Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid.

Gangliosides (GGs) represent an important class of biomolecules associated with the central nervous system (CNS). In view of their special role at a CNS level, GGs are valuable diagnostic markers and prospective therapeutic agents. By ion mobility separation mass spectrometry (IMS MS), recently implemented by us in the investigation of human CNS gangliosidome, we previously discovered a similarity between GG profiles in CSF and the brain. Based on these findings, we developed IMS tandem MS (MS/MS) to characterize rare human CSF glycoforms, with a potential biomarker role. To investigate the oligosaccharide and ceramide structures, the ions detected following IMS MS separation were submitted to structural analysis by collision-induced dissociation (CID) MS/MS in the transfer cell. The IMS evidence on only one mobility feature, together with the diagnostic fragment ions, allowed the unequivocal identification of isomers in the CSF. Hence, by IMS MS/MS, GalNAc-GD1c(d18:1/18:1) and GalNAc-GD1c(d18:1/18:0) having both Neu5Ac residues and GalNAc attached to the external galactose were for the first time discovered and structurally characterized. The present results demonstrate the high potential of IMS MS/MS for biomarker discovery and characterization in body fluids, and the perspectives of method implementation in clinical analyses targeting the early diagnosis of CNS diseases through molecular fingerprints.

Galabiosylceramide is present in human cerebrospinal fluid.

Cerebrospinal fluid (CSF) contains glycosphingolipids, including lactosylceramide (LacCer, Galß(1,4)Glcß-ceramide). LacCer and its structural isomer, galabiosylceramide (Gb2, Galα(1,4)Galß-ceramide), are classified as ceramide dihexosides (CDH). Gb2 is degraded by α-galactosidase A (GLA) in lysosomes, and genetic GLA deficiency causes Fabry disease, an X-linked lysosomal storage disorder. In patients with Fabry disease, Gb2 accumulates in organs throughout the body. While Gb2 has been reported to be in the liver, kidney, and urine of healthy individuals, its presence in CSF has not been reported, either in patients with Fabry disease or healthy controls. Here, we isolated CDH fractions from CSF of patients with idiopathic normal pressure hydrocephalus. Purified CDH fractions showed positive reaction with Shiga toxin, which specifically binds to the Galα(1,4)Galß structure. The isolated CDH fractions were analyzed by hydrophilic interaction chromatography (HILIC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS). HILIC-ESI-MS/MS separated LacCer and Gb2 and revealed the presence of Gb2 and LacCer in the fractions. We also found Gb2 in CSF from neurologically normal control subjects. This is the first report to show Gb2 exists in human CSF.

Cerebrospinal fluid: Profiling and fragmentation of gangliosides by ion mobility mass spectrometry.

The proximity of cerebrospinal fluid (CSF) with the brain, its permanent renewal and better availability for research than tissue biopsies, as well as ganglioside (GG) shedding from brain to CSF, impelled lately the development of protocols for the characterization of these glycoconjugates and discovery of central nervous system biomarkers expressed in CSF. Currently, the investigation of CSF gangliosides is focused on concentration measurements of the predominant classes and much less on their profiling and structural analysis. Since we have demonstrated recently the high performance of ion mobility separation mass spectrometry (IMS MS) for compositional and structural determination of human brain GGs, in the present study we have implemented for the first time IMS MS for the exploration of human CSF gangliosidome, in order to generate the first robust mass spectral database of CSF gangliosides. IMS MS separation and screening revealed 113 distinct GG species in CSF, having similar compositions to the species detected in human brain. In comparison with the brain tissue, we have discovered in CSF several components containing fatty acids with odd number of carbon atoms and/or short glycan chains. By tandem MS (MS/MS) we have further analyzed the structure of GD3(d18:1/18:0) and GD2(d18:1/18:0), two glycoforms exhibiting short carbohydrate chains found in CSF, but discovered and characterized previously in brain as well. According to the present results, human CSF and brain show a similar ganglioside pattern, a finding that might be useful in clinical research focused on discovery of ganglioside species associated to neurodegenerative diseases and brain tumors.

Evaluation of biomarkers for Sanfilippo syndrome.

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, GM2 and GM3, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.

Diagnosis value of membrane glycolipids biochemistry index in intracranial and gastrointestinal tumors.

