NATURAL MOISTUR[ZING AGENT IN SKIN 355 Table II Moisture-Binding Ability of 2-Pyrrolidone-5-Carboxylie Acid-- Its Sodium Salt and Glycerol Per Cent Moisture Uptake at Compound 31% R.H. 58% R.H. Pyrrolidone carboxylic acid 1 1 Sodium pyrrolidone carboxylate 20 61 Glycerol 13 35 higher humidities, even dissolved in its own water of hydration. Table II compares the humectancy of PCA with that of its sodium salt and with the commonly used humectant, glycerol. It thus appeared that the ionic form of PCA could certainly partially explain the high humect- ancy of the callus water extractables. Experiments were, therefore, in- itiated to explore in a more definite manner the role of PCA as a naturally occurring moisturizing agent in skin. ANALYSIS OlV SKIN SAMPLES FOR PCA Samples of stratum corneum and callus were analyzed for pyrrolidone carboxylic acid using paper chromatographic techniques. Samples of stratum corneum were obtained by first clipping the hair from the back of the hand using an electric razor. Then, using a Spencer microtome knifeholder containing a razor blade, the skin could be carefully scraped from the backs of the hands, yielding up to 20 mg of stratum corncure. The skin scrapings were submitted to two one-hour extractions at room temperature with ethyl ether. Five-tenths milliliter of water was then added to the dry scrapings, and an overnight extraction was performed. After centrifugation, 50X to 200X of aqueous extract was used for two dimensional descending paper chromatography. The solvent systems used were butanol-acetic acid-water (12:3:5) and phenol-water-ammonia (prepared by adding 1000 g phenol to 250 ml water 2 ml of concentrated ammonium hydroxide is then added to 200 ml of the aforementioned solution). Generally, the first solvent was employed overnight and the second solvent for seven hours. The amino acids were identified with a 0.5% ninhydrin in butanol spray. In these studies the PCA was identified with a more sensitive and specific test. The chromatogram was placed in a chlorine atmosphere for twenty minutes, then blown free of residual chlorine and sprayed with a starch-iodine reagent (21). PCA appears as a brown spot. For comparison, water extracts of callus were also run.

356 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The paper chromatograms from stratum corneum and callus were quite similar. PCA was found in both chromatograms. Identification of amino acids and PCA were accomplished by establishing reference chromatograms. Thus, in agreement with Pascher (6), PCA was dem- onstrated to be a normal component of stratum corneum. While PCA can be shown to be a naturally occurring moisturizing agent in skin, its actual role in maintaining the skin in a hydrated state remained to be determined. In order to establish whether a relationship exists between PCA content of the skin and the skin's moisture binding ability, an analytical method to determine the amount of PCA in skin scrapings was developed. Aliquots of the water extract of skin scrapings were submitted to ion- exchange chromatography using a 95 cm X 1.5 cm column of Dowex 50--X4, 100-200 mesh resin in an 0.1N citrate buffer at pH 3.0, using the general method of Moore and Stein (22). On this basis, the first 125 ml of effluent contained all of the PCA and, possibly, a small amount of aspartic acid. Aliquots of this effluent were analyzed with ninhydrin reagent (23) before and after hydrolysis with HC1. Since PCA produces no color with ninhydrin until after it is hydrolyzed with HC1, the difference between the ninhydrin value before vs. that after HC1 hydrolysis yields the amount of PCA. Typical recoveries of standard PCA samples were in the range of 93-95%. A fourteen-member panel was formed, and skin scrapings from the backs of the hands were obtained from each subject. The moisture- binding abilities of the scrapings were determined at 95% relative hu- midity. Water extracts of the samples were then prepared, and total ninhydrin-positive material was determined on an aliquot. The re- mainder of the extract was submitted to ion-exchange chromatography and PCA assay. The results are presented in Table III. Statistical analysis of the data indicated that a reliable linear rela- tionship is exhibited between moisture-binding ability and the PCA con- tent of the skin scrapings. The product moment correlation coefficient, calculated from the data, gives r = 0.652. This value differs signifi- cantly from zero at the 0.98 level and thus points to the existence of a re- lationship between the variables studied. Some idea of the predictive ability of this relationship is gained by noting that 42.5% (= r s) of the observed variation in the data is attributable to linear regression (24). The fourteen pairs of values are presented together with the two regres- sions in Fig. 1.