Urinary Folate Excretion Research Articles

Urinary Excretion of Deuterium-Labeled Folate and the Metabolite p-Aminobenzoylglutamate in Humans1–4

Stable isotopic methods were employed to determine the proportion of ingested folate excreted as the metabolite p-aminobenzoylglutamate (pABG, free and N-acetyl) in urine. Adult male subjects (n = 7) were maintained on a 4.54 mumol/d (2 mg/d) folate saturation regimen. After 7 d, subjects were given a mixed oral dose containing 0.67 mumol (300 micrograms) each of bideuterofolic acid (d2) and tetradeutero-folic acid (d4). Urine was collected for the following 48 h and analyzed for folate and pABG. The extent of deuterium labeling of urinary folate and pABG was determined by gas chromatography-mass spectrometry. Urinary total pABG excretion increased less than twofold as a result of the folate saturation, whereas urinary folate increased over 10-fold. Urinary d2 and d4 folates each contained 14-15% of the respective oral doses of labeled compounds, whereas urinary d2 and d4 pABG comprised only 0.98-1.15% of the labeled doses. Molar ratios (d2/d4) of excreted folate and pABG indicated that there was no in vivo isotopic discrimination between the labeled folates. Urinary pABG accounted for 5.1 +/- 0.6% of total ingested folate, whereas labeled pABG was about 6.7-7.6% of the excretion of labeled compounds (i.e., labeled folate + pABG). This study indicated that pABG is not a major excretory product during folate supplementation, but its relative importance may increase in conditions of reduced folate nutriture.

Urinary excretion of B-group vitamins reflects the nutritional status of B-group vitamins in rats.

We have reported previously that the urinary excretion of B-group vitamins reflects recent dietary intakes of these vitamins. We also proposed reference values for the urinary levels of B-group vitamins for human subjects, and used these for evaluating human nutritional status. However, the question arises as to whether the urinary excretion of B-group vitamins in animals or human subjects decreases immediately before they become B-group vitamin insufficient or when fed a diet low in vitamins. In the present study, rats were fed a vitamin-free diet for 5d, and changes in the levels of B-group vitamins in urine and blood were monitored. Urinary excretion of vitamin B1, vitamin B2, 4-pyridoxic acid (a catabolite of vitamin B6), pantothenic acid, folate and biotin steeply decreased, and all of the values reached zero within 1-2d. With respect to blood, the concentrations of only three of the eight B-group vitamins (vitamin B1, pyridoxal phosphate and biotin) decreased to 15% (P<0·0001), 7% (P<0·0001) and 2% (P<0·0001) on day 5, respectively, compared with the values at the beginning of the experiment. The decrease was more rapid and the changes were greater in the urine samples than in the blood samples. The present data complement our previous proposal that the urinary excretion of B-group vitamins reflects the nutritional status of these vitamins.

Folate Deficiency Is Associated With Oxidative Stress, Increased Blood Pressure, and Insulin Resistance in Spontaneously Hypertensive Rats

The role of folate deficiency and associated hyperhomocysteinemia in the pathogenesis of metabolic syndrome is not fully established. In the current study, we analyzed the role of folate deficiency in pathogenesis of the metabolic syndrome in the spontaneously hypertensive rat (SHR). Metabolic and hemodynamic traits were assessed in SHR/Ola rats fed either folate-deficient or control diet for 4 weeks starting at the age of 3 months. Compared to SHRs fed a folate-replete diet, SHRs fed a folate-deficient diet showed significantly reduced serum folate (104 ± 5 vs. 11 ± 1 nmol/L, P < 0.0005) and urinary folate excretion (4.3 ± 0.6 vs. 1.2 ± 0.1 nmol/16 h, P < 0.0005) together with a near 3-fold increase in plasma total homocysteine concentration (4.5 ± 0.1 vs 13.1 ± 0.7 μmol/L, P < 0.0005), ectopic fat accumulation in liver, and impaired glucose tolerance. Folate deficiency also increased systolic blood pressure by approximately 15 mm Hg (P < 0.01). In addition, the low-folate diet was accompanied by significantly reduced activity of antioxidant enzymes and increased concentrations of lipoperoxidation products in liver, renal cortex, and heart. These findings demonstrate that the SHR model is susceptible to the adverse metabolic and hemodynamic effects of low dietary intake of folate. The results are consistent with the hypothesis that folate deficiency can promote oxidative stress and multiple features of the metabolic syndrome that are associated with increased risk for diabetes and cardiovascular disease.

