Arthur Kammeyer

Natural moisturizing factor components in the stratum corneum as biomarkers of filaggrin genotype: evaluation of minimally invasive methods

Background  The carriers of loss‐of‐function mutations in the filaggrin gene (FLG) have reduced levels of natural moisturizing factor (NMF) in the stratum corneum. The concentration of NMF components which are formed by filaggrin protein breakdown in the stratum corneum might therefore be useful as a biomarker of the FLG genotype.

Increased Sensitivity of Histidinemic Mice to UVB Radiation Suggests a Crucial Role of Endogenous Urocanic Acid in Photoprotection

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Photoisomerization spectrum of urocanic acid in human skin and in vitro: effects of simulated solar and artificial ultraviolet radiation

Summary Ultraviolet (UV) irradiation of trans‐urocanic acid (UCA), a major UV absorbing component of the epidermis, leads to the formation of cis‐UCA, which mediates immunosuppressive effects. In this study, the net yield of cis‐UCA was measured after the photoisomerization of urocanic acid by narrow UV wavebands (spectral range 295–405 nm), with the irradiation doses related to solar irradiance at sea level. The formation of cis‐UCA in Caucasian skin (in vivo), as well as in aqueous solution (in vitro), was determined by HPLC analysis. The same irradiation conditions were met in both components of the study. The in vivo experiments showed high efficiency of cis‐UCA formation in the spectral region of 305–341 nm, whereas high efficiency in vitro was found at 305 and 326 nm. At 350 and 363 nm, cis‐UCA was formed in vivo, but not in vitro. At longer test wavelengths up to 405 nm. no significant formation of cis‐UCA was detectable. The established partition between UVB and UVA at 320 nm is not relevant for the isomerization pattern of UCA. Additional studies revealed substantial cis‐UCA formation in human skin by UVA phototherapy lamps. Furthermore, raised levels of 295 nm irradiation doses, a possible effect of stratospheric ozone depletion, were found to increase the cis‐UCA yield. Our results demonstrate that the formation of cis‐UCA in the skin with common exposures takes place over a broad spectrum range of UVB and UVA, up to at least 363 nm. These findings emphasize the potency of UVA to isomerize UCA, and they may contribute to further elucidation of the effects of phototherapy and sunbathing.

Prolonged Increase of cis-Urocanic Acid Levels in Human Skin and Urine after Single Total-body Ultraviolet Exposures

Cis-urocanic acid (cis-UCA), a mediator of immunosuppression, is formed from trans-UCA upon UV-exposure of the skin. This study describes a liquid chromatographic method for the simultaneous quantification of cis- and trans-UCA in skin, urine and plasma of nonirradiated volunteers. It also describes cis- and trans-UCA kinetics in UV-irradiated volunteers. New procedures to remove interfering substances from urine and plasma are reported. Normal levels of cis-UCA in skin, urine and plasma of nonirradiated volunteers were 0.5 nmol/cm2, 0.03 mumol/mmol creatinine (median 0.00) and undetectable and those of trans-UCA were 17.1 nmol/cm2, 1.36 mumol/ mmol creatinine and 0.5 microM, respectively. Upon single total body UVB (290-320 nm) exposures of 250 J/m2, epidermal cis-UCA levels immediately reached a maximum and returned to basic levels 3 weeks later. The cis-UCA levels in urine reached a maximum in 5-12 h postirradiation and reached baseline values in 8-12 days. Additionally, a single total body UVA (320-400 nm) irradiation of 200 kJ/m2 yielded a similar pattern. The kinetics of cis-UCA in plasma could not be followed due to low concentrations; however, that of skin and urine was informative in relation to solar exposures and phototherapy.

Determination of catechol O-methyltransferase activity in relation to melanin metabolism using high-performance liquid chromatography with fluorimetric detection

A new sensitive method for the determination of catechol O-methyltransferase activity has been developed. The method is based on the O-methylation of the indolic intermediates of melanin metabolism. The substrate, 5,6-dihydroxyindole-2-carboxylic acid, is converted by the enzyme to two O-methylated products, which can be separated by high-performance liquid chromatography and measured with fluorimetric detection. The physiological presence of both substrate and products could be detected in crude melanoma cell extracts. The limit of sensitivity for detection of the O-methylated products is less than 0.5 pmol per injection. The method was compared with an earlier described HPLC method which makes use of uv detection of O-methylated products of 3,4-dihydroxybenzoic acid. The described method will be used to study the importance of catechol O-methyltransferase as a protective enzyme in (malignant) melanocytes.

