Keiko Tsuchihashi

Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes

Novel mono-O-acetylated GM3s, one containing 9-O-acetylN-glycolyl neuraminic acid and another containing 6′-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of theO-acetyl residue was identified by the downfield shift of the methylene protons at C-9 ofN-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6′-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6′-O-Ac GM3, theO-acetylated GM3 mixture was desialylated withArthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1.

Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine

A novel O‐acetylated GM3 containing 3‐O‐acetyl 4‐sphingenine was isolated with one having a non‐acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one‐ and two‐dimensional proton nuclear magnetic resonance, and fast atom bombardmentmass spectrometries. In addition, the O‐acetyl showed higher immunological activity toward anti‐melanoma antibody in the presence of non‐acetylated GM3 in complement‐dependent liposome lysis than did non‐acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O‐acetyl GM3.

Antigenicity of the carbohydrate moiety of ganglioside GM3 having 3-O-acetyl ceramide

To elucidate the effect of a modification of ceramide on antigenicity of the carbohydrate of ganglioside, the reactivity of O-acetyl GM3 having 3-O-acetyl ceramide, which has been characterized as a gliomarelated ganglioside, with monoclonal antibody M2590 was examined in comparison to that of non-acetylated GM3, by means of quantitative enzyme-linked immunosorbent assay, TLC-immunostaining and liposome immune lysis assay. In all these assay systems, O-acetyl GM3 showed less activity than GM3 as follows: GM3 was detected till 0.1 nmol in TLC-immunostaining, whereas O-acetyl GM3 could not be detected even at 0.25 nmol; the GM3 reaction was approximately twofold that of O-acetyl GM3 at each diluted point in the enzyme-linked immunosorbent assay; and 20% of the liposomes containing GM3 were lysed at 6 mol%, while liposomes containing O-acetyl GM3 did not lyse at that concentration. The lesser antigenicity of the sugar moiety of O-acetyl GM3 could be ascribed to the presence of an acetyl group in the ceramide at the 3-position of sphingosine.

New blocking method for the hydroxyl group on carbohydrate. Determination of the O-acylated position of the modified glycolipid

To determine O-esterified positions, a rapid and complete acetalization to prepare an intermediate was established using ethyl vinyl ether as a new reagent. The new method was applied to O-esterified glycolipids followed by GC-MS analysis of the monosaccharide derivatives after methylation and methanolysis, revealing the derivatives with correctly substituted positions. This method was superior in terms of its shorter reaction time and complete acetalization, particularly of the N-glycolyl hydroxyl residue, to previously reported methods using methyl vinyl ether.

The mechanism of high-density lipoprotein cholesterol elevation in patients treated with simvastatin

Fourteen patients with untreated hypercholesterolemia were given simvastatin 5 mg/d for 12 weeks. Cholesteryl ester transfer protein (CETP) activity and lecithin-cholesterol acyltransferase (LCAT) activity were determined every 4 weeks during treatment. Total cholesterol (TC) decreased from 279.1 ± 55.1 mg/dL before treatment to 224.6 ± 30.7 mg/dL after 12 weeks, and high-density lipoprotein cholesterol (HDL-C) rose from 56.2 ± 13 mg/dL to 61.2 ± 15.2 mg/dL. These changes were statistically significant. CETP activity decreased significantly from 26.8 ± 6.4% before treatment to 23.6 ± 6.3% after 12 weeks, and LCAT activity at 37 °C increased significantly from 43.3 ± 33.5 nmol/mL per hour to 68.4 ± 32.6 nmol/mL per hour. The changes in TC correlated with those in CETP activity at each time point (r = .674), but not with those in LCAT activity. The HDL-C level was negatively correlated with CETP activity at each time point (r = .655) but not with LCAT activity. These findings show that CETP activity may play a more important role than LCAT activity in simvastatin-induced HDL-C elevation and suggest that simvastatin increases HDL-C by reducing CETP activity secondary to low-density lipoprotein cholesterol reduction.