The Journal of Nutrition Vol. 128 No. 5 May 1998, pp. 903-907

Increased Dermal Carotenoid Levels Assessed by Noninvasive Reflection Spectrophotometry Correlate with Serum Levels in Women Ingesting Betatene^1,2

Wilhelm Stahl³, Ulrike Heinrich^*, Holger Jungmann, Jutta von Laar, Michael Schietzel, Helmut Sies, and Hagen Tronnier^*

Institut für Physiologische Chemie I and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, D-40001 Düsseldorf, Germany; ^* Institut für Experimentelle Dermatologie, Universität Witten-Herdecke, D-58453 Witten, Germany; and  Krebsforschung Herdecke e.V., D-58313 Herdecke, Germany

	ABSTRACT

Abstract Introduction Methods Results Discussion References

-Carotene is being used as an oral sun protectant, and evidence indicates that carotenoids may protect human skin from light-induced lesions. However, limited information is available on the distribution and accumulation of -carotene in skin, especially with respect to various skin regions. With the use of reflection spectroscopy, we investigated the accumulation of total carotenoids in human skin after repeated supplementation of 12 women with -carotene from a natural source Betatene, an algal extract. After daily ingestion of 24 mg -carotene (in Betatene) for 12 wk, an increase in carotenoid skin levels was observed. Highest basal values were measured in skin of the forehead, palm of the hand and dorsal skin, with lower levels measured in skin of the arm and back of the hand. Upon treatment, increases in carotenoid skin levels were found in all areas as follows: 2.4-fold in forehead, 0.7-fold in dorsal skin, 2.2-fold in the palm of the hand, 17-fold on the back of the hand and 1.7-fold on the inside of the arm. After cessation of treatment, the carotenoid levels decreased in all skin areas. Serum -carotene levels were elevated upon treatment and correlated with carotenoid skin levels. Correlations for serum vs. skin from the palm of the hand (r = 0.94) and skin from the forehead (r = 0.89) were calculated, indicating that serum levels appeared to be a suitable indicator for carotenoid accumulation in specific regions of the skin. With doses of ~20-25 mg carotenoids/d, it is possible to raise dermal carotenoid levels.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Long-term supplementation with -carotene leads to a significant increase in the blood levels of -carotene, and within 4 wk, a plateau is reached (Mathews-Roth 1990, von Laar et al. 1996). Plateau concentrations differ among individuals, ranging from 2 to 18 µmol/L at intake levels of ~100 mg -carotene/d; -carotene blood levels in unsupplemented persons are considerably lower, ~0.2-0.5 µmol/L (for review see Stahl and Sies 1996).

The absorption of -carotene and the influence of several factors on its bioavailability have been investigated (Erdman et al. 1993, Olson 1994, Parker 1996, Wang 1994). Less is known about its distribution and accumulation in tissues. -Carotene has been found in liver, adrenal glands, testes, fat, pancreas, lung, kidney and skin (Kaplan et al. 1990, Schmitz et al. 1991, Stahl et al. 1992). Dermal accumulation of -carotene has attracted attention, because of the speculation that carotenoids contribute to protection against acute and chronic exposure to UV light (Biesalski et al. 1996).

UV irradiation of the skin results in sunburn reactions, photoaging and an increased risk for skin cancer (Taylor et al. 1990). The adverse effects of UV light might be due to the formation of reactive oxygen species, which are capable of damaging biological macromolecules (Pathak and Carbonare 1992) or interfering with regulation of gene expression (Schreck and Baeuerle 1994). Carotenoids are efficient scavengers of singlet molecular oxygen and of peroxyl radicals (Rice-Evans et al. 1997, Sies and Stahl 1995) and might thus exhibit protection against UV-induced skin lesions.

Although the protective effects of carotenoids towards skin lesions are still under investigation (Biesalski et al. 1996, Garmyn et al. 1995, Ribaya-Mercado et al. 1995), -carotene supplements are widely used as oral sun protectants, generally recommended to be taken over a period of 4-6 wk before sun exposure. -Carotene levels in human skin increase after single and repeated doses (Biesalski et al. 1996, Prince and Frisoli 1993), but no information is available on the distribution of -carotene into various dermal areas. The correlation between blood levels of carotenoids and their content in various skin areas is of interest. In this study, we describe the accumulation of -carotene in serum and skin after long-term supplementation with -carotene from an extract (Betatene) of the alga, Dunaliella salina, as a natural source of carotenoids. A noninvasive method, reflection photometry, was used for the determination of carotenoid levels in skin.

