Bifunctional DEGS2 has higher hydroxylase activity toward substrates with very-long-chain fatty acids in the production of phytosphingosine ceramides.

Phytosphingosine (PHS) is a sphingolipid component present mainly in epithelial tissues, including the epidermis and those lining the digestive tract. DEGS2 is a bifunctional enzyme that produces ceramides (CERs) containing PHS (PHS-CERs) via hydroxylation and sphingosine-CERs via desaturation, using dihydrosphingosine-CERs as substrates. Until now, the role of DEGS2 in permeability barrier functioning, its contribution to PHS-CER production, and the mechanism that differentiates between these two activities have been unknown. Here, we analyzed the barrier functioning of the epidermis, esophagus, and anterior stomach of Degs2 KO mice and found that there were no differences between Degs2 KO and WT mice, indicating normal permeability barriers in the KO mice. In the epidermis, esophagus, and anterior stomach of Degs2 KO mice, PHS-CER levels were greatly reduced relative to WT mice, but PHS-CERs were still present. We obtained similar results for DEGS2 KO human keratinocytes. These results indicate that although DEGS2 plays a major role in PHS-CER production, another synthesis pathway exists as well. Next, we examined the fatty acid (FA) composition of PHS-CERs in various mouse tissues and found that PHS-CER species containing very-long-chain FAs (≥C21) were more abundant than those containing long-chain FAs (C11-C20). A cell-based assay system revealed that the desaturase and hydroxylase activities of DEGS2 toward substrates with different FA chain lengths differed and that its hydroxylase activity was higher toward substrates containing very-long-chain FAs. Collectively, our findings contribute to the elucidation of the molecular mechanism of PHS-CER production.

Phenotypic and genetic spectra of galactose mutarotase deficiency: A nationwide survey conducted in Japan.

PURPOSE: Galactose mutarotase (GALM) deficiency was first reported in 2019 as the fourth type of galactosemia. This study aimed to investigate the clinical and genotypic spectra of GALM deficiency. METHODS: This was a questionnaire-based retrospective survey conducted in Japan between February 2022 and March 2023. RESULTS: We identified 40 patients with GALM deficiency in Japan (estimated prevalence: 1:181,835). Four of 38 patients (10.5%) developed cataracts, which resolved with lactose restriction in 3 out of 4 patients. Transient transaminitis was the most common symptom (23.1%). All of the patients followed lactose restriction; discontinuation of the restriction after infancy did not cause any complications. Moreover, none of the participants experienced long-term complications. Two variants, GALM NM_138801.3: c.294del and c.424G>A, accounted for 72.5% of the identified pathogenic variants. The patients showed moderately elevated blood galactose levels with lactose intake; however, the elevation was lower than that observed in galactokinase deficiency. CONCLUSION: GALM deficiency is characterized by a similar but milder phenotype and lower blood galactose elevation than in galactokinase deficiency. Diagnosis and initiation of lactose restriction in early infancy should be essential for prevention of cataracts, especially in cases of irreversible opacity.

Promyelinating drugs ameliorate oligodendrocyte pathologies in a mouse model of Krabbe disease.

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.

Genetic ablation of Saposin-D in Krabbe disease eliminates psychosine accumulation but does not significantly improve demyelination.

Krabbe disease is an inherited demyelinating disease caused by a genetic deficiency of the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase (GALC). The Twitcher (Twi) mouse is a naturally occurring, genetically and enzymatically authentic mouse model that mimics infantile-onset Krabbe disease. The major substrate for GALC is the myelin lipid GalCer. However, the pathogenesis of Krabbe disease has long been explained by the accumulation of psychosine, a lyso-derivative of GalCer. Two metabolic pathways have been proposed for the accumulation of psychosine: a synthetic pathway in which galactose is transferred to sphingosine and a degradation pathway in which GalCer is deacylated by acid ceramidase (ACDase). Saposin-D (Sap-D) is essential for the degradation of ceramide by ACDase in lysosome. In this study, we generated Twi mice with a Sap-D deficiency (Twi/Sap-D KO), which are genetically deficient in both GALC and Sap-D and found that very little psychosine accumulated in the CNS or PNS of the mouse. As expected, demyelination with the infiltration of multinucleated macrophages (globoid cells) characteristic of Krabbe disease was milder in Twi/Sap-D KO mice than in Twi mice both in the CNS and PNS during the early disease stage. However, at the later disease stage, qualitatively and quantitatively comparable demyelination occurred in Twi/Sap-D KO mice, particularly in the PNS, and the lifespans of Twi/Sap-D KO mice were even shorter than that of Twi mice. Bone marrow-derived macrophages from both Twi and Twi/Sap-D KO mice produced significant amounts of TNF-α upon exposure to GalCer and were transformed into globoid cells. These results indicate that psychosine in Krabbe disease is mainly produced via the deacylation of GalCer by ACDase. The demyelination observed in Twi/Sap-D KO mice may be mediated by a psychosine-independent, Sap-D-dependent mechanism. GalCer-induced activation of Sap-D-deficient macrophages/microglia may play an important role in the neuroinflammation and demyelination in Twi/Sap-D KO mice.

