Abstract
Background and aimsInborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine.MethodsThe assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders.ResultsReliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens.ConclusionsA LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.
Highlights
Purine and pyrimidine bases, nucleosides and nucleotides are essential components of the nucleic acids DNA and RNA, and are associated with metabolic regulation, synthesis of numerous biomolecules and other vital processes in cell physiology [1,2,3] (S1 Fig)
Reliable determination of 27 analytes including SAICAr, 5aminoimidazole-4-carboxamide ribonucleoside (AICAr), beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation
Improved LC MS/MS assay for purine and pyrimidine disorders from urine
Summary
Nucleosides and nucleotides are essential components of the nucleic acids DNA and RNA, and are associated with metabolic regulation, synthesis of numerous biomolecules and other vital processes in cell physiology [1,2,3] (S1 Fig). Previously considered pediatric diseases, these disorders are increasingly being diagnosed in adults as milder presentations and mutations are recognized [1, 2]. This underlines the importance of analytical assays that can detect biomarkers of purine and pyrimidine metabolism with sufficient sensitivity and specificity [1, 2]. Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. We aimed to develop a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine
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