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Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency
J. Krijt, J. Kopecká, A. Hnízda, S. Moat, LA. Kluijtmans, P. Mayne, V. Kožich
Jazyk angličtina Země Nizozemsko
Typ dokumentu hodnotící studie, časopisecké články, práce podpořená grantem, validační studie
NLK
ProQuest Central
od 1999-02-01 do 2018-11-30
Medline Complete (EBSCOhost)
od 2009-08-01 do Před 1 rokem
Health & Medicine (ProQuest)
od 1999-02-01 do 2018-11-30
- 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
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.
Department of Medical Biochemistry and Immunology University Hospital of Wales
Institute of Inherited Metabolic Disorders—1st Faculty of Medicine Charles University Prague
Citace poskytuje Crossref.org
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- $a 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.
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