320 (a) 30 JOURNAL OF COSMETIC SCIENCE .......... . . .. . ........... .... · ·• . .. . . ··· ··.... E E 20 . . . . . . . : : --- a 10 0 . . . . - t f ·0··1 ... _roo .: ,..l \ • ..- .... • ••• · 0 . . . . . . . . . . . . · .... ... 1 I \ I 10 (b) I 40 x/mm I I 50 60 . . :. ...tip . ... 70 Figure 1. A schematic representation of a two-dimensional image of a curved hair fiber. (a) The plot of x-y coordinates of the points on the two-dimensional fiber image was determined at millimeter intervals from the root end of the fiber. (b) An enlarged image of the plot. Tangential vectors were approximated from the x-y coordinates of two adjacent points (solid arrows in this figure). The difference in the direction angles of the two adjacent vectors (�0) was obtained for each of three adjacent points. The angle difference (in radian/mm) is mathematically equal to the curvature (1 IR) of the fiber between the three adjacent points, where R is the curl radius for the corresponding points. decoloration was carried out at 2 5 ° C by immersing the stained hair fibers in deionized water for 24 h. The hair fibers were washed twice with deionized water for 30 s and finally air-dried at room temperature. Through this procedure, a sufficient amount of erythrosine B remains in the macrofibrils of cortical cells and in the exocuticle of cuticle cells, while the dye is washed out from the inter-macrofibrillar region, cell membrane complex (CMC), and endocuticles (9). The macrofibrils and exocuticles are, therefore, observed to be darker than surrounding cell components under TEM. The stained hair fibers were embedded in Spurr's resin (Polysciences, Inc. Spurr low­ viscosity embedding media). Thin transverse sections of the embedded hair fibers were prepared by cutting with an ultra-microtome (Reichert type Ultracut-N) equipped with a diamond knife (DiAtome type ultra 45°). The thickness of the sample sections was kept at 500 nm. These sections were very thick compared with conventional thin sections (50-100 nm) for TEM observation because the staining density of erythrosine B is relatively lower than the density achievable by conventional staining with heavy metal compounds. The prepared sections were observed with a TEM apparatus (Hitachi, type H-7000) operated at an acceleration voltage of 75 kV.

HAIR CURVATURE IN JAPANESE WOMEN 321 In the second staining method, with silver nitrate, hair fibers were reduced in an aqueous solution of 0.54 M thioglycolic acid at pH 9.0 at 25 ° C for 10-40 min. The reduced hair fibers were then washed twice with deionized water for 30 s and finally air-dried. The reduced hair fibers were embedded in Spurr's resin. Transverse and longitudinal thin sections, of 100-nm thickness, of the reduced hair fibers, were prepared with an ultra­ microtome and a diamond knife. The thin sections were immersed in an aqueous solution of 1.0 mM AgNO3 , 2.0 mM EDTA, and 100 mM Tris/HNO3 buffer at pH 7.0 at 25 ° C for 10 min, and then washed twice with deionized water and air-dried at room temperature. The prepared sections were observed by TEM as mentioned above. In the case of staining with silver nitrate, the cystine (disulfide) bonds of proteins in the hair fiber are reduced to cysteine. The free sulfhydryl (R-SH) groups of cysteine are stained with silver ions due to the high reactivity of silver cations with thiolate anions (10,11). The matrix region between intermediate filaments (IFs) in macrofibrils and exocuticles are observed to be darker than IFs and surrounding cell components under TEM because they contain larger amounts of cystine-rich proteins such as the high­ sulfur keratin-associated proteins. In the above TEM observations, we attempted to observe the relationship between the microstructure and the curved shape of the hair fiber. When a curved hair fiber was sectioned with an ultra-microtome, the cross-sectional shape of the embedding resin with the fiber was prepared as an asymmetrical, trapezoidal shape, having one set of parallel sides in relation to the direction of the hair curvature. During the TEM obser­ vation, the direction of hair curvature and the relative position of the observed area were determined, based on the asymmetrical shape of the cross section. For example, when the concave side of a curved hair fiber is oriented to the longer side of the parallel sides of the trapezoidal section, the half area from the fiber center to the longer side and the opposite half area are determined as the inner and outer regions of the curved shape, respectively. AMINO ACID ANALYSIS The amino acid compositions of keratin in the inner and outer halves of the curved hair fibers were determined. Typical highly curved fibers (curl radius: 0.7-2.5 cm) were sampled from 39 selected volunteers. Each of the curved fibers was divided into an inner half and an outer half by cutting the fiber at its midpoint along the longitudinal direction. The schematic diagrams of the preparation of the inner and outer halves are shown in Figure 2. A curved hair fiber of 1.0-cm length was put on a flat celluloid plate. An appropriate amount of ethyl acetate was applied to the celluloid plate until the hair fiber was covered with this solvent (Figure 2a: Step 1). Since the celluloid near the plate surface was partially dissolved in the ethyl acetate, the hair fiber on the plate was covered with the dissolved celluloid. After the ethyl acetate was completely evaporated from the plate, the curved hair fiber was fixed near the plate surface (Figure 26: Step 2). The fixed hair fiber was then hand cut with a sharp scalpel (Feather, Surgical Blade #11) along the midline of the fiber axis (Figure 2c: Step 3). The midline was determined by the position of porous medulla because the medulla usually exists at the center of hair fiber. The hand cutting with the scalpel was performed under a stereoscopic zoom microscope (Nikon, Type SMZ-10) because the magnification (x 10-x40) and focal depth (mm order) of the stereoscopic microscope were adequate for the hand cutting of hair fiber. Then, each half