Improved Stability of a Stable Crystal Form C of 6S-5-Methyltetrahydrofolate Calcium Salt, Method Development and Validation of an LC-MS/MS Method for Rat Pharmacokinetic Comparison.

Folate is a vitamin beneficial for humans that plays an important role in metabolism, but it cannot be well supplemented by food; it is necessary to supplement it in other ways. Based on this consideration, a novel crystal form C of 6S-5-methyltetrahydrofolate calcium salt (MTHF CAC) was obtained. To explore the difference between MTHF CAC and the crystal form Ⅰ of 6S-5-methyltetrahydrofolate calcium salt (MTHF CA) as well as an amorphous product of 6S-5-methyltetrahydrofolate glucosamine salt (MTHF GA), their stability and pharmacokinetic behaviours were compared. The results of high-performance liquid chromatography coupled with ultraviolet detection analysis indicated that MTHF CAC showed a better stability than MTHF CA and MTHF GA. After oral administration of MTHF CAC, MTHF CA, and MTHF GA to male rats, the MTHF concentrations were analysed using a validated liquid chromatography-tandem mass spectrometry, and the pharmacokinetic parameters were compared. The mean residence times (0-t) of MTHF CAC, MTHF CA, and MTHF GA were 3.7 ± 1.9 h, 1.0 ± 0.2 h (p < 0.01), and 1.5 ± 0.3 h (p < 0.05), respectively. The relative bioavailability of MTHF CAC was calculated to be 351% and 218% compared with MTHF CA and MTHF GA, respectively, which suggests that MTHF CAC can be better absorbed and utilized for a longer period of time.

Natural folates from biofortified tomato and synthetic 5-methyl-tetrahydrofolate display equivalent bioavailability in a murine model.

Folate deficiency is a global health problem related to neural tube defects, cardiovascular disease, dementia, and cancer. Considering that folic acid (FA) supply through industrialized foods is the most successful intervention, limitations exist for its complete implementation worldwide. Biofortification of plant foods, on the other hand, could be implemented in poor areas as a complementary alternative. A biofortified tomato fruit that accumulates high levels of folates was previously developed. In this study, we evaluated short-term folate bioavailability in rats infused with this folate-biofortified fruit. Fruit from tomato segregants hyperaccumulated folates during an extended ripening period, ultimately containing 3.7-fold the recommended dietary allowance in a 100-g portion. Folate-depleted Wistar rats separated in three groups received a single dose of 1 nmol of folate/g body weight in the form of lyophilized biofortified tomato fruit, FA, or synthetic 5-CH3-THF. Folate bioavailability from the biofortified tomato was comparable to that of synthetic 5-CH3-THF, with areas under the curve (AUC(0-∞)) of 2,080 ± 420 and 2,700 ± 220 pmol · h/mL, respectively (P = 0.12). Whereas, FA was less bioavailable with an AUC(0-∞) of 750 ± 10 pmol · h/mL. Fruit-supplemented animals reached maximum levels of circulating folate in plasma at 2 h after administration with a subsequent steady decline, while animals treated with FA and synthetic 5-CH3-THF reached maximum levels at 1 h. Pharmacokinetic parameters revealed that biofortified tomato had slower intestinal absorption than synthetic folate forms. This is the first study that demonstrates the bioavailability of folates from a biofortified plant food, showing its potential to improve folate deficiency.

Is 5-methyltetrahydrofolate an alternative to folic acid for the prevention of neural tube defects?

