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.

EFFECTS OF LIPID EXTRACTION AND SOAKING 15 EXPERIMENTAL PROCEDURE Prior to entry into the studies, subjects refrained from using moisturizer on their fore- arms for 72 hours. Two 2 × 2-cm treatment sites were marked on each volar forearm. Panelists acclimated to environmental conditions (temperature 21° ± 1°C and relative humidity 31% ± 5%) for 30 minutes before initial measurements were collected. Base- line measurements of transepidermal water loss (TEWL, g/m2/hr) and the rate of mois- ture accumulation (MAT, cru/sec) were made for all sites. Biophysical measurements were collected again at 0.25, 0.5, and 4 hours following treatment. TREATMENTS The skin was exposed to three treatments: (a) acetone/ether, (b) water soak, and (c) ace- tone/ether followed by a water soak. Untreated skin served as the study control. The treatments were randomized both up and down and between arms. Acetone/ether extraction. One site on each forearm was treated with a 1:1 mixture of acetone and ether (A/E) to remove surface and intercellular lipids from the outer lay- ers of the SC. The sites were exposed to A/E for ten minutes using a glass extraction cup to hold the solvent. The areas were continuously rubbed with a cotton swab dipped in the A/E mixture during this time. Following extraction, the biophysical measurements were repeated on the extracted sites to quantify the immediate post- extraction state. Soaking. Once the extraction was complete, one forearm was soaked in fresh water (tem- perature 40° ± 2°C) for ten minutes and blotted dry. The sites on the other, unsoaked, arm served as control. Biophysical measurements were repeated on all test sites 15 min- utes, 30 minutes, and four hours after soaking. NMF MEASUREMENTS Skin surface sampling. At the end of the measurement period, four hours after soaking, and after collection of all biophysical measurements, SC samples were serially col- lected on all treatment and control sites. Two different sizes of tapes were used in these studies: for sample collection, 14-mm tapes were used for desquamation be- tween the collected samples (tapes 2, 4, 6–9, and 11–14), larger, 22-mm tapes were used to help ensure that the collected samples refl ected a consistent SC depth. Each sampling tape was placed on the skin, rubbed once with the collector’s thumb, and removed immediately by tweezer. The next tape was placed in the same location but removed from a different angle to ensure overall evenness of desquamation. The col- lected sampling tapes were placed in snap-cap centrifuge tubes and stored at -18 degrees Celsius until analysis. Preparation of standards and calibration curves. The stock solution was diluted 1:100 with a further 10 mM HCl. A calibration curve for citrulline was prepared using the amino acid acidics and neutrals standards as follows: Four separate dilutions, 1/200, 1/200, 1/75, and 1/50, of the stock solution (2.5 μmol/ml) were prepared using 10 mM of HCl. A 2.5- μmol/ml solution of citrulline in 10 mM of HCl was prepared by weighing 4.38 mg of citrulline into 10 ml of 10 mM HCl and analyzed in triplicate against the acidics and