Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

Honda, Akira; Yamashita, Kouwa; Miyazaki, Hiroshi; Shirai, Mutsumi; Ikegami, Tadashi; Xu, Guowang; Numazawa, Mitsuteru; Hara, Takashi; Matsuzaki, Yasushi

doi:10.1194/jlr.d800017-jlr200

Cited by 144 publications

(145 citation statements)

References 41 publications

Supporting

Mentioning

143

Contrasting

Order By: Relevance

“…Alkali-treated lipid extract was dried, resuspended in 1 mL tetrahydrofuran (THF)/methanol/ water (2:1:2, v/v/v) containing 0.1% formic acid, and used for LC-MS/MS analyses of sphingolipids and SG. FS was analyzed by LC-MS/MS after picolinyl esterification according to Honda et al (2008).…”

Section: Preparation Of Sphingolipids and Sterolsmentioning

confidence: 99%

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

et al. 2016

View full text Add to dashboard Cite

Numerous plant defense-related proteins are thought to congregate in plasma membrane microdomains, which consist mainly of sphingolipids and sterols. However, the extent to which microdomains contribute to defense responses in plants is unclear. To elucidate the relationship between microdomains and innate immunity in rice (Oryza sativa), we established lines in which the levels of sphingolipids containing 2-hydroxy fatty acids were decreased by knocking down two genes encoding fatty acid 2-hydroxylases (FAH1 and FAH2) and demonstrated that microdomains were less abundant in these lines. By testing these lines in a pathogen infection assay, we revealed that microdomains play an important role in the resistance to rice blast fungus infection. To illuminate the mechanism by which microdomains regulate immunity, we evaluated changes in protein composition, revealing that microdomains are required for the dynamics of the Rac/ROP small GTPase Rac1 and respiratory burst oxidase homologs (Rbohs) in response to chitin elicitor. Furthermore, FAHs are essential for the production of reactive oxygen species (ROS) after chitin treatment. Together with the observation that RbohB, a defense-related NADPH oxidase that interacts with Rac1, is localized in microdomains, our data indicate that microdomains are required for chitin-induced immunity through ROS signaling mediated by the Rac1-RbohB pathway.

show abstract

Section: Preparation Of Sphingolipids and Sterolsmentioning

confidence: 99%

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Briefly, 50 ng of coprostanol was added to 5 L of serum as an internal standard, and alkaline hydrolysis in 1 N ethanolic KOH and derivatization to the picolinyl esters were carried out 14) . Then the picolinyl ester derivatives of the sterols were quantified by LC-MS/MS 15) . Coprostanol, a compound almost nonexistent in normal human serum, was used as an internal standard, because coprostanol and the sterols of interest to us should be lost to the same extent during the quantification process due to their structural similarity.…”

Section: Determination Of Sterol Concentrationsmentioning

confidence: 99%

A SNP of NPC1L1 Affects Cholesterol Absorption in Japanese

Maeda

Honda

Ishikawa

et al. 2010

JAT

Self Cite

View full text Add to dashboard Cite

Aim: Ezetimibe is known to target Niemann-Pick Type C1 Like1 (NPC1L1), a key protein in intestinal cholesterol absorption, and thus to decrease serum LDL-cholesterol (LDL-C) levels. The response of serum LDL-C levels to ezetimibe was reported to differe among NPC1L1 haplotypes. We analyzed NPC1L1 genotypes in Japanese and investigated differences in markers of cholesterol synthesis/absorption among the genotypes. Methods: Blood samples were collected from 42 adult volunteers to measure markers of cholesterol synthesis (lathosterol) and absorption (sitosterol and campesterol) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on a study by Hegele RA et al. in Canada, we selected three SNPs (1735 C G, 25342 A C and 27677 T C (numbers relative to the transcription start site)) and analyzed them using PCR-RFLP.

show abstract

“…In general, ESI is not the best ionization method for neutral steroids because of its poor ionization efficiency. However, our recent study demonstrated that the derivatization of monohydroxysterols into picolinyl esters markedly enhanced the ionization efficiency in the ESI process, and the method was much more sensitive than the assay of native monohydroxysterols by LC-APCI-MS/MS (23). In this study, we have applied our derivatization method to dihydroxy-and epoxysterols.…”

mentioning

confidence: 99%

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

Honda

Yamashita

Hara

et al. 2009

Journal of Lipid Research

Self Cite

177

154

View full text Add to dashboard Cite

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 ml) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4b-hydroxycholesterol, 7a-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio 5 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples. Supplementary key words liquid chromatography-tandem mass spec-Biological samples contain a large number of oxysterols (1), and most of them are formed from cholesterol by enzymatic oxidation (2 -6) ( Fig. 1) or autoxidation (7). By contrast, the oxysterol 24S,25-epoxycholesterol is not derived from cholesterol but is produced de novo from acetyl-CoA via a shunt in the mevalonate pathway (8).These oxysterols are important molecules for preserving lipid homeostasis in the body. 7a-Hydroxycholesterol is a product of CYP7A1, which is the rate-limiting enzyme in the classic bile acid biosynthetic pathway. 27-Hydroxycholesterol, 24S-hydroxycholesterol, 4b-hydroxycholesterol, 22R-hydroxycholesterol, and 24S,25-epoxycholesterol are effective endogenous ligands of the nuclear receptors liver X receptor a (LXRa) and LXRb (9-11). In addition, 27-hydroxycholesterol (12), 25-hydroxycholesterol (13), and 24S,25-epoxycholesterol (14) are known to downregulate the cholesterol biosynthetic pathway, presumably by blocking the processing of the sterol-regulatory element binding protein.GC-MS has historically been used for the analyses of oxysterols in serum and tissues (1, 15) because the sensitivity and specificity of conventional GC with flame ionization detector is not sufficient to quantify oxysterols in biological samples. However, GC-MS is still not an ideal method, especially for the analysis of 24S,25-epoxycholesterol, because this epoxycholesterol does not survive the temperature required for GC analysis (16). Another approach to quantifying oxysterols in biological samples was HPLC with ultraviolet (UV) detection after derivatization to the D 4 -3-ketones (16-19). Thi...

show abstract

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

Cited by 144 publications

References 41 publications

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

A SNP of NPC1L1 Affects Cholesterol Absorption in Japanese

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

Contact Info

Product

Resources

About