Women have higher requirements for folate during pregnancy. An optimal folate status must be achieved before conception and in the first trimester when the neural tube closes. Low maternal folate status is causally related to neural tube defects (NTDs). Many NTDs can be prevented by increasing maternal folate intake in the preconceptional period. Dietary folate is protective, but recommending increasing folate intake is ineffective on a population level particularly during periods of high demands. This is because the recommendations are often not followed or because the bioavailability of food folate is variable. Supplemental folate [folic acid (FA) or 5-methyltetrahydrofolate (5-methylTHF)] can effectively increase folate concentrations to the level that is considered to be protective. FA is a synthetic compound that has no biological functions unless it is reduced to dihydrofolate and tetrahydrofolate. Unmetabolized FA appears in the circulation at doses of >200 µg. Individuals show wide variations in their ability to reduce FA. Carriers of certain polymorphisms in genes related to folate metabolism or absorption can better benefit from 5-methylTHF instead of FA. 5-MethylTHF [also known as (6S)-5-methylTHF] is the predominant natural form that is readily available for transport and metabolism. In contrast to FA, 5-methylTHF has no tolerable upper intake level and does not mask vitamin B12 deficiency. Supplementation of the natural form, 5-methylTHF, is a better alternative to supplementation of FA, especially in countries not applying a fortification program. Supplemental 5-methylTHF can effectively improve folate biomarkers in young women in early pregnancy in order to prevent NTDs.

Estimation of the 5-methyltetrahydrofolate apparent volume of distribution in humans.

The fractional absorption of a stable isotope-labeled folate dose can be estimated from the subsequent short-term temporal changes in the concentration of labeled L-5-methyltetrahydrofolate (L-5-methyl-THF) in plasma using mathematical modeling. However, the model is dependent on the use of an accurate value for the apparent volume of distribution of L-5-methyl-THF. Previous studies that estimated the apparent volume of distribution of L-5-methyl-THF used large (nonphysiological) doses of unlabeled folates that are not found to any great extent in the circulatory system. The current study estimates the apparent volume of distribution at steady state in 16 healthy humans aged 18-65 y after an i.v. dose (440 nmol) of a stable isotope-labeled version of the naturally circulating plasma folate, L-5-methyl-THF. Blood was collected from 2 min to 2 h postinjection and plasma assayed by specific and sensitive liquid chromatography-tandem MS. The apparent volume of distribution for L-5-methyl-THF was 32.0 ± 11.6 L (mean ± SD; 392 ± 110 mL/kg bodyweight). There was a positive association with volunteer body weight (r = 0.64; P = 0.010), which allowed a simple linear equation to be developed relating apparent volume of distribution to body weight. This has important implications for predicting apparent absorption of labeled folates in future bioavailability studies.

Functional characterization of folic acid transport in the intestine of the laying hen using the everted intestinal sac model.

Absorption at the level of the intestine is likely a primary regulatory mechanism for the deposition of dietary supplemented folic acid into the chicken egg. Therefore, factors affecting the intestinal transport of folic acid in the laying hen may influence the level of egg folate concentrations. To this end, a series of experiments using intestinal everted sacs were conducted to characterize intestinal folic acid absorption processes in laying hens. Effects of naturally occurring folate derivatives (5-methyl and 10-formyltetrahydrofolate) as well as heme on folic acid absorption were also investigated. Folic acid absorption was measured based on the rate of uptake of (3)H-labeled folic acid in the everted sac from various segments of the small and large intestines. Folic acid concentration, incubation length, and pH condition were optimized before the performance of uptake experiments. The distribution profile of folic acid transport along the intestine was highest in the upper half of the small intestine. Maximum uptake rate (nmol·100 g tissue(-1)·min(-1)) was observed in the duodenum (20.6 ± 1.9) and jejunum (22.3 ± 2.0) and decreased significantly in the ileum (15.3 ± 1.1) and cecum (9.3 ± 0.9). Transport increased proportionately (P < 0.05) between 0.0001 and 0.1 µM folic acid. Above 0.1 µM, the slope of the regression line was not significantly different from zero (P < 0.137). Folic acid uptake in the jejunum showed a maximum rate of transport at pH 6.0, but was lowest at pH 7.5. The presence of 5-methyl and 10-formyltetrahydrofolate as well as heme impeded folic acid uptake, reducing intestinal folic acid absorption when added at concentrations ranging from 0 to 100 µM. Overall, these data indicated the presence of a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folate.

Basolateral efflux mediated by multidrug resistance-associated protein 3 (Mrp3/Abcc3) facilitates intestinal absorption of folates in mouse.

PURPOSE: This study investigated the role of an ABC transporter, Mrp3/Abcc3 in intestinal folate absorption. METHODS: Plasma concentrations of folic acid and leucovorin, given orally, were determined in wild-type and Mrp3 ( -/- ) mice. Mucosal-to-serosal transport was determined in the everted intestinal sacs. The plasma concentrations of endogenous 5-methyltetrahydrofolic acid, homocysteine and vitamin B(12), and mRNA levels of hepatic and intestinal folate metabolizing enzymes were compared between wild-type and Mrp3 ( -/- ) mice. RESULTS: C ( max ) and area-under plasma concentration-time curve of folic acid were 3.0- and 2.3-fold lower in Mrp3 ( -/- ) mice compared with wild-type mice, whereas the total body clearance was unchanged. Absorption of leucovorin was significantly delayed in Mrp3 ( -/- ) mice. Mucosal-to-serosal transport of folic acid and leucovorin was significantly decreased in the duodenum of Mrp3 ( -/- ) mice, where their PS ( serosal ) was decreased to 6.3 and 22% of that in wild-type mice, respectively. PS ( serosal ) of 5-methyltetrahydrofolic acid was moderately decreased in Mrp3 ( -/- ) mice. There was no obvious abnormality in folate homeostasis in Mrp3 ( -/- ) mice. CONCLUSIONS: Mrp3 accounts for the serosal efflux of folic acid and leucovorin, while it makes a moderate contribution to the serosal efflux of 5-methyltetrahydrofolic acid in mice. Mrp3 dysfunction does not disrupt folate homeostasis in mouse.

Comparison of (6 S)-5-methyltetrahydrofolic acid v. folic acid as the reference folate in longer-term human dietary intervention studies assessing the relative bioavailability of natural food folates: comparative changes in folate status following a 16-week placebo-controlled study in healthy adults.

Folic acid (pteroylmonoglutamic acid) has historically been used as the reference folate in human intervention studies assessing the relative bioavailability of dietary folate. Recent studies using labelled folates indicated different plasma response kinetics to folic acid than to natural (food) folates, thus obviously precluding its use in single-dose experiments. Since differences in tissue distribution and site of biotransformation were hypothesised, the question is whether folic acid remains suitable as a reference folate for longer-term intervention studies, where the relative bioavailability of natural (food) folate is assessed based on changes in folate status. Healthy adults aged 18-65 years (n 163) completed a 16-week placebo-controlled intervention study in which the relative bioavailability of increased folate intake (453 nmol/d) from folate-rich foods was assessed by comparing changes in plasma and erythrocyte folate concentration with changes induced by an equal reference dose of supplemental (6S)-5-methyltetrahydrofolic acid or folic acid. The relative increase in plasma folate concentration in the food group was 31 % when compared with that induced by folic acid, but 39 % when compared with (6S)-5-methyltetrahydrofolic acid. The relative increase in erythrocyte folate concentration in the food group when compared with that induced by folic acid was 43 %, and 40 % when compared with (6S)-5-methyltetrahydrofolic acid. When recent published observations were additionally taken into account it was concluded that, in principle, folic acid should not be used as the reference folate when attempting to estimate relative natural (food) folate bioavailability in longer-term human intervention studies. Using (6S)-5-methyltetrahydrofolic acid as the reference folate would avoid future results' validity being questioned.

Pharmacokinetics of Sodium and Calcium Salts of (6S)-5-Methyltetrahydrofolic Acid Compared to Folic Acid and Indirect Comparison of the Two Salts.

