TRANSCUTANEOUS ABSORPTION OF VITAMIN A 175 min were applied without massage. Total gains in weight during test periods.of five weeks were 57 gm. for the former group of rats and 34 gm. for rats in the latter group. . During the first four weeks of the test period, the group of rats receiving topical applications of vitamin A with massage of the test areas of skin exhibited an average gain in weight which was approxi- mately equivalent t6 the result shown in Chart II for the first group of rats which had received topical applications to their skins of petro- latum containing 2100 units of vitamin A per gram. Results of the series of experi- ments which are presented in Chart III indicate that massage of the petrolatum, containing the concen- trate of vitamin A, enhances the utilization of the vitamin for growth by young rats maintained on a vita- min-deficient diet. The liver is the principal site of storage of the vitamin A which is in excess of the animal's immediate requirements. Two independent groups of investigators have re- ported that from 90 to 95 per cent of the total vitamin A of the rat's body may be found in the liver the re- mainder was located in lung, kidney, and adrenal tissues (6, 7). One group of workers observed that the amount of vitamin A stored in the liver paralleled the quantities ad- ministered orally but only from 10 to 20 per cent could be accounted for (6). In applications of the second criterion of transcutaneous absorp- tion of vitamin A, particular atten- tion was directed to the vitamin A contents of the test rats' livers and skins. After the animals had been anesthetized, at the time of sacrifice, their livers were removed in toto and weighed. After thorough mincing, the liver tissue was extracted with ethyl and petroleum ether under nitrogen. Sections of skin, weighing from approximately 1 to 2 gm. were dissected from the anterior abdom- inal wall and, in some experiments, from the backs of the rats and im- mediately frozen with liquid carbon dioxide. These frozen specimens were pulverized and then extracted as described for liver tissue. Determinations of vitamin A in livers and skins were carried out by a modification of the method which has been described by McCoord and Luce-Clausen (7) but U.S.P. Refer- ence Oil was utilized as the standard for colorimetric comparisons in place of the solutions of copper sulfate. All values for vitamin A in livers and skins are reported as International Units. Table 1 presents data for vitamin A contents of livers and skins of three of the groups of rats which had been maintained on the vitamin A- free diet but had not received topi- cal applications of vitamin A. Five rats were sacrificed at the end of their depletion periods, i.e., after they had received no sources of vitamin for thirty-nine days and ha4 exhibited declines in weight during the preceding seven days. Vitamin A contents of their livers varied from traces to 425 with an average of 216

176 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS TABLE 1--VITAMIN A IN LIVERS OF RATS MAINTAINED ON THE U.S.P. VITAMIN A TEST DIET WITH OR WITHOUT ORAL SUPPLEMENTS OF THE VITAMIN Groups Vitamin A Units Time of Av. Change per 100 Gm. Sacrifice, No. of in Body Wt. Days After Rats in During Test Abdominal Depletion ,Group ' Period, Gin. Livers Skin Depleted* 0 5 216 0•' Negative controls in Group V 24 6 --•1 69 0:1:' Positive controls receiving oral 35 4 +28 168 3 doses of U.S.P. Reference Oil * Sacrificed at the end of a period of 39 days, from weaning age, on the vitamin A-free diet. t Four rats gave no evidence of vitamin A in their skins 1 rat showed 6 units per 100 gm. Five rats gave no evidence of vitamin A in their skins 1 rat showed 3 units per 100 gm. : units per 100 gm. Within this ß group, it was not possible to demon- strate any quantitative relationship between the amounts of vitamin stored in the livers of these rats and their gains in weight during the de- pletion period. Six negative control rats in Group ¾ (Chart II) were sacrificed in a moribund state. These rats had been continued on the vitamin-free diet for a longer period then their litter mates which were sacrificed at the time of depletion. Of the six negative controls, two gave evi- dences of only traces of vitamin A in their livers, and for the remaining four rats, the vitamin A contents of the livers varied from 80 to 112 with an average of 69 units per 100 gm. Comparisons of the vitamin A contents of the livers of the negative controls with those of so-called de- pleted rats point to the conclusion that maintenance of rats on the basal, vitamin A-deficient diet for three weeks beyond the time of beginning the test periods reduced the liver stores of vitamin A coinci- dent with losses in body weight. The four animals included in the third group of Table 1 were repre- sentative of 12 rats in a reference group which had been used in a biologic assay of a preparation of petrolatum containing a concentrate of vitamin A. These four rats were continued on supplements of the Reference Oil for one week beyond the usual period of assay for vitamin A. During a test period of thirty-five days, these rats had been fed an amount of the U.S.P. Reference Oil which was adequate to yield, during the first four weeks, total gains in weight within the Pharmacopoeial requirements of 12 and 60 gin. (5). At this point, it is deemed advis- able to emphasize the fact that this range of gains in weight, prescribed by the Pharmacopoeia of the U.S. do not represent either maximum or optimum rates of growth of young rats. It is simply a criterion of the nutritional status in which the rapidly growing young rat is most responsive to variations in intakes of vitamin A. At the time of sacrifice, the livers of the four rats, which had received oral supplements of the Reference