Abstract
The analysis of (6R)-5,6,7,8-tetrahydrobiopterin (BH4) and neopterin in cerebrospinal fluid (CSF) is often used to identify defects in the pterin biosynthetic pathway affecting monoamine metabolism that can lead to pediatric neurotransmitter diseases. Low levels of BH4 and neopterin alone may not be sufficient to determine the defect, and further testing is often required. We have developed a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of BH4, 7,8-dihydrobiopterin (BH2), neopterin, and sepiapterin in CSF, which provides a more comprehensive evaluation of the pterin pathway. The method utilizes labeled stable isotopes as internal standards and allows for a fast 10-minute analysis by LC/MS/MS over a linear working range of 3 to 200 nmol/L. Total analytical imprecision is less than 14.4% for all pterin metabolites. Accuracy for BH4 and neopterin was determined by comparing data obtained by an alternative method using HPLC with EC and fluorescence detection. Excellent correlation was demonstrated for BH4 (r = 0.9646, 1/slope = 0.9397; n = 28; concentration range 3 to 63 nmol/L) and neopterin (r = 0.9919, 1/slope = 0.9539; n = 13; concentration range 5 to 240 nmol/L). CSF specimens from patients diagnosed with inborn errors of sepiapterin reductase (SR), 6-pyruvoyl-tetrahydropterin synthase (PTPS), dihydropteridine reductase (DHPR), and guanosine triphosphate cyclohydrolase (GTPCH) have been analyzed, and distinct pterin metabolite patterns were consistent with the initial diagnosis. This method differentiates patients with DHPR and SR deficiency from other pterin defects (GTPCH and PTPS) and will be useful for the diagnosis of specific defects in the pterin biosynthetic pathway.
Competing interests: None declared
An erratum to this chapter is available at 10.1007/8904_2014_372
An erratum to this chapter can be found at http://dx.doi.org/10.1007/8904_2014_372
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Antonozzi I, Carducci C, Vestri L, Pontecorvi A, Moretti F (1988) Rapid and sensitive method for high-performance liquid chromatographic analysis of pterins in biological fluids. J Chromatogr A 459:319–324
Blau N, Thöny B, Cotton RGH, Hyland K (2001) Disorders of tetrahydrobiopterin and related biogenic amines. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Vogelstein B (eds) The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp 1725–1776
Bonafe L, Thöny B, Penzien JM, Czarnecki B, Blau N (2001) Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia. Am J Hum Genet 69:269–277
Bräutigam M, Dreesen R, Herken H (1982) Determination of reduced biopterins by high pressure liquid chromatography and subsequent electrochemical detection. Hoppe Seylers Z Physiol Chem 363(3):341–343
Fismen L, Eide T, Djurhuus R, Svardal AM (2012) Simultaneous quantification of tetrahydrobiopterin, dihydrobiopterin and biopterin by liquid chromatography coupled electrospray tandem mass spectrometry. Anal Biochem 430:163–170
Fukushima T, Nixon JC (1980a) Chromatographic analysis of pteridines. Meth Enzymol 66:429–436
Fukushima T, Nixon JC (1980b) Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 120:176–188
Fukushima T, Kobayashi K, Eto I, Shiota T (1978) A differential microdetermination for various forms of biopterin. Anal Biochem 89:71
Guibal P, Lévêque N, Doummar D, Giraud N, Roze E, Rodriguez D, Couderc R, Billette De Villemeur T, Moussa F (2014) Simultaneous determination of all forms of biopterin and neopterin in cerebrospinal fluid. ACS Chem Neurosci 5(7):533–541
Guroff G, Rhoads CA, Abramowitz A (1967) A simple radioisotope assay for phenylalanine hydroxylase cofactor. Anal Biochem 21(2):273–278
Hyland K (1985) Estimation of tetrahydro, dihydro and fully oxidized pterins by high-performance liquid chromatography using sequential electrochemical and fluorometric detection. J Chromatogr Biomed Appl 343:35–41
Hyland K, Howell DW, Smith I (1986) An isocratic high-performance liquid chromatographic system for the investigation of abnormalities of neurotransmitter amine, biopterin, and aromatic amino acid metabolism in cerebrospinal fluid using sequential coulometric electrochemical and fluorescence detection. In: Joseph MH, Fillenz M, Macdonald IA, Marsden C (eds) Monitoring neurotransmitter release during behaviour. Ellis Howard, UK, pp 233-238
Kaufman S, Holtzman N, Milstein S, Butler IJ, Krumholz A (1975) Phenylketonuria due to a deficiency of dihydropteridine reductase. N Engl J Med 293:785–789
Kim HR, Kim TH, Hong SH, Kim HG (2012) Direct detection of tetrahydrobiopterin (BH4) and dopamine in rat brain using liquid chromatography coupled electrospray tandem mass spectrometry. Biochem Biophys Res Commun 419(4):632–637
Lunte CE, Kissinger PT (1983) The determination of pterins in biological samples by liquid chromatography/electrochemistry. Anal Biochem 129(2):377–386
Nagatsu T, Yamaguchi T, Kato T, Sugimoto T, Matsuura S, Akino M, Tsushima S, Nakazawa N, Ogawa H (1981) Radioimmunoassay for biopterin in body fluids and tissues. Anal Biochem 110(1):182–189
Niederwieser A, Blau N, Wang M, Joller P, Atarés M, Cardesa-Garcia J (1984) GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia. Eur J Pediatr 141(4):208–214
Schmidt H, Tegeder I, Geisslinger G (2006) Determination of neopterin and biopterin by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in rat and human plasma, cell extracts and tissue homogenates. http://www.nature.com/protocolexchange/protocols/86
Segawa M, Hosaka A, Miyagawa F, Nomura Y, Imai H (1976) Hereditary progressive dystonia with marked diurnal fluctuation. Adv Neurol 14:215–233
Stea B, Halpern R, Smith R (1979) Separation of unconjugated pteridines by high-pressure cation-exchange liquid chromatography. J Chromatogr 188(2):363–375
Thöny B, Neuheiser F, Kierat L, Blaskovics M, Arn PH, Ferreira P, Rebrin I, Ayling J, Blau N (1998) Hyperphenylalaninemia with high levels of 7-biopterin is associated with mutations in the PCBD gene encoding the bifunctional protein pterin-4a-carbinolamine dehydratase and transcriptional coactivator (DCoH). Am J Hum Genet 62(6):1302–1311
Zorzi G, Redweik U, Trippe H, Penzien JM, Thöny B, Blau N (2002) Detection of Sepiapterin in CSF of Patients with Sepiapterin Reductase Deficiency. J Neurochem 80(2):362–364
Acknowledgements
We would like to thank the following doctors for providing CSF from subjects with inborn errors of BH4 metabolism. Dr. Jose Abdenur and Dr. Richard Chang (Children’s Hospital of Orange County, Orange, CA); Dr. Klaas Wierenga (University of Oklahoma Health Sciences Center, Oklahoma City, OK); and Dr. Kathryn Swoboda (Pediatric Motor Disorders Research Program, Department of Neurology, University of Utah, Salt Lake City, UT).
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Communicated by: Nenad Blau, PhD
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Synopsis
Newly developed LC-MS/MS method for determination of CSF pterins will improve diagnostic differentiation of patients with inborn errors of pterin metabolism.
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Erland Arning and Teodoro Bottiglieri declare that they have no conflict of interest.
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All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Appropriate informed consents were obtained from all subjects or attending pediatric neurologists. All biochemical data was obtained through the routine analysis required for regular clinical care of each subject, as approved by the attending pediatric neurologist.
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This article does not contain any studies with animal subjects performed by the any of the authors.
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Article contributions by Erland Arning: conception and design, analysis, interpretation of data, drafting, and revising manuscript. Article contributions by Teodoro Bottiglieri: conception and design, interpretation of data, and revising manuscript. Erland Arning is the guarantor for this article and accepts full responsibility for the work and conduct of the study, had access to the data, and controlled the decision to publish.
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Arning, E., Bottiglieri, T. (2014). LC-MS/MS Analysis of Cerebrospinal Fluid Metabolites in the Pterin Biosynthetic Pathway. In: Morava, E., Baumgartner, M., Patterson, M., Rahman, S., Zschocke, J., Peters, V. (eds) JIMD Reports, Volume 29. JIMD Reports, vol 29. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8904_2014_336
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DOI: https://doi.org/10.1007/8904_2014_336
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