Speculation of Sphingolipids in Capsanthin by Ultra-Performance Liquid Chromatography Coupled with Electrospray Ionization-Quadrupole-Time-of-Flight Mass Spectrometry.

Sphingolipids are constituents of cellular membranes and play important roles in cells. As nutraceutical compounds in foods, sphingolipids have been proven to be critical for human health. Therefore, the sphingolipids content of capsanthin was established based on ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole-time-of-flight mass spectrometry. A total number of 40 sphingolipids were successfully identified, including 20 Glucosylceramides and 20 Ceramides. The predominant GlcCers contain 4-hydroxy-8-sphingenine t18:1 (8) with different structures of α-OH fatty acids. For the Cers, the main long-chain bases are 4-hydroxy-8-sphingenine t18:1 (8) and 4-hydroxysphingenine (t18:0) with different structures of α-OH or α, ß-di (OH) fatty acids.

Liquid chromatography-tandem mass spectrometry with a new separation mode for rapid profiling of the Z/E isomers of plant glucosylceramides.

Plant glucosylceramide (GlcCer) is characterized by various long-chain bases (LCBs) containing a Z/E isomeric unsaturated bond at the Δ8 position. The isomer ratio of GlcCer is highly diversified among plant species and is involved in tolerance to membrane fluidity-associated stresses such as chilling and aluminum toxicity. Therefore, a plant GlcCer isomer-selective quantitative method is required, allowing further sphingolipidomic studies for crop breeding. We here report a new technique for rapid determination of the Z/E isomers of plant GlcCer. A Cholester column contains cholesteryl groups as the hydrophobic stationary phase and separated the GlcCer isomers more efficiently than a conventional C18 column. We also investigated various mobile phases and column temperatures. The optimized column, solvent, and temperature conditions provided comprehensive profiles of the Z/E ratios of GlcCer in crude extracts of plant materials in less than 20 min. This high-throughput method will facilitate the large-scale profiling of plant GlcCer isomers.

Direct LC-ESI-MS/MS analysis of plant glucosylceramide and ceramide species with 8E and 8Z isomers of the long chain base.

Glucosylceramides and ceramides with 8E and 8Z isomers of the long chain base are found in plants. These isomers have been difficult to quantify separately using liquid chromatography-tandem mass spectrometry (LC-MS/MS) because the isomers have the same retention time, their precursor and product ions have the same m/z values, and plant ceramide standards are not commercially available. Here we tested trial separations using various ODS columns and prepared plant ceramide standards generated by human glucocerebrosidase (imiglucerase) using commercially available plant glucosylceramide standards as the substrates. Consequently, we were able to quantify the isomers based on differences in retention times in a TSKgel ODS-120A column (Tosoh, Tokyo Japan) using LC-electrospray ionization-MS/MS (LC-ESI-MS/MS).

Sphingolipid Properties in Sake Rice Cultivars and Changes During Polishing and Brewing.

Sphingolipids, including ceramide (Cer) and glucosylceramide (GlcCer), have the characteristic structural units called sphingoid bases, and are constituents of cell and vacuole membranes. Plant sphingolipids bear highly diverse base structures and the base composition differs depending on the plant species. It is thought that the composition of sphingolipid classes and sphingoid bases is related to membrane fractions. However, there is little information about differences in sphingolipids among plant cultivars and the changes occurring in sphingolipids during food processing. This study investigated sphingolipids in sake rice (saka-mai) cultivars grown for sake (rice wine), and the changes in sphingolipids during polishing and brewing. In six brown rice samples, there were no large differences of the base composition among Cer or GlcCer of cultivars, whereas there were differences in their sphingolipid contents. When compared to brown rice, highly polished rice contained lower levels of sphingolipids, especially Cer. For three rice brans from different polishing steps, the Cer content was higher in the outer bran than in the inner bran. Sake and sake lees (sake-kasu) were produced by three different starter cultures (shubo preparations: the mixture of koji rice as an enzyme cocktail containing amylases, sake yeast, and adding rice as a carbohydrate source). The Cer/GlcCer ratio in sake and sake lees depended on the starter culture; Cer and GlcCer in sake lees possessed a fungi-specific base, 9-methyl-trans-4,trans-8- sphingadienine. In addition, sake lees had a higher Cer/GlcCer ratio when compared to highly polished rice as a sake source. These results suggest that the sphingolipid content of brown rice differs depending on the rice cultivar; further, the sphingolipids and the sphingolipid composition in sake and sake lees are affected by fungal sphingolipids and self-digestion during brewing.

