350 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (6) that produced much of the current knowledge concerning the composition of human sebum. Xerosis, the condition commonly referred to as dry skin, is a condition that is considered almost universal among the aged (7). We found the potential for a direct investigation of a cosmetic treatment on stratum corneum of great appeal and used small-angle x- ray diffraction techniques to investigate the effects of washing, treatment with G2, and ether extraction upon the stratum corneum of human thin skin samples. EXPERIMENTAL Fresh full thickness samples of human skin were obtained from radical mastectomy cases and prepared by a method described by Lampe et al. (8). The skin samples were from normal skin well away from the tumor site. After removal of the subcutaneous fat with a #22 blade, each sample was placed dermis side down on filter paper soaked with 0.5 % trypsin in phosphate-buffered saline (PBS). The samples were then incubated while refrigerated for 24 hours prior to removal of the stratum corneum. Sheets of stratum corneum were then thoroughly rinsed with PBS and frozen on filter paper after being blotted dry. Immediately prior to x-ray analysis, the samples were thawed and vortexed in PBS to remove any granular cells or filter paper residue. The samples, taken from the same skin specimen, were then prepared for low-angle x- ray measurements by one or more of the following techniques. "Washed" samples were vortexed in a 0.1% (by weight) Ivory © soap solution for ten minutes. Samples "treated with G2" were spread on filter paper soaked with purified G2 and allowed to remain in contact with G2 for one to thirteen hours. "Extracted" samples were vortexed in ethyl ether for 30 minutes. The prepared stratum corneum sheets were then rolled and placed in a 0.7-mm glass capillary tube and examined by small-angle x-ray diffraction. The untreated stratum corneum band was checked by multiple measurements of skin specimens from three different subjects, while the treated, washed, and extracted sam- ples were determined by duplicate measurements from the same skin specimen. Only slight differences in intensity were encountered between spectra of the multiple runs. G2 was purified as described previously (9). Trypsin (98%) and dry phosphate-buffered saline (diagnostic) were obtained from Sigma Chemical. Absolute ethyl ether (Aldrich, Reagent) was used for extraction. All glassware was thoroughly cleaned and ultimately rinsed with ether to eliminate the possibility of external lipid contamination. Small-angle x-ray diffraction spectra were collected for seven hours using a Kiessig low- angle camera from Richard Siefert. Ni-filtered Cu radiation was used and the reflections determined by a Tennelec position-sensitive detection system (Model PSD-1100). RESULTS Normal stratum corneum gave a characteristic broad diffraction peak of moderate in- tensity in the range of 50-80 • (Figure lo). No other bands were distinguishable over the coml•lete available range of 20- 160 A for collection times of twelve hours. This 50-80 A band was removed both by washing and by extraction with ether as seen in Figure 2.

STRATUM CORNEUM X-RAY DIFFRACTION 351 600 400 oøO o o o o o o o o Oo o o o o o • [] Uoo D o a a a oo_oo o o o o o ß ß ß ! sb ' eo INTERLAYER SPACING (•) Figure l. Effect of G2 on x-ray diffraction spectrum of washed skin. O, untreated normal stratum corneum [U, washed normal stratum corneum ', washed normal stratum corneum treated with G2 for one hour O, region of complete spectral overlap. Treatment with G2 produced a weak diffraction from 30-45 fk. The intensity of this diffraction increased slightly as the treatment duration increased from one hour to thirteen hours (Figure 3), while the intensity of the 50-80 /k band significantly de- creased for the same change in treatment duration. Treatment of the stratum corneum with soy oil for one hour produced a narrower, slightly shifted diffraction peak from 60-80 fk. This spectrum is compared to the normal stratum corneum pattern in Figure 4. Neat G2 produced no diffraction pattern. When washed stratum corneum was treated with G2, the spectrum from 45 to 90 • was indistinguishable from the pattern of washed stratum corneum (Figure 1), while the broad G2 peak from 30-45 fk was still present. By washing the stratum corneum after a one-hour treatment with G2, a spectrum indistinguishable from that of washed normal stratum corneum resulted for the entire 20-90 A range. DISCUSSION The 50-80 • band is due to lipid structures contained within the stratum corneum. This is based on similarities between the 50-80 •k thin skin band found in the present investigation and Swanbeck's 40-60 •k thick skin band (1). The removal of the band by ether extraction (Figure 2) supports the conclusion of the presence in both thin and thick skin of a lipid layer giving an interlayer spacing of the magnitude of 60 •. The enhanced intensity at spacings in excess of 80 •k for the ether-extracted sample was not