Effect of prenatal and postnatal exposure to ethanol on rat central nervous system gangliosides and glycosidases

We investigated the effect of maternal alcohol consumption on cell number, gangliosides and ganglioside catabolizing enzymes in the central nervous system (CNS) of the offspring. Virgin female rats of the Charles Foster strain were given 15% (v/v) ethanol in drinking water one month prior to conception and during gestation and lactation. At 21 days postnatal age, the offspring were sacrificed and the brains were separated into cerebrum, cerebellum and brain stem to investigate possible regional variations. Compared to controls, wet weight of cerebrum, cerebellum and brain stem, and of spinal cord was decreased in the pups exposed to alcohol. DNA and protein contents were also found to be lowered in all the CNS regions of the pups exposed to alcohol. Conversely, maternal alcohol consumption was found to increase the concentration and the content of total gangliosideN-acetylneuraminic (NANA) in CNS of the pups. In addition, alcohol treatment was found to induce alterations in the proportions of individual ganglioside fractions. Interestingly, these alterations are somewhat different than those observed in the neonatal brain and spinal cord of the pups subjected to prenatal alcohol exposure. The alterations in the proportions of ganglioside fractions were shown to be region-specific. Maternal alcohol consumption resulted in decreased activities of sialidase, β-galactosidase, β-glucosidase and β-hexosaminidase. The results suggest that the alcohol-associated increases in ganglioside concentration may be at least partly due to the decreased activities of ganglioside catabolizing enzymes.     

Total gangliosides,ganglioside species and the activity of neuraminidase in different brain regions and spinal cord of normal and undernourished rats

Total gangliosides, their concentrations and the distribution of individual ganglioside species were determined in the spinal cord, pons-medulla, midbrain, cerebellum, hypothalamus, cerebral cortex, olfactory lobes and the rest of the forebrain, of 18- and 33-day-old normal and food-restricted and rehabilitated rats. The activity of neuraminidase in the different regions of the brain and spinal cord was determined. Differences in total gangliosides as well as individual species in different regions of the brain were observed. Among brain regions, while total ganglioside content was significantly reduced in the case of the cerebellum, the hypothalamus was the most affected in the distribution of ganglioside species and the activity of neuraminidase was decreased. The significance of the distribution of ganglioside species in different brain regions in relation to the activity of neuraminidase is discussed.     

Enhanced β-oxidative utilization of [1-14C]Palmitate during active myelinogenesis in developing rat brain under nutritional stress

Food restriction was found to impair the incorporation of [1-¹⁴C]palmitate into myelin membrane lipids of developing rat brain. An attempt was made to determine whether this phenomenon is due to differences in the rate of utilization of the labelled precursor or to its enhanced degradationvia β-oxidative pathways. Undernutrition in pups was imposed by food restriction during gestation and lactation. β-Oxidation by brain region homogenates using [1-¹⁴C]palmitate was monitored at days 7, 14 and 21 of postnatal age. There was a significant increase in β-oxidation in the brain regions of undernourished pups, with the cerebrum and cerebellum being more affected than the brain stem. Because developing brain possesses the enzymic potential to utilize ketone bodies, the data may indicate increased usage of palmitate as an energy source in the developing brain of undernourished animals.     

Influence of dietary vitamin E,selenium and age on regional distribution of α-tocopherol in the rat brain