The diagnostic value of membrane glycolipid biochemistry index, the lipid-bound sialic acid (LSA) and total sialic acid (TSA) in cerebrospinal fluid (CSF) was evaluated in 30 intracranial and 65 gastrointestinal tumors. The plasma LSA, TSA and red cell membrane sialic acid (R-SA) in were determined according to the method of Sevenmerhulm. Our results showed that the levels of LSA and TSA in CSF of intracranial tumor patients was higher than that of normal group(p<0.01). The concentration of TSA and LSA in patients with malignant glioma was higher than that of benign meningioma patients(P<0.01). No significance was found between intracranial halmatoma patients and normal control group for levels of membrane glycolipids (p>0.05). Results also found that the plasma LSA, TSA and R-SA of gastric carcinoma were significantly higher than those of control group (p<0.05); while no significant difference was found in the plasma LSA, TSA and R-SA levels between chronic gastritis, gastrohelcoma and normal control group (p>0.05). Plasma LSA, TSA and R-SA levels of gastric carcinoma patient were significantly higher than those of chronic gastritis patients and gastrohelcoma patients(p<0.05). It was also found that plasma LSA, TSA and R-SA contents were significantly higher in large intestine carcinoma patients than in benign in stestine tumor patients (p<0.05) while no significant difference was found between intestine benign tumor and normal control group (p>0.05). The levels of LSA, TSA and R-SA were obviously higher in the patients with metastasis than in the ones without (p<0.05.) The membrane glycolipid biochemistry index LSA and TSA in CSF are sensive markers for diagnosing intracranial tumors. For gastrointestinal malignant tumors the plasma LSA TSA and red blood cell membrane SA may be considered as auxiliary indicators for diagnosis. They can be used for distinguishing benign from malignant tumors.

Detection of the GD2+/CD56+/CD45- immunophenotype by flow cytometry in cerebrospinal fluids from a patient with retinoblastoma.

Triple-color flow cytometry with a panel of antibodies comprising GD2, CD56, and CD45 was performed to analyze cerebrospinal fluids (CSF) from a patient with retinoblastoma who was suspicious of meningeal metastasis based on clinical presentation. Our results showed that the cells in CSF demonstrated the immunophenotype positive for GD2 and CD56 but negative for CD45 antigen, which suggested the presence of CSF metastasis of retinoblastoma. At the end of eight cycles of intrathecal chemotherapy, CSF specimen was analyzed with Flow cytometry immunophenotyping (FCI) again and the result showed no detectable malignant cells with the same immunophenotype. Our conclusion is that FCI can be a quick and reliable method for the diagnosis of CSF metastasis of retinoblastoma and the immunophenotype (GD2+, CD56+, and CD45-) can be used to recognize residual retinoblastoma cells in CSF.

Dose- and duration-dependent alterations by tellurium on lipid levels: differential effects in cerebrum, cerebellum, and brain stem of mice.

The effect of various doses of sodium tellurite (1/50 LD50=0.4 mg/kg, 1/25 LD50=0.8 mg/kg, and 1/10 LD50=2.0 mg/kg body weight orally) on the lipid levels (cholesterol, triglycerides, phospholipids, esterified fatty acids, gangliosides, and total lipids) in the cerebrum, cerebellum, and brainstem of male albino mice was studied after 7 and 15 d of treatment. Sodium tellurite (2.0 mg/kg body weight) for 7 d has an apparent effect on the depletion of cholesterol, triglycerides, phospholipids, esterified fatty acids, and total lipids. The cholesterol content was decreased significantly in the cerebrum, cerebellum, and brainstem after 7 d of treatment with a 2.0-mg/kg dose compared to the control. On the other hand, treatment for 15 d with doses of 0.4, 0.8, and 2.0 mg/kg body weight resulted in a significant and dose-dependent increment in cholesterol level in the cerebrum, cerebellum, and brainstem. The triglycerides content was decreased significantly in the cerebrum, cerebellum, and brainstem with the 2.0-mg/kg dose after 7 d of treatment. The doses of 0.4, 0.8, and 2.0 mg/kg orally for 15 d resulted in a significant and dose-dependent depletion of triglycerides in the cerebrum, cerebellum, and brainstem. All the doses of tellurium (0.4, 0.8, and 2.0 mg/kg) both for 7 and 15 d have depleted the level of phospholipids in varying degrees of significance in the cerebrum, cerebellum, and brainstem. However, the level of esterified fatty acids was decreased significantly with the 2.0-mg/kg dose of tellurium for 7 d but increased with the 0.4-mg/kg dose for 15 d in the cerebrum and cerebellum. The level of gangliosides was depleted in the cerebrum but elevated in the cerebellum and brainstem after receiving a 2.0-mg/kg dose of sodium tellurite for 7 d. The content of gangliosides was increased with doses of 0.4 and 0.8 mg/kg but decreased with 2.0 mg/kg for 15 d in the cerebrum, cerebellum, and brainstem. The total lipids content was depleted significantly and dose dependently after 7 and 15 d of treatment in the cerebrum, cerebellum, and brainstem. These results suggest that sodium tellurite affects the lipids content differentially in various parts of the mice brain.

