Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.

• Klíčová slova
• ADSL, ADSLD, DNA damage, SAICAR, cell biology, chicken, cilia, developmental biology, human, microcephaly, zebrafish,
• MeSH
• adenylsukcinátlyasa nedostatek   metabolismus   MeSH
• aminoimidazolkarboxamid analogy a deriváty   metabolismus   MeSH
• autistická porucha metabolismus   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• ciliopatie metabolismus   MeSH
• dánio pruhované metabolismus   MeSH
• fenotyp MeSH
• fosfoproteiny metabolismus   MeSH
• kur domácí metabolismus   MeSH
• lidé MeSH
• mikrocefalie metabolismus   MeSH
• neurogeneze * MeSH
• poruchy autistického spektra metabolismus   MeSH
• poruchy metabolismu purinů a pyrimidinů metabolismus   MeSH
• poškození DNA MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• puriny metabolismus   MeSH
• ribonukleotidy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• CCP110 protein, human MeSH Prohlížeč
• fosfoproteiny MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny buněčného cyklu MeSH
• purine MeSH Prohlížeč
• puriny MeSH
• ribonukleotidy MeSH
• SAICAR MeSH Prohlížeč

OBJECTIVES: Stable isotope dilution coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the sensitive method for screening for various inherited metabolic disorders using dried blood spots (DBSs). We present a method for LC-MS/MS determination of succinyladenosine (SAdo) and succinylaminoimidazole carboxamide riboside (SAICAr), biomarkers for adenylosuccinate lyase deficiency (dADSL), in DBS. DESIGN AND METHODS: SAICAr and SAdo were separated on a Symmetry-C18 column and detected using positive electrospray ionisation in selected reaction monitoring mode. The quantification was performed using the isotopically labelled internal standards SAdo-(13)C4 and SAICAr-(13)C4, which were prepared via ADSL-catalysed reactions of fumarate-(13)C4 with adenosine monophosphate and aminoimidazole carboxamide ribotide, respectively, and subsequent alkaline phosphatase-catalysed dephosphorylation of the resulting products. RESULTS: The detection of SAICAr and SAdo in DBS was linear over the range of 0-25μmol/L. The respective intra-assay and inter-assay imprecision values were less than 10.7% and 15.2% for SAICAr and 4.7% and 5.7% for SAdo. The recoveries from DBS spiked with different concentrations of SAICAr and SAdo were between 94% and 117%. The concentrations of SAICAr and SAdo were higher in the archived DBS from dADSL patients (SAICAr, 0.03-4.7μmol/L; SAdo, 1.5-21.3μmol/L; n=5) compared to those of the control subjects (SAICAr, 0-0.026μmol/L; SAdo, 0.06-0.14μmol/L; n=31), even after DBSs from dADSL patients were stored for 2-23years. CONCLUSIONS: We developed and validated a method of succinylpurine analysis in DBS that improves selective screening for dADSL in the paediatric population and may be used for retrospective diagnosis to aid the genetic counselling of affected families.

• Klíčová slova
• Adenylosuccinate lyase deficiency, DBS, Dried blood spots, LC–MS/MS, Purine metabolism, Screening, Tandem mass spectrometry,
• MeSH
• adenosin analogy a deriváty   krev   MeSH
• adenylsukcinátlyasa krev   nedostatek   MeSH
• aminoimidazolkarboxamid analogy a deriváty   krev   MeSH
• autistická porucha MeSH
• chromatografie kapalinová MeSH
• izotopy uhlíku MeSH
• lidé MeSH
• limita detekce MeSH
• novorozenec MeSH
• poruchy metabolismu purinů a pyrimidinů krev   diagnóza   MeSH
• referenční standardy MeSH
• ribonukleosidy krev   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• test suché kapky krve metody   MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• izotopy uhlíku MeSH
• ribonukleosidy MeSH
• succinyladenosine MeSH Prohlížeč
• succinylaminoimidazole carboxamide riboside MeSH Prohlížeč

