Summary. In order to noninvasively measure water concentration in the stratum corneum, infrared spectra were obtained using an attenuated total-reflec-.
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Arch Dermatol Res (1985) 277:489-495 9 Springer-Verlag 1985
A Noninvasive, In Vivo Technique to Quantitatively Measure Water Concentration of the Stratum Corneum Using Attenuated Total-Reflectance Infrared Spectroscopy R. O. Ports, D. B. Guzek, R. R. Harris, and J. E. McKie Pfizer Central Research, Eastern Point Road, Groton, CT 06340, USA
Summary. In order to noninvasively measure water concentration in the stratum corneum, infrared spectra were obtained using an attenuated total-reflectance technique in conjunction with Fourier transform spectroscopy. A weak water-absorbance band near 2,100 c m - 1 was detected in both in vivo and in vitro spectra. The significance o f this band is that it occurs in a region of the mid-infrared where the stratum corneum and most topically applied substances show no absorbance. In vitro spectra obtained as a function of ambient relative humidity showed an increase in the absorbance near 2,100 c m - 1 with increasing water concentration in the stratum corneum. The combined in vivo and in vitro results lead to a quantitative assessment of water concentration in the uppermost layers of the stratum corneum.
Key words: Infrared spectra - Stratum corneum Water concentration
the absorbance spectrum of water can be uniquely identified, and the water concentration can be quantitatively measured from the absorbance. Numerous investigators have utilized the technique of attenuated total-reflectance infrared (ATR-IR) spectroscopy to noninvasively detect water in the stratum corneum [1, 7, 16]. Quantitative interpretation of these data has been difficult, however, due to overlap of the water spectrum with that of other tissue components and topically applied substances. Due to these difficulties, we chose to measure the water concentration via the detection of a combination band of water near 2,100 cm -1 (4.8 gm). This absorbance is distant from the absorbances of either the stratum corneum or commonly applied substances and, thus, leads to a quantitative assessment of the water concentration in the surface layers of the stratum corneum.
Materials and Methods Introduction Changes in the water concentration o f the stratum corneum have important consequences for the functional and cosmetic properties o f h u m a n skin. There have been many recent advances in our ability to noninvasively measure the hydration of the stratum corneum. These techniques include measurement of the electrical [4, 15], microwave [9], mechanical [3, 11], thermal [6], and spectroscopic properties of the skin. Many of these techniques, however, suffer from the fact that, while the property measured is influenced by water content, the theoretical relationship between the two is not always understood. Infrared (IR) spectroscopy has great potential for the measurement o f stratum corneum hydration, since Offprint requests to: R. O. Potts, Ph.D. (address see above)
Infrared spectra were obtained using an Analect FX-6200 Fourier Transform Infra Red. (FTIR) spectrophotometer (Laser Precision, Irvine, CA, USA) equipped with an Analect FXA525 ATR sampling device and a liquid-nitrogen-cooled mercurycadmium-telluride detector. Due to digital signal processing, a very sensitive detection system, high energy throughput, and simultaneous interaction with the sample at all IR energies, the FTIR system enables the rapid acquisition and analysis of IR spectra. All spectra presented here represent an average of 64 scans obtained in about 2 min. The FTIR system not only reduces the data acquisition time but, as a result, minimizes the effect of water build-up in the stratum corneum due to occlusion of the site during the experiment. The internal reflection element (IRE) used in this study was a zinc sulfide trapezoid (dimensions, 50 x 20 x 2 mm) having 45~ entrance and exit faces. In Vivo Spectra
The ATR sampling device was designed with the IRE surface parallel to the optical bench so that the sample could be placed directly upon the IRE. For in vivo experiments, this design was ideal, since it allowed the lower leg to rest directly on the ATR
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R.O. Ports et al.: Quantitatively Measuring Water Concentration in the Stratum Corneum
ZnSIRE
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MIRRORSj Fig. 1. A schematic diagram of the modified ATR sampling device
sampling device. The leg was supported by a platform flush with the upper surface of the IRE and separated from both the ATR sampling device and interferometer so that no mechanicaldistortion of the optics occurred during the experiment. This platform was constructed to enclose the optical path so that dry-nitrogen purging could be maintained at all times. All in vivo spectra were obtained under ambient laboratory conditions of approximately 35% relative humidity and 23~C.
In Vitro Spectra Human and porcine epidermis were obtained by immersing fullthickness skin in a 60~C water bath for 1 and 6 rain, respectively. Epidermal cells were then removed from the overlying stratum corneum by digestion with 0.1% trypsin in phosphate-buffered saline, pH 7.4, for 1.5 h at 37~C. The resulting stratum corneum sheet was then rinsed with water, incubated in 0.1% soybean trypsin inhibitor for 30 rain, rinsed in water again, and stored over a CaSO4 desiccant at room temperature until needed. Prior to experimentation, the dried stratum corneum sheet was subjected to a 1-min wash in cold hexane ( - 20~C) to remove surface sebaceous lipids. The stratum corneum sheet was floated on a water bath until pliant and then spread evenly across the IRE, taking care to eliminate trapped air. The IRE and surrounding support surface were then enclosed in an air-tight plastic chamber (10 x 7.5 x 5.5 cm) with a removable lid. The relative humidity was varied by the use of saturated-salt solutions placed in the chamber. Alternatively, the relative humidity could be varied over a broad range by passing the appropriate mixture of dry and water-saturated air through the chamber. In this case, the relative humidity was measured with dry and wet bulb thermocouples placed in the chamber. The sample was allowed to equilibrate for many hours at each relative humidity tested. Throughout this period, IR-absorbance measurements of the stratum corneum sample were made until the readings remained constant. After the ATR-IR measurements had been completed, the same sample underwent gravimetric analysis to determine its water content as a function of relative humidity. Using the same saturated-salt solutions as used for the ATR-IR measurements, the piece of stratum corneum was allowed to equilibrate to constant weight in a sealed chamber. From published values [13] of stratum corneum volume as a function of relative humidity, the experimentally determined water content (mass) could be converted to water concentration. In this manner, it was possible to relate water concentration and IR absorbance of the same sample at various relative humidities.
