Alpha-mannosidosis: characterization of CNS pathology and correlation between CNS pathology and cognitive function.

Alpha-mannosidosis (AM) (OMIM 248500) is a rare lysosomal storage disease. The understanding of the central nervous system (CNS) pathology is limited. This study is the first describing the CNS pathology and the correlation between the CNS pathology and intellectual disabilities in human AM. Thirty-four patients, aged 6-35 years, with AM were included. Data from 13 healthy controls were included in the analysis of the magnetic resonance spectroscopy (MRS). Measurements of CNS neurodegeneration biomarkers in cerebrospinal fluid (CSF), CSF-oligosaccharides, and performance of cerebral magnetic resonance imaging (MRI) and MRS were carried out. On MRI, 5 of 10 patients had occipital white matter (WM) signal abnormalities, and 6 of 10 patients had age-inappropriate myelination. MRS demonstrated significantly elevated mannose complex in gray matter and WM. We found elevated concentrations of tau-protein, glial fibrillary acidic protein and neurofilament light protein in 97 patients, 74% and 41% of CSF samples, respectively. A negative correlation between CSF-biomarkers and cognitive function and CSF-oligosaccharides and cognitive function was found. The combination of MRS/MRI changes, elevated concentrations of CSF-biomarkers and CSF-oligosaccharides suggests gliosis and reduced myelination, as part of the CNS pathology in AM. Our data demonstrate early neuropathological changes, which may be taken into consideration when planning initiation of treatment.

Administration of sulfatide to ameliorate type I diabetes in non-obese diabetic mice.

The endogenous glycosphingolipid sulfatide is a ligand for CD1d-restricted type II natural killer T (NKT) lymphocytes. Through the action of these cells,sulfatide treatment has been shown to modulate the immune response in mouse models for autoimmune diseases, infections and tumour immunity. Sulfatide exists naturally in different organs including the pancreas, where sulfatide colocalizes with insulin within the Langerhans islet b-cells, targets for the immune destruction in type 1 diabetes (T1D). Human T1D patients, but not patients with type 2 diabetes nor healthy individuals, have autoantibodies against sulfatide in serum, suggesting that sulfatide induces an immune response in the natural course of T1D in humans. Here, we investigate sulfatide as an autoantigen and a modulator of autoimmune disease in the murine model forT1D, the non-obese diabetic (NOD) mice. We demonstrate that aged NOD mice displayed serum autoantibody reactivity to sulfatide; however, this reactivity did not correlate with onset of T1D. Repeated administration of sulfatide did not result in an increase in serum reactivity to sulfatide. Moreover, a multidose sulfatide treatment of female NOD mice initiated at an early (5 weeks of age),intermediate (8 weeks of age) or late (12 weeks of age) phase of T1D progression did not influence the incidence of disease. Thus, we demonstrate that a fraction of NOD mice develop autoantibody reactivity to sulfatide; however, we fail to demonstrate that sulfatide treatment reduces the incidence of T1D in this mouse strain.

Children born extremely preterm show significant lower cognitive, language and motor function levels compared with children born at term, as measured by the Bayley-III at 2.5 years.

AIM: To assess developmental outcomes of children aged 2.5 years born extremely preterm. METHODS: As a part of the population-based Extremely Preterm Infants in Sweden Study (EXPRESS), 399 children born before 27 weeks of gestation and 366 control children born at term were assessed with the Bayley Scales of Infant and Toddler Development, third edition (Bayley-III), assigning scores for cognition, receptive and expressive communication, fine and gross motor functions. Based on control group means, prevalences of developmental delay in the preterm group were calculated. Mean score differences between subtests constituting the overall Bayley-III indices were analysed within both groups. RESULTS: After controlling for socio-demographic, child and assessment variables, analyses showed significantly lower performances of the preterm group compared with the control group on the Bayley-III subtests. Prevalence of moderate-severe delay was 10.8% in cognitive, 14.9% in receptive communication, 14.5% in expressive communication, 12.4% in fine motor and 7.0% in gross motor functions. Significant differences between performances on subtests included in the same indices were detected. CONCLUSION: Extremely preterm children show significant lower cognitive, communicative and motor function levels at 2.5 years compared with children born at term. Bayley-III assessments permit the acquisition of nuanced information about development following extreme prematurity.

