Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

Shane, Barry; Stokstad, E. L. R.

doi:10.1099/00221287-103-2-249

Cited by 17 publications

(9 citation statements)

References 30 publications

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“…A major advantage of assays with L. casei is that L. casei responds equally well to all folate monoglutamate forms ( Table 1 ) (8). Although the affinity for the folate transporter varies with different monoglutamates, growth promotion is essentially identical because intracellular one-carbon metabolism, rather than transport, limits growth on monoglutamates (13, 14). …”

Section: Microbiological Assay Methodsmentioning

confidence: 99%

Folate status assessment history: implications for measurement of biomarkers in NHANES

Shane

2011

The American Journal of Clinical Nutrition

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This article presents a historical perspective on the different methods used to measure folate status in populations and clinical settings. I discuss some of the advantages and limitations of these procedures. For >50 y researchers have used microbiological assay methods to assess folate status in clinical settings and in population-based studies, such as NHANES. Serum and red blood cell folate values obtained with the Lactobacillus casei assay have formed the basis for current ranges and cutoffs for the establishment of folate sufficiency and for the current dietary reference intakes for folate. Over the past 30 y competitive folate protein binding assays, which are available in kit form, have supplanted microbiological assays in many clinical laboratories because of their ease of use. Several NHANES cycles have used these assays. Folate concentrations obtained with these kits are lower than those from microbiological assays and show a wide variation between different protein binding assay kits. This variation has complicated the setting of values for normal ranges of folate status and the comparison of status changes between different NHANES cycles. The recent development of mass spectrometry methods for folate opens up the possibility of measurement of individual folate vitamers such as folic acid. Past experience with microbiological and competitive protein binding assays indicates some of the technical problems that research will need to address before this promise becomes reality.

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Section: Microbiological Assay Methodsmentioning

confidence: 99%

Folate status assessment history: implications for measurement of biomarkers in NHANES

Shane

2011

The American Journal of Clinical Nutrition

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“…However, menadione does not affect the reaction rate for the oxidation of 5-methyl-H4PteGlu,, it only prevents the 5, I o-methylene-H,PteGlu, formed from being reduced back to 5-methyl-H4PteGlu,. The kinetic constants obtained for the L. casei reductase adequately accounted for the observed metabolism of 5-[Me-14C]methyl-H,PteGlu by this organism and the consequent incorporation of the labelled one-carbon moiety into purine and thymidylate derivatives (Shane & Stokstad, 1977). The L. casei auxotrophic requirement for purines and thymine has been known for some time (Rogers & Shive, 1948;Stokstad, 1941).…”

Section: Discussionmentioning

confidence: 91%

Rate-limiting Steps in Folate Metabolism by Lactobacillus casei

Shane¹,

Stokstad²

1977

Journal of General Microbiology

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Oxidation of 5-methyltetrahydrofolate to 5,Io-methylenetetrahydrofolate was the ratelimiting step in 5-methyltetrahydrofolate metabolism by Lactobacillus casei. The limiting steps in the utilization of suboptimal levels of folate by L. casei were related to the ability of folates to function in purine and/or thymidylate biosynthesis. Folates with glutamate chains of up to at least seven residues were substrates for these biosynthetic enzymes, and comparisons of bacterial growth yields with transport rates for these folates indicated that the polyglutamates were more effective substrates in purine and thymidylate synthesis than the corresponding pteroylmonoglutamates. Lactobacillus casei contained low levels of a B,,-independent, pteroylpolyglutamate-specific methionine synthetase. Its methylenetetrahydrofolate reductase also functioned more effectively with pteroylpolyglutamate substrates. I N T R O D U C T I O NThe major route for 5-methyltetrahydrofolate (5-methyl-H4PteGlu) metabolism in Lactobacillus casei is oxidation with the incorporation of the one-carbon moiety of 5-methyl-H4PteGlu into thymidylate and purine derivatives (Shane & Stokstad, I 977).In this study, the growth requirement of L. casei for folates in media containing purines or thymine and the effects of products of one-carbon metabolism on 5-methyl-H4PteGlu metabolism were investigated to assess rate-limiting steps in folate metabolism. METHODSNomenclature. The abbreviations used are : PteGlu, pteroylglutamic acid, folk acid; PteGlu,, pteroylmono-to pteroyloligo-y-L-glutamic acid, n indicating the number of glutamic acid residues ; H,PteGlu,, 5,6,7,8-tetrahydropteroylmono-to 5,6,7,8-tetrahydropteroyloligo-y-~-glutamic acid. The symbols (l) and (d) are used to denote the natural and unnatural diastereoisomers of H,PteGlu,, respectively, due to the asymmetric centre at the C-6 position, and do not indicate optical activity. Materials. (I)-5-~Me-14C]Methyl-H4PteGlu, (specific activity, 47 mCi mmol-l), (l)-~o-~ormyl-~~C]formyl-H,PteGlu (sp. act., 47 mCi mmol-l), and PteGlu, (n = I, 3, 5 and 7) were synthesized and purified as described previously (Shane & Stokstad, 1976).[14C]Formate (sp. act., 47 mCi mmol-l) was obtained from Schwarz/Mann (Orangeburg, New Jersey, U.S.A.) and unlabelled purines and pyrimidines, nucleosides and amino acids from Sigma.Organism and growth conditions. Lactobacillus casei (~~~~7 4 6 9 ) was cultured as described previously (Tamura et al., 1972).Metabolism of (l)-5-[Me-14CJmethyl-H4PteGlu. Lactobacillus casei, harvested from growth media in late exponential phase, was washed with, and resuspended (0.2 mg dry wt ml-l) in 50 ~M-K~HPOJIOO msodium acetate, adjusted to pH 6 with H3P04, and containing I % (w/v) glucose and 5 m-mercaptoethanol.The bacteria were preincubated at 37 "C for z h before addition of (l)-5-[Me-14C]methyl-H,PteGlu (0.45 ,uM). After a further 15 min, the bacteria were filtered, resuspended in fresh buffer (0.4 mg dry wt ml-l) and reincubated at 37 "C for 2 h. Intracellular labelled metabolites were extr...

