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The aim of this study was to localize and visualize aminopeptidase activity within freshly excised, dermatomed human skin without perturbation of its histologic integrity. The use of confocal laser scanning microscopy (CLSM) is introduced as a novel approach by which to monitor the degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase probe bis-Leu-rhodamine 110 (Leu2-R11O) was interpreted to reflect the local aminopeptidase activity in the tissue. To separate the kinetics of diffusion and degradation of Leu2-R110, a lateral application mode was introduced: the probe was applied at the cutting plane of a mechanical cross-section of the sample, and optical cross-sections were made parallel to the cutting plane of the mechanical section. By this means, simultaneous and equal access of the substrate to the various strata and domains of the skin was achieved. The observations revealed that the fluorescence, i.e., aminopeptidase activity, was evenly distributed throughout the viable part of the epidermis, with enhanced fluorescence ("hot spots") in the upper layers of the stratum granulosum, while dermis and stratum corneum showed considerably less aminopeptidase activity. Independent studies with hair follicles (obtained from trypsin-separated stratum corneum) also showed aminopeptidase activity, mostly at the root sheath. Because of the advantage of direct visualization and localization of enzymatic activity in intact tissue, the lateral application mode of substrate administration in combination with CLSM may be beneficial to further elucidate the location and intensity of metabolic activity in other living tissues as well.

HaCaT cell culture sheets were recently demonstrated to be a useful tool to study epidermal metabolism. Here we report on a mechanistic and quantitative correlation between the kinetics of aminopeptidase-based cleavage of L-Ala-4-methoxy-2-naphthylamide (Ala-MNA) in HaCaT sheets versus stripped human skin. Fresh human skin (breast or abdominal) was obtained from cosmetic surgery, tape-stripped, and dermatomed. HaCaT sheets were cultured on porous membranes. Diffusion and concurrent metabolism were studied under reflection and permeation conditions. Numerical simulations of simultaneous diffusion and saturable Michaelis-Menten metabolism were based on a physical model and a fixed set of independently obtained parameters (diffusion coefficient D, distance x, partition coefficient P, Michaelis constant Km, maximum metabolic rate Vmax). Under reflection conditions, cleavage of Ala-MNA in HaCaT sheets was very close to stripped skin. In contrast, in permeation studies substrate only permeated through HaCaT whereas passage through stripped skin led to full cleavage of Ala-MNA to MNA. All experimental data were in reasonable to excellent agreement with numerically generated data. Differences between HaCaT and stripped skin could be quantitatively and mechanistically explained by the thickness of the metabolically active layer, i.e., approximately 10 microm in HaCaT and approximately 40 microm in stripped skin. Full cleavage of permeating Ala-MNA in stripped skin was predicted to occur within the upper approximately 20 microm of viable epidermis. Thus epidermal aminopeptidase activity may act as an efficient metabolic barrier to fully block the permeation of aminopeptidase labile xenobiotics. Within the settings of this study the kinetics of metabolism in the viable epidermis of skin is predictable from HaCaT sheets.

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