Enzyme replacement therapy for alpha-mannosidosis: 12 months follow-up of a single centre, randomised, multiple dose study.

BACKGROUND: Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disease (LSD) caused by alpha-mannosidase deficiency. Manifestations include intellectual disabilities, facial characteristics and hearing impairment. A recombinant human alpha-mannosidase (rhLAMAN) has been developed for weekly intravenous enzyme replacement therapy (ERT). We present the preliminary data after 12 months of treatment. METHODS: This is a phase I-II study to evaluate safety and efficacy of rhLAMAN. Ten patients (7-17 y) were treated. We investigated efficacy by testing motor function (6-minutes-Walk-Test (6-MWT), 3-min-Stair-Climb-Test (3-MSCT), The Bruininks-Oseretsky Test of Motor Proficiency (BOT2), cognitive function (Leiter-R), oligosaccharides in serum, urine and CSF and Tau- and GFA-protein in CSF. RESULTS: Oligosaccharides: S-, U- and CSF-oligosaccharides decreased 88.6% (CI -92.0 -85.2, p < 0.001), 54.1% (CI -69.5- -38.7, p < 0,001), and 25.7% (CI -44.3- -7.1, p < 0.05), respectively. Biomarkers: CSF-Tau- and GFA-protein decreased 15%, p < 0.009) and 32.5, p < 0.001 respectively. Motor function: Improvements in 3MSCT (31 steps (CI 6.8-40.5, p < 0.01) and in 6MWT (60.4 m (CI -8.9 -51.1, NS) were achieved. Cognitive function: Improvement in the total Equivalence Age of 4 months (0.34) was achieved in the Leiter R test (CI -0.2-0.8, NS). CONCLUSIONS: These data suggest that rhLAMAN may be an encouraging new treatment for patients with alpha-mannosidosis.The study is designed to continue for a total of 18 months. Longer-term follow-up of patients in this study and the future placebo-controlled phase 3 trial are needed to provide greater support for the findings in this study.

Altered distribution of the gangliosides GM1 and GM2 in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder where ß-amyloid tends to aggregate and form plaques. Lipid raft-associated ganglioside GM1 has been suggested to facilitate ß-amyloid aggregation; furthermore, GM1 and GM2 are increased in lipid rafts isolated from cerebral cortex of AD cases. AIM/METHOD: The distribution of GM1 and GM2 was studied by immunohistochemistry in the frontal and temporal cortex of AD cases. Frontotemporal dementia (FTD) was included as a contrast group. RESULTS: The distribution of GM1 and GM2 changes during the process of AD (n = 5) and FTD (n = 3) compared to controls (n = 5). Altered location of the GM1-positive small circular structures seems to be associated with myelin degradation. In the grey matter, the staining of GM1-positive plasma membranes might reflect neuronal loss in the AD/FTD tissue. The GM1-positive compact bundles were only visible in cells located in the AD frontal grey matter, possibly reflecting raft formation of GM1 and thus a pathological connection. Furthermore, our results suggest GM2 to be enriched within vesicles of pyramidal neurons of the AD/FTD brain. CONCLUSION: Our study supports the biochemical finding of ganglioside accumulation in cellular membranes of AD patients and shows a redistribution of these molecules.

Spatial and temporal predictions of moose winter distribution.

