Non-targeted metabolomics by high resolution mass spectrometry in HPRT knockout mice

Tschirner, Sarah K.; Bähre, Heike; Kaever, Alexander; Schneider, Erich H.; Seifert, Roland; Kaever, Volkhard

doi:10.1016/j.lfs.2016.05.031

Cited by 6 publications

(7 citation statements)

References 38 publications

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“…As mentioned previously, erythrocytes lack an intact PDNS pathway but are capable of metabolizing exogenous ribosides to their corresponding mono-, di-, and triphosphates and accumulating the metabolites [35]. Several studies reported increased AICAR contents in the brains of HGPRT-deficient mice [37, 38]. We detected AICAR in cell lysates and AICAr in both the cell lysates and media of CR-HGPRT cells.…”

Section: Resultssupporting

confidence: 57%

“…The bifunctional enzyme ATIC, responsible for the last two steps of the pathway, is strongly inhibited by xanthosine-5'-monophosphate, which is accumulated in patients with Lesch-Nyhan syndrome because of the massive flux to uric acid production [39]. Another possible hypothesis for the accumulation of AICAR is enhanced histidine biosynthesis in HGPRT patients; however, there are no experimental data to support this hypothesis [38, 40]. As a result of shifted enzyme equilibria, labeled SAICAR was also detected.…”

Section: Resultsmentioning

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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Section: Resultssupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Mass spectrometric analysis of purine de novo biosynthesis intermediates

et al. 2018

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“…This finding suggests that ZMP might accumulate in the brain, because AICAr normally arises from dephosphorylation of ZMP. Although we have not measured purine nucleotides in the brain of patients with LND, it is interesting to note that HPRT1 knockout mice accumulate ZMP in the brain (19).…”

Section: (F)mentioning

confidence: 95%

“…Presumably, erythrocytes accumulate ZNT because they use metabolic precursors synthesized by other tissues. Interestingly, ZMP accumulation has been detected recently in the brain of HPRT1 knockout mice (19).…”

Section: Significancementioning

confidence: 99%

Physiological levels of folic acid reveal purine alterations in Lesch-Nyhan disease

López

Outtrim

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Lesch-Nyhan disease (LND), caused by a deficient salvage purine pathway, is characterized by severe neurological manifestations and uric acid overproduction. However, uric acid is not responsible for brain dysfunction, and it has been suggested that purine nucleotide depletion, or accumulation of other toxic purine intermediates, could be more relevant. Here we show that purine alterations in LND fibroblasts depend on the level of folic acid in the culture media. Thus, physiological levels of folic acid induce accumulation of 5-aminoimidazole-4-carboxamide riboside 5′-monophosphate (ZMP), an intermediary of de novo purine biosynthetic pathway, and depletion of ATP. Additionally, Z-nucleotide derivatives (AICAr, AICA) are detected at high levels in the urine of patients with LND and its variants (hypoxanthine-guanine phosphoribosyltransferase [HGprt]-related neurological dysfunction and HGprt-related hyperuricemia), and the ratio of AICAr/AICA is significantly increased in patients with neurological problems (LND and HGprt-related neurological dysfunction). Moreover, AICAr is present in the cerebrospinal fluid of patients with LND, but not in control individuals. We hypothesize that purine alterations detected in LND fibroblasts may also occur in the brain of patients with LND.

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“…It is now possible to detect and identify thousands of metabolites in a single run [1,2,3]. Although, initially, metabolomics had been applied mostly as a data-driven hypothesis generating approach in an un-targeted manner, recently both targeted and un-targeted metabolomics are becoming more popular to resolve many research questions [3,4,5,6]. Among different techniques of metabolite analysis, metabolite profiling is the oldest and most widely used in life sciences, and focuses on the study of a large group of metabolites related to different metabolic pathways [7].…”

Section: Introductionmentioning

confidence: 99%

Fully Automated Trimethylsilyl (TMS) Derivatisation Protocol for Metabolite Profiling by GC-MS

et al. 2016

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Gas Chromatography-Mass Spectrometry (GC-MS) has long been used for metabolite profiling of a wide range of biological samples. Many derivatisation protocols are already available and among these, trimethylsilyl (TMS) derivatisation is one of the most widely used in metabolomics. However, most TMS methods rely on off-line derivatisation prior to GC-MS analysis. In the case of manual off-line TMS derivatisation, the derivative created is unstable, so reduction in recoveries occurs over time. Thus, derivatisation is carried out in small batches. Here, we present a fully automated TMS derivatisation protocol using robotic autosamplers and we also evaluate a commercial software, Maestro available from Gerstel GmbH. Because of automation, there was no waiting time of derivatised samples on the autosamplers, thus reducing degradation of unstable metabolites. Moreover, this method allowed us to overlap samples and improved throughputs. We compared data obtained from both manual and automated TMS methods performed on three different matrices, including standard mix, wine, and plasma samples. The automated TMS method showed better reproducibility and higher peak intensity for most of the identified metabolites than the manual derivatisation method. We also validated the automated method using 114 quality control plasma samples. Additionally, we showed that this online method was highly reproducible for most of the metabolites detected and identified (RSD < 20) and specifically achieved excellent results for sugars, sugar alcohols, and some organic acids. To the very best of our knowledge, this is the first time that the automated TMS method has been applied to analyse a large number of complex plasma samples. Furthermore, we found that this method was highly applicable for routine metabolite profiling (both targeted and untargeted) in any metabolomics laboratory.

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Non-targeted metabolomics by high resolution mass spectrometry in HPRT knockout mice

Cited by 6 publications

References 38 publications

Mass spectrometric analysis of purine de novo biosynthesis intermediates

Mass spectrometric analysis of purine de novo biosynthesis intermediates

Physiological levels of folic acid reveal purine alterations in Lesch-Nyhan disease

Fully Automated Trimethylsilyl (TMS) Derivatisation Protocol for Metabolite Profiling by GC-MS

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