Natural moisturising factor constituents in South African nursing students.

BACKGROUND: The majority of South African healthcare workers are Black Africans with dark-pigmented skin. Studies on how the markers of skin barrier function and natural moisturising factor (NMF) compare between dark and light-pigmented skin are limited. Quantifying NMF in a nursing student population during their practical training at university may provide valuable insight into their potential susceptibility to skin conditions associated with low NMF. OBJECTIVES: The objectives of this study were to quantify and compare NMF content of Black African, Mixed Race and White nursing students from their dominant dorsal hand. METHODS: Forty-nine White, 32 Black African and 5 Mixed Race nursing students participated in this study. Tape strip samples were collected from the participants' dominant dorsal hand and NMF content was measured, including histidine (HIS), pyrrolidone carboxylic acid (PCA), trans-urocanic acid (t-UCA) and cis-urocanic acid (c-UCA), as well as cytokines interleukin-1 alpha (IL-1α) and interleukin-1 receptor antagonist (IL-1RA). RESULTS: No statistically significant differences in PCA, t-UCA, c-UCA, IL-1α or IL-1RA were found between Black African and White nursing students. HIS was significantly (p = 0.001) higher in White nursing students when compared to Black African students. The ratio of tot-UCA/HIS was significantly higher in Black Africans (p = 0.0002) when compared to White nursing students. CONCLUSION: No significant differences were established in NMF content between White and Black African nursing students, other than HIS which was significantly higher in White students than in Black African students. Different HIS levels between the racial groups suggest variation in histidase activity which may be related to skin pH and pigmentation. This finding may suggest that nursing students at the beginning of their careers may have similar susceptibility to skin diseases related to NMF.

Analytical tools for urocanic acid determination in human samples: A review.

Urocanic acid is a chromophore found in the skin that has been identified as an important immunosuppressant and carcinogenesis mediator through its photoisomerization from trans to cis form induced by ultraviolet radiation. Research on analytical methods that explore urocanic acid isomerization is indispensable to fully understand the deleterious effects mediated by this biomarker. In this context, the current relevant analytical methods for determination of these isomers in human samples are summarized in this review. The methods presented here are applicable to human samples collected by noninvasive methods (or minimally invasive), encompassing an array of analytical techniques, including high-performance capillary electrophoresis, confocal Raman spectroscopy, gas chromatography, high-performance liquid chromatography, and mass spectrometry, among others. Developed high-performance liquid chromatography methods have proven to be advantageous, allowing noninvasive collections for in vivo analysis and the confocal Raman, specially, for real-time analysis. Among all these methods, high-performance liquid chromatography is the most investigated one with mass spectrometry or ultraviolet detector, and the mass spectrometry detector being the most studied in the last years, demonstrating high sensitivity, very low detection limits, and accurate identification, especially for clinical investigations.

Handheld confocal Raman spectroscopy (CRS) for objective assessment of skin barrier function and stratification of severity in atopic dermatitis (AD) patients.

BACKGROUND: We developed the first-of-its-kind handheld confocal Raman spectroscopy (CRS) system to quantify the concentration of natural moisturizing factors in the skin. OBJECTIVE: To evaluate the feasibility of our handheld CRS system and propose a novel quantitative index to measure skin barrier function. METHODS: This prospective study included 30 atopic dermatitis (AD) patients and 14 healthy volunteers. All AD participants were assessed using the Scoring Atopic Dermatitis (SCORAD) severity instrument, a vapometer for trans-epidermal water loss and a moisture meter for skin surface moisture. A handheld CRS operating at 785 nm laser was used to measure the biochemical constituents of the skin up to a depth of ∼100 µm. We trained a linear kernel-based support vector machine (SVM) model for eczema classification based on the water, ceramide and urocanic acid content. A novel Eczema Biochemical Index (EBI) was then formulated using the skin constituents measured from the AD participants to stage disease severity. RESULTS: The SVM model used to classify healthy participants and AD patients obtained high cross-validated area under the curve of 0.857 and accuracy of 0.841, with high sensitivity and specificity values of 0.857 and 0.833 respectively. EBI can be used to stratify AD patients of varying severity, based on the biochemical constituents in the skin. CONCLUSION: As compared to the standard CRS system, the handheld CRS offers higher portability and provides Raman measurements at various body regions with similar sensitivity. This suggests that a handheld CRS device could be a valuable point-of-care resource in both research and clinical use.

