Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

Hdl Handle:
http://hdl.handle.net/10147/263374
Title:
Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.
Authors:
Krijt, Jakub; Kopecká, Jana; Hnízda, Aleš; Moat, Stuart; Kluijtmans, Leo A J; Mayne, Philip; Kožich, Viktor
Affiliation:
Institute of Inherited Metabolic Disorders--1st Faculty of Medicine, Charles University in Prague, Praha, Czech Republic.
Citation:
Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency. 2011, 34 (1):49-55 J. Inherit. Metab. Dis.
Journal:
Journal of inherited metabolic disease
Issue Date:
Feb-2011
URI:
http://hdl.handle.net/10147/263374
DOI:
10.1007/s10545-010-9178-3
PubMed ID:
20821054
Abstract:
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.
Item Type:
Article
Language:
en
MeSH:
Blood Chemical Analysis; Calibration; Case-Control Studies; Chromatography, Liquid; Cystathionine beta-Synthase; Enzyme Stability; Homocystinuria; Humans; Immunoenzyme Techniques; Plasma; Pyridoxal Phosphate; S-Adenosylmethionine; Tandem Mass Spectrometry
ISSN:
1573-2665

Full metadata record

DC FieldValue Language
dc.contributor.authorKrijt, Jakuben_GB
dc.contributor.authorKopecká, Janaen_GB
dc.contributor.authorHnízda, Alešen_GB
dc.contributor.authorMoat, Stuarten_GB
dc.contributor.authorKluijtmans, Leo A Jen_GB
dc.contributor.authorMayne, Philipen_GB
dc.contributor.authorKožich, Viktoren_GB
dc.date.accessioned2012-12-20T11:41:34Z-
dc.date.available2012-12-20T11:41:34Z-
dc.date.issued2011-02-
dc.identifier.citationDetermination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency. 2011, 34 (1):49-55 J. Inherit. Metab. Dis.en_GB
dc.identifier.issn1573-2665-
dc.identifier.pmid20821054-
dc.identifier.doi10.1007/s10545-010-9178-3-
dc.identifier.urihttp://hdl.handle.net/10147/263374-
dc.description.abstractCystathionine 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.en_GB
dc.language.isoenen
dc.rightsArchived with thanks to Journal of inherited metabolic diseaseen_GB
dc.subject.meshBlood Chemical Analysis-
dc.subject.meshCalibration-
dc.subject.meshCase-Control Studies-
dc.subject.meshChromatography, Liquid-
dc.subject.meshCystathionine beta-Synthase-
dc.subject.meshEnzyme Stability-
dc.subject.meshHomocystinuria-
dc.subject.meshHumans-
dc.subject.meshImmunoenzyme Techniques-
dc.subject.meshPlasma-
dc.subject.meshPyridoxal Phosphate-
dc.subject.meshS-Adenosylmethionine-
dc.subject.meshTandem Mass Spectrometry-
dc.titleDetermination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.en_GB
dc.typeArticleen
dc.contributor.departmentInstitute of Inherited Metabolic Disorders--1st Faculty of Medicine, Charles University in Prague, Praha, Czech Republic.en_GB
dc.identifier.journalJournal of inherited metabolic diseaseen_GB
dc.description.provinceLeinsteren
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