Determination of total plasma oxysterols by enzymatic hydrolysis, solid phase extraction and liquid chromatography coupled to mass-spectrometry

Mendiara, Isabel (Universidad de Zaragoza) ; Domeño, Celia (Universidad de Zaragoza) ; Nerín, Cristina (Universidad de Zaragoza) ; Geurts, Aron M. ; Osada, Jesús (Universidad de Zaragoza) ; Martínez-Beamonte, Roberto (Universidad de Zaragoza)

Resumen: 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 4ß-, 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.

Idioma: Inglés

DOI: 10.1016/j.jpba.2017.12.033

Año: 2018

Publicado en: Journal of Pharmaceutical and Biomedical Analysis 150 (2018), 396-405
ISSN: 0731-7085

Factor impacto JCR: 2.983 (2018)

Categ. JCR: PHARMACOLOGY & PHARMACY rank: 105 / 266 = 0.395 (2018) - Q2 - T2
Categ. JCR: CHEMISTRY, ANALYTICAL rank: 24 / 84 = 0.286 (2018) - Q2 - T1

Factor impacto SCIMAGO: 0.786 - Analytical Chemistry (Q1) - Clinical Biochemistry (Q1) - Spectroscopy (Q1) - Pharmaceutical Science (Q1) - Drug Discovery (Q1)

Financiación: info:eu-repo/grantAgreement/ES/DGA/T10
Financiación: info:eu-repo/grantAgreement/ES/ISCIII/CB06-03-1012
Financiación: info:eu-repo/grantAgreement/ES/MINECO/SAF2013-41651-R
Financiación: info:eu-repo/grantAgreement/ES/MINECO/SAF2016-75441-R

Tipo y forma: Article (PostPrint)

Área (Departamento): Área Química Analítica (Dpto. Química Analítica)
Área (Departamento): Área Nutrición Bromatología (Dpto. Produc.Animal Cienc.Ali.)
Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)
Área (Departamento): Área Tecnología de Alimentos (Dpto. Produc.Animal Cienc.Ali.)