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Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and waterholding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normalphase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile saltcontaining polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESItandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of ahydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C 18 )-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.-Masukawa, Y

Melanin in the epidermis determines the wide variation in skin color associated with ethnic skin diversity. Ethnic differences exist regarding melanosome loss in keratinocytes, but the mechanisms underlying these differences, and their contribution to the regulation of skin color, remain unclear. Here, we explored the involvement of autophagy in determining skin color by regulating melanosome degradation in keratinocytes. Keratinocytes derived from Caucasian skin exhibit higher autophagic activity than those derived from African American (AA) skin. Furthermore, along with the higher autophagy activity in Caucasian skin-derived keratinocytes compared with AA skin-derived keratinocytes, Caucasian skin-derived keratinocytes were more sensitive to melanosome treatment as shown by their enhanced autophagic activity, which may reflect the substantial mechanisms in the human epidermis owing to the limitations of the models. Melanosome accumulation in keratinocytes was accelerated by treatment with lysosomal inhibitors or with small interfering RNAs specific for autophagy-related proteins, which are essential for autophagy. Furthermore, consistent with the alterations in skin appearance, the melanin levels in human skin cultured ex vivo and in human skin substitutes in vitro were substantially diminished by activators of autophagy and enhanced by the inhibitors. Taken together, our data reveal that autophagy has a pivotal role in skin color determination by regulating melanosome degradation in keratinocytes, and thereby contributes to the ethnic diversity of skin color.

This article is available online at http://www.jlr.org novo pathways, such as alterations of some species of the targeted lipidomes into others. Similar to common quantitative analyses using direct MS or LC-MS that need authentic standards together with internal ones, quantitative lipidomics may also require a standard set of all lipid species targeted, because each lipid species has a different molar response in MS detection except for special cases. However, it is practically impossible to obtain all authentic species, including not only different even straight carbon chains but also odd and/or branched ones. To overcome such limitations, lipidomics researchers have contrived a novel procedure to quantify lipidomes comprehensively. Han and Gross ( 3 ) proposed a quantitative method for analyzing triglyceride molecular species using direct MS, which involves a procedure to calculate response factors for species that are not available as authentic materials, based on differences of the responses in the numbers of total carbons and double bonds among the species. For quantitative determination of phospholipid molecular species, Koivusalo et al. ( 4 ) utilized the following parameAbstract One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantifi cation of the overall CER species by improving our previously reported profi ling technique using normal-phase liquid chromatography-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm × 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. , ceramide class consisting of non-hydroxy fatty acids and 4-sphingenines; ESI, electrospray ionization; LOD, limit of detection; LOQ, limit of quantifi cation; NPLC, normal-phase liquid chromatography; RPLC, reversed-phase liquid chromatography; RSD, relative standard deviation; SC, stratum corneum; SIM, selected ion monitoring; S/N, signal to noise; TLC, thinlayer chromatography

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