The Effect of Nicotinamide on the Cholesterol Biosynthesis by Cell-free Rat Brain Extract

It has been reported that cell-free extracts of adult rat brain incubated with mevalonic acid-2-14C synthesize radioactive nonsaponifiable materials consisting largely of squalene, and the synthesis of digitonin-precipitable sterols from mevalonic acid is depressed by nicotinamide[1]. The evidence suggested that the cyclization of squalene was inhibited by nicotinamide. BOWEN et al.[2] reported that the cyclization of squalene in yeast was also inhibited by nicotinic acid. Serum cholesterol levels were reduced by nicotinic acid but not nicotinamide[3]. However, the mechanisms of this inhibition are not known. This paper reports the stimulation of formation of squalene from mevalonic acid in cell-free extracts in adult rat brain by nicotinamide. DL-Mevalonic acid-2-14C lactone (specific activity : 5.58 mCi/mM) was obtained from Daiichi Pure Chemicals Co., Ltd., and before use, lactone was converted to the acid by alkali. Squalene-14C (specific activity : 21.2 mCi/mM) used was prepared by rat liver enzyme according to the method of GOODMAN[4]. Male rats of Donryu strain weighing about 250 g were used. The rats were sacrificed by decapitation, and their brains were quickly excised. The brain homogenate and the components of incubation mixture were prepared according to the method of KELLEY et al. [1] except 0.1 μCi of mevalonic acid-14C or 0.05 μCi of squalene-14C as substrate was used. Incubation was carried out for 2 hours at 37°, and the reaction was stopped by the addition of 3.0 ml of water- ethanol (1:1) containing 15% KOH (w/v) and 20 μg each of carrier squalene and cholesterol, after which the mixture was heated for 1 hour at 90°. The mixture was added 6 ml of distilled water and extracted three times each time with 20 ml of petroleum ether. The combined petroleum ether was washed three times, each time with 20 ml of distilled water and evaporated to dryness. The dried nonsaponifiable extract obtained was dissolved in a small volume of benzene, and was applied on a silica gel plate for thin-layer chromatography. n-Hexene-ethyl acetate (9 :1) was used as an ascending solvent. After development, the plate was sprayed with 0.1 % 2, 7'-dichlorofluorescein ethanol solution to detect the carriers. The spots on the plate were determined by ultraviolet absorption. Then, the radioactive areas on the plate were determined using a thin-layer radiochromatogram scanner, and the radioactive products were scraped off and eluted from the gel by three washes with ethyl acetate. The ethyl acetate was transferred into a counting vial and dried under a stream of nitrogen. Radioactivity of each sample was assayed with a liquid scintillation counter. The results are summarized in Tables 1 and 2.The activity of the cell-free extracts of rat brain for the incorporation of mevalonic acid-2-14C into squalene-14C was markedly increased about 7 times by nictinamide. However, nicotinamide had no significant stimulatory effects on the formation of cholesterol-14C from squalene-14C.