608 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS IN VITRO METHODOLOGIES A. Gravimetric Measurement ol • Water Binding This widely used method of assessing the affinity of isolated stratum cor- neum for water consists of equilibrating corneum tissue at a fixed tempera- ture in a constant relative humidity (RH) chamber until a nonvarying weight is attained. Temperatures in the range of 0 ø to 35øC and RH in the range of 10 to 90 per cent have been commonly used (1-5). A period of 5 to 7 days is generally required for attaining constant weights at 10 to 90 per cent RH. The samples are then desiccated over a dehydrating agent until a dry weight is reached. The data are expressed as the per cent moisture uptake (i.e., weight per cent gained) with respect to the dry weight. The capacity of callus tissue to remain soft and flexible was shown by gravimetric assay of water uptake to be directly correlated with the presence of natural moisturizing factors (NMF) in the tissue (1, 2). Human callus, extracted with diethyl ether and water, and then allowed to equilibrate in chambers at 35 per cent RH, gained 5 per cent less absolute weight (i.e., moisture) than callus which was just water-soaked and equilibrated. At 80 per cent RH, this differential increased to as much as 20 per cent water up- take. Laden and Spitzer were able to identify the major humectant in NMF as being sodium 2-pyn'olidone-5-carboxylate (2). Since that study, Middle- ton has further substantiated the role of an NMF (e.g., lactic acid) in influ- encing the state of stratum corneum hydration (6). These investigations sup- port the hypotheses that NMF within the cornified cells of the epidermis maintain the flexibility of this tissue (a) through enhancing the rate of water migration from lower living cell layers and (b) by hindering the release of moisture from the skin surface by reinforcing the water retaining capacity at very low RH. B. Di•:erential Scanning Calorimetry A direct measure of the levels of "bound" (nonfreezing) and "unbound" (freezing) water in animal and human corneum strips was described by Walkey (7) in 1972 using a differential scanning calorimeter.* She was able to quantirate the level of hydration after equilibration of dried strips at vari- ous RH from latent heat of melting curves. Walkley showed that when dry human corneum attained a 45 per cent moisture regain above dry weight, ap- proximately two-thirds of that •vater (0.35 mg/mg dry corneum) was non- freezable (i.e., bound). Her results were confirmed by the findings of Ander- son et al. (5), based on proton magnetic resonance and infrared spectroscopy, which demonstrated the presence of 0.35 to 0.50 mg of bound water per mg *Perkin Elmer Corp., Norwalk, Conn. 06856.

ASSESSMENT OF SKIN MOISTURIZATION 6O9 of dry corneum. Both Walkley (7) and Anderson et al. (5) hypothesized that the freezable fraction was held only by diffusional barriers, whereas the non- freezable (i.e., bound) fraction was strongly associated with •he polar groups of corneum proteins and NMF. Walkley further found that the effect of ex- tracting lipids with diethyl ether and NMF with water allowed for an increase in the portion of bound water from 0.29 to 0.41 mg/mg of dry animal foot pad corneum. Ether-water extraction caused a dramatic lowering of corneum dif- fusional barriers and allowed for a greater proportion of sorbed water to be bound by polar residues of the remaining proteins and lipids. In a study of swelling properties of unmodified and ether-water extracted stratum corneum via biomechanical analyses as is described below, Wolfram et al. (8) have confirmed Walkley's finding. C. Biomechanical Analyses As several investigators have pointed out (4, 6, 8, 9), the elastic modulus of stratum corneum is directly correlated with the amount of water retained in the tissue. Water retention, in turn, has been demonstrated to depend on the surrounding temperature and relative humidity and on the structural integrity of the cornified cells (10-14). It is widely observed th, at, in winter, the rate of moisture replacement from beneath the comeum becomes inadequate in com- parison to the rate of transpiration from the surface. Moreover, exposure to organic solvents or aqueous detergents damages the skin and allows for de- hydration of the outermost cell layers. As these cornified layers become pro- gressively more dehydrated, they become inflexible and less extensible than the deeper layers causing the surface to stiffen, flake, and crack, while the per- son involved perceives the tight, drawn, and itching sensations of chapped and dry skin. Changes in the reversible stretching properties of animal corneum may be evaluated by the method of Elfbaum and Wolfram (9) who used the extenso- meter.* Their results have been expressed as the work index (i.e., the ratio of the work required to reversibly stretch a strip o.f corneum to a 5 per cent dis- placement in a given solvent versus preliminary 5 per cent displacement of the same strip in water). In this way, aqueous dimethyl sulfoxide (DMSO) concentrations greater than 50 per cent cause a reversible stiffening (increase in the work index) of animal corneum together with exter•sive swelling in the cells of the cornified tissue. A conco•nitant increase in the tautness and hard- ness of the samples is observed at the macroscopic level. In a similar manner, ether-delipidized tissue has been water-swollen and reversibly stretched (8). Unlike the dimethyl sulfoxide treatment, exposure of stratum corneum to *Instron Corp., Canton, Mass. 02021.