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

184 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Figure 1. Freeze-fractured lanolin showing emulsified transepidermal water. base of the desiccator held a saturated solution of calcium chloride dihydrate to provide a relative humidity of 38% within the chamber. The desiccator was kept in a constant- temperature room at 37øC. Treated skin was sampled after 16 hours. At the end of the treatment period, skin was removed from the holders, and small, roughly cubical pieces about 1.5 mm per side were cut for fixation by the standard operating procedure as follows: Primary fixation: Buffer wash: Secondary fixation: Wash: Dehydration: Infiltration: Embedding: Sectioning: Staining: Observations and microscopy: Freeze-substitution medium: 3% Glutaraldehyde solution in phosphate buffer 0.1 M Mixed phosphate buffer at pH 7.2 1% Solution of osmium tetroxide in buffer Distilled water Ethanol series 50%, 70%, 90%, 100% (twice) Propylene oxide 50:50 Propylene oxide: resin 100% Resin at 37øC 100% Resin 100% Resin in flat embedding molds Polymerized at 65øC By Reichert OMU2 ultramicrotome Some sections unstained, others stained in uranyl acetate and lead citrate solutions JEOL 1200 EX transmission electron microscope Methyl alcohol (100%) 100 ml