Concentrations of α-tocopherol (α-T) in plasma, cerebrum, cerebellum, midbrain and brain stem and activity of selenium (Se)-dependent glutathione peroxidase (GSH-Px) in plasma were measured in 1- and 15-month-old male F344 rats fed diets containing vitamin E (E, IU/kg) and Se (ppm) in the following combinations: 30 E, 0.1 Se (control diet, minimum requirements); 200E, 0.2 Se; 0.0 E, 0.2 Se; 200 E, 0.0 Se; 0.0 E, 0.0 Se for 8 or 20 weeks. α-T and GSH-Px levels in plasma were reflective of dietary treatment in young rats in which an interaction of the two nutrients was noted. A longer period of dietary vitamin E deficiency was necessary to deplete plasma α-T and depress GSP-Px activity significantly in the old rats. Among the brain regions of all ages, cerebrum and midbrain had the highest concentrations of α-T while cerebellum showed the lowest. However, cerebellum of young rats and cerebellum and brain stem of old rats had a greater α-T accumulation with doubly supplemented diets, whereas only cerebellum of young and old rats showed a marked increased of α-T with vitamin E supplementation. In old rats, vitamin E deficiency resulted in greater depletion of α-T in cerebellum and brain stem than cerebrum and midbrain regions. Se deficiency in brain stem of young and old rats significantly decreased α-T accumulation by vitamin E supplementation. Se supplementation marginally alleviates vitamin E depletion in brain. Cerebellum and brain stem of old rats fed the minimum requirements of vitamin E and Se for 20 weeks showed a significant decline in α-T. Therefore, cerebellum and brain stem appear, to have a higher turnover of α-T than cerebrum and midbrain, and older rats may require a higher level, of vitamin E in the diet to maintain steady state levels of α-T in these regions.     

Effect of dietary vitamin E and selenium on susceptibility of brain regions to lipid peroxidation

The effect of dietary vitamin E and/or selenium (Se) supplementation (200 IU and/or 0.2 ppm, respectively) or deficiency for two months on lipid peroxidation in cerebrum, cerebellum, mid-brain, and brain stem of one-month-old male F344 rats was investigated. Dietary treatment had a minimal effect on weight gain of rats for the period tested. Plasma α-tocopherol (α-T) concentration and glutathione peroxidase (GSH-Px) activity were reflective of dietary treatments. Supplementation of diets with vitamin E and/or Se increased plasma α-T and/or GSH-Px activity, while diets devoid of these nutrients reduced them significantly. Increased GSH-Px activity in Sesupplemented rats was further enhanced by vitamin E supplementation. Differential concentrations of α-T among brain regions were affected by dietary vitamin E but not by Se. In vitro lipid peroxidation of brain homogenates was inhibited by dietary vitamin E supplementation and increased by deficiency. Addition of 0.25 mM ascorbic acid or 0.1 mM of Fe²⁺ to brain homogenates markedly increased in vitro lipid peroxidation. Ascorbic acid-induced lipid peroxidation was inversely correlated with dietary vitamin E and Se in cerebrum. In vitro Fe²⁺-addition induced the greatest stimulation of lipid peroxidation, with cerebellum and brain stem of vitamin E-deficient rats showing the highest response to Fe²⁺ challenge. These findings indicate that concentrations of α-T among the brain regions are different and can be altered by dietary vitamin E treatments, cerebellum and brain stem are more susceptible to in vitro challenge by peroxidative agents than other regions, and the degree of lipid peroxidation of brain regions is partially affected by dietary vitamin E but not by Se in the levels tested.     

Partition of ketone bodies into cholesterol and fatty acids in vivo in different brain regions of developing rats

The proportions of labeled ketone bodies and glucose incorporated into cholesterol and fatty acids in different regions of the brain in developing rats were compared. In cerebrums of 15- and 18-day-old rats, the ratios of dpm cholesterol/dpm fatty acids incorporated from [3-¹⁴C] acetoacetate and [3-¹⁴C] β-hydroxybutyrate ranged from 0.4 to 0.7, or 50 to 100% higher than values obtained with [U-¹⁴C] glucose. Much higher ratios were obtained with younger animals: from 1 to 12 days of life, the values ranged from 1.0 to 1.3 with [3-¹⁴C] β-hydroxybutyrate as substrate, and, from 1 to 5 days, with [3-¹⁴C] acetoacetate, they were 1.0 or greater. During the first 12 days of life, the ratios resulting from administration of [U-¹⁴C] glucose were 0.4–0.7. Clearly, a greater proportion of acetoacetate and β-hydroxybutyrate was incorporated into cholesterol during the first week of life than the remaining suckling period. Like cerebrum, other brain regions (i.e., cerebellum, midbrain, brain stem and thalamus) yielded higher ratios of dpm cholesterol/dpm fatty acids from [3-¹⁴C] β-hydroxybutyrate during the first 12 days of life than on day 17. Brain stem was the most active region for lipid synthesis, and had the highest dpm cholesterol/dpm fatty acid ratio. Since active synthesis of cholesterol from ketone bodies during the early postnatal period coincides with a period of rapid brain growth, the results indicate that ketone bodies are more important early in the suckling period as sources of cholesterol for brain growth.     

