A new diagnostic method for detecting a rare kidney stone disease has recently been developed at the University of Iceland. Instead of using urine microscopy, which has certain disadvantages, the diagnostic method is based on mass spectrometry of plasma samples. Preliminary clinical data shows very promising results both in terms of detecting the disease and therapeutic drug monitoring. Design of Experiments (DOE) was used as a chemometric approach to optimize the assay. Below is a summary of the assay development and optimization.
Scientists used DOE as a chemometric approach to optimize an assay for mass spectrometry.
Severe consequences for patients if the disease is undetected
Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism. In patients suffering from APRT deficiency, the function of the APRT enzyme is deficient and the purine base adenine, instead of being metabolized to adenosine monophosphate (AMP), is oxidized by the enzyme xantine oxidoreductase (XOR), into 2,8-dihydroxyadenine (DHA).
DHA is poorly soluble and accumulates in the urine, forming urinary crystals and stones. The consequences for the patients are recurrent kidney stones, chronic kidney disease and in some cases even kidney failure.
Disadvantages with current diagnostic methods
The prevalence of APRT deficiency has been estimated to 0.5 to 1 per 100,000 in the Caucasian population, but far fewer are diagnosed, meaning that the disease might be underdiagnosed. APRT deficiency can be effectively treated with XOR inhibitors. Lifestyle changes such as a high fluid intake and a low-purine diet can also improve conditions for patients. Since the disease can be treated, it is important that patients are diagnosed at an early stage to avoid severe consequences.
Currently, diagnosis is performed by APRT enzyme activity assay as well as gene sequencing. Therapeutic drug monitoring is performed with urine microscopy, which lacks precision and is also dependent on the operator being able to recognize the crystals.
Using Design of Experiments to optimize a UPLC-MS/MS assay
Unnur A. Thorsteinsdóttir, PhD student at the University of Iceland, has together with her team developed and optimized a clinical mass spectrometry assay for diagnosis as well as therapeutic drug monitoring of APRT deficiency. The aims of the project were to simultaneously quantify the biomarker DHA, the XOR inhibitors as well as all the purines involved in the adenine metabolic pathway. With that information it will be possible to shed some light on the variations in the clinical expression of the disease and also to establish a safe level of the biomarker DHA, where the patients are not experiencing recurring kidney stones.
There were several reasons for choosing Design of Experiments for the optimization. The team wanted to include several compounds in the assay and they also had multiple aims, which is difficult to handle with a traditional COST approach. Furthermore, instead of looking at big data tables, Design of Experiments provides a good visualization of results.
Read more about using DOE instead of the traditional COST approach.
The image shows how Design of Experiments can assist as a visual tool when optimizing an assay.
The equipment used for the analysis and optimization
Thorsteinsdóttir and her team used an ultra-performance liquid chromatography tandem mass spectrometry, UPLC-MS/MS, with electrospray ionization for the analysis. Design of Experiments was used as chemometric approach to optimize the assay, using MODDE from the Umetrics Suite of Data Analytics Solutions for the Design of Experiments and SIMCA for multivariate data analysis.
Plasma samples from a healthy control group and APRT deficient patients were used as well as clinical data from urine samples from an earlier assay.
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Thorsteinsdóttir and her team were able to successfully optimize the UPLC-MS/MS assay for simultaneous quantification of DHA, the purine compounds and the XOR inhibitors. Preliminary clinical data from patients show that the mass spectrometry assay makes it possible to quickly detect if an individual is affected by APRT deficiency and the assay can also be used to monitor the effect of drug therapy. Thorsteinsdóttir concludes that the assay can significantly improve the lives of patients.
Unnur A. Thorsteinsdóttir holds a master’s degree in cell biology from the University of Copenhagen and is currently a PhD student at the School of Health Sciences, University of Iceland. The UPLC-MS/MS assay was developed in cooperation with The National University Hospital of Iceland and bioanalytical services company Arctic Mass Ltd.
Want to know more?
In this webinar, Unnur A. Thorsteinsdóttir gives a detailed presentation of the development and optimization of the mass spectrometry assay.