Folate, alcohol, and liver disease

Alcoholic liver disease (ALD) is typically associated with folate deficiency, which is the result of reduced dietary folate intake, intestinal malabsorption, reduced liver uptake and storage, and increased urinary folate excretion. Folate deficiency favors the progression of liver disease through mechanisms that include its effects on methionine metabolism with consequences for DNA synthesis and stability and the epigenetic regulation of gene expression involved in pathways of liver injury. This paper reviews the pathogenesis of ALD with particular focus on ethanol-induced alterations in methionine metabolism, which may act in synergy with folate deficiency to decrease antioxidant defense as well as DNA stability while regulating epigenetic mechanisms of relevant gene expressions. We also review the current evidence available on potential treatments of ALD based on correcting abnormalities in methionine metabolism and the methylation regulation of relevant gene expressions.

Folate-status response to a controlled folate intake in nonpregnant, pregnant, and lactating women

Folate dose-response studies in women of childbearing age who consumed a folic acid (FA)-containing multivitamin in the era of FA fortification are lacking. We sought to investigate folate-status response to a known folate dose comprising an FA-containing prenatal supplement (750 μg/d) plus natural food folate (400 μg/d) in third-trimester pregnant women, lactating women 5-15 wk postpartum, and nonpregnant women. Pregnant (n = 26), lactating (n = 28), and nonpregnant (n = 21) women consumed the study folate dose under controlled intake conditions for 10-12 wk. Blood, urine, and breast milk were collected at baseline, study midpoint, and study end. Study-end serum total folate concentrations averaged ~30 ng/mL and did not differ by physiologic group (P = 0.876). Study-end urinary folate excretion represented ~9-43% of total folate intake and ranged from 100 to 500 μg/d. Third-trimester pregnant women excreted less urinary folate than did lactating (P = 0.075) and nonpregnant (P < 0.001) women. Lactating women excreted less (P < 0.001) urinary FA than did nonpregnant women. Breast-milk total folate concentrations remained constant (P = 0.244; 61.8 ng/mL at study end), whereas breast-milk FA concentrations increased (P = 0.003) to 24.1 ng/mL at study end. The consumption of the study folate dose yielded a supranutritional folate status regardless of the physiologic state. Based on urinary folate excretion, folate use was greatest to least: pregnant > lactating > nonpregnant women. Breast-milk folate species were responsive to maternal folate intake, and FA made up ~40% of breast-milk total folate at study end. These findings warrant revisiting prenatal supplement FA formulation in populations exposed to FA-fortification programs.

Consumption of a folic acid‐containing prenatal vitamin yields supra‐physiologic folate status in pregnant and non‐pregnant women

With the goal of reducing neural tube defect incidence, the CDC recommends that women of childbearing age consume 400mcg/d folic acid (FA) from fortified foods and/or dietary supplements. About 40% of women use supplements containing FA; prenatal vitamins often contain ≥600mcg FA. Consumption of a prenatal vitamin plus fortified foods, which supply 200–250mcg/d FA, may result in FA intakes that exceed metabolic requirement and near or exceed the 1000mcg/d UL. Study objective measurement of folate status markers in response to a mixed diet combined with daily FA containing prenatal vitamin in nonpregnant and third trimester pregnant women. Results Total serum and RBC folates were >20 and >400ng/mL, respectively, in both the nonpregnant and pregnant groups. Mean urinary folate excretion was 460 and 250mcg/d in nonpregnant and pregnant women, respectively. Preliminary measurements characterizing folate species found un-metabolized FA in urine. Conclusion The consumption of a prenatal vitamin containing 600mcg FA in addition to food folate from a mixed diet (400mcg/d DFE) results in supra-physiologic folate status in nonpregnant and third trimester pregnant women as evidenced by reported folate biomarker concentrations. These findings warrant revisiting prenatal vitamin FA formulation. Funded in part by USDA, ENC and the Beef Checkoff; AAW is supported by an ENC Dissertation Fellowship.

Urinary excretion of vitamin B1, B2, B6, niacin, pantothenic acid, folate, and vitamin C correlates with dietary intakes of free-living elderly, female Japanese

We hypothesized that 24-hour urinary excretion of water-soluble vitamins might correlate with their intake in free-living Japanese elderly females aged 70 to 84 years. We performed a cross-sectional study composed of 37 healthy, elderly, Japanese females living freely. All foods and the corresponding weights consumed for 4 consecutive days were recorded accurately. A 24-hour urine sample was collected on the fourth day, and the urinary content of water-soluble vitamins was measured. The urinary levels of all vitamins, except for B(12) (r = 0.01; P = .936), were correlated positively with the mean intake over the recent 4 days (vitamin B1: r = 0.62; P < .001; vitamin B2: r = 0.57; P < .001; vitamin B6: r = 0.37; P < .005; niacin: r = 0.54; P < .001; niacin equivalents: r = 0.54; P < .001; pantothenic acid: r = 0.59; P < .001; folate: r = 0.55; P = .001; and vitamin C: r = 0.53; P < .001). Mean estimated intakes of water-soluble vitamins calculated using urinary concentrations and recovery rates showed 96% to 107% of their 3-day mean intake, except for vitamin B12 (65%). We conclude that urinary levels of water-soluble vitamins, except for B12, reflected their recent intake in free-living Japanese elderly females and could be used as a measure of their intake during the previous few days both for group means and for individual rankings within a group.

Acute Ethanol Exposure Inhibits Renal Folate Transport, but Repeated Exposure Upregulates Folate Transport Proteins in Rats and Human Cells

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 (≥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.

A human model to determine folate bioavailability from food: a pilot study for evaluation

Background: Knowledge about folate bioavailability from food is essential for the estimation of dietary requirements. Yet, there is a lack of data obtained from validated human studies performed with physiological folate doses. Objective: In this pilot study, a new model for the determination of folate absorption is developed and validated. Design: Under strictly standardized procedures, two healthy ileostomy volunteers consumed single portions of test foods or an oral dose of a pharmaceutical folate preparation of the natural folate diastereomer (6S)-5- methyltetrahydrofolate. Relative folate absorption from oral doses versus an intramuscular injection of the same pharmaceutical preparation was determined using postdose plasma folate concentration curves. Nonabsorbed folate was estimated by postdose folate excretion into stomal effluent. Results: Estimated by plasma areas under the curve, relative folate absorption ranged from 47 to 67% for oral doses from the te st foods strawberries and broccoli and the pharmaceutical (6S)-5-methyltetrahydrofolate preparation. During 10 h postdose, 19-44% of the dietary folate was excreted with the stomal effluent. Varying gut passage times were observed for different food matrices by determining ileostomal folate excretion in 2 h intervals. Around 90% of the folate from the oral doses was recovered in the collected body fluids, plasma and stomal effluent, by 10 h postdose, independent of the size of the administered folate doses of 200 or 400 mg (0.4 or 0.9 mmol). Conclusion: The results imply that this model provides a suitable tool to estimate folate bioavailability from foods. Keywords: Folate absorption; folate in ileostomal effluent; ileostomists; plasma folate kinetics; urinary folate excretion

A human model to determine folate bioavailability from food: a pilot study for evaluation

Background: Knowledge about folate bioavailability from food is essential for the estimation of dietary requirements. Yet, there is a lack of data obtained from validated human studies performed with physiological folate doses. Objective: In this pilot study, a new model for the determination of folate absorption is developed and validated. Design: Under strictly standardized procedures, two healthy ileostomy volunteers consumed single portions of test foods or an oral dose of a pharmaceutical folate preparation of the natural folate diastereomer (6S)-5- methyltetrahydrofolate. Relative folate absorption from oral doses versus an intramuscular injection of the same pharmaceutical preparation was determined using postdose plasma folate concentration curves. Nonabsorbed folate was estimated by postdose folate excretion into stomal effluent. Results: Estimated by plasma areas under the curve, relative folate absorption ranged from 47 to 67% for oral doses from the te st foods strawberries and broccoli and the pharmaceutical (6S)-5-methyltetrahydrofolate preparation. During 10 h postdose, 19-44% of the dietary folate was excreted with the stomal effluent. Varying gut passage times were observed for different food matrices by determining ileostomal folate excretion in 2 h intervals. Around 90% of the folate from the oral doses was recovered in the collected body fluids, plasma and stomal effluent, by 10 h postdose, independent of the size of the administered folate doses of 200 or 400 mg (0.4 or 0.9 mmol). Conclusion: The results imply that this model provides a suitable tool to estimate folate bioavailability from foods. Keywords: Folate absorption; folate in ileostomal effluent; ileostomists; plasma folate kinetics; urinary folate excretion

