Identification of intermediates after inhibition of cholesterol synthesis by aminotriazole treatment in vivo

Abstract

Cholesterol synthesis from mevalonate is inhibited by aminotriazole treatment in vivo. We tried to identify intermediates accumulated in liver of aminotriazole-treated rats. At 6 h after the aminotriazole treatment, the liver was excised. Sterols were extracted from it, and subjected to capillary gas-liquid chromatography, high-performance liquid chromatography, gas-liquid chromatography linked to mass spectrometry and gas-liquid chromatography linked to Fourier-transform infrared spectrometry. It was found that 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest- 8-en-3 beta-ol were accumulated in the liver, mainly as the free forms. The contents of the former and the latter were increased to 25- and 64-times the control values, respectively. In another experiment, [2-13C]mevalonate was injected at 2 h after aminotriazole treatment, and 4 h later the liver was excised. The sterols extracted from the liver were subjected to gas-liquid chromatography linked to mass spectrometry. Specific fragment ions reflecting the incorporation of [13C] mevalonate were detected in the mass spectra of the intermediate sterols. Accumulation of 4 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol and 4,4-dimethyl-5 alpha-cholest-8-en-3 beta-ol after aminotriazole treatment suggests that elimination of the 4 alpha-methyl group from 4-methyl intermediate sterols is inhibited by aminotriazole.