Development of an analytical method for urocanic acid isomers in fish based on reactive extraction cleanup and chaotropic chromatography techniques.

Urocanic acid (UA), existing in trans- or cis-isoform, is of fairly recent interest to food researchers because of its potential public health hazards of scombrotoxicity and immunotoxicity, as well as associating with fish spoilage. This work is among the first efforts to study the analytical chemistry of UA in fish. With 0.6 M perchloric acid UA was extracted, and co-extracted fish matrix components were efficiently removed through a reactive extraction of UA. The optimum conditions for the reactive extraction, which allowed an 80% recovery of UA, were sample pH adjustment to 9, twice extractions with 32% (w/w) di (2-ethylhexyl) phosphate in hexanol, and a back-extraction with 0.1 M hydrochloric acid at 1:1 phase ratio. A chaotropic hexafluorophosphate salt was added to acidic water-acetonitrile mobile phases to improve the reversed-phase chromatography of UA, which otherwise was poorly retained. Optimum separation conditions were obtained for fish samples and enabled a fast (10 min), convenient-to-use chromatography that clearly outperforms cumbersome legacy ion-pair chromatography. Intended for routine use in our laboratory, the proposed method passed an in-house validation test for linearity, matrix effect (on reactive extraction), accuracy, precision, and detectability.

Determination of trans- and cis-urocanic acid in relation to histamine, putrescine, and cadaverine contents in tuna (Auxis Thazard) at different storage temperatures.

Scombroid fish poisoning is usually associated with consumption of fish containing high levels of histamine. However, reports indicate that some cases have responded to antihistamine therapy while ingested histamine levels in these cases were low. Potentiation of histamine toxicity by some biogenic amines, and release of endogenous histamine by other compounds such as cis-urocanic acid (UCA) are some hypotheses that have been put forth to explain this anomaly. Very little is known about the effects of storage conditions on the production of both UCA isomers and biogenic amines in tuna. Thus, the production of trans- and cis-UCA, histamine, putrescine, and cadaverine in tuna during 15 d of storage at 0, 3, and 10 °C and 2 d storage at ambient temperature were monitored. The initial trans- and cis-UCA contents in fresh tuna were 2.90 and 1.47 mg/kg, respectively, whereas the levels of putrescine and cadaverine were less than 2 mg/kg, and histamine was not detected. The highest levels of trans- and cis-UCA were obtained during 15 d storage at 3 °C (23.74 and 21.79 mg/kg, respectively) while the highest concentrations of histamine (2796 mg/kg), putrescine (220.32 mg/kg) and cadaverine (1045.20 mg/kg) were obtained during storage at room temperature, 10 and 10 °C, respectively. Histamine content increased considerably during storage at 10 °C whereas trans- and cis-UCA contents changed slightly. The initial trans-UCA content decreased during storage at ambient temperature. Thus, unlike histamine, concentrations of trans- and cis-UCA did not result in elevated levels during storage of tuna.

Changes in urocanic acid, histamine, putrescine and cadaverine levels in Indian mackerel (Rastrelliger kanagurta) during storage at different temperatures.

Histamine, putrescine cadaverine and cis-urocanic acid (UCA) have all been implicated or suggested in scombroid fish poisoning. However, there is little information on UCA especially during storage. Changes in their contents during storage of whole Indian mackerel at 0, 3±1, 10±1 for up to 15 days and 23±2°C for up to 2 days were monitored. Fresh muscles contained 14.83 mg/kg trans-UCA, 2.23 mg/kg cis-UCA and 1.86 mg/kg cadaverine. Histamine and putrescine were not detected. After 15 days at 0 and 3°C, trans-UCA content increased to 52.83 and 189.51 mg/kg, respectively, and decreased to <2 mg/kg at the other two temperatures. Storage at 10°C also resulted in an increase in trans-UCA after 3 days, only to decrease after 6 days. The concentration of cis-UCA increased nearly 13-fold after 15 days at 0 and 3°C, decreased at 10°C and remained unchanged at 23°C. Histamine, putrescine and cadaverine levels increased significantly (P value<0.05) at all temperatures especially at 23°C.

