NATURAL MOISTURIZING AGENT IN SKIN 353 samples in small desiccator chambers, each containing an appropriate solution to maintain the desired relative humidity. Data obtained in these experiments (Table I) closely paralleled re- sults obtained by other investigators (2, 20). Thus, removing the water solubles from callus greatly diminishes the ability of the callus to hold moisture, and the water extractables can be shown to be hygroscopic. In order to study further the nature of the water-soluble materials responsible for this humectancy, the water solubles from callus were frac- tionated on ion exchange resin columns, and the fractions obtained were evaluated for humectancy. Columns containing C-G 400 Type I Amberlite resin were converted to the acetate form. Next, the water-soluble extract, dissolved in a small volume of water, was placed on the column. After washing the column thoroughly with water, the column was eluted with 11% for- mic acid, and fractions of the acidic eluant were taken to dryness and evaluated for humectancy. The humectancy of these fractions was far below that expected based on the humectancy of the total extract. One possible reason was that the fractionation procedure had converted one or more of the moisturiz- ing agents to a nonhygroscopic form. These fractions were then subjected to paper chromatography. From 202, to 50X of sample were spotted on No. 1 Whatman chroma- tographic grade filter paper, and one dimensional descending chroma- tography was run using a butanol-acetic acid-water mixture (12: 3: 5). After drying in a vacuum oven for several hours, the chromatograms were developed by spraying with a solution of ().2c•o ninhydrin in bu- Table I Moisture-Binding Ability of Callus Material Per Cent Moisture Uptake = Wt. of Water X 100 Wt. of Dry Sample 37% R.H. 70% R.H. Unextracted callus Extracted callus Callus extractables 9.2 16.8 5.8 12.3 ß.. 45.0

354 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS tanol to detect amino acids or by dipping into a solution of potassium iodide, potassium iodate, and starch. (A solution of 2% potassium iodide and 4% potassium iodate is mixed with an equal volume of 2% starch. A trace of NH4OH is added to reduce background color.) Intense blue colors appear using this test where relatively large amounts of acidic material are present on the chromatogram. The chromatograms indicated that, in addition to ninhydrin-positive amino acids, there was also present a ninhydrin-negative, potassium iodide-potassium iodate-starch positive acid. Since Pascher (9) had re- ported the existence of an acid, 2-pyrrolidone-5-carboxylic acid, in ex- tracts of skin, a known sample of this material was chromatogrammed along with the unknown acidic material. Upon developing the chroma- tograms, both the known sample of 2-pyrrolidone-5-carboxylic acid (PCA) and the unknown sample had the same Rf value. To confirm that the unknown acid material was PCA, a known sample and the un- known acid were hydrolyzed with HC1. The hydrolyzed samples were rechromatogrammed, and upon developing it was found that in both in- stances the PCA spot had disappeared and a new ninhydrin-positive spot which corresponded in Rf value to glutamic acid had appeared. Since PCA on acid hydrolysis is converted to glutamic acid, it was concluded that the unknown acid in the water-soluble fraction was PCA. Initial studies on the humectant properties of PCA indicated that this material was nonhygroscopic. There was a possibility, however, that the fractionating procedure had converted some of the moisturizing agents into a nonhygroscopic form. This possibility was investigated further with respect to PCA. Since PCA can arise as a result of cycliza- tion of glutamic acid, the humectancy of glutamic acid was evaluated but was also found to be very low. The pH of skin and of water extracts of skin are usually between pH 5-6. At this pH, amino acids are present in their zwitter-ion form. However, PCA, which has no free amino group, is present largely in the ionic form at this pH. Indeed, a titration curve of PCA rs. pH indicates that at pH 5.3 99% of the acid is in the sodium salt form. During the [ractionation procedure, the amino acids are converted to the formic acid salts, which upon drying are converted back to the free amino acids. Any salt of PCA in the aqueous extract, however, is converted to the free acid during isolation. Thus, the amino acids undergo essentially no change during isolation, while PCA is converted from the salt to the acid form. The sodium salt of PCA was, therefore, prepared and its humectancy evaluated. This compound was found to be highly hygroscopic and, at