	SUBJECTS AND METHODS

Abstract Introduction Methods Results Discussion References

Reagents.  -Carotene and -apo-8-carotenal were obtained from Fluka (Buchs, Switzerland); all other chemicals were from E. Merck (Darmstadt, Germany).

Study design.  Twelve female healthy adults, 20-45 y old, took part in the study. Smokers consuming more than three cigarettes per day were not included. Written informed consent was obtained from each participant.

Betatene, an extract of the alga Dunaliella salina (Betatene, Cheltenham, Australia) was used as a carotenoid source. It contains ~20% of a carotenoid mixture (mainly -carotene) in soybean oil; low amounts of algal sterols and algal hydrocarbons (3-5%) are also present in the supplement. Betatene, equivalent to 25 mg total carotenoids, was given daily over a period of 12 wk; capsules were taken together with the main meal. For single carotenoids, a 25 mg dose contained the following: 13.0 mg all-trans--carotene, 10.5 mg 9-cis--carotene, 0.3 mg other cis isomers of -carotene, 0.75 mg -carotene, 0.18 mg cryptoxanthin, 0.15 mg zeaxanthin and 0.12 mg lutein.

View larger version (11K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Fig 1. Spectra as obtained by reflection spectroscopy of human skin. A) Uncorrected spectrum of human skin; the spectrum was taken from dorsal skin at wk 4 of supplementation. B) -Carotene spectrum derived from the uncorrected spectrum overlaid with the spectrum of synthetic -carotene in homogeneous solution.

Blood samples were collected on d 0 and after 4, 8 and 12 wk of treatment. An additional blood sample was obtained 2 wk after cessation of the intake of Betatene. Serum was prepared from the blood samples and stored at 20°C until analyses. Total carotenoid levels in skin were determined by reflection spectroscopy at the same time points (Jungmann et al. 1996). The skin areas investigated were forehead, back, back of the hand, palm of the hand and inside of forearm. These sites were selected because differences in coloration of the skin have been observed in these areas after long-term intake of -carotene.

Serum carotenoid analyses.  The analyses of -carotene in serum were performed as described earlier (Stahl et al. 1992) with slight modifications. After addition of an appropriate amount of internal standard (-apo-8-carotenal), 150 µL of serum were diluted with 850 µL of 2 mmol/L phosphate buffer, pH 7.2 (containing 250 mg/L EDTA and 250 mg/L ascorbic acid). Ethanol (1 mL) was added and the mixture was extracted with 6 mL hexane/dichloromethane (5:1, v/v); the extraction solvent contained 250 mg butylated hydroxytoluene/L. Five milliliters of the organic layer was removed and the solvent was evaporated under nitrogen. The dry residue was dissolved in 200 µL HPLC eluent/dichloromethane (19:1, v/v) and an aliquot was injected for HPLC analyses.

The HPLC equipment included a HPLC pump Merck-Hitachi model 655A-12 (Merck); UV-Vis detector Merck-Hitachi model L-4250 (Merck); data collection integrator Merck-Hitachi model D-2500 (Merck). For spectrophotometric peak identification, we used a model 168 diode array detector (Beckman, Munich, Germany). Separation of carotenoids was performed with a column of 5-µm particles of Suplex pKb 100 (Supelco, Bellefonte, PA) with methanol/acetonitrile/2-propanol (54:44:2, v/v/v) as mobile phase. The flow rate was 1 mL/min; detection was at 450 nm.

Carotenoid concentrations in the samples were calculated from calibration curves generated from peak height ratios of the carotenoids to the internal standard. Each sample was analyzed in triplicate.

Reflection spectroscopy.  Reflection spectra were collected noninvasively between 350 and 850 nm with a Multiscan OS 20 spectrophotometer (MBR GmbH, Herdecke, Germany) coupled to an all-silica fiberoptic reflectance bundle (Top Sensor Systems, Eerbeek, Netherlands). Generally, an average spectrum consisted of 8 scans; each scan was completed within 124 ms. The spectral resolution used in the present study was ~1.2 nm; all spectra were measured against a white reference standard (titanium oxide). A 5 W (5 J/s) halogen lamp (MBR GmbH) was used for tissue illumination. Under the conditions applied in this study, the increase in skin surface temperature was <0.5°C.

