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

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.

Fig 1. Results of the ERNDIM Diagnostic Proficiency Testing (DPT) samples (n = 10).

Numbers on the cylinders show how many times a given result exceeded the respective cutoff for that biomarker. PP: purine-pyrimidine disorder. Further abbreviations are provided in the List of Abbreviations Section. Note that orotidine is not pathognomonic for HGPRT (sample E04) but is accumulated as a result of allopurinol treatment.

Fig 2. Results of urine samples from patients with known diagnosis (n = 10).

Numbers on the cylinders show how many times a given result exceeded the respective cutoff for that biomarker. Abbreviations are provided in the List of Abbreviations Section. The Y-axis has been cut at 45 for a better visibility. Note that orotidine is not pathognomonic for HGPRT (sample P05) but is accumulated as a result of allopurinol treatment.

Fig 3

LC-MS/MS chromatograms of A: the Cal06 calibrator (16 μM) and urine specimens from B: a healthy proband; C: a patient with ADSL deficiency; D: a patient with HGPRT deficiency; E: a patient with UPB1 deficiency; and F: a patient with APRT deficiency. All intensities (Y-axes) have been normalized to 1.5*10⁸ cps for better comparability and have been plotted against retention time (X-axes). Analytes have been numbered as follows: 1. URA; 2. DHU; 3. Thy; 4. DHT; 5. BUP; 6. ADN; 7. Hypo; 8. ALLOP; 9. BUIB; 10. Xan; 11. DHA; 12. dAdo; 13. AICAr; 14. ADS; 15. HMU; 16. Oro; 17. dUrd; 18. THD; 19. PSU; 20. dIno; 21. dGui; 22. Ino; 23. Guo; 24. Ord; 25. SAICAr; 26. SAdo. The ISs have been marked by an asterisk (*) after the number of the respective unlabeled analyte. Note that guanosine-IS has been measured in both ESI+ and ESI-. ADSL: Adenylosuccinate lyase deficiency; HGPRT: Hypoxanthine-guanine phosphoribosyltransferase deficiency; UPB1: beta-Ureidopropionase deficiency; APRT: Adenine phosphoribosyltransferase deficiency. Further abbreviations are provided in the List of Abbreviations Section. Chromatograms were generated by MassLynx 4.1 (Waters, Milford, MA, USA) and modified using Inkscape 0.91pre4 (Open Source Software licensed under the GNU General Public License).