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

Abstract

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 chromatographytandem 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 5 3), ?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 6 0.07 nmol/mg protein, which coincided completely with the observed X 0 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.

A novel role for thyroid-stimulating hormone: Up-regulation of hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase expression through the cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-responsive element binding protei

“…15 The method for the measurement of HMGCR activity was carried out as described previously. 16 Determination of Intracellular cAMP and TC. See Supporting Materials and Methods.…”

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS

“…2, the sensitivity of N-ESI was not suffi cient to quantify MA concentrations in a small volume of normal human serum. Recently, we derivatized another organic acid, mevalonate, into mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide and measured it using LC-P-ESI-MS/MS ( 12 ). In this method, mevalonate was lactonized into mevalonolactone and then a tertiary amine moiety was introduced by a characteristic amidation reaction with a primary alkylamine.…”

Thyroid-stimulating hormone decreases HMG-CoA reductase phosphorylation via AMP-activated protein kinase in the liver

“…Hepatocellular microsomes for assay of HMGCR activity were prepared as described by Honda et al ( 23 ). The HepG2 cells stimulated by different concentrations of bTSH (0, 1, and 4 M) were homogenized with a loose-fi tting Tefl on pestle in 4 vol of 3 mM TrisHCl buffer (pH 7.4) containing 0.25 M sucrose, 0.1 mM EDTA, and Subclinical hypothyroidism is a type of thyroid function abnormality; its characteristics are elevated serum thyroidstimulating hormone (TSH) and normal serum thyroid hormone levels, as well as an increased serum cholesterol level ( 10 ).…”