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“…Folic acid uptake by L. casei is independent of conversion to other folate forms (Cooper, 1970), and enters the folate metabolic cycle via reduction to tetrahydrofolic acid which can be subsequently metabolized to 5, 10-methylene-tetrahydrofolic acid, the one-carbon moiety of which is rapidly incorporated into thymidylate and purines. Significant metabolism of folic acid to 5-methyl-tetrahydrofolic acid does not occur, as indicated by radio-tracer experiments with tetrahydrofolic acid (Shane & Stokstad, 1977 b), nor is it metabolized to 5-methyl-tetrahydrofolate-polyglutamates .…”

Section: Discussionmentioning

confidence: 96%

“…I .13), 5-methyl-tetrahydrofolic acid-monoglutamate (like folic acid) also enters the folate metabolic cycle as tetrahydrofolic acid with the subsequent synthesis of thymidylate and purines as described previously. L. casei, on the other hand, possesses a tetrahydropteroylglutamate methyltransferase (EC 2.1.1.14) that is polyglutamate-specific which cannot metabolize 5-methyltetrahydrofolic acid-monoglutamate directly (Shane & Stokstad, 1977 b) and, since 5-methyl-tetrahydrofolic acid-monoglutamate is also an ineffective substrate for L. casei polyglutamate synthetase , it cannot metabolize it indirectly either. The metabolism of 5-methyl-tetrahydrofolic acid-monoglutamate by L. casei can only proceed through its direct oxidation by 5, 10-methylene-tetrahydrofolate reductase (FADH,) (EC 1.1.99.15) to 5, 10-methylene-tetrahydrofolic acid and its subsequent utilization in thymidylate and purine synthesis (Shane & Stokstad, 1977 a).…”

Section: Discussionmentioning

confidence: 99%

“…L. casei, on the other hand, possesses a tetrahydropteroylglutamate methyltransferase (EC 2.1.1.14) that is polyglutamate-specific which cannot metabolize 5-methyltetrahydrofolic acid-monoglutamate directly (Shane & Stokstad, 1977 b) and, since 5-methyl-tetrahydrofolic acid-monoglutamate is also an ineffective substrate for L. casei polyglutamate synthetase , it cannot metabolize it indirectly either. The metabolism of 5-methyl-tetrahydrofolic acid-monoglutamate by L. casei can only proceed through its direct oxidation by 5, 10-methylene-tetrahydrofolate reductase (FADH,) (EC 1.1.99.15) to 5, 10-methylene-tetrahydrofolic acid and its subsequent utilization in thymidylate and purine synthesis (Shane & Stokstad, 1977 a). However, the equilibrium of 5, 10-methylene-tetrahydrofolic acid reductase can be strongly against this reaction (Shane & Stokstad, 1977b) and, as this is the only step that 5-methyl-tetrahydrofolic acid metabolism does not share with folic acid metabolism, it would appear that when L. casei is in a medium at pH 6.8 with low substrate concentrations, the amount of transported folate is insufficient to activate this enzyme (thus trapping folate unmetabolized and causing a threshold effect on growth) and that oxidation only occurs when the intracellular concentration is sufficient to overcome the enzyme equilibrium.…”

Section: Discussionmentioning

confidence: 99%

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

Wright¹,

Phillips²

1985

Br J Nutr

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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 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 (PhillipsMicrobiological 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...

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Metabolism of 5-Methyltetrahydrofolate by Lactobacittus casei

Cited by 17 publications

References 30 publications

Folate status assessment history: implications for measurement of biomarkers in NHANES

Folate status assessment history: implications for measurement of biomarkers in NHANES

Rate-limiting Steps in Folate Metabolism by Lactobacillus casei

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

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