Herbivores are usually distributed unevenly across the landscape often because of variation in resource availability. We used zero-inflated generalised additive models (to account for data with a high number of zeros) that include georeferences to predict winter distribution of a large herbivore (moose Alces alces). Moose distribution was analysed in relation to forage availability and distance to neighbouring sites. Our results showed that the ability to explain moose distribution indexed by pellet count data at a local scale increased when spatial information (longitude and latitude) was added to the model compared to the model only including food availability. By using the relationship between moose and forage distribution, and the spatial information, we predicted patch choice by moose reasonably well in 2 out of 4 years. However, the distribution of moose was also influenced by weather conditions, as it was most clumped in the year with most snow. In conclusion, our study lends support for a non-linear approach using georeferences for a comprehensive understanding of herbivore distribution at a small scale. This result also indicates that the use of a certain patch by moose not only depends on the selected patch itself but is also influenced by the neighbouring patch and factors at a larger spatial scale, such as moose management influencing the density above moose home range level. The relatively high proportion of unexplained variation suggests that the use of a certain patch is also influenced by other factors such as topography, predation, competition, weather conditions, and wildlife management strategies.

The glycolipid sulfatide protects insulin-producing cells against cytokine-induced apoptosis, a possible role in diabetes.

AIMS/HYPOTHESIS: Cytokine-induced apoptosis is recognised as a major cause of the decline in ß-cell mass that ultimately leads to type 1 diabetes mellitus. Interleukin-1ß, interferon-γ and tumour necrosis factor-α in conjunction initiate a series of events that lead to ß-cell apoptosis; important among these is NO production. The glycosphingolipid sulfatide is present in ß-cells in the secretory granules in varying amounts and is secreted together with insulin. We now investigate whether sulfatide is able to protect insulin-producing cells against the pro-apoptotic effect of interleukin-1ß, interferon-γ and tumour necrosis factor-α. METHODS: INS-1E cells and genuine rat islets were incubated for 24 h exposed to interleukin-1ß, interferon-γ and tumour necrosis factor-α with or without sulfatide. The production of NO was monitored and the number of apoptotic cells was determined using terminal deoxynucleotidyl transferase-mediated dUTP Nick-End labelling and caspase-3/7 activity assays. In addition, the amount of iNOS mRNA was determined using real-time quantitative polymerase chain reaction. RESULTS: Cytokine-induced apoptosis was reduced to 27% of cytokine-treated controls with 30 µmol/L sulfatide treatment (p < 0.01). Likewise, sulfatide in concentrations of 3-30 µmol/L decreased NO production in a dose-dependent manner to 19-40% of cytokine-treated controls (overall p = 0.0007). The level of iNOS mRNA after cytokine exposure was reduced to 55% of cytokine-treated controls with 30 µmol/L of sulfatide. CONCLUSIONS/INTERPRETATION: In the present study, we report the ability of sulfatide to significantly reduce apoptosis, cellular leakage and NO production in insulin-producing cells. Data suggest this is not due to induction of ß-cell rest. Our findings indicate a possible implication for sulfatide in the pathogenesis of diabetes.

Mucopolysaccharidosis type III (Sanfilippo disease) in Sweden: clinical presentation of 22 children diagnosed during a 30-year period.

AIM: The aim of this study was to present the natural clinical course in children and adolescents with MPS III diagnosed during a 30-year period in Sweden. METHODS: The patients were identified from metabolic laboratory records between 1975 and 2004. Patient data were assessed from interviews of parents and by clinical examination and records from the patients. RESULTS: A total of 15 children, 68%, with MPS IIIA were diagnosed at a median age of 6.8 years (range 1.2-18.9 years). One boy had MPS IIIB and five children MPS IIIC, diagnosed at ages between 1.9 and 11.6 years. In one child the type was not determined. The median age of children with type IIIA who had deceased was 16.2 years (range 10.4-31.2 years). Ten individuals with MPS III are alive at ages between 5 and 29 years. In four families, two children were affected. CONCLUSION: In 22 Swedish children with Sanfilippo disease an early normal development followed by a delay in speech and an appearance of behaviour problems was found in most children during the early preschool period. Mental retardation was diagnosed in almost all individuals before starting school. Early diagnosis is important in this devastating, progressive disorder, not only for genetic counselling but also for participation in future treatments.