Determination of folate transport pathways in cultured rat proximal tubule cells

Deficiency of the vitamin folic acid has recently been linked with increased incidence of neural tube defects and of cardiovascular disease, through elevated plasma homocysteine levels. The kidney has an important role in conserving folate to counteract development of deficiency. Urinary folate excretion is regulated by the degree of reabsorption of folate by the proximal tubule cell. To evaluate an in vitro model for studies of the regulation of urinary folate excretion, the present studies examined the transport of 5-methyltetrahydrofolate (5-CH 3-H 4PteGlu), the primary form of folate in the glomerular filtrate, by normal rat proximal tubule (RPT) cells in confluent monolayer cultures. Specific binding of 5-CH 3-H 4PteGlu to the apical membrane was saturable ( K D=27 nM), but intracellular transport was not saturated up to 100 nM concentrations. 5-CH 3-H 4PteGlu transport was decreased 50% by concentrations of folic acid that completely blocked 5-CH 3-H 4PteGlu binding by the apical folate receptor. Probenecid (10 mM), an anion exchange (reduced folate carrier) inhibitor, reduced 5CH 3-H 4PteGlu transport by 50% without significantly affecting binding. Aspirin (3 mM) did not alter 5-CH 3-H 4PteGlu transport, but significantly enhanced the inhibition due to probenecid. Similarly, indomethacin (5 μM) potentiated the inhibition of 5-CH 3-H 4PteGlu transport by probenecid. These data suggest that RPT cells take up 5-CH 3-H 4PteGlu by both the folate receptor and the reduced folate carrier, implying a role for both pathways in regulating urinary folate excretion.

Folate Transport Proteins Mediate the Bidirectional Transport of 5-Methyltetrahydrofolate in Cultured Human Proximal Tubule Cells , ,

Although reabsorption across the apical (AP) membrane of the renal proximal tubule cell plays a vital role in the conservation of plasma 5-methyltetrahydrofolate, basolateral (BL) membrane–directed secretory pathways may also be important in regulating the urinary excretion of folate. Folate transport proteins, folate receptor and the reduced folate carrier have been implicated in the renal conservation of folate across the AP membrane, but their role in BL membrane–directed folate transport has not been studied. 5-Methyltetrahydrofolate transport across the AP and BL membranes of human proximal tubule cells was studied in cells grown on membrane inserts to allow optimum differentiation of AP and BL domains. Colchicine, an inhibitor of vesicular-mediated endocytosis, inhibited AP binding and AP-directed transport without affecting BL transport. Probenecid, an inhibitor of anion exchange, did not affect binding, but inhibited both AP and BL-directed transport with a greater effect on BL transport. Folic acid abolished AP binding of 5-methyltetrahydrofolate, but diminished AP-mediated transport by only 50%. These data suggest that both the folate receptor and the reduced folate carrier participate in AP uptake of folates by human kidney cells, but that BL-mediated uptake occurs primarily by the reduced folate carrier. Folate transport from the secretory direction occurred as readily as that from the reabsorptive direction, indicating that altered secretion could mediate excess urinary folate excretion.

Kinetic modeling of folate metabolism through use of chronic administration of deuterium-labeled folic acid in men

This study was conducted as an initial investigation of in vivo folate kinetics in healthy men (n = 4) and made use of a chronic-administration protocol with stable-isotope labeling. Subjects were given 0.453 mumol (200 micrograms) total folic acid in aqueous solution daily throughout the 18-wk study while they consumed self-selected folate-adequate diets. After a 2-wk pretrial period with unlabeled folic acid, subjects were given 0.227 mumol (100 micrograms) pteroyl-L-[2H4]glutamic acid/d ([2H4]folic acid) combined with 0.227 mumol nonlabeled folic acid or [2H2]pteroylhexaglutamic acid/d for the next 8 wk; then for the next 8 wk the [2H4]folic acid was withdrawn and the subjects received only nonlabeled folic acid. Little unmetabolized folic acid was excreted in urine. Isotopic enrichment of urinary folate during [2H4]folic acid administration and withdrawal was consistent with a kinetic model having a rapid turnover pool and a slow turnover pool. In contrast with previous two-pool models, provisions were made for folate turnover by urinary folate excretion (as measured here) and by fecal excretion and catabolic processes. The precision of modeling will be improved in future studies by measurement of enrichment of additional pools. However, this study shows clearly the slow turnover of the whole-body folate pool (< or = 1% per day) and the feasibility of further long-term kinetic analysis.

Effect of ethanol on folate binding by isolated rat renal brush border membranes

Deficiency of folic acid, an essential vitamin involved in critical metabolic pathways, occurs in several conditions, including alcoholism. In humans and animal models, chronic ethanol consumption leads to decreased plasma levels and increased urinary levels of folate. An isolated perfused rat kidney model has shown that ethanol produces increased urinary excretion of folate, suggesting a direct effect of ethanol on the kidney. Because the folate binding protein, located in the brush border membrane (BBM) of proximal tubule cells, is thought to be involved in renal folate reabsorption, the effects of ethanol on BBM binding of folate were assessed. Binding studies were conducted using isolated rat kidney cortex BBM preparations, incubated with 3H-labeled 5-methyltetrahydrofolate (5-CH 3H 4PteGlu) at varying concentrations (0.1–100 nM). Ethanol at 500 mg/dl did not significantly affect [ 3H]5-CH 3H 4PteGlu binding in BBM. The structural analogue, folic acid, decreased [ 3H]5-CH 3H 4PteGlu binding under similar conditions. Because of the lack of effect of ethanol on binding to isolated BBM, the effects of ethanol probably occur at other steps in the renal uptake and metabolism of folate.

The fate of [ 3H]folic acid in folate-adequate rats

To assess more fully the metabolic fate and in vivo kinetics of dietary folate, we conducted an in depth study that followed the metabolism and excretion of radiolabeled folate from a single administration, through its many forms in the organs and tissues of folate-adequate rats. Twenty-two rats were equilibrated with an amino acid diet containing 1 mg folic acid/kg diet, then given 185 kBq [ 3H]folic acid intragastrically. The isotopic label was followed through the tissues and in urine and feces for 32 days, every 8 hours for the first 48 hours. Individual folates, as their monoglutamyl forms, were separated and measured by high performance liquid chromatography (HPLC), and the peaks counted for 3H. Liver and kidney folates exhibited labeling (∼0.8 kBq/g for each) by 8 hours post-dose. These organs exhibited a peak of [ 3H]folate at 40 hours post-dose, then slowly lost label from 48 hours to 32 days, with the exception of a rise in renal radioactivity at 16 days. Heart and spleen exhibited low levels of labeling at 8 hours, a peak at 32 hours, then a gradual loss of label. Testes and muscle (hind leg) showed very low levels of labeling throughout the study. Whole blood showed labeling almost entirely associated with 5-methyl-tetrahydrofolate (THF). Urinary excretion of intact folate occurred mainly as 5-methyl-THF, although excretion of products of folate catabolism exceeded urinary excretion of intact folates. Much of the labeled dose (35%) was excreted into the urine as catabolites and intact folates by 32 days post-dose, whereas 15% of the label was lost through the feces by 32 days post-dose. Unexpected differences were observed among the specific radioactivity values among tissues (liver < kidney << testes, spleen, whole blood < heart << muscle). These may indicate different rates of clearance of labeled folates from these tissues or else the presence of kinetically slow pools comprising a significant portion of total folate in certain tissues (especially liver and kidney). The results of this study will be used to develop a compartmental model to simulate folate metabolism in folate-adequate rats.

Ethanol Acutely Impairs the Renal Conservation of 5‐Methyltetrahydrofolate in the Isolated Perfused Rat Kidney

The ethanol-induced increase in urinary folate excretion has been shown to decrease plasma folate levels and may contribute to the development of folate deficiency associated with alcoholism. The mechanism for this effect remains to be elucidated. The present studies were designed to examine the direct effect of ethanol on the renal handling of 5-methyltetrahydrofolate (5-CH3-H4PteGlu), the physiological folate. Rats were given four consecutive hourly doses of ethanol (1 g/kg) or isocaloric doses of glucose solution orally. This treatment generated an average plasma ethanol level of 305 mg/dl. Kidneys from male Sprague-Dawley rats were removed 5 hr after initial treatment and perfused in vitro to eliminate any extrarenal effects that could confound interpretation of results. Ethanol was not added to the perfusate. These treatments had no effect on 5-CH3-H4PteGlu conservation by the isolated perfused rat kidney in comparison to experiments in which the animal received no treatment. Ethanol was then added directly to the perfusate to generate average concentrations of 293 mg/dl. The in vitro addition of ethanol significantly decreased the percentage reabsorption and increased the fractional excretion of 5-CH3-H4PteGlu in comparison to controls (kidneys perfused with or without an isocaloric dose of glucose). This effect did not become significant until the renal tissue was exposed to these levels of ethanol for 1 hr. These results indicate that ethanol directly impairs the renal conservation of 5-CH3-H4PteGlu.