Development and validation of an ion-pair chromatographic method for simultaneous determination of trans- and cis-urocanic acid in fish samples.

Urocanic acid (UCA) has been reported to be a mast cell degranulator and has also been suggested as a complementary agent in implicated scombroid fish poisoning. In this research, a new method is described to extract, clean up and perform simultaneous ion-pair chromatographic analysis of trans- and cis-urocanic acid (UCA) in fish samples. UCA was extracted using 0.05 M HCl and protein was removed from the extract by precipitation with 10% trisodium citrate and 10% citric acid. The HPLC method that is developed showed a rapid, precise and sensitive method with short retention time for simultaneous separation of UCA isomers in fish samples. Estimation of trans- and cis-UCA in the muscle of Indian mackerel, tuna and sardine showed that, as expected, no cis-UCA existed in fish muscles and the highest concentration of trans-UCA was found in Indian mackerel with 118.8 mg kg(-1) while the highest concentrations of trans-UCA in tuna and sardine were 12.1 and 17.5 mg kg(-1), respectively.

Rapid, simultaneous and nanomolar determination of pyroglutamic acid and cis-/trans-urocanic acid in human stratum corneum by hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization tandem mass spectrometry.

A rapid, sensitive and specific hydrophilic interaction liquid chromatography coupled to tandem mass spectrometric (HILIC-MS/MS) method for the simultaneous determination of pyroglutamic acid, cis- and trans-urocanic acid in human skin stratum corneum (SC) were developed and validated. This method was carried out without derivatization or addition of ion-pair additives in mobile phase. The analytes were extracted by PBS buffer solution and analyzed using an electrospray positive ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. Chromatographic separation was performed on an AQUITY UPLC amide column using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 1.0-250 ng/mL with a correlation coefficient higher than 0.999 with an LLOQ of 0.5 ng/mL. The lower limits of detection (LLOD) of these analytes were lower than 0.2 ng/mL. The intra- and inter-day precisions were measured to be below 7.7% and accuracies were within the range of 94.3-102.6%. The validated method was successfully applied to determine the level of pyroglutamic acid and cis-/trans-urocanic acid in the SC samples from forearm and forehead region of 19 human volunteers.

Characterizing the composition of underarm and forearm skin using confocal raman spectroscopy.

Many studies have reported differences in skin composition as a function of body site and age. However, rarely has axilla (underarm) skin been included in these studies. This report highlights the results of a clinical study where confocal Raman spectroscopy was used to assess the differences between underarm and volar forearm skin. In the reported study, the concentration of natural moisturizing factor (NMF), urea, lactic acid, cholesterol, trans urocanic acid (t-UCA) and ceramide 3 (N-octadecanoyl-phytosphingosine) was evaluated as a function of depth, body site (underarm vs. forearm) and age. Two age groups of women were included: (i) between 20 and 30 years and (ii) older than 55 years (post-menopause). The levels of cholesterol, ceramide 3 and lactic acid were highest in the underarm regardless of the age group whereas the forearm contained higher amounts of NMF. Ceramide 3, urea and lactic acid were significantly lower for women 55 years or older in the underarm. No age-related differences were detected within the forearm site..

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. OBJECTIVES To investigate the feasibility of different sampling methods for the determination of two NMF components, 2-pyrrolidone-5-carboxylic acid (PCA) and urocanic acid (UCA), in the stratum corneum as biomarkers for the FLG genotype. METHODS: PCA and UCA from the stratum corneum were sampled by using a tape stripping technique and an extraction technique using skin patches containing potassium hydroxide (KOH). The concentrations of PCA and UCA were measured by high-performance liquid chromatography. Eleven carriers of an FLG mutation and 10 individuals wild type for the two most common FLG mutations (R501X and R2447X) [corrected] were included in the study. RESULTS: The most significant difference between the FLG genotypes was found for PCA sampled by the tape stripping technique. The mean values of PCA obtained by the tape stripping technique were, respectively, 0.18, 0.50 and 1.64 mmol g(-1) protein in homozygous (or compound heterozygous), heterozygous and wild-type genotypes (P < 0.005 homozygous vs. heterozygous; P < 0.0001 heterozygous vs. wild type). The tape stripping technique showed less intrasubject variation compared with the KOH patches, in particular when the concentrations of UCA and PCA on the tape strips were normalized for protein amount. CONCLUSIONS: The concentration of PCA in the stratum corneum collected by tape stripping showed it to be a feasible biomarker of the FLG genotype.

