Primary aldosteronism is a secondary hypertensive disease caused by autonomous aldosterone production that often caused by an aldosterone-producing adenoma (APA). Immunohistochemistry of aldosterone synthase (CYP11B2) shows the presence of aldosterone-producing cell clusters (APCCs) even in non-primary aldosteronism adult adrenal cortex. An APCC-like structure also exists as possible APCC-to-APA transitional lesions (a speculative designation) in primary aldosteronism adrenals. However, whether APCCs produce aldosterone or 18-oxocortisol, a potential serum marker of APA, remains unknown because of lack of technology to visualize adrenocorticosteroids on tissue sections. To address this obstacle, in this study, we used highly sensitive Fourier transform ion cyclotron resonance mass spectrometry to image various adrenocorticosteroids, including 18-oxocortisol, in adrenal tissue sections from 8 primary aldosteronism patients with APCC (cases 1–4), possible APCC-to-APA transitional lesions (case 5), and APA (cases 6–8). Further analyses by tandem mass spectrometry imaging allowed us to differentially visualize aldosterone from cortisone, which share identical mass-to-charge ratio value ( m/z ). In conclusion, these advanced imaging techniques revealed that aldosterone and 18-oxocortisol coaccumulated within CYP11B2-expressing lesions. These imaging outcomes along with a growing body of aldosterone research led us to build a progressive development hypothesis of an aldosterone-producing pathology in the adrenal glands.

Moisturization of the skin plays an important role in maintaining skin homeostasis. Although it is understood that skin dryness initiates the formation of fine wrinkles, there are few objective reports to support that understanding. The purpose of this study was to establish an in vitro dry epidermal model using reconstructed human epidermal equivalents (RHEEs) and to elucidate the relationship between skin dryness and alterations of the dermal matrix which is one of the causes for the formation of wrinkles. An in vitro dry epidermal model was prepared by loading a CaCl2 -filled ampoule on the surface of an RHEE. To evaluate whether the in vitro model reproduced the characteristics of in vivo skin dryness, histological studies and biological assays using a protein array were carried out. Histologically, a distinct fluorescence which originated from carbonylated protein was observed in the stratum corneum. In addition, conditioned medium from RHEEs cultured under dry conditions for 24 h revealed the secretion of several proteins, such as IL-1α, IL-1ra, IL-8, MMP-9, VEGF, M-CSF and IGFBP-2 and IGFBP-3, galectin-1, Cys-C, FGF-6, OPG, Glc and TSP4 compared with normal RHEEs. It has been reported that an increase in IL-1α and the accumulation of carbonylated proteins are observed in the intact stratum corneum in the low humidity winter season. Thus, the in vitro dry epidermal model expresses the features of in vivo skin dryness observed in the winter season. Furthermore, the conditioned medium from RHEEs cultured under dry conditions enhanced MMP-1 secretion by normal human dermal fibroblasts. Taken together, we propose the hypothesis that skin dryness contributes to alterations of the dermal matrix through the elevation of MMP-1 secretion.

Recent molecular biological, chemical, physiological and morphological studies indicate that Tri-chosporon cutaneum and related species should be reclassified. In this study, antigenic characteristics of the species were determined. The results of adsorption experiments revealed that there were at least three serological types: I, II and III. Specific factor sera I, II and III were prepared on the basis of adsorption experiments and isolates were serotyped by cell slide agglutination (CSA). Since the CSA test was difficult to read in some strains, the results of the CSA test were compared with the findings from an enzyme-linked immunosorbent assay (ELISA). For the ELISA, crude polysaccharide antigens prepared from the culture supernatant were used as the antigen. The types determined by ELISA correlated well with those determined by the CSA test. These data suggest that T. cutaneum and related species have at least three serological types, and that the typing can be done by either CSA or ELISA.

Caffeic acid (CA) is a hydrophobic polyphenol with a high antioxidant capacity and γ-cyclodextrin (γ-CD) is a cyclic polysaccharide. The current study prepared a coprecipitate (CP), a freeze-dried (FD) preparation, a ground mixture (GM), and a physical mixture (PM) of CA and γ-CD, and this study then assessed the physicochemical properties and antioxidant capacity of these preparations. PXRD patterns revealed that a PM and a GM prepared at a certain molar ratio (CA/γ-CD =1/1) produced a diffraction peak due to CA crystals. Diffractions peaks characteristic of CA and γ-CD disappeared with the CP, but new peaks were noted. In addition, an FD with CA and γ-CD at a molar ratio of 1/1 produced a halo pattern. DSC measurements revealed that the PM produced an endothermic peak at 220 º C due to the melting of CA, but the endothermic peak due to CA disappeared with the CP, FD, and GM. IR spectra revealed that the absorption peak due to the carbonyl group (C=O) of CA shifted for both the CP and the FD. The absorption peak due to C=C in the aromatic ring of CA also shifted. These findings presumably indicate molecular interaction between CA and γ-CD when the 2 substances are present at a molar ratio of 1/1 (CA/γ-CD). In the GM, molecular interaction presumably occurred as a result of heat. The preparations were compared to CA alone in dissolution testing, which revealed that the CP and FD both had a high rate of dissolution. 1 H-1 H NMR (NOESY) spectra revealed cross peaks involving protons of the γ-CD cavity and protons of the aromatic ring of CA. Thus, the formation of CA and γ-CD inclusion complexes helped to improve the dissolution of CA and γ-CD at a molar ratio of 1/1. The CP and FD had a higher antioxidant capacity than did CA alone. This presumably indicates that the formation of CA and γ-CD inclusion complexes helped to increase the electron density of CA in the CD cavity.

A method for the simultaneous determination of 25-hydroxyvitamin D 3 [25(OH)D 3 ] and its 3-sulfate [25(OH)D 3 S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. e plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). e derivatization enabled the accurate quanti cation of 25(OH)D 3 without interference from 3-epi-25(OH)D 3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quanti cation was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. is method was reproducible (intra-and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4-105.6%). e limits of quanti cation were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D 3 and 25(OH)D 3 S, respectively, when using a 20-µL sample. e developed method was applied to the simultaneous determination of plasma 25(OH)D 3 and 25(OH)D 3 S in newborns; it was recognized that the plasma concentration of 25(OH)D 3 S is signi cantly higher than that of 25(OH)D 3 , and preterm newborns have lower plasma 25(OH)D 3 S concentrations than full-term newborns.

Background/Aims: Advanced glycation end products, which are linked to both aging and hyperglycemia, cause marked functional and structural alterations in human skin. Though it is well known that the metabolism of glucose is closely associated with that of fatty acid (FA), sharing the same energy-yielding reaction pathways as glucose, its effect on the epidermis has been unclear so far. Methods: Content of ceramides, cholesterol and FA in a reconstructed epidermal model glycated by glyoxal was analyzed by high-performance thin-layer chromatography. FA species extracted from HaCaT keratinocytes was determined by gas chromatography/mass spectrometry. Regulation of FA synthesis was analyzed by real-time PCR. For physiological analysis, excised mouse skin was glycated using a vertical diffusion cell and used for the evaluation of barrier function by transepidermal water loss measurement and observation of penetration of sodium fluorescein. Results: Saturated FA content was significantly increased in glycated epidermis, and glycation upregulated mRNA expression of FA elongases 2 and 3 and FA synthase in HaCaT cells. Further, both inside-out and outside-in barriers were disrupted in glycated excised skin. Conclusion: Biological and physical change in the epidermis, especially upregulation of FA synthesis by glycation, contributed to barrier disruption, and inhibiting glycation may offer an effective treatment option for aged or glycated skin.

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