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

Journal: Journal of pharmaceutical and biomedical analysis
PMID: 29288965

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.

Authors

  • Isabel Mendiara
    Departmento de Química Analítica, Universidad de Zaragoza, Spain; Instituto Agroalimentario de Aragón, CITA-Universidad de Zaragoza, Spain.
  • Celia Domeño
    Departmento de Química Analítica, Universidad de Zaragoza, Spain.
  • Cristina Nerín
    Departmento de Química Analítica, Universidad de Zaragoza, Spain.
  • Aron M Geurts
    Human Molecular Genetics Center and Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
  • Jesús Osada
    Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto de Investigación Sanitaria de Aragón-Universidad de Zaragoza, Spain; Instituto Agroalimentario de Aragón, CITA-Universidad de Zaragoza, Spain; CIBER de Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Spain. Electronic address: Josada@unizar.es.
  • Roberto Martínez-Beamonte
    Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Instituto de Investigación Sanitaria de Aragón-Universidad de Zaragoza, Spain; Instituto Agroalimentario de Aragón, CITA-Universidad de Zaragoza, Spain; CIBER de Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Spain.

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