International Journal of Cosmetic Science, 2014, 1–7
doi: 10.1111/ics.12178
The coordination environment of copper in hair can be altered by treatment products N. Worasith* and B. A. Goodman† *Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Krungthep, 2 Nang Lin Chi Road, Soi Suan Plu, Sathorn, Bangkok, Thailand and †State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi 530004, China
Received 22 October 2014, Accepted 26 October 2014
Keywords: chemical analysis, copper coordination, hair treatment, spectroscopy
Synopsis OBJECTIVE: To determine whether the coordination environment of copper in hair is affected by the shampoo used. METHODS: Electron paramagnetic resonance (EPR) spectroscopy to discriminate between mixed oxygen/nitrogen and mixed oxygen/ sulphur coordination of copper after treatment with two different shampoos. RESULTS: Copper with mixed oxygen/nitrogen coordination could be converted to mixed oxygen/sulphur coordination by treating with the appropriate shampoo, but this was not reversible with the products tested, although copper was removed from hair at very high pH values. CONCLUSION: Commercial hair treatment products can have a profound effect on the copper coordination environment in hair, and this must be taken into account in any attempt to use hair as a health marker. sume Re OBJECTIF: Determiner si l’environnement de coordination du cuivre dans les cheveux est affecte par le shampooing utilise. METHODES: Resonance paramagnetique electronique (RPE) pour discriminer entre la coordination de cuivre mixte oxygene / azote et la coordination de cuivre mixte oxygene / soufre apres le traitement avec deux shampooings differents. RESULTATS: Le cuivre avec la coordination mixte oxygen/nitrogen peut ^etre converti en coordination mixte oxygene / soufre en traitant avec le shampooing approprie, mais ce ne fut pas reversible avec les produits testes, bien que le cuivre ait ete retire des cheveux aux tres hautes valeurs de pH. CONCLUSION: Les produits de traitement des cheveux commerciaux peuvent avoir un effet profond sur l’environnement de coordination du cuivre dans les cheveux, et cela doit ^etre pris en compte dans toute tentative d’utiliser les cheveux comme un marqueur de la sante. Introduction The possibility of using hair analysis as a marker of health status and/or environmental exposure to heavy metals and other potenCorrespondence: Bernard A. Goodman, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004 Guangxi, China. Tel.: 0086 771 3272475; fax: 0086 771 3237873; e-mail: bernard_a_ [email protected]
tially dangerous substances has been the subject of debate for many years (e.g. [1, 2]). The principal component of hair, the cysteine-rich protein keratin, is produced by specialized cells which die after they have been filled with the protein in the hair follicle. Thus the elemental composition of hair may reflect that of the blood while it is being formed and represent a historical record of variability in blood composition (e.g. [3]). In addition, the hair shaft is porous and hair can adsorb various chemicals and salts from its environment and may thus be of direct use in environmental monitoring, especially in an industrial setting. There have been many reports in the scientific literature of hair analyses being used for the identification of environmental problems that could impact on health, including passive smoking [4, 5], alcoholism [6], illicit drug use [7] and exposure to high levels of heavy metals, such as mercury [8], lead [9] and cadmium [10]. In addition, hair analyses have been used as indicators of some disease conditions which involve abnormalities in metal accumulation (e.g. [11]), and there are continual efforts to search for abnormalities in the metal contents of hair as markers of other disease states (e.g. [12, 13]). Copper is a trace element that is commonly found in normal hair, and although appreciable contributions come from tap water [14], it might be possible to use hair as a marker of copper-based genetic disorders [15], such as Menkes disease which involves copper deficiency [16], or Wilson disease, which is a copper toxicosis condition [17]. These have been shown to be caused by mutations of two closely related Cu-transporting ATPases [18], although no elevation in total copper contents of hair is observed in Wilson’s disease (e.g. [19]). However, as copper is also involved in certain types of cancer [20, 21] and the pathogenesis of neurological disorders such as Alzheimer’s disease [22], motor neuron diseases [23] and prion diseases [24], it is plausible that the chemical forms rather than the total amounts of copper in the hair could be markers of various health conditions. In addition, as shown recently by Naqvi et al. [25], the chemical forms of copper influence the free radical chemistry in hair that can produce structural damage during colouring processes. Recently, we reported an investigation of the paramagnetic components of hair using electron paramagnetic resonance (EPR) spectroscopy [26], a technique that can specifically detect and characterize paramagnetic chemical species, such as free radicals and certain transition metal ions and complexes. In that work, we observed two distinctly different types of copper complex in hair from different subjects; one was interpreted as corresponding to Cu (II) coordinated to a mixture of oxygen and nitrogen atoms,
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
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Coordination of Cu in hair
