ABSTRACT: This study sought to obtain internal wool lipid ex- tracts rich in ceramides from different wool types. Extraction methods, i.e., Soxhlet extraction with different organic solvents and supercritical fluid extraction with CO 2 using several polarity modifiers such as MeOH or EtOH, were optimized. The internal wool lipid content varied from 0.2 to 1.9% (based on wool weight) with a ceramide content ranging from 15 to 30% (based on extract weight). The Spanish and Russian Merino wool extracts were the richest in ceramide compounds. TLC–FID was used to quantify the different internal wool lipid extracts. A new experi- mental protocol that enabled us to identify most of the different ceramide types is presented. These internal wool lipid extracts, especially the ones with a high ceramide content, may be re- garded as an alternative source of animal ceramides, which could be of value in the cosmetic and dermopharmaceutical industries. Paper no. J10013 in JAOCS 79, 1215–1220 (December 2002). Wool is a natural fiber composed mainly of protein with an ex- ternal lipid content (lanolin) and with a minor internal lipid con- tent (1.5%), which could arouse considerable interest due to its high proportion of ceramides. Internal wool lipids (IWL) are rich in cholesterol, FFA, cholesteryl sulfate, and ceramides and resemble those from membranes of other keratinic tissues such as human hair or the stratum corneum (SC) of skin (1,2). This particular composition allows a highly ordered arrange- ment of lipids known as lamellar lipid bilayers. The intercellu- lar lipids of the SC, especially the ceramides, play an important role in the barrier function of the skin, preventing penetration of external agents and controlling the transepidermal water loss to maintain the physiological skin content of water (3). Recent studies have demonstrated that topical application of optimized molar ratio mixtures of SC lipids (SCL) on insulted skin accel- erates the barrier recovery process (4,5). In fact, the composition of IWL, similar to the ones present in the SC (1), allows for the formation of liposomes with a sta- ble bilayer structure (6–8). Furthermore, topical application of IWL liposomes on intact and disturbed skin has been demon- strated to improve barrier skin properties (9). Accordingly, IWL could be regarded as a new natural extract, beneficial for topical application and suitable for incorporation into pharmaceutical or cosmetic formulations in the treatment and care of skin. Most ceramides used in cosmetic or dermatological for- mulations are biotechnologically synthesized, but they do not have the identical composition or the variety of those present in keratinized tissues such as skin, hair, or wool. Recent physicochemical studies based on intermolecular and in- tramolecular lipid organization provide a basis for specula- tion on the particular role of each ceramide species in SC organization and function (10,11). This demands the full mix- ture of ceramides with the composition and proportions simi- lar to the ones present in the skin. The aim of this work was to obtain IWL extracts rich in cer- amides from different types of wool. The extraction methods, i.e., Soxhlet extraction with different organic solvent mixtures or supercritical fluid extraction (SFE) with CO 2 and several po- larity modifiers, were optimized to achieve wool lipid extracts similar in composition to SCL. Moreover, TLC–FID was used to quantify the different IWL extracts. A new experimental pro- tocol that enabled us to identify the majority of different ce- ramides is presented. EXPERIMENTAL PROCEDURES Raw Merino wool samples from Australia (19.0 μm fiber di- ameter, 56.4 mm fiber length), Spain (22.9 μm, 51.6 mm), South Africa (19.8 μm), and Russia (22.3 μm) and Romney wool samples from New Zealand (38.0 μm) were supplied by SAIPEL (Terrassa, Spain). In an earlier study, surface lipids and contaminants were re- moved from the raw, unprocessed Australian Merino wool fol- lowing a sequential extraction procedure with t-butanol (5) and heptane (2.5 h) and rinsing with water (12). In the following studies, all wool samples were industrially cleaned with sodium carbonate and a nonionic polyoxyethylene surfactant to remove lanolin before being mechanically shaken and rinsed with water to eliminate vegetable matter and dust. Prior to in- ternal lipid extraction, the wool was equilibrated in a condi- tioned room (20°C, 60% RH). Soxhlet extraction was performed for 5 h with a solvent ratio of 1:30 in pure organic solvents such as acetone (b.p. 56.2°C), methanol (b.p. 65.2°C), and the azeotropes acetone/methanol (88:12, vol/vol; b.p. 56.1°C), chloroform/ methanol (79:21, vol/vol; b.p. 53.4°C), and chloroform/ methanol/water (85:13.7:1.4, by vol; b.p. 52.3°C). The lipid extracts were concentrated and stored in chloroform/methanol (2:1, by vol) at 6°C. Aliquots were dried and weighed, and lipid extraction percentages were determined (1). SFE was performed using a dual-syringe pump (SFC 3000; Fisons, Milan, Italy) for the delivery of CO 2 (SFE 99.998%; Praxair España, Barcelona, Spain) and a modifier (MeOH or EtOH; Merck, Darmstadt, Germany) as described elsewhere Copyright © 2002 by AOCS Press 1215 JAOCS, Vol. 79, no. 12 (2002) *To whom correspondence should be addressed at Instituto de Investiga- ciones Químicas y Ambientales de Barcelona (IIQAB) (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain. E-mail: lcnesl@cid.csic.es Extraction and Analysis of Ceramides from Internal Wool Lipids L. Coderch*, J. Fonollosa, M. Martí, F. Garde, A. de la Maza, and J.L. Parra Instituto de Investigaciones Químicas y Ambientales de Barcelona (IIQAB) (CSIC), 08034 Barcelona, Spain