In vivo evaluation of the protective capacity of sunscreen by monitoring urocanic acid isomer in the stratum corneum using Raman spectroscopy.

BACKGROUND/PURPOSE: Urocanic acid (UCA) is a major ultraviolet (UV) ray-absorbing component in the epidermis, and it isomerizes from the trans to the cis form upon exposure to UV radiation. Continuous measurement of UCA isomers in the skin at the same area is not available using conventional methods. This study aimed to evaluate the protective capacity of sunscreen by non-invasively monitoring the trans-UCA (t-UCA) amount in the stratum corneum (SC) by confocal Raman spectroscopy. METHODS: In vivo Raman spectra of the skin at the cheek or volar forearm were obtained from 27 healthy Japanese volunteers of different ages (age range, 22-53 years) throughout a whole year using confocal Raman spectroscopy. Eighteen healthy male Japanese volunteers (age range, 25-52 years) were enrolled for the evaluation of the protective capacity of sunscreen. The concentration depth profile of t-UCA relative to keratin was calculated from the Raman spectra in the 400-2200 cm(-1) region. Then, the integrated amount within the depth of 0-12 mum was calculated, which represented the total amount of t-UCA in the SC. RESULTS: The integrated amount of t-UCA in the cheek skin was significantly lower than that in the volar forearm skin throughout a year. The amount of it in the volar forearm skin was significantly the lowest in summer, but not in the cheek skin. The amount of t-UCA decreased immediately after UV exposure even below 1 minimal erythema dose, remained low for 1 week, and gradually increased up to the initial level about 2 weeks after UV exposure. The decrease in the t-UCA amount was hindered by the application of sunscreen on the skin surface. There were no statistical differences in response to UV exposure between the erythema-positive and erythema-negative groups. CONCLUSIONS: The monitoring of the amount of t-UCA in the SC by confocal Raman spectroscopy is a good method to assess the efficacy of sun protective substances.

Interference-based electrochemical biosensor for the measurement of the concentration and isomer ratio of urocanic acid.

Urocanic acid (UCA) is known to be synthesized as the trans isomer (trans-UCA) in the skin, and trans-UCA is transformed by UV light to the cis isomer (cis-UCA), which may be involved in photoimmunosuppression. An electrochemical method has been developed for the measurement of the concentration and isomer ratio of UCA. A heme peptide-modified electrode (HP electrode) reduces hydrogen peroxide at +150 mV vs Ag/AgCl, and the reduction current is inhibited by UCA. Since cis-UCA is a stronger inhibitor than trans-UCA, irradiation of a sample solution with UV light increases the percent inhibition. The concentration and isomer ratio of UCA in the sample solution can be estimated from the values of percent inhibition before and after the UV irradiation.

Studies to determine the immunomodulating effects of cis-urocanic acid.

Exposure to ultraviolet (UV) radiation, particularly the UVB wavelengths, leads not only to DNA damage but also to suppression of cell-mediated immunity to antigens encountered shortly after the irradiation. One initiator of this complex process is cis-urocanic acid (cis-UCA), which is formed from the naturally occurring trans isomer in the epidermis on absorption of UV. cis-UCA has been shown to have immunomodulating properties in a variety of in vivo and in vitro experimental systems, although its mechanism of action is not yet clear. This article covers methods of preparing cis-UCA and of analyzing UCA isomers in various human and mouse tissues. Experiments that demonstrate that cis-UCA is immunosuppressive are described. The final section deals with the preparation and characterization of a monoclonal antibody with specificity for cis-UCA.

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 in patients with non-melanoma skin cancer.

