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.

Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons.

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

Metabolomic and Lipidomic Profiling of Bone Marrow Plasma Differentiates Patients with Monoclonal Gammopathy of Undetermined Significance from Multiple Myeloma.

Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.

Analysis of sphingolipid composition in human vitreous from control and diabetic individuals.

OBJECTIVE: Sphingolipids have a fundamental role in many cellular processes, and they have been implicated in insulin resistance and Diabetes Mellitus (DM) and its complications, including diabetic retinopathy (DR). Little is known about how bioactive sphingolipids relate to retinopathies in human DM. In this study, we analyzed the sphingolipid composition of type 2 diabetic (T2DM) and non-diabetic human vitreous samples. METHODS: We conducted an observational study on post-mortem human vitreous samples from non-diabetic (Controls; n = 4; age: 71.6 ±â€¯11.0 years, mean ±â€¯SD) and type 2 diabetic (T2DM; n = 9; age: 67.0 ±â€¯9.2 years) donors to identify changes in sphingolipid composition. Samples were analyzed by a triple quadrupole mass spectrometer and individual sphingolipid species were identified and quantified using established protocols. RESULTS: The total quantity (pmol/mg) of ceramide (Cer), lactosylceramide (Lac-Cer), and sphingomyelin (SM) were increased in type 2 diabetic vitreous samples. Among individual species, we found a general trend of increase in the longer chain species of ceramides, hexosylceramides (Hex-Cer), Lac-Cer, and SM. CONCLUSIONS: This study shows the presence of measurable levels of sphingolipids in human vitreous. The results indicate changes in sphingolipid composition in the vitreous due to type 2 diabetes, which could be connected to the disease pathologies of the retina, retinal vessels, vitreous and the surrounding tissues.

Compensatory occurrence of IV2Fucalpha,II3NeuAcalpha-Gg4Cer and human fetal antigen Lc4Cer in small cell carcinomas of the human lung.

By means of a thin-layer chromatography immunostaining procedure involving a human monoclonal anti-Lc4Cer antibody, which was established by hybridizing murine myeloma cells and human lymphocytes from a cancer patient, Lc4Cer was proven to be a fetal antigen of human lung and to be a cancer-related antigen in small cell carcinomas of human lung, but not of other lung cancers, i.e., large cell carcinomas, adenocarcinomas, and squamous carcinomas. With the simultaneous detection of IV2Fuc alpha,II3NeuAc alpha-Gg4Cer with rabbit anti-IV2Fuc alpha,II3NeuAc alpha-Gg4Cer antiserum, the expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer was found to be compensatory and, consequently, small cell lung carcinomas could be classified into Lc4Cer- and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer-expressing types, L-SCLC and F-SCLC, respectively, which were detected in four and 27 of 31 patients' tissues and in one and three of four nude mouse-transplanted small cell lung carcinoma tissues, respectively. The compensatory expression of Lc4Cer and IV2Fuc alpha,II3NeuAc alpha-Gg4Cer in small cell carcinomas indicated that different metabolic pathways for glycosphingolipids were activated to give the distinct glycosphingolipid compositions in the two types of small cell lung carcinomas.

Human monoclonal antibody (HMST-1) against lacto-series type 1 chain and expression of the chain in uterine endometrial cancers.

A human monoclonal antibody termed HMST-1 was produced by fusing lymphocytes from segments of human pelvic lymph nodes from an endometrial cancer patient with murine myeloma cells. The epitope recognized by HMST-1 was determined to be lacto-series type 1 chain-containing glycosphingolipid (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer) by isolating the antigen from endometrial cancer cell line SNG-II and analyzing with fast atom bombardment mass spectrometry, permethylation analysis, and exoglycosidase treatment. By the immunohistochemical avidin-biotin-peroxidase complex method, no normal endometrium and benign endometrial hyperplasia were stained with HMST-1, but HMST-1 reacted with about 35% of endometrial cancer cases. These facts indicate that the rate of expression of the antigen increases along with the course of malignancy in the endometrium. By sialidase treatment of the section, the positive rate increased to 57% in endometrial cancers and to 13% in normal endometrium, indicating that the antigen was masked with sialic acid and exposed by neuraminidase treatment. Immunohistochemistry also revealed that the antibody reacted with human fetal alimentary tract epithelium and mesothelium, indicating the oncodevelopmental nature of Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer.

