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| Guest Article | Analysis of the stratum corneum by attenuated total reflection spectroscopy using a novel mid-infrared fibre probe | H. Michael Heise, Lukas Küpper, Wolfgang Pittermann, and Manfred Kietzmann |
Summary
Infrared attenuated total reflection (ATR) spectroscopy is an established method for stratum corneum analysis. The potential for dermatology studies is increased by the development of a flexible fibre-optic probe made from infrared-transparent polycrystalline silver halide material, which eases the epidermal surface characterisation significantly. As a substitute for human in-vivo tests, the isolated perfused bovine udder skin (BUS-model) has been proposed. The chemistry of human skin and of bovine udder skin is compared on the basis of their infrared spectra. For depth profiling, subsequent stripping of corneocyte layers by adhesive tape application was carried out. An example of a topical cream application and the measurement of the penetration profile of the cream compounds in the stratum corneum by the fibre-probe ATR-measurements and subsequent tape stripping are presented.
For studies of the upper horny skin layer, attenuated total reflection (ATR) infrared spectroscopy has often been used, allowing a shallow micrometer probing depth for the surface measurement. The range of applications is increased by the development of a fiber-optic probe made from polycrystalline infrared-transparent silver halide material with ductile and non-toxic characteristics, (1) which provides more flexibility for the epidermal surface characterisation than available with conventional accessories. Largest transmittance of the fibres is observed particularly within the information-rich infrared fingerprint region (about 1500 - 600 cm-1) (2). Our aim was to study the chemistry of the human horny layer and that of the upper bovine udder skin, and a comparison was made possible on the grounds of their ATR-spectra. Further potential of the measurement technique in combination with adhesive-tape stripping for penetration studies of topically applied active substances is illustrated.
The in-vitro model of isolated perfused bovine udder skin (BUS-model) has been proposed as a substitute for human in vivo tests (3). Due to the continuous perfusion, the horny layer demonstrates active barrier and reservoir functions. The in vitro BUS model is widely used in dermatological and cosmetic research as well and exhibits hair follicles and sebaceous glands, providing the corneal compartment with sebum similar to the human in vivo situation. Histological studies also prove the similarity of the BUS-model to human skin.
In Figure 1, the measurement instrument, including the attachment of the fibre-probe, is shown.
The spectrometer was a Fourier-transform infrared (FTIR)-spectrometer. The set-up, with mirror optics, fibre-probe and fibre-detector coupling, was a development of the Institute of Spectrochemistry and Applied Spectroscopy and Infrared Fiber Sensors, which includes a semiconductive mercury-cadmium-telluride (MCT)-detector. The silver halide fibre-optic probe consisted of a shaft with one fibre for transmitting and a second one for receiving infrared radiation from the ATR-measuring head. The measurement principle, with the evanescent electrical radiation field outside the optically dense fibre, which is either air or the sample in contact with the fibre for the respective background or sample measurement, is illustrated in Figure 1 b. The transmitting and receiving fibres were 2 m in length (either of square cross-section of 750 µm x 750 µm or of circular cross-section with a diameter of 700 µm; optical numerical aperture of 0.5). The shaft length was 20 cm (12 mm in diameter), and one prototype also had means for attaching detachable short U-shaped silver halide fibre pieces for ATR measurements (see Figure 1 c).
An example for the spectral measurements of bovine udder and human forearm skin using the fibre-optic ATR-probe is provided in Figure 2.
Skin areas of less than 1 mm² were measured with a probe made from a silver halide fibre of square cross-section. Repeat skin stripping by application of adhesive tape was also performed, similar to the technique described recently (4). Besides spectra measured from the skin surface, a spectrum of the lipids within the udder skin and of sebum from a human male forehead are also shown (Figure 2 a and 2 b, respectively). Differences in the spectra are due to keratin differences (see the strong amide I bands at 1650 cm-1, mainly C=O stretching vibration, and the most striking features in the C-O and C-C-stretching region between 1150 and 1000 cm-1) and the amount of lipids observed. For the latter, the absorption band of the ester C=O stretching at 1740 cm-1 and an overlapped band at about 1460 cm-1 from CH2-deformation vibrations are characteristic. There is an astonishing similarity in both lipid spectra, giving evidence to a similar chemistry, apart from the absorption band at 1710 cm-1, which is found in the human sebum spectrum and can be assigned to a free fatty acid component.
