The threshold growth response of Lactobacillus casei to 5-methyl-tetrahydrofolic acid: implications for folate assays

A. J. A. Wright, D. R. Phillips
1985 British Journal of Nutrition  
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/lO ml basal medium at pH 6.8. 2. At concentrations of G I ng/lO ml the comparative growth response to 5-methyl-tetrahydrofolic acid was markedly depressed whereas growth was the same at 2 ng/lO ml and above. Comparative growth was unaffected by the length of assay
more » ... 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/lO 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. & 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-methylenetetrahydrofolic 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/lO ml, itisrecommended thatmicrobiologicalfolateassaysbeconductedonlyin t h e G l ng/lO 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. A previous observation (Phillips Microbiological assay in a basal medium of approximately pH 6.5-6.8 using Lactobacillus casei ATCC 7469 (NCIB 6375) is the most widely used method for the measurement of folate concentrations in foods and biological fluids. In a recent study, Phillips & Wright (1 982) demonstrated that L. casei has a reduced growth response to 5-methyl-tetrahydrofolic acid at low concentrations (0-1 ng/lO ml assay), where the initial pH of the assay medium was 6.5-6.8, when compared with other folate monoglutamates such as folic acid which is commonly used as the calibration standard for the microbiological assay. Since 5-methyl-tetrahydrofolate (as polyglutamates) is one of the most important forms of folate in foods, it was argued that this reduced response of L. casei would result in an underestimation of total food folate. The present paper describes further studies of the microbiological assay of folic acid and 5-methyl-tetrahydrofolic acid with a view to establishing the cause and extent of the reduced L. casei growth response to 5-methyltetrahydrofolic acid. E X P E R I M E N T A L Folic acid (pteroylmonoglutamic acid) and DL-5-methyl-tetrahydrofolic acid were obtained from Sigma (London) Chemical Co. Ltd, Poole, Dorset. Stock solutions were prepared with ascorbic acid (10 g/l, 57 mM) adjusted to pH 6.0 with sodium hydroxide. When making up the stock solution of DL-5-methyl-tetrahydrofolic acid, consideration was given to the fact that L. casei responds only to the biologically-active L-form (Shane et al. 1980) . Before assay, working solutions of 4 and 40 ng/ml of the two folates were freshly prepared from stock solutions by diluting portions with freshly-prepared ascorbic acid (1 g/l, 5.7 mM) adjusted to pH 6.0.
doi:10.1079/bjn19850066 pmid:3933550 fatcat:rxv3syxvirghdfmcdcqzwbqski