J. Cosmet. Sci., 61, 13–22 (January/February 2010) 13 Natural moisturizing factors (NMF) in the stratum corneum (SC). I. Effects of lipid extraction and 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 p odium l ecture at the SCC Annual Meeting and Technical Showcase, New York, December 2003. Synopsis Natural moisturizing factor (NMF) is essential for appropriate stratum corneum hydration, barrier homeosta- sis, desquamation, and plasticity. It is formed from fi laggrin proteolysis to small, hygroscopic molecules in- cluding amino acids. We hypothesized that common lipid extraction and soaking in water would alter the level of NMF in the upper SC and its biophysical properties. A novel method of measuring and quantifying the amino acid components of NMF is presented. Adhesive tapes were used to collect samples of the stratum corneum (SC) and were extracted with 6mM perchloric acid for analysis by reverse-phase HPLC. HPLC re- sults were standardized to the amount of protein removed by the tapes. An increase in NMF was found with increased SC depth. Also, the combination of extraction and soaking was found to increase NMF loss relative to control or to extraction or soaking alone. Our results indicate that common skin care practices signifi cantly infl uence the water binding materials in the upper SC. The fi ndings have implications for the evaluation and formulation of skin care products. INTRODUCTION Natural moisturizing factor (NMF) is important for maintaining proper moisture levels in the stratum corneum. It is a degradation product of fi laggrin, a histidine-rich protein found in the upper layers of the epidermis. Filaggrin aggregates keratin fi laments in the cells of the stratum granulosum to form the keratin bundles responsible for the rigid structure of the cells of the stratum corneum (SC). In the stratum corneum, fi laggrin is degraded into a number of low-molecular-weight, hygroscopic molecules such as urea, pyrrolidone carboxylic acid (1,2), glutamic acid, and other individual amino acids, which are collectively referred to as the natural moisturizing factor of the skin (NMF). Filaggrin degradation to NMF has been shown to relate to ambient humidity levels, with an opti- mum humidity range for NMF production of 80–95% (3). The role of NMF and its optimum levels in vivo are not well known, although it has been observed experimen- tally that fi laggrin levels are lower in the skin of patients with atopic dermatitis, even in non-lesional areas (1,2,4-6).

JOURNAL OF COSMETIC SCIENCE 14 Various investigators have linked mutations in fi laggrin-encoding genes to such dis- eases as atopic dermatitis (1,2,5,7) and ichthyosis vulgaris (6), and even to self-perceived frequent dry skin (8). Surprisingly, the experimental data on the effects of common practices such as bathing or soaking on NMF levels in vivo is sparse. It has been shown that increased hydration of SC ex vivo increases membrane fl uidity, thus increasing the permeability of the SC to endogenous compounds such as NMF as well as to exogenous ones (9). Our earlier reports have shown that bathing/soaking reduces skin hydration and the rate of stratum corneum moisturization in both infants and adults, while ace- tone/ether (A/E) extraction increases TEWL but does not affect MAT (10,11). Topical application of NMF reversed the dehydrating effects of soaking, but direct quantitative measures of NMF were not made. The A/E extraction procedure used in these studies was not expected to remove substantive quantities of water-soluble materials from the skin (12,13). A/E extraction, however, has been shown to disrupt the lipid lamellae of the SC in such a way as to reduce bound-water content and permit the liberation of large amounts of water-soluble material (13). Given our previous fi ndings, we hypoth- esized that exposure to water (soaking) and solvent would reduce NMF levels in the outer stratum corneum relative to normal, non-exposed skin and that the combination of both water and solvent together would create a larger effect, as it impacts both the water-handling and lipid facets of skin hydration. We report a sensitive new method to quantify the amino acid components of NMF that uses reverse-phase high-performance liquid chromatography (HPLC) in conjunction with the collection of SC samples fol- lowing in vivo exposure to water, solvent (acetone/ether), and combinations thereof. MATERIALS AND METHODS MATERIALS Sampling tapes (D-Squame®) were from CuDerm (Dallas, TX). HPLC grade acetonitrile was from Acros Organics (Geel, Belgium), HPLC grade methanol from Fisher chemi- cals (Fairlawn, NJ), and the AccQ-Tag system (containing derivatizing reagent, buf- fers, and one HPLC liquid phase) from Waters Corp (Milford, MA). α-amino-n-butyric acid (AABA), amino acid standard solution (acidic and neutral), citrulline, and sodium lauryl sulfate came from Sigma Chemical Co., (St Louis, MO). Amino acid standard H, BCA protein assay kit, and 6 N hydrochloric acid came from Pierce Biotechnology Inc. (Rockford, IL), and urea was from Ameresco (Solon, OH). Samples were run on a Wa- ters 2690 HPLC system using a C-18 reverse-phase column, 25 cm × 4.6 mm, and a Waters 470 fl uorescence detector, with Waters Millennium 32 acquisition software, Version 2.15.01. SUBJECTS Evaluations were performed on eleven healthy female subjects aged 23–55. Exclusion criteria included visually dry forearm skin and dermatological conditions such as psoriasis and eczema in the study areas. The Institutional Review Board of the Uni- versity of Cincinnati Medical Center approved the protocols. All subjects provided informed consent.