Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency
Language English Country United States Media print-electronic
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't, Validation Study
PubMed
20821054
PubMed Central
PMC3026677
DOI
10.1007/s10545-010-9178-3
Knihovny.cz E-resources
- MeSH
- Blood Chemical Analysis methods standards MeSH
- Chromatography, Liquid MeSH
- Cystathionine beta-Synthase deficiency metabolism MeSH
- Homocystinuria blood diagnosis enzymology MeSH
- Immunoenzyme Techniques methods standards MeSH
- Calibration MeSH
- Plasma chemistry enzymology metabolism MeSH
- Humans MeSH
- Pyridoxal Phosphate pharmacology MeSH
- S-Adenosylmethionine pharmacology MeSH
- Enzyme Stability MeSH
- Case-Control Studies MeSH
- Tandem Mass Spectrometry methods standards MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Validation Study MeSH
- Names of Substances
- Cystathionine beta-Synthase MeSH
- Pyridoxal Phosphate MeSH
- S-Adenosylmethionine 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.
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Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency
