irradiation and investigations of hair proteins

.J. Soc. Cosmet. Chem., 46, 85-99 (March/April 1995) Photochemical alterations in human hair. I. Artificial irradiation and investigations of hair proteins EDO HOTING, MONIKA ZIMMERMANN, and SABINE HILTERHAUS-BONG, Hans Schwarzkopf GmbH, Hamburg (E.H.), and Deutsches Wollforschungsinstitut an der TH Aachen e.v., Aachen (M.Z., S.H.-B. ), Germany. Received October 27, 1994. Synopsis Using a combination of specialamps and optical filters, equipment was constructed permitting irradiation of hair with artificial sunlight whose spectral distribution as well as its intensity corresponds to that of natural sunlight in summer months at central European latitudes. Furthermore, irradiation can be carried out selectively with the UV-B, UV-A, visible light, and IR parts of global sunlight. Black and light-brown human hair was irradiated with defined segments of the sunlight spectrum, and the extent of photodamage hair proteins was related to the radiation spectrum, to the degree of pigmentation (black or light-brown), and to the morphological region of the hair fiber (cortex or cuticle). The melanin pigment exerts a photoprotectiveffect, and thus its higher concentration in the cortex of black hair markedly retards its photodamage. On the other hand, as cuticle layers are devoid of pigment, photodegradation is similar for both the black and the light-brown hair. INTRODUCTION A well known and most obvious effect of the weathering of human hair is hair lightening, an effecthat is accelerated by moisture (1-5). The extent of this photochemically induced color change is dependent on the nature of the hair pigment and is understood to involve an oxidative attack on the eumelanin (brown-black pigment) or pheomelanin (red pigment) melanosomes (6). Hair exposed to sunlight is claimed to be more brittle, stiffer, and drier than before irradiation, and exhibits a reduced water absorption capacity (4,7). Both the tensile strength and the elongation at break are diminished in irradiated hair (8,9). These changes in mechanical properties of irradiated human hair correlate with the photodamage of the hair proteins (3,5,10). It was shown that the C-S bonds of cystine are cleaved upon UV radiation (5). The decrease in the cystine content, however, does not necessarily imply an increased swelling of the hair, since cross-linking of amino acid residues may occur as well (11). With long-term irradiation there is a progressive 85

86 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS decrease in the total recovery of amine acids after hydrolysis, except for alanine, glycine, and arginine. However, there is no clear pattern of protein degradation (11). For clarification of the action of UV light on hair proteins, model studies on keratinmelanin mixtures were carried out using electron spin resonance spectroscopy (ESR) (12). Melanin and keratin compete for the absorption of photons between 254 and 345 nm. From keratin only the cystine and the aromatic amine acid residues tyrosine, phenylalanine, and tryptephan absorb light within this wavelength range (12). Therefore, they are predestined for photochemical degradation. This finding is supported by photochemical studies on wool, a melanin-free keratin fiber, where cystine, tryptephan, and tyrosine are degraded by sunlight (13). Up to now there have been no systematic investigations the effect of the different spectral regions, i.e., UV-B, UV-A, visible light, and IR radiation found in natural sunlight, on the chemical and morphological components of human hair. In most cases the irradiation experiments have involved exposure of hair to either UV or visible light. Thus, according to Deftandre et al. (14), UV light in general is responsible for the oxidation of cystine and cleavage of protein chains. The fact that in the previously reported investigations the irradiation conditions (radiation source, wavelength filters, or duration of irradiation) have often varied makes it largely impossible to compare quantitatively conclusions made about the photochemical alterations of human hair. As a consequence, there are conflicting conclusions on the photochemically active region of the sunlight, e.g., with reference to the brightening of the color of human hair. Reese and Maak (3) attribute the bleaching action of the sunlight exclusively to the visible range of the sun's spectrum. In contrast, Tatsuda et al. (5) demonstrated a bleaching effect of UV radiation on black hair. The carbon or xenon arc lamps used to simulate sunlight emit radiation with a spectral intensity distribution that is markedly different from that of natural sunlight. The carbon arc lamp emits more intense light in the region of 350-400 nm than sunlight, while the xenon arc lamp produces the so-called green yellow hole between 475 and 600 nm. A more precise knowledge of the conditions under which hair undergoes photodamage is necessary for the development of potentially photoprotective treatments. To this end radiation equipment was designed by the Institute of Light Technology, TU Berlin, which, by a combination of specialamps and optical filters (Table I), allows for irradiation with artificial sunlight whose spectral distribution as well as intensity largely corresponds to that of natural sunlight in the summer months at central European latitudes. Furthermore, irradiation can be performed selectively with UV-B, UV-A, visible light, and IR. By irradiating human hair with specific ranges of sunlight and then analyzing the hair, the extent of photodamage of the hair proteins can be related to the range of radiation. Furthermore, by irradiating black and blond human hair, the protective function of the melanin pigments can be ascertained. MATERIALS AND METHODS MATERIAL The investigations were carried out on untreated black and light-brown European human hair (supplied by Herzig Co.).

