Folic acid compounds in romaine lettuce.

The composition of folate coenzymes in romaine lettuce was studied. Lettuce extract was purified on QAE-Sephadex A-25 and folate compounds were separated into a monoglutamate fraction and a polyglutamate fraction by chromatography on Sephadex G-15. Both the mono- and poly-glutamate fractions were resolved on DEAE-cellulose. Positive identification of DEAE peaks was made by further cochromatography with high specific activity radioactive marker folate compounds and with differential microbiological assay. The distribution of folate compounds in lettuce is as follows: 32% 5-CH3-H4PteGlu; 1% 5-CHO-H4PteGlu; 3% 5-CHO-H4PteGlu4; 9% 5-CH3-H4PteGlu4; 13% 5-CHO-H4PteGlu5; and 31% 5-CH3-H4PteGlu5.     

Preparation of (6R)-tetrahydrofolic acid and (6R)-5-formyltetrahydrofolic acid of high stereochemical purity

Commercially available 5-formyltetrahydrofolate (5-CHO-H4PteGlu) is chemically prepared in a reaction that introduces an asymmetric center at the 6 carbon, and hence is the mixture of diastereomers differing in chirality about this position. (6R)-5-CHO-H4PteGlu, the diastereomer that is not normally found in vivo, was prepared from folic acid. Folic acid was chemically reduced and (6R)-tetrahydrofolate (H4PteGlu) was obtained from the resultant (6R,S)-H4PteGlu by enzymatic consumption of the natural diastereomer of (6R,S)-5,10-CH2-H4PteGlu (reversibly formed from (6R,S)-H4PteGlu in the presence of formaldehyde) with Lactobacillus casei thymidylate synthase. The 5 position of purified (6R)-H4PteGlu was directly formylated in a carbodiimide-catalyzed reaction. The level of contamination of these preparations with the corresponding 6S diastereomers was estimated using the binding of fluorodeoxyuridylate to thymidylate synthase promoted by folate cofactor (for H4PteGlu) and by the growth of folate requiring bacteria (for 5-CHO-H4PteGlu). Purified preparations of (6R)-H4PteGlu promoted the binding of fluorodeoxyuridylate to L. casei thymidylate synthase (in the presence of formaldehyde) only at concentrations greater than 1000-fold higher than equiactive levels of (6S)-H4PteGlu. Likewise, the (6R)-5-CHO-H4PteGlu made by this method was 600 times less active as a growth factor for Pediococcus cerevisiae than was authentic (6S)-5-CHO-H4PteGlu. Hence, the minimum stereochemical purity of these preparations was 99.9% for (6R)-H4PteGlu and 99.8% for (6R)-5-CHO-H4PteGlu.     

Mouse folylpoly-gamma-glutamate synthetase isoforms respond differently to feedback inhibition by folylpolyglutamate cofactors.

Folylpoly-gamma-glutamate synthetase (FPGS) is the enzyme responsible for metabolic trapping of reduced folate cofactors in cells for use in nucleotide and amino acid biosynthesis. There are two isoforms of FPGS expressed in mouse tissues, one is expressed in differentiated tissue, principally liver and kidney, and the other in all rapidly proliferating cell types. The present study sought the functional difference that would explain the evolution of two mouse FPGS species. Recombinant cytosolic mouse isozymes were compared with respect to steady state kinetics, chain length of polyglutamate derivatives formed, and end-product inhibition by the major reduced folylpentaglutamate cofactors. Both isoforms were equally effective in catalyzing the addition of a mole of glutamic acid to reduced folate monoglutamate substrates. Each isoform was also capable of forming long chain polyglutamate derivatives of the model folate, 5,10-dideazatetrahydrofolate. In contrast, the FPGS isoform derived from rapidly proliferating tissue was much more sensitive to inhibition by (6R)-5,10-CH(2)-H(4)PteGlu(5) and (6S)-H(4)PteGlu(5) than the isoform expressed in differentiated tissues, as demonstrated by 13- and 6-fold lower inhibition constants (K(i)), respectively. Interestingly, each isozyme was equally sensitive to inhibition by (6R)-10-CHO-H(4)PteGlu(5). We drew the conclusion that the decreased sensitivity of the FPGS expressed in mouse liver and kidney to feedback inhibition by 5,10-CH(2)-H(4)PteGlu(5-6) and H(4)PteGlu(5-6) may have evolved to permit accumulation of a larger folate cofactor pool than that found within rapidly proliferating tissue.     