The Urinary Bladder is Rich in Glycosphingolipids Composed of Phytoceramides.

Glycosphingolipids (GSLs) are composed of a polar glycan chain and a hydrophobic tail known as ceramide. Together with variation in the glycan chain, ceramides exhibit tissue-specific structural variation in the long-chain base (LCB) and N-acyl chain moieties in terms of carbon chain length, degree of desaturation, and hydroxylation. Here, we report the structural variation in GSLs in the urinary bladders of mice and humans. Using TLC, we showed that the major GSLs are hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, Neu5Ac-Gal-Glc-Ceramide, and Neu5Ac-Neu5Ac-Gal-Glc-Ceramide. Our LC-MS analysis indicated that phytoceramide structures with a 20-carbon LCB (4-hydroxyeicosasphinganine) and 2-hydroxy fatty acids are abundant in hexosylceramide and Neu5Ac-Gal-Glc-Ceramide in mice and humans. In addition, quantitative PCR demonstrated that DES2 and FA2H, which are responsible for the generation of 4-hydroxysphinganine and 2-hydroxy fatty acid, respectively, and SPTLC3 and SPTSSB, which are responsible for the generation of 20-carbon LCBs, showed significant expressions in the epithelial layer than in the subepithelial layer. Immunohistochemically, dihydroceramide:sphinganine C4-hydroxylase (DES2) was expressed exclusively in urothelial cells of the urinary bladder. Our findings suggest that these ceramide structures have an impact on membrane properties of the stretching and shrinking in transitional urothelial cells.

Work beliefs and experiential learning among proficient physical therapists in Japan: developing a method to support belief formation.

[Purpose] This study aimed to elucidate the content of work beliefs related to proficiency among physical therapists in Japan. [Participants and Methods] Participants included 50 therapists who met the definition of proficiency to participate in a questionnaire survey conducted between October 2017 and March 2019. Participants were asked to freely describe their daily work beliefs, including their thoughts, values, and ideals. This content was coded and categorized using open coding; a hierarchical cluster analysis (Ward's method) was conducted of the proficient therapists with the individuals and belief categories as the variables. All belief categories were classified into three groups. [Results] Three work beliefs were identified as follows: 1) practices that emphasize building relationships in the field, 2) broad practices with physical therapist pride (responsibility and enthusiasm), and 3) practices with awareness of treatment outcomes and social benefits. [Conclusion] A better understanding of the three aforementioned work beliefs would facilitate good support for and development of physical therapists. We recommend continuing to elucidate the three work beliefs identified among proficient therapists and verify their educational effects.

Prevalence of patients with lysosomal storage disorders and peroxisomal disorders: A nationwide survey in Japan.

