NAG AS EXFOLIATION ENHANCER 425 containing TBS). These membranes were then incubated for 120 minutes with an anti- body for involucrin (Biomedical Techniques Inc., MA) and keratin K1 and K10 (Chemi- con, Temecula, CA). Following this incubation the membranes were again washed three times with TTBS. The membranes were then incubated with a secondary antibody (either goat or rabbit anti-mouse antibody) conjugated to alkaline phosphatase. The enzyme activity was then visualized by reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium tablets (Sigma, St. Louis, MO). The blots were scanned with a Hewlett-Packard ScanJet IIc and analyzed with the densitometric software package SigmaGel 1.0. CLINICAL STUDY DESIGN The subjects included in this study were 45 females between the ages of 21 and 65 years, all meeting the screening criteria of good health and not pregnant or lactating. The sub- jects reported for testing without moisturizers or any other products on their faces and hands, and their baseline measurements were taken. They were given the product to take home and self-administer for four weeks to their right hand only, twice a day, in the morning after washing and in the evening at least 15 minutes before bedtime. The left hand served as the untreated control site. The subjects were only allowed to use the test product and specifi cally log its use in a daily diary we provided. At the end of two and four weeks the subjects returned for testing without applying the product for at least 12 hours and they were re-evaluated under the same conditions. SKIN EXFOLIATION VIA THE D-SQUAME DISCS METHOD AND IMAGE ANALYSIS Skin exfoliation was evaluated by measuring the amount of fl akes removed from the skin surface using D-Squame discs and analyzing them by image analysis. Four D-Squame discs were fi rmly and evenly pressed on the face and the back of each hand with a hand-held uniform pressure device and removed by gently pulling them away from the skin. The D-Squame discs were mounted on clear microscope slides and labeled according to the panelist’s name and date of visit. Desquamation was evaluated from the D-Squame discs using the image analyzer. Skin evaluation was carried out before treatment and after two and four weeks of treatment. The OPTIMA image analyzer (Optimas 6.5 from Media Cybernetics, Bethesda, MD) was used to evaluate skin fl akiness. The D-Squame samples containing the corneocytes were placed under a camera on top of a light table and each image was imported into the image analyzer. The average gray value corresponding to the sample density was measured: the denser the sample, the higher the gray value difference. ASSESSMENT OF SKIN MOISTURIZATION WITH A DERMAL PHASE METER Skin surface capacitance measurements were made with a NOVA dermal phase meter (DPM 9003 Nova Technology, Portsmouth, NH). The DPM is an electronic instrument that non-invasively measures skin capacitance in vivo. The capacitance readings are directly related to picoFarads of capacitance in the volume of skin that is effectively measured,

JOURNAL OF COSMETIC SCIENCE 426 and are correlated to skin water content. A uniform-pressure sensor probe was placed on the surface of the hand skin for approximately fi ve seconds and a reading was taken. Four measurements were taken at each site at every time point. Measurements were automatically acquired via a computer software package, ensuring standardization of the measurements. STATISTICAL ANALYSIS Data were given as means ± SEM. Excel (Microsoft) software was used to evaluate statistical signifi cance by using the Student’s two-tailed t-test function in the software package. A p value of 0.05, at least, was considered signifi cant. RESULTS IN VITRO EFFECTS OF N-ACETYL-GLUCOSAMINE ON KERATINOCYTES IN CULTURE Human keratinocyte (HaCaT) monolayers were treated with increasing amounts of amino sugars to assess their effect on cellular morphology. HaCaT keratinocytes treated with 50 mM, 125 mM, and 250 mM of NAG appeared to be released from culture dishes and dissociated (Figure 1). Similar effects were observed with other amino sugars such as N-acetylneuraminic acid and N-acetyl-galactosamine (data not shown). Suspension or release of keratinocytes from the substrate has been reported to promote keratinocyte dif- ferentiation (10). We therefore investigated the levels of two different protein markers of keratinocyte differentiation in these N-acetyl-glucosamine treatments. Increasing con- centrations of N-acetyl-glucosamine resulted in increased accumulation of involucrin, keratin K1, and keratin K10 in cultured keratinocytes (Figure 2). These data supported the observation that the keratinocytes had reduced adhesion to the substratum. Figure 1. Effect of NAG on human keratinocyte morphology. HaCaT cells were treated for 24 hours with 50 mM, 125 mM, and 250 mM of NAG in whole media. Cell cultures were then viewed at ×100 magnifi caion by phase contrast and refl ectance microscopy with an Olympus BX60 microscope (Olympus, Melville, NY).