Yoshinori Masukawa

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding 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 normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem 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 α-hydroxy 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 C18)-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.

Comprehensive quantification of ceramide species in human stratum corneum

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 quantification of the overall CER species by improving our previously reported profiling 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. This method opens lipidomics approaches for CERs in the SC.—Masukawa, Y., H. Narita, H. Sato, A. Naoe, N. Kondo, Y. Sugai, T. Oba, R. Homma, J. Ishikawa, Y. Takagi, and T. Kitahara. Comprehensive quantification of ceramide species in human stratum corneum. J. Lipid. Res. 2009. 50: 1708–1719.

Relationship between covalently bound ceramides and transepidermal water loss (TEWL)

Abstract The stratum corneum, which is the outermost layer of the skin, functions as an important barrier to maintain biological homeostasis. The multilamellar structures formed by intercellular lipids present in the stratum corneum are considered to play an important role in barrier function. Most intercellular lipids are unbound and can be extracted by organic solvents, but some intercellular lipids are covalently bound to cornified envelope proteins. Decreases in unbound lipid levels reduce the barrier function of the stratum corneum, but the relationship between bound lipid and the barrier function of the stratum corneum is not well understood. In this study, we examined the relationship between the amount of covalently bound ceramide, the main bound lipid, and the barrier function of the stratum corneum. A single dose of UVB irradiation (2 × MED), or continuous UVB irradiation (0.5 × MED/day for 14 days) to the back, or feeding with an essential fatty acid-deficient (EFAD) diet for ¶8 weeks caused a significant elevation of TEWL and a significant reduction in covalently bound ceramides in hairless rats. Transmission electron microscopy revealed that the intercellular multilamellar structures in the stratum corneum of treated rats were incomplete (folding, defects, unclear images) compared to the structures seen in the stratum corneum of non-UVB-irradiated and non-EFAD rats. These results suggest that the amount of covalently bound ceramides is highly correlated with the barrier function of the skin, and that covalently bound ceramides play an important role in the formation of lamellar structures, and are involved in the maintenance of the barrier function of the skin.

Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair

Ceramides (CERs) play key roles in signal transduction and cell regulation, probably during the keratinization of human hair. Current methods using mass spectrometry (MS), however, are not sufficient to allow the comprehensive analysis of CER molecules, including isobaric and isomeric CERs. Therefore, a method for the comprehensive profiling of CERs was developed. The method developed is based on reversed-phase liquid chromatography (RPLC) coupled to atmospheric pressure chemical ionization (APCI)-MS. Comprehensive identification and profiling of CERs is achieved using two sets of multimass chromatograms obtained from two channel detections that monitor both molecular-related and sphingoid-related ions under two different in-source collision-induced dissociation conditions and using retention times obtained from RPLC. The application of this method revealed that human hair contains 73 species of CER molecules, which were all corroborated by structural analysis using tandem mass spectrometry. The results further revealed that the composition is characterized by predominant molecules consisting of even carbon atom-containing saturated/unsaturated nonhydroxy or α-hydroxy fatty acids and C18 dihydrosphingosine, a minor but distinct content of isobaric/isomeric and odd chain-containing CERs. This successfully developed RPLC-APCI-MS technique allows the comprehensive profiling of CER molecules in hair for the investigation of their physicochemical and physiological roles.

Protein Secondary Structure Imaging with Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering (CARS) Microspectroscopy

Protein secondary structures in human hair have been studied with ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The CARS peak-shift mapping method has been developed and applied to hair samples with and without treatments by chemical reduction and mechanical extension. It clearly visualizes the treatment induced changes in protein secondary structures and their spatial distributions. Using the new imaging technique, we found a multilayered structure in the human hair cortex.

Damage to Human Hair Caused by Repeated Bleaching Combined with Daily Weathering during Daily Life Activities

Background: Although it is well known that chemical oxidation with alkaline hydrogen peroxide damages human hair, little is known about the effects of bleaching during daily life activities which may alter hair components. Objective: To determine the effects of bleaching combined with daily weathering on lipids and amino acids in hair. Methods: Levels of lipids and amino acids were analyzed by chromatography in scalp hair fibers collected from three different females and in experimentally treated hair. Results: Bleaching combined with daily weathering induced the loss of 18-methyleicosanoic acid (18-MEA) and the conversion of half-cystine (H-CYS) to cysteic acid (CYS-A), which were the most remarkable changes among all lipids and amino acids tested. Although a single bleaching had no effect, repeated bleaching combined with daily weathering elicited significant decreases in other intrinsic endogenous lipids and alterations in amino acids that are characteristic for the cuticle. Conclusion: Hair damage, eventually leading to the worst damage such as splitting, is caused by bleaching during daily life activities and is mainly attributable to the loss of 18-MEA as well as the conversion of H-CYS to CYS-A.

Characterization of hair lipid images by argon sputter etching-scanning electron microscopy.

Hair lipid images, as visualized by argon sputter etching-scanning electron microscopy (ASE-SEM), reveal convex structures with a stitch pattern (SP) at the cell membrane complex (CMC) in the transverse hair plane. Based on interindividual variation, different features of the convex SP were classified into Types 0 to 4 with the corresponding scores 0 to 4. Observations using hair fibers collected from 27 Japanese females revealed significant positive correlations between the scores and the levels of exogenous lipids, which suggests that exogenous lipids internalized at the CMC predominantly constitute the convex SP. Intraindividual variation with different levels of exogenous lipids among hair fibers derived from individual females may be relevant to the uneven physicochemical properties of hair fibers on the scalp. Observations of 380 hair fibers collected from Japanese (Mongoloid), German and American (Caucasoid) females aged 3 to 77 yr demonstrated similar age-related changes in the lipid images, which represent an increase and then a decrease in levels of exogenous lipids with increasing age. This suggests that age-related changes in exogenous lipids are attributable to alterations in sebum excreted during aging and that this elicits age-related changes in physical parameters, which affect human hair texture.

Deletion of the Sox21 gene drastically affects hair lipids

The effects of Sox21 gene deletion on hair lipids have been studied. For the cuticle‐specific bound lipid 18‐methyl eicosanoic acid (18‐MEA), which was found to predominantly exist as the free form in Sox21−/− hair, total levels and distribution were unexpectedly unchanged. This indicates that while the biosynthesis of 18‐MEA is unaffected, its covalent attachment to the cuticle surface is disrupted by loss of keratin‐associated protein binding partners. Although the class compositions differed, the total ceramide (CER) levels were found to be comparable between Sox21+/+ and Sox21−/− hairs. Deletion of the gene was also found to increase cholesterol sulphate (CS) levels. The biosynthesis process might be associated with cuticle keratinocyte maturation, because both CS and CERs are known bioactives in keratinocyte differentiation.