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Application of XANES profiles to X-ray spectromicroscopy for biomedical specimens: Part II. Mapping oxidation state of cysteine in human hair
Abstract
Human hair fibers are primarily composed of keratin protein, characterized by a very high content of cysteine, a sulfur-containing amino acid, which ordinarily forms cystine via a disulfide bond. It is known that some cystine residues are converted to cysteic acid during permanent waving or hair coloring, although details of their distribution and extent are still unclear. In this study, by using difference in XANES profiles of cystine and cysteic acid at the S-K absorption edge, the formation of cysteic acid was confirmed for homogenized samples of permed or bleached hair. Furthermore chemical mapping of cysteic acid was performed on hair-section samples with X-ray contact microscopy. The peripheral region, cuticle, in bleached hair showed the highest content of cysteic acid compared with the other parts, while permed hair showed relatively uniform distribution. This finding suggests that perming and bleaching damage hair by different mechanisms.
... Here we would like to consider three of them namely sulfur, zinc and titanium and thus discuss health problems not connected to pathological calcifications. X-ray Sulfur detection in human hair fibers ( Figure 19) is due to the fact that hair is primarily composed of keratin proteins with a very high content of cysteine, a sulfur-containing amino acid, which commonly forms cystine via a disulfide bond [189,190]. The detection of sulfur in hair by XRF and XANES in dementia has been discussed, for example by Siritapetawee et al. [191] in the context of possible roles of calcium, chlorine, phosphorus and C. R. Chimie -Online first, 24th February 2022 83 Figure 19. ...
... sulfur in the etiology of elderly patients' dementia. Also, Inoue et al. [189] showed that it is possible to map the oxidation state of cysteine in human hair through Xanes spectroscopy. In our experiment (Figure 20), it is quite clear that the nanometer scale resolution allows us to precisely define the distribution of sulfur in hair. ...
... Finally, it is worth noting that various shampoos contain zinc; clinical studies have established that Zn pyrithione is one of the most effective anti-dandruff ingredients in shampoo formulations [189][190][191]196]. ...
Bien que de nombreuses pathologies dermatologiques soient associées à des dépôts cutanés pathologiques, endogènes ou exogènes, ceux-ci restent mal décrits. Leur taille sub-micrométrique ainsi que leur diversité de composition chimique nécessitent l’utilisation de divers outils physico-chimiques pour en permettre une caractérisation complète. Notre objectif est d’apporter une caractérisation physico-chimique multi échelle du micromètre au nanomètre, mais aussi d’explorer la genèse et les conséquences tissulaires de ces dépôts en utilisant différentes techniques afin d’en extraire un message clinique. Après avoir présenté les connaissances actuelles sur ces dermatoses calcifiantes et décrit les principes de la microscopie électronique à balayage couplée à la spectroscopie de rayons X à dispersion d'énergie et des spectroscopies vibrationnelles, nous présentons l’application de ces outils à quatre dermatoses différentes : calciphylaxie, ulcères de jambe calcifiés, calcinosis cutis sur nécrolyse épidermique et sarcoïdose. L’ensemble de nos résultats montrent que les dépôts cutanés endogènes sont principalement composés de carbapatite, dont l’aspect ultra-structural correspond à des nano-sphérules phospho-calciques s’agrégeant pour former des plaques de dimension micronique. Ces dépôts présentent un tropisme vis-à-vis des fibres élastiques que des études complémentaires par microscopie multiphoton permettront d’explorer. Ces résultats originaux permettent de mieux comprendre la physio genèse des calcifications cutanées, mais également d’envisager des méthodes de diagnostic ainsi que des pistes thérapeutiques.
... Here we would like to consider three of them namely sulfur, zinc and titanium and thus discuss health problems not connected to pathological calcifications. X-ray Sulfur detection in human hair fibers ( Figure 19) is due to the fact that hair is primarily composed of keratin proteins with a very high content of cysteine, a sulfur-containing amino acid, which commonly forms cystine via a disulfide bond [189,190]. The detection of sulfur in hair by XRF and XANES in dementia has been discussed, for example by Siritapetawee et al. [191] in the context of possible roles of calcium, chlorine, phosphorus and sulfur in the etiology of elderly patients' dementia. ...
... The detection of sulfur in hair by XRF and XANES in dementia has been discussed, for example by Siritapetawee et al. [191] in the context of possible roles of calcium, chlorine, phosphorus and sulfur in the etiology of elderly patients' dementia. Also, Inoue et al. [189] showed that it is possible to map the oxidation state of cysteine in human hair through Xanes spectroscopy. In our experiment (Figure 20), it is quite clear that the nanometer scale resolution allows us to precisely define the distribution of sulfur in hair. ...
