ANALYTICAL SCIENCES OCTOBER 1993, VOL. 9 637 Determination of Mercapto Groups in Human Hair Treated with Chlorine Oxides Takaho WATANABE,YOshiko KIMURA,Yoshihiro YOSHIMURA,Katsumi UCHIYAMA, Dairo YAMAMOTO,Kazuo IMAEDAand Keiko OHSAWA Derpartment of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Ebara, Shinagawa, Tokyo 142, Japan To evaluate the damage to human hair by treatment with chlorine oxides, the mercapto (SH) groups in human hair were measured spectrophotometrically without dissolving the hair. The procedure used the reaction of 2,2'-dithiodipyridine with a thiol giving the corresponding 2-thiopyridone, which is quantitated by measuring the absorbance at 343 nm. By incubating human hair in a solution containing 2,2'-dithiodipyridine at 60° C for 90 min, a linear relationship was found between the concentration of hair and the absorbance of 2-thiopyridone, with a relative standard deviation of 6.3%. Hypochlorite decreased the concentration of SH groups in hair, while other chlorine oxides exhibited little effect. Keywords Mercapto group, hair, spectrophotometry, 2,2'-dithiodipyridine, chlorine oxide Chlorine is used as a disinfectant for potable water and of cysteine in hair. However, since the cysteine in hair is waste water. The level of residual chlorine in drinking easily oxidized during dissolving, methods which involve water is more than 0.4 ppm in Japan. Since this hydrolyzing hair cannot determine the exact amount of disinfection generates trihalomethanes12, which induce cysteine in hair. Therefore, we employed a spectro- cancer34, the use of chlorine dioxide is preferable.5 photometric technique without dissolving the hair, and Chlorine dioxide, used as a bleaching agent and as a used 2,2'-dithiodipyridine (2-PDS) and an SH-SS disinfectant for drinking water in USA and Canada6, has exchange reaction, to investigate the damage to hair by recently been authorized as a disinfectant for pool water chlorine oxides in potable water and swimming pool in the range from 0.1 to 0.4 ppm in Japan. An aqueous water. solution of chlorine dioxide often contains a mixture of chlorine oxides. Chlorite ion, which is the main re- duction or disproportionation product of chlorine Experimental dioxide, is present in water treated with the latter.' Chlorate ion is also present as a disproportionation Reagents and samples product of chlorine dioxide.' Cysteine, chloroform, methanol, ethanol, sodium There have been few toxicological studies of chlorine hypochlorite and sodium chlorite were purchased from oxides. Functional components in mammalian tissues, Wako Pure Chemical Industries, Ltd. (Osaka, Japan), such as proteins in keratin and lipids in the skin, are and 2,2'-dithiodipyridine (2-PDS) from Dojindo oxidized by chlorine oxides. Human hair is particularly Laboratories (Kumamoto, Japan). A stock solution of vulnerable to chlorine oxides which damage keratin. 1 mM 2-PDS dissolved in a 0.1 M Tris buffer (pH 7.5) There are two factors which damage hair; physical was stored at 5° C until use. A stock solution of chlorine factors such as heat, friction and drying, and chemical dioxide was prepared by mixing 7.5% sodium chlorite factors such as acids, alkalises, oxidizing and reducing with 9.0% hydrochloric acid and was stored at 5° C in the agents. Many analytical methods have been reported dark. Chlorine dioxide solutions were analyzed ac- for elucidating chemical changes in damaged hair, cording to the amperometric titration method12 before including determination of amino acid compositions, use. Human hair used was obtained from a 21-year- alkaline consumption9 and copper compounds absorbed old female volunteer. by hair.10 When hair is treated with a strong oxidizing or reducing agent, as in permanent, hairdye or hair- Procedure bleach, the amount of cystine decreases, with con- The procedure for determining the SH groups in hair is comitant increase of cysteic acid.11 To assess the shown in Fig. 1. To remove oily substances, hair was damage to hair treated with weak oxidizing agents found rinsed successively with chloroform-methanol (1: 2, v/ v), in potable water, it is necessary to determine the amount ethanol and distilled water, and kept in a desiccator until 638 ANALYTICAL SCIENCES OCTOBER 1993, VOL. 9 Fig. 2 Effect of incubation time on the absorbance (343 nm) of 2TP. Human hair (50 mg) was incubated at 40° C (/) or 60°C (S). Fig. 1 Procedure for determination of SH groups in human Table 1 Correlation between the absorbance of 2TP and the hair. amount of hair by incubation at 60° C use. After weighing, hair treated with either chlorine oxide or distilled water (control) was soaked in 5 ml of a 1 mM 2-PDS solution for 30 min at room temperature, and then incubated at 60° C for 90 min. The