Simultaneous evaluation of HMG-CoA reductase and cholesterol 7alpha-hydroxylase activities by electrospray tandem MS.
Simultaneous evaluation of HMG-CoA reductase and cholesterol 7alpha-hydroxylase activities by electrospray tandem mass spectrometry (ES-MS-MS) was performed. The assay was based on the measurement of mevalonolactone (MVL) and 7alpha-hydroxycholesterol (7alpha-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 7alpha-OHC were analyzed, without derivatization, by ES-MS-MS. The analysis was achieved in 5 min. Calibration curves were made for MVL and 7alpha-OHC, and were linear from 0 to 100 microg. 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 7alpha-hydroxylase activities in liver microsomal preparations.
Ndong-Akoume
Marie-Yvonne
MY
Département de Pharmacologie, Université de Montréal, Montréal, Québec, Canada H3C 3J7.
Mignault
Diane
D
Perwaiz
Shahid
S
Plaa
Gabriel L
GL
Yousef
Ibrahim M
IM
D000818:Animals; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D015394:Molecular Structure; D051381:Rats; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D006903:Hydroxymethylglutaryl CoA Reductases; D002790:Cholesterol 7-alpha-Hydroxylase