Qualitative and Quantitative Study of Glycosphingolipids in Human Milk and Bovine Milk Using High Performance Liquid Chromatography-Data-Dependent Acquisition-Mass Spectrometry.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography-tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9-17.4 µg Crb/mL and 1.3-3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8-12.0 and 14.3-16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

Effects of Dietary Ethanol Extracts from Sake Rice and Sake Lees on Intestinal Impairment in Mice.

Glucosylceramide (GlcCer), a major sphingolipid in plants and fungi, is known to have food functions, such as preventing intestinal impairment and enhancing the moisture content of skin. This study investigated the influence of fermentation on the composition and function of lipophilic components containing GlcCer in plant-based foods; we compared the effects of ethanol extracts from sake rice (SR) and sake lees (SL) on colon impairment in mice. GlcCer and ceramide (Cer) levels in SL were much higher than those in SR, and GlcCer in SL contained 9-methyl-trans-4,trans-8-sphingadienine as a fungi-specific sphingoid base. 1,2-dimethylhydrazine (DMH) treatment markedly increased the formation of aberrant crypt foci (ACF) and the levels of TNF-α and lipid oxidation in mice colons. However, dietary SR or SL significantly suppressed these DMH-induced changes, and SR demonstrated stronger effects than SL. In addition, dietary SR or SL suppressed the expression of apoptotic and anti-apoptotic proteins induced by DMH treatment. This study suggests that SR or SL intake could reduce colon ACF formation via the suppression of inflammation and oxidation-induced cell cycle disturbances. When compared to SR, the weaked effects of SL rich in GlcCer may be the result of the changes in sphingolipid composition (sphingoid base and Cer) and differences in the concentration of other bioactive compounds produced or digested during fermentation.

Impact of Reduced Dietary Levels of Eicosapentaenoic Acid and Docosahexaenoic Acid on the Composition of Skin Membrane Lipids in Atlantic Salmon ( Salmo salar L.).

Membrane lipids, including sphingolipids and glycerol-phospholipids, are essential in maintaining the skin's barrier function in mammals, but their composition in fish skin and their response to diets have not been evaluated. This study investigated the impacts of reducing dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on membrane lipids in the skin of Atlantic salmon through a 26 week feeding regime supplying different levels (0-2.0% of dry mass) of EPA/DHA. Ceramide, glucosylceramide, sphingomyelin, sphingosine, and sphinganine in salmon skin were analyzed for the first time. Higher concentrations of glucosylceramide and sphingomyelin and higher ratios of glucosylceramide/ceramide and sphingomyelin/ceramide were detected in the deficient group, indicating interruptions in sphingolipidomics. Changes in the glycerol-phospholipid profile in fish skin caused by reducing dietary EPA and DHA were observed. There were no dietary impacts on epidermal thickness and mucus-cell density, but the changes in the phospholipid profile suggest that low dietary EPA and DHA may interrupt the barrier function of fish skin.

Untargeted Metabolomics Reveal Lipid Alterations upon 2-Deoxyglucose Treatment in Human HaCaT Keratinocytes.

The glucose analogue 2-deoxyglucose (2-DG) impedes cancer progression in animal models and is currently being assessed as an anticancer therapy, yet the mode of action of this drug of high clinical significance has not been fully delineated. In an attempt to better characterize its pharmacodynamics, an integrative UPLC-Q-Exactive-based joint metabolomic and lipidomic approach was undertaken to evaluate the metabolic perturbations induced by this drug in human HaCaT keratinocyte cells. R-XCMS data processing and subsequent multivariate pattern recognition, metabolites identification, and pathway analyses identified eight metabolites that were most significantly changed upon a 3 h 2-DG exposure. Most of these dysregulated features were emphasized in the course of lipidomic profiling and could be identified as ceramide and glucosylceramide derivatives, consistently with their involvement in cell death programming. Even though metabolomic analyses did not generally afford such clear-cut dysregulations, some alterations in phosphatidylcholine and phosphatidylethanolamine derivatives could be highlighted as well. Overall, these results support the adequacy of the proposed analytical workflow and might contribute to a better understanding of the mechanisms underlying the promising effects of 2-DG.

Malusides, novel glucosylceramides isolated from apple pomace (Malus domestica).

Three new glucosylceramides (GluCers) named malusides I-III (1-3) were isolated from apple (cultivars of Malus domestica) pomace (fruit material remaining after juice extraction). An unusual oxo/hydroxy group pattern within the sphingadienine (d18:2) type sphingoid base was observed. All compounds contained the same α-hydroxylated fatty acid (h16:0) and a ß-D-glucose moiety. Their structures were assigned on the basis of one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopic analyses and mass spectrometry (MS) measurements.