... Finally, it is worth noting that various shampoos contain zinc; clinical studies have established that Zn pyrithione is one of the most effective anti-dandruff ingredients in shampoo formulations [189][190][191]196]. ...
Although numerous pathologies are associated with abnormal skin deposits, these remain poorly described, as accurate characterization continues to present a challenge for dermatologists. Their submicrometer size as well as their diverse chemistry require various characterization tools.We aim to exemplify characterization of endogenous and exogenous skin deposits in some selected skin diseases using different physico-chemical techniques. We begin with a presentation of selected diseases associated with skin deposits. We then present those of our results which show their variety of structure, location and chemical composition, obtained with various tools: Field Emission Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectroscopy, X-ray fluorescence, vibrational spectroscopies, as well as techniques specific to synchrotron radiation. Our results constitute a real opportunity to improve diagnosis, and to understand the pathogenesis of many skin diseases, and opportunities for therapeutic intervention.
... The product of oxidative treatments is cysteic acid, which is an indicator of damage to the hair structure. The higher the cysteic acid content, the more serious the damage [9]. No statistical differences (p = 1) can be observed between the 3 times bleached hair tress, which is the reference, and both dyed hair tresses. ...
The aim of this work was to optimize our natural hair dyeing system which we described in our previous work and to compare with other dyeing systems. Therefore, we investigated concentration limits of matcha and mordant and compared this new dyeing method with commercial permanent systems on the market. Completely unpigmented hair tresses were dyed with matcha powder (camelia sinensis) and iron(II)-lactate. To investigate the wash fastness and concentration limits, the differently dyed hair tresses were spectrophotometrically measured. The comparison of the damage potential for which cysteic acid is an indicator was measured by NIR. The concentration of matcha and mordant are responsible for the intensity of the color results. The higher the matcha or the mordant concentration, the darker the color results of the dyed hair tresses. Hair damage of matcha mordant dyeing is comparable with results of commercial permanent hair coloration systems. Moreover, the results of wash fastness of matcha mordant dyed hair tresses is comparable and even better by tendency to permanent colored hair tresses.
... The spectra of all the feathers, both melanised and unpigmented, are dominated by two sharp peaks at 2472.3 and 2473.5 eV as well as a broader feature at ~2480.4 eV. The double peaks originate from the abundant disulfide and sulfur-carbon bonds ( Supplementary Fig. 7, oxidised glutathione standard) of keratin protein [27][28][29][30] . The key result here is a subtle difference in the spectrum of strongly pheomelanised regions of the kestrel and red-tailed hawk compared to the eumelanised and unpigmented feathers. ...
Melanin is a critical component of biological systems, but the exact chemistry of melanin is still imprecisely known. This is partly due to melanin’s complex heterogeneous nature and partly because many studies use synthetic analogues and/or pigments extracted from their natural biological setting, which may display important differences from endogenous pigments. Here we demonstrate how synchrotron X-ray analyses can non-destructively characterise the elements associated with melanin pigment in situ within extant feathers. Elemental imaging shows that the distributions of Ca, Cu and Zn are almost exclusively controlled by melanin pigment distribution. X-ray absorption spectroscopy demonstrates that the atomic coordination of zinc and sulfur is different within eumelanised regions compared to pheomelanised regions. This not only impacts our fundamental understanding of pigmentation in extant organisms but also provides a significant contribution to the evidence-based colour palette available for reconstructing the appearance of fossil organisms.
... Exposure to oxidative stress induces cysteic acid, an oxidative form of cystine (Fig. 3a). Since these two forms of amino acids have different XANES peaks at the S K absorption edge as shown in Fig. 3b [12], we could obtain the distribution of cysteic acid (oxidation map) by the subtraction of images taken at the energy (c) and (d) [13]. For a cystine map, the subtraction between energy (a) and (b) was performed. ...
Soft X-ray microscopy has great advantages over other types of microscopy on the observation of hydrated specimens at high spatial resolution below 100 nm, and on the elemental and molecular mapping using absorption characteristics. These advantages are particularly suitable for biomedical applications. A possible problem associated with observation of specimens at high spatial resolution in a hydrated environment is described and a strategy to overcome the problem is presented. The latter part of the present report deals with spectromicroscopy to obtain molecular distribution in biological specimens. DNA and protein distributions in isolated nuclei are examined following to the progression of apoptosis at high resolution, and the oxidation product is visualized in human hair in relation to calcium content.
The confocal three-dimensional micro X-ray fluorescence using polycapillary optics and a synchrotron radiation was applied to nondestructively analyze elemental compositions of minerals, and thereby obtain the three-dimensional distributions of elements in the minerals. Such confocal micro X-ray fluorescence had potential applications in the mineral prospecting, identification of jades, differentiation of stones.
