156 JOURNAL OF COSMETIC SCIENCE mining the water-soluble urea in the stratum corneum. First we determined the homo- geneity of urea distribution on the volar forearms of volunteers. Then we investigated the urea loss of stratum corneum following a washing procedure and the partial supple- mentation of lost urea by addition of urea to a cleansing product. Because of the high loss of urea in consequence of the cleansing procedure, we measured to what extent an external application of urea influences the extractable urea and the hydration of stratum corneum. EXPERIMENTAL SUBJECTS The experimental subjects were healthy female and male volunteers (age 18 to 60 years) without a history of dermatological disease and lacking visible hairs on the volar forearm. The skin area treated and examined was the volar forearm. The tests revealed no patho- logical findings. All subjects gave their informed consent. The number of subjects and the subjects themselves varied between the different tests. STRATUM CORNEUM EXTRACTS Extraction of water-soluble urea from stratum corneum was carried out using a plastic cylinder, 2.5 cm in diameter. The cylinder was fixed in position on the skin area by the volunteers. The urea was extracted with 1.5 ml distilled water for two minutes. The volunteers were asked to gently move the water within the cylinder by a movement of the forearm. Immediately after extraction, 75 lul of a sodium azide solution (4 g/100 ml distilled water) was added to each extract. UREA DETERMINATION Urea determination was carried out according to a method of Kerscher and Ziegenhorn (9), with modifications in concentrations of enzyme and NADH solutions. The enzyme solution I consisted of glutamate dehydrogenase (EC 1.4.1.3.) from bovine liver (about 15 U/mg lyophilisate 40 kU/l), ADP-disodium salt (6.8 mmol/l), 2-oxoglutarate (41.7 mmol/l), bovine serum albumin (400 mg/1), Tris base (500 mmol/1), and succinate (200 mmol/l) adjusted to a pH of 8.0 at 25øC with NaOH. The enzyme solution II consisted of seven parts of urease (EC 3.5.1.5.) from jack bean (ca. 80 U/mg lyophilisate 100 kU/l) in sodium-phosphate buffer (4 mmol/1) adjusted to a pH of 6.8 and one part ofglycerine. The NADH solution consisted of [3-NADH (5 mmol/1), ADP-disodium salt (6.8 mmol/l), 2-oxoglutarate (41.7 mmol/1), Tris base (500 mmol/l), and succinate (200 mmol/l) adjusted to a pH of 8.0 at 25øC with NaOH. One hundred forty microliters of standard or sample was mixed with 50 lul of enzyme solution I and 10 lal of NADH solu- tion in 96-well plates. After ten minutes the absorption at 365 nm was measured (A•) us- ing the spectrophotometer Spectra Max 250 (Molecular Devices Corporation, Sunnyvale, California). Fifteen minutes after the initiation of the reaction by addition of 5 lul of enzyme solution II, the absorption at 365 nm was measured (A2). Each solution was measured in quadruplicate. The urea concentration was calculated by determination of

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.