The projects is based on using tandem mass spectrometry for determination of plasma activity of selected intracellular enzymes involved in metabolism of amino acids, purines and fatty acids and verify the basic hypothesis that the enzymes expressed in tissues may be released into the bloodstream. We will investigate whether the methods of determining the plasma activity of these enzymes may be used for developing novel non-invasive diagnosis of enzyme deficiencies in patients with inborn errors of metabolism (IEM) and we will analyze whether plasma activity correlates with the clinical severity and the efficacy of the treatment. The project results will demonstrate whether it is possible to utilize the release of intracellular enzymes into circulation as a new approach for non-invasive diagnosis and prediction of treatment response in patients with IEM.

V rámci projektu zavedeme s využitím tandemové hmotnostní spektrometrie metody stanovení plazmatické aktivity vybraných intracelulárních enzymů podílejících se na metabolismu aminokyselin, purinů a mastných kyselin a prověříme základní hypotézu, že enzymy exprimované v tkáních se mohou uvolňovat do krevního oběhu. Dále budeme zjišťovat, zda je možno metody stanovení plazmatické aktivity těchto enzymů využít pro neinvazivní diagnostiku enzymového deficitu u pacientů s dědičnou metabolickou poruchou (DMP) a budeme analyzovat, zda plazmatické aktivity korelují s tíží klinického postižení pacienta a efektivitou léčby. Výsledky projektu ukáží, zda lze fenomén uvolňování intracelulárních enzymů do oběhu využít pro nové přístupy v neinvazivní diagnostice a pro predikci léčebné odpovědi u pacientů s DMP.

• MeSH
• enzymy nedostatek   MeSH
• genetické poradenství MeSH
• intracelulární tekutina enzymologie   MeSH
• klinické enzymatické testy MeSH
• plazmatické buňky enzymologie   MeSH
• plošný screening MeSH
• prediktivní hodnota testů MeSH
• prognóza MeSH
• tandemová hmotnostní spektrometrie MeSH
• vrozené poruchy metabolismu diagnóza   MeSH

• Konspekt
• Pediatrie
• NLK Obory
• pediatrie
• genetika, lékařská genetika
• biochemie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

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.

• MeSH
• adenosin analogy a deriváty   moč   MeSH
• adenylsukcinátlyasa nedostatek   moč   MeSH
• aminoimidazolkarboxamid analogy a deriváty   metabolismus   moč   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

Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS/MS. The median enzyme activity in control plasma samples was 404 nmol/h/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol/ho/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol/hour/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

• MeSH
• biochemická analýza krve metody   normy   MeSH
• chromatografie kapalinová MeSH
• cystathionin-beta-synthasa nedostatek   metabolismus   MeSH
• homocystinurie krev   diagnóza   enzymologie   MeSH
• imunoenzymatické techniky metody   normy   MeSH
• kalibrace MeSH
• krevní plazma chemie   enzymologie   metabolismus   MeSH
• lidé MeSH
• pyridoxalfosfát farmakologie   MeSH
• S-adenosylmethionin farmakologie   MeSH
• stabilita enzymů MeSH
• studie případů a kontrol MeSH
• tandemová hmotnostní spektrometrie metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity ("methylation index"). We present a rapid and sensitive LC-MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column (30 mm x 2.1 mm, 3 microm particle size) filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC-MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98-105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4 degrees C for 5 min and at 25 degrees C for 2 min, respectively. Storage of liver tissues at -80 degrees C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.

• MeSH
• chromatografie kapalinová metody   MeSH
• játra chemie   MeSH
• ledviny chemie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• S-adenosylhomocystein farmakokinetika   chemie   MeSH
• S-adenosylmethionin farmakokinetika   chemie   MeSH
• tandemová hmotnostní spektrometrie MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• hodnotící studie MeSH
• práce podpořená grantem MeSH

• MeSH
• adenosin krev   MeSH
• antirevmatika aplikace a dávkování   MeSH
• dítě MeSH
• erytrocyty metabolismus   MeSH
• juvenilní artritida farmakoterapie   krev   MeSH
• kyselina polyglutamová analogy a deriváty   krev   MeSH
• lidé MeSH
• methotrexát analogy a deriváty   krev   terapeutické užití   MeSH
• mladiství MeSH
• výsledek terapie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH

• MeSH
• fosfiny MeSH
• homocystein krev   MeSH
• oxidace-redukce MeSH
• sulfhydrylová reagencia krev   MeSH
• vysokoúčinná kapalinová chromatografie MeSH

• MeSH
• adenosin analogy a deriváty   MeSH
• mozkomíšní mok chemie   MeSH
• poruchy metabolismu purinů a pyrimidinů MeSH
• spektrofotometrie ultrafialová MeSH
• vysokoúčinná kapalinová chromatografie MeSH

• MeSH
• cytochromy metabolismus   MeSH
• herbicidy farmakologie   MeSH
• krysa rodu rattus MeSH
• mikrozomy metabolismus   účinky léků   MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• mozek účinky léků   ultrasonografie   MeSH
• oxadiazoly farmakologie   MeSH
• oxidoreduktasy antagonisté a inhibitory   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

• MeSH
• adenylsukcinátlyasa MeSH
• aminoimidazolkarboxamid MeSH
• geny recesivní MeSH
• poruchy metabolismu purinů a pyrimidinů MeSH
• ribonukleotidy MeSH
• S-adenosylmethionin MeSH