HAIR PHOTOCHEMISTRY 87 Table I Combination of Lamps and Filters Yielding Defined Partial Spectra of Natural Sunlight Spectral Results range Lamp Filter x (nm) Wavelength UV-B 14 Philips fluorescent UG 11/1 mm, 280-350 lamps, TC 20 W/12 GG 19/1 mm UV-A 2 Osram-Ultra-med, UG 11/1 mm, 320-400 400 W WG 320/! mm Visible light 2 Osram HMI 575 W Plexiglas type 201, 370-780 Blakers IR stop filter IR 2 Philips RG 780/3 mm, 750-2800 halogen-incandescent KG 4/2 mm lamps, 600 W Global light 2 Osram HMI, 575 W, 2 layers metal gauze mesh, 280-1100 1 Philips density 2 mm halogen-incandescent lamp, 600 W Optical filters from Schott Co. HAIR PURIFICATION The hair was extracted for 5 min with dichloromethane and for 30 min with diethyl ether, washed with a nonionic tenside, rinsed, and dried. The hair was stored at 65% relative humidity and 20øC. IRRADIATION OF THE HAIR The hair swatches were irradiated for six weeks (1008 h) with UV-B, UV-A, visible light, IR, or global irradiation. Approximately 10 g of hair were spread out in parallel in an area of 600 cm 2 in closed climatic boxes (RH > 70%) and turned over daily during the irradiation period. COLOR DETERMINATION The color of the irradiated hair was determined visually by comparing the color quality with non-irradiated light-brown and black samples. ISOLATION OF THE CUTICLE Approximately 500 mg of hair were shaken with 25 ml of distilled water in sample glasses in an ellipsoid shaker at 2.800 min- for 8 h. According to Swift and Bews (15) the cuticle detaches from the fiber stem under these conditions and can be subsequently separated from the stem by filtering through a 100-mesh filter. The obtained hair fragment suspension was centrifuged at 30.000 min- for 20 min and the supernatant liquid removed. The residue containing the cuticle cells was lyophilized.

88 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS AMINO ACID ANALYSIS AFTER TOTAL ACID HYDROLYSIS Approximately 10 mg of hair or isolated cuticle were placed into 4 ml of 5.7 M freshly distilled HCI and hydrolyzed in a glass tube at 105øC for 24 h. The subsequent amino acid analysis was performed according to Spackman et al. (16). CYSTINE OXIDES The cystine oxides of the outer cuticle layers were determined semiquantitatively by means of IR spectroscopy, using a technique of attenuated total internal reflection (FTIR/ATR) from a KRS-5-crystal (17). RADIATION EQUIPMENT AND IRRADIATION CONDITIONS RADIATION EQUIPMENT The arrangement of the radiation unit that simulates five different parts of the natural sunlight spectrum is sketched in Figure 1. The radiation installation consists of five open containers with inside dimensions of 50 cm x 50 cm x 60 cm. By the combination of specialamps with optical filters each radiation container produces a spectrum with defined ranges of wavelengths (Tables I and II) and emission intensities (Table II). UV-B, UV-A, visible light, IR, and global irradiation simulate the intensities of natural sunlight in summer months at central European latitudes (Table II). air shaft lamps optical filter sedir 1-1,.,,/, I I.,/ quartz glass samples glyc./water closed chamber irradiation compartment air stream Figure 1. Sketch of the radiation installation with boxes for UV-B, UV-A, visible light, IR radiation, and global light. Each radiation unit contains two closed climatic chambers in which the human hairs are irradiated under controlled humidities and temperatures.

