Oligodendroglia from ADSL-deficient patient produce SAICAribotide and SAMP

Zídková, Lenka; Krijt, Jakub; Sladkova, Jana; Hlobílková, Alice; Magner, Martin; Zikánová, Marie; Kmoch, Stanislav; Friedecký, David; Zeman, Jiřı́; Elleder, M; Adam, Tomáš

doi:10.1016/j.ymgme.2010.06.014

Cited by 1 publication

(2 citation statements)

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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%

“…The methods used for studying purine metabolism and diagnosing related metabolic diseases include proton nuclear magnetic resonance, electrophoresis, and chromatography. Chromatography dominates the field and has historically used UV absorbance, radiometric and tandem mass spectrometric detection [4, 8–10]. PDNS ribosides excreted into growth media in four genetically modified cell lines were detected in a recent study [11].…”

Section: Introductionmentioning

confidence: 99%

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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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