Ganglioside Technology Patented (original) (raw)

Rapid and sensitive LC-ESI-MS of gangliosides

Journal of Chromatography B, 2014

Gangliosides are a class of sphingolipids characterized by a ceramide lipid chain attached to an anionic oligosaccharide moiety that varies in complexity based on the level of sialylation. Heterogeneity in the oligosaccharide chain of gangliosides is a direct result of the monosaccharide structure, content, sequence, and connections. Gangliosides are highly concentrated in the central nervous system, and are cell typespecific as well as development-dependent and their quantities and species can undergo drastic changes during cell differentiation. Specific localization of gangliosides also allows for interaction with a variety of bioeffectors, including glycoproteins, antibodies, peptide hormones, and growth factors. There are currently no rapid analytical assays capable of identifying and quantifying gangliosides. The aim of this study is to establish a reliable chromatographic mass spectrometry based assay capable of profiling ganglioside levels in complex biological samples at high sensitivity. We describe here a chromatographic method using an amino column on which the separation is based on hydrophilic interaction with the sugar moiety of gangliosides. Several gangliosides, including GM1-3, GD1 a,b , GD2-3, and GT1 a,b , were efficiently separated in less than 10 min at a limit of detection ranging between 10-50 pg on column with a concentration dynamic range extending over 4 orders of magnitude. The developed method allowed the sensitive quantitation of gangliosides derived from the blood serum of patients with different esophagus diseases, including, adenocarcinoma, high-grade dysplasia, and Barrett's.

Quantification of gangliosides by microbore high performance liquid chromatography

2000

A highly sensitive analytical method was devel- oped that allows the separation of ganglioside mixtures and quantification of individual nonderivatized gangliosides in the concentration range between 2 pmol and 1 nmol. Gan- gliosides were separated with a gradient of acetonitrile/phos- phate buffer on a 1 mm diameter microbore HPLC column packed with Spherisorb-NH2. They eluted according to their number of

Derivatives of ganglioside GM1 as neuronotrophic agents: comparison of in vivo and in vitro effects

Brain Research, 1990

Exogenously administered gangliosides have been shown to behave as neuronotrophic/neuritogenic agents in a variety of cell culture systems and animal models, but it is not known whether they operate by the same mechanism in vivo and in vitro. To probe this question we have employed two derivatives of GM1 lacking the negative charge: the methyl ester (GM1-CH3) and the NaBH4 reduction product of the latter (GM1-OH) in which the carboxyl group is replaced by a primary alcohol. Both derivatives proved to be as neuritogenic as GM1 in 3 cell culture systems: neuro-2A ceis, PC12 cells and explanted dorsal root ganglia. However, GM1-OH proved ineffective when applied to two animal models involving reduction of cholinergic markers in: (a) hippocampus following lesion of the lateral fimbria and (b) nucleus basalis magnocellularis following cortical lesion; GM1-CH a showed marginal activity in (a) but more in (b), possibly owing to slow hydrolysis to GM1 which was highly active in both animal models. These results indicate the necessity of a negative charge on the ganglioside molecule for in vivo but not in vitro activity and point to different mechanisms for the trophic effects of exogenous gangliosides.

Synthesis of ganglioside Hp-s1

A simple protocol for the synthesis of a ganglioside Hp-s1 (1) starting from commercially available phytosphingosine, sialic acid, and D-glucose is described. This synthesis involved a glycosylation reaction of a phytosphingosine derived acceptor with a highly active benzyl protected glucosyl donor, a second glycosylation of a glucosyl acceptor with a sialyl donor followed by Staudinger reaction, amidation, and global deprotection as key steps.