Glycosphingolipids of guinea pig gastric mucosa.

Glycosphingolipids have beenn isolated from guinea pig gastric mucosa and their composition and content determined. The neutral glycospingolipids were found to consist of mono-, di-, tri- and pentaglycosylceramide. The acidic glycosphingolipids wee represented by galactosyl and lactosyl sulfatides, and GM4, GM3 and GD3 gangliosides. None of the analyzed glycolipids contained N-acetylglucosamine and fucose.

Gangliosides and neutral glycolipids of human adrenal medulla.

Glycolipids were isolated from human adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The lipid-bound sialic acid was about 234 microgram/g fresh tissue. The glanglioside fraction contained two major gangliosides which accounted for 93% of the total lipid-bound sialic acid. They were identified as GM3, N-acetylneuraminylgalactosylglucosylceramide and GD3, N-acetylneuraminyl N-acetylneuraminylgalactosylglucosylceramide on the basis of cochromatography with authentic standards, sugar composition analysis, and neuraminidase digestion. GM3, N-acetylneuraminylgalactosylglucosylceramide and GD3, N-acetylneuraminyl N-acetylneuraminylgalactosylglucosylceramide occurred in a ratio of approximately 3 : 2, and the ratio seemed to be rather constant irrelevant of age and sex differences. The neutral glycolipid fraction consisted of GL1a, glucosylceramide (18%), GL1b, galactosylceramide (23%), GL2a, lactosylceramide (27%), GL3, digalactosylglucosylceramide (20%), and GL4, globoside (12%). The major fatty acids of all these glycolipids were 16 : 0, 18 : 0, 22 : 0, 24 : 0 and 24 :1.

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma.

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.

Biochemical studies in Niemann-Pick disease. I. Major sphingolipids of liver and spleen.

In liver and spleen specimens of 12 patients with Niemann-Pick disease types A or B, sphingomyelin was increased 15-45-fold, total phospholipids 4-10-fold and cholesterol 3-6-fold over the normal values. The storage pattern was qualitatively similar in both types but the degree of accumulation was less in type B. In Niemann-Pick disease type C (16 cases), sphingomyelin was increased 3.5-fold in liver and 6-fold in spleen. In all forms of Niemann-Pick disease, bis(monoacylglycero)phosphate was markedly elevated. Glycosphingolipids were studied in six cases with type C, three cases with type B and two cases with type A. Glucosylceramide showed the largest increase from the normal pattern in all types of Niemann-Pick disease. Highest values were recorded in type C, 14- and 35-fold normal concentrations in liver and spleen, respectively. Other neutral glycosphingolipids, particularly lactosylceramide, were also elevated, and a 2-4-fold increase of ganglioside GM3 occurred. The fatty acid profiles of the sphingolipids showed only minor alterations. In contrast to the largely dominating sphingomyelin storage found in liver and spleen of Niemann-Pick disease types A and B, the major characteristic of the lipid storage in Niemann-Pick disease type C was the absence of any prevailing accumulation and, thus, the concept of this disorder as a primary sphingomyelin storage disease is not founded.

Lactosylceramide in lysosomal storage disorders: a comparative immunohistochemical and biochemical study.