Aberrant bone marrow vascularization patterns in untreated patients with Gaucher disease type 1.

Bone marrow (BM) in subjects with Gaucher disease (GD) displays accumulation of Gaucher cells (GC), i.e. glucocerebroside-laden macrophages. Following the assumption that macrophage proliferation and perturbation in GD modulates local inflammation-associated phenomena including angiogenesis, BM biopsies from 11 untreated GD patients and 36 controls were investigated for morphology and angiogenesis-associated features. These included microvascular density, (MVD), vessel structure and pericyte coverage, expression of VEGF-A and angiopoietins (ANGPT1 and 2). In GD BM, cellularity was higher, and GC clustered in cohesive but poorly demarcated areas, leaving irregular islands with normal hematopoiesis. MVD was 2.6-fold higher in GD marrows than in controls (p<0.001). In GC-rich areas, MVD was 1.4-fold higher (p=0.026), and vessel architecture was abnormal compared with GC-poor areas. MVD correlated with BM cellularity, particularly in GC-rich areas. Moreover, 30±17% of GD BM vessels were pericyte-coated, significantly fewer than in controls (48±16%; p<0.001). Expression of ANGPT1 and 2 was significantly higher in GD BM vessel walls than in controls (7.2- and 13.2-fold higher), whereas VEGF expression was 20-fold lower (p<0.05 for all). Thus, human GD BM shows increased angiogenesis with defective pericyte coating and skewed VEGF/ANGPT1 and 2 balances, presumably related to local accumulation of GC.

Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course.

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

Potential application of oat-derived ceramides in improving skin barrier function: Part 1. Isolation and structural characterization.

The impaired epidermal barrier and skin dryness in chronic skin conditions such as atopic dermatitis, psoriasis and aged skin are associated with the depletion of ceramides (CERs) in the stratum corneum. Previously, the beneficial effects of phyto-CERs, mainly from wheat and rice, in replenishing the depleted epidermal CERs and restoring the skin barrier have been shown. However, very few efforts have been made to exploit CERs from other plants for dermal applications. In an attempt to explore alternative plant source of CERs, glucosylceramides (GlcCERs) were isolated from the lipid extract of Ethiopian oat grain (Avena abyssinica). The GlcCER species were separated on a reversed phase HPLC and the structure of individual GlcCERs were identified by tandem MS with atmospheric pressure chemical ionization interface. The glycosidic linkage of the GlcCERs was cleaved by acid treatment and the predominant CERs species were isolated using column chromatography and preparative LC-MS. Further structural characterization of the CERs was made by HR/ESI-MS and NMR analyses. All the detected oat-derived GlcCER species consisted of C18 dihydroxy sphingoid bases amide-linked with α-hydroxylated saturated fatty acids (C16-C24). The two predominant GlcCER species consisted of sphingenine (d18:1) amide-linked to hydroxypalmitic acid (h16:0) and hydroxyarachidic acid (h20:0). The molecular formulae of the two major CERs assigned by HR/ESI-MS were identical to the ones identified by LC/APCI-MS/MS. The structural information was also supported by 1H, 13C, 1H COSY NMR and HMBC spectral analyses. The amount of GlcCERs in oat grain, quantified by HPTLC, was found to be 193.5mg/kg. The results indicated the similarity of oat CERs with commercial plant CERs (with comparable GlcCER content) suggesting its potential as source of CERs for oral (as dietary supplements) as well as topical applications.

Glucosylceramide and Glucosylsphingosine Quantitation by Liquid Chromatography-Tandem Mass Spectrometry to Enable In Vivo Preclinical Studies of Neuronopathic Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the GBA1 gene that encodes the lysosomal enzyme acid ß-glucosidase (GCase). Reduced GCase activity primarily leads to the accumulation of two substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Current treatment options have not been shown to ameliorate the neurological pathology observed in the most severe forms of GD, clearly representing an unmet medical need. To better understand the relationship between GlcCer and GlcSph accumulation and ultimately their connection with the progression of neurological pathology, we developed LC-MS/MS methods to quantify GlcCer and GlcSph in mouse brain tissue. A significant challenge in developing these methods was the chromatographic separation of GlcCer and GlcSph from the far more abundant isobaric galactosyl epimers naturally occurring in white matter. After validation of both methods, we evaluated the levels of both substrates in five different GD mouse models, and found significant elevation of brain GlcSph in all five, while GlcCer was elevated in only one of the five models. In addition, we measured GlcCer and GlcSph levels in the brains of wild-type mice after administration of the GCase inhibitor conduritol ß-epoxide (CBE), as well as the nonlysosomal ß-glucosidase (GBA2) inhibitor N-butyldeoxygalactonojirimycin (NB-DGJ). Inhibition of GCase by CBE resulted in elevation of both sphingolipids; however, inhibition of GBA2 by NB-DGJ resulted in elevation of GlcCer only. Taken together, these data support the idea that GlcSph is a more selective and sensitive biomarker than GlcCer for neuronopathic GD in preclinical models.

