Omics-Driven Systems Interrogation of Metabolic Dysregulation in COVID-19 Pathogenesis.

The coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented threat to global public health. Herein, we utilized a combination of targeted and untargeted tandem mass spectrometry to analyze the plasma lipidome and metabolome in mild, moderate, and severe COVID-19 patients and healthy controls. A panel of 10 plasma metabolites effectively distinguished COVID-19 patients from healthy controls (AUC = 0.975). Plasma lipidome of COVID-19 resembled that of monosialodihexosyl ganglioside (GM3)-enriched exosomes, with enhanced levels of sphingomyelins (SMs) and GM3s, and reduced diacylglycerols (DAGs). Systems evaluation of metabolic dysregulation in COVID-19 was performed using multiscale embedded differential correlation network analyses. Using exosomes isolated from the same cohort, we demonstrated that exosomes of COVID-19 patients with elevating disease severity were increasingly enriched in GM3s. Our work suggests that GM3-enriched exosomes may partake in pathological processes related to COVID-19 pathogenesis and presents the largest repository on the plasma lipidome and metabolome distinct to COVID-19.

Gangliosides profiling in serum of breast cancer patient: GM3 as a potential diagnostic biomarker.

Gangliosides altered during the pathological conditions and particularly in cancers. Here, we aimed to profile the gangliosides in breast cancer serum and propose potential biomarkers. LC-FTMS method was first used to identify all the ganglioside species in serum, then LC-MS/MS-MRM method was employed to quantitate the levels of gangliosides in serum from healthy volunteers and patients with benign breast tumor or breast cancer. 49 ganglioside species were determined, including GM1, GM2, GM3, GD1, GD3 and GT1 species. Compared to healthy volunteers, the levels of GM1, GM2, GM3, GD1 and GD3 displayed a rising trend in breast cancer patients. In particular, as the major glycosphingolipid component, GM3 showed excellent diagnostic accuracy in cancer serum (AUC > 0.9). PCA profile of the GM3 species showed clear distinction between normal and cancer serum. What's more, ROC curve proved great diagnostic accuracy of GM3 between cancer and benign serum. In addition, GM3 was discovered as a diagnostic marker to differentiate luminal B subtype from other subtypes. Furthermore, a positive correlation between GM3 and Ki-67 status of patients was identified. In conclusion, our results introduced the alteration patterns of serum gangliosides in breast cancer and suggested serum GM3 as a potential diagnostic biomarker in breast cancer diagnosis and luminal B subtype distinction.

Serum GM3(d18:1-16:0) and GM3(d18:1-24:1) levels may be associated with lymphoma: An exploratory study with haematological diseases.

GM3 (monosialodihexosylganglioside) is a type of ganglioside, which is a molecule composed of ceramide and oligosaccharide containing one or more sialic acids. Since GM3 is abundantly expressed in blood cells, we investigated the association between GM3 molecular species and haematological diseases. We measured the serum levels of seven GM3 molecular species in subjects with various haematological diseases (n = 52) and healthy subjects (n = 24) using a liquid chromatography tandem-mass spectrometry technique as an exploratory study. In all the subjects with haematological diseases, GM3(d18:1-16:0) were inversely correlated with the erythrocytes counts. Regarding the difference in serum GM3 molecular species levels among each haematological diseases and healthy subjects, the levels of GM3(d18:1-16:0) and GM3(d18:1-24:1) were higher in the lymphoid neoplasm group than healthy subjects. Principal component analyses also revealed that the GM3(d18:1-16:0) and GM3(d18:1-24:1) levels were significant contributing factors for discriminating the lymphoid neoplasm group. Moreover, in the lymphoid neoplasm group, the GM3(d18:1-16:0) levels were significantly and positively correlated with the levels of C-reactive protein, soluble interleukin-2 receptor, and lactate dehydrogenase. In conclusion, in our exploratory study with haematological diseases, GM3 molecular species showed different distribution among disease groups, and serum GM3(d18:1-16:0) and GM3(d18:1-24:1) might be associated with lymphoma.

Elevated GM3 plasma concentration in idiopathic Parkinson's disease: A lipidomic analysis.

