10-formyltetrahydrofolate and 5-methyltetrahydrofolate

Studies have shown that conversion of leucovorin to the metabolite 5,10-methylenetetrahydrofolate (5,10-CH2FH4) is responsible for enhancement of the antitumor effects of fluorouracil given in combination with leucovorin, but the biochemical basis of this conversion in humans is not fully understood. To determine a possible sequence of metabolic steps, we studied the pharmacokinetics of leucovorin and its reduced folate metabolites in plasma in healthy volunteers. Groups of five subjects were given two equal doses of 10, 25, 125, 250, or 500 mg/m2 leucovorin, one orally and one intravenously at a 30-day interval. A sensitive radioenzymatic method that we developed previously was used to measure plasma concentrations of [S]5-formyltetrahydrofolate, 10-formyltetrahydrofolate (10-CHOFH4), 5-methyltetrahydrofolate (5-CH3FH4), and the combined 5,10-CH2FH4 plus tetrahydrofolate (FH4) pools. Intravenous administration of leucovorin resulted in dose-dependent accumulation of 5,10-CH2FH4 + FH4 exceeding 2 microM at peak levels. After oral and intravenous administration, 10-CHOFH4 and 5,10-CH2FH4 + FH4 exhibited peak levels earlier and were eliminated more rapidly than 5-CH3FH4. Accumulation of all metabolites after intravenous administration was linearly dose dependent, while oral administration appeared to result in saturation. We propose that the host activation of leucovorin suggested by these findings could be responsible for elevation of intratumor 5,10-CH2FH4 levels, thus enhancing the antitumor effects of fluorouracil. These results also suggest that 10-CHOFH4, 5,10-CH2FH4, and FH4 are intermediate metabolites and that 5-CH3FH4 is the terminal metabolite. In addition, our results indicate that attainment of high plasma levels of the metabolites active in modulation of the therapeutic effects of fluorouracil is best achieved through intravenous administration of high doses of leucovorin. Our future studies will address the proposed sequential conversion pathway and, thus, the mechanism by which pharmacologically relevant reduced folates accumulate in plasma after leucovorin administration.

Topics: Administration, Oral; Adult; Female; Humans; Injections, Intravenous; Leucovorin; Male; Random Allocation; Tetrahydrofolates; Time Factors

Folate (vitamin B

Topics: Animals; Antioxidants; Epithelial Cells; Folic Acid; Glucose; Inflammation; Leucovorin; Oxidative Stress; Tetrahydrofolates

Topics: Chromatography, Thin Layer; Fabaceae; Folic Acid; Food Analysis; Germination; Image Processing, Computer-Assisted; Lens Plant; Leucovorin; Mass Spectrometry; Molecular Imaging; Multivariate Analysis; Reproducibility of Results; Seeds; Tetrahydrofolates

Topics: Leucovorin; Oxidation-Reduction; Pteroylpolyglutamic Acids; Stereoisomerism; Tetrahydrofolates

10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) was previously identified as a folate-binding protein in rat liver cytosol (R.J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982) by virtue of the tetrahydrofolate polyglutamate tightly bound to the partially purified enzyme. In this current study we provide evidence to show that when liver cytosol was rapidly processed to identify the protein bound folate, large amounts of both 10-formyl- and 5-formyltetrahydrofolate were present. After overnight storage of the cytosol at 5 degrees C before processing, almost no formylfolates were present and the major protein-bound form was tetrahydrofolate. This suggests that 10-formyltetrahydrofolate polyglutamates are tightly bound to the enzyme in vivo and are converted to tetrahydrofolate forms during isolation by the hydrolase activity associated with the enzyme. Covalent binding of the stable folate analogue, 5-formyltetrahydrofolate, to the purified enzyme resulted in 2 mol bound per mole of enzyme subunit. This is consistent with earlier reports suggesting the enzyme is capable of carrying out both oxidative and hydrolytic conversion of 10-formyltetrahydrofolate to tetrahydrofolate at the same time. Partial tryptic digestion of the purified enzyme selectively inhibited dehydrogenase activity of the enzyme but did not affect the hydrolase or aldehyde dehydrogenase activities.