(6S)-5-Methyltetrahydrofolic acid ((6S)-5-Methyl-THF) salts and folic acid may differ in their abilities to raise plasma (6S)-5-Methyl-THF levels. We compared the area under the curve (AUC), Cmax, and Tmax of plasma (6S)-5-Methyl-THF after intakes of (6S)-5-Methyl-THF-Na salt (Arcofolin®) and folic acid. Moreover, we compared the AUCs after intakes of (6S)-5-Methyl-THF-Na and the calcium salt, (6S)-5-Methyl-THF-Ca, that were tested against folic acid in two independent studies. The study was randomized, double blind, and cross over. Twenty-four adults (12 men and 12 women) received a single oral dose of 436 µg (6S)-5-Methyl-THF-Na and an equimolar dose of folic acid (400 µg) on two kinetic days with two weeks washout period in between. The plasma concentrations of (6S)-5-Methyl-THF were measured at 9 time points between 0 and 8 h. We found that the AUC0-8 h of plasma (6S)-5-Methyl-THF (mean (SD) = 126.0 (33.6) vs. 56.0 (25.3) nmol/L*h) and Cmax (36.8 (10.8) vs. 11.1 (4.1) nmol/L) were higher after administration of (6S)-5-Methyl-THF-Na than after the administration of folic acid (p < 0.001 for both). These differences were present in men and women. Only administration of folic acid resulted in a transient increase in plasma unmetabolized folic acid (2.5 (2.0) nmol/L after 0.5 h and 4.7 (2.9) nmol/L after 1 h). Intake of (6S)-5-Methyl-THF-Na was safe. The ratios of the AUC0-8 h for (6S)-5-Methyl-THF-Na and (6S)-5-Methyl-THF-Ca to the corresponding folic acid reference group and the delta of these AUC0-8 h did not differ between the studies. In conclusion, a single oral dose of (6S)-5-Methyl-THF-Na caused higher AUC0-8 h and Cmax of plasma (6S)-5-Methyl-THF compared to folic acid. The Na- and Ca- salts of (6S)-5-Methyl-THF are not likely to differ in their pharmacokinetics. Further studies may investigate whether supplementation of the compounds for a longer time will lead to differences in circulating or intracellular/tissue folate concentrations.

[Simultaneous determination of leucovorin and 5-methyl-tetrahydrofolate diastereoisomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry coupled with ultrafiltration centrifugation-based pretreatment and its application to a pharmacokinetic study].

A simple, sensitive, and stable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous determination of leucovorin and 5-methyltetrahydrofolate diastereomers in human plasma using methotrexate as the internal standard. The analytes and the internal standard were extracted from plasma samples by simple ultrafiltration centrifugation-based extraction. The separation was achieved on a chiral HSA column (150 mm×4 mm, 5 µm) using mobile phases containing 10 mmol pH 8.0 ammonium acetate and acetonitrile in gradient mode. The method showed good linearities in the ranges of 25-5000 µg/L and 12.5-3000 µg/L for leucovorin and 5-methyltetrahydrofolate diastereoisomers, respectively. The method was fully validated with respect to sensitivity, precision, accuracy, matrix effect, extraction recovery, and stability of analytes under various conditions. The method was successfully applied to a pharmacokinetic study of 125 mg/m2 6R,S-leucovorin and 62.5 mg/m2 6S-leucovorin. The results showed that the maximum observed concentrations (Cmax) of 6S-leucovorin and L-5-methyltetrahydrofolate were (3137.917±408.837) and (1679.633±244.132) µg/L, respectively, and the areas under the curve from the time of dosing to the last measurable concentration (AUC0-t) were (7504.883±1185.101) and (14001.214±2868.949) µg/L in the 125 mg/m2 6R,S-leucovorin dose group. The Cmax values of 6S-leucovorin and L-5-methyltetrahydrofolate were (3187.917±387.298) and (1739.204±224.755) µg/L, respectively, and AUC0-t values were (7426.664±854.825) and (14884.331±1843.353) µg/L in the 62.5 mg/m2 6S-leucovorin dose group. There were no significant diffe-rences in the main pharmacokinetic parameters between the two dose groups, and the pharmacokinetic characteristics as well as the rate and extent of absorption were consistent. This method can provide technical support for future bioequivalence studies of sodium leucovorin.

Folic acid and its metabolite codetermination for pharmacokinetics with circadian rhythms and evaluation of oral bioavailability.