HAIR PHOTOCHEMISTRY 89 Table Spectral Distribution in the Radiation Chambers and Radiation Intensities (E) of the Partial Ranges of the Sunlight Spectrum in Comparison With Natural Sunshine Global irradiation E recommended 2 E measured 3 Spectral range h. (nm) (%) (W/m 2) (W/m 2) UV-B 280-350 0.4 4 2.5 UV-A 320-400 5.7 50 48 Visible-light 370-780 51.8 585 463 IR 750-2800 43.9 511 440 Global light 280-1100 100 1140 1037 Distribution of natural sunlight in summer in Central Europe. CIE recommended distribution of radiation intensities during investigations with artificial sunlight (18). Measured radiation intensities the irradiation chambers, determined by G. Geutler, 1988 (19). The lamps of the irradiation containers are cooled by an airflow conducted through a pipe into hood shafts. The side walls of the radiation units are lined with aluminium sheets for uniform distribution of radiation. The radiation containers are open on one side. Since the photooxidative changes in hair are affected by humidity and temperature (4,11), the containers were supplemented by climatic boxes with closed gas space that allow the passage of visible light as well as UV radiation and in which the temperature and humidity can be regulated. For this purpose, steam-saturated air with a relative humidity of 74-94% is introduced into the climatic boxes with a tubing system (Table III: outer RH). A ventilator installed in the side of the boxes evenly distributes the humid air warmed by the lamps. The hair samples are irradiated while arranged in parallel over a 20-cm X 30-cm perforated table. Table II provides an overview of the energies in the individual radiation containers, which were intended and actually measured. In column 3 the radiation intensity values (E recommended) are given, which are proposed in a CIE recommendation for radiation experiments with artificial sunlight corresponding to the values occurring in natural sunlight (18). In column 4 (global irradiation) the values are listed as relative to the natural sunlight in summer at central European latitudes. The empirically determined wattage in the radiation chambers (19) in column 5 (E measured) shows an optimum matching of the simulated sunlight with the naturally occurring light. II Table Temperature and Relative Humidity in the Climatic Chambers During Irradiation With Artificial Sunlight Spectral range T inside (øc) III RH outside (%)2 Calc. RH inside (%)3 UV-B 31 74 52.2 UV-A 25 94 94 Visible light 48 77 22 IR 35 84 47.3 Global light 43 77 28.2 Temperature in climati chambers. Relative humidity of the piped-in air. Calculated relative humidity in the climatichambers applying the formula p H20/p H2Omax.

._ -- 90 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The radiation intensity values measured in the radiation chambers in Table II arise from the sum of radiation strengths dependent on the wavelength. The resulting spectral emission strengths of UV-B, UV-A, visible light, IR, and global radiation are graphically given in Figures 2-6. IRRADIATION OF HUMAN HAIR Light-brown and black untreated European hair was irradiated for six weeks (total time: 1008 h) in the radiation chambers with UV-B, UV-A, visible light, IR, or global light. The light intensity in these experiments corresponded to that present in the relevant spectral segments of natural sunlight (Table II). In the course of irradiation the temperature within the irradiation chambers varied (from 25øC in the case of UV-B to 48øC in the case of visible light,), and thus led to changes in the relative humidity within the chambers. These changes are estimated and are given in Table III. RESULTS COLOR MEASUREMENTS The resulting color changes of the irradiated light-brown and black hair are given in Table IV as a function of the radiation conditions. Light-brown hair was significantly lightened by visible light and global irradiation and perceptibly bleached by UV-B (lighter tips) and UV-A radiation (somewhat lighter) (Table IV). Reese and Maak claimed a slight lightening of dark-brown hair, a greater lightening of brown, and a very marked lightening of red-brown and blond hair after irradiating the samples for 21 days with Mediterranean sun in summer (3). They attributed the bleaching action of the sunlight only to the visible range of the sunlight and negated any bleaching effect of the UV light. 0,12. 0,10 r' 0,08 c 0,06 0.0/,, o.o2 25O 400 Wovelength [nm] Figure 2. Spectral radiation intensities in the UV-B container.