whereas the other corresponded to a mixture of oxygen and sulphur atoms. However, no information was presented as to the reasons for these differences, although environmental effects or hair care products were considered to be possibilities, as samples from the hair of one subject taken over a period of several years showed variations in the copper complexes at different times. Thus, if the composition of hair is influenced by hair care products, its use for either environmental monitoring or health status becomes increasingly complicated, especially as there is currently a huge range of hair care products openly available for a variety of uses, including shampooing, conditioning, colouring, relaxing and curling. As hair treatments might result in changes in the coordination environments of trace metals in the hair, the present experiments were undertaken with the objective of determining whether different types of hair treatment can produce major changes in the copper coordination environment. Measurements focused on hair samples from two subjects which gave well resolved EPR spectra, each corresponding to one of the two identified coordination environments in the work of Worasith and Goodman [26]. These samples were then subjected to a range of pH values, as these are encountered in some of the more extreme treatments associated with relaxing or permanent waving. Hair relaxers typically have pH values in the range 12–13, whereas perms can be either acidic or alkaline. In addition, hair samples from each subject were treated with the shampoo/conditioning regimens used by the other
subject, in order to determine the extent to which such commercially available products might directly influence the copper coordination chemistry. Materials and methods Hair samples The hair samples used in the present work were those that gave good quality EPR spectra each corresponding to one of the two types of copper signal in our original investigations [26]. In each case, the subject was a healthy middle-aged Asian female. In addition to the original hair samples which were taken in 2012, additional samples were taken from the same two subjects in 2014. All hair samples were cut from the nape of the neck. Treatments Two separate types of treatment were investigated. The first considered the effect of pH. Initial measurements were made by immersing hair samples in solutions of 1 M HCl or 1 M NaOH for 1 h, then rinsing thoroughly with deionized water and drying in air at ambient temperature. Then subsequent measurements were made using HCl or NaOH solutions at pH 2, 4, 10 or 12. In the second treatment, hair samples from each subject were immersed in the
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Magnetic field (gauss) Figure 1 Electron paramagnetic resonance (EPR) spectra of ‘untreated’ hair from Subjects A (a) and B (b) taken in 2012. Note. Peak assignments are shown as stick diagrams and the free radical signal in (b) has been cut to display the copper signal.
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© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
N. Worasith and B. A. Goodman
Coordination of Cu in hair
shampoo (The two products are marketed in Thailand as ‘Miracle Daily’ and ‘Nutrium X’.) used by the other subject for 10 min, then rinsed thoroughly with deionized water and finally dried in air. In each case, the EPR spectra were acquired from samples which were pushed into a 5-mm i.d. Spectrosil tube using a quartz glass rod, which also prevented sample movement during spectral acquisition. EPR spectroscopy Spectra were recorded in 1024 points at room temperature (~21°C) as 1st derivatives of the plots of microwave absorption versus magnetic field on a Bruker Biospin A300 spectrometer operating at X-band (~9 GHz) frequencies using Gunn diode as microwave source. All spectra were acquired using a microwave power of 10 mW, 100 kHz modulation frequency and 6 gauss modulation amplitude. Most spectra were acquired in 1024 data points, although some also used 2048 points. The centre field and sweep width were selected so that the items of interest were centred in the spectrum, and the conversion time, time constant, receiver gain and number of scans were determined according to the spectral intensity and field range. In addition, some 2nd derivative spectra were obtained by numerically differentiating 1st derivative spectra.
Results For reference, the EPR spectra of ‘untreated’ hair from Subjects A and B taken in 2012 are shown in Fig. 1. Each shows a signal from a Cu(II) complex and a free radical, but the Cu spectra are completely different. The relatively broad spectrum in Fig. 1a could be simulated with g// = 2.25, g⊥ = 2.059, A// = 17.0–17.5 mT [26]. These values are typical of copper(II) proteins coordinated to two oxygen and two nitrogen atoms (e.g. [27, 28]), and as described by Worasith and Goodman [26], a 2nd derivative recording of the ‘g⊥‘ region revealed the presence of 14N shfs. The peaks in the spectrum in Fig. 1b are much sharper, and components corresponding to the individual 63Cu and 65Cu isotopes are resolved at the low and high field ends of the spectra. The 63Cu component could be simulated with g// = 2.148, g⊥ = 2.036, A// = 18.6 mT, A⊥ = 3.5 mT [26]. The narrow lines excluded the possibility of any unresolved 14N shfs, and it was concluded that the copper environment corresponded to a mixture of oxygen and sulphur atoms, possibly a two oxygen, two sulphur complex. With the samples taken in 2014, the spectral parameters were similar to those in Fig. 1a,b, except that the Cu(II) signal in the sample from Subject A was considerably weaker. In an attempt to understand the reasons for this change, the effects of various treatments to the hair on the Cu were investigated.
