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

Abstract

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.

Fig. 1

Schematic absorption spectra of chromophores in human skin, including transitions from oxy- hemoglobin, HbO, de-oxyhemoglobin, Hb, β-carotene, and melanin. A spectral window for the detection of carotenoids with relatively weak absorptions from Hb/HbO exists in the 460–520 nm wavelength range.

Fig. 2

Portable optical devices used for skin chromophore measurements. (a) reflection spectroscopy device and typical reflection spectrum in 400–750 nm wavelength range (Veggie Meter®, Longevity Link Corp.); (b) resonance Raman spectroscopy device and typical Raman spectrum in 900–1750 cm⁻¹ range; (c) device based on diffuse reflection with integrating sphere (Model CM2500d, Konica Minolta, Inc.) with display of melanin and Hb indices.

Fig. 3

Skin carotenoid scores by RS (“reflection scores”) versus serum carotenoid concentrations by HPLC for 54 volunteer subjects. Tissue site: palm.

Fig. 4

Skin carotenoid scores by RS versus skin carotenoid scores by Resonance Raman spectroscopy (RRS) for 54 volunteer subjects. Tissue site: palm.

Fig. 5

Repeatability of RS skin carotenoid scores, comparing two subjects with relatively high score (a) and low score (b). Average scores were 460 and 200, respectively, with corresponding standard deviations of 3.4 and 4.1%. The standard deviation increases with lower score, consistent with the reduced signal to noise level.

Fig. 6

Histogram of skin carotenoid scores by RS for 569 Japanese subjects recruited from an eye clinic. In agreement with a biomarker, the skin carotenoid scores via RS vary widely between subjects, from near zero to near 900. The average score for this population is 300; the distribution is near-normal, with a slight skew to higher levels.

Fig. 7

Dependence of skin carotenoid scores (a) on gender, (b) age, and (c) tobacco smoking status. Skin carotenoid scores are higher in females compared to males, have no dependence on age, and are lower in active tobacco smokers relative to non-smokers and past smokers. Vertical bars above rectangles indicate the width of the respective subject distribution.

Fig. 8

(a) Histogram of skin carotenoid scores via RS for 111 children scanned at one of the participating child care centers in which children scored relatively high. Average score is 380, with strong skew of distribution to higher levels. (b) Screening results for a total of 947 children, measured in 24 participating child care centers, and ranked in order of percentages of children with scores below 300. In the first 6 centers, less than 25% of the children had scores below 300; in the last 7 centers, over 50% of the children had scores below 300. The results are used to promote higher V/F consumption in the child care centers.

Fig. 9

Comparison of carotenoid levels measured with RS and RRS devices at an outdoor screening event. a) 49 subjects; tissue site: palm; b) 47 subjects; tissue site: index finger. High skin score correlations are obtained between the two methods for either tissue site; (c) histogram of RS skin scores, revealing an average score of 305 and strongly skewed distribution towards higher scores. The average score of this population is similar to the Japanese population (Fig. 6), but slightly lower compared to the example in Fig. 8 for children in a day care center.

Fig. 10

Absence of melanin effects. Skin carotenoid scores by RS (a) and RRS (b) versus skin melanin indices measured, respectively, for 160 subjects with widely varying skin melanin levels. Skin melanin indices were measured with a Konica Minolta reflection instrument. The low correlation coefficients and high p-values in both plots prove that RS and RRS skin carotenoid scores for the used devices are not affected by melanin absorptions.

Fig. 11

RS skin carotenoid scores measured over the course of 12 weeks for a subject consuming four 5.5 oz cans of tomato juice per week (Campbell’s V8). Skin scores increase from a baseline level of 350 to 530 over 3 weeks, finally reaching saturation. The plot demonstrates the sensitivity of the RS device for the tracking of increasing skin carotenoid levels upon dietary supplementation with common vegetable juice.

Fig. 12

(a) Average RRS skin carotenoid scores and serum plasma concentrations measured over time in a controlled feeding intervention study (adapted from [10]). (b) and (c) Examples of RS and RRS skin carotenoid scores in depletion phase of study (day 0 corresponds to week 5 of study). RS scores are shown on solid line, RRS scores on dotted line. Tissue site for RS: thumb; tissue site for RRS: palm; b) example for subject with relatively high skin carotenoid score at the start of the depletion phase; c) subject with lowest skin carotenoid score.