ACID NUMBER AND SPREADING INDEX OF THE HUMAN SKIN SURFACE LIPIDS By I2RA•Z HERRMA•N, M.D., W. M. Coo•, M.D., L. HARBER, M.D., R. SCHEa, M.D., and L. MANDOL* Presented October 8, 1958, Seminar, New York City IN PREVIOUS extensive investigations we studied the amount ("casual level") of the skin surface lipids under various conditions with special attention to their distribution in different areas of the human body surface (1, 2, 3, 4, 5). More recently we investigated the free fatty acid contents of the respective samples, as well as the spreading index (6, 7). The latter term was introduced for the cm. 2 area of skin surface covered by 1 mcgm. of the lipid under investigation--when a given quantity of it is, under standardized conditions, placed on an aqueous surface (6). Assays of the lipid spread on water had been advocated by Jones and collaborators for determination of the lipid quantity (8, 9, 10), since they presumed that the free fatty acids control the spreading ability of the samples and that their proportion in the skin surface lipids is constant. Our investigations had revealed (1) that the spreading index differs from one individual to another for samples collected from a given site so that it is not generally feasible to utilize the index for determination of the lipid quantity: (2) that there are distinct regional differences for the lipids collected from different test areas and (3) that for certain test areas the index tended to increase during the warmer season. Our assays of the acid number which had been carried out on only a minor scale have been greatly expanded. It is the object of this presenta- tion to report on the results obtained to date in addition to those previously published. EXPERIMENTAL Method. The methods of assaying lipid quantity and spreading index were the same as described in our previous reports (1, 2, 6, 7). In order to obtain sufficient lipid quantities per sample for each of the * Dept. of Dermatology and Syphilology, New York University Post Graduate Medical School (Dr. Marion B. Sulzberger, Chairman) and the Skin and Cancer Unit of University Hospital, New York 3, N.Y. 88

HUMAN SKIN SURFACE LIPIDS 89 three pertinent assays--weight, acid number (A.N.) and spreading index (S.I.)--every sample was processed as follows. After the total volume (10 ml.) of fat free ethyl ether used to collect the material in our cylindrical receptacle had in a 10 ml. measuring flask slowly evaporated at 40øC., the residue was redissolved in 10 ml. of fat free petroleum ether. Under precautions necessary to avoid transfer of scales, etc. (6, 11), 5 ml. of this volume were used for gravimetric deter- mination of the lipid quantity,*t 3ml. for assaying the acid number and the remainder for assaying the spreading index. The A. N. was assayed in a manner similar to the procedure of Hodgson- Jones and Wheatley (12), who in turn rather closely followed the method described by Moyle, Baldwin and Scarisbruck (13). However, because of the much smaller lipid quantity obtainable from the different sites to be investigated separately, several modifications had to be introduced. Thus, for the titration it was found necessary to reduce the concentration of KOH in methanol from 0.005 N to 0.002 N.• Smaller volumes, more- over, of solvent (chloroform) and of indicator had to be employed. Briefly, we proceed in the following manner: The 3 ml. aliquot of the solution in petroleum ether is placed in a wide- necked 30 ml. Erlenmeyer flask and evaporated again at 40øC. After cooling, 2 ml. of chloroform and one drop of 0.04 per cent cresol red in methanol are added to the residue. The solution is carefully shaken. In order to remove the COs present a stream of gaseous nitrogen is carefully bubbled through the chloroform solution for thirty seconds prior to titra- tion. This is continued also through the period of titrating it must be done slowly to avoid volatilization. The titration out of a microburette (one drop equals 0.01 ml. of the KOH solution in methanol) likewise is carried out slowly, since neutralization and color change, as a rule, do not occur immediately. The turn from greenish-yellow to a reddish-yellow is the end point. The addition of one drop in excess produces a distinctly purple color. Under our laboratory conditions it is necessary to titrate about four "blanks" of chloroform, 2 ml. each, immediately prior to any assaying of samples. There are day to day variations of the blank titres from 0.02 to 0.03 or even 0.04 ml. of KOH, which must be deducted from the volumes required for neutralizing the solution under test. From time to time it has been useful to perform control titrations of' different known concentrations of stearic acid in chloroform. * TCY (Key Board) Balance, William Ainsworth & Sons, Denver, Colo., sensitivity 0.01 mg. t Weighing of the residue was performed in 6 ml. aluminum foil cups, 3 cm. in. diame- ter, "Crinkle Cups," Muller Paper Goods Co., Inc., Long Island City, N. ¾. it Prepared by courtesy of Joseph R. La Vieres, La-Mar Laboratories, Inc., New York, N. ¾.