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Magnetic field (gauss) Figure 2 Effect of Shampoo A on the hair from Subject A taken in 2012. (a) original sample and (b) after washing with Shampoo A.
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
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Magnetic field (gauss) Figure 3 Second derivative displays of the g⊥ regions of the Electron paramagnetic resonance (EPR) spectra from Fig. 2. (a) original sample and (b) after washing with Shampoo A. In each case, the spectrum was smoothed by applying a 7-point moving average three times. Note. The stick diagram shows the tentative interpretation of the superhyperfine structure (shfs) in terms of two sets of two equivalent 14N atoms.
Influence of acidic and alkaline conditions Hair samples from Subjects A and B in 2012 were treated with either 0.1 M HCl or 0.1 M NaOH. Much of the Cu signal was removed from the sample from Subject A by either 0.1 M HCl or 0.1 M NaOH, but the parameters for the residual copper were different at the low and high pH values. Such changes are common in copper peptides (e.g. [29, 30]) where pH-induced deprotonation reactions change the metal coordination behaviour; therefore, this result is consistent with the copper in this hair sample being bound to the keratin protein, as originally interpreted [26]. In addition, there was a major increase in the intensity of the free radical signal on alkaline treatment, consistent with polyphenol autoxidation (e.g. [31]); thus, this result suggests that (in this sample at least) oxidation of phenols to form melanin in the hair follicle was incomplete. With the sample from Subject B, there was only a minor effect of 0.1 M HCl on the EPR spectrum, but almost all of the signal was lost after treatment with 0.1 M NaOH. Furthermore, at this high pH, the small residual signal was similar to that in the sample from Subject A. Samples from both subjects were then investigated after treatment with weaker acid and alkali at pH values 2, 4, 10 and 12, but the results resembled those of the initial hairs. Influence of shampoo Between 2012 and 2014, Subject A changed her shampoo/conditioner regimen, whereas Subject B did not. Thus, the next
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step in this research was to investigate the effects of the shampoo on the copper EPR signal. Figure 2 shows that the use of Subject A’s 2014 shampoo on her hair sample from 2012 resulted in an appreciable reduction in the intensity of the Cu (II) EPR signal. However, there also appeared to be a better resolution of the 14N superhyperfine structure (shfs) in the latter sample; this region of the spectra was then investigated in greater detail and is shown as 2nd derivative recordings in Fig. 3. The shfs is clearly better resolved in the sample treated with the 2014 shampoo and can be tentatively interpreted in terms of two quintet signals with 1 : 2 : 3 : 2 : 1 intensity ratios, as could be obtained with a mixture of cis and trans coordination of the 14N atoms. However, the positions of the peaks are unaltered between the samples, and it would appear that the decrease in the copper signal between Fig. 2a,b is the result of the removal of a copper component with slightly different shfs parameters from that which remained after this shampoo treatment (Note the g//-, g⊥- and A//(Cu)-values, appear to be the same). Treatment of the hair from Subject B with the 2014 shampoo from Subject A had no effect on the Cu EPR spectrum (Fig. 4a), but treatment of the hair from Subject A with the shampoo from Subject B resulted in an EPR spectrum that was similar to that from Subject B. Additional treatment of this hair with the shampoo from Subject A still produced a spectrum similar to that from Subject B (Fig. 4b), and completely different from the spectrum of the hair from Subject A subjected to the shampoo from Subject A (Fig. 4c). Discussion These results show clearly that different types of shampoo can have a profound effect on the coordination chemistry of copper in the hair. Furthermore, the shampoo used by Subject B produced a permanent change in the copper coordination environment in the hair, whereas that used by Subject A did not. Although the colour of hair is derived from melanin, the protein keratin is the major component of hair fibres, which are made up of the cortex, closely packed elongated cells whose axis is parallel to that of the hair, covered by 6–8 layers of overlapping flattened cells known as the cuticle [32]. Therefore, it is probable that the copper observed in the present experiments is located primarily in the cuticle. Hair keratin is organized in chains containing a-helix (a) and b-sheet and/ or random coil (b/R) regions with neighbouring chains being held together by bridging disulphide bonds. The intimate structures of hair fibres are independent of ethnic origin, although ethnicity affects their geometry, mechanical and water swelling properties [33]. The contents of (a) and (b/R) regions