Elevated amounts of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are observed in the epidermis in different skin disorders. Thus, epidermal sensing of H2O2 should be useful to monitor the progression of skin pathologies. We have evaluated epidermal sensing of H2O2 in vitro, by visualising H2O2 permeation through the skin. Skin membranes were mounted in Franz cells, and a suspension of Prussian white microparticles was deposited on the stratum corneum face of the skin. Upon H2O2 permeation, Prussian white was oxidised to Prussian blue, resulting in a pattern of blue dots. Comparison of skin surface images with the dot patterns revealed that about 74% of the blue dots were associated with hair shafts. The degree of the Prussian white to Prussian blue conversion strongly correlated with the reciprocal resistance of the skin membranes. Together, the results demonstrate that hair follicles are the major pathways of H2O2 transdermal penetration. The study recommends that the development of H2O2 monitoring on skin should aim for pathway-specific epidermal sensing, allowing micrometre resolution to detect and quantify this ROS biomarker at hair follicles. Graphical abstract

BACKGROUND: Interferon-gamma (IFN-γ) represents a potent inducer for keratinocyte inflammatory and immune activation in vitro. Since tryptophan (trp) conversion to kynurenine (kyn) is involved in inflammation, the topical kyn/trp ratio may serve as a biomarker of skin inflammation. However, the trp metabolism in keratinocytes exposed to IFN-γ is not yet fully understood.

OBJECTIVE: The aim of this study was to establish a human epidermis model in order to quantify cytokine and kyn/trp secretion from IFN-γ stimulated cells and tissues. Moreover, to compare the cell response of 2D-cultured keratinocytes and the 3D epidermis model.

METHODS: Polycarbonate filters were used on which primary keratinocytes could attach and stratify in order to form the typical layers of reconstructed human epidermis (RHE). After IFN-γ treatment, secretion of kyn/trp was measured by high performance liquid chromatography. Gene and protein expression of indoleamine 2,3-dioxygenase 1 (IDO) was analyzed with real-time PCR and immunohistochemistry. The secretion of cytokines was quantified with ELISA.

RESULTS: Trp catabolism to kyn was significantly increased (P < 0.01) in the 2D culture in response to IFN-γ treatment. Before kyn secretion, IDO was strongly upregulated (P < 0.001). IFN-γ treatment also increased the secretion of IL-6 and IL-8 from the keratinocytes. In the RHE, IDO was upregulated by IFN-γ, and kyn secretion could be detected. Interestingly, IDO expression was only present in the basal cells of the RHE.

CONCLUSION: Our results suggest that IFN-γ acts as an inducer of trp degradation preferentially in undifferentiated keratinocytes, indicated by the IDO expression in the basal layer of the RHE.

The tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

Monitoring of low-molecular weight cancer biomarkers, such as tryptophan (Trp) and its derivative kynurenine (Kyn), might be advantageous to non-invasive skin cancer detection. Thus, we assessed several approaches of topical sampling of Trp and Kyn, in relation to phenylalanine (Phe) and tyrosine (Tyr), on the volar forearm of six healthy volunteers. The sampling was performed with three hydrogels (made of agarose or/and chitosan), hydrated starch films, cotton swabs, and tape stripping. The biomarkers were successfully sampled by all approaches, but the amount of collected Kyn was low, 20 ± 10 pmol/cm2. Kyn quantification was below LOQ, and thus, it was detected only in 20% of topical samples. To mitigate variability problems of absolute amounts of sampled amino acids, Tyr/Trp, Phe/Trp, and Phe/Tyr ratios were assessed, proving reduced inter-individual variation from 79 to 45% and intra-individual variation from 42 to 21%. Strong positive correlation was found between Phe and Trp, pointing to the Phe/Trp ratio (being in the 1.0–2.0 range, at 95% confidence) being least dependent on sampling materials, approaches, and sweating. This study leads to conclusion that due to the difficulty in quantifying less abundant Kyn, and thus the Trp/Kyn ratio, the Phe/Trp ratio might be a possible, alternative biomarker for detecting skin cancers.

The molecular composition of human skin is altered due to diseases, which can be utilized for non-invasive sampling of biomarkers and disease diagnostics. For this to succeed, it is crucial to identify a sampling formulation with high extraction efficiency and reproducibility. Highly hydrated skin is expected to be optimal for increased diffusion of low-molecular-weight biomarkers, enabling efficient extraction as well as enhanced reproducibility as full hydration represents a well-defined endpoint. Here, the aim was to explore water-based formulations with high water activities, ensuring satisfactory skin hydration, for non-invasive sampling of four analytes that may serve as potential biomarkers, namely tryptophan, tyrosine, phenylalanine, and kynurenine. The included formulations consisted of two hydrogels (chitosan and agarose) and two different liquid crystalline cubic phases based on the polar lipid glycerol monooleate, which were all topically applied for 2 h on 35 healthy subjects in vivo. The skin status of all sampling sites was assessed by electrical impedance spectroscopy and transepidermal water loss, enabling explorative correlations between biophysical properties and analyte abundancies. Taken together, all formulations resulted in the successful and reproducible collection of the investigated biomarkers. Still, the cubic phases had an extraction capacity that was approximately two times higher compared to the hydrogels.