OBJECTIVES: Adenylosuccinate lyase deficiency (dADSL) is a rare inherited metabolic disorder. Biochemical diagnosis of the disease is based on the determination of enormously elevated urinary levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) and succinyladenosine (SAdo). We report a case of false negative screening for dADSL caused by deribosylation of the urinary biomarkers SAICA-riboside and SAdo. DESIGN AND METHODS: A thin-layer chromatography (TLC) method with Pauly reagent detection of SAICA-riboside was used as a screening method. High-performance liquid chromatography with diode-array detection (HPLC-DAD) and LC-MS/MS methods were used for the identification and quantitative determination of SAICA-riboside, SAdo, succinylaminoimidazole carboxamide (SAICA) and succinyladenine (SA). RESULTS: Following a negative TLC screening in a known case of dADSL, we analyzed urine using HPLC-DAD. The concentration of SAICA-riboside was 2.7mmol/mol creatinine (below the TLC detection limit), and we detected the two abnormal metabolites identified by LC-MS/MS as SAICA and SA. We showed that SAICA and SA were produced by deribosylation of SAICA-riboside and SAdo in the patient's urine. Studies performed by monitoring the production of SAICA and SA after the addition of SAICA-riboside and SAdo to the patient's urine and to urine samples from patients with urinary tract infections suggested that deribosylation is facilitated by bacterial enzymes. CONCLUSIONS: Screening methods for the diagnosis of dADSL may be falsely negative due to bacteria-mediated deribosylation of SAICA-riboside and SAdo. HPLC-DAD or LC-MS/MS analyses allowing for simultaneous detection of SAICA-riboside, SAdo and their deribosylation products SAICA and SA should be preferentially used for the diagnosis of dADSL in urine.

• Klíčová slova
• ADSL, Adenylosuccinate lyase deficiency, HPLC-DAD, LC–MS/MS, Purine metabolism, SA, SAICA, SAICA-riboside, SAdo, Succinyladenosine, Succinylaminoimidazole carboxamide riboside, Succinylpurines, TLC, adenylosuccinate lyase, adenylosuccinate lyase deficiency, dADSL, high-performance liquid chromatography with diode array detection, liquid chromatography–tandem mass spectrometry, succinyladenine, succinyladenosine, succinylaminoimidazole carboxamide, succinylaminoimidazole carboxamide riboside, thin-layer chromatography,
• MeSH
• adenosin analogy a deriváty   moč   MeSH
• adenylsukcinátlyasa nedostatek   moč   MeSH
• aminoimidazolkarboxamid analogy a deriváty   metabolismus   moč   MeSH
• autistická porucha MeSH
• bakteriální proteiny metabolismus   MeSH
• chromatografie na tenké vrstvě metody   MeSH
• Enterococcus faecalis MeSH
• enzymy metabolismus   MeSH
• falešně negativní reakce MeSH
• Klebsiella pneumoniae MeSH
• lidé MeSH
• moč mikrobiologie   MeSH
• poruchy metabolismu purinů a pyrimidinů diagnóza   moč   MeSH
• předškolní dítě MeSH
• ribonukleosidy metabolismus   moč   MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• bakteriální proteiny MeSH
• enzymy MeSH
• ribonukleosidy MeSH
• succinyladenosine MeSH Prohlížeč
• succinylaminoimidazole carboxamide riboside MeSH Prohlížeč