Results and Discussion The A T R p h e n o m e n o n occurs when radiation p r o p agating t h r o u g h a m e d i u m o f refractive index nl strikes an interface with a n o t h e r m e d i u m o f lower refractive index (n2). I f the incident b e a m strikes the interface at an angle that is greater than the critical angle, defined as 0c = sin -1 (nz/nO, the b e a m will penetrate slightly into the m e d i u m o f lower refractive index as it is being totally reflected. I f the m e d i u m o f lower refractive index has a b s o r p t i o n b a n d s in the frequency range o f the incident radiation, each penetration will result in an energy loss due to absorbance. E n e r g y losses due to scattering m a y also occur. These c o m b i n e d energy losses are amplified by successive reflections within the I R E . T h e ability o f A T R spectroscopy to detect absorbance a n d scattering depends u p o n a n u m b e r o f factors, including the intensity, wavelength, and entry angle o f the incident radiation, the a b s o r p t i o n coefficent o f the absorber, the degree o f c o n t a c t between the two media, the n u m b e r o f internal reflections, and the ratio o f rt2 to n~. A c c o r d i n g to A T R theory [8], the sensitivity o f the technique is especially dependent u p o n energy coupling between the two media a n d the depth o f b e a m penetration into the m e d i u m o f lower refractive index. C o u p l i n g can be increased by c h o o s i n g an I R E with a refractive index close to, but greater than, the sample (i.e., index matching), while the depth o f penetration can be increased by c h o o s i n g an incident angle close to, but greater than, the critical angle. The particular A T R c o n f i g u r a t i o n utilized in this study uses zinc sulfide (nl = 2.24) as the I R E , since it provides high coupling with b o t h skin (nz = 1.6 [14]) and water (n2 = 1.33) as c o m p a r e d to other n o n t o x i c I R E s c o m m o n l y used for in vivo skin studies [1, 5, 7, 16], such as g e r m a n i u m (nl = 4.0). The critical angle for the zinc-sulfide/skin interface is 45.6 ~, a n d thus, the angle o f b e a m incidence was set just a b o v e this
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R. O. Potts et al. : Quantitatively Measuring Water Concentration in the Stratum Corneum
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0.95) The extrapolated, zero-time values were obtained by a linear fit of water content vs sampling-period data
This A T R - I R technique was used to measure the in vivo changes in the water content of the stratum corneum due to occlusion with petrolatum. The I R spectrum o f a site on the back o f the lower leg was measured in six individuals. Three successive spectra of 64 scans were obtained over about 6 min. The site was then evenly coated with 90 mg petrolatum spread over an area of 3 x 6 cm and then covered with a dressing. After 5 h, the dressing was removed, the petrolatum was wiped away with a soft cloth, and the I R spectra were immediately recorded. The same site was again measured 3 0 - 4 5 rain after the removal of the petrolatum. Even though small absorbances due to the petrolatum were apparent after wiping the test site, there was no contribution to absorbance in the range of 1 , 8 0 0 - 2 , 3 0 0 cm -1. Thus, under all experimental conditions, the water content of the test site could be
quantitatively determined from the measured absorbance ratio using the data shown in Fig. 7. The results shown in Table l lead to several interesting conclusions. Under all conditions tested, the water content of the test site increased with each successive spectral measurement due to occlusion of the site by continuous contact with the IRE. Because of this temporal increase in water content, it is important to compare results obtained during similar sampling times. Alternatively, one may extrapolate water content values to zero time to obtain time-independent results. Regardless of the method of comparison, however, the results show that, immediately after the removal o f the petrolatum from the occluded site, there was an approximately two-fold increase in water content at all three sampling times. Finally, when measured 3 0 - 45 rain after removal, the water content had already returned to pretreatment values.
R. O. Potts et al.: Quantitatively Measuring Water Concentration in the Stratum Corneum
o n e x p e r i m e n t a l needs a n d the I R i n s t r u m e n t used. I n a d d i t i o n , this A T R - I R t e c h n i q u e h o l d s g r e a t p r o m i s e for the m e a s u r e m e n t o f the w a t e r c o n c e n t r a t i o n at v a r y i n g d e p t h s , since the p e n e t r a t i o n c h a n g e s with the i n c i d e n t angle. Thus, " o p t i c a l s e c t i o n i n g " o f the s t r a t u m c o r n e u m m a y be a c h i e v e d w i t h v a r i a b l e - a n g l e optics. These i n v e s t i g a t i o n s a r e c u r r e n t l y u n d e r w a y .
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