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BACKGROUND: Adequate folate status supports endothelial structure and function. Folic acid (FA), an oxidized synthetic folate, which is present in the plasma of patients consuming fortified food or FA supplements, may impair cellular uptake of physiological, reduced folates. We studied the effect of FA on uptake of the dominant circulatory folate, 5-methyltetrahydrofolate (5MTHF) in endothelial cells. METHODS AND RESULTS: For short-term effects of FA, primary human umbilical vein endothelial cells (HUVECs) were maintained in growth medium containing 200 nM 5MTHF and preincubated with 20 nM FA 10 minutes before the 5MTHF uptake assessment. For long-term effects, HUVECs were cultured for 3 passages in growth medium containing either 200 nM 5MTHF, or a combination of 100 nM 5MTHF and 100 nM FA. 5MTHF uptake was assessed after exposing cells to 200 nM [C5]-5MTHF, after which intracellular [C5]-5MTHF was quantified using liquid chromatography/tandem mass spectrometry. Acute FA exposure caused a 57% reduction in 5MTHF uptake compared with control conditions (51 ± 12 vs. 22 ± 7 fmol·min·mg protein; P = 0.01). Long-term exposure to FA reduced 5MTHF uptake by 41% (51 ± 12 vs. 30 ± 11 fmol·min·mg protein; P = 0.05) and reduced total cellular 5MTHF levels by 47 ± 21% in HUVEC (P = 0.02). CONCLUSION: Unmetabolized FA, which appears in the plasma after consumption of fortified food or FA supplements, may impair uptake of 5MTHF, the dominant bioactive form of folate, in HUVEC.

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Deficiency of folate in heavy-drinking alcoholic populations can occur partly because of an increased urinary folate excretion. Ethanol directly reduces the reabsorption of folate in the renal proximal tubule (PT) by acting on either of 2 folate transport proteins, the reduced folate carrier (RFC) and the folate receptor (FR). This study was designed to determine the effects of ethanol on the transport of folate by PT cells and to examine the effects of ethanol on RFC and the FR protein expression. Normal human PT (HPT) cells were cultured on membrane inserts to study intracellular transport of 5-methyltetrahydrofolate from the apical or basolateral direction in the presence of ethanol [11-109 mmol/L (50-500 mg/dL)]. The long-term effect of ethanol on the renal folate transport protein content was determined by western blot in treated HPT cells and in vivo in rats pair-fed control diets or ethanol-containing liquid diets. A 1-h treatment of HPT cells with ethanol (> or = 65 mmol/L) reduced the apically directed transport of folate by 20-25% without affecting the basolateral transport. A 5-d exposure of HPT cells to ethanol dose-dependently increased the content of both the FR and RFC proteins, with a greater effect on the RFC. Similarly, a 14-d exposure of rats to ethanol increased the in vivo expression of both the RFC and FR. These studies demonstrate that ethanol decreases the reabsorptive transport of folate by renal PT cells, which would increase urinary folate excretion. In contrast, subchronic exposure of PT cells, both in vivo and in vitro, to folate-depleting concentrations of ethanol leads to an upregulation of the 2 folate transport proteins. The increase in folate transporters partly counteracts the inhibitory effects of ethanol on folate transport activity, which explains the lower magnitude of ethanol's effect on transport with subchronic exposure compared with that with acute exposure.

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Folylpoly-gamma-glutamate synthetase (FPGS) catalyzes the addition of several equivalents of glutamic acid to the gamma-carboxyl group in the side chain of folate cofactors and analogs. Folylpoly-gamma-glutamate synthetase has three functions in folate homeostasis in mammals: polyglutamation prevents efflux of folate cofactors from the cell, it increases the binding of folate cofactors to some of the enzymes of folate interconversion and biosynthesis, and it appears to allow the accumulation of folates in the mitochondria that are required for glycine synthesis. The efficient substrate activity of the newer generations of tetrahydrofolate analogs results in levels of intracellular accumulation of cytotoxic drug in any cell expressing FPGS in which the enzyme activity is not suppressed by feedback, and the binding of folate inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase is substantially increased by polyglutamation. Resistance to these drugs appears to be most frequently due to mutations that change the level of polyglutamation of parent compound, a clear indication of the centrality of the process to the cytotoxicity of these drugs. Folylpoly-gamma-glutamate synthetase is widely expressed in human tumors and is tightly linked either to proliferation or to a lack of differentiation. The cytotoxicity of both thymidylate synthase and purine inhibitors requires continued inhibition of target for greater than one generation time, so that the integrative function of FPGS adds considerably to the efficiency of folate antimetabolites.