are independent of age, but the disulphide contents decrease with age [34], and this is probably a reason for increasing damage and hair loss in the elderly, as the thiol contents are related to the mechanical properties of hair fibres [35]. In the context of the current work, the hair from Subject A with Cu in mixed N, O coordination was noticeably more brittle than that from Subject B with Cu in mixed S, O coordination. Copper plays an important role in melanin formation as the metal cofactor in the enzyme tyrosinase, which is responsible for the transformation of tyrosine to dihydroxyphenylalanine [36], the 1st stage of melanin synthesis. The observation of a large increase in the EPR free radical signal in the hair sample from Subject A treated with 1 M NaOH is consistent with phenol
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
N. Worasith and B. A. Goodman
Coordination of Cu in hair
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Magnetic field (gauss) Figure 4 Electron paramagnetic resonance (EPR) spectra of hair samples taken in 2012 after shampoo treatments. (a) Subject B hair treated with Shampoo A, (b) Subject A hair treated with Shampoo B, followed by Shampoo A, and (c) Subject A hair treated with Shampoo A.
autoxidation [31] and suggests that appropriate treatment of this hair could produce phenol oxidation and increase the melanin content. However, the large Cu(II) EPR signal in this sample indicates that any problem is unlikely to be associated with copper deficiency.
Conclusions These results demonstrate clearly that the environment of Cu(II) in hair can be influenced in a major way by chemical treatment of the hair. Strongly alkaline or acidic conditions are able to remove
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
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copper from the hair, but the mixed oxygen/sulphur coordination of the hair from Subject B was less sensitive to strong acid treatment than the mixed oxygen/nitrogen coordination of Subject A. However, the copper coordination in the hair was insensitive to pH values normally encountered with shampoos and most other types of hair treatments. Nevertheless, the coordination environment of the copper was shown to be influenced by the shampoo; it was possible to change the EPR spectrum of the hair from Subject A to that of the hair of Subject B by treating it with the shampoo of Subject B, but it was not possible to change the spectrum of the hair from Subject B to that of Subject A by treating it with the shampoo/conditioner of Subject A. Thus, the copper in hair can be strongly influenced by the nature of any treatments applied
to the hair, and it is necessary to be aware of such effects with copper and possibly other trace metals when attempting to use hair analyses for health monitoring or environmental exposure to heavy metals. However, the sensitivity of the EPR spectra to the shampoo used may have value in certain forensic investigations. Acknowledgements Rajamangala University of Technology Krungthep is acknowledged for funding this work. We are also grateful to the Office of Atoms for Peace, Bangkok, Thailand for the use of their EPR spectrometer for this work.
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superoxide dismutase transgenes with amyotrophic lateral sclerosis: associated mutations develop motor neuron disease. J. Neurosci. 21, 9246–9254 (2001). Pauly, P.C. and Harris, D.A. Copper stimulates endocytosis of the prion protein. J. Biol. Chem. 273, 33107–33110 (1998). Naqvi, K.R., Marsh, J.M., Godfrey, S., Davis, M.G., Flagler, M.J., Hao, J. and Chechik, V. The role of chelants in controlling Cu(II)-induced radical chemistry in oxidative hair colouring products. Int. J. Cosmet. Sci. 35, 41–49 (2013). Worasith, N. and Goodman, B.A. Determination of the coordination environment of Cu(II) in human hair and its possible relevance to health and hair care treatments. Int. J. Cosmet. Sci. 35, 424–429 (2013). Peisach, J. and Blumberg, W.E. Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins. Arch. Biochem. Biophy. 165, 691–708 (1974). Addison, A.W. Spectroscopic and redox trends from model systems. In: Copper Coordination Chemistry; biochemical and inorganic perspectives (Karlin, K.D. and Zubieta, J.A., eds.), pp. 109–128. Adenine Press, Guilderland, New York (1983). McPhail, D.B. and Goodman, B.A. An electron paramagnetic resonance (EPR) study of copper(II)- diglycine complexes. J. Chem. Res., M2901-M2923, S276–S277 (1985). McPhail, D.B. and Goodman, B.A. An electron spin resonance investigation of the nature of the complexes formed between copper (II) and glycylhistidine. J. Chem. Soc. Faraday Trans. 83, 3683–3692 (1987). Severino, J.F., Goodman, B.A., Kay, C.W.M., Stolze, K., Tunega, D., Reichenauer, T.G. and Pirker, K.F. Free radicals generated during oxidation of green tea polyphenols: electron paramagnetic resonance spectroscopy combined with density functional theory calculations. Free Radic. Biol. Med. 46, 1076–1088 (2009).
