Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride
We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-G...
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doaj-317ec88c09cc494fa2ff32aaa2287c962021-04-27T04:38:50ZengElsevierJournal of Lipid Research0022-22752003-07-0144714131419Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydrideJacques Bodennec0Selena Trajkovic-Bodennec1Anthony H. Futerman2Department of Biological Chemistry, Weizmann Institute of Science, 76100, Rehovot, IsraelDepartment of Biological Chemistry, Weizmann Institute of Science, 76100, Rehovot, IsraelDepartment of Biological Chemistry, Weizmann Institute of Science, 76100, Rehovot, IsraelWe describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an ∼40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation.The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.http://www.sciencedirect.com/science/article/pii/S0022227520311056glucosylceramidegalactosylceramidelactosylceramideGaucher diseaseKrabbe's disease |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jacques Bodennec Selena Trajkovic-Bodennec Anthony H. Futerman |
spellingShingle |
Jacques Bodennec Selena Trajkovic-Bodennec Anthony H. Futerman Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride Journal of Lipid Research glucosylceramide galactosylceramide lactosylceramide Gaucher disease Krabbe's disease |
author_facet |
Jacques Bodennec Selena Trajkovic-Bodennec Anthony H. Futerman |
author_sort |
Jacques Bodennec |
title |
Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride |
title_short |
Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride |
title_full |
Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride |
title_fullStr |
Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride |
title_full_unstemmed |
Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride |
title_sort |
simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by n-acetylation with [3h]acetic anhydride |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
2003-07-01 |
description |
We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [3H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an ∼40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation.The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate. |
topic |
glucosylceramide galactosylceramide lactosylceramide Gaucher disease Krabbe's disease |
url |
http://www.sciencedirect.com/science/article/pii/S0022227520311056 |
work_keys_str_mv |
AT jacquesbodennec simultaneousquantificationoflysoneutralglycosphingolipidsandneutralglycosphingolipidsbynacetylationwith3haceticanhydride AT selenatrajkovicbodennec simultaneousquantificationoflysoneutralglycosphingolipidsandneutralglycosphingolipidsbynacetylationwith3haceticanhydride AT anthonyhfuterman simultaneousquantificationoflysoneutralglycosphingolipidsandneutralglycosphingolipidsbynacetylationwith3haceticanhydride |
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1721506998267150336 |