Department of Dermatology and AllergyCharité - Universitätsmedizin Berlin

Overview of noninvasive methods for determination of carotenoid concentrations in

mammalian skin

M.E. Darvin, J. Lademann

Charité-Universitätsmedizin Berlin, Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Berlin,

Germany

Carotenoids are known to be powerful antioxidants acting effectively against reactive oxygen species. The ability of carotenoids to quench oxygen radicals (including singlet oxygen) is related to the conjugated carbon double-bond system, and maximum protection is provided by those having nine or more double bonds.

Most prominent carotenoids being accumulated in human skin are beta-carotene (9 double bonds) and lycopene (11 double bonds) and their isomers.

High-performance liquid chromatography (HPLC) is a widely used „gold standard“ method for determination of carotenoids. This method is highly invasive, time-consuming and expansive.Moreover, the oxidation of carotenoids during the sample preparation cannot be excluded.

For analyzing the kinetics of carotenoids in the skin noninvasive methods are irreplaceable and should be performed.

Carotenoids cannot be detected in the skin using fluorescence analyses because their fluorescence efficiency is very low (10-

4-10-5).

Optical methods for noninvasive determination of carotenoids in mammalian skin:

1. resonance Raman spectroscopy2. Raman microscopy

3. reflection spectroscopy4. skin color measurements

Resonance Raman spectroscopy

1 – Ar+ laser; 2 – lens system; 3 – filter (488nm/514.5nm);4, 9 – optical fiber; 5 – excitation channel; 6 – optical imaging system; 7 – skin; 8 – receiving channel; 10 – spectrometer; 11 – CCD; 2 – computer; 13 – photo detector

Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

Brandon et al. Cancer Prev. Res. 3(4): 529-538, 2010

Excitation wavelength 488 nm

Fiber optic-based resonance Raman spectroscopy

for endoscopic measurement of carotenoid oxidative breakdown in living tissue

Advantages:

- measurement quickness- high sensitivity- selectivity between beta-carotene and lycopene- increasing the measurement accuracy using photobleaching effect- measurement stability- transportability

Limitations:

- influence of other carotenoids- reabsorption of Raman signal at 527.2 nm by lycopene

Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

Resonance Raman spectroscopy

Raman microscopy

1 – laser (NIR or VIS); 2 – short-pass filter; 3 – objective; 4 – mirrors;5 – skin; 6 – laser rejection filter; 7 – lens; 8 – optical fiber; 9 – spectrometer; 10 – CCD; 11 – computer

Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

Advantages:

- measurement of axial distribution of carotenoids in the skin

Limitations:

- low and very low carotenoid concentrations are not detectable under non-resonant excitation

- bulky size- high price

Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

Raman microscopy

Reflectance spectroscopy

1 – LED (440 - 490 nm); 2 – skin; 3 – focusing system;4 – replicated holographic grating spectrometer; 5 – Bluetooth

Darvin et al. J. Biophotonics 5(7): 550-558, 2012

„Scanner“ in use

Advantages:

- low price- measurement stability- compact size (easy to transport)- independence from electric mains

Limitations:

- low carotenoid concentrations are not detectable

- could be applied only on thenal and plantar skin areas where the epidermis is thick enough and influence of melanin and blood

chromophores is less pronounced

Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

Reflectance spectroscopy

Darvin et al. J. Biophotonics 5(7): 550-558, 2012

Skin color measurements

Bersha. Master Thesis, University of Eastern Finland, 21 June 2010

b*-value characterizing yellowness of the skin serves as a measuring parameter for epidermal carotenoids

Alaluf et al. Journal of Nutrition 132(3), 399-403, 2002

b*-value was found to correlatewith concentration of carotenoids in human epidermis

Advantages:

- low price- compact size (easy to transport)- independence from electric mains

Limitations:

- low carotenoid concentrations are not detectable

- could be applied only on thenal and plantar skin areas where the epidermis is thick enough and influence of melanin and blood

chromophores is less pronounced- measurement stability is not high enough

Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

Skin color measurements

  Resonance Raman spectroscopy

Raman microscopy Skin color measurements Reflection spectroscopy

Size/weight compact to bulky bulky compact compact

Transportability yes no yes yes

Price middle to high high low low to middle

Measurement time  3 to 90 sec

 around 60 seconds

 around 30 sec

 around 60 sec

Measurement stability  better than 10%

 better than 10%

 no data

 better than 10%

Light source laser, LED laser lamp, LED LED

Measurement depth  up to 200 µm

 up to 40 µm

 up to 200 µm

 up to 200 µm

Selectivity to lycopene no no no

Electric mains needed needed don’t needed don’t needed

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ConclusionsComparison of optical methods

for measurement of carotenoids in human skin

Thank you for your attention