Elevated cerebrospinal fluid levels of prostaglandin E2 and 15-(S)-hydroxyeicosatetraenoic acid in multiple sclerosis.

OBJECTIVE: To test the hypothesis that the arachodinic acid metabolites prostaglandin E2 (PGE2) and 15-(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in cerebrospinal fluid (CSF) are elevated and reflect neuroinflammation and degenerative changes in multiple sclerosis (MS). PATIENTS AND METHODS: We measured PGE2 and 15(S)-HETE concentrations, as well as markers of axonal and astroglial injury in CSF from 46 MS patients, 46 healthy siblings and 50 controls. RESULTS: We found elevated levels of both PGE2 and 15(S)-HETE in MS compared with the control and sibling groups. Siblings had lower PGE2 levels and higher 15(S)-HETE levels than controls. There were no correlations between either PGE2 or 15(S)-HETE and clinical scores of MS severity or biochemical markers of axonal or astroglial injury. CONCLUSION: These data suggest no direct involvement of PGE2 and 15(S)-HETE in the MS disease process. Rather, the elevated levels reflect a general up-regulation of arachidonic acid metabolism and neuroinflammation.

The g.1170C>T polymorphism of the 5' untranslated region of the human alpha-galactosidase gene is associated with decreased enzyme expression--evidence from a family study.

Subnormal leukocyte α-galactosidase (α-Gal) activity was found during evaluation of an adolescent male with cryptogenic cerebrovascular small-vessel disease. The only molecular abnormality found was the g.1170C>T single-nucleotide polymorphism (SNP) in the 5' untranslated region of exon 1 in the α-Gal gene (GLA). Historically, this polymorphism has been considered to be biologically neutral. To test the hypothesis that the g.1170T allele might be associated with lower α-Gal expression, we genotyped GLA exon 1 and measured leukocyte and plasma α-Gal in the parents, brother and sister of the index case. The g.1170T allele co-segregated with a subnormal leukocyte α-Gal activity in the three siblings. Although plasma enzyme activities were within the normal range in all five relatives, the ranking of their values suggested a dosage effect of the g.1170T allele. Western blotting assays of leukocyte protein extracts showed that the relative expression of α-Gal in both the patient and his sister was significantly lower than in sex-matched hemizygous or homozygous controls for the g.1170C allele, either normalized to the ß-actin immunoblot expression or standardized to a known amount of recombinant human α-Gal. These family data, in combination with results from a recent GLA SNP screening study among healthy Portuguese individuals, suggest that the g.1170C>T SNP may be co-dominantly associated with a relatively decreased GLA expression at the transcription and/or translation level. Larger population studies are needed to confirm these findings and to test the hypothesis that the GLA g.1170C>T may contribute to the multifactorial risk of ischaemic small-vessel cerebrovascular disease.

Effect of single-nucleotide polymorphisms of the 5' untranslated region of the human α-galactosidase gene on enzyme activity, and their frequencies in Portuguese caucasians.

BACKGROUND: The α-galactosidase gene (GLA) has three single-nucleotide polymorphisms in the 5' untranslated region of exon 1, respectively g.1150G>A, g.1168G>A, g.1170C>T. The g.1150A allele is associated with increased plasma α-galactosidase (α-Gal) activity in hemizygotes, while the others are regarded as biologically neutral. The primary goal of this investigation was to test the hypothesis, raised by a clinical observation and results of a family study, that the g.1170T allele polymorphism is associated with lower α-Gal expression. SUBJECTS AND METHODS: Plasma and leukocyte α-Gal activities were assayed in unrelated healthy young adults of both sexes, who had been genotyped for GLA exon 1, and enzyme activity values in carriers of any of the polymorphisms were compared to those of individuals with the standard genotype; GLA exon 1 was genotyped in males who had α-Gal activity in dried blood spots lower than 2 SD below the cohort average. RESULTS AND CONCLUSIONS: Mean α-Gal leukocyte activity was ∼ 25% higher in subjects with the g.1170C or CC genotype than in those with the alternative genotypes (p < 0.05). The frequency of the g.1170T allele in subjects with low α-Gal activity in dried blood spots was 4-fold higher (p < 0.05) than in the general population. As in hemizygotes, the g.1150A heterozygote identified in this study had plasma α-Gal activity more than 2-fold above the normal mean. The g.1168A allele did not affect enzyme activity. Surprisingly, females with the standard GLA exon 1 genotype had significantly higher plasma α-Gal activity than genetically comparable males.

