THE CEMENTING SUBSTANCE OF HUMAN HORNY LAYERS* By PETER FLESCH, M.D., PH.D.• Presented November 28, 1961, New York City I• PREVIOUS reports (1-3) we have postulated the existence of a cementing substance between the epidermal cells. We suggested that this substance was an acid mucoprotein. The theory was advanced that during normal keratinization this material is largely decomposed, while in patho- logic scales its degradation was incomplete. It is postulated that the nor- mal process of desquamation leads to the shedding of invisible particles, while pathologic horny layers fail to disintegrate and are cast off as large, coherent scales. We ascribed this anomaly to the persistence of a mucoid cementing substance in the horny layer. The evidence for this theory was based on histochemical observations and chemical analyses. Psoriatic scales are strongly basophilic this basophilia can be correlated with their hexosamine content and is reduced by elastase, a mucolytic enzyme. In chemical analyses it was possible to isolate from aqueous extracts of whole human epidermis with a thin horny layer a com- ponent with a 5 per cent hexosamine content, which characterizes it as a mucoprotein. On the other hand, the same purification process of aqueous extracts of ether-treated callus yielded a component with a 12-fold reduced (0.4 per cent) hexosamine content. Similar material from patients with psoriasis and exfoliative erythroderma fell between these two extremes, with a hexosamine content of 2.5-4 per cent. Recently, from ether- treated scales of sunburn erythema we have recovered a substance with a hexosamine content of $ per cent such scales may be considered the end products of an accelerated keratinization process. Although these data strongly supported our view of an incompletely de- composed cementing material in some pathologic scales, recent observations could not be explained with these findings. Using x-ray diffraction meth- ods, Swanbeck (4, 5) showed that the individual keratin filaments in nor- mal horny layers aggregated to form bundles, while in psoriasis this aggrega- tion was incomplete. Between the fibrils in all horny layers he found a sub- * This study was supported by Grant RG-7533-C from the National Institutes of Health, Bethesda, Md. t Dept. of Dermatology, University of Pennsylvania School of Medicine, Philadelphia 4, Pa. 113

114 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS stance extractable with a mixture of ether and alcohol (4), believed to be a lipid (5). Equally novel were the findings of a study of hair in Great Britain (6). Exhaustive treatment of human hair with polar solvents, such as alcohol, yielded a fatty acid-protein complex. This material, con- sisting of 20 to 30 per cent fatty acids and 60 to 70 per cent protein, pro- tected the hair against enzymatic digestion by papain. Very suggestive are also recent electron microscopic pictures of human epidermis, which have finally resolved the ultrastructure of the horny layer (7). They have revealed the existence of a strongly electron dense, osmiophilic substance between the keratin filaments which could be a lipid. In view of these data, we undertook a study of the alcohol-extractable substance of normal and pathologic horny layers. It has been generally assumed that exhaustive treatment of horny layers with ether or acetone removed all fatty substances. This belief is unfounded. From presum- ably "defatted" (i.e. ether-treated) horny layers we were able to remove a fatty substance by prolonged extraction with alcohol. Although chemical analyses of this substance are in a preliminary stage, the data were thought to be of sufficient interest to cosmetic chemists to warrant their presenta- tion at this time. EXPERIMENTAL Five samples of callus and scales from patients with various dermatoses (five psoriatics, two with congenital ichthyosiform erythroderma and one with exfoliative dermatitis) were ground in a Wiley mill to 60 mesh size and exhaustivel? extracted and washed with ether. This cleaning process lasted for several days with numerous changes of ether. The scales were dried and extracted for twenty-four hours with water in a shaking appa- ratus. After drying, 2 to 6 gm. samples of this powder were placed into absolute alcohol and incubated for two weeks at room temperature with fre- quent shaking. Preliminary analyses of aliquots taken at various intervals have shown that two weeks were needed to complete the extraction. The alcohol was filtered and evaporated to dryness. Upon concentra- tion, the alcoholic extract turned yellow. After drying, a fatty residue was obtained. The amounts of this material, as related to dry scales, were about 2 per cent in callus and psoriatic scales. Chemical analyses of this material included estimations of protein, free amino nitrogen, choline, phosphorus and hexosamine before and after hy- drolysis. Purification was attempted in a mixture of ethanol, chloroform and water. RESULTS From all scales analyzed, alcohol extracted a yellowish lipid which was soluble in ethanol, chloroform or pyridine, somewhat less soluble in meth-