Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS

Akira Honda, Yasushi Matsuzaki, Yuji Mizokami, Tadashi Ikegami, Mikio Doy, and Hiroshi Miyazaki

Ibaraki Prefectural Institute of Public Health, Mito, Ibaraki 310-0852

Corresponding Author: AkiraHonda{at}aol.com

We have developed a new sensitive and specific non-radioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. The present method was based upon a stable isotope-dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (S/N = 3), which was approximately 833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by the present method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77 – 2.31 nmol/mg protein of MVL and were validated by polynomial equation. The results showed that the estimated concentration within a 95% confidence limit was 0.47 ± 0.07 nmol/mg protein, which coincided completely with the observed X₀ ± SD = 0.47 ± 0.01 nmol/mg protein with a mean recovery of 94.6%. The present method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to the conventional RI method.

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