Sulfatide isoform pattern in cerebrospinal fluid discriminates progressive MS from relapsing-remitting MS.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Several biomarkers including proteins and lipids have been reported in MS cerebrospinal fluid (CSF), reflecting different aspects of the pathophysiology particularly of relapsing-remitting MS (RRMS). Sulfatide, abundant in the myelin sheath and a proposed target for autoimmune attack in MS, has been reported altered in MS CSF. Here, we investigated the potential of CSF sulfatide and its isoforms as biomarkers in MS. A highly sensitive and quantitative mass spectrometry method was employed to determine levels of sulfatide isoforms in CSF from RRMS and progressive MS (PMS) patients, and healthy donors (HD). We demonstrate that levels of total CSF sulfatide and C24:1, C26:1, and C26:1-OH isoforms were significantly increased in PMS compared with RRMS patients and HD, while C23:0-OH was significantly decreased in CSF from PMS patients compared to the other two groups. Multivariate discriminant analysis showed that CSF sulfatide isoform pattern in PMS patients was distinct and non-overlapping with that of RRMS patients and HD. Sulfatide levels did not correlate with tested biomarkers or clinical parameters. The results suggest that CSF sulfatide isoform levels may be used to discriminate the phenotype of MS and might play a role in the progression of the disease.

High-throughput analysis of sulfatides in cerebrospinal fluid using automated extraction and UPLC-MS/MS.

Sulfatides (STs) are a group of glycosphingolipids that are highly expressed in brain. Due to their importance for normal brain function and their potential involvement in neurological diseases, development of accurate and sensitive methods for their determination is needed. Here we describe a high-throughput oriented and quantitative method for the determination of STs in cerebrospinal fluid (CSF). The STs were extracted using a fully automated liquid/liquid extraction method and quantified using ultra-performance liquid chromatography coupled to tandem mass spectrometry. With the high sensitivity of the developed method, quantification of 20 ST species from only 100 µl of CSF was performed. Validation of the method showed that the STs were extracted with high recovery (90%) and could be determined with low inter- and intra-day variation. Our method was applied to a patient cohort of subjects with an Alzheimer's disease biomarker profile. Although the total ST levels were unaltered compared with an age-matched control group, we show that the ratio of hydroxylated/nonhydroxylated STs was increased in the patient cohort. In conclusion, we believe that the fast, sensitive, and accurate method described in this study is a powerful new tool for the determination of STs in clinical as well as preclinical settings.

Cerebrospinal fluid sulfatide isoforms lack diagnostic utility in separating progressive from relapsing-remitting multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system. The glycosphingolipid sulfatide, a lipid particularly enriched in the myelin sheath, has been shown to be involved the maintenance of this specific membrane structure. Sulfatide in cerebrospinal fluid (CSF) may reflect demyelination, a dominating feature of MS. We investigated the diagnostic utility of CSF sulfatide isoform levels to separate different courses or phenotypes of MS disease. MATERIAL AND METHODS: This was a mono-center, cross-sectional study of relapsing-remitting MS (RRMS) (n = 45) and progressive MS (PMS) (n = 42) patients (consisting of primary PMS (n = 17) and secondary PMS (n = 25)) and healthy controls (n = 19). In total, 20 sulfatide isoforms were measured in CSF by liquid chromatography-mass spectrometry. RESULTS: CSF total sulfatide concentrations, as well as CSF sulfatide isoform distribution, did not differ across the study groups, and their levels were independent of disease course/phenotype, disease duration, time to conversion to secondary PMS, age, and disability in MS patients. CONCLUSION: CSF sulfatide isoforms lack diagnostic and prognostic utility as a biomarker for progressive MS.

Low cerebrospinal fluid sulfatide predicts progression of white matter lesions: The LADIS study.

