HAIR BLEACHING AND WAVING 163 Table II Changes in the Amino Acid Composition of the Melanoprotein (tool%) After Alkaline Bleaching for Different Times Bleached Melanin Residues Untreated Time of Bleaching Melanin 5' 10' 15' Decreasing.' LYS 7.6 7.7 6.7 6.4 HIS 2.5 2.6 2.2 2.2 ARG 15.8 14.4 12.9 12.0 PHE 4.0 3.2 3.1 3.1 (CYS)2 3.1 2.5 2.2 1.3 Increasing.' CYSO3H 0.5 1.9 2.2 4.5 ASP 6.1 6.5 6.9 7.1 GLU 6.6 7.1 7.7 8.0 VAL 4.6 4.7 5.2 5.2 ORN 0 0 0.2 0.2 Unchanged THR, SER, PRO, GLY, ALA, MET, ILE, LEU, TYR Protein content in % 23.1 22.0 18.3 18.2 Bleaching conditions: solution of 1% H202, adjusted to pH 10 with ammonia, room temperature mel- anin:liquor ratio/1 g:100 ml (3). This scheme suggests that larger amounts of cysteic acid than actually present would be determined by amino acid analysis following conventional acid hydrolysis. An indirect method for demonstrating the presence of cystine oxides has been developed in our laboratories (13). It compares the cysteic acid levels in hydrolysates prepared with and without the addition of thioglycollic acid as reducing agent. The "true" values in Table Ill obtained by hydrolyzing in the presence of thioglycol!ic acid represent the true amount of cysteic acid in hair. They are obviously smaller than those of comparable samples obtained after hydrolysis without thioglycollic acid, and these differences in cysteic acid content are explained as being due to the presence of cystine oxides. Although this method is an approximation, it nevertheless allows a routine check for the determination of cystine oxides in keratins. Investigations concerning the reaction mechanisms of cystine oxides were carried out at DWI on a model substance by Schumacher-Hamedat (14), using N,N'-bisacetyl-L-cys- tine-bismethylamide (ACM) because of the relative stability of its oxides (Figure 3). By application of high performance thin layer chromatography for the separation of the reaction products after peroxide bleaching, Schumacher-Hamedat (14) found that even during treatments at pH 9.5, cystine monoxides are formed (Figure 4). These findings supplement earlier results based on acidic treatments (15). Since the stability of par- tially oxidized cystine-containing peptides increases with molecular weight, an op- timum stability for protein-combined cystine oxides in human hair can be assumed.

164 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 100- 50- I I I ! •" 5 10 15 20 [ mini BI ec[ching lime Figure 2. Oxidative treatment on isolated melanin. Correlation between oxidized disulfide bridges in % ( ..... ) and loss of weight of the granules in % ( ) (Giesen (3)). A method for direct measurement of cystine oxides in hair is via infrared spectroscopy. Fourier transform infrared spectroscopy (FTIR) offers the possibility of demonstrating the existence of cystine oxides and cysteic acid non-destructively in one step by ob- serving the sulfur-oxygen vibrations. Application of this technique to the investigation of oxidative damage on human hair has already been described in the literature (16). Schumacher-Hamedat found during her work on this subject an increase in monoxide and dioxide concentration after per- formic acid oxidation of human hair. Using peroxide-containing bleaching solutions, however, only the monoxides are found (Figure 5) these findings are identical to those obtained in studies of the model compound ACM (17). The oxides even appear in untreated hair, as does cysteic acid, presumably due to weathering. TWO-DIMENSIONAL GEL ELECTROPHORESIS OF UNTREATED AND BLEACHED HAIR SAMPLES The application of two-dimensional gel electrophoresis techniques in the field of human Table III Cysteic Acid Values (mol%) in Total Hydrolysates of Hair in Two Different Media (FiShles et al. (13)) 6N-HC1 + 3 Vol% Hair Sample 6N-HC1 Thioglycollic Acid Untreated 0.60 0.44 47 h irradiation • 0.73 0.51 126 h irradiation • 0.81 0.58 Oxidized • 3.22 2.70 Xenotest apparatus Model 150--Filter system 6 IR/1 UV. Bleached under unknown conditions in a hair dresser's salon.