Parkinson's disease (PD) is a common neurodegenerative disease whose pathological hallmark is the accumulation of intracellular α-synuclein aggregates in Lewy bodies. Lipid metabolism dysregulation may play a significant role in PD pathogenesis; however, large plasma lipidomic studies in PD are lacking. In the current study, we analyzed the lipidomic profile of plasma obtained from 150 idiopathic PD patients and 100 controls, taken from the 'Spot' study at Columbia University Medical Center in New York. Our mass spectrometry based analytical panel consisted of 520 lipid species from 39 lipid subclasses including all major classes of glycerophospholipids, sphingolipids, glycerolipids and sterols. Each lipid species was analyzed using a logistic regression model. The plasma concentrations of two lipid subclasses, triglycerides and monosialodihexosylganglioside (GM3), were different between PD and control participants. GM3 ganglioside concentration had the most significant difference between PD and controls (1.531±0.037 pmol/µl versus 1.337±0.040 pmol/µl respectively; p-value = 5.96E-04; q-value = 0.048; when normalized to total lipid: p-value = 2.890E-05; q-value = 2.933E-03). Next, we used a collection of 20 GM3 and glucosylceramide (GlcCer) species concentrations normalized to total lipid to perform a ROC curve analysis, and found that these lipids compare favorably with biomarkers reported in previous studies (AUC = 0.742 for males, AUC = 0.644 for females). Our results suggest that higher plasma GM3 levels are associated with PD. GM3 lies in the same glycosphingolipid metabolic pathway as GlcCer, a substrate of the enzyme glucocerebrosidase, which has been associated with PD. These findings are consistent with previous reports implicating lower glucocerebrosidase activity with PD risk.

Anti-NeuGcGM3 reactivity: a possible role of natural antibodies and B-1 cells in tumor immunosurveillance.

While not naturally expressed in normal human tissues, N-glycolylated (NeuGc) gangliosides are overexpressed in several tumors and have immunosuppressive capacity, which contributes to cancer progression. Naturally occurring antibodies against NeuGcGM3 exist in healthy donors that specifically recognize and kill tumor cells expressing the antigen by complement-dependent and -independent mechanisms, the latter resembling an oncotic necrosis-type of cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with these natural antibodies decrease with age. Our work has shown that anti-NeuGcGM3 antibodies are not detected in the sera of non-small cell lung cancer (NSCLC) patients, compared to age- and sex-matched healthy donors, which have anti-NeuGcGM3. Interestingly, the level of serum total IgM, but not IgG, was significantly lower in cancer patients than in healthy donors. Screening of immortalized mouse splenic and peritoneal-derived hybridomas showed that peritoneal B-1 cells secrete anti-NeuGcGM3 with tumor cytotoxic capacity. Defects in the natural surveillance against tumor antigens could increase the risk of elderly donors developing cancer and affect the capacity of cancer patients to effectively fight against tumor cells.

Identification of Ganglioside GM3 Molecular Species in Human Serum Associated with Risk Factors of Metabolic Syndrome.

Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 ¼ d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).

Bimodal plasma metabolomics strategy identifies novel inflammatory metabolites in inflammatory bowel diseases.

INTRODUCTION: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characterized by variable phenotypes. Metabolites are signatures of biochemical activity that can reveal unknown pathogenic pathways. We employed untargeted mass spectrometry (MS) based metabolomics to identify novel inflammatory mechanisms in IBD and a targeted assay to quantify metabolites of the auto-immunomodulating kynurenine pathway (KP) in IBDs and health. MATERIALS AND METHODS: Metabolome analysis of CD, UC, and control plasmas was performed on a Liquid Chromatography (LC)-MS/MS system. KP metabolites quinolinic acid (QA) and picolinic acid (PA) were quantified by gas chromatography/MS. RESULTS: Nineteen UC, 25 CD, and 9 control plasmas were analyzed: 34 metabolites exhibited abundance profiles associated with CD by global metabolome analysis (P≤0.05, false discovery rate q≤0.01). Notably, inflammatory-implicated metabolites angiotensin IV (P=0.049, q<0.001), diphthamide (P=0.018, q<0.001), and GM3 gangliosides (P<0.001, q<0.001) were increased in CD. By targeted kynurenine metabolites assay, QA (73.53 ng/mL ± 23.40 SD) and combined kynurenine metabolites (CKM) were increased in CD (120.19 ± 39.71) compared to controls (QA 50.14 ± 15.04; P<0.01; CKM 92.73 ± 26.30; P<0.01). CD QA positively correlated with CDAI (r=0.85; P<0.01), CRP (r=0.46; P=0.01), and ESR (r=0.42; P=0.03), while CKMs correlated with CDAI (r=0.615; P<0.01) and CRP (r=0.615; P=0.02). CONCLUSIONS: Associations of angiotensin IV, diphthamide, and GM3 gangliosides with CD implicate novel pathways in activating a Th1/Th17 inflammatory profile. Increased QA concentrations in CD may indicate a defective auto-immunomodulation mechanism.