Sulfur 1s NEXAFS spectroscopy is sensitive to sulfur speciation; however, the overlapping energy position of the white line in the spectra of thiols and disulfides can be problematic for speciation. We have acquired highly resolved sulfur 1s NEXAFS spectra of a group of disulfides with aliphatic and unsaturated ligand groups. Spectral transitions were assigned by the aid of Improved Virtual Orbital (IVO) ab initio calculations. These results confirm how molecular symmetry and the ligand group can affect the NEXAFS spectra, and show how the NEXAFS spectra of thiols and disulfides can be used for chemical speciation.
High-resolution sulfur 1s NEXAFS spectra of a series of sulfoxides and sulfones have been obtained, and are assigned with the aid of ab initio improved virtual orbital (IVO) calculations. Spectral differences between sulfones and sulfoxides are attributed to the sulfur oxidation state and the energy spacing between unoccupied molecular orbitals. The character of the white line in sulfones depends on the sulfur ligand groups, where an aromatic ligand causes a red shift and a broadening of the white line. These effects can pose challenges for speciation when the white line of sulfones overlaps with that of sulfoxide.
Many inorganic species are now recognized as being essential for life, including many forms of sulfur, phosphate and numerous classes of metal ions. For example, recent progress in the fields of biochemistry and biology has pointed out the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. Special attention has also been drawn to the diverse roles of alkaline (Na⁺, K⁺) and alkaline earth (Mg²⁺, Ca²⁺) metal ions in mediating the activity of RNA, proteins and many processes in living cells. While the general effect of these ions in biology is mostly understood, information on their detailed role is deficient. Here the application of softer X-ray absorption spectroscopy (XAS) to probe the local structural and electronic environment of such ions within their biological complexes and during physiological reactions is discussed. In addition, the required experimental set-up and the difficulties associated with conducting softer XAS experiments on biological samples are presented.
For the first time, infrared microspectroscopy with a synchrotron source has been used to characterize human hair before and after bleaching. This high-brightness source allows a lateral resolution approaching the diffraction limit (a few microns) to be achieved. Thus, 4-micron diameter areas have been analyzed, and chemical imaging of hair cross sections obtained with good contrast. In particular, the variations of the integrated absorbances of the amide stretching vibration (3290 cm-1) and CH stretching vibrations (between 3000 and 2800 cm-1) as a function of sample position have allowed chemical imaging of the medulla and cuticle. We also studied bleaching of the hair, and we checked different chemical effects as a function of position in the hair cross section. We also observed a non-homogeneous distribution of bonded water. The transformation, due to breakage of a specific chemical group, was observed and spatially characterized. The sulphonate contributions were given special attention and showed non-homogeneous distributions within the hair.
Synopsis--The color of mammalian HAIRS is due mainly to the inclusion of discrete, darkly colored MELANIN granules in the keratinized cytoplasmic protein of the fiber-forming cells. During BLEACHING the melanin pigment undergoes irreversible physicochemical changes which result either in the toning down or complete elimination of the original fiber color. The modification of the fiber protein (KERATIN) attendant upon bleaching is largely confined to the oxidation of combined CYSTINE. The cysteic acid residues formed in this reaction cause a significant change in the distribution of electrostatic cross links.
We have developed computer-assisted procedures for mapping minor light elements and their chemical bond information from a set of soft X-ray transmission images of biological specimens. Minor light elements such as Ca, S and P play an important role in the biological function and structure in a cell. Contact X-ray microscopy with an electric zooming tube covering wide wavelength range from the K absorption edge of S to the L absorption edge of P is applied to elemental mapping. The procedure of elemental mapping depends basically on the subtraction of the contribution of major elements such as C, O and N in intracellular absorption spectra to find the presence of the absorption edges of minor elements. The results of simulation experiments were presented. Chemical bond imaging utilizes a XANES spectrum corresponding to a specific chemical bond. Since the intensity is relatively low for minor elements, the ratio between absorption at the peak and at the bottom, which represents the chemical bond distribution, becomes small. We developed the computer program for the enhancement of very small ratio value. This procedure was applied to the S-S and S-C bond imaging at the S-K absorption edge.
Amino acid analysis, photometry and polarography were selected as analytical methods for the determination of thiol and disulphide groups in untreated human hair and the results are discussed. The pre-treatments necessary for the different analytical methods, e.g., hydrolysis, to some extent already induce chemical changes of the cysteine and cystine derivatives leading to method-dependent differences in the results. In many cases the partial oxidation products of cystine are supposed to be responsible for this effect. Electron Spectroscopy for Chemical Analysis (ESCA), as an analytical method for the determination of the cystine oxides, was found to be inapplicable due to insufficient resolution and sensitivity, whereas by the use of Fourier Transform Infrared (FTIR) Spectroscopy the sulphur-oxygen vibrations could be analysed and cystine monoxide and cystine dioxide were detected.
