HAIR DAMAGE WITH S100A3 ELUTION 19 from hair fiber and cuticle fragments all migrated mainly in monomeric form (10 kDa band), but only a small amount of the polymeric form was observed (25 kDa band Figure lB). By utilizing efficient separation on tricine/SDS/PAGE, the precise quanti- fication of S100A3 was possible. The S100A3 contents of natural hair were estimated to be 0.10% (w/w) and 1.3% (w/w) of total mass from whole hair fiber and cuticle fragments, respectively. Total protein extraction yielded approximately 60% (w/w) of the total mass from whole hair fiber, while only 11% (w/w) was from the cuticle fragments. Thus, the proportion of S100A3 was more than 10% (w/w) in the cuticle extract, while only 0.2% (w/w) was in whole hair, indicating that S100A3 is one of the major soluble components of cuticle. Subsequently, we adopted tricine/SDS/PAGE for the analyses of effluents obtained as a result of permanent waving and reducing conditions (see Materials and Methods). The effluent obtained from permanent waving agents showed a smear protein pattern (Figure 1A, lane 4), in which a sharp band of S100A3 detected by western blot analysis is underlain (Figure lB, lane 4). The effluents under the reducing conditions showed a distinguishable 10 kDa protein band (Figure 1A, lane 5), which reacted with an anti- body for S100A3 (Figure lB, lane 5). In addition, we observed several other protein bands for both effluents. The identification study of the bands is currently underway. ELUTION OF S100A3 WITH A PERMANENT WAVING LOTION The protein eluted with permanent waving lotions was analyzed (13,14), but its com- ponents have not been yet identified. Through western blot analyses we identified S100A3 as one of the components eluted with permanent waving lotions. The elution profile of S100A3 during three exposures to waving agent and neutralizing agent is shown in Figure 2A. The total amounts of eluted S100A3 with both waving lotions decreased in consecutive treatments. Both S100A3 and protein were eluted with the waving agent in relatively large quantities. They were also found in smaller amounts in a solution of a neturalizing agent. The proportion of S100A3 to protein was 1-2% (w/w) and 3-8% (w/w) in the solutions of waving and neutralizing agents, respectively. These values correspond to approximately 5-40 times the content of S100A3 in the total protein extract of the whole hair fiber. 6 (A). 1st 2nd 3rd 250 '• 200 ._c 1 oo so (B) o o 1 st 2nd 3rd Figure 2. Elution profiles of S100A3 and protein in permanent waving lotion. Permanent treatment was performed with consecutive applications of waving agent (open box) and neutralizing agent (hatched box) three times. Amounts of eluted S100A3 (A) and protein (B) were estimated through western blot analyses and protein assay, respectively.

20 JOURNAL OF COSMETIC SCIENCE ELUTION OF S100A3 FROM DAMAGED HAIR WITHOUT A REDUCTANT Virgin, UV-irradiated, and permed hair were subjected to the analyses for S100A3 under non-reducing conditions (see Materials and Methods). We adopted slot-blot analyses to quantify low concentrations of S100A3 in these effluents. This technique is capable of detecting 0.5 ng of S100A3 in 200 pl of the effluent. The amounts of S100A3 eluted from UV-irradiated and permed hair were greater than those from virgin hair (Figure 3). UV-damaged or permed hair gradually lost S100A3 during daily washings, even in the absence of reductant. EFFECT OF WEATHERING ON S100A3 IN HAIR Several aspects of hair damage may be attributed to weathering. For example, it was shown that the tip end of hair is generally more damaged than the root end (15,16). In this study, we attempted to estimate the effect of weathering through the comparative analyses of S 100A3 elution from samples derived from the root, middle, and tip sections of hair. All employed hair samples were not chemically treated in order to exclude any influence from chemical damage. The results of experiments showed that the amounts of the eluted S100A3 from the middle and tip sections of hair were significantly smaller than that eluted from the root part (p 0.001 Figure 4A). In addition, a significant difference between the middle and tip parts (i.e., the root and tip ends of long-hair sample) was obtained with a paired t-test (p 0.05). In contrast to this, the amount of eluted protein from the tip part of hair was significantly greater than that from the root part (p 0.01 Figure 4B). The above-described hair samples were further subjected to total protein extraction. The performed analysis indicated that the amounts of S100A3 and protein gradually de- creased as a particular of dislocation from the root end (Figure 4C,D). The proportion of eluted S100A3 obtained under reducing conditions in relation to the total extract (0.6-4.3%) was higher than that of the protein (0.05-0.2%). 0.4 =tO2 •.• . o0.1 Cont UV Per Figure 3. Comparison of the S100A3 elution from natural, UV-irradiated, and permed hair. The elution test was performed under non-reducing condition (See Materials and Methods). The amounts of eluted S100A3 were estimated through slot-blot analyses.