Behavior of gangliosides on dialysis

Mixed brain gangliosides or individual ganglioside GM₁ were dissolved in one of the following solvents: (a) water, (b) 0.1 M aqueous KC1, or (c) methanol-aqueous 0.1 M KC1 at concentrations ranging from 10⁻⁸ to 10⁻³ M, and were submitted to dialysis against distilled water for up to 4 days. No significant loss of gangliosides on dialysis was observed when gangliosides were dissolved in water or 0.1 M aqueous KC1, but a loss occurred when the ganglioside solution contained methanol; the loss was greater at the lowest ganglioside concentration. However, losses ceased to occur after 1 day when methanol was removed from the dialysis sac.     

Lactational changes in the fatty acid composition of human milk gangliosides

The objectives of this work were to study the FA composition of milk gangliosides, as well as to gain further insight into the characterization of human milk gangliosides. The potential capacity of human milk gangliosides to adhere to human enterotoxigenic Escherichia coli (ETEC-strains) was also studied. Human milk gangliosides were isolated and identified by high-performance TLC or immunoassay. The latter also was used to assay bacterial adhesion. The FA composition of gangliosides was studied by GC. The presence of O-acetyl GD3 (Neu5,9Ac₂α2–8 NeuAcα2–3Galβ1–4GlcCer) and trace amounts of GM1 [Galβ1–3GalNAcβ1,−3(NeuAcα2–3)Galβ1–4GlcCer] in human milk was confirmed. Medium-chain FA were almost absent in colostrum, whereas in the subsequent stages they rose to 20%. The levels of long-chain FA decreased after colostrum. With respect to the degree of saturation, gangliosides from colostrum were richer in monounsaturated FA than gangliosides synthesized during the rest of the lactation period, opposite to the pattern for PUFA. A human-ETEC colonization factor antigen II-expressing strain showed binding capacity to human milk GM3 (NeuAcα2–3Galβ1–4GlcCer). New data on human milk gangliosides have been gathered. A thorough knowledge of their composition is needed since they may have important biological implications in regard to newborns' defense against infection. The ganglioside nomenclature of Svennerholm (34) is followed.     

Lipids of retina: I. Analysis of gangliosides in beef retina by thin layer chromatography

Lipids were extracted from beef retina by chloroform-methanol (2∶1); the gangliosides were removed from the total lipid extract by partitioning into water and chromatographing on thin layer plates coated with silica gel. The analytical methods are described for estimating ganglioside components, i.e., N-acetyl neuraminic acid, hexoses, hexosamine, sphingosine and fatty acids, in the presence of silica gel. Major gangliosides present in beef retina have been tentatively identified as follows: a ganglioside containing two N-acetyl neuraminyl groups but no hexosamine; two gangliosides containing two N-acetyl neuraminyl groups and one hexosamine; and a ganglioside with three N-acetyl neuraminyl moieties and one hexosamine.     

Behavior of sugar derivatives in procedure for ganglioside isolation

A common method of studying ganglioside metabolism is to measure the amounts of radioactivity incorporated into ganglioside from a radiolabeled precursor. This requires that radioactive nonganglioside material be completely removed from the ganglioside fraction. Nucleotide sugars and aminosugars comprise an important source of such contaminants. Therefore, we have studied their behaviors in several procedures currently employed to isolate gangliosides. Over 50% of the radioactivity associated with several nucleotide sugars added to a brain homogenate is extracted with chloroform/methanol (2∶1, v/v), and most of this is recovered in the upper phase of a Folch partition. Dialysis against water removes almost all of the free aminosugar but only 70% of nucleotide sugar. Treatment with alkaline phosphatase, phosphodiesterase and alkaline methanol followed by dialysis removes almost all of the nucleotide diphosphate sugars but only 88% of cytidine 5′-monophosphate sialic acid (CMP-NeuAc). Nucleotide sugars cannot be separated from gangliosides by Unisil or latrobead chromatography, but nucleotide diphosphate sugars and gangliosides are resolved with Sephadex LH-20 chromatography following treatment with phosphodiesterase and alkaline phosphatase. CMP-NeuAc was not satisfactorily separated from gangliosides using any of the procedures.     

