title: 5,10-Methylenetetrahydrofolate reductase (MTHFR) assay in the forward direction: residual activity in MTHFR deficiency.
abstract: BACKGROUND
  Assay of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in homocysteine metabolism, is important for the study of severe and mild deficiency states. Because the conventional assay measures in the reverse direction, lacks sensitivity, and uses nonphysiologic substrates, the exact measurement and characterization of residual activity in easily accessible tissues have been difficult.
  
  
  METHODS
  To measure MTHFR in the physiologic direction, we determined the NADPH-dependent conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate by use of HPLC with fluorescence detection.
  
  
  RESULTS
  MTHFR activity in control fibroblast in the presence of FAD was maximal between pH 6.3 and 6.9, increased linearly up to 40 min and 80 microg protein/assay, and showed K(m)s of 30 micromol/L for NADPH and 26 micromol/L for 5,10-methylenetetrahydrofolate. Intraassay variation (CV) was 10%, interassay variation was 7.2%, and variation among 10 subcultures of the same cell line was 18%. Mean (SD) control activity was 431 (150) microU/mg protein (range, 242-910; n = 75), which is 2.5-fold higher than that with the reverse assay. After heat treatment (46 degrees C for 5 min), the activity showed a trimodal distribution corresponding to the 677TT (thermolabile; 15%), 677CT (35%), and 677CC (51%) genotypes. We found clearly measurable activity ranging from 2.6% to 25.6% of the mean control value in 15 patients with MTHFR deficiency, including 11 cell lines with zero activity in the reverse assay. Ten patients had complete enzyme deficiency.
  
  
  CONCLUSIONS
  This assay allows reliable determination of residual activity in mutant fibroblasts and characterization of kinetic parameters for natural substrates.
authors.affiliation: University Children's Hospital, CH-4005 Basel, Switzerland. terttu.suormala@unibas.ch
authors.forename: Terttu
authors.initials: T
authors.lastname: Suormala
authors.forename: Gertraud
authors.initials: G
authors.lastname: Gamse
authors.forename: Brian
authors.initials: B
authors.lastname: Fowler
mesh_terms: D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000201:enzymology; D005838:Genotype; D006361:Heating; D006801:Humans; D007700:Kinetics; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D009249:NADP / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000172:deficiency / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D009249:NADP; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2)