BACKGROUND/AIMS: Demyelination and axonal degeneration are the hallmarks of established white matter lesions (WML). The neurochemistry of ongoing WML is only partially known. We explored cerebrospinal fluid (CSF) substances as markers of brain tissue damage in relation to progression of WML rated on magnetic resonance imaging. METHODS: CSF from elderly individuals with WML was analyzed for amyloid markers, total τ, hyperphosphorylated τ, neurofilament protein light subunit, sulfatide and CSF/serum-albumin ratio. After 3 years, a follow-up magnetic resonance imaging was performed. Progression of WML was rated using the Rotterdam Progression Scale (RPS). RESULTS: 37 subjects (age 73.6 ± 4.6 years) were included. Subjects with more pronounced progression (RPS > 2; n = 15) had lower mean sulfatide concentration at baseline as compared to subjects with no or minimal progression (RPS 0-2; n = 22) according to univariate analyses (p = 0.009). Sulfatide was the only biomarker that predicted the RPS score according to regression analysis, explaining 18.9% of the total variance (r = 0.38, p = 0.015). CONCLUSION: The correlation of CSF sulfatide levels and RPS scores may reflect a remyelination response to the demyelination process associated with WML. Furthermore, the results strengthen the notion that WML pathology is different from that of Alzheimer's disease.

Cerebrospinal Fluid Sulfatide Levels Lack Diagnostic Utility in the Subcortical Small Vessel Type of Dementia.

BACKGROUND: Sulfatides (STs) in cerebrospinal fluid (CSF), as well as magnetic resonance imaging (MRI)-detected white matter hyperintensities (WMHs), may reflect demyelination. Here, we investigated the diagnostic utility of CSF ST levels in the subcortical small vessel type of dementia (SSVD), which is characterized by the presence of brain WMHs. OBJECTIVE: To study the diagnostic utility of CSF ST levels in SSVD. METHODS: This was a mono-center, cross-sectional study of SSVD (n = 16), Alzheimer's disease (n = 40), mixed dementia (n = 27), and healthy controls (n = 33). Totally, 20 ST species were measured in CSF by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS: CSF total ST levels, as well as CSF levels of hydroxylated and nonhydroxylated ST species, did not differ across the study groups. In contrast, CSF neurofilament light chain (NFL) levels separated the patient groups from the controls. CSF total ST level correlated with CSF/serum albumin ratio in the total study population (r = 0.64, p < 0.001) and in all individual study groups. Furthermore, CSF total ST level correlated positively with MRI-estimated WMH volume in the total study population (r = 0.30, p < 0.05), but it did not correlate with CSF NFL level. CONCLUSION: Although there was some relation between CSF total ST level and WMH volume, CSF ST levels were unaltered in all dementia groups compared to the controls. This suggests that CSF total ST level is a poor biomarker of demyelination in SSVD. Further studies are needed to investigate the mechanisms underlying the marked correlation between CSF total ST level and CSF/serum albumin ratio.

Cerebrospinal fluid biomarkers of white matter lesions - cross-sectional results from the LADIS study.

BACKGROUND AND PURPOSE: White matter lesions (WMLs) caused by small vessel disease are common in elderly people and contribute to cognitive impairment. There are no established biochemical markers for WMLs. We aimed to study the relation between degree of WMLs rated on magnetic resonance imaging of the brain and cerebrospinal fluid (CSF) levels of structural biomarkers associated with Alzheimer's disease (AD) and subcortical vascular dementia. METHODS: Fifty-three non-demented elderly individuals with WMLs were subjected to lumbar puncture. Degree of WMLs was rated using the Fazekas scale. Volumetric assessment of WMLs was performed. CSF samples were analyzed for the 40 and 42 amino acid fragments of amyloid beta, alpha- and beta-cleaved soluble amyloid precursor protein, total tau (T-tau), hyperphosphorylated tau (P-tau(181)), neurofilament light protein (NFL), sulfatide and CSF/Serum-albumin ratio. RESULTS: Fifteen subjects had mild, 23 had moderate and 15 had severe degree of WMLs. CSF-NFL levels differed between the groups (P < 0.001) and correlated with the volume of WMLs (r = 0.477, P < 0.001). CSF sulfatide concentration displayed similar changes but less strongly. T-tau, P-tau(181) and the different amyloid markers as well as CSF/S-albumin ratio did not differ significantly between the groups. CONCLUSIONS: The association of increased CSF-NFL levels with increasing severity of WMLs in non-demented subjects suggests that NFL is a marker for axonal damage in response to small vessel disease in the brain. This manifestation may be distinct from or earlier than the neurodegenerative process seen in AD, as reflected by the lack of association between WMLs and AD biomarkers.