5-methyltetrahydrofolate urinary excretion: modeling by cultured human kidney cells.

Initial attempts to model urinary folate reabsorption using cultures of HPT cells on porous filter inserts produced disappointing results in that large amounts of 5M were transported across the epithelial monolayer in a nonspecific manner. Since the impermeable molecule inulin was also transported, there apparently existed a significant leakage pathway in the way that the cultured cells were used for transport studies. 5M was bound to the AP membrane and taken up into the HPT cell by specific processes, while inulin was excluded, suggesting that the HPT cells were nevertheless operating functionally. TER values from cultured HPT cells plateaued at a high level when cells became confluent, suggesting an epithelial layer with functional tight junctions. However, when growth media were removed and replaced with transport buffers, there was an immediate loss of TER that fully recovered if the transport buffers were preincubated for 60 min. Under these conditions, transport studies showed the expected results--no movement of inulin through the cell layer and much reduced transfer of folate through the paracellular pathway. These results suggest that a transient opening of tight junctions occurs when growth media are replaced with biological buffers (or with fresh growth media), but that recovery of tight junction function occurs with time.

Monitoring Cobalamin Inactivation During Nitrous Oxide Anesthesia by Determination of Homocysteine and Folate in Plasma and Urine

ERMENS, A. A. M.; REFSUM, H.; RUPREHT, J.; SPIJKERS, L. J. M.; GUTTORMSEN, A. B.; LINDEMANS, J.; UELAND, P. M.; ABELS, J. Author Information

Effects of ethanol on tissue folate incorporation and recovery from folate deficiency in rats.

Studies in folate-deficient alcoholics suggest that ethanol interferes with the recovery of folate status and the hematopoietic response to folate. Previous animal studies have suggested diverse effects of ethanol on intestinal absorption, hepatic metabolism, and urinary excretion of folate. In order to examine the effects of ethanol on folate distribution during folate deficiency, tissue incorporation of a tracer dose of folate was studied in rats chronically fed ethanol-containing and/or folate-deficient diets. Rats fed these diets were also used to study the effect of chronic ethanol consumption on the dietary reversal of folate deficiency by changing the diets (adding folate or replacing ethanol) from 12 to 16 weeks. After 16 weeks, tissue folate depletion was severe in rats fed folate-deficient diets. Plasma and whole body retention of the tracer dose of folate was decreased in folate-deficient rats consuming ethanol. In folate-deficient rats, ethanol consumption increased the incorporation of folate by the kidney and brain, but had no effect in other tissues (liver, lung, spleen, intestine, testis). In ethanol-fed folate-deficient rats that continued to consume ethanol, but with added folate in their diets, urine, plasma, liver, and kidney folate levels returned to control levels in 4 weeks. In the rats that stopped ethanol, but continued low folate diet consumption, no recovery of tissue folate levels was seen in 4 weeks. These results suggest that chronic ethanol consumption can exacerbate folate requirements by inhibiting body retention of small doses of folate. However, these effects are minor because ethanol consumption does not block recovery from folate deficiency when rats are fed sufficient amounts of folate.

In Vivo Folate Kinetics during Chronic Supplementation of Human Subjects with Deuterium-Labeled Folic Acid

Six healthy men (22–31 y) were supplemented for 4 wk with folic acid labeled with deuterium [3’,5’-2H2; 3.6 μmol/d (1.6 mg/d)] to permit evaluation of in vivo kinetics of this vitamin. Total folate in urine, serum and erythrocytes was determined by microbiological assay, and isotopic labeling of urinary and erythrocyte folate was determined by gas chromatographymass spectrometry. During supplementation, serum folate reached maximal concentration in ∼18 d, whereas excretion of total and deuterium-labeled folates increased rapidly and reached isotopic steady state in 1–2 wk. Isotopic labeling of erythrocyte folate increased continually over the entire supplementation period. Upon cessation of supplementation, red blood cell folate and urinary folate excretion (total and labeled) decreased linearly. The decline in total serum folate could be described with a biexponential model that yielded a slow-phase half-life of 18.7 ± 2.3 d. This model also indicated a turnover of 4.5% of the total body folate pool per day. Pool sizes of total body folate before and after supplementation (at steady state) were calculated to be 10 μmol (4.4 mg) and 98.9 μmol (43.7 mg), respectively. These kinetic data and stable isotope methodology may be used to address a wide range of experimental questions related to folate metabolism.