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32. Dawber, R. Hair: its structure and response to cosmetic preparations. Clin. Dermatol. 14, 105–112 (1996). 33. Franbourg, A., Hallegot, P., Baltenneck, F., Toutaina, C. and Leroy, F. Current research on ethnic hair. J. Am. Acad. Dermatol. 48 (Suppl.), S115–S119 (2003).
34. Kuzuhara, A., Fujiwara, N. and Hori, T. Analysis of internal structure changes in black human hair keratin fibers with aging using Raman spectroscopy. Biopolymers 87, 134–140 (2007). 35. Robinson, M.S. and Rigby, B.J. Thiol differences along keratin fibers: stress/strain and
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stress-relaxation behavior as a function of temperature and extension. Text. Res. J. 55, 597–600 (1985). 36. Hearing, V.J. and Jimenez, M. Mammalian tyrosinase – The critical regulatory control point in melanocyte pigmentation. Int. J. Biochem. 19, 1141–1147 (1987).
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doi: 10.1111/ics.12178
The coordination environment of copper in hair can be altered by treatment products N. Worasith* and B. A. Goodman† *Department of Chemistry, Faculty of Science and Technology, Rajamangala University of Technology Krungthep, 2 Nang Lin Chi Road, Soi Suan Plu, Sathorn, Bangkok, Thailand and †State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, Guangxi 530004, China
Received 22 October 2014, Accepted 26 October 2014
Keywords: chemical analysis, copper coordination, hair treatment, spectroscopy
Synopsis OBJECTIVE: To determine whether the coordination environment of copper in hair is affected by the shampoo used. METHODS: Electron paramagnetic resonance (EPR) spectroscopy to discriminate between mixed oxygen/nitrogen and mixed oxygen/ sulphur coordination of copper after treatment with two different shampoos. RESULTS: Copper with mixed oxygen/nitrogen coordination could be converted to mixed oxygen/sulphur coordination by treating with the appropriate shampoo, but this was not reversible with the products tested, although copper was removed from hair at very high pH values. CONCLUSION: Commercial hair treatment products can have a profound effect on the copper coordination environment in hair, and this must be taken into account in any attempt to use hair as a health marker. sume Re OBJECTIF: Determiner si l’environnement de coordination du cuivre dans les cheveux est affecte par le shampooing utilise. METHODES: Resonance paramagnetique electronique (RPE) pour discriminer entre la coordination de cuivre mixte oxygene / azote et la coordination de cuivre mixte oxygene / soufre apres le traitement avec deux shampooings differents. RESULTATS: Le cuivre avec la coordination mixte oxygen/nitrogen peut ^etre converti en coordination mixte oxygene / soufre en traitant avec le shampooing approprie, mais ce ne fut pas reversible avec les produits testes, bien que le cuivre ait ete retire des cheveux aux tres hautes valeurs de pH. CONCLUSION: Les produits de traitement des cheveux commerciaux peuvent avoir un effet profond sur l’environnement de coordination du cuivre dans les cheveux, et cela doit ^etre pris en compte dans toute tentative d’utiliser les cheveux comme un marqueur de la sante. Introduction The possibility of using hair analysis as a marker of health status and/or environmental exposure to heavy metals and other potenCorrespondence: Bernard A. Goodman, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004 Guangxi, China. Tel.: 0086 771 3272475; fax: 0086 771 3237873; e-mail: bernard_a_ [email protected]
tially dangerous substances has been the subject of debate for many years (e.g. [1, 2]). The principal component of hair, the cysteine-rich protein keratin, is produced by specialized cells which die after they have been filled with the protein in the hair follicle. Thus the elemental composition of hair may reflect that of the blood while it is being formed and represent a historical record of variability in blood composition (e.g. [3]). In addition, the hair shaft is porous and hair can adsorb various chemicals and salts from its environment and may thus be of direct use in environmental monitoring, especially in an industrial setting. There have been many reports in the scientific literature of hair analyses being used for the identification of environmental problems that could impact on health, including passive smoking [4, 5], alcoholism [6], illicit drug use [7] and exposure to high levels of heavy metals, such as mercury [8], lead [9] and cadmium [10]. In addition, hair analyses have been used as indicators of some disease conditions which involve abnormalities in metal accumulation (e.g. [11]), and there are continual efforts to search for abnormalities in the metal contents of hair as markers of other