Measurement of turnover time of stratum corneum using

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1 j. Soc. Cosmet. Chem., 38, (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, 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 assesskin 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 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 cosmeti chemists because it is important for epidermal 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

2 322 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS This paper describes a more quantitative method to measure stratum corneum turnover and provideseveral 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 fluorometer (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 adjacento 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 Seve normal healthy subjects (5 male, 2 female), ages 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,

3 MEASURING TURNOVER TIME F1 /Quartz glass F2 I IS2 I D splay Skin Fli12 Interference F,Iter Sl,S2 Photomultiplier Figure 1. Diagrams to show construction of the fiuorometer. background) was measured daily. During this experiment, volunteers were not allowed to bathe. CIRCADIAN RHYTHM Sevenormal healthy male volunteer subjects (21-36 years) were studied. After applying 5% DC in WSP to the forearm (under occlusion) for 24 hours, the fluorescence intensity was measured twice daily (9:00-10:00 in the morning and 5:00-6:00 in the evening) to examine the difference in desquamation rate during day and night. Volun- teers were not allowed to bathe during the experiment.

4 324 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS EFFECTS OF SKIN PROTECTION AND OCCLUSION Seven normal healthy male volunteer subjects (21-36 years) had 5% DC in WSP applied to the flexor aspect of each forearm for 24 hours, using Finn chambers. After 24 hours, the Finn chambers were removed and one site covered with woven cotton gauze fastened in place with adhesive plaster. The gauze was renewed twice daily after readings had been taken. The other site was left unprotected. In a second group of six normal volunteers (23-46 years), the above experiment was repeated; instead of gauze being used to protect one of the sites, Finn chamber occlusion was used, the chamber being renewed twice daily after fluorescence readings had been taken. During these experiments, volunteers were not allowed to take a bath. EFFECT OF KERATOLYTIC AGENT After 5 % DC in WSP was applied to four sites on the back of depilated guinea pig skin (under occlusion) for 24 hours, experimental sites were treated daily with 0.1 ml/4 cm 2 of a preparation containing 6% each of salicylic acid, sulphur, and resorcinol (in WSP), and readings were taken at the same time each day. RESULTS PENETRATION OF DC INTO THE STRATUM CORNEUM Figure 2 shows the relationship between DC application time and the number of tape strippings required for fluorescence to disappear. The number of strippings increased with increasing application time. In 3-hour DC applications, fluorescence disappeared " lo -.,,.5 Z 0 I I I I Application time (hr) Figure 2. Depth of penetration of dansyl chloride in WSP under occlusion, with time. Results are shown as mean and SD (n = 7).

5 MEASURING TURNOVER TIME 325 after an average of 9 strippings, indicating that about half of the stratum corneum was stained. However, in 24-hour applications, an average of 20 strippings was required and the glistening layer appeared in every subject when the fluorescence disappeared. This means that whole stratum corneum was completely stained after 24 hours' application. This result was confirmed by the fluorescent microscopic observation of the skin tissue taken by snip biopsy (Figure 3). The equality of fluorescence intensity in each stratum corneum layer was checked by progressive stripping of a fully stained stratum corneum. As depicted in Figure 4, the fluorescence intensity declined almost linearly with strippings. From this result, stratum corneum appeared to be uniformly stained with dansyl chloride. FOREARM STRATUM CORNEUM TURNOVER TIME Figure 5 shows changes in the fluorescence intensity of skin after DC application. As can be seen, the decrease in fluorescence was linear against time. The turnover time of stratum corneum was obtained by reading the time at which the declining fluorescence intersected with the background fluorescence of a nearby area. Table I shows the turnover time of forearm stratum corneum obtained from 14 volunteer subjects. The turnover time was 12.9 days for those in their twenties and 15.1 days for those in their thirties and forties, suggesting that it increaseslightly with age. Figure 3. Cryostat section of dansyl chloride stained skin immediately after 24-hour application (phase contrast under UV lighting).