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Inhibition of clonogenic potential by the glycinamideribonucleosyl transformylase inhibitor 5,10-dideazatetrahydrofolic acid (DDATHF, Lometrexol) was evaluated in vitro in a human ovarian carcinoma cell line, SW626. Drug-induced inhibition of clonogenic potential is a function of the dose and time of exposure and is independent of the formation of DNA single-strand breaks or de novo synthesis of protein. Simultaneous treatment with 100 microM hypoxanthine completely prevented the inhibition of clonogenic potential caused by 0.5 microM DDATHF. DDATHF blocked cells in the early-middle S-phases of the cell cycle, and there was a corresponding marked reduction in the rate of DNA synthesis after drug withdrawal. The cytotoxic potential of DDATHF was modulated by the folic acid concentration present in the medium. In a medium containing 0.22 microM folic acid, DDATHF cytotoxicity was at least 100 times that in a regular medium containing 2.22 microM folic acid, levels which, however, are about 100 times those found in human plasma. DDATHF cytotoxicity differed moderately when folic acid concentrations varied between 0.22 and 0 microM, suggesting that folic acid does not necessarily antagonise DDATHF anti-tumour activity. Folinic acid at a concentration as low as 0.1 microM can completely rescue cells when given simultaneously with 0.5 microM DDATHF. When folinic acid was given 24 h after DDATHF, a reversal of cytotoxicity was observed at 0.5 and 1 microM, but to a much lesser extent than simultaneous treatment. When folinic acid was added after 48 or 72 h of DDATHF washout, even at a high concentration and for a long time, no reduction in DDATHF cytotoxicity was found. In conclusion, the study highlights the modulation of DDATHF cytotoxicity by folic acid or by folinic acid and provides further rationale for in vivo clinical investigation with these combinations.

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Feeding rats a semiliquid ethanol diet for a period of four weeks produced a hepatic accumulation of the methylating agent N5-methyltetrahydrofolate (N5CH3THF). When the ethanol-containing diet was supplemented with 0.5% betaine, an agent known to promote the generation of methionine and S-adenosylmethionine (SAM) in ethanol-fed animals, the accumulation of N5CH3THF was prevented. One index that the methyl folate trap exists is the hepatic accumulation of N5CH3THF, and a second index is that the N5CH3THF accumulation can be relieved by methionine administration. Since ethanol is shown to produce N5CH3THF accumulation in this study, and since betaine (a generator of methionine and SAM) acts to eliminate this accumulation, it is suggestive that ethanol can contribute to the impairing hepatic condition referred to as the "methyl folate trap."

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We examined 5-methyltetrahydrofolate transport across the basolateral membrane (BLM) of rat liver using isolated membrane vesicles. Uptake of 5-methyltetrahydrofolate by BLM vesicles (BLMV) was found by osmolarity and initial uptake studies to be mostly the result of transport of the substrate into an active, intravesicular space with some binding (approximately 25%) to membrane surfaces. Uptake was similar in the presence of an inwardly directed Na+ or K+ gradient, indicating that transport is not dependent on Na+. Uptake of 5-methyltetrahydrofolate was linear for the first 30 s and was more rapid when an initial pH gradient was imposed across the vesicular membrane [extravesicular pH (pHo) = 5.0, intravesicular pH (pHi) = 7.5]. Under pH gradient conditions, uptake at 3-5 min was about twofold higher than at equilibrium (60 min). The initial rate of uptake at pHo = pHi = 5.0 was more rapid than at pHo = pHi = 7.5, but in neither case was uptake above equilibrium observed. Uptake under pH gradient conditions and at pH 5.0 (no pH gradient) was saturable (apparent Michaelis constants of 0.58 and 0.36 microM, respectively; maximum uptake rates of 1.25 and 0.79 pmol.10 s-1.mg protein-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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Developmental aspects of the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4-PteGlu) were studied in suckling (14-day-old), weanling (22-day-old), and adult (90-day-old) rats by use of the intestinal everted-sac technique. Mucosal-to-serosal transport of 0.5 microM 5-CH3H4PteGlu was linear with time for 40-min incubation and occurred at a rate of 0.035, 0.032, and 0.010 nmol X g initial tissue wet wt-1 X min-1 for suckling, weanling, and adult rats, respectively. The transport of 5-CH3H4PteGlu in all age groups was pH dependent (maximal at pH 6) and was higher in the jejunum than in the ileum. In all age groups the transport of 5-CH3H4PteGlu occurred by an active carrier-mediated system. The system was saturable; energy, temperature, and Na dependent; inhibited by structural analogues; and capable of accumulating the substrate against a concentration gradient. Kinetic parameters of the transport process, however, showed some difference. A progressive decrease in Vmax was observed from suckling to weanling to adult rats (5.1, 3.7, and 0.8 nmol X g initial tissue wet wt-1 X 30 min-1, respectively), while apparent Kt was similar (2.2, 1.73, and 1.79 microM, respectively). This study demonstrates that the transport system of 5-CH3H4PteGlu in the rat is fully developed at the suckling age. The results also suggest that the activity and/or the number, but not the affinity, of the transport carriers decrease with maturation.

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