherosclerosis plaques and the brain tissues of Alzheimer's disease patients, implicating them in the pathogenesis of cardiovascular and neurodegenerative diseases. We have recently reported that when cholesterol is oxidized with an ozone-like oxidant generated by activated mouse neutrophils, secosterol-A is generated which is then converted to secosterol-B by an aldol reaction. To investigate further pathophysiological roles of secosterols, we have developed a highly sensitive method to detect secosterol-A and -B as derivatives with 2-hydrazino-1-methylpyridine (HMP) by LC-ESI-MS/MS. The limits of detection for the HMP derivatives of secosterol-A and secosterol-B were 0.05 and 0.01fmol, respectively, which were approximately 400 and 2000 times better than those for underivatized secosterol-A and -B. We also developed a highly reproducible and accurate method to extract, purify and derivatize secosterol in small volumes of biological specimens. Using this method, we determined the levels of secosterol-A and -B as 1.4 ± 0.7 and 4.3 ± 0.8 nM, respectively, in the plasma of normal C57BL/6 mice, and in the range of 10.4 ± 16.3 to 40.7 ± 20.1 pmol/g and 110.9 ± 10.6 to 161.5 ± 56.3 pmol/g, respectively, in the brain, liver and lung tissues.
authors.affiliation: Laboratory of Biochemistry, Graduate School of Nutritional and Environmental Sciences, and Global COE Program at University of Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Tomono
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Miyoshi
authors.forename: Megumi
authors.initials: M
authors.lastname: Ito
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ohshima
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000031:analogs & derivatives / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D006834:Hydrazines / Q000737:chemistry*; D016014:Linear Models; D013058:Mass Spectrometry; D051379:Mice; D008810:Mice, Inbred C57BL; D010126:Ozone / Q000737:chemistry*; D011725:Pyridines / Q000737:chemistry*; D015203:Reproducibility of Results; D012632:Secosteroids / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014018:Tissue Distribution
chemical_list: C506217:2-hydrazino-1-methylpyridine; D006834:Hydrazines; D011725:Pyridines; D012632:Secosteroids; D010126:Ozone; D002784:Cholesterol