j. Soc. Cosmet. Chem., 37, 191-198 (May/June 1986) !n vivo cutaneous distribution of linoleic acid following topical application in the hairless rat J. WEPIERRE and M. CORROLLER, Laboratoire de Pharmacologie, Universitg Paris-Sud, rue J. B. Clgment, 92290 Chatenay Malabry, France D. DUPUIS, A. ROUGIER, and C. BERREBI, Laboratoire d'Absorption percutange et d'Histologie, Centre de Recherche Fondamentale de l'Oreal, 1 avenue Saint Germain, 93601 Aulnay sous Bols, France. Received December 30, 1985. Synopsis Hairless rats were treated by single or iterative applications on the skin with an emulsion containing 153 of '4C labeled linoleic acid in order to test whether this compound becomes localized in epidermal and derreal layers. The distribution of radioactivity within the skin was investigated in the horny layer by stripping and in the epidermis and the dermis by cutting slices parallel to the cutaneous surface. In the horny layer, total amounts of labeled products remained constant 24 h after single application and 24 and 72 h after iterative applications. Thus a reservoir of linoleic acid was demonstrated in the stratum corneum. In the epidermis and dermis, the concentration of linoleic acid increased with time. It was always smaller in the epidermis than in the dermis where storage in the vicinity of the sebaceous glands appears to Occur. The retention of linoleic acid in the horny layer and in the sebaceous area of the dermis might explain in part the findings of others that it has a specific localized action in the skin appearing to correct the cutaneous symptoms in essential fatty acid deficient animals. INTRODUCTION Linoleic acid is an important essential fatty acid (EFA). Its bioconversion to •/linolenic acid is the first absolute step leading to arachidonic acid synthesis (1). In some patho- logical cases, this bioconversion is defective so that chronic squamous dermatosis has been observed in man (2). In EFA-deprived animals, several disorders of the keratiniza- tion processes appear in the epidermis such as acanthosis, hyperkeratosis, and a high level of transepidermal water loss (3-6) while DNA synthesis increases (3,6,7). More- over, sebaceous glands are hypertrophied. (!,6). Topical application of linoleic acid (or its metabolites) may correct these deficiency effects (8-11). It has been shown that percutaneously applied •/ linolenic acid in the hairless rat in vivo (12) or linoleic acid in man in vitro (13) were locally concentrated in epidermal and dermal tissues while their plasma and urine levels stayed very low. In order to more completely test EFA-specific action, we have investigated the linoleic 191

192 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS acid distribution in the cutaneous tissue of hairless rats, in vivo, after single and repeti- tive applications. MATERIALS AND METHODS ANIMALS Hairless Sprague Dawley male rats, IOPS (IFFA CREDO), weighing from 300 to 350 g, were used. The animals, provided with A03 food (UAR), were kept at 22øC with light provided for 12-h periods. PREPARATION OF THE LINOLEIC ACID EMULSION An isotopic dilution in toluene of 1-14C labeled linoleic acid (56 mCi/mM, Amersham France) was performed with vitamin F • containing 53% of linoleic acid. The radio- chemical purity of the labeled compound, checked before use by thin layer chromatog- raphy, was greater than 98%. An oil/water emulsion (Nutribel ©, Lanc6me) containing 2% (w/w) of the labeled vitamin F was prepared so its final radioactive concentration reached 125 IxCi/g. DISTRIBUTION OF 14C LINOLEIC ACID UNDER THE APPLICATION AREA Single application procedure. 100 mg of emulsion were spread upon 2.54 cm 2 of the dorsal skin of the animal and allowed to remain for 2, 6, or 24 h. To prevent contamination by self-licking or scratching, a metallic protection device was fixed above the applica- tion area, on the animal which was previously anesthetized by an IP injection of Nem- butal (30 mg/kg). Six rats were used for each time of application. Iterative applications procedure. Fifty mg of emulsion were applied on six rats according to the single application procedure. The applications were repeated daily for six days with no product rinse off or wipe off between applications. After the last treatment, the animals were divided into two groups kept respectively for 24 h and 72 h longer before sacrifice. Distribution studies. After each defined period, the rats were sacrificed by decapitation and the treated skin was cut off. The excess emulsion was wiped off, taking care not to spread it out of the application area. The underlying stratum corneum was removed by twenty strippings with adhesive tape. Three biopsies (0.3 cm 2) were punched out from the frozen skin and fixed on a freeze microtome (Cryo-cut, American Optical). Using the method previously described by Schaefer and Stuttgen (14), tissue slices of 20-lxm thickness or 40-lxm thickness when the dermis was reached were cut parallel to the cutaneous surface. The strips and slices were each placed in counting vials filled with 15 ml of toluene scintillator (Packard) and shaken for 12 h to extract radioactive material which was assayed in a liquid scintilla- tion counter (Packard Tricarb 3330). Essential fatty acids mixture.