Contribution of individual adiponectin isoforms to lipolysis regulation remains unknown. We investigated the impact of full-length, trimeric and globular adiponectin isoforms on spontaneous lipolysis in subcutaneous abdominal (SCAAT) and visceral adipose tissues (VAT) of obese and non-obese subjects. Furthermore, we explored the role of AMPK (5'-AMP-activated protein kinase) in adiponectin-dependent lipolysis regulation and expression of adiponectin receptors type 1 and 2 (AdipoR1 and AdipoR2) in SCAAT and VAT. Primary adipocytes isolated from SCAAT and VAT of obese and non-obese women were incubated with 20 µg/ml of: A) full-length adiponectin (physiological mixture of all adiponectin isoforms), B) trimeric adiponectin isoform or C) globular adiponectin isoform. Glycerol released into media was used as a marker of lipolysis. While full-length adiponectin inhibited lipolysis by 22% in non-obese SCAAT, globular isoform inhibited lipolysis by 27% in obese SCAAT. No effect of either isoform was detected in non-obese VAT, however trimeric isoform inhibited lipolysis by 21% in obese VAT (all p<0.05). Trimeric isoform induced Thr172 p-AMPK in differentiated preadipocytes from a non-obese donor, while globular isoform induced Ser79 p-ACC by 32% (p<0.05) and Ser565 p-HSL by 52% (p = 0.08) in differentiated preadipocytes from an obese donor. AdipoR2 expression was 17% and 37% higher than AdipoR1 in SCAAT of obese and non-obese groups and by 23% higher in VAT of obese subjects (all p<0.05). In conclusion, the anti-lipolytic effect of adiponectin isoforms is modified with obesity: while full-length adiponectin exerts anti-lipolytic action in non-obese SCAAT, globular and trimeric isoforms show anti-lipolytic activity in obese SCAAT and VAT, respectively.

• MeSH
• adiponektin krev   chemie   metabolismus   MeSH
• aminoimidazolkarboxamid analogy a deriváty   farmakologie   MeSH
• dospělí MeSH
• exprese genu účinky léků   MeSH
• hypoglykemika farmakologie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipolýza účinky léků   MeSH
• multimerizace proteinu účinky léků   MeSH
• nitrobřišní tuk cytologie   MeSH
• obezita patologie   MeSH
• podkožní tuk cytologie   MeSH
• protein - isoformy krev   chemie   metabolismus   MeSH
• proteinkinasy aktivované AMP metabolismus   MeSH
• receptory adiponektinu genetika   metabolismus   MeSH
• ribonukleotidy farmakologie   MeSH
• tukové buňky cytologie   účinky léků   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adiponektin MeSH
• ADIPOR1 protein, human MeSH Prohlížeč
• ADIPOR2 protein, human MeSH Prohlížeč
• AICA ribonucleotide MeSH Prohlížeč
• aminoimidazolkarboxamid MeSH
• hypoglykemika MeSH
• protein - isoformy MeSH
• proteinkinasy aktivované AMP MeSH
• receptory adiponektinu MeSH
• ribonukleotidy MeSH

Succinylpurines accumulate in the body fluids of patients with adenylosuccinate lyase (ADSL) deficiency but their source in the cerebrospinal fluid remains obscure. Study based on the incorporation of 13C-stable isotope-labeled glycine into cultured oligodendroglia from ADSL-deficient patient and the measurement of labeled products by LC/MS/MS showed total intracellular concentrations of succinylpurines from 45 to 99μmol/l and so these results suggest that these cells can be the source of the compounds in vivo.

• MeSH
• adenosinmonofosfát analogy a deriváty   biosyntéza   MeSH
• adenylsukcinátlyasa nedostatek   MeSH
• aminoimidazolkarboxamid analogy a deriváty   MeSH
• fatální výsledek MeSH
• lidé MeSH
• novorozenec MeSH
• oligodendroglie metabolismus   MeSH
• ribonukleosidy biosyntéza   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosinmonofosfát MeSH
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• ribonukleosidy MeSH
• succinyladenosine monophosphate MeSH Prohlížeč
• succinylaminoimidazole carboxamide riboside MeSH Prohlížeč

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

• MeSH
• aminoimidazolkarboxamid analogy a deriváty   metabolismus   MeSH
• bazální metabolismus MeSH
• dietní tuky aplikace a dávkování   metabolismus   MeSH
• kosterní svaly metabolismus   fyziologie   MeSH
• kyseliny mastné neesterifikované krev   MeSH
• multienzymové komplexy metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• náhodné rozdělení MeSH
• nepřímá kalorimetrie MeSH
• novorozená zvířata MeSH
• obezita etiologie   metabolismus   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinkinasy aktivované AMP MeSH
• ribonukleotidy metabolismus   MeSH
• spotřeba kyslíku fyziologie   MeSH
• tělesná hmotnost fyziologie   MeSH
• tělesná teplota fyziologie   MeSH
• termogeneze fyziologie   MeSH
• triglyceridy krev   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AICA ribonucleotide MeSH Prohlížeč
• aminoimidazolkarboxamid MeSH
• dietní tuky MeSH
• kyseliny mastné neesterifikované MeSH
• multienzymové komplexy MeSH
• protein-serin-threoninkinasy MeSH
• proteinkinasy aktivované AMP MeSH
• ribonukleotidy MeSH
• triglyceridy MeSH

