An Enzymatic-HPLC Assay to Monitor Endogenous d-Serine Release from Neuronal Cultures
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.
Key wordsd-Serine High-performance liquid chromatography N-tert-butyloxycarbonyl-l-cysteine o-Phthaldialdehyde l-Serine dehydratase Glutaminase d-Serine deaminase Serine racemase
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