Extended diagnosis of purine and pyrimidine disorders from urine: LC MS/MS assay development and clinical validation

PLoS One. 2019 Feb 28;14(2):e0212458. doi: 10.1371/journal.pone.0212458. eCollection 2019.

Abstract

Background and aims: 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. 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.

Methods: The 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.

Results: Reliable 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.

Conclusions: A 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adenine / analogs & derivatives
  • Adenine / urine
  • Adolescent
  • Adult
  • Aminoimidazole Carboxamide / analogs & derivatives
  • Aminoimidazole Carboxamide / urine
  • Biomarkers / urine
  • Child
  • Child, Preschool
  • Chromatography, Liquid / methods*
  • Chromatography, Liquid / standards
  • Chromatography, Liquid / statistics & numerical data
  • Female
  • Humans
  • Infant
  • Male
  • Purine-Pyrimidine Metabolism, Inborn Errors / diagnosis*
  • Purine-Pyrimidine Metabolism, Inborn Errors / urine*
  • Quality Control
  • Reference Values
  • Ribonucleotides / urine
  • Tandem Mass Spectrometry / methods*
  • Tandem Mass Spectrometry / standards
  • Tandem Mass Spectrometry / statistics & numerical data
  • Urea / analogs & derivatives
  • Urea / urine
  • Uridine / analogs & derivatives
  • Uridine / urine

Substances

  • Biomarkers
  • Ribonucleotides
  • beta-ureidoisobutyric acid
  • 2,8-dihydroxyadenine
  • Aminoimidazole Carboxamide
  • Urea
  • orotidine
  • AICA ribonucleotide
  • Adenine
  • SAICAR
  • Uridine

Grant support

This study was generously supported by the Dietmar Hopp Foundation, St. Leon-Rot, Germany (http://dietmar-hopp-stiftung.de) grant NGS2020. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.