_ HAIR PHOTOCHEMISTRY 91 2.0 1.6 1.2 0.8 0.4. o, --I-F ]oo 3 o ' 400 Wo, ve[engt'h [ nm] Figure 3. Spectral radiation intensities in the UV-A container. 1.4 0.8-0.6 0.4 o 5o 400 450 500 550 600 650 700 750 Wovelength [ nm] Figure 4. Spectral radiation intensities in the container with visible light. [ 8OO Black hair was lightened only after six weeks of irradiation with visible light or global irradiation. There was no lightening effect by UV-A, UV-B, or IR light. Leroy et al. (20) have already shown a bleaching action of the visible segment of the sun's radiation on melanin pigments. However, the employed UV filters allowed also for the passage of part of UV-A radiation in addition to visible light. On the other hand, Tatsuda et al. (5) claimed that UV light lightens black hair. In these experiments the hair was exposed to a xenon arc lamp, which emitted radiation in both visible and UV-A segments. Our data suggest strong correlation with the pigment content of the hair. By irradiation of light-brown and black human hair it was shown that photobleaching of human hair is dependent on the hair color as well as the range of the sun's spectrum. Not only visible light, but also UV-A and UV-B lighten light-brown hair. Black hair is bleached only to a small degree by the visible range of sunlight.

-- ß 92 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 1.0 0.8 0.6 0.6 L 0.2,00 900 1200 1500 1800 2100 2/,00 2700 3000 WQve[engfh [nm] Figure 5. Spectral radiation intensities in the IR containerß 1.6 'E 1.2 ' 4.0 1,-,, -- 0.8 1,-,,._o 0.6 0.6 4--- 0.2 0 200.00 600 800 Wc veiengfh [nm] ' 10 0 1200 Figure 6. Spectral radiation intensities the container with global light. AMINO ACID ANALYSES The cuticle cells, the outer shingle-like arranged cells of the hair are most exposed to weathering. Therefore, we pursued the question as to whether different extents of photooxidative alteration on the proteins in the cuticle and cortex can be established by analysis of the amino acids. For this purpose, amino acid analyses of the acid hydrolysate of isolated cuticle and complete hair fibers were compared with each other. The concentrations of the photolabile amino acids are given separately for the cuticle and the whole fiber in Tables V-VIII. The results can be interpreted only qualitatively since the yield of proteins from the samples differed greatly with different types of radiation. A comparison of the values in Table V and VI reveals no essential differences between the protein composition of the cuticle of light-brown and black hair for either the untreated (column 1) or the irradiated (columns 2-6) samples.