On a sélectionné comme méthodes analytiques l'analyse d'amino-acide, la photométrie et la polarographie pour le dosage des groupements thiol et disulfure dans les cheveux non traités. Les résultats sont discutés. Les prétraitements nécessaires pour les differentes méthodes analytiques, pour exemple lors de l'hydrolyse, provoquent déjà des changements chimiques ses dérivés de la cysteine et de qui peuvent conduire à des résultats differents. Dans plusieurs cas, l'oxidation partielle des produits de la cystine est supposée ětre responsable des différences. La méthode analytique Electron Spectroscopy for Chemical Analysis (ESCA) pour la détermination des oxydes de cystine n'a pas été satisfaisante parce que la résolution et la sensibilité sont insuffisantes, par contre par spectroscopie FTIR des vibrations soufre-oxygène du monoxyde de la cystine et dioxyde de la cystine sont déterminés.
Sulfur is an essential biological element, yet its biochemistry is only partially understood because there are so few tools for studying this element in biological systems. X-ray absorption spectroscopy provides a unique approach to determining the chemical speciation of sulfur in intact biological samples. Different biologically relevant sulfur compounds show distinctly different sulfur K-edge X-ray absorption spectra, and we show here, as an example, that this allows the deconvolution of the sulfur species in equine blood.
An X-ray contact microscopy system has been developed for the study of molecular and elemental distributions in biological specimens based on X-ray absorption characteristics. The system consists of a chamber for measuring XANES (X-ray absorption near-edge structure) of biomolecules, and a contact microscopy system for dried specimens with an electronic zooming tube. With this system the elemental distribution of carbon, nitrogen, oxygen, Ca and Fe in an HeLa cell has been studied, and the DNA-related image using a peak from the XANES profile of the DNA at the K-absorption edge of phosphorus has been obtained.
To investigate the influence of bleaching treatments on keratin fibers, the structure of cross‐sections at various depths of bleached human hair (black and white human hair) was directly analyzed without isolating the cuticle and cortex, using Raman spectroscopy. The S–S band intensity existing from the cuticle region to the center of cortex region of virgin white human hair decreased, while the S–O band intensity at 1040 cm ⁻¹ , assigned to cysteic acid, increased by performing the bleaching treatment. Especially, the S–O band intensity of the cuticle region increased remarkably compared with that of the cortex region. Also, the amide III (unordered) band intensity in the cortex region increased, indicating that some of the proteins existing throughout the cortex region changed to the random coil form. Moreover, it has been found that the S–S band intensity existing from the cuticle region to the center of the cortex region of the virgin black human hair decreased remarkably, while the S–O band intensity increased significantly compared with that of the virgin white human hair by performing the bleaching treatment. From these experiments, we concluded that the melanin granules including metal ions act as a decomposition accelerator for the oxidizing agent, thereby leading to a higher level of disulfide (–SS–) group cleavage in the black human hair compared with that of the white human hair. © 2006 Wiley Periodicals, Inc. Biopolymers 81: 506–514, 2006
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Synchrotron-based infrared microscopic measurements have been performed on various hair transverse sections, sampled either from the heads of Caucasian or Afro-American subjects. Lipid content of various virgin hair transverse sections was established, with an unprecedented resolution. The variations in shape and intensity of the CH2, CH3, amide I and amide II bands, before and after lipid removal by solvent extraction, were profiled, showing clearly that Caucasian hair often contains lipids localized inside the medulla and to a lesser extent inside the cuticle. This statement does not hold for the Afro-American hair analysed. For this, the FT-IR spectra do not change within the hair section and are insensitive to solvent extraction. The importance of the origin of hair on its physical and chemical properties has to be taken into account in future investigations.
Par utilisation de la microscopie infra-rouge utilisant le rayonnement synchrotron, la répartition des lipides sur la section de cheveux vierges (caucasiens et afro-américains), a pu être comparée avec une résolution spatiale jamais égalée. La forme et l'intensité des bandes CH2, CH3, amide I et amide II, avant et après délipidation par solvants ont été obtenues. Il apparaît que pour le cheveu caucasien, les lipides sont majoritairement localisés dans la médule et dans la cuticule en quantité moindre. Ceci n'est pas le cas pour les cheveux afro-américains: leur spectre infra-rouge est le même sur toute la section et est insensible à la délipidation. L'origine des cheveux est donc particulièrement importante pour l'étude de leurs propriétés physico-chimiques.