HAIR LIPID COMPOSITION 3 from 1 to 81 years. About 200 fibers of 5-cm length taken from the proximal root end per individual (ca. 100 mg) were collected. The volunteers had not been exposed to any chemical treatments such as perming or bleaching for at least six months. Hair fibers were washed with plain shampoo twice and were then incubated with hexane for 5 min prior to extraction of hair lipids. EXTRACTION OF HAIR LIPIDS Each hair bundle washed with shampoo and hexane was cut into small pieces using scissors. They were immersed into a series of CHCliCH 3 OH 2:1, 1:1, 1:2 (v/v) and CHCliCH 3 OH/water 18:9:1 (v/v/v) for 24 hr each, at room temperature. The mixture of solvents was filtered through a solvent-resistant 0.5-µm Millipore filter. The filtered solution was taken to dryness, and was then used for analysis of extractable lipids. Delipidized hair fibers were saponified by heating for 2 hr at 60 ° C in lN KOH in 90% CH 3 OH (16). After water and CHC1 3 were added, the mixture was shaken in a sepa­ ratory funnel. The CHC1 3 phase was transferred to a flask, and the upper phase including hair residue was acidified by the addition of 6N HCl. The acidified upper phase was shaken again with CHC1 3 . The combined CHC1 3 phase was washed twice with saturated NaCl aqueous solution. The washed CHC1 3 phase was then filtered through a solvent­ resistant 0.5-µm Millipore filter. The filtered solution was taken to dryness, and was then used for analysis of integral lipids, which are defined as the hair lipids that can be extracted by alkali saponification following solvent extraction (16). IDENTIFICATION OF CERs WITH GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS) This procedure was performed with slight modifications of methods previously reported (19). CERs were isolated from the extracted lipids by preparative TLC (silica gel 60, Merck, Darmstadt, Germany). The isolated CERs were silylated with TMS-HT at 60 ° C for 20 min. Mass spectra were obtained by GC/MS with a Hewlett-Packard 5988A mass spectrometer coupled to a Hewlett-Packard 5890 Series GC and a Hewlett-Packard 59970C workstation (Hewlett-Packard, Palo Alto, CA). An Ultra ALLOY-l(HT) 30-m x 0.25-mm x 0.15-µm metal capillary column (Frontier-Labo, Fukushima, Japan) was used with a temperature program of 150°C to 360°C at 5°C/min, which was held for 5 min. Aliquots of the sialylated CER solutions were injected via splitless mode at 3 70°C. Helium was used as a carrier gas at a flow rate of 1.16 ml/ min. The mass spectrometer scanned from 50 to 1000 amu in 0.78 sec, with an electron ionization of 70 eV. The source and the transfer line temperatures were kept at 300°C and 320 ° C, respectively. DETERMINATION OF HCs, SQ, WEs, TGs, AND FAs WITH HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY (HPTLC)/DENSITOMETR Y These procedures were performed with slight modifications of methods previously re­ ported (16,17). Thus, for separation, HPTLC plates (silica gel 60, 20 x 10 cm, Merck) were used. Aliquots of the extractable lipid solutions were applied 2 cm from the bottom edge of the plate, which was developed first to 3 cm with CHCliCH 3 OH/water (100:10:0.5) twice, second to 12 cm with hexane/acetic acid (80:10), and third to 15 cm with petroleum ether. After drying, the plate was sprayed with 10% copper sulfate and

4 JOURNAL OF COSMETIC SCIENCE 8% phosphoric acid solution and was charred by heating at 180°C for 5 min. Each spot was densitometrically determined with a Shimadzu CS-900 photodensitometer (Kyoto, Japan) and was compared to a calibration reference curve. Duplicate analyses were performed for each measurement. DETERMINATION OF MEA WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) This procedure was performed with slight modifications of methods previously reported (6). Thus, tricosanoic acid as the IS of MEA and ADAM were added to the integral lipid solution (CHC1 3 /CH 3 OH 1: 1) and the solution was then derivatized for 1 hr at room temperature. Aliquots of the derivatized integral lipid solution were injected into an HPLC apparatus equipped with a fluorescence detector (Hitachi 1-6000 series, Hitachi, Ibaragi, Japan) and a 12.5-cm x 4.0-mm Superspher 60 RP-Se (Merck, Darmstadt, Germany) kept at 40°C. The derivatives were detected at EX.365 nm and EM.412 nm. Two solvents of (A) acetonitrile/water 85: 15 and (B) acetonitrile were used for separa­ tion at a flow rate of 1.5 ml/min with a gradient elution program, which included first (A) 100%, second (B) 2.5%/min to (B) 100% (40 min), and finally holding of (B) 100% for 20 min. The content of MEA was calibrated based on an equivalent molar sensitivity of MEA to the IS. Duplicate analyses were performed for each measurement. DETERMINATION OF CH WITH GC This procedure was performed with slight modifications of previously reported methods (7). Thus, analyses were performed on a Hewlett-Packard 5890 series GC equipped with a flame ionization detector, an Ultra #2 25-m x 0.25-mm x 0.33-µm fused silica capillary column (Hewlett-Packard), and a Hewlett-Packard 3396 Series II integrator. Aliquots of the lipid solution were injected via split mode in a ratio of 1 :50 at 340°C. The temperature of the detector was maintained at 340°C. Helium was used at a flow rate of 1.0 ml/min. The column temperature was programmed from 200°C to 320 ° C at 20°C/min, which was held for 10 min. The content of CH was determined using a calibration curve. Duplicate analyses were performed for each measurement. DETERMINATION OF CERs WITH HPLC Procedures for alkaline hydrolysis of CERs for the isolation of sphingoids were per­ formed, based on a slight modification of a method reported for CERs of the stratum corneum (24). Thus, the lipids were saponified by heating for 18 hr at 65°C in 10 ml 1 N KOH in CH 3 OH/hexane 5:1. Ten micrograms of threo-dihydrosphongosine was then added as the IS. By means of liquid-liquid extraction, CHC1 3 extracts were ob­ tained. The sphingoids extracted were isolated by preparative TLC (silica gel 60) because hydrolyzed fatty acids interrupted the reaction between sphingoid and OPA. The sphin­ goids isolated were derivatized at room temperature for 30 min with an added solution of OPA methanol, a 2-ME/methanol solution, and a sodium tetraborate aqueous solu­ tion. Aliquots of the derivatives were injected into the same HPLC equipment used for the MEA analysis with a 15-cm x 4.6-mm 1-Column ODS (Kagakuhin-Kensa-Kyoukai, Tokyo, Japan) kept at 40°C. The derivatives were detected at EX.344 nm and EM.433 nm. Two solvents of (A) CH 3 OH/water 85:15 and (B) CH 3 OH were used for separation