Reflectance Measurement of Carotenoids in Skin

Objective noninvasive optical methods to measure carotenoids are challenging since skin is a semiopaque tissue that is prone to considerable light scattering. Jungmann and colleagues developed a reflectometry-based system (85) that has been useful for monitoring relative changes in skin carotenoid content in small groups of volunteer subjects after supplementation (86). In this method, a region of the skin is illuminated with a 5-W halogen lamp via a fiberoptic reflection bundle, and the reflected light is analyzed between 350 and 850 nm using a spectrophotometer. This generates an uncorrected reflectance spectrum of the skin that is dominated by the spectrum of oxygenated hemoglobin with absorption maxima at 450, 540, and 570 nm. Estimation of the carotenoid levels in the skin is then performed using a nonlinear mapping procedure that links the initially recorded inhomogeneous reflection spectrum of the skin at discrete wavelengths (l) to the absorption spectrum of a ^-carotene solution in a cuvette. The basis for this estimation is the following equation in which s(l) is the unknown scattering coefficient of the tissue, k0 (l) is the background absorption, c,(A) are the molar concentrations of the various tissue absorbers, s,(A) are their absorption coefficients, and R1(1) is the skin reflectance (85):

The degree of inhomogeneity can be calculated in this way, and a corrected spectrum can be obtained that is compensated for the heterogeneous distribution of carotenoids in the tissue and the unknown pathlength of the reflected light in the tissue. The spectrum is further corrected for the influence of other light absorption and scattering components on the skin reflectance spectrum using a partial component regression and a partial least-square multivariate algorithm to determine the deviation due to skin carotenoids. From this derived spectrum, an estimate of skin carotenoid concentration can be determined that is in the same range as reports using skin biopsies and HPLC analyses (86). They also found a significant correlation between baseline skin and serum carotenoid levels in a 12-week ^-carotene supplementation study, and they were able to document an apparent rise in response to supplementation (86).

More recently, some of these same investigators have compared their reflectance method with measurements from a more commonly used Minolta Tristimulus Chroma Meter (87) (Fig. 7). In this technique, the colors of the measured skin surfaces are assigned numerical values (L*, a*, b*) in color space, where L* is the luminance quantifying the relative brightness ranging from total black (L* = 0) to total white (L* = 100), a* is a value representing the balance between the reds (positive values) and the greens (negative values), and b* is a value representing the balance between the yellows (positive values) and the blues (negative values). In agreement with the expected behavior for a substance that absorbs in the blue wavelength range, they found that the b* values correlated with the carotenoid reflectance readings whereas the L* and a* values did not. Furthermore, skin carotenoid levels measured by either method correlated positively with minimal erythemal dose levels, an indication of resistance to UV-induced skin damage (87).

300 Chroma Meter

Figure 7 Schematic of the optical head of a Minolta CR-300 Chroma Meter. A pulsed xenon arc lamp inside a mixing chamber provides diffuse, uniform lighting over the specimen area (8 mm diameter). Only the light reflected perpendicular to the specimen surface is collected by the optical fiber cable for color analysis. (Redrawn from http:// www.minoltausa.com/).

Figure 7 Schematic of the optical head of a Minolta CR-300 Chroma Meter. A pulsed xenon arc lamp inside a mixing chamber provides diffuse, uniform lighting over the specimen area (8 mm diameter). Only the light reflected perpendicular to the specimen surface is collected by the optical fiber cable for color analysis. (Redrawn from http:// www.minoltausa.com/).

C. Raman Measurement of Carotenoids in Skin

Recently, we applied resonance Raman spectroscopy to carotenoid measurements in skin and oral mucosal tissue (67,88) (Fig. 8). This method is an appealing alternative to reflectance due to its high sensitivity and specificity that obviates the need for complex correction models. Also, this method allows one to measure absolute carotenoid levels in these tissues, so the method does not have to rely on induced concentration changes. Although absolute levels of carotenoids are much lower in the skin relative to the macula of the human eye, laser power can be much higher, and acquisition times can be much longer to

Focused Beam Reflectance Measurement

Figure 8 (A) Schematic of basic Raman scattering instrumentation used for detection of carotenoid pigments in human tissue. Excitation light from an argon laser is routed via optical fiber, beam expanding lens L3, laser bandpass filter F2, dichroic mirror BS, and lens L2 to the tissue. The Raman shifted back-scattered light is collimated by lens L2, directed through BS, filtered by holographic rejection filter F1, focused by lens L1 onto a fiber, and sent to a spectrograph. The wavelength-dispersed signals are detected by a charge-coupled detector CCD and displayed on a computer monitor PC. (B) Typical Raman spectra for human ventral forearm skin, measured in vivo. Illumination conditions: 488 nm laser wavelength, 10 mW laser power, 20 s exposure time, 2 mm spot size. Spectrum shown at top is spectrum obtained directly after exposure and reveals broad, featureless, and strong fluorescence background of skin with superimposed sharp Raman peaks characteristic for carotenoid molecules. Spectrum at bottom is difference spectrum obtained after fitting fluorescence background with a fifth-order polynomial and subtracting it from the top spectrum. The main characteristic carotenoid peaks are clearly resolved with good signal-to-noise ratio at 1159 and 1524 cm"1. (From Ref. 67.)