6 326 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS background Number of Tape Strips Figure 4. Change in the fluorescence intensity of depilated skin from back of guinea pig using tape strippings. Results are shown as mean and SD (n = 7). CIRCADIAN RHYTHM Table II shows the declining rate of DC fluorescence expressed in comparator units per hour during day (9:00-18:00) and night (18:00-9:00). These data show that the desquamation rate is twice as fast during the day as during the evening and night. 141 Forearm (, 36) Irl = (, : test ß ee O Oo QQ ß: background OO QQ Q Q 70 0 I 2 I I 4 I t 6 I t 8 I t 10 I I 12 I I 14 I I 1 I 6 Days Figure 5. Decline of fluorescence intensity at forearm site of male subject age 36 years.

7 MEASURING TURNOVER TIME 327 Table Turnover Time of Stratum Corneum at Forearm Site I Turnover time Turnover time Subjects (days) Subjects (days) M, M, 33 M, M, 34 M, M, 36 M, M, 37 M, M, 37 M, M, 46 M, M, Mean ñ SD ñ 2.0 EFFECT OF SKIN PROTECTION Figure 6 shows the change in fluorescence intensity when the skin was protected with gauze. Similar to the non-protected sites, the fluorescence intensity at the protected sites declined linearly, though at a slower rate. The turnover time of the stratum corneum was obtained by extrapolating the line of declining fluorescence to a point which met the background fluorescence. Table III shows the results of stratum corneum turnover times at gauze-protected sites. The mean turnover time was 14.5 days at control sites, and it was prolonged to 23.7 days by gauze protection. Table IV shows the results obtained by Finn chamber occlusion. The mean turnover time at occluded sites was 29.1 days, and it appears that Finn chamber occlusion prolonged turnover time further than gauze. EFFECT OF KERATOLYTICS The effects of salicylic acid, sulphur, and resorcinol were studied using the depilated skin of guinea pig backs. The fluorescence intensity decreased linearly in guinea pig (Figure 7), and the desquamation rate was obtained in the same way as for human skin. The results are shown in Table V. The rate of exfoliation of stratum corneum became faster with all keratolytics in comparison to the controls (WSP application), and the effect of salicylic acid was especially noteworthy. Also, the thickness of epidermis in the skin treated with salicylic acid or resorcinol was similar to that of control skin, though it was thickened by sulphur treatment (Figure 8). Table II Circadian Rhythm of Turnover Rate of Stratum Corneum Mean SD (n = 7). *,0.05 <p<0.1. Day (9 AM-6 PM) Night (6 PM-9 AM) Decrease (comparator units) 3.00 ñ ñ 1.37 Decrease/hour (comparator units) 0.33 ñ ' ñ 0.09

8 328 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 130 Forearm (9, 21) Z 00 0 non-protected skin ß protection by soft gauze O ' (} backgrøund E o -øo%o Days Figure 6. Decline of fluorescence intensity at forearm site of male subject age 21 years. DISCUSSION It is well known that the dansyl chloride fluorescence test is very useful to investigate the turnover time of stratum corneum. However, there have been a few inconveniences in determining the end point by eye observation under Wood's lamp. First of all, the end point can be difficult to determine, as hair follicles may continue to fluoresce after the fluorescing stratum corneum has all been shed. Secondly, the duration of an experiment becomes long because observation is continued to the point of extinction, i.e., for the number of days equivalento the turnover time. Such disadvantages can be reduced by using the instrument mentioned in this study. The turnover time can be obtained accurately from the point of intersection of the declining fluorescence with the background fluorescence of a nearby area and may be easily extrapolated from a few readings even when the fluorescence measurement is not continued to extinction. Stratum cor- neum seems to be stained uniformly with dansyl chloride, judging from the result that Table III Effect of Skin Protection by Soft Gauze on Turnover Time of Stratum Corneum Non-protection Protection Subjects (days) (days) Ratio M, M, M, M, M, M, M, Mean _ SD *, estimated by extrapolation.

