A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism--AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry.
- MeSH
- aminoimidazolkarboxamid analogy a deriváty chemie farmakologie MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- erytrocyty cytologie metabolismus MeSH
- hmotnostní spektrometrie metody MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- nukleotidy analýza chemie izolace a purifikace MeSH
- puriny chemie MeSH
- ribonukleosidy chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acadesine MeSH Prohlížeč
- aminoimidazolkarboxamid MeSH
- nukleotidy MeSH
- puriny MeSH
- ribonukleosidy MeSH
Two inherited deficiencies have been described in purine de novo synthesis pathway. Both the defects are diagnosed by detecting ribosides--dephosphorylated substrates of the enzymes--in patient's urine. We describe here a synthesis and mass spectrometric fragmentation of ribosides potentially of diagnostic importance for defects in the second part of the pathway. All the species, except 5-amino-4-imidazolesuccinocarboxamideriboside can be synthesized from the commercially available 5-amino-4-imidazolecarboxamideriboside by chemical methods. Fragmentation spectra of the compounds were obtained by the ion trap mass spectrometry. During fragmentation an opening of the imidazole ring was not observed for any of the compounds but loss of its substituents in the form of small molecules (NH3, CO2, CO) is the major route of fragmentation. The ribose moiety cleaves off molecule(s) of water, undergoes a cross-ring cleavage or breaks away as a whole.
- MeSH
- aminoimidazolkarboxamid analogy a deriváty chemie MeSH
- biochemie metody MeSH
- chemické modely MeSH
- hmotnostní spektrometrie metody MeSH
- ionty MeSH
- molekulární struktura MeSH
- puriny chemie MeSH
- ribonukleosidy chemická syntéza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acadesine MeSH Prohlížeč
- aminoimidazolkarboxamid MeSH
- ionty MeSH
- puriny MeSH
- ribonukleosidy MeSH
The enzyme adenylosuccinate lyase (ADSL) intervenes twice in the biosynthesis of adenine nucleotides. ADSL deficiency is an inherited metabolic disease characterized by various degrees of psychomotor retardation and accumulation of dephosphorylated enzyme substrates 5-amino-4-imidazole-N-succinocarboxamide riboside (SAICAr) and succinyladenosine (SAdo) in body fluids. Severity of symptoms seems to correlate with residual activity of mutant enzyme and with SAdo/SAICAr concentration ratio in cerebrospinal fluid. To better understand the pathogenetic mechanisms of the disease symptoms, studies of catalytic properties of mutant enzymes together with in vitro and in vivo experiments utilizing SAICAr and SAdo must be performed. Such studies require availability of both ADSL substrates, 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR) and succinyladenosine 5'-monophosphate (SAMP) and their dephosphorylated products in sufficient amounts and purity. Except for SAMP, none of these compounds is commercially available and they must therefore be synthesized. SAICAR was prepared by recombinant human ADSL-catalysed reaction of AICAR (5-aminoimidazole-4-carboxamide) with fumarate and isolated by thin-layer chromatography. SAICAr and SAdo were prepared by calf intestine alkaline phosphatase-catalysed dephosphorylation of SAICAR and SAMP and isolated on cation- and anion-exchange resin columns. The procedures described are easily scalable and provide high yields of sufficiently pure products for use in experiments related to studies of pathogenetic mechanisms in ADSL deficiency.
- MeSH
- adenosin analogy a deriváty chemie MeSH
- adenylsukcinátlyasa nedostatek MeSH
- aminoimidazolkarboxamid analogy a deriváty chemie izolace a purifikace MeSH
- biochemie metody MeSH
- časové faktory MeSH
- chromatografie iontoměničová MeSH
- fosforylace MeSH
- kationty MeSH
- klinická chemie metody MeSH
- klonování DNA MeSH
- komplementární DNA metabolismus MeSH
- lidé MeSH
- mutace MeSH
- poruchy metabolismu purinů a pyrimidinů diagnóza metabolismus MeSH
- rekombinantní proteiny chemie MeSH
- ribonukleotidy chemie izolace a purifikace MeSH
- substrátová specifita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosin MeSH
- adenylsukcinátlyasa MeSH
- AICA ribonucleotide MeSH Prohlížeč
- aminoimidazolkarboxamid MeSH
- kationty MeSH
- komplementární DNA MeSH
- rekombinantní proteiny MeSH
- ribonukleotidy MeSH
- SAICAR MeSH Prohlížeč
- succinyladenosine MeSH Prohlížeč
