Inhibition of cholesterol biosynthesis by Δ22-unsaturated phytosterols via competitive inhibition of sterol Δ24-reductase in mammalian cells

Dietary phytosterols are cholesterol-lowering agents that interfere with the intestinal absorption of cholesterol. In the present study, we have studied their effects on cholesterol biosynthesis in human cells, particularly in the sterol-conversion pathway. For this, both Caco-2 (intestinal mucosa) and HL-60 (promyelocytic) human cell lines were incubated with [¹⁴C]acetate, and the incorporation of radioactivity into sterols was determined using HPLC and radioactivity detection online. Sterols containing a double bond at C-22 in the side chain (stigmasterol, brassicasterol and ergosterol) dramatically inhibited the activity of sterol Δ²⁴-reductase, as indicated by the decrease in radioactivity incorporation into cholesterol and the accumulation of its precursors (mainly desmosterol). Phytosterols with the saturated side chain (β-sitosterol and campesterol) were inactive in this regard. The inhibition of sterol ²⁴-reductase was confirmed in rat liver microsomes by using ¹⁴C-labelled desmosterol as the substrate. The ²²-unsaturated phytosterols acted as competitive inhibitors of sterol ²⁴-reductase, with K_i values (41.1, 42.7 and 36.8μM for stigmasterol, brassicasterol and ergosterol respectively) similar to the estimated K_m for desmosterol (26.3μM). The sterol 5,22-cholestedien-3β-ol, an unusual desmosterol isomer that lacks the alkyl groups characteristic of phytosterols, acted as a much stronger inhibitor of ²⁴-reductase (K_i = 3.34μM). The usually low intracellular concentrations of the physiological substrates of ²⁴-reductase explains the strong inhibition of cholesterol biosynthesis that these compounds exert in cells. Given that inhibition of sterol ²⁴-reductase was achieved at physiologically relevant concentrations, it may represent an additional mechanism for the cholesterol-lowering action of phytosterols, and opens up the possibility of using certain ²²-unsaturated sterols as effective hypocholesterolaemic agents.