Data analyses.  The spectroscopic data were transformed to a log (1/R) scale, mean-centered and normalized. The law of Lambert-Beer is not suitable for the calculation of carotenoid levels in tissues on the basis of data obtained with reflection photometry for the following reasons: 1) heterogenous distribution of carotenoids in tissues; 2) other substances present in the tissue that contribute to the reflection spectra; and 3) the unknown pathlength of the reflected light in the tissue. Thus further data analyses were performed with software developed for this purpose (Jungmann et al. 1996, Postaire et al. 1997).

View this table: [in this window] [in a new window]   Table 1. Carotenoid concentrations in different areas of the skin and serum -carotene concentrations in women who consumed carotenoids from the algal extract Betatene for 12 wk1

View larger version (22K): [in this window] [in a new window]   Fig 2. Time course of carotenoid levels in skin, forehead, or palm of the hand of women who consumed 24 mg -carotene/d for 12 wk as determined by reflection spectrophotometry, in comparison to the level of -carotene in serum determined by HPLC. Values are means ± SD, n = 12.

The distribution of the chromophore was determined as follows. If S () denotes the measured reflection spectrum of the skin and E () the absorption spectrum of a -carotene solution in a cuvette, then a mapping M exists with: E () = M (S []); M is a nonlinear one-to-one mapping (Jungmann et al. 1996). This mapping M depends on the degree of inhomogeneity, not on the type of inhomogeneity (Wodick and Lübbers 1973), and is scale-invariant. Therefore, the degree of inhomogeneity can be calculated and an inhomogeneous spectrum can be corrected. The spectra obtained in this study were processed in this way.

Correction for the influence of other components on reflection spectra. In multivariate analyses, information about every single compound is not required (Martens and Naes 1991). Because the statistical methods are sensitive to changes in measuring conditions, it is possible to assign the deviation to the compound of interest (Jochum et al. 1981). Applying partial component regression and a partial least-square multivariate algorithm, the deviation due to the known -carotene spectrum can be assessed, if the nonlinear distortion of this spectrum has been previously corrected (Marbach 1993). Because an inverse mapping of M exists, it has been demonstrated (Lübbers and Hoffmann 1980) that it is possible to alter M' in such a way that the estimated square of the deviation approaches a minimum and thus a corrected spectrum is obtained. For each corrected spectrum, multivariate analytical methods were applied (Hruschka and Norris 1982).

A CV of ~10% was calculated for repeated measurements of skin carotenoids at the same site; for multiple measurements within the same general anatomical area, the CV was ~20%. In addition to -carotene, other carotenoids with similar absorption spectra may contribute to the levels found in skin. Therefore the term carotenoids is used for skin levels.

Carotenoid levels in skin and -carotene serum were correlated by using linear regression analyses.

	RESULTS

Abstract Introduction Methods Results Discussion References

Figure 1A shows an example of an uncorrected spectrum obtained upon application of reflection spectroscopy to human skin. The spectrum is dominated by the absorption from oxygenated hemoglobin with maxima at about 450, 540 and 570 nm. The absorption maximum of -carotene and analog carotenoids is at 450 nm; the compounds contribute to the reflection spectrum, appearing at the descending part of the first maximum of oxygenated hemoglobin. The carotenoid spectrum in the range of 400-510 nm, derived from the uncorrected spectrum according to the data analyses described in the Subjects and Methods section, is presented in Figure 1B. For comparison, a spectrum of synthetic -carotene is overlaid (dotted line); there is satisfactory agreement between the two spectra.

