J. Cosmet. Sci., 61, 23–29 (January/February 2010) 23 Natural moisturizing factors (NMF) in the stratum corneum (SC). II. Regeneration of NMF over time after soaking MARISA ROBINSON, MARTY VISSCHER, ANGELA LaRUFFA, and RANDY WICKETT, The James L. Winkle College of Pharmacy, University of Cincinnati, 3225 Eden Avenue, Cincinnati, OH 45267 (M.R., R:W), and Skin Sciences Institute, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229 (M.V., A.L.R). Accepted for publication June 22, 2009. Presented as a podium lecture at the SCC Annual Meeting and Technical Showcase, New York, December 2003. Synopsis The detrimental effects of prolonged water exposure on skin are well known. Information on the effects of short-term exposure, e.g., during bathing, showering, and hand washing, on NMF levels is limited. In an attempt to isolate the effects of soaking on the NMF, a time course for skin sample collection was devised in which adhesive tapes were applied 0.5 and four hours after soaking of the treated arm. Signifi cant decreases in NMF levels, quantifi ed by HPLC analysis of serial tape strips, were observed 0.5 hours after soaking, with a replacement of NMF occurring by the four-hour mark. This replacement corresponds to a parallel rebound in skin pH also observed at these times. Stratum corneum hydration values, measured instrumentally as the rate of moisture accumulation (MAT), however, were depressed 0.5 hours post-soaking and remained low four hours later. In addition, signifi cant increases in skin pH were observed during the immediate post-soak period. These fi ndings suggest that short-term exposure to water alone produces signifi cant changes in the stratum corneum. INTRODUCTION The detrimental effects to the skin of long water exposure are well known. Extended water exposure causes maceration of the skin, disruption of the skin barrier and structure, and a dry fl aky appearance once the excess water evaporates (1–6). The effects of ordinary water exposures, such as bathing or soaking, on the skin and its biophysical properties have been explored over years of research in many laboratories. Soak baths of relatively brief dura- tion, up to 20 minutes in length, have been shown to temporarily increase skin hydration and reduce scaling (7). Studies of long-term exposures indicate that the skin looks well hydrated and scale-free immediately after the exposure, but that within two to three days, hyperplasia and infl ammation occur (1). The boundary between the therapeutic and damag- ing effects of water exposure is unknown, as is the etiology of the resulting damage. Increased stratum corneum (SC) hydration due to soaking has been shown to increase skin permeability to surfactants and other irritants (8,9). Even brief tap water washing increases

JOURNAL OF COSMETIC SCIENCE 24 pH and skin dryness in infants (10). As has been recently shown, merely maintaining a low skin pH helps prevent chemically induced atopic dermatitis and can repair moderate age-related barrier defects (11,12). Increased SC pH has been shown to directly cause SC swelling and a change in lipid transition temperatures, increasing the reactivity of the skin (13). Surprisingly, experimental data on the effects of common practices such as bathing and relatively brief soaking on NMF levels is sparse. Earlier reports from our group (Visscher, Tolia, Fugitt, Hoath, Wickett) have shown that bathing/soaking reduces skin hydration and the rate of stratum corneum moisturization in both infants and adults (14,15). Topical application of NMF reversed the effects of soaking, but direct quantita- tive measures of NMF were not made. A direct connection between NMF levels, skin moisturization, and water handling properties in normal skin has not been well estab- lished experimentally. Biophysical measurements such as TEWL or MAT are commonly used in cosmetic indus- try laboratories. Despite the frequency with which these tests are used, a complete under- standing of what these instruments really measure in the skin is still lacking. We hypothesized that exposure to water (soaking) would reduce NMF levels in the outer stratum corneum relative to normal, non-exposed skin. We also hypothesized that there is a relationship between NMF levels and commonly used biophysical measurements such as MAT and skin pH. We have also explored the relationship between NMF levels and biophysical instru- ment readings during the hours post-soak. The preceding paper (this issue) reported a new chromatographic method for quantitation of the free amino acid components of NMF and demonstrated its application for differentiating skin treatments that do not cause barrier damage. In this paper, we have used our method to quantify NMF following exposure to water alone, and to further explore the relationship between NMF and various commonly used biophysical measurements of skin moisture and acidity. MATERIALS AND METHODS SUBJECTS Evaluations were performed on twenty-seven healthy female subjects aged 23-60 in two studies: Study 1 in September of 2003 (n = 8) and Study 2 in February of 2003 (n = 19). Exclusion criteria included visually dry forearm skin and dermatological conditions such as psoriasis and eczema on the study areas. The Institutional Review Board of the Univer- sity of Cincinnati Medical Center approved the protocols. All subjects provided informed consent. EXPERIMENTAL PROCEDURE Prior to entry into the studies, subjects refrained from using moisturizer on their fore- arms for 72 hours. One 2 × 2-cm treatment site was marked on each volar forearm. Panelists acclimated to environmental conditions (temperature 21° ± 1°C and rela- tive humidity 31% ± 5%) for 30 minutes before initial measurements were collected. Baseline measurements of MAT, sorption/desorption, and skin pH were made for all