Immunohistochemical studies of the presence of lactosylceramide (LacCer) in lysosomal storage disorders (LSDs) were done using anti-LacCer monoclonal antibody of the CDw 17 type (clone MG-2). No sign of an association between LacCer and the lysosomal system in normal cells was observed, except for histiocytes active in phagocytosis. A comparative study of a group of LSDs showed a general tendency for LacCer to increase in storage cells in Niemann-Pick disease type C (NPC), and types A and B, GM1 gangliosidosis, acid lipase deficiency, glycogen storage disease type II and mucopolysaccharidoses. LacCer accumulated in storage cells despite normal activity of relevant lysosomal degrading enzymes. The accumulation of LacCer displayed variability within storage cell populations, and was mostly expressed in neurons in NPC. An absence of the increase in LacCer in storage cells above control levels was seen in neuronal ceroid lipofuscinoses (neurons and cardiocytes) and in Fabry disease. Gaucher and Krabbe cells showed significantly lower levels, or even the absence, of LacCer compared with control macrophages. Results of immunohistochemistry were corroborated by semiquantitative lipid thin-layer chromatography (TLC). It is suggested that different associations of LacCer with the lysosomal storage process may reflect differences in glycosphingolipid turnover induced by the storage-compromised lysosomal/endosomal system.

Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria.

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three-fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long-chain bases found in free ceramides were d18:1 sphingosine, d18:0 3-ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3-ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long-chain base of glucosylceramide of outer membranes.

Localization of verotoxin receptors in nervous system.

We use immunohistochemistry to show the existence of verotoxin receptor in small sensory neurons in DRG of human, rabbit, rat and mouse. In capillary in nervous system, the verotoxin receptor exists in human and rabbit, but the receptor could not be demonstrated in rat and mouse, by this method. The receptors in sensory neuron of rat and in capillary in rabbit brain are determined as galactosylglobotriaosylceramide (GalGb3) and globotriaosylceramide (Gb3,), respectively. Although verotoxin was reported to bind to glycolipid receptors that possess the terminal disaccharide Galalpha1-4Galbeta (galactobiose), the binding to toxin to galabiosylceramide was half of that of GalGb3 which has galactobiose internally.

Enzymatic sulfation of glycosphingolipids in rat parotid salivary glands.

The results of this study demonstrate that sulfatoglycosphingolipids of rat parotid salivary glands consist of galactosylceramide and lactosylceramide sulfates. The enzyme activity which catalyzes the formation of these compounds in vitro via transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate to galactosyl- and lactosylceramide is located in the microsomal fraction of parotid glands. This sulfotransferase exhibits its optimum activity at pH 6.8 and requires the presence of Triton X-100, Mg2+, and F1-.

Identification of GM3 as a marker of therapy-resistant periradicular lesions.

The purpose of this study was to analyze the profile of glycosphingolipids (GSLs) in periradicular lesions refractory to endodontic treatment. Sixteen periapical lesions were removed surgically from patients (experimental group) and compared with 10 samples of periodontal ligament removed from extracted intact third molars (control group). After the GSLs extraction and purification procedures were performed the neutral and acidic GSL fractions were analyzed by high-performance thin-layer chromatography and quantified by densitometry. Data reported herein show that: (i) tissues in the experimental group presented about twice as much GSLs as the control group; (ii) lesion tissues express lactoneotetraosylceramide, and lactofucopentaosyl (IV) ceramide, whereas these neutral GSLs are absent in normal tissues; and (iii) normal tissues express GT1b, whereas lesions cells do not express this ganglioside. In contrast lesion tissues express GM3, which is conspicuously absent in normal tissues.

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle.