9 ._ MEASURING TURNOVER TIME 329 Table Effect of Finn Chamber Occlusion on Turnover Time of Stratum Corneum Untreated IV Occlusion* Subjects (days) (days) Ratio M, M, M, M, M, M, Mean SD estimated by extrapolation. the fluorescence intensity decreased linearly with progressive tape strippings. Therefore, stratum corneum turnover can be determined in the above manner. The turnover time of the stratum corneum depends on environmental conditions. Normal routines (bathing, type of clothing worn, etc.) can markedly affect the rate of desquamation. In this paper, it was shown that skin protection by gauze prolonged turnover time by 75%, but gauze protection may not affect skin hydration. Therefore, this result seems to reflect turnover time in the absence of physical stimulation. However, Finn chamber protection should increase skin hydration. Consequently, the turnover time of stratum corneum may be prolonged further by Finn chamber occlusion than by gauze protection. There are conflicting reports regarding circadian variation in cell proliferation of human epidermis. It would have been very difficult to detect a circadian rhythm by normal fluorescence extinction. However, by this procedure, it was found that the desquamation rate was twice as fast in the daytime as at night. This result provides some support to the previous observation (6) that there is relative suppression of epidermal proliferative activity in human skin at night. left side of guinea pig back o DC application 100- right side of guinea pig back e background 50-0 background Vo lr1=0.996 o -- la. 0- ø o DC apphcation Days Days Figure 7. Decline of fluorescence intensity of depilated skin from back of guinea pig.

10 330 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table Effects of Keratolytic Agents on Disappearance Time (days) of Dansyl Chloride on Guinea Pig Stratum Corneum WSP SA 6% Sulphur 6% Resorcinol 6% Guinea pig no. (days) (days) (days) (days) Mean + SD ** ** ' *, p < 0.05; **, p < 0.01 compared with WSP (paired t-test). WSP: white soft paraffin. SA: salicylic acid. V The mode of action of keratolytic agents on stratum corneum has remained obscure. Salicylic acid, sulphur, and resorcinol all accelerated the desquamation rate of the stratum corneum; however, different actions are suggested for the three. Sulphur seems to enhance the rate of desquamation by accelerating cell proliferation in the epidermis, judging from the thickened epidermis. On the other hand, the effect of salicylic acid may be due to a direct action on the intercellular cement substance of the corneocytes because the epidermis was not thickened or irritated. These results indicate that a comparator and a fluorometer are very useful for measuring the turnover time of stratum corneum and for evaluating the effects of keratolytics or cosmetics.!..... :u'iphu r 6%.....:'!...; ir 'sor:c..ino[ '6,/_';...,.. Figure 8. Histological study of skin from back of guinea pig treated with keratolytic agents for 2 weeks H&E.

11 MEASURING TURNOVER TIME 331 REFERENCES (1) R. Marks, D. Black, I. Hamami, A. Caunt, and R. J. Marshal, A simplified method for measurement of desquamation using dansyl chloride fluorescence, Brit. J. Dermatol., 111, (1984). (2) A. Y. Finlay, R. J. Marshal, and R. Marks, A fluorescence photographic photometric technique to assesstratum corneum turnover rate and barrier function in vivo, Brit. J. Dermatol., 107, (1982). (3) D. Roberts and R. Marks, The determination of regional and age variations in the rate of desquamation, J. Invest. Dermatol., 74, (1980). (4) L. H. Jansen, T. Hojyo, and A.M. Kligman, Improved fluorescence staining technique for estimating turnover of the human stratum corneum, Brit. J. Dermatol., 90, 9-12 (1974). (5) M. Takahashi, Y. Machida, and R. Marks, A new apparatus to measure rate of desquamation using dansyl chloride fluorescence, Arch. Dermatol. Res., in press. (6) L. Rowe and W. J. Dixon, Clustering and control of mitotic activity in human epidermis, J. Invest. Dermatol., 58, (1972).

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