Optical assessment of skin carotenoid status as a biomarker of vegetable and fruit intake.

Resonance Raman spectroscopy (RRS) and reflection spectroscopy (RS) are optical methods applicable to the non-invasive detection of carotenoids in human skin. RRS is the older, more thoroughly validated method, whereas RS is newer and has several advantages. Since collective skin carotenoid levels serve as a biomarker for vegetable and fruit intake, both methods hold promise as convenient screening tools for assessment of dietary interventions and correlations between skin carotenoids and health and disease outcomes. In this manuscript, we describe the most recent optimized device configurations and compare their use in various clinical and field settings. Both RRS and RS devices yield a wide range of skin carotenoid levels between subjects, which is a critical feature for a biomarker. Repeatability of the methods is 3-15% depending on the subject's skin carotenoid level and the uniformity of its local distribution. For 54 subjects recruited from an ophthalmology clinic, we first checked the validity of the relatively novel RS methodology via biochemical serum carotenoid measurements, the latter carried out with high performance liquid chromatography (HPLC). A high correlation between RS skin and serum HPLC carotenoid levels was established (R = 0.81; p < 0.001). Also, a high correlation was found between RS and RRS skin levels (R = 0.94 p < 0.001). Subsequent comparisons of skin carotenoid measurements in diverse age groups and ethnicities included 569 Japanese adults, 947 children with ages 2-5 screened in 24 day care centers in San Francisco, and 49 predominantly Hispanic adults screened at an outdoor health fair event. Depending on the particular subject group, correlation coefficients between the RRS and RS methods ranged between R ∼0.80 and R ∼0.96. Analysis of the Japanese screening showed that, on average, skin carotenoid levels are higher in women compared to men, skin levels do not depend on age, and tobacco smokers have reduced levels versus non-smokers. For the two most ethnically diverse groups with widely varying melanin levels, we investigated the effect of dermal melanin on RS and RRS skin carotenoid levels. The analysis revealed that large variations in skin carotenoid levels remain detectable independent of the particular melanin index. This behavior is consistent with the absence of melanin effects on the skin carotenoid levels generated with the instrument configurations. The RS method has an advantage over RRS in its relative simplicity. Due to its detection of skin reflection over a wide spectral range from the near UV to the near IR, it has the unique ability to quantify each of the major tissue chromophores and take them into account in the derivation of skin carotenoid levels.

Correlations Between Macular, Skin, and Serum Carotenoids.

Purpose: Ocular and systemic measurement and imaging of the macular carotenoids lutein and zeaxanthin have been employed extensively as potential biomarkers of AMD risk. In this study, we systematically compare dual wavelength retinal autofluorescence imaging (AFI) of macular pigment with skin resonance Raman spectroscopy (RRS) and serum carotenoid levels in a clinic-based population. Methods: Eighty-eight patients were recruited from retina and general ophthalmology practices from a tertiary referral center and excluded only if they did not have all three modalities tested, had a diagnosis of macular telangiectasia (MacTel) or Stargardt disease, or had poor AFI image quality. Skin, macular, and serum carotenoid levels were measured by RRS, AFI, and HPLC, respectively. Results: Skin RRS measurements and serum zeaxanthin concentrations correlated most strongly with AFI macular pigment volume under the curve (MPVUC) measurements up to 9° eccentricity relative to MPVUC or rotationally averaged macular pigment optical density (MPOD) measurements at smaller eccentricities. These measurements were reproducible and not significantly affected by cataracts. We also found that these techniques could readily identify subjects taking oral carotenoid-containing supplements. Conclusions: Larger macular pigment volume AFI and skin RRS measurements are noninvasive, objective, and reliable methods to assess ocular and systemic carotenoid levels. They are an attractive alternative to psychophysical and optical methods that measure MPOD at a limited number of eccentricities. Consequently, skin RRS and MPVUC at 9° are both reasonable biomarkers of macular carotenoid status that could be readily adapted to research and clinical settings.

Optical detection of carotenoid antioxidants in human bone and surrounding tissue.

Carotenoids are known to play an important role in health and disease state of living human tissue based on their antioxidant and optical filtering functions. In this study, we show that carotenoids exist in human bone and surrounding fatty tissue both in significant and individually variable concentrations. Measurements of biopsied tissue samples with molecule-specific Raman spectroscopy and high-performance liquid chromatography reveal that all carotenoids that are known to exist in human skin are also present in human bone. This includes all carotenes, lycopene, ß-cryptoxanthin, lutein, and zeaxanthin. We propose quantitative reflection imaging as a noncontact optical method suitable for the measurement of composite carotenoid levels in bone and surrounding tissue exposed during open surgeries such as total knee arthroplasty, and as a proof of concept, demonstrate carotenoid measurements in biopsied bone samples. This will allow one to establish potential correlations between internal tissue carotenoid levels and levels in skin and to potentially use already existing optical skin carotenoid tests as surrogate marker for bone carotenoid status.

