Examination of lipids in human sebum by disk chromatography.

Collection of sebum. Samples of the sebum were obtained (Miescher & Schonberg, 1949) from the forehead of healthy white adults 16-60 years of age. Samples were analysed from 21 persons (11 men, 10 women) not suffering from skin diseases. To collect the sample, a strip of filter paper (Whatman no. 1, 3 cm. x 12 cm.), previously defatted by extraction with boiling light petroleum, was placed on the skin of the forehead lightly cleaned by a cotton wad soaked in ethyl ether. The paper was attached to the skin and kept there for 2 hr., then replaced by a new one for another 2 hr. The strips of paper with samples of sebum were kept at 10° in an atmosphere of nitrogen, then on the same day extracted in a micro-extractor (Bloor, 1943) with 20 ml. of boiling chloroform-methanol (4:1, v/v). The extract, after removal of the solvents, was weighed and stored at -10° in an atmosphere of nitrogen. On the average 21-5 mg. of sebum was obtained, i.e. 0-596 mg./ cm.2. Preparation of chromatogram. For analysis the sample was dissolved in the above-mentioned mixture of chloroform-methanol, at a concentration of 1 mg./0.2 ml. About 30 d. of this solution was chromatographed as described by Hack (1953) on a 6 cm. diameter disk of Whatman no. 1 paper, previously extracted by a mixture of chloroformmethanol (4:1, v/v) and eluted first by chloroformmethanol mixture to a distance of 0-8 cm. (subsequently called 'the inner circle'), then by acetone to 1-5 cm. ('the middle circle') and finally by methanol to 2-5 cm. ('the outer circle'). Before addition of each further eluent the chromatogram was dried in a current of nitrogen. When separation was finished the disk chromatogram was cut into parts, each of which was treated separately by different reagents for detection of individual components. Reaction8 used for identification of individual component8. As an orientational test for the distribution of lipids in the chromatogram, elution by a solution of Sudan II in 50% ethanol has proved very useful. Staining is visible after about 30 min. The tests described by Hack (1953) for neutral fats (Nile blue), for cholesterol (acetic and sulphuric acid), for double bonds (osmic acid), for amino lipids (ninhydrin) and for phosphoric esters (ammonium