disease states (e.g. [12, 13]). Copper is a trace element that is commonly found in normal hair, and although appreciable contributions come from tap water [14], it might be possible to use hair as a marker of copper-based genetic disorders [15], such as Menkes disease which involves copper deficiency [16], or Wilson disease, which is a copper toxicosis condition [17]. These have been shown to be caused by mutations of two closely related Cu-transporting ATPases [18], although no elevation in total copper contents of hair is observed in Wilson’s disease (e.g. [19]). However, as copper is also involved in certain types of cancer [20, 21] and the pathogenesis of neurological disorders such as Alzheimer’s disease [22], motor neuron diseases [23] and prion diseases [24], it is plausible that the chemical forms rather than the total amounts of copper in the hair could be markers of various health conditions. In addition, as shown recently by Naqvi et al. [25], the chemical forms of copper influence the free radical chemistry in hair that can produce structural damage during colouring processes. Recently, we reported an investigation of the paramagnetic components of hair using electron paramagnetic resonance (EPR) spectroscopy [26], a technique that can specifically detect and characterize paramagnetic chemical species, such as free radicals and certain transition metal ions and complexes. In that work, we observed two distinctly different types of copper complex in hair from different subjects; one was interpreted as corresponding to Cu (II) coordinated to a mixture of oxygen and nitrogen atoms,
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
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N. Worasith and B. A. Goodman
Coordination of Cu in hair
whereas the other corresponded to a mixture of oxygen and sulphur atoms. However, no information was presented as to the reasons for these differences, although environmental effects or hair care products were considered to be possibilities, as samples from the hair of one subject taken over a period of several years showed variations in the copper complexes at different times. Thus, if the composition of hair is influenced by hair care products, its use for either environmental monitoring or health status becomes increasingly complicated, especially as there is currently a huge range of hair care products openly available for a variety of uses, including shampooing, conditioning, colouring, relaxing and curling. As hair treatments might result in changes in the coordination environments of trace metals in the hair, the present experiments were undertaken with the objective of determining whether different types of hair treatment can produce major changes in the copper coordination environment. Measurements focused on hair samples from two subjects which gave well resolved EPR spectra, each corresponding to one of the two identified coordination environments in the work of Worasith and Goodman [26]. These samples were then subjected to a range of pH values, as these are encountered in some of the more extreme treatments associated with relaxing or permanent waving. Hair relaxers typically have pH values in the range 12–13, whereas perms can be either acidic or alkaline. In addition, hair samples from each subject were treated with the shampoo/conditioning regimens used by the other
subject, in order to determine the extent to which such commercially available products might directly influence the copper coordination chemistry. Materials and methods Hair samples The hair samples used in the present work were those that gave good quality EPR spectra each corresponding to one of the two types of copper signal in our original investigations [26]. In each case, the subject was a healthy middle-aged Asian female. In addition to the original hair samples which were taken in 2012, additional samples were taken from the same two subjects in 2014. All hair samples were cut from the nape of the neck. Treatments Two separate types of treatment were investigated. The first considered the effect of pH. Initial measurements were made by immersing hair samples in solutions of 1 M HCl or 1 M NaOH for 1 h, then rinsing thoroughly with deionized water and drying in air at ambient temperature. Then subsequent measurements were made using HCl or NaOH solutions at pH 2, 4, 10 or 12. In the second treatment, hair samples from each subject were immersed in the
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Magnetic field (gauss) Figure 1 Electron paramagnetic resonance (EPR) spectra of ‘untreated’ hair from Subjects A (a) and B (b) taken in 2012. Note. Peak assignments are shown as stick diagrams and the free radical signal in (b) has been cut to display the copper signal.
