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

Abstract

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.

Keywords: Betaine; Folate cycle; High-performance liquid chromatography; Homocysteine; Methionine cycle; S-adenosylmethionine.

Fig. 1

Folate metabolic pathway; connection between the folate cycle and methionine cycle. MTHFR catalyzes the reduction of 5,10-methylene THF to the active form of folate, 5-methyl THF, using NADPH as a cofactor. In MTHFR deficiency, 5-methyl THF levels are reduced, leading to decreased methionine and SAM levels along with homocysteine accumulation. DHF, Dihydrofolate; THF, Tetrahydrofolate; 5,10-methylene THF, 5,10-methylenetetrahydrofolate; MTHFR, 5,10-methylenetetrahydrofolate reductase; 5-methyl THF, 5-methyltetrahydrofolate; FAD, flavin adenine dinucleotide; SAM, S-adenosyl-methionine; SAH, S-adenosyl-homocysteine; BHMT, Betaine-Homocysteine Methyltransferase; DMG, Dimethylglycine; CBS, Cystathionine β-synthase; CTH, Cystathionine γ-lyase.

Fig. 2

Kinetics of 5-methyl THF production over reaction time. Changes in 5-methyl-THF production were observed over various reaction times (2.5 to 80 min). The substrate (5,10-methylene THF) concentration was maintained at 100 µmol/L, whereas a fixed amount of enzyme extract protein ranging from 10 to 70 µg was used. 5-methyl THF, 5-methyltetrahydrofolate; 5,10-methylene THF, 5,10-methylenetetrahydrofolate.

Fig. 3

Enzyme assay velocity as a function of enzyme extract protein amount. The enzymatic assay velocity (pmol/min) as a function of the enzyme extract protein content varied from 2 to 200 µg. The reaction time was fixed at 20 min, with a substrate (5,10-methylene THF) at a concentration of 100 µmol/L. V, enzymatic assay velocity; 5,10-methylene THF, 5,10-methylenetetrahydrofolate.

Fig. 4

Michaelis-Menten kinetics curve. MTHFR activity as a function of substrate (5,10-methylene THF) concentration. MTHFR activity was analyzed at substrate concentrations ranging from 5 to 400 µmol/L. The reaction time was fixed at 20 min, and the enzyme extract protein amounts were fixed at 10–70 µg. 5,10-methylene THF, 5,10-methylenetetrahydrofolate.

Fig. 5

LC-MS/MS chromatographic separation of 5-methyl THF. (a) A mass chromatogram in a control fibroblast containing 20.8 ng/mL 5-methyl THF. (b) A mass chromatogram in a MTHFR-deficient fibroblast (Case 3) containing 0.38 ng/mL 5-methyl THF. The x-axis represents time (min), and the y-axis represents the ion counts in the MRM channel normalized to the maximum signal (100%).