Normal pressure hydrocephalus triggers intrathecal production of TNF-alpha.

Normal pressure hydrocephalus (NPH) is associated with periventricular white matter lesions and demyelination. The aim of the present study was to examine the cerebrospinal fluid (CSF) levels of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine mediating myelin damage, in patients with NPH. TNF-alpha levels were analyzed by ELISA and measured before and after shunt operation in 35 patients with NPH. The levels of this cytokine were related to the symptomatology and to magnetic resonance imaging (MRI) verified white matter lesions. They were also related to intrathecal levels of sulfatide, a marker for white matter degradation and to levels of neurofilament, a marker for neuronal degeneration. The preoperative levels of TNF-alpha were increased in the CSF of NPH patients compared to controls, and correlated to the levels of sulfatide. The intrathecal TNF-alpha levels were higher in NPH patients with impairment of wakefulness than in those without this symptom. The preoperative TNF-alpha levels were significantly correlated to the improvement of psychometrical test scores, and of wakefulness and to the overall improvement of the patients following shunt operation. Importantly, shunt operation led to complete disappearance of intrathecal TNF-alpha. We conclude that NPH is correlated with intrathecal TNF-alpha production being reversed following shunt operation in parallel with the clinical improvement. The positive correlation between preoperative TNF-alpha and sulfatide levels in the CSF suggest that intrathecal TNF-alpha may contribute to the damage of the white matter known to occur in patients with NPH.

High levels in serum, but no signs of intrathecal synthesis of anti-sulfatide antibodies in HIV-1 infected individuals with or without central nervous system complications.

Myelin degeneration is commonly found in the central nervous system (CNS) of individuals infected with human immunodeficiency virus type 1 (HIV-1), especially in patients with HIV-1-associated dementia. We analysed cerebrospinal fluid (CSF) and serum samples from 25 HIV-1 infected individuals for the presence of antibodies directed against sulfatide, the major acidic glycosphingolipid in myelin. Nine of the patients had CNS complications, including 3 with HIV-1-associated dementia, and 16 had no neurological symptoms. Elevated titres of anti-sulfatide antibodies were found in serum from 24/25 HIV-1-infected individuals but in none of them in the CSF. Although the vast majority of HIV-1-infected individuals harbour autoantibodies directed against sulfatide in serum, the lack of detectable intrathecal production indicates that anti-sulfatide antibodies are not a major component in the pathogenesis of CNS myelin damage in HIV-1 infection.

Elevated cerebrospinal fluid sulfatide concentrations as a sign of increased metabolic turnover of myelin in HIV type I infection.

Cerebrospinal fluid (CSF) sulfatide concentrations were analyzed in 18 patients with asymptomatic HIV-1 infection, in 16 patients with AIDS who were free from opportunistic infections in the central nervous system (CNS), in 12 HIV-1-infected patients with opportunistic CNS infections or lymphoma, and in 19 HIV-negative controls, by thin-layer chromatography overlay technique using an antisulfatide antibody to estimate the metabolic turnover of myelin. The majority of asymptomatic HIV-1-infected patients had normal CSF sulfatide concentrations, but the mean CSF sulfatide concentration was still elevated compared to that in HIV-negative controls (152 compared to 99 nmol/liter, p < 0.05). The CSF sulfatide concentrations in the AIDS group (mean 395 nmol/liter) were significantly increased compared to those in asymptomatic HIV-1-infected patients (p < 0.01) and in HIV-negative controls (p < 0.001), but did not differ significantly between patients with and without dementia. Increased CSF sulfatide concentrations were also found in patients with opportunistic infection or lymphoma in the CNS. In the entire study population, the sulfatide levels were associated with blood-brain barrier function, but not with intrathecal immunoglobulin production or with positive HIV isolations from CSF. Thus, signs of white matter changes, measured as increased CSF sulfatide concentrations, could be found in some asymptomatic HIV-1-infected patients, but the highest levels were seen in patients with AIDS.

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.

Determination of gangliosides and sulfatide in human cerebrospinal fluid with a microimmunoaffinity technique.

