j. Soc. Cosmet. Chem., 38, 321-331 (September/October 1987) Measurement of turnover time of stratum corneum using dansyl chloride fluorescence MOTOJI TAKAHASHI, YASUHIKO MACHIDA, and RONALD MARKS, Shiseido Laboratories, 1050 Nippa-cho, Kohoku-ku, Yokohama, Japan 223 (M. T., Y.M.), and Department of Medicine, University of Wales College of Medicine, Cardiff, CF4 4XN, UK (R.M.). Received January 27, 1987. Synopsis The change in turnover time of stratum corneum with age and circadian rhythm was examined using new instruments, a comparator and a fluorometer, to quantitatively assess skin fluorescence due to applied dansyl chloride. The effects of skin protection and keratolytic agents on turnover time were also studied. In two groups of 8 males ages 21-28 years and 6 males between 33 and 46 years of age, stratum corneum renewal times were 12.9 days and 15.1 days, respectively, suggesting that turnover time was prolonged with age. This study demonstrated that the rate of desquamation was considerably reduced in protected sites compared to normal unprotected sites. Furthermore, by taking readings twice daily it was found that the rate of dansyl chloride clearance was twice as fast in the daytime (9 AM-6 PM) as in the nighttime (6 PM-9 AM). The rate of desquamation was accelerated by keratolytic or inflammatory agents, especially salicylic acid. This technique may prove to be very useful to assess the efficacy of cosmetics and drugs. INTRODUCTION It is significant to study functional or structural changes of the skin due to aging for development of suitable cosmetics for different aged persons. However, most testing procedures for skin aging require biopsies or some other surgical manipulation, and simple, noninvasive methods are rarely used. Recently, the dansyl chloride (DC) fluorescence method has been used in dermatology for determination of stratum corneum renewal time (1-4), which can measure turnover time conveniently and noninvasively. The turnover time of stratum corneum is a useful parameter for dermatologists and cosmetic chemists because it is important for epi- dermal cell kinetic considerations and to investigate the effects of cosmetics or drugs on the epidermal metabolic rate. However, in previous work which used dansyl chloride, the end point of turnover time can be difficult to determine because of purely subjective visual methods. 321

322 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS This paper describes a more quantitative method to measure stratum corneum turnover and provides several examples to demonstrate its applicability. EXPERIMENTAL MATERIALS AND METHODS Dansyl chloride (DC) dispersed in white soft paraffin (WSP) was applied to the skin occlusively, using a Finn chamber (10-mm diameter). The depilation of guinea pig skin was accomplished using a commercial depilatory which contained wax and rosin. To confirm that the stratum corneum was completely stained by dansyl chloride, snip biopsies were obtained after staining. Frozen sections of skin tissue were prepared and examined under ultraviolet light using a Zeiss universal microscope. APPARATUS Fluorescence intensity was measured with a "split field" comparator (1) and a fluorom- eter (5). A comparator is described in a previous paper (1). It possesses a grey wedge that can be moved over half the field and a standard fluorescing strip. In use, the instrument is placed over the DC-treated test area of skin with the standard fluorescence adjacent to it, while both are illuminated by Wood's light. The grey wedge is adjusted to attenuate the brightness of the standard and to match it in intensity. The degree of fluorescence is recorded in arbitrary units on a scale attached to the grey wedge. Any results expressed by comparator units in this study were measured by this apparatus. Figure 1 shows a schematic drawing of a fluorometer, which contains a mercury lamp accompanied by a fluorescence body (Hamatsu Photonics Co., Ltd., L1549) at the center of a tube (3.5 x 17 cm). UV radiation with a peak at 338 nm is applied to the skin through an interference filter, F 1. The intensity of DC fluorescence is measured by a photomultiplier tube through an interference filter, F2. The fluorescence intensity depends on the nature of the incident UV radiation, but the UV-lamp specificity changes with time. Therefore, the strength of the DC fluorescence is expressed as a ratio of the incident ray strength. Output was calibrated with fluorescing paper before each experiment. PENETRATION OF DC INTO THE STRATUM CORNEUM Seven normal healthy subjects (5 male, 2 female), ages 24-56 years, were studied. Five per cent DC in WSP was applied under occlusion to the middle of the flexor aspect of the forearm for 3, 6, 12, and 24 hours, using a Finn chamber. The depth of DC penetration was examined by counting the number of adhesive tape strippings of stratum corneum required for the extinction of fluorescence. DC penetration was also examined by observing skin tissue taken by snip biopsy with a fluorescent microscope. MEASUREMENT OF TURNOVER TIME Fourteen normal healthy male volunteer subjects (21-46 years) had 5% DC in WSP applied to the flexor aspect of their forearm for 24 hours, using a Finn chamber. The fluorescence intensity at the application site and a nearby non-application site (control,