Glycolipid analysis of different tissues and cerebrospinal fluid in type II Gaucher disease.

The lipid composition or the liver, spleen, brain, cerebellum and cerebrospinal fluid of a Gaucher disease type II patient who died at the age of 5 months was examined. The glycolipid analysis demonstrated a marked increase of total amounts not only in the peripheral tissues but also in the brain cerebellum and cerebrospinal fluid, with a prevalence of glucosylceramide. A reduction in gangliosides was observed in all the analysed tissues with a relative increase of GD3 in the nervous tissue. The fatty acid composition of glucosylceramide showed a prevalence of stearic acid in the central nervous system, while in the peripheral tissues palmitic acid was prevalent. This result suggests a different origin of the glucosylceramide stored in different tissues. The generalized reduction of gangliosides and their modified distribution together with the central nervous system GD3 increment represent a new observation. These data could be useful in the effort to clarify the pathophysiological mechanism of brain damage in neuronopathic Gaucher disease.

Cerebrospinal fluid markers in children with cerebral white matter abnormalities.

Disorders of the cerebral white matter in children constitute a heterogeneous group and the diagnostic work is often complicated. Clinical and radiological characteristics can provide diagnostic clues but there is a need for further diagnostic methods. This study focused on assessing neurochemical "markers" in the cerebrospinal fluid considered to reflect damage to white matter components such as myelin and glial cells as well as neurones with their axons and synapses. The aim was to evaluate whether they contributed to the elucidation of pathogenic processes and the direction of further diagnostic efforts. Seventeen of the 26 cases had increased levels of the glial cell marker ganglioside GD3, indicating gliosis, or of the CNS myelin marker sulfatide, indicating myelin disturbance. As signs of disturbed maturation or sustenance, the nerve cell markers GD1 b, GT1 b and total gangliosides were reduced, as was the synapse marker GD1a. Increased 5-HIAA indicated increased serotonergic turnover. Children with an increased level of the axonal marker Tau protein had a progressive disease whereas GD1a was reduced in the progressive group (n = 11). In contrast, GD3 and HVA were increased in the non-progressive group (n = 15). The chemical profiles were found to be useful, in combination with clinical and radiological findings, when investigating children with white matter abnormalities.

Cerebrospinal fluid gangliosides in patients with Rett syndrome and infantile neuronal ceroid lipofuscinosis.

Concentrations of the four major brain gangliosides, GM1, GD1a, GD1b and GT1b, biochemical markers of neuronal membranes, were determined in cerebrospinal fluid from a large series of patients with classical Rett syndrome, aged 1.5-21 years at sampling, and from 11 patients with infantile neuronal ceroid lipofuscinosis, aged 1.5-11 years. The results were compared with age-matched healthy controls. Compared with fluid from the control group, the cerebrospinal fluid samples from Rett patients contained significantly reduced levels of gangliosides GD1a and GT1b. In cerebrospinal fluid of the infantile neuronal ceroid lipofuscinosis patients, even the very young ones, all four major brain gangliosides were significantly reduced compared with controls and the concentration levels also differed significantly from those in patients with Rett syndrome. The ganglioside pattern in the brain is reflected in the cerebrospinal fluid early in the course of the disease in Rett syndrome and infantile neuronal ceroid lipofuscinosis.

Gangliosides in cerebrospinal fluid in children with autism spectrum disorders.

