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

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.

[1]  R. Touraine,et al.  AICA‐ribosiduria due to ATIC deficiency: Delineation of the phenotype with three novel cases, and long‐term update on the first case , 2020, Journal of inherited metabolic disease.

[2]  S. Lyonnet,et al.  PAICS deficiency, a new defect of de novo purine synthesis resulting in multiple congenital anomalies and fatal outcome. , 2019, Human molecular genetics.

[3]  J. Bierau,et al.  Extended diagnosis of purine and pyrimidine disorders from urine: LC MS/MS assay development and clinical validation , 2019, PloS one.

[4]  J. Václavík,et al.  Mass spectrometric analysis of purine de novo biosynthesis intermediates , 2018, PloS one.

[5]  S. Kmoch,et al.  CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation. , 2016, Molecular genetics and metabolism.

[6]  B. Behnam,et al.  Adenosine kinase deficiency with neurodevelopemental delay and recurrent hepatic dysfunction: A case report , 2016, Advances in rare diseases.

[7]  H. Blom,et al.  Adenosine kinase deficiency: expanding the clinical spectrum and evaluating therapeutic options , 2016, Journal of Inherited Metabolic Disease.

[8]  L. Fairbanks,et al.  Modern diagnostic approach to hereditary xanthinuria , 2015, Urolithiasis.

[9]  Stanislav Kmoch,et al.  Adenylosuccinate lyase deficiency , 2014, Journal of Inherited Metabolic Disease.

[10]  J. Christodoulou,et al.  Inborn errors of pyrimidine metabolism: clinical update and therapy , 2014, Journal of Inherited Metabolic Disease.

[11]  J. Christodoulou,et al.  Inborn errors of purine metabolism: clinical update and therapies , 2014, Journal of Inherited Metabolic Disease.

[12]  M. Baum,et al.  Developmental changes in renal tubular transport—an overview , 2015, Pediatric Nephrology.

[13]  J. Cocho,et al.  Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism. , 2012, Rapid communications in mass spectrometry : RCM.

[14]  C. Mercer,et al.  Miller (Genee-Wiedemann) syndrome represents a clinically and biochemically distinct subgroup of postaxial acrofacial dysostosis associated with partial deficiency of DHODH. , 2012, Human molecular genetics.

[15]  J. Lundeberg,et al.  Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function. , 2011, American journal of human genetics.

[16]  J. Reiss,et al.  Molybdenum cofactor deficiency: Mutations in GPHN, MOCS1, and MOCS2 , 2011, Human mutation.

[17]  A. Burlina,et al.  Quality of analytical performance in inherited metabolic disorders: the role of ERNDIM , 2008, Journal of Inherited Metabolic Disease.

[18]  S. Kmoch,et al.  Clinical, biochemical and molecular findings in seven Polish patients with adenylosuccinate lyase deficiency. , 2008, Molecular genetics and metabolism.

[19]  J. Sass,et al.  Comprehensive detection of disorders of purine and pyrimidine metabolism by HPLC with electrospray ionization tandem mass spectrometry. , 2006, Clinical chemistry.

[20]  U. Hofmann,et al.  Comprehensive analysis of pyrimidine metabolism in 450 children with unspecific neurological symptoms using high–pressure liquid chromatography–electrospray ionization tandem mass spectrometry , 2005, Journal of Inherited Metabolic Disease.

[21]  F. Bellia,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. , 2005, Clinical biochemistry.

[22]  H. Simmonds,et al.  Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features. , 1999, European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society.

[23]  W. Nyhan,et al.  Developmental disorder associated with increased cellular nucleotidase activity. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Seegmiller,et al.  A specific enzyme defect in gout associated with overproduction of uric acid. , 1967, Proceedings of the National Academy of Sciences of the United States of America.