Gangliosides in membranes fromTorpedo electric organ

The electric organ membrane has been the subject of many studies, due principally to its rich content of nicotinic acetylcholine receptor (AChR). Knowing its lipid composition is clearly important. Although its major membrane lipids have been characterized, its ganglioside composition has not been as well-described. In this study, gangliosides were characterized in membranes prepared from two species of electric organ,Torpedo californica andT. nobiliana. The ganglioside content of total electric organ membranes and AChR-enriched membranes was similar in both species, accounting for from 0.9 to 1.5% of membrane lipid by weight. However, the AChR-enriched membranes contained significantly less ganglioside relative to AChR than did the total membrane preparations. Five major gangliosides were purified fromT. californica and identified as II³NeuNAc-GgOse₃ (GM2); II³(NeuNAc)₂-GgOse₃ (GD2), IV³NeuNAc, II³NeuNAc-GgOse₄ (GD1a), IV³NeuNAc, II³(NeuNAc)₂−GgOse₅ (GT1b), and IV³(NeuNAc)₂, II³(NeuNAc)₂−GgOse₄ (GQ1b). Together these five gangliosides accounted for over 90% of the total ganglioside present in the two membrane preparations from both species. The most abundant ganglioside by far was GM2, which accounted for about one-half of the ganglioside content, followed by GD2. Determination of the N-fatty acid composition was performed on gangliosides purified fromT. nobiliana. The lower-order gangliosides, GM2, GD2, and GD1a, contained substantial amounts of very long chain fatty acids (>20 carbons), including α-hydroxynervonic acid (15–21% of total). In contrast, unsubstituted, 14–18 carbon chains accounted for about 90% of the fatty acids on the two higher-order gangliosides, GT1b and GQ1b.     

Desmosterol in developing rat brain

The brain of the young rat contains appreciable amounts of desmosterol (24-dehydrocholesterol). The peak desmosterol concentration is seen during the first week of life and only traces of this sterol are found at 21 days. The spinal cord also contains some desmosterol. Rat brain desmosterol is distributed in the white matter, gray matter and cerebellum and occurs in the same proportion to cholesterol in each of these brain fractions. Rat brain contains a small amount of sterol ester but no appreciable amounts of desmosterol are present in this fraction. Studies carried out in intact animals injected either intraperitoneally or intracerebrally with mevalonic acid-2-¹⁴C or glucose-U-¹⁴C indicate the biosynthetic origin or brain desmosterol. Rat brain slices (1–20 day old) incubated in suitably fortified medium convert sodium acetate-2-¹⁴C and glucose-U-¹⁴C to desmosterol, whereas brain slices from adult rats yielded no radioactive desmosterol under similar conditions. When labeled desmosterol was incubated with young rat brain slices, it was converted to cholesterol. When pregnant rats were treated with triparanol (20 mg/kg/day) during the course of their pregnancy, they either resorbed the fetuses or gave birth to small, stillborn litters. The brains of the progeny of triparanol treated mothers contained large amounts of desmosterol as well as another sterol which may be Δ^7,24-cholestadiene-3β-ol.     

Characterization of gangliosides of porcine erythrocyte membranes: Occurrence of ganglioside GD3 as major ganglioside

Four major ganglioside species were isolated from porcine erythrocyte membranes by DEAE-Sephadex and Iatrobeads column chromatography. Treatment of the lipids with graded neuraminidase and β-galactosidase, gas chromatographic analysis of their carboyhydrates, sphingosine bases and molecular species of sialic acid revealed that the structure of these gangliosides were G_M3(NeuAc), G_M3(NeuGc), G_D3(NeuAc) and G_D3(NeuGc), each of which was 16±2 μg, 304±42 μg, 30±3 μg and 240±26 μg, respectively, per gram of the dry erythrocyte stroma. The amount of G_M3 and G_D3 accounted for more than 95% of total gangliosides of the erythrocytes. Porcine erythrocytes may provide a good source for large scale preparation of ganglioside G_D3 which recently was identified as a human melanoma-associated antigen. Gangliosides are named according to Svennerholm (1) and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature (2).     

