UREA ANALYSIS OF STRATUM CORNEUM 157 the difference between A• and A 2 and by comparison with a standard calibration curve between 10 and 200 nmol/ml urea. MEASUREMENT OF UREA CONCENTRATION ON THE VOLAR FOREARM Four tests sites per volar forearm of 30 volunteers were extracted. The subjects included 15 women and 15 men between ages 21 and 73 (43.8 yr + 17.3 yr). MEASUREMENT OF UREA DISTRIBUTION ON THE VOLAR FOREARM Three test sites per volar forearm of four volunteers (two women and two men of ages 27, 54, 41, and 47 yr) were extracted on two different non-consecutive days. MEASUREMENT OF REPEATED EXTRACTION FROM THE SAME SKIN AREA One skin site per volar forearm of 15 volunteers was extracted four times in quick succession. The subjects included eight women and seven men between ages 30 and 57 (45.1 yr + 9.6 yr). TREATMENT OF SKIN Washing procedure. The experimental subjects were five volunteers (three women and two men of ages 29, 53, 26, 40, and 46 yr). A wet skin area of approximately 25 cm 2 was treated with 750 lnl of cleansing product for 45 seconds. The product was rinsed off with water (30øC) for 30 seconds. Each volar forearm was separated into two areas: distal and proximal. Two areas of each volunteer were used as test areas, with one area used as a control. The particular allocation of each area was randomized. The skin extracts were taken immediately after the washing procedure. Skin care procedure. Twelve volunteers were instructed to apply the skin care formulation twice a day, in the morning and the evening. The subjects included eight women and four men between ages 27 and 56 (35.1 yr 5 9.7 yr). Each test volunteer was instructed to apply a volume equivalent to 150 lnl of product to each test area of 5 cm x 5 cm, estimated by themselves. Each test area had product applied for seven days. Each volar forearm was divided into distal and proximal sites. Two sites per volunteer were used as test areas, one as a control area. The fourth area was not used. The test area of the control product without urea, and the untreated area, were located on the same forearm. The localization of the test sites was randomized with this constraint. The volunteers were asked to avoid contact of test areas with water after the final product application on day 7. The skin extracts were taken 24 hours after the last product application. Products. The cleansing products were sodium lauryl sulphate solution (4 g/100 ml distilled water), a standard cleansing product with and without a supplementation of 10 g urea/100 ml product, and water as a control. The skin care formulations were Laceran © Spezial Creme and Laceran © Spezial Creme 5% Urea. ESTIMATION OF SKIN HYDRATION The skin hydration was estimated (in arbitrary units) with the Corneometer CM 820 (Courage and Khazaka, Cologne, Germany) 24 hours after the last product application.

158 JOURNAL OF COSMETIC SCIENCE The probe was gently positioned onto the skin area. On each site, sixfold determinations were performed. STATISTICS Differences between products were calculated with the two-sided Wilcoxon U-test. Values with p • 0.05 were considered as significant. RESULTS UREA CONCENTRATION OF FOREARM SKIN EXTRACTS OF NORMAL SKIN The urea concentrations of skin extracts of the volar forearms of 30 volunteers comprised a range of 10-1000 nmol/ml. REPRODUCIBILITY OF EXTRACTION We observed no statistically significant differences when we compared samples taken from the same volar sites on the right and left forearms of the same individual. Minor fluctuations were observed for three volunteers, with only a little more fluctuation for one volunteer when comparing the results from a single individual (n = 4) taken on two different days (Figure 1). 800 700 E 600 o l= 500 .,•.J "" 400 o 300 • 200 [] day 1, left -- [] day 1, right [] day 2, left .... [] day 2, right 100 ....... ' I i I• A B C T volunteer Figure 1. Comparison of the urea content of stratum corneum from the right and left forearm at two different days of extraction. Means and standard deviations were calculated from three extracts from each volar forearm. Subjects A-D.