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© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
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Coordination of Cu in hair
shampoo (The two products are marketed in Thailand as ‘Miracle Daily’ and ‘Nutrium X’.) used by the other subject for 10 min, then rinsed thoroughly with deionized water and finally dried in air. In each case, the EPR spectra were acquired from samples which were pushed into a 5-mm i.d. Spectrosil tube using a quartz glass rod, which also prevented sample movement during spectral acquisition. EPR spectroscopy Spectra were recorded in 1024 points at room temperature (~21°C) as 1st derivatives of the plots of microwave absorption versus magnetic field on a Bruker Biospin A300 spectrometer operating at X-band (~9 GHz) frequencies using Gunn diode as microwave source. All spectra were acquired using a microwave power of 10 mW, 100 kHz modulation frequency and 6 gauss modulation amplitude. Most spectra were acquired in 1024 data points, although some also used 2048 points. The centre field and sweep width were selected so that the items of interest were centred in the spectrum, and the conversion time, time constant, receiver gain and number of scans were determined according to the spectral intensity and field range. In addition, some 2nd derivative spectra were obtained by numerically differentiating 1st derivative spectra.
Results For reference, the EPR spectra of ‘untreated’ hair from Subjects A and B taken in 2012 are shown in Fig. 1. Each shows a signal from a Cu(II) complex and a free radical, but the Cu spectra are completely different. The relatively broad spectrum in Fig. 1a could be simulated with g// = 2.25, g⊥ = 2.059, A// = 17.0–17.5 mT [26]. These values are typical of copper(II) proteins coordinated to two oxygen and two nitrogen atoms (e.g. [27, 28]), and as described by Worasith and Goodman [26], a 2nd derivative recording of the ‘g⊥‘ region revealed the presence of 14N shfs. The peaks in the spectrum in Fig. 1b are much sharper, and components corresponding to the individual 63Cu and 65Cu isotopes are resolved at the low and high field ends of the spectra. The 63Cu component could be simulated with g// = 2.148, g⊥ = 2.036, A// = 18.6 mT, A⊥ = 3.5 mT [26]. The narrow lines excluded the possibility of any unresolved 14N shfs, and it was concluded that the copper environment corresponded to a mixture of oxygen and sulphur atoms, possibly a two oxygen, two sulphur complex. With the samples taken in 2014, the spectral parameters were similar to those in Fig. 1a,b, except that the Cu(II) signal in the sample from Subject A was considerably weaker. In an attempt to understand the reasons for this change, the effects of various treatments to the hair on the Cu were investigated.
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Magnetic field (gauss) Figure 2 Effect of Shampoo A on the hair from Subject A taken in 2012. (a) original sample and (b) after washing with Shampoo A.
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
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(a) 14N
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Magnetic field (gauss) Figure 3 Second derivative displays of the g⊥ regions of the Electron paramagnetic resonance (EPR) spectra from Fig. 2. (a) original sample and (b) after washing with Shampoo A. In each case, the spectrum was smoothed by applying a 7-point moving average three times. Note. The stick diagram shows the tentative interpretation of the superhyperfine structure (shfs) in terms of two sets of two equivalent 14N atoms.
Influence of acidic and alkaline conditions Hair samples from Subjects A and B in 2012 were treated with either 0.1 M HCl or 0.1 M NaOH. Much of the Cu signal was removed from the sample from Subject A by either 0.1 M HCl or 0.1 M NaOH, but the parameters for the residual copper were different at the low and high pH values. Such changes are common in copper peptides (e.g. [29, 30]) where pH-induced deprotonation reactions change the metal coordination behaviour; therefore, this result is consistent with the copper in this hair sample being bound to the keratin protein, as originally interpreted [26]. In addition, there was a major increase in the intensity of the free radical signal on alkaline treatment, consistent with polyphenol autoxidation (e.g. [31]); thus, this result suggests that (in this sample at least) oxidation of phenols to form melanin in the hair follicle was incomplete. With the sample from Subject B, there was only a minor effect of 0.1 M HCl on the EPR spectrum, but almost all of the signal was lost after treatment with 0.1 M NaOH. Furthermore, at this high pH, the small residual signal was similar to that in the sample from Subject A. Samples from both subjects were then investigated after treatment with weaker acid and alkali at pH values 2, 4, 10 and 12, but the results resembled those of the initial hairs. Influence of shampoo Between 2012 and 2014, Subject A changed her shampoo/conditioner regimen, whereas Subject B did not. Thus, the next
