Multiphoton FLIM Analyses of Native and UVA-Modified Synthetic Melanins.

To better understand the impact of solar light exposure on human skin, the chemical characterization of native melanins and their structural photo-modifications is of central interest. As the methods used today are invasive, we investigated the possibility of using multiphoton fluorescence lifetime (FLIM) imaging, along with phasor and bi-exponential fitting analyses, as a non-invasive alternative method for the chemical analysis of native and UVA-exposed melanins. We demonstrated that multiphoton FLIM allows the discrimination between native DHI, DHICA, Dopa eumelanins, pheomelanin, and mixed eu-/pheo-melanin polymers. We exposed melanin samples to high UVA doses to maximize their structural modifications. The UVA-induced oxidative, photo-degradation, and crosslinking changes were evidenced via an increase in fluorescence lifetimes along with a decrease in their relative contributions. Moreover, we introduced a new phasor parameter of a relative fraction of a UVA-modified species and provided evidence for its sensitivity in assessing the UVA effects. Globally, the fluorescence lifetime properties were modulated in a melanin-dependent and UVA dose-dependent manner, with the strongest modifications being observed for DHICA eumelanin and the weakest for pheomelanin. Multiphoton FLIM phasor and bi-exponential analyses hold promising perspectives for in vivo human skin mixed melanins characterization under UVA or other sunlight exposure conditions.

5,6-Dihydroxyindole eumelanin content in human skin with varying degrees of constitutive pigmentation.

Human skin contains two distinct components: brown to black, insoluble eumelanin and light colored, alkaline-soluble pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) moieties, while pheomelanin consists of benzothiazine (BT) and benzothiazole (BZ) moieties. These melanin monomer units can be quantitatively analyzed through specific degradation products by high-performance liquid chromatography (HPLC). Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin gives rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) as specific degradation products of the DHICA and DHI moieties, respectively. BZ moiety in pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA). By reductive hydrolysis with hydroiodic acid, BT moieties in pheomelanin can be analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP). As a recently improved AHPO-HPLC method enabled a better characterization of PDCA, this prompted us to address the question of DHI to DHICA ratio in human skin samples with varying degrees of constitutive pigmentation ranging from very light to dark. Results showed for the first time the ratio of 4 moieties: DHI 35%, DHICA 41%, BZ 20%, and BT 4%. The ratio is constant regardless of the degree of pigmentation. The high content of DHICA moiety may impart an antioxidant property to the epidermis melanin.

Improved HPLC Conditions to Determine Eumelanin and Pheomelanin Contents in Biological Samples Using an Ion Pair Reagent.

Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin and pheomelanin, two major classes of melanin pigments, affords pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) from eumelanin and thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA) from pheomelanin. Quantification of these five markers by HPLC provides useful information on the quantity and structural diversity of melanins in various biological samples. HPLC analysis of these markers using the original method of 0.1 M potassium phosphate buffer (pH 2.1):methanol = 99:1 (85:15 for PTeCA) on a reversed-phase column had some problems, including the short lifetime of the column and, except for the major eumelanin marker PTCA, other markers were occasionally overlapped by interfering peaks in samples containing only trace levels of these markers. These problems can be overcome by the addition of an ion pair reagent for anions, such as tetra-n-butylammonium bromide (1 mM), to retard the elution of di-, tri- and tetra-carboxylic acids. The methanol concentration was increased to 17% (30% for PTeCA) and the linearity, reproducibility, and recovery of the markers with this improved method is good to excellent. This improved HPLC method was compared to the original method using synthetic melanins, mouse hair, human hair, and human epidermal samples. In addition to PTCA, TTCA, a major marker for pheomelanin, showed excellent correlations between both HPLC methods. The other markers showed an attenuation of the interfering peaks with the improved method. We recommend this improved HPLC method for the quantitative analysis of melanin markers following AHPO because of its simplicity, accuracy, and reproducibility.

Research Techniques Made Simple: Cutaneous Colorimetry: A Reliable Technique for Objective Skin Color Measurement.

Skin color evaluation contributes to assessment of an individual's cutaneous phenotype. Skin color changes provide important clues to disease progression or treatment response. Skin color is also a predictor of skin cancer risk. Melanin pigment, blood flow, skin thickness, and photoaging contribute to skin color. Melanin, hemoglobin, bilirubin, and carotene are the primary chromophores of skin color. Their concentrations vary depending on the individual's phenotype, anatomic location, external insults of chemical irritants and UVR, and physiological changes. The evaluation and perception of skin color are often subjective. Objective quantification of skin color can be achieved with colorimetric devices such as tristimulus colorimeters. These devices compute the intensity of light reflected from skin and correlate with pigmentation and erythema. Cutaneous color and color changes can be quantified under color organization systems, such as the CIELAB color space, which is standardized by the Commission Internationale de l'Eclairage (CIE). The CIELAB expresses color's lightness, red/green intensity, and yellow/blue intensity, as L*, a*, and b* values, respectively. Additionally, skin color's full spectral characteristics and cutaneous physiology can be measured with spectrophotometers. This article outlines basic principles of the CIELAB color system and how to optimally use colorimetric devices as a skin research tool.

Clinical and Biological Characterization of Skin Pigmentation Diversity and Its Consequences on UV Impact.

Skin color diversity is the most variable and noticeable phenotypic trait in humans resulting from constitutive pigmentation variability. This paper will review the characterization of skin pigmentation diversity with a focus on the most recent data on the genetic basis of skin pigmentation, and the various methodologies for skin color assessment. Then, melanocyte activity and amount, type and distribution of melanins, which are the main drivers for skin pigmentation, are described. Paracrine regulators of melanocyte microenvironment are also discussed. Skin response to sun exposure is also highly dependent on color diversity. Thus, sensitivity to solar wavelengths is examined in terms of acute effects such as sunburn/erythema or induced-pigmentation but also long-term consequences such as skin cancers, photoageing and pigmentary disorders. More pronounced sun-sensitivity in lighter or darker skin types depending on the detrimental effects and involved wavelengths is reviewed.

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio.

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene-350 and high-performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole-type pheomelanin) as well but not 4-AHP (degradation product of benzothiazine-type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.

New insights in photoaging, UVA induced damage and skin types.

UVA radiation is the most prevalent component of solar UV radiation; it deeply penetrates into the skin and induces profound alterations of the dermal connective tissue. In recent years, the detrimental effects of UVA radiation were more precisely demonstrated at cellular and molecular levels, using adequate methods to identify biological targets of UVA radiation and the resulting cascade impairment of cell functions and tissue degradation. In particular gene expression studies recently revealed that UVA radiation induces modulation of several genes confirming the high sensitivity of dermal fibroblasts to UVA radiation. The major visible damaging effects of UVA radiation only appear after years of exposure: it has been clearly evidenced that they are responsible for more or less early signs of photoageing and photocarcinogenesis. UVA radiation appears to play a key role in pigmented changes occurring with age, the major sign of skin photoaging in Asians. Skin susceptibility to photoaging alterations also depends on constitutive pigmentation. The skin sensitivity to UV light has been demonstrated to be linked to skin color type.