Development of a novel liquid chromatography-tandem mass spectrometry based enzymatic assay of 5,10-methylenetetrahydrofolate reductase.

5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common folate metabolism disorder. MTHFR is a key enzyme in the folate cycle that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (5-methyl THF). A definitive diagnosis of MTHFR deficiency relies on enzymatic studies of fibroblasts and/or molecular genetic analyses. The accurate measurement of the MTHFR enzyme activity is crucial for diagnosing and predicting disease severity, which is traditionally performed using high-performance liquid chromatography with fluorescence detection. We developed a novel method for assessing the MTHFR enzymatic activity using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MTHFR enzymatic reaction was conducted in fibroblasts, and the product, 5-methyl THF, was quantified by LC-MS/MS. The MTHFR activity was evaluated in 13 control individuals and five individuals with MTHFR deficiency. The 5-methyl THF concentration was successfully measured in all cases, and the validation trial demonstrated adequate accuracy and precision. Control fibroblasts exhibited an MTHFR activity ranging from 240.1 to 624.0 pmol/min/mg protein (mean = 338.5 pmol/min/mg), while MTHFR-deficient fibroblasts showed a markedly lower activity (2.99-51.3 pmol/min/mg protein). Although our study is associated with some limitations, we present a sensitive and reliable LC-MS/MS based assay for diagnosing MTHFR deficiency.