Comprehensive quantification of ceramide species in human stratum corneum

Abstract

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

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

“…4, it can be concluded that the mass range of this newly identifi ed CER [EOdS] ranges from m/z 1015.0 to 1181.1 amu and corresponds exactly with a CER [EOdS], having a total chain length ranging from 66 to 78 carbon atoms long. Besides, it is known from literature that the CERs containing an even number of carbon atoms are more abundantly present than odd chain length CERs ( 21,23 ), which is also the case in our mass spectrum of Fig. 4 , again confi rming that the unknown CER subclass is CER [EOdS].…”

“…Future studies will include several of these standards to make this assay (semi-) quantitative. The use of several nonisotopically labeled internal standards to correct for chain length and CER subclass regarding human SC is described in the literature only once ( 23 ).…”

“…To circumvent these issues, we decided to use poly(phenylene sulfi de) tape strips obtained from Nichiban. These tape strips were also used in studies for CER analysis by Masukawa et al ( 23,39 ). After lipid extraction and the additional concentration step (see Material and Methods), samples were injected and the 3D CER profi le is shown in Fig.…”

“…However, these approaches are very time consuming and a high quantity of material is required, which is not always available. The application of LC/MS results in a much more detailed profi le of each individual subclass using only small quantities of material (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Besides their important role in human skin, CERs are also key molecules with respect to cell signaling, growth, differentiation, and apoptosis ( 29-38 ).…”

Improvement in Skin Conditions by Consumption of Traditional Japanese Miso Soup and Its Mechanism

“…As described above, ceramides in the normal stratum corneum play an important role in maintaining the moisture and barrier functions of the skin [15,16]. Ceramides in the stratum corneum are classified into phytosphingosine-type, sphingosine-type and dihydrosphingosinetype [17][18][19]. Therefore, we investigated the effect of miso extracts on the expression of enzymes for ceramide synthesis and on the activity of the enzymes that were specifically induced by miso extracts.…”