Enzymatic diagnosis of homocystinuria by determination of cystathionine-beta-synthase activity in plasma using LC-MS/MS
Abstract
Background: Cystathionine beta-synthase (CBS) is released into plasma from organs expressing this enzyme. Decreased plasma CBS activity has been demonstrated in CBS-deficient patients with 16 different genotypes.
The aim of this study was to determine plasma CBS activity in patients carrying 11 additional genotypes using two LC-MS/MS methods. Patients and methods CBS activity was measured in EDTA or heparin plasma using either a previously described or a newly developed LC-MS/MS method optimized for analysis of the reaction product, 3,3-H-2(2)-cystathionine, as its butyl ester derivative.
We analyzed plasma samples from 26 CBS-deficient patients with known genotypes and 57 controls. Results: We developed a new LC-MS/MS method for simple and sensitive determination of CBS activity.
Plasma CBS activity was low (i.e., 0.001-0.036 of the multiples of median control values, MOM) in patients homozygous for the prevalent Hispanic mutation c572C>T (p.T191M) but was highly elevated (2.95 MoM) in a single patient homozygous for the c.1330G>A (p.D444N) mutation. Patients with the remaining nine genotypes exhibited decreased activities (0.00-0.22 MoM), which did not overlap with the controls (0.29-2.10 MoM).
Conclusions: The determination of CBS activity in plasma is a rapid and non-invasive procedure for detecting a subgroup of CBS-deficient patients with distinct genotypes.