SKIN CREAM FOR DRY AND FLAKY SKIN 521 greater and more reproducible. A further objective was to incorporate this material into hand lotion and compare the effect on dry and flaky skin with that of lotions containing no humectant or lotions containing humectants which were not retained by the corneum. EXPERIMENTAL Studies on isolated corneum--stratum corneum Corneum was obtained from the rear footpads of guinea pigs. It was separated by incubating the whole footpad in 0.1 mol tris buffer, pH 7.2, containing 2 mol urea and 0.5•o trypsin (BDH) for 18 h at 37øC (2). After this the soft underlying tissues could be scraped away and the resulting corneum was washed for 2 h in distilled water with only one change of water. It was solvent-damaged by immersion in diethyl ether at ambient temperature for 18 h, followed by immersion in distilled water at ambient temperature for 6 h, This procedure removed lipids and hygroscopic sub- stances and reduced the water content and extensibility in humid atmo- spheres (2). This corneum was then used in experiments designed to increase water content and extensibility towards the levels for intact corneum. Measurement of water content in humid atmospheres The technique for measuring water content or water holding capacity has been described previously (2). Briefly, it consisted of equilibrating pieces of corneum at 81•o r.h. and ambient temperature over saturated potassium bromide solution (9) to constant weight. The pieces of corneum were then transferred to a dry atmosphere over molecular sieve (type 4A) and re- equilibrated before weighing. Both equilibria take about 6 days to attain. The water holding capacity was expressed as mg water held 100 mg -x dry weight of corneum at 81•o r.h. Measurement of extensibility in humid atmospheres The technique for measuring extensibility has also been described pre- viously (2). From each piece of footpad corneum a strip measuring 0.6 x 1.5 cm was cut with a stainless steel punch. Extensibility was measured on an Instron Tensile Tester. The strip of corneum was clamped between two pairs of jaws and was stretched at a constant rate of extension. The load

522 JOURNAL OF THE SOCIETY OF COSMETICr CHEMISTS required to do this was measured with a tension cell and the load-extension curve was plotted automatically on a recorder. Extensibility was calculated from the initial part of the curve and expressed as percentage extension 100 g4 load. Measurements were carried out at ambient temperature and in a controlled atmosphere at 81•o r.h. This was achieved by enclosing the jaw assembly of the Instron in a polythene cabinet and recirculating air which was pre-equilibrated at 81 •o r.h. by passing it through columns of saturated potassium bromide solution. After measuring extensibility, the water holding capacity was measured with the same pieces of corneum. The two pieces of corneum from the same animal were kept together. One was subjected to the treatment under investi- gation and the other served as a control. For statistical analysis the corneum from a number of animals was used. Each animal was taken as a statistical block and significant differences between treatment and control were assessed by analysis of variance. Adsorption studies The adsorption of materials by corneum was determined by measuring the decrease in concentration of an aqueous solution of the material in contact with corneum. Weighed pieces of dry corneum were immersed in solutions of the material under investigation using a solid-solution ratio of approximately 10 mg in 1 cm a of solution. At the end of the experimental period the mix- tures were centrifuged and the supernatants analysed for the material under investigation. Sorbitol and glycerol were analysed by a periodate titration method (10) and aromatic carboxylic acids by measuring their ultra-violet absorption at the wavelength of their absorption maxima using an Optica CF 4R spectrophotometer. Lactate was determined by the method of Barker and Summerson (11) or by using sodium DL lactate-2C x4 (Radiochemical Centre, Amersham) and determining the concentration by comparing with standards in a Packard Liquid Scintillation $pectrophotometer model 3224 with 1 cm a of lactate solution added to 10 cm a of Bruno-Christain scintil- lator (12). Control aliquots which had not been in contact with corneum were analysed using the same solution in each case. The results were expressed as mg of material adsorbed mg 4 dry weight of corneum under the conditions of the experiment.