HAIR PHOTOCHEMISTRY 93 Table IV Alterations in the Color of Light-Brown and Black Human Hair After Six weeks of Irradiation With Simulated Ranges of the Sunlight Spectrum Color alteration Spectral range Light-brown Black UV-B Tips lighter Unaltered UV-A Somewhat lighter Unaltered Visible light Significantly lighter Somewhat lighter IR Unaltered Unaltered Global Lighter Somewhat lighter Table ¾ Amino Acid Composition (AA) of the Isolated Cuticle From Light-Brown Hair, Untreated and Irradiated for Six Weeks With UV-B, UV-A, Visible Light, IR, or Global Light AA (mol/100 mol) Non-irradiated UV-B UV-A Visible light IR Global CySO3H 1.32 1.40 1.70 1.96 1.15 1.62 Cys 2 8.73 5.36 9.19 8.23 9.70 7.54 Gly 9.73 10.82 9.14 9.58 9.41 10.71 Ala 5.53 6.71 5.29 5.83 5.82 5.59 Tyr 1.55 1.66 1.96 1.27 1.67 1.61 Phe 1.70 1.36 1.56 1.49 1.44 1.35 Orn 0.34 0.22 0.16 0.18 0.27 0.28 Lys-Ala -- 0.13 0.10 -- -- Traces His 0.89 0.41 0.44 0.48 0.53 0.37 Table ¾I Amino Acid Composition (AA) of the Isolated Cuticle From Black Hair, Untreated and Irradiated for Six Weeks With UV-B, UV-A, Visible Light, IR, or Global Light AA (mol/100 mol) Non-irradiated UV-B UV-A Visible light IR Global CySO3H 1.03 1.03 1.59 2.32 1.22 1.65 Cys 2 9.19 5.84 9.47 8.43 10.06 7.28 Gly 9.39 17.74 9.71 10.21 9.71 9.93 Ala 5.81 7.88 6.06 6.32 6.02 5.66 Tyr 1.55 1.32 1.65 1.10 1.78 1.77 Phe 1.74 1.35 1.33 1.48 1.38 1.46 Orn 0.30 0.20 0.18 0.41 0.31 0.27 Lys-Ala 0.05 -- -- Traces His 0.61 0.45 0.35 0.58 0.47 0.57 Amino acid composition of the cuticle. The cuticle of either light-brown or black hair is most markedly modified by UV-B irradiation (Tables V and VI). The most obvious change results from the cleavage of disulfide bridges, which is indicated by a decrease in amino acid cysteine. Additionally, the cleavage occurs heterolytically between the C-C or C-S bonds of cysteine, and accordingly the cleavage products glycine, alanine, and

94 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS dehydroalanine result. Subsequently, the latter can react to form lysinoalanine or lan- thionine. The cystine content of the cuticle of light-brown and black hair is reduced from 8.8-9.2 mol/100 mol to 5.4-5.8 mol/100 mol. This decrease does not correlate with an increase in cysteic acid (11), but is accompanied by an increase glycine (q- 100%) and alanine (q- 12% by UV-B irradiation of light-brown hair, q- 13.5% by UV-B irradiation of black hair). This unexpecte discrepancy can be resolved by a disproportioning of the photochemically formed cystine oxides to cysteic acid and cysteine; in part, they could be completely destroyed (21). Furthermore, we support a decomposition of cystine to glycine and alanine, induced by a free radical reaction (22). Both mechanisms underlying the lack of cystine oxides in irradiated keratin fibers are not established, however. Evidence for photochemical cross-linking of keratin can be deduced from the presence of lysinoalanine, which was identified in UV-B- and UV-A-irradiated samples. The aromatic amino acids phenylalanine and histidine are markedly reduced upon UV-B, visible light, IR, and global irradiation. Damage to hair proteins by irradiation with visible light is recognizable only by a decrease in the aromatic amino acid tyrosine in both light-brown and black hair. Its content decreases from 1.6 to 1.3 and 1.1 mol/100 mol amino acids, respectively. It is somewhat unexpected, as the tyrosine absorbs only in the UV region and should not be affected under conditions of the experiment. Amino acid composition of the whole fiber. Differences in the amino acid composition of the whole fibers (Tables VII and VIII) depend on the spectral characteristics of sunlight as well as on the type of pigmentation. The cystine content of light-brown hair is drastically reduced upon UV-B and UV-A irradiation, accompanied by an increase of glycine; the concentration of cystine in black hair is reduced only by UV-B light. Proline and valine are degraded by UV-A and UV-B to a higher degree in light-brown hair than in black hair. Comparison of the results of the cuticle and the total fiber. Differences in the amino acid composition of irradiated black and light-brown total hair correlate both with the nature and concentration of melanin pigments in black and light-brown hair. The lesspronounced photochemical alterations of the proteins of the whole hair in comparison to Table Amino Acid Composition (AA) of Whole Light-Brown Hair, Untreated and Irradiated for Six Weeks With UV-B, UV-A, Visible Light, IR, or Global Light AA (mol/100 mol) Non-irradiated UV-B UV-A Visible light IR Global CySO3H 1.17 1.07 0.76 1.19 1.14 1.02 Cys 2 8.95 4.32 5.79 8.55 9.17 5.75 Gly 5.47 8.21 7.60 5.93 6.17 6.39 Ala 4.54 5.84 5.51 4.78 5.12 5.45 Pro 5.9 9.21 8.99 8.01 7.98 7.45 Val 5.08 3.43 3.39 5.93 6.37 6.70 Tyr 1.27 1.79 1.91 2.16 2.23 2.18 Phe 1.26 1.88 2.43 1.97 1.89 1.69 His 0.55 0.51 0.43 0.60 0.81 0.60 VII

