Zymosterol

Liposomes were prepared from natural yeast polar lipid extracts of S. cerevisiae (Avanti Polar Lipids) using the film hydration method [23 (link)]. The phospholipids and the different sterols (zymosterol, cholesta-5,7,24-trienol (Avanti Polar Lipids), and ergosterol (Sigma)) were dissolved in chloroform. The organic solvent was evaporated to form a film that was dried under a stream of nitrogen for 1 h. This film was then hydrated with degassed PBS to obtain multilamellar vesicles. PBS was degassed of oxygen by nitrogen bubbling for 12 h. We prepared large unilamellar vesicles from the multilamellar ones using the extrusion technique. The suspension of multilamellar vesicles was transferred into a Liposofast small-volume extrusion device (Avestin Inc., Ottawa, ON, Canada) with a polycarbonate membrane with a 200 nm pore size, designed to obtain a homogeneous population of large unilamellar liposomes. The lipid concentration of the liposomes made only with yeast lipid extracts was 2.5 mM. For liposomes containing sterols, zymosterol, cholesta-5,7,24-trienol, or ergosterol was added to obtain a final concentration of 0.83 mM. This corresponds to a sterol/phospholipid molar ratio of 1/3. A control was also performed by adding tocopherol, a lipophilic antioxidant, in the same ratio of the one of sterols.

Deuterium labeled water (D₂O, 99.8% pure) was purchased from Sigma. Cholesterol, lanosterol, dihydrolanosterol, ff-MAS, t-MAS, zymosterol, desmosterol, 7-dehydroCholesterol, d₅-zymosterol, d₆-lanosterol, and d₆-sitosterol were obtained from Avanti Polar Lipids (Alabaster, AL). 25-hydroxyCholesterol (25-OHC) was obtained from Steraloids, Inc. (Newport, RI). Methyl-cyclodextrin (MCD) was obtained from Cyclodextrin Technologies (High Springs, FL). d₅-zymosterol, d₆-lanosterol were bound to MCD in a 1:10 and 1:20, respectively, stoichiometric ratio for solubilization as previously described (Brown et al., 2002 (link)).