Carotenoid levels in selected parts of the skin and serum detected at various time points under Betatene treatment are presented in Table 1; the time course for serum, forehead and palm of the hand are given in Figure 2. The basal levels in skin are distinctively different within different regions. Relatively high basal levels were found in the skin of the forehead, palm of the hand and dorsal skin, with lower levels found in the skin of the arm and back of the hand. An increase in carotenoid levels was observed in all parts of the skin after treatment with Betatene. Maximum values were reached after 12 wk of treatment in all areas. However, the accumulation of carotenoids was different at specific sites. On the basis of the starting levels, the increase in skin carotenoids was 2.4-fold in forehead, 0.7-fold in dorsal skin, 2.2-fold in the palm of the hand, 17-fold for the back of the hand and 1.7-fold for the inside of the arm after 12 wk of supplementation.

After cessation of supplementation, the carotenoid levels in skin decreased in all dermal areas. Two weeks after the end of treatment, the levels in skin were decreased by 56% in forehead, 14% in dorsal areas, 31% for the palm of the hand, 35% for the back of the hand and 47% inside of the arm. In parallel with skin, serum levels of -carotene increased upon supplementation. Starting from a mean base value of 0.44 µmol/L, mean peak levels were detected after 12 wk of treatment with 1.8 µmol/L representing a 3.1-fold increase. Mean serum levels decreased to 1.15 µmol/L, corresponding to a 36% loss, 2 wk after the end of treatment.

The correlation coefficents between -carotene serum levels and the amount of carotenoids in various parts of the skin are given in Table 1. The highest correlation coefficients were found for serum vs. skin from the palm of the hand and serum vs. skin from the forehead.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Various spectroscopic methods have been applied to determine biologically relevant molecules and follow biochemical processes in living organisms [see Sies and Brauser (1980) for review]. Reflection spectroscopy yields spectroscopic data from surfaces of tissues and is a suitable and convenient method to determine carotenoid levels in accessible tissues such as skin. The levels determined in this study are similar to data reported in the literature. In facial skin (free of visible adipose tissue), mean -carotene values of ~0.1-0.2 nmol/g wet tissue have been measured by means of HPLC (Peng et al. 1993). This is rather close to the basal level determined here, varying from 0.03 to 0.4 nmol/g depending on the skin area. Higher levels at ~1.5 nmol/g wet tissue were found when subcutaneous fat was included in sample anlayses (Ribaya-Mercado et al. 1995). Using a halogen lamp with 5 W (5 J/s) energy, the penetration depth of the skin at wavelengths between 200 and 800 nm is ~0.1-0.85 mm. Thus the content of carotenoids from subcutaneous fat does not contribute significantly to the measurement obtained with reflection spectroscopy under the conditions applied in this study. The mean base-line value for -carotene serum levels was 0.44 µmol/L, which is also within the range described in the literature (for review see Stahl and Sies 1996) .

Carotenoids are not homogeneously distributed in skin. Rather, high levels are found in the forehead, back or palm of the hand; skin samples from other parts of the body contain less. The reason for this is unknown. Differences in skin vascularization at various sites as well as differences in long-term UV-light exposure might play a role. It has been shown that exposure to a single dose of UV light hardly affects -carotene levels in skin (Ribaya-Mercado et al. 1995); other carotenoids such as lycopene are destroyed when skin is subjected to UV-light exposure.

The levels of carotenoids in human skin increased upon supplementation; no plateau was reached within 12 wk. The accumulation of carotenoids in skin differed among various body sites. A 0.7- to 2.4-fold rise over the basal levels was determined in skin from the forehead, dorsal areas, palm of the hand and arm, whereas the increase in skin areas of the back of the hand was 17-fold. The reason for this finding is also unknown. It should be noted that the data obtained from the back of the hand have the highest standard deviation of all data. Thus, more information is required to substantiate this observation.

The levels in skin decreased within 2 wk after cessation of supplementation. The highest losses were observed in light-exposed tissues such as forehead (56%) and arm (47%). In dorsal skin, which is usually protected from light, carotenoid levels decreased by only 14%. Illumination with light may contribute to the loss of carotenoids in human skin and thus point to an interaction of carotenoids with reactive species derived from light relevant for photobleaching.

An increase in serum -carotene was observed in parallel with carotenoid skin levels, which correlated in specific areas such as skin from the forehead and palm of the hand. The correlation was less significant with other skin areas. These data suggest that serum levels are an indicator for carotenoid accumulation in the skin.

	FOOTNOTES

Manuscript received 7 November 1997. Initial reviews completed 12 December 1997. Revision accepted 26 January 1998.

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