DAMAGED HAIR AND CERAMIDE-RICH LIPOSOMES 567 HAIR CHEMICAL TREATMENTS The hair sample used in this work was a virgin natural brown hair and was supplied by De Meo Brothers (New York). The hair was chemically damaged by different cosmetic treatments such as perming, bleaching, and relaxing: Permed hair. Hair (0.5 g) was placed in a perming solution (8% thioglycollate, pH 8) for three hours on a rocking table. It was then rinsed with water and placed in a neu- tralizing solution (2.5% H2O2, pH 3) for 30 minutes. It was then rinsed again and dried in air. Bleached hair. Hair (0.5 g) was placed in a bleaching solution (9% H2O2, pH 8.3, 1% am- monium persulfate) for three hours on a rocking table. It was then rinsed with water and dried in air. Relaxed hair. Hair (0.5 g) was placed in a 2.5% NaOH solution for 30 minutes on a rocking table and then rinsed with water for fi ve minutes. Next, it was placed in a 9.5% citric acid solution for fi ve minutes and then rinsed with water for ten minutes. IWL APPLICATION Internal wool lipids obtained from Spanish Merino wool fi bers were submitted to metha- nol extraction at 56°C as described in reference 11. The amount of lipids extracted was not very high (1.4% on wool fi ber), but this may be suffi cient for cosmetic utilization due to the high amount of wool processed for textile purposes. Aliquots were dissolved in chlo- roform/methanol 2:1 (v/v) and evaporated to dryness under a stream of dry nitrogen to form a thin fi lm on the fl ask. The fi lm was hydrated with 0.9% NaCl solution to give a fi nal lipid concentration of 10 mg/ml. Liposomes were formed by sonication of the sus- pension in a sonicator, Labsonic 1510 (B. Braun, Melsungen, Germany), at 100W for about 15 minutes. The temperature was maintained at 60°C by a thermostatic bath, Ul- traterm 6000383 (Selecta SA, Barcelona, Spain). These IWL liposomes (1%) were applied to untreated and chemically treated hair as fol- lows: 1-g samples of hair were soaked in a volume of 10 ml of 1% IWL liposomes for ten minutes at 40°C. Then the hair fi bers were washed with distilled water and dried. This procedure was repeated ten times. LIPID EXTRACTION Lipids of untreated and chemically treated hair samples that were subjected to IWL lipo- some application and those that were not subjected were extracted. The extraction was made at room temperature for two hours with mixtures of chloroform-methanol (2:1, 1:1, and 1:2, v/v). These extractions were repeated for one hour with the same mixtures and with methanol overnight. The different extracts were then combined, concentrated, and dissolved in chloroform-methanol (2:1) prior to analysis. To evaluate the total amount of lipids extracted, 1 ml of each of the extracts was evaporated to dryness in a P2O5 desicca- tor and weighed to a constant weight.

JOURNAL OF COSMETIC SCIENCE 568 HAIR LIPID ANALYSES Lipid analyses of the different extracts were performed by thin-layer chromatography cou- pled to an automated fl ame ionization detector (TLC-FID), Iatroscan MK-5 analyzer (Iatron, Tokyo, Japan), following the analysis methodology referred to in earlier works (12,18). Sam- ples (15–20 μg) were spotted on Silica gel S-III Chromarods by means of a precision 2-μl Hamilton syringe coupled to an SES (Nieder-Olm, Germany) 3202/15-01 sample spotter. An analysis of apolar and polar compounds was performed by developing the rods ini- tially to a distance of 10 cm with n-hexane/diethyl ether/formic acid (53:17:0.3, by vol) to separate the apolar and polar lipids. After a partial scan of 72% to quantify and elimi- nate the apolar lipids, a second development, again to a distance of 10 cm, was performed with chloroform/n-hexane/ methanol/acetone (55:5:3:7, by vol) to separate the ceramides. Following a partial scan of 85% to quantify and eliminate the ceramides, a third develop- ment, again to a distance of 10 cm, was performed with chloroform/methanol/formic acid (57:12:0.3, by vol) to separate and quantify, after a total scan of 100%, the glycosilcer- amides and sterol sulfate. After each elution, the rods were heated for fi ve minutes at 60°C to dry the remaining solvent. The experimental conditions were: air fl ow 2000 ml/ min, hydrogen fl ow 160–180 ml/min, and scanning speed 2–3 mm/s. Data were pro- cessed with Boreal version 2.5 software. These procedures were applied to the following standard compounds: palmitic acid and cholesterol from Fluka Chemicals (Buchs, Switzerland), and type II ceramides, cholesterol ester, galactoceramides, and sodium cholesteryl sulfate from Sigma (St. Louis, MO), to determine the corresponding calibration curves for quantifi cation of each compound. MOISTURE RETENTION BY THERMOGRAVIMETRIC ANALYSIS (TGA) TGA provides a measurement of the weight loss of the sample as a function of time and temperature. Before measuring, the hair was kept in a humidity-controlled room (55% RH) for 24 hours. All investigations were performed on a TGA instrument (TG-50, Met- tler Toledo, Spain). Samples consisted of short fi ber snippets (approx. 2 mm in length). Approximately 6 mg of the hair samples were packed into a 70-μl TGA pan. Samples were transferred to an aluminum crucible, weighted, and sealed for an elapsed time of 30 sec- onds. The crucible was then placed in the TGA balance where it was pierced. The heating rate used in this study was 20°C/min, with a fl ow rate of nitrogen gas of 200 ml/min. The internal and external water contents were evaluated separately. The hair sample was heated from 25° to 65°C, and this temperature was maintained for 40 minutes, which is assumed to be the normal temperature used by a hair dryer, and the water content found corre- sponded to the external water content (19). Again the temperature was increased, from 65° to 180°C, and was kept for 30 minutes to measure all the water contained in the hair (in- ternal water content). TGA curves showed the amount of water in the samples. STRENGTH MEASUREMENTS Stress-strain test. Five fi bers were randomly taken from samples previously conditioned for 48 hours in a standard atmosphere (20°C, 65% RH) and centrally attached to a pair of