Startseite Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application
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Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application

  • Alessio Cremonesi EMAIL logo , David Meili , Anahita Rassi , Martin Poms , Barbara Tavazzi ORCID logo , Václava Škopová , Johannes Häberle , Marie Zikánová und Martin Hersberger ORCID logo
Veröffentlicht/Copyright: 4. April 2023
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Clinical Chemistry and Laboratory Medicine (CCLM)
Aus der Zeitschrift Clinical Chemistry and Laboratory Medicine (CCLM) Band 61 Heft 10

Abstract

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.

Keywords: diagnosis; LC-MS/MS; purines; pyrimidines; reference intervals; stability
Corresponding author: Alessio Cremonesi, Division of Clinical Chemistry and Biochemistry, University Children’s Hospital Zurich, University of Zurich, Steinwiesstrasse 75, 8032 Zürich, Switzerland, Phone: +41 44 266 75 44, Fax: +41 44 266 71 69, E-mail:

Funding source: National Institute for Neurological Research

Award Identifier / Grant number: LX22NPO5107

Acknowledgments

The authors would like to thank Dr. Brian Fowler (University Children Hospital Basel, Basel, Switzerland), Dr. Jorgen Bierau (Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands) and Dr. George Ruijter (Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands) for kindly providing confirmed pathological samples for method validation.

  1. Research funding: The work of Václava Škopová and Marie Zikánová was supported by the Cooperation Program, research area Medical Diagnostics and Basic Medical Sciences and the programme National Institute for Neurological Research (Programme EXCELES, ID Project No. LX22NPO5107) funded by the European Union – Next Generation EU.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The local Institutional Review Board deemed the study exempt from review.

References

1. 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.Suche in Google Scholar PubMed

2. 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.Suche in Google Scholar PubMed

3. 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.Suche in Google Scholar PubMed

4. 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.Suche in Google Scholar PubMed

5. 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.Suche in Google Scholar PubMed

6. 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:1005361408031.10.1023/A:1005361408031Suche in Google Scholar

7. 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.Suche in Google Scholar PubMed

8. 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.Suche in Google Scholar PubMed

9. 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.Suche in Google Scholar PubMed

10. 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.Suche in Google Scholar PubMed

11. 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.Suche in Google Scholar PubMed

12. 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.Suche in Google Scholar PubMed PubMed Central

13. 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.Suche in Google Scholar PubMed

14. 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.Suche in Google Scholar PubMed PubMed Central

15. 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.Suche in Google Scholar PubMed

16. 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.Suche in Google Scholar PubMed

17. 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.Suche in Google Scholar PubMed PubMed Central

18. 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.Suche in Google Scholar PubMed

19. 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.Suche in Google Scholar PubMed PubMed Central

20. 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.Suche in Google Scholar PubMed

21. 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.Suche in Google Scholar PubMed PubMed Central

22. 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;65:152–7.10.14712/fb2019065030152Suche in Google Scholar

23. 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.Suche in Google Scholar PubMed

24. 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:1005356806329.10.1023/A:1005356806329Suche in Google Scholar

25. 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.Suche in Google Scholar PubMed PubMed Central

26. 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.Suche in Google Scholar PubMed

27. 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.Suche in Google Scholar PubMed

28. 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.Suche in Google Scholar PubMed PubMed Central

29. 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.Suche in Google Scholar PubMed

30. 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.Suche in Google Scholar PubMed PubMed Central

31. 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.Suche in Google Scholar PubMed PubMed Central

32. 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.Suche in Google Scholar PubMed PubMed Central

33. 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.Suche in Google Scholar PubMed

34. 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.Suche in Google Scholar

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2022-1236).

Received: 2022-12-02
Accepted: 2023-03-27
Published Online: 2023-04-04
Published in Print: 2023-09-26

© 2023 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2022-1236
Schlagwörter für diesen Artikel
diagnosis; LC-MS/MS; purines; pyrimidines; reference intervals; stability

Artikel in diesem Heft

  1. Frontmatter
  2. Editorial
  3. Laboratory Medicine: from just testing to saving lives
  4. Reviews
  5. Serum biomarkers of remodeling in severe asthma with fixed airway obstruction and the potential role of KL-6
  6. Safety monitoring of drug-induced muscle injury and rhabdomyolysis: a biomarker-guided approach for clinical practice and drug trials
  7. Mini Review
  8. Concise review on the combined use of immunocapture, mass spectrometry and liquid chromatography for clinical applications
  9. Opinion Paper
  10. Recommendation for the design of stability studies on clinical specimens
  11. General Clinical Chemistry and Laboratory Medicine
  12. Assessment of WHO 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays
  13. veRification: an R Shiny application for laboratory method verification and validation
  14. Impact of storage temperature and time before analysis on electrolytes (Na+, K+, Ca2+), lactate, glucose, blood gases (pH, pO2, pCO2), tHb, O2Hb, COHb and MetHb results
  15. The stability of blood gases and CO-oximetry under slushed ice and room temperature conditions
  16. Elevated levels of renal function tests conferred increased risks of developing various pregnancy complications and adverse perinatal outcomes: insights from a population-based cohort study
  17. Poor comparability of plasma renin activity measurement in determining patient samples: the status quo and recommendations for harmonization
  18. Salivary cortisol and cortisone in diagnosis of Cushing’s syndrome – a comparison of six different analytical methods
  19. Improved diagnostics of purine and pyrimidine metabolism disorders using LC-MS/MS and its clinical application
  20. Analytical evaluation of a GAD65 antibodies chemiluminescence immunoassay for CSF in neurological syndromes
  21. Reference Values and Biological Variations
  22. Evaluation of low-density lipoprotein cholesterol equations by cross-platform assessment of accuracy-based EQA data against SI-traceable reference value
  23. Highly sensitive tandem mass spectrometric measurement of serum estradiol without derivatization and pediatric reference intervals in children and adolescents
  24. Cancer Diagnostics
  25. Practical delta check limits for tumour markers in different clinical settings
  26. Comparison between free β subunit of human chorionic gonadotropin (hCG) and total hCG assays in adults with testicular cancer
  27. Hematology and Coagulation
  28. Value of monocyte distribution width for predicting severe cholecystitis: a retrospective cohort study
  29. Performance of digital morphology analyzer Medica EasyCell assistant
  30. Validation of non-invasive point of care blood content analysis using the TensorTip™ MTX device: a method comparison study
  31. Infectious Diseases
  32. Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration
  33. Letters to the Editor
  34. The first case of VEXAS syndrome in Austria
  35. Acetylcholine receptor and muscle-specific tyrosine kinase antibodies detection: is it time for a change?
  36. Performance of the 2009 CKDEPI, 2021 CKDEPI, and EKFC equations among high-risk patients in Denmark
  37. Biotin interference in immunoassays: water under the bridge?
  38. The new synthetic benzimidazole opioid etonitazepipne: an emerging fatal harm and a challenge for laboratory medicine
  39. Unexplained increase of serum carcinoembryonic antigen: don’t forget the thyroid!
  40. Falsely elevated cortisol serum levels in preterm infants due to use of immunoassay
  41. Misdiagnosis of Hb Bart’s disease: prenatal screening and diagnosis of thalassemia in special population
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