Gangliosides: Treatment Avenues in Neurodegenerative Disease

Frontiers in Neurology

Gangliosides are cell membrane components, most abundantly in the central nervous system (CNS) where they exert among others neuro-protective and-restorative functions. Clinical development of ganglioside replacement therapy for several neurodegenerative diseases was impeded by the BSE crisis in Europe during the 1990s. Nowadays, gangliosides are produced bovine-free and new pre-clinical and clinical data justify a reevaluation of their therapeutic potential in neurodegenerative diseases. Clinical experience is greatest with monosialo-tetrahexosyl-ganglioside (GM1) in the treatment of stroke. Fourteen randomized controlled trials (RCTs) in overall >2,000 patients revealed no difference in survival, but consistently superior neurological outcomes vs. placebo. GM1 was shown to attenuate ischemic neuronal injuries in diabetes patients by suppression of ERK1/2 phosphorylation and reduction of stress to the endoplasmic reticulum. There is level-I evidence from 5 RCTs of a significantly faster recovery with GM1 vs. placebo in patients with acute and chronic spinal cord injury (SCI), disturbance of consciousness after subarachnoid hemorrhage, or craniocerebral injuries due to closed head trauma. In Parkinson's disease (PD), two RCTs provided evidence of GM1 to be superior to placebo in improving motor symptoms and long-term to result in a slower than expected symptom progression, suggesting disease-modifying potential. In Alzheimer's disease (AD), the role of gangliosides has been controversial, with some studies suggesting a "seeding" role for GM1 in amyloid β polymerization into toxic forms, and others more recently suggesting a rather protective role in vivo. In Huntington's disease (HD), no clinical trials have been conducted yet. However, low GM1 levels observed in HD cells were shown to increase cell susceptibility to apoptosis. Accordingly, treatment with GM1 increased survival of HD cells in vitro and consistently ameliorated pathological phenotypes in several murine HD models, with effects seen at molecular, cellular, and behavioral level. Given that in none of the clinical trials using GM1 any clinically relevant safety issues have occurred to date, current data supports expanding GM1 clinical research, particularly to conditions with high, unmet medical need.

Simple micro-method for the isolation of gangliosides by reversed-phase chromatography

Journal of Chromatography B: Biomedical Sciences and Applications, 1981

A simple and convenient technique has been developed for the isolation of gangliosides from small amounts of tisues or cells. A ganglioaide fraction obtained by chromatography of the total lipid extract of DEAE-Sephadex was subjected to alkaline hydrolysis and salts and other non-lipid contaminants were removed by reversed-phase chromatography on a C,, Sep-Pak cartridge_ The purified ganglioaidea were then obtained by chromatography on a small Iatrobeads or Uniail column. This procedure yields a quantitative recovery of ganglioaides that are free of contaminants which interfere with thin-layer chromatographic analysis. The procedure was used for the quantitative isolation of gangliosides from human brain white matter and human erythrocytes.

Assay Development and Screening for the Identification of Ganglioside GM3 Synthase Inhibitors

Biochemistry, 2020

Ganglioside GM3 is a sialylated membrane-based glycosphingolipid that regulates insulin receptor signaling via direct association with the receptor. Expression of GM3 synthase (GM3S) and GM3 is increased in the tissues of patients with diabetes and murine models of diabetes, and obesityinduced insulin resistance is attenuated in GM3S-deficient mice. Therefore, GM3S has been considered a therapeutic target for type II diabetes; however, no GM3S inhibitors have been

Rapid Separation of Gangliosides Using Strong Anion Exchanger Cartridges

Journal of Oleo Science, 2008

Ganglioside is a sphingoglycolipid that contains one or more sialic acid residues. It is known that various molecular species exist. In recent years, biochemical research on ganglioside has been developed and its physiological functions such as cell-cell interaction, cell proliferation, differentiation, cell-substrate interaction, carcinogenesis, bacteria or virus infection, immune response, and inflammation have been reported 1). To purify gangliosides from tissues, total gangliosides were generally separated from other lipids by extraction with chloroform-methanol using reversed phase chromatography on octadecyl-bonded (C18) silica gel 2). Isolation of individual species of ganglioside is performed by preparative thin-layer chromatography (TLC) 3) , high-performance liquid chromatography (HPLC) using anion-exchange column such as DEAE-Sephadex 4) and MonoQ column 5). However, preparative TLC is cumbersome and time-consuming for recovery of glycolipids. Silicic acid column chromatography usually requires large amounts of solvents and samples. HPLC is effective with high resolution but this strategy requires considerable amounts of solvents. For isolation of sphingolipid classes from complex lipid mixtures, aminopropyl-bonded silica gel (LC-NH2) and weak cation exchanger (LC-WCX) cartridge are used. These cartridges are efficiently used to isolate sphingoid bases, which is obtained after hydrolysis of sphingolipids, from other hydrolysis products such as fatty acids and unhydrolyzed parent compounds 6,7). Solid phase extraction (SPE) is an interesting tool that has been used for prepative isolation of lipid compounds. However, SPE has not been used for isolation of gangliosides from complex lipid mixtures. Compounds that are similar in chemical nature may be separated by selecting suitable SPE and varying the solvent environment (pH, polarity, salt concentration, etc). This paper describes the procedures for the use of strong anion exchanger cartridges to isolate and purify gangliosides from complex lipid mixtures. We present evidence that the gangliosides can be isolated easily with greater than 95% recovery rate. 2 EXPERIMENTAL 2 1 All organic solvents and rat brain tissue were of analytical grade and purchased from Nacalai Tesque, Kyoto, Japan. Anion exchanger cartridges (InertSep SAX and InertSep PSA, InertSep MA1, InertSep MA2) were purchased from GL Science