Gangliosides are sialic acid-containing glycolipids found in all cells, especially abundant in nerve cells and mainly situated on outer-membrane surfaces. The aim of this study was to provide data on the concentration of gangliosides in the CSF of children and adolescents with autism spectrum disorders (ASD) - 66 with autistic disorder, and 19 with other autism spectrum disorders. The comparison group consisted of 29 children and adolescents, whose CSF had been sampled to exclude acute infectious CNS disorder. The concentrations of the gangliosides GM1, GD1a, GD1b, and GT1b were determined using a microimmunoaffinity technique. The ASD group had a significantly higher concentration of ganglioside GM1 compared with the comparison group. The GM1 increase could not be explained as secondary to other clinical factors. Mean ganglioside levels did not differentiate subgroups with autistic disorder and those with a more atypical clinical picture, nor subgroups with known medical disorders and those with idiopathic autism. Altered patterns of gangliosides in the CNS might reflect important correlates of pathogenesis in autism.

A new procedure for determining ganglioside GD3 a potential glial cell activation marker in cerebrospinal fluid.

Increased amounts of ganglioside GD3 [II3 (NeuAc)2-LacCer], associated with reactive gliosis, have been documented in a variety of neurodegenerative disorders. GD3 expression has also been reported in microglial cells, not only during development but also in reactive states, where the glial activation is considered to be part of the repair process. It is important to find markers in cerebrospinal fluid that will enable us to identify damage and register changes in pathological processes within the brain. A sensitive and practically applicable method for determination of GD3 ganglioside in cerebrospinal fluid has been developed. The procedure, which includes extraction, purification on silica gel and thin-layer enzyme-linked immunostaining, also allows determination of sulphatide, a marker of demyelinating processes, in the same portion of CSF. The method has been applied to CSF samples from 101 normal individuals aged 2-83 years. The GD3 concentration was found to be significantly correlated to age and reflecting the concentrations within the brain. GD3 ganglioside analysis by means of this method might be useful for studying glial changes during brain maturation as well as in brain disorders.

Increased cerebrospinal fluid ganglioside GD3 concentrations as a marker of microglial activation in HIV type 1 infection.

Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early in the infectious course. The predominant, productively infected cell type within the CNS is the microglial cell. We have analyzed the cerebrospinal fluid (CSF) levels of the ganglioside GD3, a microglia/macrophage and astrocyte marker, in 22 HIV-1-infected individuals at different stages of the disease, and in 44 age-matched HIV-negative, healthy controls. To distinguish between microglial/macrophage and astroglial involvement, the GD3 levels were compared with CSF levels of the glial fibrillary acidic protein (GFAp), which is expressed exclusively in astrocytes. A significantly higher mean CSF concentration of GD3 was found in HIV-1-infected patients compared to controls (56.7 and 40.1 nmol/L, respectively, p < 0.001). Seven of 22 HIV-1-infected patients had increased CSF levels of GD3 (above mean + 2 SD in controls), all but one of these had normal levels of GFAp, indicating a microglial activation or proliferation as the major source of the increased GD3 levels.

Glycosphingolipids as potential diagnostic markers and/or antigens in neurological disorders.

Glycosphingolipids are most abundant in the nervous system within which are developmental, regional, structural and cellular differences regarding their composition. The are shedded to the cerebrospinal fluid and thus potential markers for pathogenic alterations in the brain, such as developmental abnormalities, demyelination, gliosis, neuronal cell destruction. The glycosphingolipids have also been found to be antigens in autoimmune processes involving the nervous system, in particular in peripheral neuropathies like Guillain Barré syndrome, multifocal motor neuropathy etc. The immune response might have been triggered by infectious agents with an antigen epitope which mimic the glycosphingolipid or by a primary nerve tissue damage leading to release of glycosphingolipids. There is a series of support for a clinical significance of cerebrospinal fluid glycosphingolipid determinations and the presence of anti-glycosphingolipid antibodies but this has to be further explored. This paper is a mini review of the state of the art and discuss methodological aspects and improvements that might help to explore the relevance of glycosphingolipids in neurological disorders.

Gangliosides and sulfatide in cerebrospinal fluid in leukoaraiosis.