A Lipidomics Atlas of Selected Sphingolipids in Multiple Mouse Nervous System Regions

Many lipids, including sphingolipids, are essential components of the nervous system. Sphingolipids play critical roles in maintaining the membrane structure and integrity and in cell signaling. We used a multi-dimensional mass spectrometry-based shotgun lipidomics platform to selectively analyze the lipid species profiles of ceramide, sphingomyelin, cerebroside, and sulfatide; these four classes of sphingolipids are found in the central nervous system (CNS) (the cerebrum, brain stem, and spinal cord) and peripheral nervous system (PNS) (the sciatic nerve) tissues of young adult wild-type mice. Our results revealed that the lipid species profiles of the four sphingolipid classes in the different nervous tissues were highly distinct. In addition, the mRNA expression of sphingolipid metabolism genes—including the ceramidase synthases that specifically acylate the N-acyl chain of ceramide species and sphingomyelinases that cleave sphingomyelins generating ceramides—were analyzed in the mouse cerebrum and spinal cord tissue in order to better understand the sphingolipid profile differences observed between these nervous tissues. We found that the distinct profiles of the determined sphingolipids were consistent with the high selectivity of ceramide synthases and provided a potential mechanism to explain region-specific CNS ceramide and sphingomyelin levels. In conclusion, we portray for the first time a lipidomics atlas of select sphingolipids in multiple nervous system regions and believe that this type of knowledge could be very useful for better understanding the role of this lipid category in the nervous system.     

A rapid method for the preparation of ganglioside Glac2 (GD3)

A method is described for the preparation of ganglioside G_lac2 [(II³(NeuAc)₂-LacCer, GD3] from cream of bovine milk using liquid-phase extraction with methanol or ethanol followed by anion exchange chromatography. The method is rapid and inexpensive; 1 kg cream, centrifuged from 14–15 L of bovine milk, yields approximately 70 mg of pure ganglioside G_lac2. The sialic acid constituent of ganglioside G_lac2 isolated from bovine milk cream consists solely of theN-acetylneuraminic acid derivative. The major components of its ceramide consist of octadecasphing-4-enine and the 22∶0 (behenic acid) and 23∶0 fatty acids. Short hand notations for gangliosides are according to Wiegandt (Ref. 1). G_lac2, (II³(NeuAc)₂-LacCer) is NeuAcα,8NeuAcα,-3Galβ,4Glcβ,1Cer; G_tet1, (II³NeuAc-Gg₄Cer) is Gal\,3GalNAcβ,-4(NeuAcα,3)Galβ,4Glcβ,1Cer.     

Comparative Analysis of Brain Lipids in Mice,Cats, and Humans with Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid (GSL) storage disease that arises from an autosomal recessive mutation in the gene for the β-subunit of β-Hexosaminidase A (Hexb gene), which catabolizes ganglioside GM2 within lysosomes. Accumulation of GM2 and asialo-GM2 (GA2) occurs primarily in the CNS, leading to neurodegeneration and brain dysfunction. We analyzed the total lipids in the brains of SD mice, cats, and humans. GM2 and GA2 were mostly undetectable in the normal mouse, cat, and human brain. The lipid abnormalities in the SD cat brain were generally intermediate to those observed in the SD mouse and the SD human brains. GM2 comprised 38, 67, and 87% of the total brain ganglioside distribution in the SD mice, cats, and humans, respectively. The ratio of GA2–GM2 was 0.93, 0.13, and 0.27 in the SD mice, cats, and humans, respectively, suggesting that the relative storage of GA2 is greater in the SD mouse than in the SD cat or human. Finally, the myelin-enriched lipids, cerebrosides and sulfatides, were significantly lower in the SD brains than in the control brains. This study is the first comparative analysis of brain lipids in mice, cats, and humans with SD and will be important for designing therapies for Sandhoff disease patients.     

Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers

Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross‐sectional sample of Chinese mothers over an 8‐month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast‐feeding period of 1?6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM₃ molecular species containing long‐chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long‐chain acyl fatty acid GD₃ molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long‐chain acyl fatty acid molecular species were more varied. The major GM₃ and GD₃ molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.     

The major gangliosides of the bovine pineal body

Acetone powders of fresh-frozen pineals were extracted with chloroform/methanol mixtures. By column chromatography on silicic acid, mild alkaline methanolysis, ion-exchange high performance liquid chromatography and a final thin layer chromatography on silicic acid, the major glycosphingolipids were purified from the extracts of a total of 300 bovine pineal bodies. Chromatographically purified fractions were characterized by gas chromatographic analysis. The most prominent glycosphingolipid appeared to be cerebroside. In addition, five different gangliosides were found in detectable levels. The two major gangliosides have the chromatographic and component characteristics of GD₃ and GM₃, with disialoganglioside predominating. Gangliosides indistinguishable from purchased standards of GM₁ and GD_1a were third and fourth, respectively, in amount. The fatty acid profiles of the two lactosyl gangliosides are similar and significantly different from those of the two gangliotetraose gangliosides. The fifth most prominent ganglioside, present at a level of 1.09% of total recovered ganglioside sialic acid, appears to be a novel trisialoganglioside, called GT_x. This new molecule has a component ratio of gal:glc:sialic acid:amino sugar of approximately 1∶2∶3∶1. Similarities between bovine pineal and rod outer segments are discussed.     

Liquid Chromatography–High-Resolution Mass Spectrometry for Quantitative Analysis of Gangliosides

Gangliosides are a large family of glycosphingolipids that are abundant in the brain, and have been shown to affect neuronal plasticity during development, adulthood, and aging. We developed a fast, efficient, and sensitive liquid chromatography–mass spectrometry method to quantify eight different classes of gangliosides (GM₁, GM₂, GM₃, GD₃, GD_1a, GD_1b, GT_1b, GQ_1b) in the brains of 2-day-old and 80-day-old Wistar rats. The gangliosides were extracted from rat brain using a modified Svennerholm and Fredman method. After ganglioside class separation using a hydrophilic high performance liquid chromatography (HPLC) column, the resolving power of the LTQ-Orbitrap™ mass spectrometer was used to extract and sum the major species of each ganglioside class, generating fully resolved extracted ion current peaks for both standards and samples. The flexibility and the specificity of this method are such that it can be applied to the analysis of other ganglioside species/classes not discussed in this paper, provided appropriate standards are available. The method had good repeatability (coefficient of variation 4.8–12.3%) and mean recoveries in the range 92–107%.     

A comparative study of gangliosides from the brains of various species

A comparison is made of the sialic acid and hexose content of crude gangliosides from the brains of 13 species, including mammals, a bird, and amphibian, a reptile and fish. The sialic acid content is relatively constant from species to species. Gangliosides are less abundant in tissues other than the brain. Four species (cat, dog, pig and sheep) were selected for the determination of their major ganglioside subfractions. The ganglioside subfractions were isolated (after extraction and partition dialysis) using descending thin layer chromatography. The population of ganglioside molecules varied from species to species. From dog and sheep a mono-, a di- and a trisialoganglioside were obtained; from cat a mono-, a di- and two trisialogangliosides; and from pig a mono- and two disialogangliosides. Each ganglioside subfraction was found to contain glucose, galactose and galactosamine in the ratio of approximately 1∶2∶1. The fatty acid moieties consisted of more than 80% stearic acid with lesser amounts of arachidic, palmitic and behenic acid. Sphingosine analyses indicated ratios of sphingosine to icosisphingosine of 7∶3 for the monosialo-, 1∶1 for the disialo- and 3∶7 for the trisialogangliosides.