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Simultaneous evaluation of HMG-CoA reductase and cholesterol 7α-hydroxylase activities by electrospray tandem MS

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Lipids

Abstract

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.

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Abbreviations

CN:

cyanopropyl

DMES:

dimethylethylsilyl

DMESI:

DMES imidazole

ES-MS-MS:

electrospray tandem mass spectrometry

MVA:

mevalonic acid

MVL:

mevalonolactone

6β-OHC:

6β-hydroxy-cholestanol

7α-OHC:

7α-hydroxycholesterol

SIM:

selected ion monitoring

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Authors and Affiliations

  1. Centre de Recherche de l'Hôpital Sainte-Justine, H3T 1C5, Montréal, Canada

    Marie-Yvonne Ndong-Akoume, Diane Mignault, Shahid Perwaiz & Ibrahim M. Yousef

  2. Département de Pharmacologie, Université de Montréal, Succ. Centre-ville, C.P. 6128, H3C 3J7, Montréal, Québec, Canada

    Marie-Yvonne Ndong-Akoume, Diane Mignault, Shahid Perwaiz, Gabriel L. Plaa & Ibrahim M. Yousef

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  1. Marie-Yvonne Ndong-Akoume
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  2. Diane Mignault
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  3. Shahid Perwaiz
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  4. Gabriel L. Plaa
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  5. Ibrahim M. Yousef
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Correspondence to Ibrahim M. Yousef.

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Ndong-Akoume, MY., Mignault, D., Perwaiz, S. et al. Simultaneous evaluation of HMG-CoA reductase and cholesterol 7α-hydroxylase activities by electrospray tandem MS. Lipids 37, 1101–1107 (2002). https://doi.org/10.1007/s11745-002-1006-z

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  • DOI: https://doi.org/10.1007/s11745-002-1006-z

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