MSACL 2019 EU Abstract

UPLC-MS/MS-based Assays for Diagnosis and Therapeutic Monitoring in Patients with APRT Deficiency

Unnur A. Thorsteinsdottir (1,2), Finnur F. Eiriksson (1,2), Hrafnhildur L. Runolfsdottir (1), Vidar O Edvardsson (1,3), Runolfur Palsson (1,3), Margret Thorsteinsdottir (1,2)
(1) University of Iceland, Reykjavik, Iceland (2) ArcticMass, Reykjavik, Iceland (3) Landspitali – The National University Hospital of Iceland, Reykjavik, Iceland

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Unnur Arna Thorsteinsdottir (Presenter) University of Iceland

Presenter Bio: I hold a master’s degree in cell biology from the University of Copenhagen (graduated in 2015). I’m currently a PhD student at the Faculty of Pharmaceutical Sciences at the University of Iceland (2016-current). In my PhD project I’m developing a clinical diagnostic method for a rare kidney stone disease using UPLC-MS/MS.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction
Adenine phosphoribosyltransferase deficiency (APRTd) is an inborn error of adenine metabolism, characterized by excessive production and renal excretion of a poorly soluble compound, 2,8-dihydroxyadenine (DHA) that leads to kidney stone formation and chronic kidney disease (CKD). Treatment with the xanthine oxidoreductase (XOR) inhibitors allopurinol and febuxostat reduces urinary DHA excretion and decreases stone formation. The aim of this study was to use UPLC-MS/MS-based assay for quantification of the biomarker DHA, metabolites involved in the adenine metabolic pathway and XOR inhibitors in urine and plasma samples for the purpose of improving the diagnosis and monitoring of pharmacotherapy in APRTd patients.

Methods
The UPLC-MS/MS assay was optimized by design of experiments (DoE) using the chemometric software MODDE 12 (Sartorius Stedim Data Analytics AB, Umeå, Sweden). D-optimal design was used for experimental screening of the variables and significant variables optimized with central composite face design (CCF) and related to sensitivity and resolution utilizing partial least square (PLS) regression.
Urine and plasma samples from APRTd patients (enrolled in the APRT Deficiency Registry and Biobank of the Rare Kidney Stone Consortium), both those receiving treatment with allopurinol or febuxostat and untreated patients, were analyzed as well as samples from heterozygotes and healthy individuals.
Protein and phospholipid removal plate was used for sample preparation of plasma samples (ISOLUTE® PLD+ Protein and Phospholipid Removal Plate, Biotage, Uppsala, Sweden) and urine samples were diluted 1:15 (v/v) prior to analysis.

Results, Discussion & Conclusions
Preliminary data from the UPLC-MS/MS plasma assay revealed that the DHA concentration in untreated patients ranged from 456-741 ng/mL and 27-130 ng/ml in patients treated with allopurinol. DHA was not detected in plasma samples from patients treated with febuxostat and in healthy controls. DHA was detected in all urine samples from untreated patients but not in any specimens from heterozygotes and healthy controls. The UPLC-MS/MS urinary assay revealed that DHA excretion in APRTd patients decreased from 75–289 mg in untreated patients to 13–112 and 10.0-13.4 mg with conventional doses of allopurinol and febuxostat, respectively.
Future work will focus on comparing the concentration of DHA, purine metabolites and XOR inhibitors in plasma and urine with the long-term clinical outcome of patients in order to establish a plasma DHA level and urine excretion of DHA that must be achieved in order to prevent new kidney stone formation and CKD progression in APRTd patients. Furthermore, the effect of the disease and the treatment on the adenine metabolic pathway will be investigated.