182 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 4. The penetration of topically applied lanolin down as far as the stratum lucidum has been demonstrated by tape stripping and chemical analysis (5). 5. Lanolin has some components in common with the natural intercellular lipids, such as free and esterified cholesterol and free and esterified fatty acids some lanolic acids are hydroxy acids, and others are esterified with diols to form diesters with two long acyl chains, as in the case of ceramides. This earlier work led to the hypothesis that the moisturizing effect of lanolin was a result of absorption into the stratum corneum, where it reduced TEWL by partial occlusion of the internal water pathways or other means, and possibly also of lanolin within the stratum corneum absorbing natural epidermal moisture and thus acting as a reservoir, releasing water if the moisture gradient across the stratum corneum should favor such loss. The present work explores this hypothesis in three separate stages. EXPERIMENTAL STAGE 1 This was an initial in vitro exploratory stage involving the application of lanolin to excised skin, which, after suitable fixing and sectioning, was examined by transmission electron microscopy. The lanolin was insufficiently electron-opaque, however, to be differentiated from other structures within the stratum corneum specimens. Therefore, this approach was discontinued and details are not reported here. STAGE 2: OUTLINE This stage of the study was subdivided into two sections involving different method- ologies. Section 1 consisted of treating the surface of human skin in vivo with anhydrous lanolin, and after a time removing the surface lanolin and examining it by freeze-fracture and scanning electron microscopy in order to study the microstructural characteristics. Section 2 involved the doping of lanolin with lead to increase its electron opacity before application to human skin in vitro at a specific loading. Prepared specimens of treated skin sections were then examined by transmission electron microscopy. The principle of lead doping to increase electron opacity had been previously used by Ghadially and co-workers (6) in studies of skin penetration by petrolatum. In those studies, doping was carried out by adding lead nitrate in the form of a physical sus- pension. Lead nitrate is soluble in water but insoluble in petrolatum and other lipids, and thus there is the possibility of migration of some of the lead salt out of the petrolatum into hydrous areas. In this part •)f our work, such a potential problem was avoided by doping lanolin with lead lanolate, which is the lead salt of the naturally occurring fatty acids of lanolin, at the level of 1.3% of lead as Pb. Lead lanolate has good solubility in lanolin but negligible solubility in water, and the possibility of migration into hydrous areas may be assumed to be negligible. There is at least the possibility of selective migration of lead salt into other lipids, but there seems to be no reason why these should have a greater affinity for lead salt than in the case of lanolin. Moreover, in a later section of

MOISTURIZING FUNCTION OF LANOLIN 183 this work, a different lead salt was used (lead oleate). Despite the fundamental differ- ences between oleate and lanolate salts, results appeared to be similar. In all stages of our work, the biological treatment and electron microscopy of specimens was carried out by Dr. Ashley J. Wilson at the Centre for Cell and Tissue Research, University of York, England, in cooperation with and under the sponsorship of West- brook Lanolin Company. Lanolin used in the work was anhydrous lanolin Eur. Pharm. the lead lanolate was prepared in house by base exchange between aqueous solutions of sodium lanolate and lead nitrate. STAGE 1, SECTION 1 Rlethodology. A 50-ram square of inner forearm of a male volunteer subject was lightly delineated and 50 mg of undoped lanolin was gently rubbed into the test area for one minute. After five minutes, small portions of lanolin were carefully scraped from the surface of the skin, avoiding any abrasion, and prepared for freeze-fracture studies. As controls, specimens of the original lanolin and of lanolin containing 25% w:w of water (as a mechanically prepared w/o emulsion) were similarly examined. Samples were put into a pair of hollow rivets held together by special forceps, the whole assembly being rapidly frozen by quenching in subcooled nitrogen at 69 K. Samples were fractured and replicated in a Leybold Heraeus Biotech 2005 freeze-fracture apparatus. Platinum car- bon was evaporated at an angle of 45 ø , with carbon from above to strengthen the replicas. These were then cleaned of lanolin in trichloroethylene, followed by 5% sodium hypochlorite solution, and examined in a JEOL 1200 EX transmission electron microscope. Results. Specimens of original anhydrous lanolin showed fracture surfaces composed of a mix of relatively amorphous solid lanolin and areas of crystalline lamellae thought to be fractions of lanolin that had been liquid at room temperature. Freeze-fractured 25% hydrous lanolin showed fracture surfaces of similar general ap- pearance but superimposed on it were spherical water droplets varying in diameter from 0.5 to 3 Ixm. Lanolin removed from skin after five minutes likewise showed spherical water droplets of 50 to 300 nm in diameter (Figure 1), presumably epidermal water which had passed from the skin surface into the lanolin layer where it formed a w/o emulsion. These droplets formed in vivo are of the same order of magnitude as the water spontaneously emulsified in lanolin in vitro, as previously reported (2). STAGE 1, SECTION 2 Rlethodology. Full-thickness skin resulting from breast reduction operations was used, after removing subcutaneous fat down to the dermis. Prior to actual use, the skin samples were kept moist by saline-soaked swabs. From each piece of skin, three circular specimens of 20-mm dlameter were punched, and two of these were clamped in holders with the dermis side of the skin held in contact with a wad of three discs of filter paper soaked in saline solution. The top plate of the holder ensured that a defined area of 1 sq cm was exposed to the atmosphere. About 4 mg of lanolin doped with lead lanolate was applied to each exposed area and spread evenly by means of a piece of surplus skin. The third disc of skin was also mounted in a holder but left untreated as a control. All three holders were placed in a 25-cm diameter desiccator fitted with a hair hygrometer. The