Strategies to assess and optimize stability of endogenous amines during cerebrospinal fluid sampling

Abstract

IntroductionMetabolic profiling of cerebrospinal fluid (CSF) is a promising technique for studying brain diseases. Measurements should reflect the in vivo situation, so ex vivo metabolism should be avoided.ObjectiveTo investigate the effects of temperature (room temperature vs. 4 °C), centrifugation and ethanol, as anti-enzymatic additive during CSF sampling on concentrations of glutamic acid, glutamine and other endogenous amines.MethodsCSF samples from 21 individuals were processed using five different protocols. Isotopically-labeled alanine, isoleucine, glutamine, glutamic acid and dopamine were added prior to sampling to trace any degradation. Metabolomics analysis of endogenous amines, isotopically-labeled compounds and degradation products was performed with a validated LC–MS method.ResultsThirty-six endogenous amines were quantified. There were no statistically significant differences between sampling protocols for 31 out of 36 amines. For GABA there was primarily an effect of temperature (higher concentrations at room temperature than at 4 °C) and a small effect of ethanol (lower concentrations if added) due to possible degradation. O-phosphoethanolamine concentrations were also lower when ethanol was added. Degradation of isotopically-labeled compounds (e.g. glutamine to glutamic acid) was minor with no differences between protocols.ConclusionMost amines can be considered stable during sampling, provided that samples are cooled immediately to 4 °C, centrifuged, and stored at − 80 °C within 2 h. The effect of ethanol addition for more unstable metabolites needs further investigation. This was the first time that labeled compounds were used to monitor ex vivo metabolism during sampling. This is a useful strategy to study the stability of other metabolites of interest.Electronic supplementary materialThe online version of this article (10.1007/s11306-018-1333-0) contains supplementary material, which is available to authorized users.

Longitudinal Analysis of Multiple Neurotransmitter Metabolites in Cerebrospinal Fluid in Early Parkinson's Disease

“…Despite efforts to standardize CSF collection, processing, and measurement, 11,12 potential confounding factors on catecholamine levels remain (eg, diurnal changes, total CSF volume) and may impede the reliable quantification. [13][14][15][16] Although high-performance liquid chromatography with electrochemical detection (HPLC-ECD) and gas chromatography-mass spectrometry were previously Movement Disorders, Vol. 36, No.…”

“…Despite efforts to standardize CSF collection, processing, and measurement, 11,12 potential confounding factors on catecholamine levels remain (eg, diurnal changes, total CSF volume) and may impede the reliable quantification. [13][14][15][16] Although high-performance liquid chromatography with electrochemical detection (HPLC-ECD) and gas chromatography-mass spectrometry were previously considered gold standards for analyzing dopamine and its metabolites, 7,11 LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) has evolved during the last two decades with comparable sensitivity to HPLC-ECD and greatly improved selectivity. 17,18 LC-MS/MS reduces the complexity of preanalytical processing 17 and is now considered the gold standard for quantitative analytics.…”

Impact of sample preparation upon intracellular metabolite measurements in 3D cell culture systems

“…The addition of ISTDs, which were not present endogenously in the samples, enabled evaluation of any changes, including signal suppression or enhancement caused by sample preparation or analysis, in each extract (Broadhurst et al 2018 ; Noga et al 2018 ). As reported by Broadhurst et al another advantage of including ISTDs in all samples was to enable real-time tracking of sample responses during data collection (mass accuracy and resolution, retention time, peak intensity, and peak shape).…”

“…3 Assay procedure to evaluate sample preparation methods by measuring sample preparation effect (b/a), matrix factor (c/b), and PE (c/a). Adapted from Mathon et al ( 2013 ) and Noga et al ( 2018 ) …”

From bedside to bench—practical considerations to avoid pre-analytical pitfalls and assess sample quality for high-resolution metabolomics and lipidomics analyses of body fluids

“…Cooling of centrifuged CSF below 8 °C for up to 72 h showed only minor effects on metabolite levels, as did storage at −20 °C for 2 months [ 74 ]. In a profile of 36 amines, the levels of 31 were stable for up to 2 h when refrigerated immediately after CSF collection [ 76 ]. Furthermore, up to three freeze-and-thaw cycles of centrifuged CSF resulted in a relatively unaffected metabolome [ 74 ].…”