No abstract

Cholesterol 5 alpha,6 alpha-epoxide (alpha-epoxide) and cholesterol 5 beta,6 beta-epoxide (beta-epoxide) were individually suspended in simulated gastric juice (pH 1.2) at 37 C, and their reaction was followed by gradient high performance liquid chromatography (HPLC) with flame ionization (FID) detection. Both epoxides reacted rapidly in the aqueous acid medium. The alpha-epoxide formed 6 beta-chlorocholestane-3 beta,5 alpha-diol (alpha-chlorohydrin) and 5 alpha-cholestane-3 beta,5,6 beta-triol (triol), while the beta-epoxide formed 5 alpha-chlorocholestane-3 beta,6 beta-diol (beta-chlorohydrin) and triol. The isomeric chlorohydrins reacted further to form the triol. In mildly alkaline aqueous medium, each chlorohydrin reverted to the epoxide from which it was formed. The data suggest that both epoxides, which have been reported to have adverse health effects in animals, would be largely hydrolyzed in the stomach and to the triol, which also has been reported to have biological activity. The data further suggest that residual chlorohydrins surviving stomach residence can be expected to revert to epoxide in the more alkaline intestinal environment.

The substrate requirement of the H*-ATPase in purified com root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg2' or Mn2+. The presence of Ca2+ or Ba2+ did not significantiy affect the coupled activities. The addition of Cd2', Co2+, Cu2+, and Zn2' inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu2+ and Co2+ were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co2+, Cu2+, and Zn2+ were effective inhibitors. However, this inhibition could be further modulated by free Co2+, Cu2+, and Zn2 . While the equilibrium concentrations of Cd-ATP and free Cd2' were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H -ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in "indirect-link" mechanism.Based on the studies of isolated membrane vesicles, the tonoplast of plant cells has been shown to contain two inwardly directed, electrogenic H+-pumps, one powered by ATP and the other by PPi (1,2,6,14). The proton electrochemical potential, AUH+, which contains a ApH (acidic inside) and a A'I (positive inside) generated by these pumps serves as the driving force for the secondary transport of other solutes across tonoplast (3,16,17). The tonoplast H+-ATPase requires Mg-ATP as the substrate and can be inhibited by nitrate ions (7,19). The properties of this enzyme, e.g. vanadate insensitivity, anion dependence, and the generation of proton electrochemical potential, are similar to the Mg-ATPase associated with isolated vacuoles (29).Unlike the plant plasma membrane H+-ATPase, which contains only one polypeptide (molecular mass =100 kD) and forms a phosphorylated intermediate during catalysis (5,15), the tonoplast H+-ATPase is composed of at least three different peptide subunits and does not involve phosphorylation of the enzyme in the reaction pathway (4, 1 1). While the exact molecular process leading to proton pumping remains to be established, the kinetics of proton gradient generation by the H+-ATPase in tonoplast vesicles has been studied. Based on initial rate measurements, a fixed stoichiometry of 2 H+/ATP was reported for the tonoplast ATPase of red beet microsomal membranes (1). This observation is consistent with the notion that the proton pumping is direc...

Several products of cholesterol oxidation have been found in and isolated from foods and have been reported to have adverse biological activity. The successful separation and quantitation by isocratic HPLC of a wide polarity range of cholesterol oxidation products has eluded chromatographers. This study reports the development of an effective HPLC method for the separation and quantitation of a complex mixture of cholesterol and nine oxidation products by use of a binary solvent system of hexane and 2-propanol in a three-part gradient with detection by an HPLC flame ionization detector. Response factors relative to cholesterol were determined, and coefficients of variation were calculated. Each product gave a linear response (R > 0.99) over a concentration range of 100-0.39 pg/lOO pL.