absorbance of 2-thiopyridone (2TP), generated by an SH-SS exchange between 2-PDS and cysteine, was determined at 343 nm by use of a Hitachi spectrophotometer Model absorbance of 2TP and the L-cysteine as a standard of SH 150-20. groups over the range of 3 to 200 µM. The relative standard deviation of five measurements with 50 mg of hair was 6.3%. Although the within-run reproducibility Results and Discussion for measuring the SH groups in hair was good, the day- to-day reproducibility was poor. Therefore, to Effect of incubation time elucidate the problem, the reaction between the SH Figure 2 shows the effect of incubation time on the group in human hair and the 2-PDS reagent was absorbance of 2TP, reflecting the concentration of SH discussed below. groups in hair. Incubation at room temperature A hair is so hygroscopic that it takes up water, such as produced only a negligible increase in absorbance. 2-PDS solution. The 2-PDS uptake into hair is Incubating 50 mg of hair at 40° C or 60° C gave an influenced by the form of the hair cuticle and the water increase in the absorbance of 2TP with the incubation content in hair. In human hair, SH groups are present time. The absorbance of 2TP was about three times in the hair medulla or bulb, and hide in the hair medulla. greater at 60° C than at 40° C. In view of this, sub- As shown in Fig. 2, the absorbance at 343 nm increases sequent experiments were done at 60° C. with an increase in temperature. These results suggest that the 2-PDS permeated into the hair medulla can more Correlation between the amount of hair and the absorbance easily react with SH groups when hair medulla is of 2TP denatured by heat. In addition, it is thought that this Table 1 summarizes the relationship between the reaction of 2-PDS with SH groups depends on the absorbance of 2TP, reflecting the concentration of SH amount of 2-PDS permeated into the hair medulla. groups in human hair, and the amount of hair. Between Therefore, to enhance the permeation of 2-PDS reagent 10 and 70 mg of hair, a linear relationship is seen between into hair, hair was soaked in 2-PDS reagent for 30 min at the two. The slope of the regression line between the room temperature before incubation. Consequently, absorbance and the amount of hair increased as the the day-to-day reproducibility for measuring the SH incubation time increased. The correlation coefficient groups in hair improved to 7.3%. was 0.99 with an incubation period longer than 90 min. Therefore we fixed the incubation time at 90 min to Change in the concentration of SH groups in hair treated minimize the analysis time. Under optimum con- with chlorine oxides ditions, a linear relationship is found between the Under the optimum conditions described above, a ANALYTICAL SCIENCES OCTOBER 1993, VOL. 9 639 Fig. 4 Changes in the absorbance of 2TP after soaking hair (10 mg) in 10 ml of 100 ppm chlorite (•), chlorine dioxide (•) and hypochlorite (A) solutions. The broken line represents the control soaked in water. Fig. 3 Changes in the concentration of SH groups in human hair (20 mg) treated with chlorine oxides. The concentra- tion of SH groups in the control (broken line) was 4.3 µmol/ g hair. change in the concentration of SH groups in hair was observed after treating it with chlorine oxide such as hypochlorite, chlorite and chlorine dioxide (Fig. 3). One hundred milligrams of hair was soaked in 50 ml of a 1,10 or 100 ppm chlorine oxide solution for 48 h at 5°C. The hair was then rinsed with distilled water and dried in a desiccator, and the concentration of SH groups in 20 mg of hair was determined. When hair was soaked in Fig. 5 Effect of residual concentration of chlorite (•), the chlorite solution, the absorbance of 2TP was similar chlorine dioxide (•) or hypochlorite (A) on the absorbance to that in the control. Treatment with chlorine dioxide of 2TP. tended to increase the 2TP absorbance as the con- centration of the solution increased. On the other hand, the absorbance of 2TP in hair which had been treated chlorite treatment produced the largest decrease in the with hypochlorite decreased as the concentration of absorbance among the chlorine oxides tested. hypochlorite increased. After the hair was soaked in These results indicate that the proposed simple 100 ppm hypochlorite solution, the absorbance of 2TP procedure can be used to determine the SH groups in was lower than in the control. This suggests that human hair. By applying this procedure, a significant residual hypochlorite, or a chloramine, reacted with damaging effect of hypochlorite on hair was thus amino acids from keratin to produce a decrease in the demonstrated. absorption at 343 nm. Figure 4 shows the changes in the absorbance of 2TP in human hair treated with chlorine oxides.