Membrane vesicles were prepared from rabbit skeletal muscle, separated by sucrose density gradient centrifugation and characterized by their specific marker enzymes, ligand binding, and ion flux activities. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M). Their glycosphingolipid compositions were analyzed by biochemical and immunochemical methods with specific antibodies (TLC immunostaining) and characteristic patterns were obtained from respective membrane fractions, expressed on a protein basis. Glucosylceramide, the main neutral glycosphingolipid of rabbit muscle, was found in SL and TT fractions, whereas SR and Tr/M vesicles lack this compound. Lactosylceramide was selectively recovered in the SR1 fraction. GM3(Neu5Ac), the main ganglioside in rabbit muscle, was found to account for 64% in the SL, 13% in the TT, 7% in the SR1, 3% in the SR2 and 13% in the Tr/M fractions. IV3Neu5Ac-nLcOse4Cer was mostly abundant in SL and decreased in the order SL > TT, Tr/M > SR1, SR2. IV6Neu5Ac-nLcOse4Cer was only detected in the SL and Tr/M fractions in noteworthy quantities. Ganglioseries gangliosides GM1, GD1a, GD1b and GT1b displayed homogeneous distribution patterns in each membrane preparation. They were expressed only in small amounts but mainly in SL, TT and Tr/M vesicles and to less extent in SR1 and SR2 fractions. The presence of GM3(Neu5Ac) in the SL as well as on subcellular level was confirmed in transverse muscle cryosections by means of indirect immunofluorescence microscopy. The SL was brightly stained, but considerable intracellular fluorescence was observed as expected from the biochemical analyses. Thus, the neutral GSL and ganglioside expression of the SL and the intracellular membraneous network is different in skeletal muscle both in terms of quantitative and qualitative GSL composition as demonstrated in details by means of biochemical and immunochemical techniques. The modulatory functions of GM3 and gangliosides of the neolacto- and ganglio-series towards the voltage dependent Ca(2+)-channel, largely preponderant in the triads-containing Tr/M fraction, is the subject of the accompanying paper (J. Müthing, U. Maurer, and S. Weber-Schürholz, Carbohydr. Res., 307 (1998) 147-157).

[Analysis of glycosphingolipids from human myeloid leukemias]. / Glykosphingolipid-Analyse von menschlichen myeloischen Leukämien.

Neutral glycosphingolipids of human myeloid leukemia cells in different differentiation stages from nine patients were analyzed by high performance liquid chromatography. All cells contained ceramide monohexoside, lactosylceramide and neutral glycosphingolipids of the lacto-series. However, some of the leukemic cells contained neutral glycosphingolipids of the globo-series. Especially myelomonocytic cells (French-American-British or FAB classification M4) and poorly differentiated cells (FAB M0 and M1) were found to contain globo glycosphingolipids. On myeloblast cells, (FAB M2), globo glycosphingolipids were missing or present only in very low amounts. It seemed, therefore, that on myeloid leukemic cells the glycosphingolipid composition may be dependent on their differentiation stage.

Carbohydrate-containing molecules as potential biomarkers in colon cancer.

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages.

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.

Glycosphingolipids in human pancreatic juice. Unique fatty acid compositions of glucosylceramides and lactosylceramides.

Glycosphingolipids in human pancreatic juice were isolated and purified by DEAE and silica gel column chromatographs, and further by HPLC on silica gel and reversed-phase columns. The structures of the glycosphingolipids were determined to be glycosylceramides and lactosylceramides by means of fast atom bombardment mass spectrometry, 1H-NMR spectroscopy, and component analysis involving GLC-mass spectrometry. The ceramide portions of the glucosylceramides consist of palmitic, tetracosanoic, and alpha-hydroxy tetracosanoic fatty acids, and d18:1 and t18:0 sphingosines. The ceramide portions of the lactosylceramides consist of palmitic, teracosanoic, tetracosenoic, and tetracosadienoic fatty acids, and d18:1 sphingosine. The fatty acid compositions are different from the free fatty acid compositions of serum and pancreatic juice. The predominance of saturated, unsaturated, and hydroxy tetracosanoic fatty acid is quite unique, and the possibility that these glycosphingolipids may play a specific role in pancreatic juice deserves consideration. These glycosphingolipids were documented to be major components of the pancreatic juice from 15 patients with a variety of pancreatic diseases.