Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.
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.
Krijt
Jakub
J
Institute of Inherited Metabolic Disorders--1st Faculty of Medicine, Charles University in Prague, Praha, Czech Republic.
Kopecká
Jana
J
Hnízda
Aleš
A
Moat
Stuart
S
Kluijtmans
Leo A J
LA
Mayne
Philip
P
Kožich
Viktor
V
D001774:Blood Chemical Analysis / Q000379:methods / Q000592:standards; D002138:Calibration; D016022:Case-Control Studies; D002853:Chromatography, Liquid; D003541:Cystathionine beta-Synthase / Q000172:deficiency* / Q000378:metabolism*; D004795:Enzyme Stability; D006712:Homocystinuria / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods / Q000592:standards; D010949:Plasma / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012436:S-Adenosylmethionine / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
D011732:Pyridoxal Phosphate; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase
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