Isolation and structural characterization of glucosylceramides from Ethiopian plants by LC/APCI-MS/MS.

Chronic skin conditions such as atopic dermatitis, psoriasis and aged skin are characterized by defective skin barrier and dryness which are associated with reduced levels of skin ceramides (CERs). The beneficial effects of plant-derived CERs for skin hydration and skin barrier recovery have been shown in several studies. Although plenty of glucosylceramide (GlcCER)-based dietary supplements meant for skin barrier improvement have been marketed, there are limited commercial sources of plant GlcCERs. In an attempt to explore alternative GlcCER sources, a reversed phase LC-MS/MS method with atmospheric pressure chemical ionization (APCI) interface was developed for separation and structural identification of GlcCERs isolated from three plants. The GlcCERs were extracted from the seeds of grass pea (Lathyrus sativus L.), Ethiopian mustard (Brassica carinata) and haricot bean (Phaseolus vulgaris) and purified by column chromatography and preparative LC-MS. The individual GlcCER species were further separated and qualitatively analyzed by LC/APCI-MS/MS. The amount of GlcCERs in each plant was quantified by HPTLC. All GlcCER species detected in the three plants consisted of C18 di/trihydroxy sphingoid bases amide linked with hydroxy fatty acids (C14-C24). The trihydroxy SBs were acylated with very long chain FAs (C22-C24). The major GlcCERs derived from grass pea, Ethiopian mustard and haricot bean are composed of sphingenine (d18:1) linked to hydroxypalmitic acid (h16:0), 4-hydroxy-8-sphingenine (t18:1) coupled with hydroxynervonic acid (h24:1) and sphingadienine (d18:2) joined with h16:0, respectively. The GlcCERs contents in haricot bean (161.2mg/kg) and grass pea (130.0mg/kg) were found to be higher compared to Ethiopian mustard (71.8mg/kg). This qualitative and quantitative information suggests that the two plants of the Fabaceae family (haricot bean and grass pea) are potential alternative sources of GlcCERs for their use in products meant for the recovery of skin barrier function. The LC/APCI-MS/MS method described here has proven to be reliable for the screening of other potential plants containing GlcCERs.

Lipid composition of microdomains is altered in neuronopathic Gaucher disease sheep brain and spleen.

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase activity that leads to accumulation of glucosylceramide and glucosylsphingosine. Membrane raft microdomains are discrete, highly organized microdomains with a unique lipid composition that provide the necessary environment for specific protein-lipid and protein-protein interactions to take place. In this study we purified detergent resistant membranes (DRM; membrane rafts) from the occipital cortex and spleen from sheep affected with acute neuronopathic Gaucher disease and wild-type controls. We observed significant increases in the concentrations of glucosylceramide, hexosylsphingosine, BMP and gangliosides and decreases in the percentage of cholesterol and phosphatidylcholine leading to an altered DRM composition. Altered sphingolipid/cholesterol homeostasis would dramatically disrupt DRM architecture making them less ordered and more fluid. In addition, significant changes in the length and degree of lipid saturation within the DRM microdomains in the Gaucher brain were also observed. As these DRM microdomains are involved in many cellular events, an imbalance or disruption of the cell membrane homeostasis may impair normal cell function. This disruption of membrane raft microdomains and imbalance within the environment of cellular membranes of neuronal cells may be a key factor in initiating a cascade process leading to neurodegeneration.

Clonal Immunoglobulin against Lysolipids in the Origin of Myeloma.

Antigen-driven selection has been implicated in the pathogenesis of monoclonal gammopathies. Patients with Gaucher's disease have an increased risk of monoclonal gammopathies. Here we show that the clonal immunoglobulin in patients with Gaucher's disease and in mouse models of Gaucher's disease-associated gammopathy is reactive against lyso-glucosylceramide (LGL1), which is markedly elevated in these patients and mice. Clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC). Substrate reduction ameliorates Gaucher's disease-associated gammopathy in mice. Thus, long-term immune activation by lysolipids may underlie both Gaucher's disease-associated gammopathies and some sporadic monoclonal gammopathies.