Eliglustat, an investigational oral therapy for Gaucher disease type 1: Phase 2 trial results after 4 years of treatment.

Eliglustat is an investigational, oral substrate reduction therapy for Gaucher disease type 1 (GD1). Nineteen treatment-naïve patients have now completed 4years of an open-label study (NCT00358150). Mean hemoglobin level and platelet count increased by 2.3±1.5g/dL (baseline: 11.3±1.5g/dL) and 95% (baseline: 68,700±21,200/mm(3)), respectively. Mean spleen and liver volumes (multiples of normal, MN) decreased by 63% (baseline: 17.3±9.5 MN) and 28% (baseline: 1.7±0.4 MN), respectively. Median chitotriosidase and CCL-18 each decreased by 82%; plasma glucosylceramide and GM3 normalized. Mean bone mineral density T-score for the lumbar spine increased by 0.8 (60%) (baseline: -1.6±1.1). Femur dark marrow, a reflection of Gaucher cell infiltration into bone marrow, was reduced or stable in 17/18 patients. There were no bone crises. Most adverse events were mild and unrelated to treatment. These results extend the safety and efficacy of eliglustat reported at 1 and 2 years to 4 years.

A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma.

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-ß glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.

GM3 and diabetes.

We demonstrated the molecular pathogenesis of type 2 diabetes and insulin resistance focusing on the interaction between insulin receptor and GM3 ganglioside in adipocytes and propose a working hypothesis "metabolic disorders, such as type 2 diabetes, are membrane microdomain disorders caused by aberrant expression of gangliosides". It is expected that the development of novel diagnosis of metabolic syndrome by identifying the specific ganglioside species and a therapeutic strategy "membrane microdomain ortho-signaling therapy".

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody.

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.

Prominent increase in plasma ganglioside GM3 is associated with clinical manifestations of type I Gaucher disease.

BACKGROUND: Patients with Gaucher disease show signs of insulin resistance. The ganglioside GM3 has recently shown to be a negative regulator of insulin sensitivity. In fibroblasts of Gaucher patients, deficient in degradation of glucosylceramide, an increased anabolism of this lipid to gangliosides occurs. The goal of the current study was to establish whether GM3 is elevated in plasma of type I Gaucher disease patients, and is related to disease manifestations. METHODS: Plasma GM3, glucosylceramide, and ceramide were determined and compared to overall severity of disease, hepatomegaly, and plasma chitotriosidase activity. RESULTS: The ceramide concentration in plasma of untreated Gaucher patients was slightly but not significantly lower than in controls (median: 9.8 micromol/L, range: 5.7-14.7 micromol/L, (n=40) vs. median: 11.0 micromol/L, range: 5.1-18.0 micromol/L, (n=30)). Glucosylceramide was significantly (p<0.0001) elevated. GM3 was also significantly (p<0.0001) increased (median: 10.2 micromol/L, range: 4.3-19.1 micromol/L, (n=40) vs. median: 3.6 micromol/L, range: 2.7-5.4 micromol/L, (n=30)). Plasma GM3 concentrations correlated with those of plasma chitotriosidase activity (rho=0.45, p=0.0036), overall severity of disease (rho=0.39, p=0.012), and hepatomegaly (rho=0.49, p=0.0015). CONCLUSIONS: GM3 is strikingly elevated in plasma of most Gaucher patients. The increase is comparable to that of glucosylceramide, the primary storage lipid. The marked elevations in GM3 may play a role in the insulin resistance of Gaucher patients.

Type I Gaucher disease, a glycosphingolipid storage disorder, is associated with insulin resistance.