BACKGROUND: Cis-urocanic acid (cis-UCA), formed from the naturally occurring trans-isomer in the epidermis on ultraviolet (UV) radiation, initiates some of the changes leading to UV-induced immunosuppression, but its role in cutaneous carcinogenesis has not been fully investigated. OBJECTIVES: To measure the concentration of UCA isomers in the photoexposed and non-photoexposed skin of patients with multiple non-melanoma skin cancer (NMSC), enrolled in different periods of the year, in comparison with control subjects. PATIENTS/METHODS: UCA isomers were determined by high-performance liquid chromatography analysis in samples from the outer arm (photoexposed site) and buttock (non-photoexposed site) obtained from 20 patients and 19 controls during the winter period (October to April), and from five patients and 11 controls during the summer period (June to September). RESULTS: In the winter months, no difference was found between patients and controls in the concentration of UCA or the percentage of cis-UCA in either site. In the summer months, the percentage of cis-UCA in the buttock of patients and controls was similar but it was significantly higher in the arm of the controls (42%) than in the patients (17%). CONCLUSIONS: We conclude that different behaviour regarding sun exposure is the most likely explanation for these results, and that the concentration of UCA and its isomers does not reflect a tendency for individuals to develop NMSC.

Determination of histidine and urocanic acid isomers in the human skin by high-performance capillary electrophoresis.

Histidine was baseline separated from histamine, 1-methylhistamine and cis- and trans-urocanic acid using high-performance capillary electrophoresis (HPCE) on a fused-silica column (50 cm x 75 microm) with 0.05 M NaH2PO4 buffer, pH 5.0, and 12 kV. The detection limit of histidine, trans- and cis-urocanic acid was 10(-6) M at a wavelength of 214 nm. The detection limit of the urocanic acid isomers was slightly enhanced to 5 x 10(-7) M at 267 nm. The transformation of the trans-urocanic acid standard in vitro into the cis-isomer was dependent on the time of exposure and the energy of the light source. UVB light induced a significantly faster conversion than UVA light. The HPCE method was used for the characterization and measurement of histidine and urocanic acid in human skin eluates. The concentrations of histidine, trans- or cis-urocanic acid in ethanol washes from the skin of healthy, non-allergic volunteers were 2.22+/-0.40 x 10(-5) 0.96+/-0.26 x 10(-5) and 1.04+/-0.30 x 10(-5) M, respectively, (mean+/-SEM, n=8). The results obtained by HPCE correlated well with data obtained by HPLC. Correlation coefficients of r2=0.981, r2=0.814 and r2=0.956 were found for histidine, trans- and cis-urocanic acid, respectively.

Photobiology of Monodelphis domestica.

The gray, short-tailed opossum, Monodelphis domestica, has been used for photobiologic studies since 1984. The presence of a light-activated DNA repair pathway in the tissues of Monodelphis has been used to identify pyrimidine dimers in DNA as initiating events for a number of ultraviolet radiation (UVR)-induced pathologies of the skin and cornea. Furthermore, Monodelphis, unlike common laboratory rodents, is susceptible to the induction of melanoma by UVR alone.

Determination of histidine and related compounds in rumen fluid by liquid chromatography.

A liquid chromatographic procedure was developed for quantitative determination of histidine (His), histidinol (HDL), histamine (HTM), urocanic acid (URA), imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) in rumen fluid. The method is based on direct injection analysis by UV absorbance detection at 220 nm. The separation was performed under 2 different chromatographic conditions on a LiChrospher 100 NH2 column. In the first chromatographic system, the mobile phase used for isocratic elution was 67 mM potassium phosphate buffer (monobasic and dibasic) pH 6.45-90% acetonitrile in water (21 + 79); in the second system, an acetonitrile gradient in 63 mM potassium phosphate buffer (monobasic) pH 3.0, obtained by addition of 60 mM phosphoric acid, was used. Analyses of both systems were completed within 32 and 25 min, respectively. The limits of detection of these compounds were (microM): His, 2.8; HDL, 3.7; HTM, 4.0; URA, 0.75; ImPA, 4.7; ImAA, 1.2; and ImLA, 1.3. Recovery of these compounds added to rumen fluid was 97.4-103.0% within a 1-day study and 95.4-99.0% on different day studies. Detectable levels of His were found in the deproteinized rumen fluid of goats, with average concentrations of 16.10, 10.43, 11.14, and 13.62 microM in the rumen fluid collected before the morning feeding and 2, 4, and 6 h after feeding, respectively. HDL, HTM, URA, ImPA, ImAA, and ImLA were not detected in the rumen fluid before and after feeding. Trp, Phe, and Tyr were also identified in the rumen fluid, with average concentrations of 8.25, 29.04, and 12.6 microM, respectively, before the morning feeding.