AMMONIA/PEROXIDE BLEACHING OF HAIR 401 1 oo 80 • 60 40 20 -- emission ........ excitation 0 I I I I 200 300 400 500 600 Wavelength (nm) Figure 3. Fluorescence emission and excitation spectra of Sepia melanin (0.5 mg/ml) after 45 min of oxidation with 2% H202 and 0.75% NH 3 at pH 10.0. Fluorescence intensity is shown in arbitrary units. Sepia melanosomes. Figure 6 shows a representative image of a suspension of isolated hair melanosomes in water. It is evident from the pictures that black hair melanosomes are elongated in shape, typically 0.8-1.2 lam in length and 0.3-0.6 lam in width. Isolated hair melanosornes retain their morphology, as in hair, except that the granules form larger aggregates of rice-shaped particles in aqueous suspensions. Interestingly, it is found that these aggregates are surrounded by an amorphous material clearly evident in the TEM image. Since the melanosomes were isolated using protease enzymes, this material may not be a simple protein. Unlike Sepia, suspension of hair melanosomes did not break into nanoparticles upon sonication. Ultrastructural features were also not clearly evident in the TEM images of hair melanosomes. TEM STUDIES ON THE EFFECT OF AMMONIA A time-dependent TEM imaging study was performed to monitor the morphological and structural changes of hair melanosomes in the presence of aqueous ammonia. This study was designed to understand the specific role of ammonia in the bleaching process of hair. Isolated hair melanosomes (0.05 mg/ml) were treated with 1% NH 3 (pH 10.0) in aqueous solution. Aliquots were taken every 30 min for TEM analysis. TEM images show that ammonia treatment removes the amorphous material surround- ing the melanosomes. As a result, the melanosomes appear very well dispersed. Figure 7 shows a TEM image of ammonia-treated hair melanosomes after 30 min. It is evident from Figure 7 that ammonia treatment ruptures the melanosomal membrane, re!eaging melanin nanoparticles (30-60 nm) out of the sac. This study also demonstrates that hair melanosomes are comprised of melanin nanoparticles encapsulated in a membrane sac. However, a similar melanosomal membrane sac is absent in Sepia melanosomes. Sodium

402 JOURNAL OF COSMETIC SCIENCE 25 2O 15 10 Hair ß Sepia 0 20 40 60 8O 100 Time (min) Figure 4. Time-dependent fluorescence study for the bleaching of hair and Sepia melanin using NH3/H202 at pH 10.0. A suspension of melanosomes (0.5 mg/ml) was treated with 2% H202 and 1.0% ammonia, and aliquots were taken every 5 min for time-dependent measurements. Intensity of fluorescence emission is plotted as a function of time. hydroxide or sodium carbonate treatment at pH 10 does not effect similar structural changes to hair melanosomes as ammonia. Thus, it is confirmed that the observed melanosomal changes are not merely due to alkaline pH but are, rather, specific to ammonia. Sepia melanosomes under identical conditions of ammonia treatment did not show such changes, probably due to a slow rate of bleaching, as evident from the time-dependent fluorescence studies. Prolonged treatment of hair melanosomes with NH3 induced a complete destruction of characteristic melanosome morphology and a shrinking of the granule size (Figure 8). As shown in Figure 8, the melanosomes appeared as fibril-type structures devoid of the melanosomal membrane sac. After 3 hr of treatment, no particles were seen by TEM, except an amorphous material. The ammonia-treated melanin suspension showed a lighter color compared to the untreated suspension, and a decrease in longer wavelength absorbance in the UV-Vis spectrum. Thus, it is evident that ammonia could possibly induce lightening of hair melanosomes. However, the overall process of particle break- down leading to an amorphous material is slow and took more than 3 hr. TEM STUDIES ON THE EFFECT OF HYDROGEN PEROXIDE Isolated hair melanosome suspension was treated with 2% n20 2 solution at pH 7.0 and pH 8.0 to see how peroxide alone degrades melanosomes. The degradation of melano- somes by n202 at neutral pH is a very slow process, and even after 60 min of treatment,