Morphological characterization of solar lentigines by in vivo reflectance confocal microscopy: a longitudinal approach.

Solar lentigines are benign hyperpigmented skin lesions. Despite their widespread distribution, knowledge on the mechanisms of development is largely unknown. A clinical study was designed in which solar lentigines were characterized using various non-invasive clinical techniques. A subset of solar lentigines was followed over a 5-year time period. One hundred and twenty-eight solar lentigines were evaluated using in vivo reflectance confocal microscopy (RCM) for the evaluation of the length and density of their dermal papillae as well as the deformation of the alignment pattern of hyperrefractive basal cells. Skin colour, colour contrast, the size of the solar lentigo, epidermal proliferation rate, melanin and haemoglobin content were quantified. RCM imaging of solar lentigines revealed a profound structural deformation of the dermal papillae, as the alignment pattern of hyperrefractive basal cells shifted from a circle in non-lesional skin to an irregular non-circular shape in solar lentigines. There was a rise in the number of dermal papillae, and these dermal papillae were significantly longer. Solar lentigines had increased melanin and haemoglobin levels and a higher rate of epidermal proliferation. For a subset of nineteen solar lentigines, a longitudinal study was set-up in which these measurements were repeated 5 years after the first evaluation. The deformation and the number of the hyperrefractive dermal papillary rings increased significantly over the 5-year time span. The size of the lesion increased, and the skin colour became darker. RCM is a useful non-invasive clinical tool for the characterization of solar lentigines, in particular the compressive deformation of the dermal papillae. This deformation became more severe over a time period of 5 years. To our knowledge, this is the first time that the in vivo time-dependent progression of solar lentigines was supported by RCM images, contributing to an improved understanding of the formation and progression of solar lentigines.

Glycation associated skin autofluorescence and skin elasticity are related to chronological age and body mass index of healthy subjects.

Glycation is the non-enzymatic reaction between reducing sugars and proteins that leads to the formation of advanced glycation end products (AGEs). In vivo skin autofluorescence (lambda(ex)/lambda(em)=370/440 nm) was used as a non-invasive clinical tool to study skin AGE accumulation in healthy panellists. Using multiple linear regression analysis, it was shown that for panellists below the age of 40, glycation associated in vivo skin fluorescence intensity increased as a function of chronological age and body mass index (BMI). Above the age of 40, the fluorescence was associated to age but not to BMI, suggesting that the effect of age became dominant over BMI. Since the accumulation of AGEs is expected to affect the biomechanical properties of the skin, in vivo skin elasticity data were gathered on a second panel. It was found that skin elasticity depended on age and BMI in a similar fashion as to what we observed for the skin fluorescence data. It is hypothesised that skin AGE accumulation contributes to the loss of skin elasticity in aged and/or overweight people.

Prevention of oxidative damage that contributes to the loss of bioenergetic capacity in ageing skin.

Skin ageing is a complex biological process related to a decline in physiological and biochemical performance. A decline in the mitochondrial energy production is a feature of ageing at the cellular level. This is partially attributed to excessive production of reactive oxygen species such as superoxide and hydrogen peroxide in aged individuals. We have investigated the effect of (glyc)oxidative stress on two biochemical targets relevant for the energy metabolism of the skin. First, we showed an age dependent decline in the activity of the hydrogen peroxide detoxifying antioxidant catalase in stratum corneum on a chronically sun-exposed site. Furthermore catalase was sensitive to peroxynitrite-induced in vitro inactivation. Catalase mimetics as well as peroxynitrite scavengers are proposed to maintain hydrogen peroxide detoxification pathways. The second target was creatine kinase, an enzyme that controls the creatine-creatine phosphate shuttle. Creatine kinase lost activity after in vitro glycation by methylglyoxal. This activity loss could be prevented by antiglycation actives. These data suggest that biomolecules involved in energy homeostasis become damaged by different sources of stress. Actives specifically selected for optimal protection against these stress situations will decrease skin vulnerability and prevent the premature loss of skin function.

Use of the synthetic superoxide dismutase/catalase mimetic EUK-134 to compensate for seasonal antioxidant deficiency by reducing pre-existing lipid peroxides at the human skin surface.

The generation of reactive oxygen species (ROS) in UV-exposed skin is believed to contribute to the photoaging process. The stratum corneum (SC) contains a variety of enzymatic and non-enzymatic antioxidants to protect against various environmental sources of free radicals. We have previously shown a seasonal variation in SC catalase activity with strong deactivation in sun-exposed skin in the summer, whereas SC superoxide dismutase (SOD) activity remained intact in those conditions. This potentially leads to the local overproduction of H(2)O(2). The oxidized lipid squalene hydroperoxide accumulates at the surface of sun-exposed skin in the summer and upon exposure to ultravoilet A (UVA) doses as low as 0.1 J cm(-2) and adequate protection against excessive lipid peroxidation at times of UV exposure should be aimed for. We have been using the induction of lipid hydroperoxides at the skin surface by a single dose of UVA (1 J cm(-2)) as a model system to evaluate the protective effect of antioxidants in vivo. Topical treatment with the synthetic SOD/catalase mimetic molecule (EUK-134) 1 h before UVA exposure reduced the level of lipid peroxides at the surface of UVA-exposed skin but also baseline peroxide levels on non-irradiated skin were reduced in a dose-dependent fashion. In contrast to alpha-tocopherol, EUK-134 even reduced the level of lipid peroxides at the surface of UVA-exposed skin when it was applied after irradiation. We confirmed that this salen-manganese complex was able to reduce squalene hydroperoxide levels in vitro, suggesting peroxidase-like activity towards organic peroxides. These data support the concept that the synthetic SOD/catalase mimetic EUK-134 might be able to compensate for seasonal deficiencies in antioxidant defense capacity at the skin surface, thereby contributing to an optimal protection of the skin against the accumulation of oxidative damage.

Affinity capillary electrophoresis.

The application of affinity capillary electrophoresis (ACE) to the study of molecular interactions is reviewed. ACE appears to be a sensitive, versatile and convenient tool to obtain reliable data on binding constants and stoichiometries of interacting systems using the Hummel-Dreyer method and variants thereof. A powerful feature is the possibility to analyze simultaneously the affinity of a large number of compounds for the same ligand, making it a promising tool for the screening of large combinatorial libraries.

Variation of the pH of the background electrolyte due to electrode reactions in capillary electrophoresis: theoretical approach and in situ measurement.

Electrode reactions during the electrophoretic process may change the pH of the buffer and subsequently the migration behavior of solutes with resultant loss of reproducibility. A theoretical treatment of pH variations due to electrolytic processes is presented. The choice of buffer appears to have a dramatic influence on the pH variations observed, even if substantial buffer action is expected at the pH chosen. The experimental evaluation of the separation of 4-hydroxy-3-methoxycinnamic acid and 3-hydroxybenzoic acid reveals that the quality of the separation decreases continuously from a baseline separation observed in the first experiment to a comigration of the two solutes (resolution = 0) in the ninth experiment. A pH decrease of about 0.05 pH units accounts for the observed changes in mobility. A novel in situ pH measurement approach is presented, in which the mobility, peak area, and peak height of an indicator dye are related to the pH in the capillary. This enables the identification and quantitation of pH variations during electrophoretic runs: the pH decreases at the anodic side already after the first experiment and pH variations as small as 0.02 pH units can be measured. The variations in peak height appear to be less suited. The calculated pH variations are in close agreement with the ones obtained experimentally.