HAIR PHOTOCHEMISTRY 95 Table VIII Amino Acid Composition (AA) of Whole Black Hair, Untreated and Irradiated for Six Weeks With UV-B, UV-A, Visible Light, IR, or Global Light AA (mol/100 mol) Non-irradiated UV-B UV-A Visible light IR Global CySO3H 1.14 0.96 0.74 0.90 0.49 0.97 Cys 2 7.00 5.7! 8.95 9.02 9.22 8.32 Gly 7.34 7.77 6.39 6.08 5.85 5.72 Ala 5.47 5.95 4.56 4.83 5.84 4.54 Pro 8.26 8.68 8.53 7.72 8.13 7.67 Val 3.02 3.33 6.05 6.07 6.24 4.70 Tyr!. 96 2.00 2.63 2.24 2.24!. 09 Phe 2.86 2.60 1.91 1.80 1.67 1.27 His 0.74 0.72 0.66 0.75 0.84 0.55 the cuticle is suggestive of the photoprotectiveffect of the melanin pigment, which resides predominantly the fiber cortex of human hair (23). As the extent of changes in the amino acid composition of the cuticle of light-brown and black hair is similar, this can be readily explained by absence of pigment in these cells. It appears that the pigments of black hair protecthe cortex proteins of the fiber better against photodamage than does the melanin of light-brown hair. INFRARED SPECTROSCOPIC INVESTIGATIONS By means of infrared spectroscopic investigations (FTIR/ATR), photochemically created cystine oxides were determined semiquantitatively in the outer cuticle layers. The IR spectra were standardized using the amide-i band, allowing a semiquantitative comparison of the absorptions of the cystine oxides (17). In Figures 7 and 8 the --1 vibrations of cysteic acid and cystine monoxide are depicted in the range of 1136 cm to 940 cm-1 for both light-brown and black hair. In the spectra the bands 1042 cm- 1 of sulfonic acid R-SO3H (peak 8), 1073 cm- of the cystine monoxide R-SOS-R (peak 7), and 1125 cm- of cystine dioxide R-SO2S-R (peak 6) have been identified (Table IX and Figure 9). The IR spectrum of untreated, non-irradiated hair (Figure 9) is characterized by similar intensities of absorption at 1073 cm-1 (peak 7) and 1042 cm-1 (peak 8). In the untreated light-brown and black hair used in the present investigation (Figures 7 and 8, untreated sample) a markedly higher absorption of the sulfonic acid at 1042 cm- 1 (peak 8) than of the cystine monoxide (1073 cm- 1) is already seen, suggesting oxidative damage of the source material, e.g., by natural exposure to weather. IR irradiation of light-brown and black hair does not result in an increase in the cysteic acid peaks (Figures 7 and 8). The spectra even show decreased absorptions for cystine monoxide and cysteic acid (absorbance of peak 8 = 0.22) compared to the spectra of the untreated samples (absorbance of peak 8 = 0.29 in the case of black hair and 0.30 in the case of light-brown hair). Apparently the photooxidation is not the dominant mode of photodegradation in this case.

96 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 0,5 0,3-0,2 0,1 i i i 1136 1097 1058 1019 980 9/,1 Wo, venumber [cm - ] Figure 7. Alteration in the absorption intensities of cysteic acid (peak 8) and cystine monoxide (peak 7) by irradiation of light-brown human hair for six weeks with parts of the sunlight spectrum and globalight. Measurement of the surface was performed with the FTIR/ATR technique (16). 0, s t // gtobo[ / /... vis. I. ighf 0,3.. ß 0,2 0,1 0 1136 i 1097 1058 1019 980 9/+1 Wovenumber [cm - ] Figure 8. Alteration in the absorption intensities of cysteic acid (peak 8) and cystine monoxide (peak 7) by irradiation of black human hair for six weeks with parts of the sunlight spectrum and global light. Measurement of the surface was performed with the FTIR/ATR technique (16). i

