160 JOURNAL OF COSMETIC SCIENCE 350 300 250 200 150 100 [] volunteer 1 B volunteer 2 - [] volunteer 3 [] volunteer 4 control water SDS 4% Figure 3. Urea content of stratum corneum skin extracts (nmol/ml) after washing procedure with water and with a 4% sodium lauryl sulphate solution in comparison with an unwashed skin area. EFFECTS OF UREA-CONTAINING SKIN CARE FORMULATIONS The skin care and stratum corneum urea-content-influencing effect of prolonged appli- cation of the urea-containing cream Laceran © Spezial Creme 5% Urea and of the cream Laceran © Spezial Creme without urea as a control were compared. The skin care efficacy of both products was inferred from measurements of skin hydration. Treatment with both care products apparently caused a significant increase of skin hydration (Figure 5a). The skin hydration efficacy indicated for Laceran © Spezial Creme 5% Urea is signifi- cantly higher than that indicated for Laceran © Spezial Creme. An only slightly decreased amount of urea in skin extracts after treatment with Laceran © Spezial Creme was detected. The urea content of stratum corneum after a one-week application of Laceran © Spezial Creme 5% Urea is significantly higher than that fol- lowing the application of Laceran © Spezial Creme and that of the untreated area (Figure 5b). DISCUSSION Because of the high relevance of urea for skin physiology, a rapid method of urea determination is of great value. Therefore, we established a noninvasive method for determination of the urea content of stratum corneum. The concentration of urea in skin extracts was measured by a rapid enzymatic assay. This method allows the simultaneous determination of up to 20 skin extracts by using 96-well microtiter plates. Our extrac- tion method has no obvious influence on stratum corneum integrity, in contrast to the

UREA ANALYSIS OF STRATUM CORNEUM 161 11o - lOO c 90 ,,.. 80 c 70 P 60 c 50 c 40 o 30 • 20 lO [] volunteer A [] volunteer B [] volunteer C [] volunteer D [] volunteer E SDS-solution 4% shower gel shower gel with 10 % urea cleansing product Figure 4. Comparison of urea content (in percent of control) of stratum corneum after treatment with different cleansing products. method described by Schwarz (10-12) and Kloss and Schwarz (13). Schwarz examined the urea content of stratum corneum in samples scraped from the skin surface. A similar extraction method was introduced by Koyama et aL (14). However, in our protocol, water is the only extraction solvent instead of a sequence of an organic and an inorganic solvent. The rapidity and the safety of our method offers the possibility of using it routinely on human volunteers when testing skin care formulations and cleansing prod- ucts. We observed a good reproducibility of the urea concentration in different skin extracts taken from the volar forearms of an individual volunteer even when taken on separate days. Therefore, it appears possible to compare different volar forearm skin sites when the testing of a number of products is required. Repeated extractions from an identical skin area resulted in a tenfold lower urea concentration in extracts when compared to the initial extraction. The extraction procedure causes a swelling of stratum corneum that might cause a slightly enhanced mobilization of urea in repeated extracts. The extension of extraction time will therefore produce only a slight increase of urea extracted from stratum corneum. From these results we conclude that the amount of extracted urea by the given method is representative of the urea content of stratum corneum. Treatment of skin with water or cleansing products dramatically decreases the urea content and, therefore, one important component of the NMF of stratum corneum. This might be a possible factor in the development of a dry and scaly skin after prolonged skin cleansing with water exclusively (15). In our experiments neither the presence nor the actual concentration of detergents in cleansing products had any influence on the de- crease of urea content in stratum corneum. However, a supplementation of cleansing product with 10% urea has a measurable positive effect on the urea amount that can be extracted from the skin surface. This positive effect on extractable urea might be related