OBJECTIVES: Pharmacokinetics of vitamins is still a challenge. In this study, folic acid (FA) was used as a model drug and aimed at investigating a reliable method for its detailed pharmacokinetic evaluations. METHODS: An high-performance liquid chromatography-tandem mass spectrometry method was developed and performed to determinate the FA and 5-methyltetrahydrofolic acid (5-methylTHF) simultaneously, which was applied to characterize the circadian rhythms as well as the pharmacokinetics of different preparations. KEY FINDINGS: The plasma concentration of 5-methylTHF in fasted state was twofold higher than that in fed state. The circadian rhythms were studied before the pharmacokinetics and revealed that free FA was almost undetected in blank plasma, while 5-methylTHF had a slight decrement at 12:00. Hence, the pharmacokinetics of FA was conducted and showed that the administration of FA solution resulted in enhancing bioavailability of 5-methylTHF comparing with FA raw material suspension, whereas the free FA level in plasma was similar. The mechanism could be that FA was rapidly metabolized to 5-methylTHF in intestinal epithelial cell after absorption, which revealed that intestinal metabolism would affect its bioavailability. CONCLUSION: A suitable method was established considering the baseline level, circadian rhythms and intestinal metabolism to investigate the pharmacokinetics of FA for guiding the further research of vitamins.

Quantitation of 5-methyltetraydrofolic acid in plasma for determination of folate status and clinical studies by stable isotope dilution assays.

Folates play a key role in the prevention of neural tube defects in newborns. Thus, it is important to reliably determine the bioavailability of folates from various foods. Accurate analytical methods are essential for quantifying blood-folates, especially in human studies. Here, we present the development and validation of a sensitive method using stable isotope dilution liquid chromatography coupled with mass spectrometry for determining various folates in plasma. Moreover, this study reports the applicability of the developed method to a human pilot study using strawberries as a test food. Validation of the assay revealed the precision, sensitivity, and accuracy of the method in determining the predominant 5-methyltetrahydrofolate in plasma. This method was also applicable for the screening of individual folate status using finger prick blood and for monitoring the post-absorptive plasma-concentration curve. Moreover, the human study revealed a high recovery of strawberry folates with a calculated relative bioavailability of 96.2%. Thus, the developed method enables prospective bioavailability studies. This work also confirmed, via human studies, that strawberries are a rich and natural source of folates that are available for human metabolism.

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model.

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor alpha (FRalpha) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro. Two distinct processes are apparent; one that is FRalpha dependent and one that is independent of the receptor. FRalpha binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRalpha independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRalpha -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRalpha remains non-metabolized, supporting the hypothesis that FRalpha may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRalpha-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRalpha.

L-ascorbic acid improves the serum folate response to an oral dose of [6S]-5-methyltetrahydrofolic acid in healthy men.

OBJECTIVE: To investigate the effect of simultaneous administration of [6S]-5-methyltetrahydrofolic acid ([6S]-5-CH(3)H(4)PteGlu) with L-ascorbic acid (L-AA) on serum folate concentrations in healthy male subjects. SUBJECTS AND METHODS: A total of nine healthy male volunteers were recruited. Serum folate concentrations were measured before and up to 8 h after administration of each treatment (1) placebo, (2) 343 microg [6S]-5-CH(3)H(4)PteGlu), (3) 343 microg [6S]-5-CH(3)H(4)PteGlu) with 289.4 mg L-AA and (4) 343 microg [6S]-5-CH(3)H(4)PteGlu) with 973.8 mg L-AA (n=10 samples per treatment). RESULTS: Serum folate concentrations significantly increased compared with baseline values, starting from 30 min after [6S]-5-CH(3)H(4)PteGlu administration and remained significantly higher than baseline values during the first 6 h for treatments 3 and 4, and during the first 4 h for treatment 2. Maximal serum folate responses were observed between 0.5 and 1.5 h after [6S]-5-CH(3)H(4)PteGlu consumption and significantly differed between treatments 2 and 4 (P<0.05). When [6S]-5-CH(3)H(4)PteGlu was concurrently administered with 289.4 or 973.8 mg L-AA, the total serum folate response, calculated as the area under the curve (AUC), was significantly improved (46.5+/-4.0 and 53.0+/-4.0 vs 34.3+/-3.8 h nmol/l, P<0.05). No significant difference in AUC was found between the 289.4 and the 973.8 mg L-AA treatments. CONCLUSIONS: Administration of a physiological dose of [6S]-5-CH(3)H(4)PteGlu with L-AA significantly improved the measured serum folate response in folate saturated healthy men.