HAIR PHOTOCHEMISTRY 97 Table Characteristic Bands in the IR Spectrum of Keratin Fibers IX Oscillation (cm- ) Molecular group Peak number 1628 Amide-I 1 1513 Amide-II 2 1449 CH 3 1232 Amide-III 4 1176 Sulfonic acid, RSO3H, asymmetrical oscillation 5 1125 Cystine dioxide, RSO2SR 6 1073 Cystine monoxide, RSOSR 7 1042 Sulfonic acid, RSO3H, symmetrical oscillation 8 1850 1750 1650 1550 1/+50 1350 1250 1150 1050 950 Wovenumber [ cm - ] Figure 9. Keratin spectrum of untreated, non-irradiated human hair in the range of 1850 to 950 cm- Measurement of the surface was performed with the FTIR/ATR technique (16). The classification corresponds to Table IX. UV irradiation and, to a somewhat greater extent, irradiation with visible light lead to a slight increase cysteic acid content of the cuticle of both light-brown and black hair (absorbance of 0.35-0.38 in irradiated hair in relation to absorbance of 0.29 resp. 0.30 in untreated hair, Figures 7 and 8). The highest content of cysteic acid in the cuticle of the hair samples is attained by irradiation with global light (absorbance of 0.47 resp. 0.49), which, as the sum of irradiation energies, gives rise to the most extensive photochemically induced reactions. The extent of the photochemically induced cystine oxidation in the cuticle of lightbrown hair is similar to that of black hair. Since only a few melanin granules are present in the cuticle cells of human hair, they lack this sunlight-protective component, which may reduce the cystine oxidation of proteins caused by UV or visible light. The IR spectroscopic investigations partly supporthe results of the amino acid analyses on cuticle proteins. Both determinations reveal the highest cysteic acid concentrations

98 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS in the samples irradiated with visible light, and no differences in the content of cystine oxidation of light-brown and black hair are discernible. SUMMARY Hair samples of two different melanin contents have been subjected to irradiation with selective ranges of sunlight. Their physical and chemical alterations brought about by the photoexposure have been evaluated and can be summarized as follows: 1. Sunlight-induced color changes in hair are more extensive in light-brown than in black hair. The most obvious photobleaching effect occurs within the visible range of sunlight. Furthermore, UV-A and UV-B also cause a lightening in light brown hair. 2. The proteins of the cuticle are especially impaired by UV-B and UV-A, but only slightly by visible light. The composition of the amino acids in the cuticle is more strongly altered than in the cortex since greater intensities of radiation can act in the outer cell layers. The changes in the cuticle proteins do not show any correlation with the hair color since only a small number of melanin granules are present here. In contrast, differences between the irradiated proteins of light-brown and black hair cortex are readily detectable (melanin-rich areas). In particular, the cystine, proline, and valine residues in whole light-brown hair are more markedly degraded by UV-A and UV-B than in whole black hair. 3. Infrared spectroscopic investigations (FTIR) support the conclusions drawn from the amino acid analyses with respecto the radiation-induced formation of cysteic acid in the cuticle. The highest contents are reached by irradiation with visible light and UV-A to the same extent in light-brown as in black hair cuticle layers. CONCLUSIONS The present results suppor the assertion that black hair is more photostable than blond hair. Black hair seems well protected against UV-light, and light-brown hair is obviously damaged by a wide range of the natural sunlight. The protective action of melanin granules is limited to the melanin-rich cortex of black hair, which shows only a slight modification of fiber proteins under irradiation; the melanin-poor cuticle proteins of black as well as of light-brown hair are modified to a similar extent. The photodegradation of cystine is the most conspicuous alteration in the amino acid residues of the whole fiber, to which additional consequences for the hair are attributable: loss of fiber strength and water infiltration that subsequently creates favorable conditions for further photooxidative reactions by the dissolved oxygen (11). For these reasons it would be very useful to employ sunscreens for all types of human hair, to preserve primarily the cuticle from extensive photodamage and also the whole fiber from photoinduced cystine and melanin degradation. REFERENCES (1) V. H. Price, "The Role of Hair Care Products," in Hair Research.' Status and Future Aspects, Orfanos, Montagna, and Stiittgen, Eds. (1981). (2) L. J. Wolfram and L. Albrecht, Chemical and photo-bleaching of brown and red hair, J. Soc. Cosmet. ½hem., 82, 179 (1987).

