Effects of Free Sterols, Steryl Ester, and Steryl Glycoside on Membrane Permeability

Abstract

The physiological importance of sterols in higher plants has never been fully established. It has been suggested that they are structural components of membranes (7, 11), and that they might be involved in controlling the permeability of membranes (6). Cholesterol was found to be more effective than CaCl2, the classic membrane stabilizer, in preventing the methanol-induced leakage of betacyanin from red beet disks (6). Other plant sterols, such as /3-sitosterol and stigmasterol, were found to be less effective. It was suggested that only those sterols that have a flat molecular configuration, similar to that of cholesterol, are physiologically active because only they can penetrate the phospholipids of the membranes (6). Sterols in higher plants occur in at least three different forms: as free sterols, steryl esters, and steryl glycosides, and the steryl glycosides may or may not be acylated (1, 4). Quantitatively , these sterol forms vary with tissue and species. In potato tuber the steryl glycosides are the major form (4), while in barley shoots the free sterols are highest in concentration (1). Only the free sterols have been tested for their membrane stabilizing effectiveness (6). It, therefore, seemed of interest to compare the effectiveness of free sterol, steryl ester, and steryl glycoside on membrane permeability. MATERIALS AND METHODS Barley Root Material. Barley (Hordeumn vulgare, var. Bar-soy) was germinated on cheesecloth over continuously aerated 0.5 mM CaSO, at room temperature in the dark. Three-day-old barley roots were harvested and placed in 0.5 mm CaSO4 for 30 min, transferred to 5 mM KCl for 1 hr, and washed with 500 ml of distilled water in a funnel layered with cheesecloth. The CaSO4 and KCl solutions were continuously aerated. Chemicals. Cholesterol, campesterol, stigmasterol, fl-sito-sterol, and cholesteryl palmitate were purchased from Applied Science Laboratories, Inc., State College, Pa. The synthesis of cholesteryl glucoside was by the method of Meystre and Miescher (13). Five grams of cholesterol and 6 g of silver carbonate were dissolved in 200 ml of benzene. This mixture was brought to boiling under continuous stirring, and 13.3 g of acetobromo-D-glucose dissolved in 300 ml of benzene were added drop-wise with continuous boiling and stirring. The mixture was filtered, taken to dryness under vacuum, and re-dissolved in 200 ml of methanol. It was cooled in an ice bath and 250 ml of sodium alcoholate were added. The mixture was allowed to stand for 3 hr and then neutralized with dilute IThis paper (No. 71-3-84) is part of a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director. HCI. The cholesteryl glucoside precipitated. The precipitate was washed with alcohol and ether, boiled in water, cooled, filtered, and washed with cold water. The residue was dissolved in 50 ml of pyridine, heated, and filtered, and water was added till a precipitate was obtained. The precipitate was washed with alcohol and ether, and applied in n-hexane to a 5-g silica gel column (1-cm diameter). The column was washed with 150 ml of 40% benzene in n-hexane, 50 ml of benzene, and 100 ml of chloroform; all were discarded. The cholesteryl glucosides were eluted with 150 ml of 5% methanol in chloro-form. This fraction gave a single spot by thin layer chromatography. Analysis of Sterols. About 50 g of fresh barley roots were homogenized in acetone and extracted in a Soxhlet apparatus for 24 hr. At the same time three samples were removed for dry weight determination. One gram of fresh roots equaled 0.0543 g of dry weight. The acetone extract was taken to dryness under vacuum, redissolved in 50 ml of acetone, and dried under vacuum in the presence of 5 g of silica gel. This procedure allowed for a more exact transfer of the n-hexane suspended sample to the silica gel column. A 1.5-cm diameter column packed with 25 g of silica gel (70 to 325 mesh) in n-hexane was used for serial elution (5, 14). Passed through the column were 150 ml of 10% benzene in n-hexane. This fraction was discarded. The steryl esters were eluted with 700 ml of 40% benzene in n-hexane. The free sterols were eluted next with 150 ml of 100% benzene followed by 800 ml of chloro-form. The acylated steryl glycosides were eluted with 700 ml of 2% methanol in chloroform, and the steryl glycosides were eluted with 600 ml of 5% methanol in chloroform. The four sterol fractions were taken to dryness under vacuum. The esters were hydrolyzed with 5% KOH in 95% methanol for 30 min, and the glycosides were hydrolyzed with 0.5% H-SO, in 95% methanol for 12 hr. The above fractions were neutralized and the sterols were extracted 3 times with n-hexane, dried, and precipitated with digitonin (7). The qualitative and quantitative sterol analysis was performed by gas chromatography using a 1.80-m U-shaped glass column, 6-mm internal diameter, packed with 5% OV-101 on Anakrom ABS 80/90 mesh. The column temperature was 255 C and the nitrogen carrier flow rate was 60 ml/min. Cholestane was used as the internal standard. For more details refer to a previous report (8). Permeability Experiments. Barley root samples of 0.25 ± 0.01 g were transferred to glass sample chambers (2-cm diameter) fitted with a circular magnetic stirrer. The samples were continually aerated in a 30 C constant temperature water bath. The test medium consisted of 14.25 ml of distilled water (conductance <5 micromhos) to which was added 0.75 ml of ethanol containing 20 times the desired sterol concentration. A 653