The pharmacokinetic advantage of 5-methyltetrahydrofolate for minimization of the risk for birth defects.

Despite efforts to increase folic acid (FA) intake, even within countries mandating FA fortification, there remain pregnant women with folate levels inadequate to minimize congenital disorders (e.g., of the neural tube, heart, and lip/palate). The pharmacokinetics of FA and [6S]-5-methyltetrahydrofolate (5-MTHF) were examined to find a reliable and minimal dose for rapidly rescuing folate status prior to critical periods of embryonic development. Serum total folate increased much more rapidly over the first four days in insufficient women given 7.5 mg doses of 5-MTHF than the same regimen of FA (P for trend <0.0001). Nearly all women given 7.5 mg 5-MTHF (every 12 hours, five doses total) almost immediately reached 50 nM serum total folate. Moreover, this level could be maintained by subsequent administration of 0.4 mg/d of folic acid. Thus, 5-MTHF enables repletion of folate stores more quickly and uniformly than FA and without exposure to unmetabolized FA.

Down-regulation of placental folate transporters in intrauterine growth restriction.

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th-75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (-34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (-38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation.

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.

Dose-dependent effects of (anti)folate preinjection on 99mTc-radiofolate uptake in tumors and kidneys.

INTRODUCTION: The folate receptor (FR) is frequently overexpressed in tumors and can be targeted with folate-based (radio)pharmaceuticals. However, significant accumulation of radiofolates in FR-positive kidneys represents a drawback. We have shown that preadministration of the antifolate pemetrexed (PMX) significantly improved the tumor-to-kidney ratio of radiofolates in mice. The aim of this study was to investigate the dose dependence of these effects and whether the same results could be achieved with folic acid (FA) or 5-methyl-tetrahydrofolate (5-Me-THF). METHODS: Biodistribution was assessed 4 h postinjection of the organometallic (99m)Tc-picolylamine monoacetic acid folate in nude mice bearing FR-positive KB tumor xenografts. PMX (50-400 microg/mouse) was injected 1 h previous to radioactivity. The effects of FA and 5-Me-THF (0.5-50 microg/mouse) were investigated likewise. Tissues and organs were collected and counted for radioactivity and the values tabulated as percentage of injected dose per gram tissue (% ID/g). RESULTS: PMX administration reduced renal retention (<1.6% ID/g vs. control: >10% ID/g), while the tumor uptake (average 1.35%+/-0.40% ID/g vs. control: 1.79%+/-0.49% ID/g) was only slightly affected independent of the PMX dose. Replacement of PMX by FA or 5-Me-THF (50 microg/mouse) resulted in a significant renal blockade (<0.1% ID/g) but at the same time in an undesired reduction of tumor uptake (<0.2% ID/g). CONCLUSIONS: Selective reduction of radiofolate uptake in kidneys under retention of high tumor accumulation could be achieved in combination with PMX over a broad dose range but not with FA or 5-Me-THF.

Simulation of Food Folate Digestion and Bioavailability of an Oxidation Product of 5-Methyltetrahydrofolate.

Generating bioavailability data from in vivo studies is time-consuming and expensive. In vitro simulation can help to investigate factors influencing bioavailability or facilitate quantifying the impact of such factors. For folates, an efficient deconjugation of polyglutamates to the corresponding monoglutamates is crucial for bioavailability and highly dependent on the food matrix. Therefore, the bioaccessibility of folates of different foodstuffs was examined using a simulated digestion model with respect to folate stability and the efficiency of deconjugation. For realistic simulated deconjugation, porcine brush border membrane was used during the phase of the simulated digestion in the small intestine. For a better understanding of folate behaviour during digestion, single folate monoglutamates were also investigated with this in vitro digestion model. The results for bioaccessibility were compared with data from a human bioavailability study. They support the idea that both stability and deconjugation have an influence on bioaccessibility and thus on bioavailability. Tetrahydrofolate is probably lost completely or at least to a high extent and the stability of 5-methyltetrahydrofolate depends on the food matrix. Additionally, 5-methyltetrahydrofolate can be oxidised to a pyrazino-s-triazine (MeFox), whose absorption in the human intestinal tract was shown tentatively.

