j. Cosmet. Sci. 1 56, 1-16 Qanuary/February 2005) Characterization of the lipid composition at the proximal root regions of human hair YOSHINO RI MASUKA WA, HIROFUMI NARIT A, and GENJI IMOKAWA, Tochigi Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi, 321-3497 Japan. Accepted for publication December 3 1 2004. Synopsis The hair lipid composition collected from 44 Japanese females between 1 and 81 years of age was examined for eight lipids including hydrocarbons (HCs), squalene (SQ), wax esters (WEs), triglycerides (TGs), fatty acids (FAs), cholesterol (CH), ceramides (CERs), and 18-methyl eicosanoic acid (MEA). In this study, the 5-cm length from the proximal root end of hair fibers, which had never been exposed to any chemical treatment, was used after 5-min incubation with hexane following shampooing. Hair lipids were extracted with solvent and subsequent alkali-solvent and were then analyzed by a combination of chromatography. Although the average contents of the lipids showed great fluctuations among individuals, there were significant correlations between the levels of each lipid, which allowed for the classification of the hair lipids into four groups: group A: SQ, WEs, TGs, and FAs (designated as endogenous lipids based upon their sebum origin) group B: CH and CERs (designated as endogenous lipids) group C: HC (unknown origin) and group D: MEA (the other endogenous lipid). A principal component analysis for eight lipids revealed that the hair lipid composition was characterized by a predominant negative correlation between each lipid for groups A and B. This negative correlation suggests that the endogenous lipids in group B serve as a barrier against the penetration of predominantly sebum-derived exogenous lipids (group A). Endogenous lipids consisting of CH and CERs (group B) and MEA (group D) should be designated as intrinsic internal lipids of human hair. INTRODUCTION A fine structure, called the cell membrane complex (CMC), exists between cortical or cuticle cells of human hair as well as wool ( 1,2). The CMC is visualized by transmission electron microscopy as a tram-line structure including a densely stained 8-layer sand­ wiched between lightly stained 13-layers on both sides. The main components of the 8-layer and the 13-layers have been thought to consist of proteins and lipids, respectively (1,3). The fact that lipid extracts from wool or from human hair form liposomes suggests that lipids might comprise a lipid bilayer at the 13-layers of the CMC (4,5 ). A charac­ teristic lipid, 18-methyl eicosanoic acid (MEA), is chemically bound to the surface of Address all correspondence to Genji Imokawa. 1

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