Screening for adenylosuccinate lyase deficiency using tandem mass spectrometry analysis of succinylpurines in neonatal dried blood spots

Zikánová, Marie; Krijt, Jakub; Škopová, Václava; Krijt, Matyáš; Barešová, Veronika; Kmoch, Stanislav

doi:10.1016/j.clinbiochem.2014.10.004

Cited by 7 publications

(11 citation statements)

References 23 publications

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“…Enzyme kinetics experiments, as well as theoretical calculations, have proposed that only the first step of PDNS is limiting for the pathway and that no change in other steps has an impact on the overall flux [26–28]. AICAr and SAICAr are the only PDNS intermediates detectable in the bodily fluids of healthy humans by current analytical technologies [10, 17, 29, 30]. These observations imply that other regulatory places might exist within the PDNS pathway.…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometric analysis of purine de novo biosynthesis intermediates

et al. 2018

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Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.

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Section: Resultsmentioning

confidence: 99%

Mass spectrometric analysis of purine de novo biosynthesis intermediates

et al. 2018

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“…Indeed, the sensitivity of S-Ado detection in plasma is much lower than that in urine and CSF [19] . Several simple screening procedures [20] , [21] were developed for detection of disorders of purine metabolism; most of the methods are based on the detection of abnormal metabolites, SAICAr and S-Ado, in the urine and CSF through HPLC and tandem mass spectrometry [2] , [19] .…”

Section: Discussionmentioning

confidence: 99%

“…1 ). Biochemically, ADSL deficiency can be identified by the presence of SAICAr and succinyladenosine (S-Ado) in biologic fluids [2] , which are otherwise not detected or not elevated [3] .…”

Section: Introductionmentioning

confidence: 99%

Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum

Donti

Cappuccio

Hubert

et al. 2016

Molecular Genetics and Metabolism Reports

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Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive neurometabolic disorder that presents with a broad-spectrum of neurological and physiological symptoms. The ADSL gene produces an enzyme with binary molecular roles in de novo purine synthesis and purine nucleotide recycling. The biochemical phenotype of ADSL deficiency, accumulation of SAICAr and succinyladenosine (S-Ado) in biofluids of affected individuals, serves as the traditional target for diagnosis with targeted quantitative urine purine analysis employed as the predominate method of detection. In this study, we report the diagnosis of ADSL deficiency using an alternative method, untargeted metabolomic profiling, an analytical scheme capable of generating semi-quantitative z-score values for over 1000 unique compounds in a single analysis of a specimen. Using this method to analyze plasma, we diagnosed ADSL deficiency in four patients and confirmed these findings with targeted quantitative biochemical analysis and molecular genetic testing. ADSL deficiency is part of a large a group of neurometabolic disorders, with a wide range of severity and sharing a broad differential diagnosis. This phenotypic similarity among these many inborn errors of metabolism (IEMs) has classically stood as a hurdle in their initial diagnosis and subsequent treatment. The findings presented here demonstrate the clinical utility of metabolomic profiling in the diagnosis of ADSL deficiency and highlights the potential of this technology in the diagnostic evaluation of individuals with neurologic phenotypes.

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“…Urinary excretion profiles are most commonly used but application of plasma, serum or cerebrospinal fluid have also been published [1, 2]. Only very few disorders were reported to be detectable from dried blood spots which could possibly allow their inclusion in newborn screening [14–16].…”

Section: Discussionmentioning

confidence: 99%

“…However, this color reaction test is neither quantitative nor specific as it may give positive results with a number of structurally related purine intermediates [10]. Previously, only few assays were reported for the quantitative measurement of SAICAr [14; 23; 24] which, in turn, allowed the detection of a much lower number of additional analytes.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2019

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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.

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Screening for adenylosuccinate lyase deficiency using tandem mass spectrometry analysis of succinylpurines in neonatal dried blood spots

Cited by 7 publications

References 23 publications

Mass spectrometric analysis of purine de novo biosynthesis intermediates

Mass spectrometric analysis of purine de novo biosynthesis intermediates

Diagnosis of adenylosuccinate lyase deficiency by metabolomic profiling in plasma reveals a phenotypic spectrum

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

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