Red blood cell folate concentrations increase more after supplementation with [6S]-5-methyltetrahydrofolate than with folic acid in women of childbearing age.

BACKGROUND: For the primary prevention of neural tube defects (NTDs), public health authorities recommend women of childbearing age to take 400 mug folic acid/d 4 wk before conception and during the first trimester. The biologically active derivate [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF) could be an alternative to folic acid. OBJECTIVE: We investigated the effect of supplementation with [6S]-5-MTHF compared with that of folic acid on red blood cell folate concentration, an indicator of folate status. DESIGN: The study was designed as a double-blind, randomized, placebo-controlled intervention trial. Healthy women (n = 144) aged 19-33 y received 400 microg folic acid, the equimolar amount of [6S]-5-MTHF (416 microg), 208 microg [6S]-5-MTHF, or placebo as a daily supplement for 24 wk. Red blood cell and plasma folate concentrations were measured at baseline and at 4-wk intervals. RESULTS: The increase in red blood cell folate over time was significantly higher in the group receiving 416 microg [6S]-5-MTHF/d than in the groups receiving 400 microg folic acid/d or 208 microg [6S]-5-MTHF/d (P < 0.001). No plateau was reached in red blood cell folate concentration in the 3 treatment groups during 24 wk of intervention; however, plasma folate plateaued after 12 wk. CONCLUSIONS: We showed that administration of [6S]-5-MTHF is more effective than is folic acid supplementation at improving folate status. In addition, the study indicates that the recommended period for preconceptional folic acid supplementation should be extended to >4 wk for maximal prevention of NTDs based on folate concentrations. [6S]-5-MTHF might be an efficient and safe alternative to folic acid.

In vivo and in vitro metabolism of 5-deazaacyclotetrahydrofolate, an acyclic tetrahydrofolate analogue.

5-Deazaacyclotetrahydrofolate is a cytotoxic tetrahydrofolate analogue which inhibits glycinamide ribonucleotide transformylase (Kelley et al., J. Med. Chem., 33: 561-567, 1990). Cultured mouse L-cells and human MCF-7 and MOLT-4 cells concentrated the drug several hundred-fold after 24 h of continuous exposure to a cytotoxic level (100-200 nM) of radiolabeled drug. High performance liquid chromatography analysis revealed that each cell type metabolized greater than or equal to 80% of the internalized drug to polyglutamated forms, which are more potent glycinamide ribonucleotide transformylase inhibitors. In L-cells, 45% of the polyglutamated metabolites were also N-formylated. The pharmacokinetics and distribution of [14C]-deazaacyclotetrahydrofolate were studied in C57BL/6 male mice. Its plasma half-life was 2.15 h. Radiolabel was concentrated to well above plasma level in the kidney, pancreas, and liver. Metabolism was examined in tumor-bearing and in normal mice. Twenty-four h after a single i.p. injection (50 mg/kg), drug equivalents were 0.6 nmol/g (83% polyglutamated) in colon-38 adenocarcinoma carried s.c., 2.4 nmol/g (100% polyglutamated) in ascitic P388 cells, and 3.7 nmol/g (76% polyglutamated and approximately 20% formylated) in mouse liver. Elimination was mostly in the urine as unmetabolized drug. Feces contained 5-deazaacyclotetrahydropteroate (parent compound less glutamate). In conclusion, 5-deazaacyclotetrahydrofolate was shown to be concentrated to well above the extracellular level and metabolized to more active polyglutamated forms by transformed cells grown in culture and in mice.