Figure 8 (A) Schematic of basic Raman scattering instrumentation used for detection of carotenoid pigments in human tissue. Excitation light from an argon laser is routed via optical fiber, beam expanding lens L3, laser bandpass filter F2, dichroic mirror BS, and lens L2 to the tissue. The Raman shifted back-scattered light is collimated by lens L2, directed through BS, filtered by holographic rejection filter F1, focused by lens L1 onto a fiber, and sent to a spectrograph. The wavelength-dispersed signals are detected by a charge-coupled detector CCD and displayed on a computer monitor PC. (B) Typical Raman spectra for human ventral forearm skin, measured in vivo. Illumination conditions: 488 nm laser wavelength, 10 mW laser power, 20 s exposure time, 2 mm spot size. Spectrum shown at top is spectrum obtained directly after exposure and reveals broad, featureless, and strong fluorescence background of skin with superimposed sharp Raman peaks characteristic for carotenoid molecules. Spectrum at bottom is difference spectrum obtained after fitting fluorescence background with a fifth-order polynomial and subtracting it from the top spectrum. The main characteristic carotenoid peaks are clearly resolved with good signal-to-noise ratio at 1159 and 1524 cm"1. (From Ref. 67.)

Beta Carotene Hplc

Figure 8 Continued.

1000 1200 1400 1600 1800 Wavenumber, cm'1

Figure 8 Continued.

compensate. Since the bulk of the skin carotenoids are in the superficial layers of the dermis (67), the thin-film Raman equation given in Sec. II.E is still valid. Background fluorescence of the tissue can be quite high, but baseline correction algorithms are still adequate to yield carotenoid resonance Raman spectra with excellent signal-to-noise ratios. The Raman method exhibits excellent precision and reproducibility (67,88). Deep melanin pigmentation likely interferes with penetration of the laser beam, so measurements are standardly performed on the palm of the hand where pigmentation is usually quite light even in darkly pigmented individuals. As with reflectometry, relatively high levels of skin carotenoids are measured by the Raman method on the forehead and on the palm of the hand, while other body areas are significantly lower (67,87). Quantitative validation studies to correlate skin Raman readings with HPLC analysis of biopsy specimens are in progress.

Measurements of large populations with the Raman device have demonstrated a bell-shaped distribution of carotenoid levels in the palm of the hand (89). Field studies have recently been carried out where a population of 1375 healthy subjects could be screened within a period of several weeks (90). Preliminary analysis of the data confirmed that smokers had dramatically lower levels of skin carotenoids as compared to nonsmokers. Furthermore, the study showed that people with habitual high sunlight exposure have significantly lower skin carotenoid levels than people with little sunlight exposure, independent of their carotenoid intake or dietary habits. When analyzed by a chemical assay based on urinary malondialdehyde excretion, an indicator of oxidative lipid damage, people with high oxidative stress had significantly lower skin carotenoid levels than people with low oxidative stress. Again, this relationship was not confounded by dietary carotenoid intakes that were similar in both groups. These observations provide evidence that skin carotenoid resonance Raman readings might be useful as a surrogate marker for general antioxidant status (89). Studies are also underway to determine whether low skin Raman measurements may be associated with increased risk of various skin cancers. Initial studies have demonstrated that lesional and perilesional Raman carotenoid intensities of cancerous and precancerous skin lesions are significantly lower than in region-matched skin of healthy subjects (67).

The larger number of conjugated carbon bonds in lycopene compared to the other carotenoids in skin produces an absorption band shift that can be used to measure lycopene independently of the other carotenoids (88). It is possible in this way to assess this carotenoid independently from the other dietary carotenoids. There is considerable interest in a specific role for lycopene in prevention of prostate cancer and other diseases (83,91), and a noninvasive biomarker for lycopene consumption would be of tremendous utility.

A Disquistion On The Evils Of Using Tobacco

A Disquistion On The Evils Of Using Tobacco

Among the evils which a vitiated appetite has fastened upon mankind, those that arise from the use of Tobacco hold a prominent place, and call loudly for reform. We pity the poor Chinese, who stupifies body and mind with opium, and the wretched Hindoo, who is under a similar slavery to his favorite plant, the Betel but we present the humiliating spectacle of an enlightened and christian nation, wasting annually more than twenty-five millions of dollars, and destroying the health and the lives of thousands, by a practice not at all less degrading than that of the Chinese or Hindoo.

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Responses

  • edna
    Can beta carotene be measured using reflection spectroscopy?
    7 years ago

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