Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS.
We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This 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 (signal-to-noise ratio=3), 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 this 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 MVL/mg protein and were validated by a polynomial equation. These 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 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.
Honda
Akira
A
Department of Internal Medicine, Tokyo Medical University, Kasumigaura Hospital, Ami, Ibaraki 300-0395, Japan. akirahonda-gi@umin.ac.jp
Mizokami
Yuji
Y
Matsuzaki
Yasushi
Y
Ikegami
Tadashi
T
Doy
Mikio
M
Miyazaki
Hiroshi
H
D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000032:analysis* / Q000378:metabolism*; D008297:Male; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
C015367:mevalonolactone; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid