OBJECTIVES: To develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 41 different purine and pyrimidine (PuPy) metabolites in human urine to allow detection of most known disorders in this metabolic pathway and to determine reference intervals. METHODS: Urine samples were diluted with an aqueous buffer to minimize ion suppression. For detection and quantification, liquid chromatography was combined with electrospray ionization, tandem mass spectrometry and multiple reaction monitoring. Transitions and instrument settings were established to quantify 41 analytes and nine stable-isotope-labeled internal standards (IS). RESULTS: The established method is precise (intra-day CV: 1.4-6.3%; inter-day CV: 1.3-15.2%), accurate (95.2% external quality control results within ±2 SD and 99.0% within ±3 SD; analyte recoveries: 61-121%), sensitive and has a broad dynamic range to quantify normal and pathological metabolite concentrations within one run. All analytes except aminoimidazole ribonucleoside (AIr) are stable before, during and after sample preparation. Moreover, analytes are not affected by five cycles of freeze-thawing (variation: -5.6 to 7.4%), are stable in thymol (variation: -8.4 to 12.9%) and the lithogenic metabolites also in HCl conserved urine. Age-dependent reference intervals from 3,368 urine samples were determined and used to diagnose 11 new patients within 7 years (total performed tests: 4,206). CONCLUSIONS: The presented method and reference intervals enable the quantification of 41 metabolites and the potential diagnosis of up to 25 disorders of PuPy metabolism.

Division of Clinical Chemistry and Biochemistry University Children's Hospital Zurich University of Zurich Zurich Switzerland

Division of Metabolism and Children's Research Center University Children's Hospital Zurich University of Zurich Zurich Switzerland

Research Unit for Rare Diseases Department of Paediatrics and Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

UniCamillus Saint Camillus International University of Health and Medical Sciences Rome Italy

Zobrazit více v PubMed

Balasubramaniam, S, Duley, JA, Christodoulou, J. Inborn errors of pyrimidine metabolism: clinical update and therapy. J Inherit Metab Dis 2014;37:687–98. https://doi.org/10.1007/s10545-014-9742-3 . DOI

Balasubramaniam, S, Duley, JA, Christodoulou, J. Inborn errors of purine metabolism: clinical update and therapies. J Inherit Metab Dis 2014;37:669–86. https://doi.org/10.1007/s10545-014-9731-6 . DOI

Van Kuilenburg, AB, Vreken, P, Abeling, NG, Bakker, HD, Meinsma, R, Van Lenthe, H, et al.. Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency. Hum Genet 1999;104:1–9. https://doi.org/10.1007/pl00008711 . DOI

Jurecka, A, Zikanova, M, Tylki-Szymanska, A, Krijt, J, Bogdanska, A, Gradowska, W, et al.. Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency. Mol Genet Metabol 2008;94:435–42. https://doi.org/10.1016/j.ymgme.2008.04.013 . DOI

Kohler, M, Assmann, B, Brautigam, C, Storm, W, Marie, S, Vincent, MF, et al.. Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features. Eur J Paediatr Neurol 1999;3:3–6. https://doi.org/10.1053/ejpn.1999.0172 . DOI

Sebesta, I, Krijt, J, Kmoch, S, Hartmannova, H, Wojda, M, Zeman, J. Adenylosuccinase deficiency: clinical and biochemical findings in 5 Czech patients. J Inherit Metab Dis 1997;20:343–4. https://doi.org/10.1023/a:1005361408031 .

Sumi, S, Kidouchi, K, Ohba, S, Wada, Y. Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography. J Chromatogr B Biomed Appl 1995;672:233–9. https://doi.org/10.1016/0378-4347(95)00228-b . DOI

Vidotto, C, Fousert, D, Akkermann, M, Griesmacher, A, Muller, MM. Purine and pyrimidine metabolites in children’s urine. Clin Chim Acta 2003;335:27–32. https://doi.org/10.1016/s0009-8981(03)00291-2 . DOI

Hartmann, S, Okun, JG, Schmidt, C, Langhans, CD, Garbade, SF, Burgard, P, et al.. Comprehensive detection of disorders of purine and pyrimidine metabolism by HPLC with electrospray ionization tandem mass spectrometry. Clin Chem 2006;52:1127–37. https://doi.org/10.1373/clinchem.2005.058842 . DOI

Rebollido-Fernandez, MM, Castineiras, DE, Boveda, MD, Couce, ML, Cocho, JA, Fraga, JM. Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism. Rapid Commun Mass Spectrom 2012;26:2131–44. https://doi.org/10.1002/rcm.6325 . DOI

Schmidt, C, Hofmann, U, Kohlmuller, D, Murdter, T, Zanger, UM, Schwab, M, et al.. Comprehensive analysis of pyrimidine metabolism in 450 children with unspecific neurological symptoms using high-pressure liquid chromatography-electrospray ionization tandem mass spectrometry. J Inherit Metab Dis 2005;28:1109–22. https://doi.org/10.1007/s10545-005-0133-7 . DOI

Monostori, P, Klinke, G, Hauke, J, Richter, S, Bierau, J, Garbade, SF, et al.. Extended diagnosis of purine and pyrimidine disorders from urine: LC MS/MS assay development and clinical validation. PLoS One 2019;14:e0212458. https://doi.org/10.1371/journal.pone.0212458 . DOI

Tavazzi, B, Lazzarino, G, Leone, P, Amorini, AM, Bellia, F, Janson, CG, et al.. Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism. Clin Biochem 2005;38:997–1008. https://doi.org/10.1016/j.clinbiochem.2005.08.002 . DOI

Madrova, L, Krijt, M, Baresova, V, Vaclavik, J, Friedecky, D, Dobesova, D, et al.. Mass spectrometric analysis of purine de novo biosynthesis intermediates. PLoS One 2018;13:e0208947. https://doi.org/10.1371/journal.pone.0208947 . DOI

Fowler, B, Burlina, A, Kozich, V, Vianey-Saban, C. Quality of analytical performance in inherited metabolic disorders: the role of ERNDIM. J Inherit Metab Dis 2008;31:680–9. https://doi.org/10.1007/s10545-008-1025-4 . DOI

Rainger, J, Bengani, H, Campbell, L, Anderson, E, Sokhi, K, Lam, W, et al.. Miller (Genee-Wiedemann) syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH. Hum Mol Genet 2012;21:3969–83. https://doi.org/10.1093/hmg/dds218 . DOI

Kelley, WN, Rosenbloom, FM, Henderson, JF, Seegmiller, JE. A specific enzyme defect in gout associated with overproduction of uric acid. Proc Natl Acad Sci USA 1967;57:1735–9. https://doi.org/10.1073/pnas.57.6.1735 . DOI

Mraz, M, Hurba, O, Bartl, J, Dolezel, Z, Marinaki, A, Fairbanks, L, et al.. Modern diagnostic approach to hereditary xanthinuria. Urolithiasis 2015;43:61–7. https://doi.org/10.1007/s00240-014-0734-4 . DOI

Page, T, Yu, A, Fontanesi, J, Nyhan, WL. Developmental disorder associated with increased cellular nucleotidase activity. Proc Natl Acad Sci USA 1997;94:11601–6. https://doi.org/10.1073/pnas.94.21.11601 . DOI

Reiss, J, Hahnewald, R. Molybdenum cofactor deficiency: mutations in GPHN, MOCS1, and MOCS2. Hum Mutat 2011;32:10–8. https://doi.org/10.1002/humu.21390 . DOI

Ichida, K, Amaya, Y, Kamatani, N, Nishino, T, Hosoya, T, Sakai, O. Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria. J Clin Invest 1997;99:2391–7. https://doi.org/10.1172/jci119421 . DOI

Krijt, M, Souckova, O, Baresova, V, Skopova, V, Zikanova, M. Metabolic tools for identification of new mutations of enzymes engaged in purine synthesis leading to neurological impairment. Folia Biol (Praha) 2019;65:152–7.

Baresova, V, Krijt, M, Skopova, V, Souckova, O, Kmoch, S, Zikanova, M. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation. Mol Genet Metabol 2016;119:270–7. https://doi.org/10.1016/j.ymgme.2016.08.004 . DOI

van Gennip, AH, Abeling, NG, Vreken, P, van Kuilenburg, AB. Inborn errors of pyrimidine degradation: clinical, biochemical and molecular aspects. J Inherit Metab Dis 1997;20:203–13. https://doi.org/10.1023/a:1005356806329 .

Gattineni, J, Baum, M. Developmental changes in renal tubular transport-an overview. Pediatr Nephrol 2015;30:2085–98. https://doi.org/10.1007/s00467-013-2666-6 . DOI

Pelet, A, Skopova, V, Steuerwald, U, Baresova, V, Zarhrate, M, Plaza, JM, et al.. PAICS deficiency, a new defect of de novo purine synthesis resulting in multiple congenital anomalies and fatal outcome. Hum Mol Genet 2019;28:3805–14. https://doi.org/10.1093/hmg/ddz237 . DOI

Ramond, F, Rio, M, Heron, B, Imbard, A, Marie, S, Billiemaz, K, et al.. AICA-ribosiduria due to ATIC deficiency: delineation of the phenotype with three novel cases, and long-term update on the first case. J Inherit Metab Dis 2020;43:1254–64. https://doi.org/10.1002/jimd.12274 . DOI

Jurecka, A, Zikanova, M, Kmoch, S, Tylki-Szymanska, A. Adenylosuccinate lyase deficiency. J Inherit Metab Dis 2015;38:231–42. https://doi.org/10.1007/s10545-014-9755-y . DOI

Bollee, G, Harambat, J, Bensman, A, Knebelmann, B, Daudon, M, Ceballos-Picot, I. Adenine phosphoribosyltransferase deficiency. Clin J Am Soc Nephrol 2012;7:1521–7. https://doi.org/10.2215/cjn.02320312 . DOI

Kaartinen, K, Hemmila, U, Salmela, K, Raisanen-Sokolowski, A, Kouri, T, Makela, S. Adenine phosphoribosyltransferase deficiency as a rare cause of renal allograft dysfunction. J Am Soc Nephrol 2014;25:671–4. https://doi.org/10.1681/asn.2013090960 . DOI

Bjursell, MK, Blom, HJ, Cayuela, JA, Engvall, ML, Lesko, N, Balasubramaniam, S, et al.. Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function. Am J Hum Genet 2011;89:507–15. https://doi.org/10.1016/j.ajhg.2011.09.004 . DOI

Shakiba, M, Mahjoub, F, Fazilaty, H, Rezagholizadeh, F, Shakiba, A, Ziadlou, M, et al.. Adenosine kinase deficiency with neurodevelopmental delay and recurrent hepatic dysfunction: a case report. Adv Rare Dis 2016;3. https://doi.org/10.12715/ard.2014.3.2 . DOI

Staufner, C, Lindner, M, Dionisi-Vici, C, Freisinger, P, Dobbelaere, D, Douillard, C, et al.. Adenosine kinase deficiency: expanding the clinical spectrum and evaluating therapeutic options. J Inherit Metab Dis 2016;39:273–83. https://doi.org/10.1007/s10545-015-9904-y . DOI

Raida, M, Schwabe, W, Hausler, P, Van Kuilenburg, AB, Van Gennip, AH, Behnke, D, et al.. Prevalence of a common point mutation in the dihydropyrimidine dehydrogenase (DPD) gene within the 5′-splice donor site of intron 14 in patients with severe 5-fluorouracil (5-FU)-related toxicity compared with controls. Clin Cancer Res 2001;7:2832–9.

Expanding clinical spectrum of PAICS deficiency: Comprehensive analysis of two sibling cases