title: Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.
abstract: Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.
authors.affiliation: Disease Glycomics, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakajima
authors.forename: Shinobu
authors.initials: S
authors.lastname: Kitazume
authors.forename: Takashi
authors.initials: T
authors.lastname: Angata
authors.forename: Reiko
authors.initials: R
authors.lastname: Fujinawa
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Ohtsubo
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D002241:Carbohydrates / Q000737:chemistry*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002241:Carbohydrates; D008562:Membrane Glycoproteins; D009711:Nucleotides