A rapid method using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry (PTV-LP-GC-HR-TOF-MS) for the analysis of multiple pesticide residues in fruit-based baby food was developed. The fast and inexpensive buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction method and "conventional" approach that employs ethyl acetate extraction followed by gel permeation chromatography (GPC) cleanup were employed for sample preparation. A PTV injector in solvent venting mode was used to reduce volume of acetonitrile and acetic acid (from the buffered QuEChERS extracts) that caused higher column bleed without their elimination. Otherwise, the time-to-digital converter would become saturated in HR-TOF-MS. For fast GC separation allowing analysis of 100 analytes within a 7 min runtime, both a high temperature programming rate and vacuum conditions in a megabore GC column were employed. The use of HR-TOF-MS allowed the unbiased identification and reliable quantification of target analytes through the application of a narrow mass window (0.02 Da) for extracting analyte ions and the availability of full spectral information even at very low levels. With only a few exceptions, the lowest calibration levels for the pesticides tested were

• MeSH
• aminoimidazolkarboxamid analogy a deriváty   analýza   MeSH
• analýza potravin metody   MeSH
• časové faktory MeSH
• endosulfan analýza   MeSH
• hydantoiny analýza   MeSH
• isomerie MeSH
• kyselina octová chemie   MeSH
• ovoce chemie   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí metody   MeSH
• pufry MeSH
• reprodukovatelnost výsledků MeSH
• rezidua pesticidů analýza   izolace a purifikace   MeSH
• teplota * MeSH
• tlak MeSH
• volatilizace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminoimidazolkarboxamid MeSH
• endosulfan MeSH
• hydantoiny MeSH
• iprodione MeSH Prohlížeč
• kyselina octová MeSH
• pufry MeSH
• rezidua pesticidů MeSH

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

AICA-ribosiduria is a recently discovered inherited metabolic disease caused by a defect in final steps of purine de novo biosynthesis-5-amino-4-imidazolecarboxamide ribotide (AICAR)-transformylase/inosinemonophosphate (IMP)-cyclohydrolase (ATIC). A rapid and selective capillary electrophoretic method for screening of patients with AICA-ribosiduria is described. The method is based on direct ultraviolet detection of 5-amino-4-imidazolecarboxamide (AICA) and 5-amino-4-imidazolecarboxamide riboside (AICAr) in untreated urine. Background electrolyte consists of 100mM malonic acid adjusted with gamma-aminobutyric acid (pH 2.7). Under the given separation conditions both compounds of interest are well separated from other substances with separation efficiency of 1020000 and 130000 theoretical plates/m for AICA and AICAr, respectively. Total analysis time is 3 min with the limits of detection of 3.6 microM and 4.5 microM for AICA and AICAr, respectively. The usefulness of the presented method for screening of patients with ATIC deficiency is demonstrated on samples of Chinese hamster ovary cell line defective in ATIC activity, spiked urine samples and urine samples from patients treated with high-dose MTX which do not excrete increased amounts of AICA and AICAr compared to untreated controls (p<0.05). The described method is fast and effective enough for diagnostic applications.

• MeSH
• aminoimidazolkarboxamid analogy a deriváty   moč   MeSH
• dospělí MeSH
• elektroforéza kapilární metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• ribonukleosidy moč   MeSH
• vrozené poruchy metabolismu diagnóza   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acadesine MeSH Prohlížeč
• aminoimidazolkarboxamid MeSH
• ribonukleosidy MeSH