Cerebrospinal fluid tau and amyloid beta42 protein in patients with myotonic dystrophy type 1.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is associated with brain morphology changes including neurofibrillary degeneration. METHODS: We have examined cerebrospinal fluid (CSF) markers indicative of neuronal degeneration and amyloidogenesis; total tau (T-tau), phosphorylated tau (P-tau) and beta amyloid 1-42 (Abeta42), in 32 patients with DM1. RESULTS AND CONCLUSIONS: Associations between CSF markers and CTG repeat expansion size, brain MRI findings, and neuropsychological test results were analysed. As compared with matched controls Abeta42 was significantly decreased (P = 0.001), whilst levels of T-tau were increased (P < 0.001). No difference was found between measures considering P-tau levels. At present the clinical implications of these findings is unclear, because of an overlap between CSF values of DM1 patients and healthy controls, but also regarding modest associations between CSF markers and other measures. However notably, the Tau pathology, as seen in DM1, differs from Alzheimers disease, considering the lack of increased levels of P-tau.

Ventricular cerebrospinal fluid neurofilament protein levels decrease in parallel with white matter pathology after shunt surgery in normal pressure hydrocephalus.

Normal pressure hydrocephalus (NPH) is characterized by disturbed cerebrospinal fluid (CSF) dynamics and white matter lesions (WML). Although the morphology of these lesions is described, little is known about the biochemistry. Our aim was to explore the relationship between ventricular CSF markers, periventricular WML and postoperative clinical outcome in patients with NPH. We analysed lumbar and ventricular concentrations of 10 CSF markers, 12 clinical symptoms and signs, magnetic resonance imaging (MRI) periventricular white matter hyperintensities (PVH) and ventricular size before and 3 months after shunt surgery in 35 patients with NPH. Higher ventricular CSF neurofilament protein (NFL), an axonal marker, correlated with more extensive PVH. A larger postoperative reduction in NFL correlated with larger reduction in PVH and a more pronounced overall improvement. Albumin ratio, HMPG, NPY, VIP and GD3 increased postoperatively whereas NFL, tau and HVA decreased. Variations in ventricular size were not associated with CSF concentrations of any marker. We conclude that NPH is characterized by an ongoing periventricular neuronal dysfunction seen on MRI as PVH. Clinical improvement after shunt surgery is associated with CSF changes indicating a restitution of axonal function. Other biochemical effects of shunting may include increased monoaminergic and peptidergic neurotransmission, breakdown of blood brain barrier function, and gliosis.

Modest phenotypic improvements in ASA-deficient mice with only one UDP-galactose:ceramide-galactosyltransferase gene.

BACKGROUND: Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice. RESULTS: ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice. CONCLUSION: Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology.

Five new epitope-defined monoclonal antibodies reactive with GM2 and human glioma and medulloblastoma cell lines.