An immunostaining procedure has been developed for the assay of the gangliotetraose gangliosides and sulfatide in cerebrospinal fluid. Gangliosides of the gangliotetraose series were individually determined with cholera toxin B-subunit (CT-B) and an anti CT-B monoclonal antibody after chromatography and sialidase hydrolysis to GM1 on high performance thin-layer plates. Sulfatide was determined by thin-layer chromatography using an anti-sulfatide antibody. The method was applied to normal cerebrospinal fluid from 20 adults and 30 children. The concentration of the gangliotetraose series gangliosides in adults varied from 100-300 nmol/l with a mean value of 230 +/- 56 nmol/l. Corresponding values for sulfatide were 30-225 nmol/l and 140 +/- 46 nmol/l. The values for gangliosides and sulfatide in children increased during development. The major gangliosides in cerebrospinal fluid of adults were GT1b and GD1b and in children GD1a and GT1b.

Increased CSF sulfatide levels and serum glycosphingolipid antibody levels in healthy siblings of multiple sclerosis patients.

A proportion of healthy siblings of multiple sclerosis (MS) patients have an oligoclonal immunological reaction in their cerebrospinal fluid (CSF) termed the "MS oligoclonal trait". The CSF levels of the major myelin glycosphingolipid sulfatide and serum antibodies against the glycosphingolipids sulfatide and galactosylceramide were recently reported to be increased in MS patients. We studied the levels of these substances in pairs of 46 patients and their 46 healthy siblings and 50 unrelated healthy blood donors (HBD). The sulfatide concentration in CSF was assayed by thin layer chromatography and immunostaining, and the concentration of galactosylceramide by densitometry after thin layer chromatography. Anti-glycosphingolipid antibody levels were assayed by ELISA. In the healthy siblings, the CSF sulfatide concentrations were markedly increased (p<0.001, age adjusted p=0.025), and the serum IgM anti-GalCer antibodies were increased in healthy siblings compared with HBD (p=0.02). The increased sulfatide or antibody levels did not co-segregate with the "MS oligoclonal trait" or the HLA-DR15 phenotype. In conclusion, a proportion of healthy siblings of MS patients have increased CSF sulfatide and anti-glycosphingolipid antibody levels, which may, analogous to the "MS oligoclonal trait", constitute an "MS glycosphingolipid endophenotype". Endophenotypes could potentially simplify the genetics of complex disorders.

Late cerebral graft versus host reaction in a bone marrow transplanted girl with Hurler (MPS I) disease.

A girl with Hurler disease (MPS IH) underwent allogeneic stem cell transplantation at 13 months of age with her one HLA-B antigen mismatch mother as donor. The procedure was complicated by cerebral hemorrhage and a ventricular-peritoneal shunt device was inserted. Mild GVH reactions were rapidly reversed. One year after transplantation ventriculitis was suspected and the shunt was replaced by a ventricular drainage catheter. Antibiotics had no effect and graft-versus-host disease (GVHD) was diagnosed. All symptoms were reversed by prednisolone and cyclosporine. Increased albumin and pleocytosis in the cerebrospinal fluid (CSF) normalized concomitantly. Electron microscopy of the CSF sediment showed debris consisting of numerous complex aggregates of thin lamellae and electron dense fragments with a tight lamellar texture. Biochemical analysis of the CSF sediment proved that the debris contained galactosylceramide and sulfatide. The electron microscopic and biochemical findings were interpreted to represent stripping of central myelin as a result of subacute GVHD in the central nervous system and its desquamation from the brain parenchyma into the ventricular CSF through the post-hemorrhage defect. From reversal of the GVHD at 2 years of age until follow-up at 10 years of age the clinical condition remained stable with no recurrence or deterioration.

CSF biomarkers in the evaluation of idiopathic normal pressure hydrocephalus.

