Abstract

(1, 2) gradually increased. The in-crease exceeded the expected varia-tion reported for a reference method (3). Chohesterylacetatehas been re-ported to yield a greater absorptivity with the Liebermann-Burchard re-agent than cholesterol (4); and we suspected that the cholesterol had gradually esterified. To test this hy-pothesis, we prepared a fresh solution of 100 mg of thrice-recrystallized (from absolute ethanol) cholesterol in 100 ml of glacial acetic acid and allowed it to stand at room tempera-ture. Immediately after preparation and seven days, 24 days, and 53 days thereafter, 0.1-mi ahiquots of the solu-tion were removed and dried under decreased pressure at room tempera-ture. When all the dried samples had been collected, they were redissolved in 0.1 ml of isopropanol, and 10-id aliquots were spotted on a silica gel thin-layer chromatographic plate. At the same time, lO-zl ahiquots of fresh-ly prepared isopropanol solutions of cholesterol and chromatographicahly pure cholesteryl acetate were also spotted, as was a lO-zl aliquot of a solution of cholesterol in isopropanol that had been standing for 53 days at room temperature. The plate was de-veloped in chloroform and the spots were made visible by spraying with sulfuric acid:water (1:1 by vol) and heating briefly in an oven at 110 #{176}C. As Figure 1 shows, within seven days of preparation and progressively thereafter, cholesteryl acetate was in-creasingly present in the solution. Al-though exactly when esterification began cannot be ascertained from this experiment, another experiment, with use of larger aliquots of the cho-lesterol solution, suggested that with-in 72 h of preparation some cholester-yl acetate was qualitatively detecta-ble. No cholesteryl ester was present in the 53-day old cholesterol solution in isopropanol. Further supporting evi-dence was obtained by submitting al-iquots from these same samples to gas-liquid chromatography on a