CONTEXT: Complex glycosphingolipids, in majority the ganglioside GM3, surround the insulin receptor in a special membrane compartment (raft) and modulate signaling through this receptor. Increased levels of GM3 in rafts impair insulin signaling, resulting in insulin resistance. Gaucher disease is a lysosomal storage disorder in which impaired breakdown of glucosylceramide leads to its accumulation in macrophages. Secondary to this defect, GM3 concentrations, for which glucosylceramide is the precursor, in plasma and several cell types are elevated. OBJECTIVE: We studied the influence of glycosphingolipid storage on whole body glucose and fat metabolism by measuring insulin-mediated (IMGU) and noninsulin-mediated glucose uptake (NIMGU) and suppression of free fatty acids by insulin. DESIGN AND MAIN OUTCOME MEASURES: We studied six Gaucher patients, either naive to treatment or with considerable remaining burden of disease, and six matched healthy control subjects in the basal state, during an euglycemic and a hyperglycemic clamp with somatostatin measuring NIMGU and during an euglycemic hyperinsulinemic clamp measuring IMGU, using stable isotopes. RESULTS: NIMGU (both during euglycemia and hyperglycemia) did not differ between patients and control subjects. IMGU was lower in Gaucher patients, compared with controls. Suppression of lipolysis by insulin tended to be less effective in Gaucher patients. CONCLUSION: Gaucher disease, a lysosomal glycosphingolipid storage disorder, is associated with (peripheral) insulin resistance, possibly through the influence of glycosphingolipids on insulin receptor functioning.

Diet-induced changes in membrane gangliosides in rat intestinal mucosa, plasma and brain.

OBJECTIVES: The objective of this study was to determine if dietary gangliosides induce changes in the ganglioside content of intestinal mucosa, plasma and brain and to identify where GM3 and GD3 are localized in the enterocyte membrane. METHODS: Male 18-day-old Sprague-Dawley rats were fed a semipurified diet containing 20% (w/w) fat. The control diet contained triglyceride, reflecting the fat formulation of an existing infant formula. Two experimental diets were formulated by adding sphingomyelin (1% w/w of total fat) or a ganglioside-enriched lipid (0.1% w/w of total fat) to the control diet fat. The ganglioside fraction of ganglioside-enriched lipid diet contained more than 80% GD3. After 2 weeks of feeding, the total and individual ganglioside and cholesterol content was measured in small intestinal mucosa, plasma and brain. RESULTS: The ganglioside-enriched lipid diet significantly increased total gangliosides in the intestinal mucosa, plasma and brain compared with the control diet. The ganglioside-enriched lipid diet significantly increased the level of GD3 (7.5% w/w) in the intestine compared with control (3.2% w/w) while decreasing the level of GM3, the major ganglioside in the intestine. The ratio of cholesterol to ganglioside in the intestinal mucosa, plasma and brain decreased significantly in rats fed the ganglioside-enriched lipid diet compared with controls. Confocal microscopy showed that GM3 is exclusively localized in the apical membrane of the enterocyte whereas GD3 is primarily localized in the basolateral membrane. CONCLUSIONS: : The authors conclude that dietary ganglioside is absorbed in the small intestine and transported to different membrane sites, altering ganglioside levels in the intestinal mucosa, plasma and brain and thus possibly having the potential to change developing enterocyte function (and possibly that of other cell lines).

Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase.

We identified an autosomal recessive infantile-onset symptomatic epilepsy syndrome associated with developmental stagnation and blindness. Assuming a founder effect in a large Old Order Amish pedigree, we carried out a genome-wide screen for linkage and identified a single region of homozygosity on chromosome 2p12-p11.2 spanning 5.1 cM (maximum lod score of 6.84). We sequenced genes in the region and identified a nonsense mutation in SIAT9, which is predicted to result in the premature termination of the GM3 synthase enzyme (also called lactosylceramide alpha-2,3 sialyltransferase). GM3 synthase is a member of the sialyltransferase family and catalyzes the initial step in the biosynthesis of most complex gangliosides from lactosylceramide. Biochemical analysis of plasma glycosphingolipids confirmed that affected individuals lack GM3 synthase activity, as marked by a complete lack of GM3 ganglioside and its biosynthetic derivatives and an increase in lactosylceramide and its alternative derivatives. Although the relationship between defects in ganglioside catabolism and a range of lysosomal storage diseases is well documented, this is the first report, to our knowledge, of a disruption of ganglioside biosynthesis associated with human disease.

Serum ganglioside patterns in multiple sclerosis.

