title: An enzymatic-HPLC assay to monitor endogenous D-serine release from neuronal cultures.
abstract: D-Serine is a transmitter-like molecule that physiologically activates NMDA receptors in the brain. Although D-serine was thought to be exclusively released by astrocytes, we recently demonstrated endogenous D-serine release from neurons in cultures and slices. So far high-performance liquid chromatography (HPLC) has been the standard technique to monitor D-serine and other amino acids. This method employs pre-column derivatization with a chiral reagent to produce fluorescence derivatives that can be further separated on a reversed-phase column. Due to the close retention times of L-serine, L-glutamine, and D-serine, the quantification of low levels of endogenous D-serine synthesis and release from cell cultures and tissues can be challenging. We here describe an enzymatic treatment method to specifically destroy L-glutamine and L-serine by glutaminase and L-serine dehydratase enzymes, respectively, allowing accurate determination of nanomolar D: -serine concentrations by subsequent HPLC analysis.
authors.affiliation: Department of Biochemistry, Technion - Israel Institute of Technology, Haifa, Israel.
authors.forename: Inna
authors.initials: I
authors.lastname: Radzishevsky
authors.forename: Herman
authors.initials: H
authors.lastname: Wolosker
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D004798:Enzymes / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004798:Enzymes; D012694:Serine