Abstract
Lipids were extracted from the skin of normal rats with chloroform-methanol (1:1) and the solvent evaporated. The resulting residue was saponified, and the unsaponifiable matter extracted with benzene was purified by subjecting the extract successively to hydroxy-alkoxypropyl (HAP) Sephadex column chromatography, silica-gel column chromatography and preparative thin-layer chromatography (TLC). The resulting purified provitamin D fraction was confirmed to contain 7-dehydrocholesterol (7-DHC) based on data of ultraviolet (UV) spectrum, high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC) and gas-liquid chromatography-mass spectro-metry (GC-MS). Based on the results of GC-MS and mass chromato-graphy, it was also confirmed that the fraction contained small amounts of cholesterol, lathosterol and two other unknown sterols besides 7-DHC. Then, the determination of 7-DHC in rat skin was investigated and a HPLC method was proposed. This method included extraction of lipids from small pieces of rat skin with chloroform-methanol (1:1), saponifica-tion, isolation of the unsaponifiable matter and application to HPLC. The estimated average values of 7-DHC in normal rat skin (20 weeks old), with and without UV irradiation, were 107.7 and 103.8μg/g, respectively. These were significantly higher than the corresponding values of vitamin D-deficient rat skin. UV irradiation of rat skin did not affect the change of 7-DHC content in the tissue.
