Homocystinuria

Abstract

A B S T R A C T We have compared in vivo pyridoxineresponsiveness with in vitro cystathionine /8-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5'-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five-and sevenfold stimulation of cystathionine ,B-synthase activity with added pyridoxal-5'-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two-and fivefold with the added cofactor. Cystathionine ,8-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5'-phosphate were estimated and thermostability (54°C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine 83-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5'phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activDr.

Cystathionine β-synthase deficiency: Of mice and men

“…A large number of in vitro studies, using either cell extracts or purified mutant proteins, have failed to consistently show an increase in CBS activity in the presence of excess pyridoxine or pyridoxal phosphate [74, 75]. The most consistent finding is that the presence of some level of detectable residual activity is required for pyridoxine response [15]. …”

“…Some patients can achieve lowering by giving pharmacologic doses of pyridoxine (vitamin B 6 ), the precursor to pyridoxal phosphate, which is a co-factor for the CBS enzyme [14]. These so-called “B 6 -responders” generally produce mutant CBS proteins that have some residual enzyme activity [15], but the exact mechanism behind pyridoxine response is not well understood. For patients that do not respond to pyridoxine, a low-methionine diet in combination with B-vitamins is used to control tHcy levels [16].…”

A liquid chromatography mass spectrometry method for the measurement of cystathionine β-synthase activity in cell extracts

“…This type of method is characterized by high sensitivities with a limit of quantification ∼30 pmol/h/mg protein. [18] Our LC-MS/MS method and 2 other recently published methods offer comparable sensitivity [14,15]. However, to be able to measure small changes due to for example therapy or diet, a good precision is also required.…”

“…The fact that CBS activity in most cell types is low, warrants a sensitive method for measuring the enzyme activity. To date several methods have been published for the measurement of CBS activity in cell extracts, ranging from radioisotopes to LC-MS/MS [13][14][15][16][17][18]. However, most of them have proven to be rather laborious and not sensitive or specific enough.…”

Detection of a novel deletion in the cystathionine β‐synthase (CBS) gene using an improved genomic DNA based method1

“…Skin biopsy fibroblast cultures were established from both patients for enzyme activity measurements according to [8]. CBS activity was measured in patient fibroblasts as described elsewhere [9]. Assays were performed with and without addition of 1 mM PLP to the incubation mixture, and after stimulation with 100 μM S ‐adenosylmethionine (AdoMet).…”