In order to investigate GM2 expression in gliomas, the GM2-positive human glioma cell line (HGL) D-54 MG, which contains 0.6 nmol GM2/mg protein, representing 77% of the total monosialoganglioside fraction, was used as an immunogen for the production of anti-GM2 monoclonal antibodies. For ganglioside designations, see IUPAC-IUB (Eur. J. Biochem., 79: 11-21, 1977) and Svennerholm (J. Neurochem., 10: 613-623, 1963). Five IgM monoclonal antibodies (DMAb-1 through DMAb-5) specifically recognizing the GalNAc beta1-4(NeuAc alpha 2-3)Gal-terminal epitope common to GM2 and GalNAC-GD1a are reported. The antibodies did not react with GM1, GM3, GD2, GD3, GD1a, GD1b, and GQ1b. Purified anti-GM2 MAbs were used to define the expression of the "GM2" terminal epitope by cultured human malignant and normal cells by radioimmunoassay and membrane immunofluorescence. Among neuroectodermal tissue-derived cell lines, DMAb-3, at an optimal concentration of 5 micrograms/ml, showed high reactivity (radioimmunoassay binding ratios greater than 20) with 9 of 19 HGLs, 3 of 5 medulloblastoma, 4 of 5 neuroblastoma, and 1 of 3 melanoma lines. Moderate reactivity (binding ratio, 10-20) was exhibited by 3 HGL, 2 medulloblastoma, and 1 neuroblastoma lines and low reactivity (binding ratio, 3-10) by 5 HGL lines; no reactivity was detected with 2 HGL and 2 melanoma lines. Densitometric evaluation of monosialoganglioside extracts from human glioma and medulloblastoma cell lines in conjunction with immunostaining on thin-layer chromatograms showed that GM2 represents the major monosialoganglioside in 8 of 10 HGL and in 3 of 4 Med lines. In these lines the amount of GM2 ranged from less than 0.1 to 0.6 nmol/mg protein. These results indicate that GM2 represents a proportionally increased ganglioside of most glioma, medulloblastoma, and neuroblastoma cells in vitro.

Isolation of complex gangliosides from human brain.

A simple procedure that enables the isolation of ganglioside GQ1b and other complex gangliosides from the human brain is described. The tissue was extracted with a mixture of chloroform, methanol, and water, and the extract purified twice by means of silica gel column chromatography and preparative HPTLC. Phase partition and ion exchange chromatography were omitted. The silica gel chromatography was based on a two step developing system, which provided an efficient separation of oligosialogangliosides. The yields of chromatographically homogenous fractions of ganglioside GQ1b isolated from the whole cerebrum, cerebellar cortex and occipital grey matter of a 60-year-old woman were 62, 138 and 110 nmol SA per g of fresh tissue. The problem of co-extraction of protein-positive material with gangliosides into the organic solvents is discussed. Chromatographic search of gangliosides in different regions of the human brain revealed the presence of small quantities of more complex gangliosides than GQ1b.

Characterization of glucosylceramide from plasma membranes of plant root cells.

Plasma membranes of oat root cells were isolated from intracellular membranes by subfractionation of the microsomal fraction using an aqueous polymer two-phase system. The plasma membranes originated from oat plants which were acclimated to dehydration by exposure to a repeated water-deficit stress program. Glucosylceramides was a major component of the plasma membrane lipids and amounted to 9% of the lipid of control plants and 5% of the lipid of acclimated plants. Structural analysis using FAB-MS showed only one type of glucosylceramides. The constituent monosaccharide was exclusively glucose and the sphingosine base was 4,8-sphingadienine. The fatty acid composition was determined to 24:1-OH, with only trace levels of non-hydroxy acids. The decrease in the level of glucosylceramides during acclimation to dehydration was accompanied by a corresponding decrease in phospholipids and increase in free sterols.

Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung.

Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor-associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1.

Characterization of the binding epitope of the monoclonal antibody DMAb-1 to ganglioside GM2.

Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.

Phosphatidylethanol formation in rat organs after ethanol treatment.

An abnormal acidic phospholipid was found in high concentration in kidney and brain, and also in other organs of rats exposed to ethanol by i.p. injection or by a liquid diet. The compound could be identified as phosphatidylethanol. Phosphatidylethanol is probably formed in cell membranes by a phospholipase D-catalyzed transphosphatidylation reaction.