The threshold growth response ofLactobacillus caseito 5-methyl-tetrahydrofolic acid: implications for folate assays

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Abstract
1. The comparative and absolute growth response of Lactobacillus casei was measured nephelometrically for time periods of 17–23 h in the microbiological assay of folic acid and 5-methyl-tetrahydrofolic acid at concentrations of 0–8 ng/10 ml basal medium at pH 6.8. 2. At concentrations of 0–1 ng/10 ml the comparative growth response to 5-methyl-tetrahydrofolic acid was markedly depressed whereas growth was the same at 2 ng/10 ml and above. Comparative growth was unaffected by the length of assay incubation, depressed growth being due to differences in log-phase growth rates with the rate-plot for 5-methyl-tetrahydrofolic acid being sigmoidal and for folk acid being a rectangular hyperbola with linearity only in the 0–1 ng/10 ml range. The reciprocal rate-plot for folic acid was linear whereas that for 5-methyl-tetrahydrofolic acid was coincidental only in part, giving rise to the same estimate of maximum velocity and substrate concentration for half-maximum velocity, with the exhibition of a strong threshold at low concentration. 3. A previous observation (Phillips & Wright, 1982) that the L. casei growth response to 5-methyl-tetrahydrofolic acid may be significantly less than that to folic acid is confirmed as is the long-established view that the response to both folates may be equal. In the light of current knowledge regarding folate-binding, transport and metabolism by L. casei, it is argued that the intracellular oxidation of 5-methyl-tetrahydrofolic acid to 5, 10-methylene-tetrahydrofolic acid is a rate-limiting step at low substrate concentrations, subsequently giving rise to a threshold growth response peculiar to 5-methyl-tetrahydrofolic acid. Since the rate of L. cusei growth with folk acid is not linearabove 1 ng/10 ml, it is recommended that microbiological folate assays be conducted only in the 0–1 ng/10 ml range and at a pH that elicits the same growth response from L. casei to 5-methyl-tetrahydrofolic acid as to folic acid and other folate monoglutamates.