EXOGENOUS DISULFIDE IN HAIR REDUCTION 101 REDUCTION METHOD Four commercially available perming lotions were studied. Two of the lotions were ammonium thioglycolate based (A, B) and two were glyceryl monothioglycolate based (C, D). One from each of these categories contained only mercaptan reducing reagent (A, C), while the other two (B, D) contained both a mercaptan reducing reagent and disulfide. These waving lotions were used to perm 152.5-mm hair tresses weighing approximately 2.0 g each. The hair swatches were then either immersed in a large excess of perming solution (50:1 w/w) or saturated with a limited amount of waving lotion (5:1 or 0.7:1 w/w). After exposure to the reducing medium for varying periods of time, the reduced fibers were immersed into a cold 1% iodoacetic acid solution to prevent further reduction from occurring. The tresses were immediately reacted further with sodium iodoacetate at pH 8.3 to form carboxymethyl derivatives of all of the cysteine residues. Treated fibers were then rinsed, dried to constant weight, and hydro- lyzed with 6 N HCI by a modification of a procedure appearing in the literature (6). From amino acid analyses of the acid hydrolysate, percent cystine cleaved was calculated by comparing the value of cystine obtained after reduction with that of an unreduced control. In one series of experiments, an attempt was made to duplicate salon conditions by placing ("implanting") several previously wound rods, containing 2.0 g of hair, onto the head of live models. Waving lotion-to-hair ratio under these practical conditions was approximately 0.7:1. The cosmetologist then proceeded to perm the model's head in accordance with the use directions. Periodically these reduced "implants" were re- moved and treated with iodoacetate, as described above. To further substantiate results obtained on hair tresses, reducing experiments were run on hair fiber bundles. Each bundle contained twelve hair fibers. These bundles were stress relaxed in water at 1.5% strain, for 30 minutes, and then treated with 500-ml solutions of 0.8 N ammonium thioglycolate at pH-9.2 or a mixture of 0.8 N am- monium thioglycolate and ammonium dithiodiglycolate at pH-9.2. Reduction of each fiber bundle was terminated after different periods of reaction time and treated with iodoacetate in a manner identical to that previously described for hair tresses. Treated fiber bundles were hydrolyzed and subjected to amino acid analysis in the usual manner. RESULTS AND DISCUSSION EXCESS LOTION--FIBER BUNDLE METHOD In order to reduce experimental variations, such as temperature and concentrations, reduction studies were performed on fiber bundles. These bundles, consisting of twelve strands of hair fiber, were stress relaxed in water for thirty minutes (1.5% strain) at ambient temperature and then reduced with 500 ml of an aqueous mercaptan reducing solution. All of the solutions contained ammonium thioglycolate at a concentration of 0.8 N-RSH and pH-9.2. However, concentration of added dithiodiglycolic acid, cal- culated as the acid, varied from zero to 1.0 N with intermediate levels of 0. 125 N, 0.25 N and 0.5 N. After the strands were reduced for different periods of time, they

102 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS were reacted with iodoacetate, acid hydrolyzed, and assayed for cystine content as de- scribed earlier in this paper. Results obtained are plotted in Figure 1. Not only are effects noted near equilibrium, but rate effects, caused by the addition of dithio compounds to a reducing mercaptan, are readily observed from these curves. When fiber bundles were reduced with am- monium thioglycolate (0.8 N-RSH, pH-9.2) for fifteen minutes, approximately 80% of the cystine bonds were reduced. However, in the presence of 0.125 N dithiodigly- colic acid, the amount of cystine reduced is only 60%. For 0.25 N, 0.5 N and 1.0 N, cystine reducton is approximately 40, 20 and 9 percent respectively. Even after sixty minutes of fiber immersion, levels of fiber reduction with solutions containing dithio- diglycolic acid are from 20% to 70% lower than reduction achieved after 15 minutes by an ammonium thioglycolate solution without dithiodiglycolic acid. EXCESS LOTION--IMMERSION METHOD Table I summarizes the data obtained when 152.5-mm, 2.0-g tresses of hair were immersed into 100 ml of perming lotion, at ambient temperature, for the indicated period of time. The reduced keratin cystine (cysteine) was then reacted with iodoacetate to form the carboxymethyl cysteine derivative. From amino acid analyses on the acid hydrolyzed portions of these hair strands, percent disulfide (cystine) reduction was cal- culated. These data indicate that perm solution B (1.0 N-RSH, pH-9.2), containing diam- monium dithiodiglycolate, achieves maximum keratin reduction (56.3%) in approxi- % C 90 y 8o s 70 t i 6o n 50 e 40 c I 3o e 2o a lO v e o d [ CUR•E # CO•lC. DTD(•A I 0.000 N f 2 0.125 N 3 0.250 N CURVE #1 CURVE #2 4 0.500 N 5 1.000 N ?? ou.w • • cu.w ,• • • • CURVE •5 • • 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (Min.) Figure 1. Cystine cleavage in hair fiber bundles stress-relaxed in water (1.5% strain) and immersed in 0.8 N ammonium thioglycolate containing various amounts of dithiodiglycolate.