High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver

Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100°C in 2% ($\text{w}\text{v}$) sodium ascorbate, 0.2 m 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H₄PteGlu), 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5-methyl-H₄PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H₂PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H₄PteGlu was quantitatively recovered as H₄PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H₄PteGlu, 37.2%; H₄PteGlu, 32.7%; 10-formyl-H₄PteGlu, 22.6%; and 5-formyl-H₄PteGlu, 7.7%. Experiments with [³H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.     

The metabolism of 5-methyltetrahydropteroyl-L-glutamic acid and its oxidation products in the rat.

Folate metabolism in the rat was investigated using radiolabelled 5-methyltetrahydropteroylglutamate (5-CH3-H4PteGlu) and its oxidation products. 5-CH3-H4PteGlu is absorbed completely from the intestine, although in some preparations it is an equimolecular mixture of C-6 epimers, only one of which is naturally present in biological systems. The methyl group is incorporated into non-folate compounds, including methionine and creatine. No evidence was observed for the oxidation of the methyl group of 5-CH3-H4PteGlu to form other folate types. The tetrahydrofolate moiety of 5-CH3-H4PteGlu is metabolized in a similar manner to folic acid, forming formyl folates and tissue polyglutamates, and is catabolized by scission. The triazine oxidation product of 5-CH3-H4PteGlu is not metabolized by the rat or its gut microflora. 5-Methyl-5,6-dihydropteroylglutamate, however, is assimilated into the folate pool, but is substantially broken down by passage through the gut. The possible implication of this in scorbutic diets is discussed.     

Carrier-Mediated Transport of Folate in a Mutant of Pediococcus cerevisiae

A mutant strain of Pediococcus cerevisiae (P. cerevisiae/PteGlu) was isolated which grows on low-folate (PteGlu) concentrations (200 pg/ml). The growth response of the parent and mutant strains to folinate (5-CHO-H(4)PteGlu) was the same. The transport of (14)C-PteGlu by P. cerevisiae/PteGlu was temperature-dependent (Q(10) between 27 C and 37 C was about 2), energy-dependent, and pH-dependent and was inhibited by iodoacetate, 2,4-dinitrophenol, potassium fluoride, and sodium azide. The uptake obeyed saturation kinetics with an apparent K(m) of 6.6 x 10(-6) M and V(max) of 4.0 x 10(-10) mol per min per mg (dry weight). At the steady state the intracellular concentration of PteGlu was 120-fold higher from that of the medium. Reduced folates like 5-CHO-H(4)PteGlu and methyl-tetrahydrofolate (5-CH(3)-H(4)PteGlu) as well as 2,4-diaminoanalogues (amethopterin and aminopterin) were shown to compete for the PteGlue-carrier.     

Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography

A recent report from this laboratory described the use of affinity chromatography for the isolation of pure folates from tissue extracts (J. Selhub, B. Darcy-Vrillon, and D. Fell (1988) Anal. Biochem. 168, 247-251). The present study was undertaken to develop chromatographic procedures for quantitative analysis of the individual folates in the affinity-purified mixture. Methods were devised whereby mixtures containing pteroylglutamates (PteGlu1-7) were batch reduced to the dihydro, H2PteGlu1-7, and tetrahydro, H4Pte-Glu1-7, forms. The 5-methylH4PteGlu1-7 and the 10-formylH4PteGlu1-7 series were prepared from H4Pte-Glu1-7. These compounds were used to calibrate a liquid chromatographic system for the resolution of folate mixtures. This system included reverse-phase ion pair chromatography and a diode array detector. A mixture containing oxidized and reduced PteGlu1-7, a total of 35 derivatives, was separated into seven clusters arranged in an order of increasing number of glutamate residues. Each cluster was represented by two or more peaks which were due to folates that differed in the pteridine ring structure but had the same number of glutamate residues. In clusters containing mono and diglutamyl derivatives the 10-formyltetrahydro-, the tetrahydro-, and the dihydrofolate forms appeared as separate peaks while those representing folic acid and 5-methyl-tetrahydrofolate derivatives eluted in coinciding peaks. This hierarchy was maintained in the following clusters except for increasing tendency of the former three forms of folates to elute in the same peak. The number of glutamate residues of any eluting folate can be determined on the basis of retention time in relation to those of the clusters. The pteridine ring structure of that same folate can be determined on the basis of its elution position within that cluster and spectral characteristics determined by the diode array detection system. If that position is common for more than one derivative then identification is based on differential spectral properties. Using uv absorption signals at 280 nm to determine indiscriminate folate activity, absorption signals at 350 nm are used to identify folic acid and dihydrofolate derivatives and signals at 258 nm are used to identify 10-formyltetrahydrofolate derivatives. These principles were incorporated into mathematic expressions which were used for quantitative resolution of simulated mixtures containing oxidized and reduced PteGlu5 and for the analysis of folate composition in rat liver, human milk, and cows milk.     

Endogenous folate of normal fibroblasts using high-performance liquid chromatography and modified extraction procedure

The endogenous levels of the various folate monoglutamate compounds in cultured human fibroblasts were determined using high-performance liquid chromatography for the separation of folate monoglutamate. Endogenous folates were converted to monoglutamate forms using conjugase enzyme present in rat serum and incubation was carried out at pH 6.5. This minimized folate coenzyme interconversion during processing. Using methanol for precipitation of protein instead of heat minimized degradation of labile folates. Recovery of all folates except 10-formyltetrahydrofolic acid (10-CHO H4PteGlu) using this procedure was more than 90%. Disruption of cells by boiling appeared to cause less postextraction changes of cell folates than did freezing and thawing or sonication. When heat to release endogenous folate, conjugase treatment with rat serum at pH 6.5, and precipitation of protein with methanol were used, more than half of the intracellular folate of normal fibroblasts in confluent growth was 5-methyltetrahydrofolic acid (5-CH3 H4PteGlu), and 10-CHO H4PteGlu and tetrahydrofolic acid (H4PteGlu) comprised 29 and 6%, respectively.     

Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism

Most mammalian cells receive exogenous folate from the bloodstream in the form of 5-methyltetrahydropteroylmonoglutamate (CH3-H4PteGlu1). Because this folate derivative is a very poor substrate for folylpolyglutamate synthetase, the enzyme that adds glutamyl residues to intracellular folates, CH3-H4PteGlu1 must first be converted to tetrahydropteroylmonoglutamate (H4PteGlu1), 10-formyltetrahydropteroylmonoglutamate (CHO-H4PteGlu1), or dihydrofolate (H2folate), which are excellent substrates for folylpolyglutamate synthetase. Polyglutamylation is required both for retention of intracellular folates and for efficacy of folates as substrates for most folate-dependent enzymes. Two enzymes are known that will react with CH3-H4PteGlu1 in vitro, methylenetetrahydrofolate reductase and methyltetrahydrofolate-homocysteine methyltransferase (cobalamin-dependent methionine synthase). These studies were performed to assess the possibility that methylenetetrahydrofolate reductase might catalyze the conversion of CH3-H4PteGlu1 to CH2-H4PteGlu1. CH2-H4PteGlu1 is readily converted to CHO-H4PteGlu1 by the action of methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, and these enzyme activities show very little preference for folypolyglutamate substrates as compared with folylmonoglutamates. We conclude from in vitro studies of the enzyme that methylenetetrahydrofolate reductase cannot convert CH3-H4PteGlu1 to CH2-H4PteGlu1 under physiological conditions and that uptake and retention of folate will be dependent on methionine synthase activity.     