In Figure 3 the exemplary results from experiments with the application of a lamellar cream applied to udder skin are shown.
In Figure 3 a, the ATR-spectrum of a dry film of the cosmetic cream is also given, which eases the interpretation of the in-situ measurements that were carried out after subsequent tape-stripping. The disappearance of cream constituents is obvious after 10 tape-strippings. Complementary information is obtained from the measurements on the adhesive tapes used (Figure 3 b), which can be exploited also for a quantitative determination of the stripped corneal material and cream constituents.
Conclusion
The non-invasive infrared spectroscopic measurement technique can be applied to human studies as well as in vitro studies using skin models with fully functional horny layers and natural sebum excretion. The technique, based on probes made from fibres, in particular of square cross-section, is very promising, since it opens the field for new medical and cosmetic applications that were not possible with conventional ATR-crystals and bulky sampling compartment-based accessories.
Acknowledgements:
H.M. Heise and L. Küpper from the Institute of Spectrochemistry and Applied Spectroscopy acknowledge gratefully the financial support by the Ministerium für Schule, Wissenschaft und Forschung des Landes Nordrhein-Westfalen and the Bundesministerium für Bildung und Forschung.
References
1. L. Küpper, H.M. Heise, L.N. Butvina, Novel developments in mid-IR fiber optic spectroscopy for analytical applications, J. Mol. Struct. 563/564, 173-181 (2001)
2. L. Küpper, H.M. Heise, W. Pittermann, L.N. Butvina, New tool for epidermal and cosmetic formulation studies by attenuated total reflection spectroscopy using a flexible mid-infrared fiber probe, Fresenius J. Anal. Chem. 371, 753-757 (2001)
3. Th. Förster, W. Pittermann, M. Schmitt, M. Kietzmann, Skin penetration properties of cosmetic formulations using a perfused bovine udder model, J. Cosmet. Sci. 50, 147-157 (1999)
4. C. Laugel; C. Do Nascimento; D. Ferrier; J.P. Marty; A Baillet, Contribution of ATR/FT-IR spectroscopy for studying the in vivo behavior of octylmethoxycinnamate (OMC) after topical application, Appl. Spectrosc. 55, 1173-1180 (2001)
Author
Dr. H. Michael Heise
Dr. H. Michael Heise
Institut für Spektrochemie und Angewandte Spektroskopie
Bunsen-Kirchhoff-Str. 11
D-44139 Dortmund
Tel.: +49 231 1392215
Fax: +49 231 1392120
E-mail: heise@isas-dortmund.de
Dipl.-Chem. Dr. rer. nat. H. Michael Heise took up his current appointment at the Institute of Spectrochemistry and Applied Spectroscopy at the University of Dortmund in 1982. Current research interests are in near- and mid-infrared spectroscopy and chemometrics, especially with analytical applications to clinical chemistry and medical diagnostics. After obtaining his PhD in 1976 he carried out postdoctoral work at the University of Western Australia in Perth, at the Federal Biological Research Centre and the Physikalisch-Technische Bundesanstalt in Braunschweig (Germany). He is the author and coauthor of more than 130 publications, books, and book chapters.
This article was coautored by Dr. Lukas Küpper, Infrared Fiber Sensors (IFS), Aachen, Germany, Dr. Wolfgang Pittermann, Henkel KGaA, Düsseldorf, Germany and Prof. Dr. M. Kietzmann, Institute for Pharmacology, Toxicology and Pharmacy, Veterinary School Hannover, Hannover, Germany.
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