REDUCTION-INDUCED HAIR SURFACE MODIFICATION 3 EXPERIMENTAL MATERIALS AND TECHNIQUES (a) Unaltered hair samples. A tress of 14˝-long, unaltered, European dark brown hair from DeMeo Bros., New York, was used for this study. (b) Hair sample preparation. Eight inches of the root portions of individual hair fi bers were taken and numbered 1 to 30. These root portions were then cut into six equal segments. Starting with the top section of each hair fi ber, the segments were numbered 30, 15, 10, 5, 2, and 0 minutes. (c) Reduction treatment. The numbered segments were then subjected to reduction with ~0.5 M ammonium thioglycolate (TGA at pH 9.4 with ammonium hydroxide) for 30, 15, 10, 5, and 2 minutes, while the bottom section (numbered “0”) served as an unaltered, “not reduced” control. The reduced fi ber segments were thoroughly rinsed in lukewarm run- ning tap water for ten minutes and blotted between paper towels. The fi ber segments were air-dried. (d) Fluorochrome. A 0.020% aqueous solution of the cationic Rhodamine B (CI Basic Vio- let 10), (Aldrich Chemical Co., Milwaukee, WI) was used as labeling agent to highlight, characterize, and quantify the oxidative damage infl icted upon the scale surface (1). (e) Tagging of the hair with Rhodamine B. The untreated and reduced hair fi bers were treated for one minute with 0.020% aqueous Rhodamine B solution, actively rinsed for 15 sec- onds in warm running tap water, blotted between paper towels, blow-dried, and stored in the dark at ambient temperature. (f ) Instrumental settings for microfl uorometric scanning. A Leitz MPV 1.1 microspectropho- tometer (Ernst Leitz Wetzlar, GmbH, Wetzlar, Germany) with a Vertical Ploem Illumi- nator, a microfl uorometry unit, was used for this study. Instrumental settings for the spectral and spatial (cross-sectional) microfl uorometric measurements (scans) of the Rho- damine B-labeled unaltered and “reduced” hair fi bers were as follows: • Green excitation beam: 515-560 nm KP = 580 nm LP = 580 nm • λm: 608 nm (fi lter: 37.5 mm) • Objective: 40 X • Accel. voltage: 1.2 kV • Measuring sensor: (5×60) units2 for spectral and distance (spatial) scans • Scanning speed: 72 μm/s for distance scans (high-speed scans) The dried, RB-tagged hair segments were mounted in parallel on microscope slides for spatial scanning. From the fl uorescence emission spectrum of a Rhodamine B-tagged hair fi ber (1), the wavelength of maximum fl uorescence emission had been established at λm ~ 608 nm. All spatial scans were carried out at this wavelength under identical instrumental settings. (g) Wettability scanning. Single-fi ber wettability scanning was carried out using the Wilhelmy technique (8) (using our TRI/scan apparatus). (h) XPS analysis. XPS analysis of the samples was done at an outside analytical facility for an appropriate number of hair fi bers to assure confi dence and reliability in the obtained results.

JOURNAL OF COSMETIC SCIENCE 4 RESULTS AND DISCUSSION BACKGROUND In earlier work (9), we characterized and quantifi ed the extent of cuticle cell ablation/ abrasion and complete erosion along the human hair fi ber caused by physical means. We demonstrated, with the help of a fl uorochrome (Rhodamine B in this case), how everyday standard grooming practices severely damage the physical nature of the surface structures (the cuticula) of hair fi bers (9). In other studies (1,2), we attempted to characterize and quantify photochemically and chemically induced oxidative damage to the outer β-layer on the exposed cuticle cell surface. These earlier studies have provided some interesting results, indicating distinctly different phases of hydrolysis-induced 18-MEA scission. The highlights of these studies will be briefl y summarized for the ease of comparing the effects of photochemical and chemical oxidation of earlier studies with the effects of re- duction on the outer β-layer of the exposed scale faces (our current research). (a) Photochemical oxidation. Photochemical oxidation is apparently a “two-phase” process as clearly shown in Figure 1a showing the interfi ber averages (~1200 data points per scan) of progressively UV-exposed segments of 30 different hair fi bers. As can be seen in the plot, there are two distinct phases of photodegradation of the cuticula: (1) short-term light exposure, which is an initiation period of physical changes, especially at the scale edge (as observed in the SEM), preceding lipid removal on the scale faces, and (2) long- term light exposure, during which lipid scission (delipidation) and formation of acid functionalities (sulfonic acid groups) on the scale faces take place. The kinetics of photodegradation, (see Figure 1a), may be explained as follows: The ini- tial and rather constant fl uorescence intensity (FI) of up to 48 hours suggests that this may be an induction period during which photodegradation is suppressed by free-radical generation in the sample. The source of these free radicals could be the ferrous iron in the hair that can generate free radicals by the well known Fenton’s reaction. These free radi- cals (mainly OH and OOH) are very active and mobile and terminate faster than propa- gate free-radical chain reactions. When all the iron is converted to ferric iron, the internal source of free radicals is exhausted. This seems to occur by the end of 48 hours, after which time span the photochemical degradation reaction by direct photolysis of keratin takes over. In addition, free radicals generated by the high-energy photons in combina- tion with water and oxygen may also contribute to overall photolysis. This strongly sug- gests that photochemical oxidation occurs through a free-radical mechanism, leading ultimately to negatively charged cysteic acid groups, which are tagged with RB. (b) Chemical oxidation (bleaching). Bleaching with alkaline hydrogen peroxide, on the other hand, involves thioester hydrolysis at high pH, leading to delipidation, combined with some cystine disulfi de cleavage. Both these reactions lead to formation of cysteic acid at the end, which adsorbs RB. This leads to a monotonic increase in fl uorescence as shown in Figure 1b. CURRENT STUDY: REDUCTION-INDUCED DAMAGE TO THE OUTER β-LAYER OF THE EXPOSED SCALE FACES The present study again uses microfl uorometry to establish how reduction with ammo- nium thioglycolate spontaneously initiates damage to the scale faces by attacking/breaking