Tandem Mass Spectrometry Multiplex Analysis of Glucosylceramide and Galactosylceramide Isoforms in Brain Tissues at Different Stages of Parkinson Disease.

Previous studies demonstrated that Parkinson disease (PD) is associated with a decreased activity of the glucocerebrosidase (GCase) enzyme in brain tissues. The objective of this study was to determine if GCase deficiency is associated with the accumulation of its glucosylceramide (GluCer) substrate in PD brain tissues. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed, optimized, and validated for the multiplex analysis of GluCer isoforms (C18:0, C20:0, C22:0, C24:1, and C24:0) in brain tissue samples. These molecules were chromatographically separated from their isobaric galactosylceramide (GalCer) counterparts using normal phase chromatography. The analysis was performed by tandem mass spectrometry in the multiple reaction monitoring (MRM) acquisition mode. Limits of detection ranging from 0.4 to 1.1 nmol/g brain tissue were established for the different GluCer isoforms analyzed. For the first time, GluCer isoform levels were analyzed in temporal cortex brain tissue samples from 26 PD patients who were divided into three PD disease stages (IIa, III, and IV) according to the Unified Staging System for Lewy Body Disorders. These specimens were compared with brain tissue samples from 12 controls and 6 patients with Incidental Lewy Body Disease. No significant GluCer concentration differences were observed between the 5 sample groups. The GluCer isoform levels were also normalized with their matching GalCer isoforms. The normalized results showed a trend for GluCer levels which increased with PD severity. However, the differences observed between the groups were not significant, owing likely to the high standard deviations measured.

AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides.

The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer.

Sphingolipid metabolism is strikingly different between pollen and leaf in Arabidopsis as revealed by compositional and gene expression profiling.

Although sphingolipids are essential for male gametophytic development in Arabidopsis thaliana, sphingolipid composition and biosynthetic gene expression have not been previously examined in pollen. In this report, electrospray ionization (ESI)-MS/MS was applied to characterization of sphingolipid compositional profiles in pollen isolated from wild type Arabidopsis Col-0 and a long-chain base (LCB) Δ4 desaturase mutant. Pollen fractions were highly enriched in glucosylceramides (GlcCer) relative to levels previously reported in leaves. Accompanying the loss of the Δ4 unsaturated LCB sphingadiene (d18:2) in the Δ4 desaturase mutant was a 50% reduction in GlcCer concentrations. In addition, pollen glycosylinositolphosphoceramides (GIPCs) were found to have a complex array of N-acetyl-glycosylated GIPCs, including species with up to three pentose units that were absent from leaf GIPCs. Underlying the distinct sphingolipid composition of pollen, genes for key biosynthetic enzymes for GlcCer and d18:2 synthesis and metabolism were more highly expressed in pollen than in leaves or seedlings, including genes for GlcCer synthase (GCS), sphingoid base C-4 hydroxylase 2 (SBH2), LCB Δ8 desaturases (SLD1 and SLD2), and LOH2 ceramide synthase (LOH2). Overall, these findings indicate strikingly divergent sphingolipid metabolism between pollen and leaves in Arabidopsis, the significance of which remains to be determined.

CNS, lung, and lymph node involvement in Gaucher disease type 3 after 11 years of therapy: clinical, histopathologic, and biochemical findings.

A Caucasian male with Gaucher disease type 3, treated with continuous enzyme therapy (ET) for 11 years, experienced progressive mesenteric and retroperitoneal lymphadenopathy, lung disease, and neurological involvement leading to death at an age of 12.5 years. Autopsy showed significant pathology of the brain, lymph nodes, and lungs. Liver and spleen glucosylceramide (GluCer) and glucosylsphingosine (GluS) levels were nearly normal and storage cells were cleared. Clusters of macrophages and very elevated GluCer and GluS levels were in the lungs, and brain parenchymal and perivascular regions. Compared to normal brain GluCer (GC 18:0), GluCer species with long fatty acid acyl chains were increased in the patient's brain. This profile was similar to that in the patient's lungs, suggesting that these lipids were present in brain perivascular macrophages. In the patient's brain, generalized astrogliosis, and enhanced LC3, ubiquitin, and Tau signals were identified in the regions surrounding macrophage clusters, indicating proinflammation, altered autophagy, and neurodegeneration. These findings highlight the altered phenotypes resulting from increased longevity due to ET, as well as those in poorly accessible compartments of brain and lung, which manifested progressive disease involvement despite ET.