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Simultaneous evaluation of HMG‐CoA reductase and cholesterol 7α‐hydroxylase activities by electrospray tandem MS
Author(s) -
NdongAkoume MarieYvonne,
Mignault Diane,
Perwaiz Shahid,
Plaa Gabriel L.,
Yousef Ibrahim M.
Publication year - 2002
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-002-1006-z
Subject(s) - chromatography , chemistry , reductase , derivatization , electrospray , microsome , hmg coa reductase , tandem mass spectrometry , clinical chemistry , lipidology , biochemistry , enzyme , mass spectrometry
Simultaneous evaluation of HMG‐CoA reductase and cholesterol 7α‐hydroxylase activities by electrospray tandem mass spectrometry (ES‐MS‐MS) was performed. The assay was based on the measurement of mevalonolactone (MVL) and 7α‐hydroxycholesterol (7α‐OHC) produced by the incubation of HMG‐CoA with hepatic microsomes in the presence of NADPH and glucose‐6‐phosphate dehydrogenase. Following extraction and purification using a cyanopropyl cartridge, MVL and 7α‐OHC were analyzed, without derivatization, by ES‐MS‐MS. The analysis was achieved in 5 min. Calibration curves were made for MVL and 7α‐OHC, and were linear from 0 to 100 μg. The recovery was >97%. The procedure was validated under similar calibration and recovery experiments, by measuring the above mentioned products as dimethylethylsilyl ether derivatives using the classical technique of GC‐MS. Data obtained by ES‐MS‐MS and GC‐MS showed a good correlation, with no significant differences. ES‐MS‐MS is a simple and reliable method for the evaluation of HMG‐CoA reductase and cholesterol 7α‐hydroxylase activities in liver microsomal preparations.