The aim of the study was to evaluate gangliosides and sulfatide in cerebrospinal fluid (CSF) as markers for neuronal degeneration, gliosis, and demyelination in leukoaraiosis (LA). Lumbar CSF samples were taken from 37 elderly subjects with LA on computed tomography (CT). Patients with other pathology than LA or infarction on CT were excluded. In addition, CSF samples were collected from 16 elderly reference subjects without any neurological disease. Gangliosides GM1, GD1a, GD1b, GT1b, GD3, and sulfatide were determined. This concentration of the individual gangliosides and sulfatide showed no correlation with age. Gangliosides GD1b, GT1b, and GD3 were elevated in patients with mild LA compared to controls and patients with moderate or severe LA. GD1a was elevated in patients with mild LA compared to those with moderate LA. The concentration of sulfatide did not differ between the groups. When the patients were grouped in accordance to whether they had had cerebral infarction or not, differences between the groups were not found in the concentrations of any gangliosides and sulfatide. In conclusion, the analysis of CSF markers suggests that neuronal degeneration and gliosis predominate in the early stage of LA.

Increased cerebrospinal fluid ganglioside GM1 concentrations indicating neuronal involvement in all stages of HIV-1 infection.

UNLABELLED: Measurements of cerebrospinal fluid (CSF) concentrations of gangliosides can be used as markers of central nervous system (CNS) neuronal involvement. We have analysed the CSF concentrations of the four major brain gangliosides GM1, GD1a, GD1b, and GT1b at different stages of HIV-1 infection. CSF samples were collected from 44 HIV-1-infected patients and from 24 HIV-negative, healthy controls. A significantly higher mean CSF concentration of the ganglioside GM1 was found in HIV-1-infected patients than in HIV-negative controls (27 and 19 nmol/l, respectively, P<0.01). The HIV-infected patients also had a higher mean GM1 proportion of the total ganglioside concentration (11% compared with 8.5%, P < 0.01). Nine out of 27 patients with asymptomatic HIV-1 infection, three of ten with AIDS without neurological complications, and three of seven with AIDS dementia complex had CSF GM1 concentrations above the mean+2SD in the HIV-negative control group. CONCLUSION: Biochemical signs of ongoing neuronal involvement could be found in about one third of HIV-1-infected patients. The same frequency was found regardless of stage, although the highest levels of CSF gangliosides were found in patients with AIDS.

Detection of medulloblastoma and astrocytoma-associated ganglioside GD3 in cerebrospinal fluid.

Shedding of gangliosides by tumor cells may enhance tumor development. We recently showed that cells of the human brain tumor, medulloblastoma, shed gangliosides in vitro and have therefore examined ganglioside shedding by pediatric brain tumors into the cerebrospinal fluid (CSF). GD3, a major ganglioside in medulloblastoma and astrocytoma, was the target for detection in the CSF by immunostaining using the monoclonal antibody R24 and enhanced chemiluminescence detection. Mean CSF GD3 levels in patients with medulloblastomas (n = 9) and astrocytomas (n = 10) were significantly higher than those of controls (mean +/- SD 44.7 +/- 8.4 versus 18.2 +/- 1.9 pmol/ml, n = 20, P < 0.0002). Mass spectrometric analysis showed that tumor-derived ganglioside GD3 contained heterogeneous ceramide structures and, interestingly, the ceramide subspecies with shorter fatty acyl chains were selectively shed. The elevated CSF GD3 concentrations in patients with medulloblastoma and astrocytoma support the concept that ganglioside shedding, which may have significant biological consequences, is characteristic of human brain tumors.

Gangliosides in children with autism.

Concentrations of the four major brain gangliosides, GM1, GD1a, GD1b and GT1b, biochemical markers of neuronal membranes, were determined in the cerebrospinal fluid (CSF) of 20 children with autism and in 25 controls. In addition, the gangliosides were determined in children with different forms of non-progressive neurological disorders lacking clinical features of autism. GM1, GD1a, GD1b and GT1b were significantly increased in patients with autism compared with age-matched controls and children with non-progressive neurological disorders. The gangliosides have previously been shown to have a function in synaptic transmission and increased synaptic activity leads to added release of gangliosides. Our finding of increased CSF levels of gangliosides in autism suggests increased synaptic activity in this disorder.