Determination of 5-methyltetrahydrofolate (13C-labeled and unlabeled) in human plasma and urine by combined liquid chromatography mass spectrometry

The association of folates with the prevention of neural tube defects and reduced risk of other chronic diseases has stimulated interest in the development of techniques for the study of their bioavailability in humans. Stable isotope protocols differentiate between oral and/or intravenous test doses of folate and natural levels of folate already present in the body. An liquid chromatography/mass spectrometry (LC/MS) procedure is described that has been validated for the determination of [13C]5-methyltetrahydropteroyl monoglutamic acid ([13C]5-CH3H4PteGlu) in plasma and urine, following oral dosing of volunteers with different labeled folates. Folate binding protein affinity columns were used for sample purification prior to LC/MS determination. Chromatographic separation was achieved using a Superspher 100RP18 (4 microm) column and mobile phase of 0.1 mol/L acetic acid (pH 3.3):acetonitrile (90:10; 250 microL/min). Selected ion monitoring was conducted on the [M-H](-) ion: m/z 458 and 459 for analyzing 5-CH3H4PteGlu; m/z 464 [M+6-H](-) to determine 5-CH3H4PteGlu derived from the label dose; m/z 444 for analysis of 2H4PteGlu internal standard, and m/z 446 and 478 to confirm that there was no direct absorption of unmetabolized compounds. Calibration was linear over the range 0-9 x 10(-9) mol/L; the limits of detection and quantification were 0.2 x 10(-9) and 0.55 x 10(-9) mol/L, respectively. The mean coefficient of variation of the ratios (m/z 463/458) was 7.4%. The method has potential applications for other key folates involved in one-carbon metabolism.     

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.     

Purification and properties of pancreatic glycine N-methyltransferase.

Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine. It is very abundant in liver cytosol and earlier studies have shown it to be present in high concentrations in the pancreas. We have previously reported that liver GNMT is allosterically inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-H4PteGlu5), and proposed that this represents a metabolic control mechanism which links the de novo synthesis of methyl groups to the methylating ability of the liver. We now report that pancreatic GNMT also contains bound folate in vivo. Purified pancreatic GNMT is inhibited by reduced folate polyglutamates in vitro. The KI for the synthetic (R,S)5-CH3-H4PteGlu5 is 2.4 x 10(-7) M. The natural (S) form of 5-CH3-H4PteGlu5 is tightly bound and has a Kd of 1.3 x 10(-7) M. One mole is bound per enzyme tetramer. These studies suggest that GNMT is important in the regulation of methyl group metabolism in the pancreas as well as in the liver.     

Na+ and pH dependence of 5-methyltetrahydrofolic acid and methotrexate transport in freshly isolated hepatocytes

The dependence of the high-affinity transport systems for 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) and methotrexate on sodium ions and on pH was examined in freshly isolated rat hepatocytes. Previous studies indicated that transport of these folate derivatives was sodium-dependent. Experiments to determine the Km for sodium of 5-CH3-H4PteGlu transport showed no dependence on extracellular sodium. However, uptake was sodium-dependent when hepatocytes were preincubated for 30 min in sodium-free medium, a treatment which resulted in an increase in the transmembrane pH gradient (delta pH = pH out-pH in) and a decrease in the uptake of 5-CH3-H4PteGlu. Uptake of methotrexate displayed a linear dependence on extracellular sodium ions. Uptake of 5-CH3-H4PteGlu increased linearly as the transmembrane pH gradient decreased; i.e., as the medium became more acid with respect to the cytosol. Lineweaver-Burk and Scatchard plots of 5-CH3-H4PteGlu uptake indicated an apparent Km for H+ of about 24 nM, equivalent to a pH of 7.6. Hill-plots suggested a stoichiometry of 1:1 for the interaction of protons with the 5-CH3-H4PteGlu transport system. Both the Km and Vmax for 5-CH3-H4PteGlu transport were increased at pH 5.5 compared to pH 7.4, suggesting that extracellular protons increased the number of and/or the activity of the membrane carrier. In contrast, methotrexate transport was maximal at pH 7 where the transmembrane pH gradient was zero. These results suggest the possibility that 5-CH3-H4PteGlu may be cotransported along with H+ ions in hepatocytes, although they do not rule out a 'catalytic coupling' whereby protons interact with the carrier to stimulate substrate flux without concomitant H+ transport.     

Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue.     

Phosphorylation modulates the activity of glycine N-methyltransferase, a folate binding protein. In vitro phosphorylation is inhibited by the natural folate ligand

Glycine N-methyltransferase (EC 2.1.1.20) was recently identified as a major folate binding protein of rat liver cytosol (Wagner, C., and Cook, R. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3631-3634). Activity of the enzyme is inhibited when the natural folate ligand, 5-methyltetrahydropteroylpentaglutamate (5-CH3-H4PteGlu5), is bound. It has been suggested that glycine N-methyltransferase plays a role in regulating the availability of methyl groups in the liver. Purified transferase was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase. If 5-CH3-H4PteGlu5 was first bound to the transferase, phosphorylation was inhibited. Phosphorylation of glycine N-methyltransferase in vitro increased its activity approximately 2-fold. 5-CH3-H4PteGlu5 inhibited the activity of newly phosphorylated enzyme as well as native enzyme. Freshly isolated rat hepatocytes incorporated 32P-labeled inorganic phosphate into this folate binding protein. Chemical analysis of purified enzyme showed about 0.55 mol of phosphate present per mol of glycine N-methyltransferase subunit. These results indicate that phosphorylation of glycine N-methyltransferase may provide a mechanism for modulating the activity of this enzyme and support its role in regulating the availability of methyl groups.     

Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate

Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Application of stable isotope dilution assays based on liquid chromatography-tandem mass spectrometry for the assessment of folate bioavailability

A pilot study was performed to prove the suitability of stable isotope dilution assays for assessing the bioavailability of endogenous folates in foods. By using [2H(4)]folic acid, [2H(4)]tetrahydrofolate, [2H(4)]5-methyltetrahydrofolate, [2H(4)]5-formyltetrahydrofolate and [2H(4)]10-formylfolic acid as internal standards, folates in spinach, apple juice and blood plasma were quantified by liquid chromatography coupled to tandem mass spectrometry. To liberate the pteroyl monoglutamates, sample extracts of foods were treated by rat plasma. Sample clean-up was achieved by solid-phase extraction on anion-exchange cartridges, which proved to be sufficient to obtain mass chromatograms devoid of matrix interferences. The bioavailability study was designed as a short-time protocol with three meals, the first consisting of 600 g spinach (meal A), the second consisting of 600 g apple sauce with additionally 400 microg synthetic folic acid (meal B) and the third consisting solely of 600 g apple sauce (meal C). Prior to the meals, the participating volunteer's tissue was saturated with folates to achieve a significant response of plasma folate to the meals. After consumption of meals A and B a significant rise in folate plasma level compared to meal C (mean level at 28 microg/ml) was observed. The relative bioavailability of folate following meal A exceeded significantly the suggested value of 50% for food folates by taking the dose-normalized area under the curve (AUC) following ingestion of meal B as reference.     

Pediococcus cerevisiae mutant with altered transport of folates.

A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.     

Methionine dependency of cultured human lymphocytes

Human peripheral blood lymphocytes stimulated with phytohemagglutinin and a lymphocyte model consisting of the RPMI 6410 cell, a human virus-transformed B cell, required added methionine (Met) for growth of the cultures. This failure to meet all needs for Met via endogenous synthesis, which is characteristic of oncogenic transformation, occurred even in the presence of adequate homocysteine, methylfolate (5-CH3-H4PteGlu) and cobalamin (Cbl)-dependent methionine synthetase activity. Folinic acid (5-CHO-H4PteGlu), which provides available folate independently of Cbl, improved growth only slightly in the absence of Met. Free Cbl at 222 nM, an amount great enough to alter other intracellular events, failed to increase growth in the absence of Met, but 0.22 nM Cbl bound to transcobalamin II did, however, enhance growth.