Topics: Animals; Cytosol; Folic Acid; Leucovorin; Ligands; Liver; Oxidoreductases Acting on CH-NH Group Donors; Pteroylpolyglutamic Acids; Rats; Tetrahydrofolates; Trypsin

This report describes an analytical method for the measurement of tetrahydrofolate (H4PteGlu), 10-formyltetrahydrofolate (10-HCO-H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4-PteGlu) in rat bile by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). After diluting the bile sample with 0.2% sodium ascorbate solution, the sample was analyzed under the following conditions; (a) phenyl bonded phase column as an analytical column; (b) mobile phase consisting of 20 mM acetate buffer (pH 5.0) containing 0.1 mM EDTA; (c) an applied ECD potential of +300 mV; (d) 0.8 ml/min of flow rate. Under the above conditions, peaks of H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu in rat bile were well separated on the ECD-chromatogram. Detection limits of H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu were 0.13, 0.11 and 0.10 ng/ml, respectively, at S/N = 3. Bile excretion rates for H4PteGlu, 10-HCO-H4PteGlu and 5-CH3-H4PteGlu, which were analyzed by this method in rats, were 314 +/- 181, 321 +/- 179 and 449 +/- 198 ng/hr, respectively. Bile concentrations of the folates were more than 5,000 times higher than the detection limits for this method. This HPLC-ECD method is, therefore, a useful tool for bile folate analysis.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Electrochemistry; Evaluation Studies as Topic; Female; Leucovorin; Rats; Rats, Sprague-Dawley; Tetrahydrofolates

A pharmacokinetic study of folic acid and its metabolites was conducted to provide a basis to consider folic acid as a therapeutic alternative to leucovorin. Leucovorin has been used in various folate antagonist rescue regimens and to modulate fluorouracil activity in the treatment of solid tumors. Although leucovorin is typically administered intravenously in fluorouracil modulation therapy, limited oral administration trials have yielded equivalent responses. Because metabolites rather than leucovorin are the predominant circulating species after oral administration, these clinical results indicate that metabolites themselves can be modulating agents. Folic acid could be an attractive alternative to leucovorin provided it effectively elevates the same plasma metabolites. Hence, folic acid at doses of 25 and 125 mg/m2 was administered orally and intravenously to normal volunteers. Plasma folic acid and its reduced folate metabolites were monitored over a 24-hour period by use of a previously developed radioenzymatic method. The metabolites that accumulated--5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, tetrahydrofolate, and 10-formyltetrahydrofolate--were the same metabolites that were observed previously after leucovorin administration. Folic acid metabolites accumulated more slowly and persisted longer than leucovorin metabolites, which can be attributed to slower metabolism of the fully oxidized vitamin. Based on these results, it is concluded that folic acid could be an attractive therapeutic alternative to leucovorin for fluorouracil modulation.

Topics: Adult; Female; Folic Acid; Humans; Leucovorin; Male; Reference Values; Tetrahydrofolates

Reduced folate derivatives in rat bile were examined using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Three peaks of folate compounds were observed on the chromatograms. From the retention-time profiles and hydrodynamic voltammograms, and the profiles of ultraviolet (UV) absorbance spectra obtained by HPLC with photodiode array detection, these 3 peaks were identified as 10-formyltetrahydrofolate (10-HCO-H4PteGlu), tetrahydrofolate (H4PteGlu) and 5-methyltetrahydrofolate (5-CH3-H4PteGlu). The rates of bile secretion of 10-HCO-H4PteGlu, H4PteGlu and 5-CH3H4PteGlu were 314 +/- 181, 321 +/- 179 and 449 +/- 198 ng/h (mean +/- S.D.), respectively. 10-HCO-H4PteGlu and H4PteGlu together wtih 5-CH3-H4-PteGlu are found to be the major folate derivatives in rat bile. The nonmethylated folates, 10-HCO-H4PteGlu and H4PteGlu, may also play an important role in folate homeostasis.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Female; Folic Acid; Hydrogen-Ion Concentration; Leucovorin; Rats; Rats, Sprague-Dawley; Spectrophotometry, Ultraviolet; Tetrahydrofolates; Tritium

The folic acid content of grain, cereal products (including beer), bakery products and legumes was determined by means of high-performance liquid chromatography (HPLC). Free folate (monoglutamate forms) and total folate (monoglutamate + polyglutamate forms) were differentiated. Of the grain analysed, rye, with a mean value of 143 micrograms/100 g, contained more total folate than wheat (mean = 91 micrograms/100 g). The total folate content of bakery products ranged from 14 micrograms/100 g (whole grain rye bread) to 88 micrograms/100 g (crispbread). Beer had a very low total folate content (mean = 3 micrograms/100 ml). The mean of the free folate portion was 76.5% in grain and cereal products and 65.6% in bakery products. Of the legumes analysed, beans (mean = 128 micrograms/100 g) had the highest content of total folate, followed by lentils (103 micrograms/100 g) and peas (57 micrograms/100 g). The mean value of the free folate portion in legumes (73.1%) was comparable with the values of grain, cereal products and bakery products. In addition to tetrahydrofolic acid (THF), 5-methyl-THF and 5-formyl-THF, pteroylglutamic acid (PteGlu) and 10-formyl-PteGlu were determined in all products (except beer). Their proportion (PteGlu + 10-formyl-PteGlu) of the total folate content ranged from 23.5% to 44.4%.