HAIR PHOTOCHEMISTRY 99 (3) G. Reese and N. Maak, Die Bestiindigkeit von Haafarben unter dem Einflu[3 von Sonne, Wasser, Salz, ß alrztl. Kosmetol., 12, 373-379 (1982). (4) E. Tolgyesi, Weathering of hair, Cosmet. Toiletr., 98, 29-33 (1983). (5) M. Tatsuda, M. Uemura, K. Torii, and M. Matsuoka, Studies on hair damage and demelanization by ultra violet light, J. Soc. Cosmet. Chem. Japan., 21, 43-49 (1987). (6) R. Crippa, V. Horak, G. Prota, P. Svoronos, and L. Wolfram, "Chemistry of Melanins," in The Alkaloids, Vol. 36, A. Brossi, Ed. (Academic Press, 1989). (7) T. Horiuchi, Nature of damaged hair, Cosmet. Toiletr., 93, 65 (1978). (8) S. Kanetaka, K. Tomizawa, H. Iyo, and Y. Nakamura, The effect of UV radiation on human hair concerning physical properties and fine structure of protein, International Federation Societies of Cosmetic Chemists, Yokohama (1992). (9) R. Beyak, G. S. Kass, and C. F. Meyer, Elasticity and tensile properties of human hair. II. Light radiation effects, J. Soc. Cosmet. Chem., 22, 667 578 (1971). (10) F. G. Lennox and R. J. Rowlands, Photochemical degradation of keratins, Photochem. Photobiol., 9, 359-367 (1969). (11) L. J. Wolfram, "Reactivity of Human Hair, a Review," in Hair Research: Status and Future Aspects, Organos, Montagna, and Stiittgen, Eds. (1981), pp. 479-500. (12) C. R. Robbins and M. Bahl, Analysis of hair by electron spectroscopy for chemical analysis, J. Soc. Cosmet. Chem., 35, 379-390 (1984). (13) K. RiSper and E. Finnimore, Chemical structure of chromophores formed during photoyellowing of wool, Int. Wool Text. Res. Conf. Tokyo, IV, 21-31 (1985). (14) A Deftandre, J. C. Garson, and F. Leroy, Photoaging and photoprotection of natural hair, International Federation Societies of Cosmetic Chemists Congress, New York, October 1990. (15) J. A. Swift and B. Bews, The chemistry of human hair cuticle. I. A new method for the physical isolation of cuticle, J. Soc. Cosmet. Chem. 25, 13-22 (1974). (16) D. H. Spackman, W. H. Stein, and S. Moore, Automatic recording apparatus for use in the chromatography of amino acids, Anal. Chem., 30, 1190-1206 (1958). (17) U. Schumacher-Hamedat, J. FiShles, and H. Zahn, Intermediate steps in the oxidation of cystine in the bleaching process, Textilveredlung, 21, 121-125 (1986). (18) Commision Internationale de L'Eclairage (CIE): Empfehlungen fiir die Gesamt-bestrahlungsstiirken und die spektrale Verteilung kiinstlicher Sonnenstrahlung fiir Priifzwecke, CIE-Publication No. 20 (1972). (19) G. Geutler, MeJ3gutachten Lichttechnik TU Berlin (1988). (20) F. Leroy, A. Deftandre, and J. C. Garson, Photoaging of human hair, Haarwissenschaftl. Symp. Bad Neuenahr (Nov. 1990). (21) U. Schumacher-Hamedat, Modellstudien zur Entstehung von partiell oxidierten Cystin-derivaten bei oxidativer Veredlung von Wolle, Thesis RWTH Aachen (1986). (22) This radical reaction mechanism is suggested by the reviewer. (23) N. L. Sakina and A. E. Dontsov, Inhibition oflipid photooxidation by melanin, Biochem. USSR, 51, 744-747 (1986).