Metabolomic analysis of amino acids and lipids in human hair altered by dyeing, perming and bleaching (2016)

Chemical treatment of hair including perming, dyeing and bleaching is very popular. The manipulations involve oxidizing and reducing steps that necessitate the use of reactive chemicals, such as hydrogen peroxide, (di-, tri, and polynuclear indo dyes and resorcinol formed from the oxidation of hydrogen peroxide and para dye precursors) reducing agents (sodium pyrosulphite, thioglycolate) and alkalizing agents (ammonia, monoethanolamine) 1. Accordingly, hair damage and compositional alterations of hair are unavoidable, especially for amino acids (AAs) and lipids. We investigated the comprehensive alteration of 18 AAs and a cysteine (Cys) derivative, cysteic acid (Cya) and lipids (34 ceramides (CERs), 14 free fatty acids (FFAs), 18-methyl eicosanoic acid (18-MEA) and cholesterol (CH)) of human hair following chemical treatments with sensitive ultra-performance liquid chromatography–photodiode array (UPLC-PDA) and UPLC–tandem mass spectrometry (UPLC-MS/MS). Metabolomic tools, principle component analysis (PCA) and orthogonal projection to latent structures dataset analysis (OPLS-DA), which are widely used to identify novel biomarkers 2, were employed to recognize global alterations of AAs and lipids, and to identify markers characteristic to damaged hair. Natural virgin hairs of Asians that had never been chemically treated were purchased (Beaulax Co., Saitama, Japan). They were cleansed by soaking in de-ionized water, rinsing several times and air drying for 3 min at 50–75°C. Each hair tress was divided into four. Individual bundle underwent perming, dyeing or bleaching. Detailed methods are provided in the supplement. Hairs of other ethnic groups were not studied. Macroscopic and microscopic observation of hairs revealed that substantial damage occurred after chemical treatments (Fig. 1a), but the severity was indistinguishable. PCA of AA contents (Fig. 1b, N = 4), however, revealed separate clustering of bleached hairs from untreated along the y-axis. Loading plot (Fig. 1c) indicated that chemically treated hairs could be distinguished by Cys/Cya, tryptophan (Trp), Cya and methionine (Met). Further analysis with a larger number of hair tresses (N = 10, Fig. 1d–f) confirmed this pattern. Levels of AAs and Cya are presented in Table S1. Quantitation of hair lipids, including endogenous CH, CERs (CER-AP, CER-NP, CER-NDS and CER-NS), saturated and unsaturated FFAs, and chemically bound 18-MEA, was achieved by UPLC-MS/MS 3. PCA (Fig. 2a) showed an apparent orthogonal separation of bleached hairs from untreated. Further analysis of hair lipids employing OPLS-DA clearly separated untreated hairs from bleached (Fig. 2b). Figure 2c depicts a VIP plot, which displays the importance of the variables both to explain X and to correlate with Y. 18-MEA, C22:1FA, C24:1FA, C22FA, C26FA and C24FA were identified as important (VIP > 1.5) for discriminating treated hairs. Graphical representation of C24:1FA, 18-MEA (Fig. 2d), C22FA and C22:1FA (Fig. 2e) also corroborated the significant depletion of these hair lipids by chemical treatments. Levels of CERs and FFAs are presented in Table S2. We investigated the overall alteration of AAs, Cya and lipids during chemical treatment of hairs. PCA and OPLS-DA revealed substantial alterations of AAs and lipids of hairs following chemical treatments. Importantly, they presented a simplified pattern for the alteration of hair AAs and lipids after chemical treatments, which could be instrumental for the evaluation of hair damage and its prevention. Representative AA component of hairs in the aspect of hair damage was Cys owing to its integral role in imparting mechanical strength to hairs through forming disulphide bridge in keratin. Disulphide bond of cystine is susceptible to oxidative stress occurring during oxidative hair bleaching 1. Oxidation of sulphur atom results in the formation of Cya, which has been used as a marker for bleach-induced hair damage 4, which is consistent with our results. Interestingly, as well as bleaching, perming or hair dyeing also increased Cya, the extent of which matched the known degrees of hair damage (s2). This may be due to the difference in the time and extent of hydrogen peroxide application during perming, hair dyeing or bleaching. Another sulphur-containing amino acid, Met, which is sensitive to the addition of oxygen to its sulphur atom, was decreased, consistently with the recent report revealing the oxidation of Met during bleaching (s3). Trp, an amino acid that is susceptible to reactive oxygen species (s4) or thermal reaction 5, was depleted by the chemical treatments of hair. In addition to the individual alteration of these AAs, multivariate analysis using PCA revealed that combined assessment of Trp, Met, Cya and Cys/Cya may be useful in grading hair damage associated with chemical treatments. High levels of Trp, Met and Cys/Cya suggested intact or healthy hairs, while their decreases indicated hair damage. Along with the recently reported proteomic approach 6, this methodology may overcome the limitation of morphological or mechanical evaluation by providing information on the compositional changes, which is crucial to the prevention or repairing of damaged hairs. Hair lipids are important components of hair cuticle. Hair lipids are mainly composed of FFAs, CH, cholesteryl sulphate, wax esters, 18-MEA, triglyceride and CERs 7. Of these hair lipids, FFAs like C22:1FA (erucic acid), C22FA (behenic acid), C24FA (lignoceric acid), C24:1FA (nervonic acid), C26FA (cerotic acid) and an integral hair lipid, 18-MEA, were depleted in proportion to the severity of hair damage. FFAs originated from sebum secretion coat the outside of hairs 8. Therefore, it is plausible that these FFAs are easily depleted during chemical treatment of hair. 18-MEA, which is biosynthesized in hair matrix cells, is linked by a cysteine thioester bond within the cuticle at the surface of human hairs (s5), which makes it susceptible to the breakage of cross-links during chemical treatment as well as in diseases 9. In conclusion, we demonstrate that hair AAs and lipids are markedly altered during chemical treatments of hair. The alteration of these AAs and lipids that are critical to impart mechanical strength and barrier function on hair may ultimately deteriorate the integrity or fibre quality of hair. Metabolomic analysis revealed AA or lipid biomarkers specific to hair damage, which may be useful for the quantitative grading of hair damage, and ultimately would be instrumental for the development of safer and undamaging hair care products. KMJ, BMK, HKL, JHL and KML designed the study; KMJ, ARK and SNK involved in the research; KMJ, KML and SJB analysed the data; and KMJ and KML wrote and revised the manuscript. None Table S1 Change in hair amino acid contents caused by chemical treatment (μg/g hair, mean ± SD, N = 10). Table S2 Change in hair lipid concentration caused by chemical treatment (ng/g for CERs, μg/g for FFAs, mean ± SD, N = 10). Data S1 Detailed methods and references. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.