Topics: Beer; Bread; Edible Grain; Fabaceae; Folic Acid; Leucovorin; Plants, Medicinal; Secale; Tetrahydrofolates; Triticum

The therapeutic activity of FUra alone or combined with [6RS]LV doses ranging from 50 to 1,000 mg/m2 was examined in eight colon adenocarcinoma xenografts, of which five were established from adult neoplasms (HxELC2, HxGC3, HxVRC5, HxHC1, and HxGC3/c1TK-c3 selected for TK deficiency) and three were derived from adolescent tumors (HxSJC3A, HxSJC3B, and HxSJC2). The growth-inhibitory effects of FUra were potentiated by higher doses of [6RS]LV (500-1,000 mg/m2) in three lines (HxGC3/c1TK-c3, HxSJC3A, and HxSJC3B) and by a low dose of [6RS]LV in only one tumor (HxVRC5). Expansion of pools of CH2-H4PteGlun+H4PteGlun (greater than or equal to 2.4-fold) in response to higher doses of [6RS]LV was obtained in all lines except HxHC1. Metabolism of [6RS]LV was high in HxVRC5, with high levels of 5-CH3-H4PteGlu being detected, but not in HxHC1, in which levels of 5-CH3-H4PteGlu and CH = H4PteGlu+10-CHO-H4PteGlu remained relatively low. In the adolescent tumors, levels of CH = H4PteGlu+10-CHO-H4PteGlu were consistently higher than those of 5-CH3-H4PteGlu following [6RS]LV administration, and in HxSJC3A, in which pools of CH2-H4PteGlun+H4PteGlun were significantly expanded, 5-CH3-H4PteGlu concentrations were lower than those observed in the other two lines. The sensitivity of tumors to FUra +/- [6RS]LV and the characteristics of [6S]LV metabolism did not correlate with the activity of CH = H4PteGlu synthetase, the enzyme responsible for the initial cellular metabolism of [6S]LV to CH = H4PteGlu. Thus, no single metabolic phenotype correlated with the [6RS]LV-induced expansion of CH2-H4PteGlun+H4PteGlun pools. Potentiation of the therapeutic efficacy of FUra by [6RS]LV was observed in HxGC3/c1TK-c3 xenografts but not in parent HxGC3 tumors, demonstrating the influence of dThd salvage capability in the response to FUra-[6RS]LV combinations. Plasma dThd concentrations in CBA/CaJ mice were high (1.1 microM). The present data therefore demonstrate the importance of (1) higher doses of [6RS]LV, (2) expansion of pools of CH2-H4PteGlun+H4PteGlun, and (3) dThd salvage capability in potentiation of the therapeutic efficacy of FUra in colon adenocarcinoma xenografts. The plasma levels of FUra achieved in mice are presented.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Colonic Neoplasms; Fluorouracil; Humans; Leucovorin; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Tetrahydrofolates

The carcinogenic effects of methyl-deficient, amino acid-defined diets have been attributed to alterations in cellular methylation reactions. These diets contain no choline, and methionine is replaced by homocysteine. Hence, all methyl groups needed for methionine biosynthesis with subsequent formation of S-adenosylmethionine and polyamines must be formed de novo utilizing folate-dependent reduction of one-carbon units. In rats fed the methyl-deficient diet, there was a marked decrease in total liver folate levels. This decrease was apparent in the levels of the individual forms of folate: 10-HCO-H4folate, 5-HCO-H4folate, 5-CH3-H4folate and H4folate. The percent of the total folate pool made up by 5-CH3-H4folate did not change, however, until after the rats had been fed the methyl-deficient diet for 4 wk, and then an increase was seen. After the methyl-deficient rats were switched to a nutritionally adequate control diet containing methionine and choline, all values rapidly reversed. Increased use of folate for methyl group biosynthesis may be responsible for the loss of folates from the liver.

Topics: Amino Acids; Animals; Choline Deficiency; Chromatography, High Pressure Liquid; Diet; Folic Acid; Leucovorin; Liver; Male; Methionine; Methylation; Rats; Rats, Inbred F344; Tetrahydrofolates