Faculty Collaboration Database

Determination of total plasma oxysterols by enzymatic hydrolysis, solid phase extraction and liquid chromatography coupled to mass-spectrometry. J Pharm Biomed Anal 2018 Feb 20;150:396-405

Date

12/31/2017

Pubmed ID

29288965

DOI

10.1016/j.jpba.2017.12.033

Scopus ID

2-s2.0-85039461303 (requires institutional sign-in at Scopus site)   5 Citations

Abstract

The potential use of cholesterol esterases was tested to avoid alkaline hydrolysis for cleavage of plasma esterified oxysterols. The enzymatic hydrolysis was optimized by testing two sources of enzyme-Pseudomonas and bovine pancreas, presence of surfactants, incubation time and amount of enzyme. Free forms of 4β-, 7-, 24-, 25- and 27-hydroxycholesterol (HC) as well 7-ketocholesterol (7-KC) were analyzed by liquid chromatography and mass-spectrometry using the deuterated internal standard, 25-HC(d6). Enzymatic hydrolysis was more effective using the Pseudomonas enzyme and in presence of surfactants. Compared to alkaline hydrolysis, it generated a cleaner chromatographic baseline and better recovery of the internal standard. Oxysterols were assayed with detection limits between 7 and 31 pg/mL. Interassay coefficients of variation were lower than 10% and extraction recovery efficiencies, higher than 90%. The procedure was used to characterize plasma levels of Cyp7b1-deficient rat, where it showed increased plasma levels of 7, 24 and 25-HC. Due to the low volume of sample required, it may be used in other animal models, particularly rodents, as well as in pediatric samples where sample amount is always a problem. Thus, the proposed new method offers mild enzymatic processing that greatly facilitates oxysterol determinations to delineate their role in physiopathology.

Author List

Mendiara I, Domeño C, Nerín C, Geurts AM, Osada J, Martínez-Beamonte R

Author

Aron Geurts PhD Professor in the Physiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Animals, Genetically Modified
Bacterial Proteins
Calibration
Chromatography, Liquid
Cytochrome P450 Family 7
Humans
Hydrolysis
Male
Oxysterols
Pseudomonas aeruginosa
Rats, Inbred F344
Reference Standards
Reproducibility of Results
Solid Phase Extraction
Steroid Hydroxylases
Sterol Esterase
Tandem Mass Spectrometry