2 JOURNAL OF COSMETIC SCIENCE cuticle cells by a thio-ester linkage (6). Many studies have suggested that hair lipids can contribute to physicochemical phenomena such as diffusion, cell cohesion, and mechani­ cal strength (3,7-10), although lipids occur at a much lower content (1-9% dry weight) than proteins (90%). Many analytical studies on the lipids of human hair have been performed (6,7, 11-20). Hair fibers have not only surface lipids originating from sebum (11,14), but also lipids within the hair (11-13). Curry and Golding reported that hair yields its lipids to successive Soxhlet extraction, suggesting the existence of lipids within hair (11). Using hair fibers treated with successive Soxhlet extraction, Sakamoto et al. (12) first demon­ strated the existence of lipids within them such as fatty acids (FAs) and wax esters (WEs). They designated them as "internal lipids" of human hair (12-14). Subsequently, hydrocarbons (HCs) (15 ), squalene (SQ) (7 ,15 ,20), wax esters (WEs) (15, 17 ,20), triglyc­ erides (TGs) (15,17), FAs (7,15-18,20), cholesterol (CH) (7,15-17,20), cholesterol sul­ fate (CS) (16), MEA chemically bound to hair fibers (6,16), and ceramides (CERs) (19) have been found as hair lipids. However, in all previous studies described above, hair samples were not from the proximal root ends or were not clearly described. Since hair lipids are gradually lost or altered as hair fibers grow, the proximal root ends of hair fibers or the nearest regions should be used to characterize the precise hair lipid com­ position. In addition, all previous studies have not focused on comprehensive hair lipid composition, including its individual variation, although a great deal of knowledge on the intercellular lipid composition of the stratum corneum has accumulated (e.g., 21- 23). The aim of the present study is to clarify the precise hair lipid composition at the proximal root regions of hair fibers by a combination of chromatography. MATERIALS AND METHODS CHEMICALS HPLC grade chloroform, methanol, hexane, and acetonitrile were purchased from Kanto-Kagaku (Tokyo, Japan). Biochemistry grade o-phtalaldehyde (OPA) and 2-mer­ captoethanol (2-ME) were from Wako (Tokyo, Japan), 9-anthryldiazomethane (ADAM) was from Funakoshi (Tokyo, Japan), and TMS-HT was from GL Science (Tokyo, Japan). All other chemicals were analytical grade. REFERENCES FOR LIPID ANALYSIS Tetracosane for HCs was purchased from Kanto-Kagaku. Squalene for SQ, tripalmitin for TGs, tricosanoic acid for an internal standard (IS) of MEA, D-sphingosine, DL-threo­ dihydrosphingosine, DL-erythro-dihydrosphingosine, and N-palmi toy 1-DL-dihydro­ sphingosine for CERs were purchased from Sigma (St. Louis, MO). Cetyl palmitate for WEs, palmitic acid for FAs, and cholesterol for CH were purchased from Tokyo-Kasei (Tokyo, Japan). All references were used without further purification. MATERIALS Hair fibers were obtained from 44 healthy Japanese female volunteers whose ages ranged

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