BACKGROUND - To evaluate cerebrospinal fluid (CSF) markers for neuronal degeneration and demyelination in idiopathic normal pressure hydrocephalus (INPH), subcortical arteriosclerotic encephalopathy (SAE), and neurologically healthy subjects. METHODS - Lumbar CSF concentrations of sulfatide, neurofilament protein light (NFL), total-tau (T-tau), hyperphosphorylated tau (P-tau), and beta-amyloid(1-42) (Abeta42) were analyzed in 62 INPH patients, 26 SAE patients, and 23 neurologically healthy controls. In INPH patients, samples before and after shunt surgery were analysed. RESULTS - The CSF concentration of NFL was elevated in INPH and SAE compared with the controls, and levels of T-tau, P-tau, and Abeta42 were lower in INPH compared with SAE and controls. No difference was seen for sulfatide. All markers except Abeta42 were significantly elevated after shunt surgery. CONCLUSIONS - The most striking finding was the power of the combined pattern of NFL, P-tau, and Abeta42 in distinguishing between the clinical diagnoses of INPH, SAE, and neurologically healthy elderly.

Temporarily controlled HIV-1 replication after intravenous immunoglobulin treatment of Guillain-Barré syndrome.

HIV establishes a latent infection in resting CD4(+) T-lymphocytes. A possible strategy to eliminate cellular reservoirs in long-lived, HIV-1-infected quiescent CD4(+) T-lymphocytes might be to add T-cell-activating agents to potent antiretroviral therapy. In this report we describe a patient with Guillain-Barré syndrome treated with high dose intravenous immunoglobulin (IVIG) in addition to antiretroviral therapy. A transiently increased viral load and immunoactivation during the IVIG treatment suggest activation of latently infected cells and increased turnover rate of the latent viral reservoir. HIV replication was controlled with plasma viral load <20 copies/ml, for at least 3 months after antiretroviral treatment interruption. CSF neural markers reflecting degenerative processes in the brain during the symptomatic period and follow-up were also analysed. Very high CSF sulfatide concentrations were found indicating that the pathology involves severe demyelination.We hypothesize that IVIG in this case contributed to an activation of latently infected cells, which led to a transient increase in plasma HIV-1 RNA during the IVIG treatment and a long period of undetectable viral load after antiretroviral treatment interruption. Further, this is the first time, to our knowledge, that detailed CSF findings are described in HIV-1 associated GBS.

Gangliosides and sulphatide in human cerebrospinal fluid: quantitation with immunoaffinity techniques.

A sensitive micromethod involving extraction, purification and thin-layer chromatography (TLC)-enzyme immunostaining was developed for the quantation of gangliosides and sulphatide, as markers for neuronal disorders and myelin disturbances, in individual samples of less than 5 ml of cerebrospinal fluid. The gangliosides of the gangliotetraose series were individually determined with cholera toxin subunit B by TLC-enzyme-linked immunosorbent assay (ELISA) after chromatography and subsequent sialidase hydrolysis to II3NeuAc-GgOse4Cer (GM1). Other gangliosides and sulphatide were determined with specific monoclonal antibodies by TLC-ELISA. The total ganglioside content varied between 100 and 230 nmol/l in ten normal cerebrospinal fluid samples from adults. The major gangliosides were of the gangliotetraose series, represented by GM1, IV3NeuAc,II3NeuAc-GgOse4Cer, (GD1a), II3(NeuAc)2-GgOse4Cer (GD1b) and IV3NeuAc,II3 (NeuAc)2-GgOse4Cer (GT1b) of which the b-series gangliosides dominated, i.e., GD1b and GT1b.

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.

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.

Sulfatide as a biochemical marker in cerebrospinal fluid of patients with vascular dementia.

The myelin-associated glycosphingolipid sulfatide in cerebrospinal fluid (CSF) was investigated in 20 patients with vascular dementia (VAD), 43 with Alzheimer's disease (AD) and 20 age-matched controls. The sulfatide concentration in the VAD group (307 +/- 118 nmol/l) was significantly (p less than 0.0001) higher than that in controls (145 +/- 86 nmol/l) and the AD group (178 +/- 79 nmol/l). Among the VAD patients, 8/20 had a significantly increased concentration of sulfatide (greater than mean + 2 S.D.), as compared with controls, while only 2/43 of the AD patients had a sulfatide concentration above this level. It is suggested that the elevated concentration of sulfatide in CSF from VAD patients reflects demyelination. Furthermore, sulfatide determinations, when combined with clinical findings, may be of diagnostic value, for discriminating between VAD and AD.