SC WATER-HANDLING PROPERTIES 297 humidity) and during the later stages of barrier recovery (33). Treffel and Gabard (18) reported TEWL and MAT values for skin sites treated with moisturizer or exposed to surfactant irritation. TEWL changed only after long-term exposure, whereas MAT dif- ferences were detected with shorter treatment times. In addition, control skin sites evaluated in both July and November were statistically different for MAT, but not for TEWL. Van Neste (34) used a dynamic measure of water movement in combination with TEWL to investigate surfactant effects. He found that surfactant-damaged skin had an increased TEWL and an increased rate of moisture accumulation. The two measure- ments correlated better for damaged skin than for normal skin. Soaking the skin in water resulted in an immediate increase in TEWL, followed by a reduction, and produced a significant effect on MAT, yielding very low values (Table VI, Figure 1). While TEWL returned to baseline after five hours, MAT remained signifi- cantly lower than the pre-soak value (Table VI). This result indicates that recovery of the SC barrier from the effects of soaking was not immediate. The decreased MAT values suggest that the upper layers of the stratum corneum became significantly drier as a result of water exposure. Solvent extraction followed by a water soak increased TEWL and decreased MAT (Table VI). In an investigation of the molecular mechanisms responsible for SC elastic prop- erties, Jukura et al. (14) reported that acetone/ether extraction did not change molecular mobility within the stratum corneum. However, treatment of the SC with water and the subsequent release of water-soluble materials significantly reduced molecular motion and eliminated the SC elasticity. Depletion of the water-soluble materials also decreased bound water and increased free water. Replacement of the neutral and basic amino acids increased the molecular mobility of the SC, whereas the addition of water alone had no effect. It was hypothesized that removal of water-soluble materials increased molecular interaction between keratin fibers. The water-soluble components of the SC, and not water alone, were responsible for molecular mobility and, therefore, for SC elasticity. Application of NMF consistently lowered TEWL, following A/E extraction, soaking, and extraction-plus-soaking. These effects were seen after thirty minutes and were maintained for another four hours (Table VI). We hypothesize that hygroscopic NMF components bind associated water, including water within the SC, and thereby reduce the rate of evaporative loss, i.e., TEWL. In an investigation of the skin effects of urea-containing moisturizers, Serup (35) reported that the reduction in the rate of evaporative loss (TEWL) indicated an improvement or restoration of SC barrier function. Addition of NMF to all skin sites resulted in a significant increase in the rate of moisture accumulation after thirty minutes (Table VI). For the site that was extracted, soaked, and treated with NMF, the MAT value was elevated four hours later (Table VI). In contrast, the MAT of the soak + NMF site was significantly lower after four hours than it had been 30 minutes after NMF application and significantly lower than the initial value (Table VI). The two sites behaved differently, and the findings suggest that the soaked site could not retain the water associated with the NMF amino acids. Bulgin and Vinson (36) used calorimetric techniques to examine water in the SC and reported three types of water: tightly bound water (primary), readily releasable bound water (secondary) and free bulk water. Free water existed only in very highly hydrated skin states. Takenouchi et al. (37) investigated the bound water in scaly skin conditions. Primary bound water was specified as water not readily lost, even at 0% RH, and was

298 JOURNAL OF COSMETIC SCIENCE reported to be approximately 5 mg water/100 mg of dry SC. Secondary water, held loosely by molecular bonds to the SC components, was measured using DSC techniques after subtracting the primary component. Normal skin had a 20-30% higher level of secondary water than xerotic and psoriatic skin, due to its higher secondary water- holding capacity. Takenouchi eta/. showed that normal skin had substantially increased levels of amino acids, i.e., hygroscopic NMF, and significantly higher water-holding capacity than scaly skin. Secondary bound water was associated with primary bound water through hydrogen-bonding interactions and exhibited rapid hydration and dehy- dration with environmental changes. Previous investigations in our laboratory have demonstrated that the water-holding capacity of normal skin, measured with the sorp- tion-desorption technique of Tagami, was significantly reduced following fresh water bathing (38,39). Based on the current study, we suggest that water soaking removes some water-soluble amino acids, i.e., NMF, from skin and thereby reduces the amount of secondary bound water. The decreased rate of moisture accumulation observed after soaking might be due to a decrease in the NMF-dependent bound water that gives rise to the higher capaci- tance reading prior to the soak. We further speculate that, in the presence of a normal SC barrier (i.e., normal TEWL, no damage) and the absence of eccrine sweating, the rate of moisture accumulation provides a dynamic, functional assessment of the loosely bound water ascribed to the hygroscopic NMF. In contrast to the single-point measurements of baseline hydration, the MAT technique differentiated the effects caused by soaking and NMF application (Tables IV, V). Therefore, MAT is preferred over single-point deter- minations. The results of the water soaking experiment suggest that SC recovery to normal is relatively slow. The findings raise questions about the effects on the skin of cumulative repeated wet-dry-wet exposures, such as those encountered in the diapering environment or in multiple hand washing situations (for health care workers, parents, child care providers). Additional studies are required to examine the kinetics of the depletion/ restoration and to confirm the removal of NMF by direct quantitation of the amino acids. Finally, investigations of the mechanisms by which the epidermis responds to common environmental effects (e.g., bathing) are warranted in order to develop appro- priate skin care practices and products. REFERENCES (1) I. H. Blank, Factors which influence the water content of the stratum corneum,J. Invest. Dermato/., 18, 433-440 (1952). (2) A. V. Rawlings, I. R. Scott, C. R. Harding, and P. A. Bowser, Stratum corneum moisturization at the molecular level, J. Invest. DermatoL, 103, 731-740 (1994). (3) M. Gloor, J. Bettinger, and W. Behring, Modification of stratum corneum quality by glycerin- containing external ointments, Hautarzt., 49, 6-9 (1998). (4) J. W. Fluhr, M. Gloor, L. Lehmann, S. Lazzerini, F. Distante, and E. Berardesca, Glycerol accelerates recovery of barrier function in vivo, Acta Derm. VenereoL, 79, 418-421 (1999). (5) R. Ghadially, L. Halkier-Sorensen, and P.M. Elias, Effects of petrolatum on stratum corneum struc- ture and function, J. Am. Acad. Dermatol., 26, 387-396 (1992). (6) I. Willis, The effects of prolonged water exposure on human skin, J. Invest. Dermatol., 60, 71 (1973).