Determination of l-serine-O-phosphate in rat and mouse brain tissue using high-performance liquid chromatography and fluorimetric detection

doi:10.1016/j.ab.2010.06.024

Analytical Biochemistry

Volume 405, Issue 2, 15 October 2010, Pages 260-262

https://doi.org/10.1016/j.ab.2010.06.024 Get rights and content

Abstract

l-Serine-O-phosphate (L-SOP), the precursor of l-serine, is an agonist at group III metabotropic glutamate receptors. Despite the interest in L-SOP, very few articles have reported its brain levels. Here we report a convenient and reproducible method for simultaneous analysis of L-SOP and several other important amino acids in brain tissue using high-performance liquid chromatography (HPLC) with fluorimetric detection after derivatization with o-phthaldialdehyde and N-isobutyl-l-cysteine. Analyses were carried out in rat whole brain and cerebellum and in mouse whole brain, forebrain, amygdala, and prefrontal cortex. The method should be useful for future comprehensive neurochemical and pharmacological studies on neuropsychiatric disorders.

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Acknowledgments

The authors are grateful to Sara Tomlinson for preparation of the manuscript and to Jordyce van Muyden for assistance with figures. Funding was provided by the Canadian Institutes of Health Research and the Canada Research Chairs and Canada Foundation for Innovation programs.

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Cited by (5)

Selectivity and evolutionary divergence of metabotropic glutamate receptors for endogenous ligands and G proteins coupled to phospholipase C or TRP channels
2014, Journal of Biological Chemistry
Citation Excerpt :
We focused the analysis on the transmembrane region of the protein as defined by current mGluR1 structure PDBID 4OR2. Ten amino acids that are known to be present at substantial concentrations in cerebrospinal fluid (17) and d-SOP were applied to HEK 293 cells transfected with representative members for all subtypes of mGluR. mGluR1 naturally couples to Gαq so it was transfected alone but the other mGluR, which are Gαi/o coupled receptors, were co-transfected with a promiscuous G protein, either Gα15, or Gαqo.
To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G_i/o proteins coupled to the ion channel TRPC4β. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is l-serine-O-phosphate (l-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas l-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of l-glutamate and l-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both l-glutamate and l-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of l-SOP. Experiments with pertussis toxin and dominant-negative Gα_i/o proteins revealed that mGluR1 couples strongly to TRPC4β through Gα_i/o, in addition to coupling to PLC through Gα_q/11.
Analysis of l-serine-O-phosphate in cerebrospinal spinal fluid by derivatization-liquid chromatography/mass spectrometry
2014, Analytical Biochemistry
l-Serine-O-phosphate (l-SOP), the precursor of l-serine, is a potent agonist against the group III metabotropic glutamate receptors (mGluRs) and, thus, is of interest as a potential biomarker for monitoring modulation of neurotransmitter release. So far, no reports are available on the analysis of l-SOP in cerebrospinal fluid (CSF). Here a novel method is presented to determine l-SOP levels in CSF employing precolumn derivatization with (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) coupled to liquid chromatography/mass spectrometry (derivatization–LC/MS, d-LC/MS).
Searching for an endogenous anti-alzheimer molecule: Identifying small molecules in the brain that slow alzheimer disease progression by inhibition of β-amyloid aggregation
2013, Journal of Psychiatry and Neuroscience
Alzheimer disease is a neurodegenerative disorder that progresses with marked interindividual clinical variability. We postulate the existence of endogenous molecules within the human brain exerting an antiaggregant activity that will prevent/slow Alzheimer disease progression.
We performed in silico studies to determine if the small endogenous molecules L-phosphoserine (L-PS) and 3-hydroxyanthranilic acid (3-HAA) could bind to the target region of β-amyloid responsible for protein misfolding. In vitro assays measured the antiaggregation effect of these molecules at varying concentrations.
In silico studies demonstrated that L-PS and 3-HAA, both endogenous brain molecules, were capable of binding to the histidine₁₃–histidine–glutamine–lysine₁₆ (HHQK) region of β-amyloid involved in misfolding: these interactions were energetically favoured. The in vitro assays showed that both L-PS and 3-HAA were capable of inhibiting β-amyloid aggregation in a dose-dependent manner, with 3-HAA being more potent than L-PS.
Studies were performed in silico and in vitro but not in vivo.
We successfully identified 2 endogenous brain molecules, L-PS and 3-HAA, that were capable of binding to the region of β-amyloid that leads to protein misfolding and neurotoxicity. Both L-PS and 3-HAA were able to inhibit β-amyloid aggregation in varying concentrations; levels of these compounds in the brain may impact their effectiveness in slowing/preventing β-amyloid aggregation.
Determination of amino acid neurotransmitters in mouse brain tissue using high-performance liquid chromatography with fluorescence detection
2013, Journal of Chinese Pharmaceutical Sciences
Modulation of glutamate release from parallel fibers by mGlu4 and pre-synaptic GABA <inf>A</inf> receptors
2012, Journal of Neurochemistry

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Notes & TipsDetermination of l-serine-O-phosphate in rat and mouse brain tissue using high-performance liquid chromatography and fluorimetric detection

Abstract

Section snippets

Acknowledgments

J. Chromatogr. Biomed. Appl.

J. Chromatogr. B

l-Serine-O-phosphate in the central nervous system

Brain Res.

Biochemistry and the Central Nervous System

Notes & Tips
Determination of l-serine-O-phosphate in rat and mouse brain tissue using high-performance liquid chromatography and fluorimetric detection