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Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC

Abstract

The presence of monoamines and their cofactors (the pterins and vitamin B6 (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors. This protocol describes step-by-step procedures for CSF sample preparation for the analysis of different molecules, HPLC calibration and analysis, and data quantification and interpretation. Unlike plasma/tissue/blood samples, CSF requires minimal sample preparation: in this protocol, only the analysis of PLP requires mixing with trichloroacetic acid to release the protein-bound vitamin, centrifugation, and mixing of the supernatant with phosphate buffer and sodium cyanide for derivatization in alkaline conditions. Monoamines are analyzed by HPLC with coulometric electrochemical detection (ED), pterins are analyzed by HPLC with coupled coulometric electrochemical and fluorescence detection, and PLP is analyzed by HPLC with fluorescence detection. The quantification of all compounds is achieved by external calibration procedures, and internal quality control and standards are analyzed in each run. We anticipate that investigation of dopamine and serotonin disturbances will be facilitated by measurements of these compounds in human CSF and other biological samples. The estimated time for the different procedures primarily depends on the electrochemical detector stabilization. Overnight stabilization of this detector is advised, and, after that step, preanalytical equilibration rarely exceeds 3 h.

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Figure 1: Chromatograms with different monoamine-related molecules (standard mixture).
Figure 2: Hydrodynamic voltammogram for 5HIAA and HVA in CSF.
Figure 3: A representation of a column, the in-line graphite filter and two in-line electrochemical detector cells.
Figure 4: Chromatograms of monoamines from the standard mixture and CSF samples from pediatric patients.
Figure 5: Chromatograms of pterins from the standard mixture and CSF samples from pediatric patients.
Figure 6: Chromatograms of PLP from the standard mixture and CSF samples from pediatric patients.
Figure 7: Experiment that requires troubleshooting to improve the analytical quality.

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Acknowledgements

This work was supported by grants from the Instituto de Salud Carlos III (ISCIII; FIS PI15/01082 and PI14/00032) and the FEDER Funding Program from the European Union. R.A. was supported by 'programa de intensificación de la actividad investigadora', from the ISCIII. The CIBERER is an initiative of the ISCIII.

Author information

Authors and Affiliations

Authors

Contributions

M.B., M.M.-L., A.O., M.C. and C.S. performed the experiments and collected all technical data regarding the monoamine and PLP determinations. S.P. managed the technical aspects of the pterin analysis and prepared the quality-control scheme. A.G.-C. and M.K. directed the sample collection and the management and establishment of the preanalytical protocols and clinical discussions. S.J.H. and R.A. designed the methods and planned the strategies for further methodological developments. M.B., M.M.-L., A.O. and R.A. wrote the manuscript. All authors critically reviewed the content of the manuscript.

Corresponding author

Correspondence to Rafael Artuch.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Metabolic pathways for the synthesis and catabolism of monoamines, pterins and pyridoxal phosphate.

The key metabolites for neurotransmitters and pterins are marked in bold and capital letters. Enzymes appear in italics. Tyrosine and tryptophan are the amino acid precursors of dopamine and serotonin, respectively. After a common rate-limiting enzymatic step, catalysed by two tetrahydrobiopterin (BH4)-dependent hydroxylases, L-dopa and 5-hydroxytryptophan are synthesized. After a common decarboxylation step, catalysed by L-aromatic amino acid decarboxylase (whose cofactor is pyridoxal-phosphate), the active neurotransmitters—dopamine and serotonin—are formed. Finally, several catabolic steps lead to the generation of the main end-stable metabolites, homovanillic acid (HVA) and 5-hydroxyindoleacetic (5HIAA) acid, which are the most useful diagnostic biomarkers. Adapted with permission from ref. [29], Elsevier.

Abbreviations

AADC: aromatic L-amino acid decarboxylase; 7,8-BH2: 7,8-dihydrobiopterin; BH4: tetrahydrobiopterin; COMT: catechol O-methyltransferase; CR: carbonyl reductase; DHFR: dihydrofolate reductase; DHPR: dihydropteridine reductase; DβH: dopamine betahydroxylase; GABA: Gamma-aminobutyric acid; GABA-T: Gamma-aminobutyric acid-transaminase; GAD: glutamate decarboxylase; GHB: 4-hydroxybutyric; GTP: guanosine triphosphate; Gln: glutamine; Glu: glutamate; GTPCH: GTP cyclohydrolase I; 5HIAA: 5-hydroxyindoleacetic acid; 5HTP: 5-hydroxytryptophan; 5-MTHF: 5-methyltetrahydrofolate; HCys: homocysteine; HVA: homovanillic acid; L-dopa: 3,4-dihydroxyphenylalanine; MAO: monoamine oxidase; Met: methionine; MHPG: 3-methoxy-4-hydroxyphenylglycol; n.e.: non-enzymatic; 3OMD: 3-O-methyldopa; OH-BH4: hydroxy-tetrahydrobiopterin; PCD: pterin-4a-carbinolamine dehydratase; PLK: pyridoxal kinase; PLP: pyridoxal phosphate; PNMT: phenylethanolamine N-methyltransferase; PNPO: pyridoxine-5'-phosphate oxidase; PTPS: 6-pyruvoyl-tetrahydropterin synthase; q-BH2: quinoide-dihydrobiopterin; SAH: S-adenosylhomocysteine; SAM: S-adenosylmethionine; SR: seapiapterin reductase; SSADH: succinic semialdehyde dehydrogenase; TCA: tricarboxylic acid cicle; TPH: tryprophan-5-hydroxylase; TH: tyrosine 3-hydroxylase; VLA: vanillactic acid; VMA: vanillmandelic acid; vB6: vitamin B6.

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Batllori, M., Molero-Luis, M., Ormazabal, A. et al. Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC. Nat Protoc 12, 2359–2366 (2017). https://doi.org/10.1038/nprot.2017.103

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