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step in this research was to investigate the effects of the shampoo on the copper EPR signal. Figure 2 shows that the use of Subject A’s 2014 shampoo on her hair sample from 2012 resulted in an appreciable reduction in the intensity of the Cu (II) EPR signal. However, there also appeared to be a better resolution of the 14N superhyperfine structure (shfs) in the latter sample; this region of the spectra was then investigated in greater detail and is shown as 2nd derivative recordings in Fig. 3. The shfs is clearly better resolved in the sample treated with the 2014 shampoo and can be tentatively interpreted in terms of two quintet signals with 1 : 2 : 3 : 2 : 1 intensity ratios, as could be obtained with a mixture of cis and trans coordination of the 14N atoms. However, the positions of the peaks are unaltered between the samples, and it would appear that the decrease in the copper signal between Fig. 2a,b is the result of the removal of a copper component with slightly different shfs parameters from that which remained after this shampoo treatment (Note the g//-, g⊥- and A//(Cu)-values, appear to be the same). Treatment of the hair from Subject B with the 2014 shampoo from Subject A had no effect on the Cu EPR spectrum (Fig. 4a), but treatment of the hair from Subject A with the shampoo from Subject B resulted in an EPR spectrum that was similar to that from Subject B. Additional treatment of this hair with the shampoo from Subject A still produced a spectrum similar to that from Subject B (Fig. 4b), and completely different from the spectrum of the hair from Subject A subjected to the shampoo from Subject A (Fig. 4c). Discussion These results show clearly that different types of shampoo can have a profound effect on the coordination chemistry of copper in the hair. Furthermore, the shampoo used by Subject B produced a permanent change in the copper coordination environment in the hair, whereas that used by Subject A did not. Although the colour of hair is derived from melanin, the protein keratin is the major component of hair fibres, which are made up of the cortex, closely packed elongated cells whose axis is parallel to that of the hair, covered by 6–8 layers of overlapping flattened cells known as the cuticle [32]. Therefore, it is probable that the copper observed in the present experiments is located primarily in the cuticle. Hair keratin is organized in chains containing a-helix (a) and b-sheet and/ or random coil (b/R) regions with neighbouring chains being held together by bridging disulphide bonds. The intimate structures of hair fibres are independent of ethnic origin, although ethnicity affects their geometry, mechanical and water swelling properties [33]. The contents of (a) and (b/R) regions are independent of age, but the disulphide contents decrease with age [34], and this is probably a reason for increasing damage and hair loss in the elderly, as the thiol contents are related to the mechanical properties of hair fibres [35]. In the context of the current work, the hair from Subject A with Cu in mixed N, O coordination was noticeably more brittle than that from Subject B with Cu in mixed S, O coordination. Copper plays an important role in melanin formation as the metal cofactor in the enzyme tyrosinase, which is responsible for the transformation of tyrosine to dihydroxyphenylalanine [36], the 1st stage of melanin synthesis. The observation of a large increase in the EPR free radical signal in the hair sample from Subject A treated with 1 M NaOH is consistent with phenol
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
N. Worasith and B. A. Goodman
Coordination of Cu in hair
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Magnetic field (gauss) Figure 4 Electron paramagnetic resonance (EPR) spectra of hair samples taken in 2012 after shampoo treatments. (a) Subject B hair treated with Shampoo A, (b) Subject A hair treated with Shampoo B, followed by Shampoo A, and (c) Subject A hair treated with Shampoo A.
autoxidation [31] and suggests that appropriate treatment of this hair could produce phenol oxidation and increase the melanin content. However, the large Cu(II) EPR signal in this sample indicates that any problem is unlikely to be associated with copper deficiency.
Conclusions These results demonstrate clearly that the environment of Cu(II) in hair can be influenced in a major way by chemical treatment of the hair. Strongly alkaline or acidic conditions are able to remove
© 2014 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie International Journal of Cosmetic Science, 1–7
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Coordination of Cu in hair
copper from the hair, but the mixed oxygen/sulphur coordination of the hair from Subject B was less sensitive to strong acid treatment than the mixed oxygen/nitrogen coordination of Subject A. However, the copper coordination in the hair was insensitive to pH values normally encountered with shampoos and most other types of hair treatments. Nevertheless, the coordination environment of the copper was shown to be influenced by the shampoo; it was possible to change the EPR spectrum of the hair from Subject A to that of the hair of Subject B by treating it with the shampoo of Subject B, but it was not possible to change the spectrum of the hair from Subject B to that of Subject A by treating it with the shampoo/conditioner of Subject A. Thus, the copper in hair can be strongly influenced by the nature of any treatments applied
to the hair, and it is necessary to be aware of such effects with copper and possibly other trace metals when attempting to use hair analyses for health monitoring or environmental exposure to heavy metals. However, the sensitivity of the EPR spectra to the shampoo used may have value in certain forensic investigations. Acknowledgements Rajamangala University of Technology Krungthep is acknowledged for funding this work. We are also grateful to the Office of Atoms for Peace, Bangkok, Thailand for the use of their EPR spectrometer for this work.
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