Resonance Raman spectroscopy and the preterm infant carotenoid status.

OBJECTIVE: The aim of the study was to validate the noninvasive resonance Raman spectroscopy (RRS) method in infants in comparison with the high-performance liquid chromatography (HPLC) method, and to evaluate the carotenoid status in preterm infants fed with mother's milk or formula. METHODS: In the first phase of the study, resonance Raman measurements were made on male term infants' skin and correlated with tissue harvested at the time of circumcision. Each baby's foreskin was weighed, enzymatically digested, and the total carotenoids were extracted and quantitated by the HPLC. Next, to evaluate the carotenoid status of preterm infants (BW <1500 g), the skin and serum carotenoids in infants fed with either human milk or preterm formula were studied from the start of feedings and every 2 weeks until hospital discharge. Skin carotenoids were measured by RRS and the serum total carotenoids by HPLC. RESULTS: Foreskin carotenoid levels measured by RRS correlated with HPLC measurements of total serum carotenoids (R = 0.52, P < 0.01, n = 16). Forty preterm infants were studied for their carotenoid status. Thirty-two infants were fed mother's milk, whereas 8 were fed a preterm infant formula that was not enriched with carotenoids. The gestation and birth weight of the 2 feeding groups were similar. The infants fed human milk had a higher serum total carotenoid concentration and skin Raman counts than formula-fed infants. The skin Raman counts and total serum carotenoid correlated (R = 0.44, P = 0.01). The human milk-fed infants' serum total carotenoid concentrations and Raman values did not change during the study period; however, the formula-fed group's total serum and skin carotenoid decreased significantly during the study. CONCLUSIONS: RRS of infant's skin reliably assesses total carotenoid status noninvasively. Human milk-fed preterm infants have higher serum and skin carotenoids than formula-fed infants suggesting that formula-fed infants may benefit from carotenoid supplementation.

Resonance Raman based skin carotenoid measurements in newborns and infants.

We describe Resonance Raman based skin carotenoid measurements in newborns and infants. Skin- and serum carotenoid levels correlate with high statistical significance in healthy newborns and infants, and with reduced accuracy also in prematurely born infants, who in general feature very low carotenoid levels and thin transparent skin giving rise to large background absorption effects. Skin carotenoid levels can be easily compared among subjects and/or tracked in longitudinal studies with the highly molecule-specific Raman method. It therefore holds promise as a rapid, non-invasive, carotenoid antioxidant assessment method for newborns and infants in the field of pediatrics.

Simple Raman instrument for in vivo detection of macular pigments.

Raman spectroscopy holds promise as a novel noninvasive technology for the quantification of the macular pigments (MP) lutein and zeaxanthin. These compounds, which are members of the carotenoid family, are thought to prevent or delay the onset of age-related macular degeneration, the leading cause of irreversible blindness in the elderly. It is highly likely that they achieve this protection through their function as optical filters and/or antioxidants. Using resonant excitation in the visible region, we measure and quantify the Raman signals that originate from the carbon double bond (C=C) stretch vibrations of the pi-conjugated molecule backbone. In this manuscript we describe the construction and performance of a novel compact MP Raman instrument utilizing dielectric angle-tuned band-pass filters for wavelength selection and a single-channel photo-multiplier for the detection of MP Raman responses. MP concentration measurements are fast and accurate, as seen in our experiments with model eyes and living human eyes. The ease and rapidity of Raman MP measurements, the simplicity of the instrumentation, the high accuracy of the measurements, and the lack of significant systematic errors should make this technology attractive for widespread clinical research.

Resonance Raman detection of carotenoid antioxidants in living human tissue.

Increasing evidence points to the beneficial effects of carotenoid antioxidants in the human body. Several studies, for example, support the protective role of lutein and zeaxanthin in the prevention of age-related eye diseases. If present in high concentrations in the macular region of the retina, lutein and zeaxanthin provide pigmentation in this most light sensitive retinal spot, and as a result of light filtering and/or antioxidant action, delay the onset of macular degeneration with increasing age. Other carotenoids, such as lycopene and beta-carotene, play an important role as well in the protection of skin from UV and short-wavelength visible radiation. Lutein and lycopene may also have protective function for cardiovascular health, and lycopene may play a role in the prevention of prostate cancer. Motivated by the growing importance of carotenoids in health and disease, and recognizing the lack of any accepted noninvasive technology for the detection of carotenoids in living human tissue, we explore resonance Raman spectroscopy as a novel approach for noninvasive, laser optical carotenoid detection. We review the main results achieved recently with the Raman detection approach. Initially we applied the method to the detection of macular carotenoid pigments, and more recently to the detection of carotenoids in human skin and mucosal tissues. Using skin carotenoid Raman instruments, we measure the carotenoid response from the stratum corneum layer of the palm of the hand for a population of 1375 subjects and develop a portable skin Raman scanner for field studies. These experiments reveal that carotenoids are a good indicator of antioxidant status. They show that people with high oxidative stress, like smokers, and subjects with high sunlight exposure, in general, have reduced skin carotenoid levels, independent of their dietary carotenoid consumption. We find the Raman technique to be precise, specific, sensitive, and well suitable for clinical as well as field studies. The noninvasive laser technique may become a useful method for the correlation between tissue carotenoid levels and risk for malignancies or other degenerative diseases associated with oxidative stress.

Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis.

A rapid nondestructive estimation of carotenoid levels in intact fruits and vegetables and their juices could have great value when selecting nutritionally valuable crops for further propagation and commercial use. Carotenoid levels of a variety of agricultural products and juices were measured using resonance Raman spectroscopy and compared to levels determined by extraction and high-pressure liquid chromatography. A strong correlation was observed between the two methods when evaluating juices and when comparing different strains of intact tomatoes at the same stage of ripening.

Noninvasive selective detection of lycopene and beta-carotene in human skin using Raman spectroscopy.

The predominant long-chain carotenoids found in human skin are lycopene and beta-carotene. They are powerful antioxidants and thought to act as scavengers for free radicals and singlet oxygen formed by normal metabolism as well as excessive exposure of skin to sunlight. The specific importance of the particular representatives of the carotenoid antioxidants regarding skin defense mechanisms is of strong current interest. We demonstrate fast and noninvasive detection of beta-carotene and lycopene concentrations in living human skin using Raman detection of the molecules' carbon-carbon double bond stretch vibrations. Employing excitation with suitable blue and green laser lines, and taking advantage of differing Raman cross sectional profiles for beta-carotene and lycopene, we determine the relative concentration of each carotenoid species. This novel technique permits the quantitative assessment of individual long-chain carotenoid species rather than their composite level in human skin. The obtained results reveal significant differences in the carotenoid composition of the subjects' skin and show that the ratio between beta-carotene and lycopene concentration can vary from 0.5 to 1.6. The technique holds promise as a method for rapid screening of carotenoid compositions in human skin in large populations and should be suitable for clinical studies correlating carotenoid status with risk for cutaneous diseases.

Macular pigment Raman detector for clinical applications.

Clinical studies of carotenoid macular pigments (MP) have been limited by the lack of noninvasive, objective instruments. We introduce a novel noninvasive optical instrument, an MP Raman detector, for assessment of the carotenoid status of the human retina in vivo. The instrument uses resonant excitation of carotenoid molecules in the visible wavelength range, and quantitatively measures the highly specific Raman signals that originate from the single- and double-bond stretch vibrations of the pi-conjugated carotenoid molecule's carbon backbone. The instrument is a robust, compact device and suitable for routine measurements of MP concentrations in a clinical setting. We characterized and tested the instrument in clinical studies of human subjects to validate its function and to begin to establish its role as a possible screening test for macular pathologies. We also show that the MP Raman spectroscopy technology has potential as a novel, highly specific method for rapid screening of carotenoid antioxidant levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness of the elderly in the developed world.

Resonant Raman quantification of zeaxanthin production from Flavobacterium multivorum.

Resonant Raman scattering was used as a novel, rapid, non-destructive optical technique to measure zeaxanthin levels in Flavobacterium multivorum ATCC 55238. Culture broth, after bacterial growth for 40 h, exhibited characteristic resonance Raman vibrational modes at 1159 cm(-1) (C-C stretch) and 1525 cm(-1) (C=C stretch) upon excitation at 488 nm. A striking correlation was observed between the carotenoid level as estimated by HPLC and by resonance Raman spectroscopy.

In vivo resonant Raman measurement of macular carotenoid pigments in the young and the aging human retina.

We have used resonant Raman scattering spectroscopy as a novel, noninvasive, in vivo optical technique to measure the concentration of the macular carotenoid pigments lutein and zeaxanthin in the living human retina of young and elderly adults. Using a backscattering geometry and resonant molecular excitation in the visible wavelength range, we measure the Raman signals originating from the single- and double-bond stretch vibrations of the pi-conjugated molecule's carbon backbone. The Raman signals scale linearly with carotenoid content, and the required laser excitation is well below safety limits for macular exposure. Furthermore, the signals decline significantly with increasing age in normal eyes. The Raman technique is objective and quantitative and may lead to a new method for rapid screening of carotenoid pigment levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness in the elderly in the United States.