The relative distribution of gangliosides was determined in the serum of 37 patients with multiple sclerosis (MS) and of 30 healthy subjects. There was a significant increase of GM1 and GD1a, and a decrease of GM3 proportion in the serum of relapsing-remitting MS patients (RRMS) during their first MS attack. The RRMS patients in relapse with a long duration of the disease had a significant decrease of GM1 and an increase of GD1a portion in the serum. An increase of GD1a, one of the major brain neuron ganglioside fraction, suggested the neuron injury in the early and with a long duration RRMS. The finding of an increase of GM1, the main human myelin ganglioside, during the first MS attack in RRMS patients confirms previous evidence for the possible involvement of gangliosides in the early pathological course of demyelination in MS.

Alteration of acidic lipids in human sera during the course of pregnancy: characteristic increase in the concentration of cholesterol sulfate.

In this study, we determined the concentrations of acidic lipids, including cholesterol sulfate (CS), sulfatide and GM3 ganglioside, in human sera of non-pregnant state and during the course of pregnancy. In human sera of non-pregnant women, GM3 was present at a concentration of 8 nmol/ml and the concentrations of CS and sulfatides were less than 20% of that of GM3. The concentration of sulfatides in sera at the second trimester of gestation was decreased, but CS gradually increased from the first to the third trimester of gestation with a correlation coefficient of 0.66, and a correlation between the concentration of CS and weeks of gestation (p<0.01). CS was also contained in the placental villi, and its concentration increased from the first to the third trimester of gestation, suggesting that placental CS is one of the source of CS in the blood by shedding.

Overexpression of monosialoganglioside GM3 on lymphocyte plasma membrane in patients with HIV infection.

SUMMARY: This study was undertaken to analyze both the GM3 expression on peripheral blood lymphocytes of HIV-infected patients and the relationship between ganglioside content and anti-GM3 reactivity. GM3 expression was determined as a percentage of lipid-bound sialic acid and by cytofluorimetric analysis in 25 AIDS patients, 20 anti-HIV+ asymptomatic subjects, 25 patients with different viral disease, and 25 healthy donors. GM3 distribution was analyzed by immunofluorescence and immunoelectron microscopy. A follow-up study to detect anti-lymphocytic GM3 antibodies was performed in progressive and nonprogressive anti-HIV+ subjects. Lymphocytes from HIV-infected patients showed a significant increase of plasma membrane GM3 content; no difference was found between CD4+ and CD8+ cells. Immunofluorescence and immunoelectron microscopic analysis showed that GM3 was distributed in large clusters over the cell plasma membrane. The follow-up study revealed that the occurrence of anti-lymphocytic GM3 antibodies was significantly higher in patients with progressive disease, compared with asymptomatic non-progressive subjects. These findings revealed that (1) the increased GM3 content in HIV-infected patients is detected at the plasma membrane level, (2) GM3 overexpression is able to induce an increased reactivity with anti-GM3 antibodies, and (3) the appearance of anti-lymphocytic GM3 antibodies in asymptomatic anti-HIV+ subjects could have prognostic relevance for the risk of developing AIDS.

Evaluation of the efficiency of an assay procedure for gangliosides in human serum.

An efficiency assessment of a ganglioside assay procedure was carried out on human serum gangliosides from healthy subjects of different sex and age. The analysis of the gangliosides, extracted with chloroform/methanol and purified by lipid partitioning, ion exchange column chromatographic separation and desalting procedures as described by Senn et al. (1989) Eur J Biochem 181: 657-62, was performed by HPTLC followed by densitometric quantification. The yield of the procedure, expressed as radioactivity recovery, was determined by adding GM3 ganglioside, tritium labelled at the sialic acid acetyl group and at the C3 position of sphingosine, to the lyophilized serum or by associating it with the serum lipoproteins. In spite of the fact that the extraction and purification procedures were performed exactly as described we found the radioactivity recovery to be variable (25-50%) and much lower than that proposed. Much of the radioactivity was found in the organic phase after lipid partitioning, whilst all the ganglioside purification steps led to some further loss. After the introduction of some modifications to the procedure the recovery improved, reaching 67-79%. The analyses on 33 samples of 5 ml showed a human serum ganglioside content of about 10 nmol ml-1 (as corrected for the recovery), and confirmed that GM3 ganglioside is the main component of the total serum ganglioside mixture.

Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes.

Novel mono-O-acetylated GM3s, one containing 9-O-acetyl N-glycolyl neuraminic acid and another containing 6'-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of the O-acetyl residue was identified by the downfield shift of the methylene protons at C-9 of N-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6'-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6'-O-Ac GM3, the O-acetylated GM3 mixture was desialylated with Arthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1.