Determination of Total Folates in Complex Nutritional Drinks and Supplements Using a Tri-Enzyme Microbiological Method and Michaelis-Menten Kinetics.

Background: Recent development of LC methods for the determination of total folates (vitamin B9) in complex matrixes have been hindered by vitamer interconversion and yield variability. The official microbiological method (AOAC Official Methods of Analysis 944.12 and 960.46) uses an end point turbidity reading to determine folate concentration. However, when measuring complex matrixes, shifts are observed in the growth curves of the microorganism and inaccuracies are introduced to this quantification method. Objective/Methods: In addition to the tri-enzyme digestion of the standard microbiological method, we have applied enzyme modeling of the initial velocity of bacterial growth using Michaelis-Menten kinetics to achieve more accurate and reproducible determinations of total folates. Results/Conclusions: Accuracy determined through spike recovery in Infant/Adult Nutritional Drink and a complex vitamin matrix gave values acceptable to AOAC standards of 85-110%. Repeatability of the low mass fraction analyte measured at micrograms per 100 g yielded relative standard deviations <15% for all matrixes tested, including three standard reference materials.

Microbiological assay on microtitre plates of folate in serum and red cells.

AIMS: To develop a simple microbiological assay for serum and red cell folates on 96-well microtitre plates, suitable for use in routine clinical diagnosis. METHODS: Use of a chloramphenicol resistant organism (NCIB 10463) saved time by avoiding aseptic precautions. Use of plate sealers facilitated mixing. Evaluation of assay performance included estimations of folate recovery, assay reproducibility, and response to reduced folate. Results obtained on sera (193) and red cell folates (150) were compared with those obtained using a traditional microbiological assay. RESULTS: Good recovery of folic acid added to serum and also good interassay and intra-assay precision were obtained with both serum (CV% of less than 5) and red cell folate pools (CV% of less than 5). Equimolar assay responses were obtained with folic acid, 5-formyltetrahydrofolate (L-form), and 5-methyltetrahydrofolate (L-form). The microassay correlated well with a traditional assay for estimation of folate in both serum (n = 193, r = 0.975) and red cells (n = 150, r = 0.96). CONCLUSIONS: This assay is more compact and less time consuming than the traditional assay. It is extremely economical and is easy to perform in a routine clinical laboratory.

A rapid and specific extraction procedure for folates determination in rat liver and analysis by high-performance liquid chromatography with fluorometric detection.

The native fluorescence of the main biological derivatives of folic acid is maximal at acidic pH. The intensities obtained with 5-methyltetrahydrofolic acid, tetrahydrofolic acid, and formyltetrahydrofolic acid are in the ratio 10/5/1. After separating the metabolites by reversed phase high-performance liquid chromatography, the fluorometric detection limit varied between 0.4 and 20 pmol per injection. Rat liver extract (about 2 g/50 ml of 1% ascorbate solution) could be analyzed after incubation at 37 degrees C overnight, deproteinizing with acetone, and purification and concentration in an ion exchange column. The concentrations obtained varied from 1.27 to 7.96 nmol/g depending on the derivative. Recovery varied from 89 to 113%.

A radioenzymatic method for the determination of tissue 10-formyltetrahydrofolate.

A radioenzymatic method for the determination of tissue 10-formyltetrahydrofolate is described based on the stable ternary complex formed from methylenetetrahydrofolate, tritiated fluorodeoxyuridylate and thymidylate synthase. Tissue extract 10-formyltetrahydrofolate is deacylated with 10-formyltetrahydrofolate deacylase and the tetrahydrofolate formed is converted to methylenetetrahydrofolate using formaldehyde. Mouse tissue 10-formyltetrahydrofolate levels and their stability to extraction procedures are described.

Studies on the response of Lactobacillus casei to different folate monoglutamates.

1. The response of Lactobacillus casei was measured for a number of the monoglutamyl forms of folate derivatives. 2. At the concentrations of folate commonly used in the assay of folate vitamin in foods the response of L. casei to folic acid, (pteroylglutamic acid) and 5-formyl-tetrahydrofolic acid was similar, but 5-methyl-tetrahydrofolic acid gave as little as half the response of folic acid. 3. The response was modified by altering pH but not by concentration of ascorbate. 4. These results have implications for the assays of foods for folate where mixtures of folate derivatives are present. 5. A modified procedure is suggested in which the monoglutamates give similar responses.