Epidermal cis-Urocanic Acid Levels Correlate with Lower Specific Cellular Immune Responses After Hepatitis B Vaccination of Ultraviolet B–exposed Humans¶

Abstract Urocanic acid (UCA) is a major UV-absorbing chromophore in the epidermis and has been suggested to act as one of the initiators of UV-induced immunosuppression. cis-UCA, the isomer from UCA that is formed upon UV exposure, has been shown to impair some cellular immune responses. cis-UCA levels were determined in a study in which the influence of ultraviolet B (UVB) exposure on immune responses after hepatitis B vaccination in human volunteers was established. A significant increase in cis-UCA levels was found in the skin of UVB-exposed volunteers compared with controls. cis-UCA levels, calculated as the percentage of the total UCA amount, in UVB-exposed volunteers correlated significantly with the cumulative UVB dose received in 5 consecutive days, i.e. the higher the UVB dose (J/m2), the higher the cis-UCA levels (until a cis-UCA plateau was reached in the so-called photostationary state). Correlations between skin cis-UCA levels and immune responses were determined, and they revealed no statistically significant correlations among lymphocyte proliferation responses after either mitogenic stimulation or stimulation with recall antigens. No correlation was found between cis-UCA levels and hepatitis B–specific antibody titers. However, we found a statistically significant negative correlation between cis-UCA levels and hepatitis B–specific lymphocyte proliferation responses when volunteers were irradiated with UVB before hepatitis B vaccination. In other words, volunteers with high cis-UCA levels caused by UVB exposure showed lower cellular immune responses against hepatitis B antigen after hepatitis B vaccination.

Evaluation of an HPLC Method for the Determination of Natural Moisturizing Factors in the Human Stratum Corneum

The levels of natural moisturizing factors in the skin can be used as a biomarker of hydration, for studying the effect of skin irritants, or as a biomarker of loss-of-function mutations in the filaggrin gene which are the main risk factor for atopic dermatitis. In this study the chromatographic performance and recovery of natural moisturizing factors and proteins from the skin were investigated using different extraction solvents and adhesive tapes. The uppermost layer of the skin stratum corneum, collected by using commercially available D-squame and corneofix adhesive tapes, was extracted by ammonia or potassium hydroxide. Protein levels used to correct for a variable stratum corneum amount on a tape were assessed by measuring optical density of a tape or indirectly by measuring proteins by a spectrophotometric assay. The measured natural moisturizing factors, pyrrolidone-5-carboxylic acid, histidine, tyrosine, trans-urocanic acid, and cis-urocanic acid were determined by ion pair reverse phase HPLC. Sample preparation and chromatographic performance were favorable when ammonia was used as an extraction solvent. Extraction of the natural moisturizing factors with ammonia avoids a time consuming neutralization step as required with extraction procedures using strong base or acid. The only drawback of the ammonia method is incomplete extraction of proteins from the tapes; however this can be avoided by measuring the optical density of stratum corneum-loaded tapes. The sensitivity of the method was sufficiently high to quantify the analytes even in homozygous filaggrin gene carriers. Reduced natural moisturizing factors levels found in the individuals with filaggrin gene mutation or after exposure to a skin irritant sodium lauryl sulfate were consistent with the previously reported studies.

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.

Urocanic acid isomers are good hydroxyl radical scavengers: a comparative study with structural analogues and with uric acid

UV-exposure of the epidermis leads to the isomerisation of trans-UCA into cis-UCA as well as to the generation of hydroxyl radicals. This study shows by means of the deoxyribose degradation test that UCA isomers are more powerful hydroxyl radical scavengers than the other 4-(5-)substituted imidazole derivatives, such as histidine, though less powerful than uric acid. UCA, present in relatively high concentrations in the epidermis, may well be a major natural hydroxyl radical scavenger.

Postelicitation model of allergic contact dermatitis for predicting the efficacy of topical drugs

Abstract:  To evaluate the anti‐inflammatory efficacies of topical drugs, models of contact hypersensitivity (CHS) can be used, but the conventional murine models of CHS need revision in this respect. These models utilize sensitized mice to study suppression of sensitization or elicitation by test compounds. To mimick the events occurring in allergic contact dermatitis (ACD), a modification of the murine model of CHS is needed in a way that a chronic postelicitation phase of CHS is maintained for studies of anti‐inflammatory effects of topical drugs, typically relevant for ACD therapy, not for ACD prevention. A method for the quantification of the suppression of ACD by a test compound is presented here. Two experimental drugs for topical use, imidazole‐4‐carboxylate and imidazole‐4‐acetate, were tested in parallel with the corticosteroid prednisolone. We found that prednisolone showed strong suppressive effects, while imidazole‐4‐carboxylate and imidazole‐4‐acetate showed mild suppressive effects during persistent ACD simulation. Multiple elicitations on the mouse ears led to scratching and the formation of abrasions and scabbings with, presumably, worsening of discomfort. Clear reduction of these side‐phenomena was achieved by tailoring the topical amount of contact sensitizer, while the ability of the ACD model to test anti‐inflammatory compounds, was not affected. By focussing on a prolonged postelicitation phase of CHS, a simulation of ACD has been established. We demonstrated that this model may provide an improved predictability for the clinical efficacies of (experimental) mild or strong anti‐inflammatory drugs.