INTRODUCTION: Lysosomal storage disorders and peroxisomal disorders are rare diseases caused by the accumulation of substrates of the metabolic pathway within lysosomes and peroxisomes, respectively. Owing to the rarity of these diseases, the prevalence of lysosomal storage disorders and peroxisomal disorders in Japan is unknown. Therefore, we conducted a nationwide survey to estimate the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. METHODS: A nationwide survey was conducted following the "Manual of nationwide epidemiological survey for understanding patient number and clinical epidemiology of rare diseases (3rd version)". A questionnaire asking for detailed information, such as disease phenotypes and medical history, was created and sent to 504 institutions with doctors who have experience in treating patients with lysosomal storage disorders and peroxisomal disorders. Result A total of 303 completed questionnaires were collected from 504 institutions (response rate: 60.1%). The number of patients was estimated by calculating the rate/frequency of overlap. The estimated number of patients was 1658 (±264.8) for Fabry disease, 72 (±11.3) for mucopolysaccharidosis I, 275 (±49.9) for mucopolysaccharidosis II, 211 (±31.3) for Gaucher disease, 124 (±25.8) for Pompe disease, 83 (±44.3) for metachromatic leukodystrophy, 57 (±9.4) for Niemann-Pick type C, and 262 (±42.3) for adrenoleukodystrophy. In addition the birth prevalence was calculated using the estimated number of patients and birth year data for each disease, and was 1.25 for Fabry disease, 0.09 for mucopolysaccharidosis I, 0.38 for mucopolysaccharidosis II, 0.19 for Gaucher disease, 0.14 for Pompe disease, 0.16 for metachromatic leukodystrophy, 0.16 for Niemann-Pick type C, and 0.20 for adrenoleukodystrophy. DISCUSSION: Among the diseases analyzed, the disease with the highest prevalence was Fabry disease, followed by mucopolysaccharidosis II, adrenoleukodystrophy, Gaucher disease and metachromatic leukodystrophy. In particular, the high prevalence of mucopolysaccharidosis II and Gaucher disease type II was a feature characteristic of Japan. CONCLUSION: We estimated the number of patients with lysosomal storage disorders and peroxisomal disorders in Japan. The details of the age at diagnosis and treatment methods for each disease were clarified, and will be useful for the early diagnosis of these patients and to provide appropriate treatments. Furthermore, our results suggest that supportive care and the development of an environment that can provide optimal medical care is important in the future.

Variants in saposin D domain of prosaposin gene linked to Parkinson's disease.

Recently, the genetic variability in lysosomal storage disorders has been implicated in the pathogenesis of Parkinson's disease. Here, we found that variants in prosaposin (PSAP), a rare causative gene of various types of lysosomal storage disorders, are linked to Parkinson's disease. Genetic mutation screening revealed three pathogenic mutations in the saposin D domain of PSAP from three families with autosomal dominant Parkinson's disease. Whole-exome sequencing revealed no other variants in previously identified Parkinson's disease-causing or lysosomal storage disorder-causing genes. A case-control association study found two variants in the intronic regions of the PSAP saposin D domain (rs4747203 and rs885828) in sporadic Parkinson's disease had significantly higher allele frequencies in a combined cohort of Japan and Taiwan. We found the abnormal accumulation of autophagic vacuoles, impaired autophagic flux, altered intracellular localization of prosaposin, and an aggregation of α-synuclein in patient-derived skin fibroblasts or induced pluripotent stem cell-derived dopaminergic neurons. In mice, a Psap saposin D mutation caused progressive motor decline and dopaminergic neurodegeneration. Our data provide novel genetic evidence for the involvement of the PSAP saposin D domain in Parkinson's disease.

Mesoporous Carbon Fibers with Tunable Mesoporosity for Electrode Materials in Energy Devices.

To improve the properties of mesoporous carbon (MC), used as a catalyst support within electrodes, MC fibers (MCFs) were successfully synthesized by combining organic-organic self-assembly and electrospinning deposition and optimizing heat treatment conditions. The pore structure was controlled by varying the experimental conditions. Among MCFs, MCF-A, which was made in the most acidic condition, resulted in the largest pore diameter (4-5 nm), and the porous structure and carbonization degree were further optimized by adjusting heat treatment conditions. Then, since the fiber structure is expected to have an advantage when MCFs are applied to devices, MCF-A layers were prepared by spray printing. For the resistance to compression, MCF-A layers showed higher resistance (5.5% change in thickness) than the bulk MC layer (12.8% change in thickness). The through-plane resistance was lower when the fiber structure remained more within the thin layer, for example, +8 mΩ for 450 rpm milled MCF-A and +12 mΩ for 800 rpm milled MCF-A against the gas diffusion layer (GDL) 25BC carbon paper without a carbon layer coating. The additional advantages of MCF-A compared with bulk MC demonstrate that MCF-A has the potential to be used as a catalyst support within electrodes in energy devices.

Endosomal sorting by Semaphorin 4A in retinal pigment epithelium supports photoreceptor survival.

Photoreceptor cell death is the hallmark of a group of human inherited retinal degeneration. Although the causative genetic mutations are often known, the mechanisms leading to photoreceptor degeneration remain poorly defined. Here, we show that Semaphorin 4A (Sema4A), a member of axonal guidance molecule semaphorin, plays a role in Rab11/FIP2-mediated endosomal sorting in retinal pigment epithelial cells to support photoreceptor function. In response to oxidative stress, Sema4A switches the endosomal sorting of the lysosomal precursor protein prosaposin from the lysosome to the exosomal release, which prevents light-induced photoreceptor apoptosis. In the absence of oxidative stress, Sema4A sorts retinoid-binding proteins with retinoids between the cell surface and endoplasmic reticulum, by which 11-cis-retinal, a chromophore for phototransduction, is regenerated and transported back to photoreceptors. Owing to defects in these processes, Sema4A-deficient mice exhibit marked photoreceptor degeneration. Our findings therefore indicate that Sema4A regulates two distinct endosomal-sorting pathways that are critical for photoreceptor survival and phototransduction during the transition between daylight and darkness.

Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease.

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.

Monoclonal immunoglobulin heavy chain gene rearrangement in Fuchs' uveitis.

BACKGROUND: Fuchs' uveitis (FU) is occasionarlly complicated with heavy vitreous opacity. We have performed vitrectomy procedures to remove vitreous opacity in affected patients as part of differential diagnosis for primary vitreoretinal lymphoma (PVRL). CASE PRESENTATION: We retrospectively reviewed the clinical records of five patients who first visited the Uveitis Clinic of the University of Tokyo Hospital between 2009 and 2013, were diagnosed with FU and underwent a vitrectomy for removal of dense vitreous opacity. All were diagnosed as FU by ocular findings and elevation of Goldmann-Witmer coefficient (GWC) value for the rubella virus (RV) antibody. In examinations of the vitreous body, cytological diagnosis, elevation of IL-10/IL-6 ratio, and the kappa/lambda ratio in flow cytometry findings were negative in all cases, whereas monoclonal immunoglobulin heavy chain (IgH) gene rearrangement was positive in 4 cases and negative in 1 case. CONCLUSIONS: Although monoclonal IgH gene rearrangement is thought to be a reliable biomarker for PVRL, a high percentage of vitreous specimens from our FU patients showed pseudo-positive results. Ophthalmologists must take care regarding possible pseudo-positive findings when performing differential diagnosis between FU and PVRL. Combinations of results of cytological diagnosis, IL-10/IL-6 ratio, kappa/lambda ratio, and IgH gene rearrangement may be necessary for a definitive diagnosis of PVRL and differentiation from FU.

Combined intravitreal methotrexate and immunochemotherapy followed by reduced-dose whole-brain radiotherapy for newly diagnosed B-cell primary intraocular lymphoma.

Primary intraocular lymphoma (IOL) has a propensity for central nervous system (CNS) relapse within 2 years of initial diagnosis, affecting clinical outcome. To reduce CNS relapse, we performed the combination treatment protocols of intravitreal methotrexate injections, methotrexate-based systemic induction chemotherapy and consolidation high-dose cytarabine and reduced-dose whole brain radiation therapy (rdWBRT, 23·4 Gy) for B-cell primary IOL with or without newly diagnosed CNS involvement. All patients underwent longitudinal brain magnetic resonance imaging (MRI) and cognitive assessment for evaluation of treatment-induced leucoencephalopathy. Seventeen patients initiated and 16 completed the protocol treatment. CNS relapse occurred in 2 patients and intraocular relapse in 3. Four-year progression-free survival (PFS) was 74·9% and 4-year overall survival (OS) was 86·3%, with a median follow-up period of 48·9 months. Of 11 patients without CNS involvement, 1 had CNS relapse and 3 intraocular relapse, and 4-year PFS and OS was 72·7% and 88·9%, respectively. Although white matter abnormalities shown by MRI were significantly increased at 4 years after rdWBRT, only one patient developed mild cognitive impairment. The combination of intravitreal chemotherapy, prophylactic systemic chemotherapy and rdWBRT for primary IOL showed a potential to reduce CNS relapse rate and improved 4-year PFS and OS without increase of cognitive dysfunction.

Ni-Fe Nitride Nanoplates on Nitrogen-Doped Graphene as a Synergistic Catalyst for Reversible Oxygen Evolution Reaction and Rechargeable Zn-Air Battery.

Obtaining bifunctional electrocatalysts with high activity for the oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) is a main hurdle in the application of rechargeable metal-air batteries. Earth-abundant 3d transition metal-based catalysts have been developed for the OER and ORR; however, most of these are based on oxides, whose insulating nature strongly restricts their catalytic performance. This study describes a metallic Ni-Fe nitride/nitrogen-doped graphene hybrid in which 2D Ni-Fe nitride nanoplates are strongly coupled with the graphene support. Electronic structure of the Ni-Fe nitride is changed by hybridizing with the nitrogen-doped graphene. The unique heterostructure of this hybrid catalyst results in very high OER activity with the lowest onset overpotential (150 mV) reported, and good ORR activity comparable to that for commercial Pt/C. The high activity and durability of this bifunctional catalyst are also confirmed in rechargeable zinc-air batteries that are stable for 180 cycles with an overall overpotential of only 0.77 V at 10 mA-2 .

Mesotrypsin and caspase-14 participate in prosaposin processing: potential relevance to epidermal permeability barrier formation.

A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A-D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A-D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.

In situ TEM studies on hydrogen-related issues: hydrogen storage, hydrogen embrittlement, fuel cells and electrolysis.

Hydrogen is attracting attention as an energy carrier for realizing a low-carbon society, because it can directly convert the energy obtained from chemical reactions into electrical energy without carbon dioxide emissions. This paper presents in situ transmission electron microscopy (TEM) observations related to hydrogen storage in metal and metal hydrides, hydrogen embrittlement of metallic materials used for storing and transporting hydrogen in containers and pipes, and fuel cells and water electrolysis using metal catalysts and oxides as electrode materials. All of these processes are important for practical applications of hydrogen. Numerous in situ TEM studies have revealed the microscopic structural changes when hydrogen reacts with the materials, when hydrogen is solidly dissolved in the materials and during the operation of the material. This review is expected to facilitate further development of TEM operando observations of hydrogen-related materials.

Reticular Imine-linked Coordination Polymers Based on Paddlewheel Diruthenium/Dirhodium Nodes: Synthesis and Metal-site Dependent Photocatalytic Reduction of CO2.

The paddlewheel-type dimetal core ([M2]) is a ubiquitous motif in the nodes in coordination polymers (CPs) and metal-organic frameworks (MOFs). However, their preparation has relied on ligand-substitution-labile metal ions owing to challenges associated with crystallization. Consequently, examples featuring ligand-substitution-inert metal ions, such as Ru or Rh, are scarce. This study presents the synthesis of novel reticular imine-linked CPs incorporating the paddlewheel-type diruthenium(II, II) ([Ru2II,II]; 1-Ru) or dirhodium(II, II) ([Rh2II,II]; 1-Rh) subunits. The synthetic approach involved a Schiff base dehydration condensation reaction between p-formylbenzoate-bridged [Ru2II,II] or [Rh2II,II] precursors (i.e., CHO-Ru and CHO-Rh, respectively) and 2,5-dimethyl-1,4-phenylenediamine in a 1:2 ratio. The catalytic activities of 1-Ru and 1-Rh for the photochemical reduction of CO2 in a heterogeneous system depended on the metal site. The 1-Rusystem exhibited exceptional selectivity, generating 3.0 ´ 104 mmol g-1 of CO after 24 h of irradiation, whereas the 1-Rhsystem generated a lower amount of CO (3.2 ´ 103 mmol g-1). The catalytic activity of 1-Ru ranked with that of all relevant catalytic systems. This study paves the way for the exploration of [Ru2II,II]- or [Rh2II,II]-based polymers with open metal site-dependent functional properties.

Improved high-fat diet-induced glucose intolerance by an oral administration of phytosphingosine.

We have previously reported that phytoceramide and phytosphingosine (PHS) stimulated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in cells. PPARγ is a therapeutic target for type 2 diabetes. We found in this study that an oral administration of PHS improved diet-induced glucose intolerance in mice. Since PHS is highly expressed in yeast, PHS in fermented foods may improve diabetes.

Mass Spectrometry of Neutral Glycosphingolipids.

This chapter describes the protocols for mass spectrometry (MS) applied to the structural characterization of neutral glycosphingolipids (GSLs) and the determination of neutral GSL contents in biological materials. The structural characterization is performed by thin layer chromatography-matrix assisted laser desorption ionization/mass spectrometry (TLC-MALDI/MS) and liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) with reversed phase separation. The content determination is carried out by LC-ESI/MS with multiple reaction monitoring (MRM). These protocols provide clues for the functions of neutral GSLs at the level of a single GSL molecular species.

High-performance thin-layer chromatography/mass spectrometry for the analysis of neutral glycosphingolipids.

This mini-review summarizes the protocol we have developed for the analysis of neutral glycosphingolipids (GSLs) by high-performance thin layer chromatography (HPTLC)-mass spectrometry (MS). We also present results obtained using this glycolipidomic approach to study neutral GSLs from mouse kidney, spleen, and small intestine. Finally, we discuss what is required for further development of this method, as well as what is expected for the future of glycolipid biology.