Biochemical and molecular aberrations in the rat colon due to folate depletion are age-specific

Elder adulthood and diminished folate status are each associated with an enhanced risk of colorectal carcinogenesis. We therefore examined whether these two factors are mechanistically related. Weanling male Sprague-Dawley rats (n = 44) and 1-y-old rats (n = 44) were each divided into three groups and fed diets containing 0, 4.5 or 18 micro mol folic acid/kg (deplete, replete and supplemented groups, respectively). Rats were killed at 0, 8 and 20 wk. The folate concentrations, the distribution of the different coenzymatic forms of folate, uracil incorporation into DNA and genomic DNA methylation were measured in the colonic mucosa. Folate-deplete and folate-replete elder rats had 30-45% lower colonic folate concentrations than young rats. Furthermore, 5-methyltetrahydrofolate was uniformly depleted in colons of the elder, folate-deplete rats, whereas this depletion occurred in only a minority of the younger rats. By the end of the experiment, the folate-deplete and folate-replete elder rats had approximately 50% more uracil incorporated into their colonic DNA than the corresponding young groups (P < 0.05). In elder rats, this uracil misincorporation was incremental across the three diet groups (P-test for trend < 0.05), whereas no excess uracil incorporation was observed in young rats. Neither age nor dietary folate affected genomic DNA methylation in the colon. In conclusion, the colon of elder rats is more susceptible to biochemical and molecular consequences of folate depletion than that of young rats. However, folate supplementation is as effective at sustaining adequate colonic folate status in elder rats as it is in the young.     

Folate supplementation increases genomic DNA methylation in the liver of elder rats

The availability of folate is implicated as a determinant of DNA methylation, a functionally important feature of DNA. Nevertheless, when this phenomenon has been examined in the rodent model, the effect has not always been observed. Several reasons have been postulated for the inconsistency between studies: the rodent is less dependent on folate as a methyl source than man; juvenile animals, which most studies use, are more resistant to folate depletion than old animals; methods to measure genomic DNA methylation might not be sensitive enough to detect differences. We therefore examined the relationship between folate and genomic DNA methylation in an elder rat model with a newly developed method that can measure genomic DNA methylation sensitively and precisely. Thirty-nine 1-year-old rats were divided into three groups and fed a diet containing 0, 4.5 or 18 mumol folate/kg (folate-deplete, -replete and -supplemented groups, respectively). Rats were killed at 8 and 20 weeks. At both time points, mean liver folate concentrations increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend <0.001) and by 20 weeks hepatic DNA methylation also increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend=0.025). At both time points folate-supplemented rats had significantly increased levels of DNA methylation compared with folate-deplete rats (P<0.05). There was a strong correlation between hepatic folate concentration and genomic DNA methylation in the liver (r 0.48, P=0.004). In the liver of this animal model, dietary folate over a wide range of intakes modulates genomic DNA methylation.     

Biological activity of racemic folate mixtures fed to folate-depleted rats.

Most commercially available tretrahydro and substituted folates are racemic mixtures of R and S isomers about pteridine-carbon 6. Some reports show the unnatural stereoisomers modify specific aspects of folate metabolism, whereas others show no significantly different biologic effects between isomers. The extent to which unnatural folate stereoisomers are inert in vivo is unclear. Possible interactions between isomers at normal dietary folate intakes were tested by comparing growth, liver and serum folate concentrations, and congener patterns of folate-depleted rats fed amino acid-based diets with 283, 566 or 1132 nmol folic acid (standards), or 566 nmol racemic 5-methyltetrahydrofolate, 5-formyltetrahydrofolate or 5,10-diformyltetrahydrofolate/kg diet for 3 wk. Growth and tissue folate levels increased with each increment in dietary folic acid. Growth and liver and serum folate concentrations of rats fed 566 nmol 5-formyltetrahydrofolate or 5-methyltetrahydrofolate/kg diet were similar to those of rats fed 283 nmol folic acid/kg diet. Rats fed 5,10-diformyltetrahydrofolate lost weight and had depressed serum folate levels, and most died. Results show that biologic activity of racemic 5-formyltetrahydrofolate and 5-methyltetrahydrofolate was half that of folic acid, and the R isomer did not affect growth at the levels fed. Addition of a second formyl group destroyed folate bioactivity. The folate-depleted rat model is useful for testing biologic effects of individual folate stereoisomers in vivo as they become commercially available.     

DNA stability and genomic methylation status in colonocyte isolated from methyl-donor-deficient rats

Summary Background: Epidemiological studies report an inverse relationship between intake of the B vitamine folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways. Aims of the study: The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated. Methods: Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionne and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples. Results: Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocytes DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls. Conclusions: Folate and methyl deficiency in vivo primarily afects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation. Received: 16 February 2000, Accepted: 25 April 2000     

Folic acid supplementation for 4 weeks affects liver morphology in aged rats

Several countries have approved universal folic acid (FA) fortification to prevent neural tube defects and/or high homocysteine levels; this has led to a chronic intake of FA. Traditionally, the vitamin is considered to be safe and nontoxic, except for the potential masking of vitamin B-12 deficiency. Recent reports from our laboratories showed several effects of high-dose folate supplementation in rats. In this work, we compared the effect of FA on the liver of weanling (3 wk) and aged (18 mo) male rats fed either a diet supplemented with 40 mg FA/kg diet or a control diet (1 mg FA/kg diet) for 4 wk. FA supplementation did not alter serum aspartate aminotransferase, alanine aminotransferase, urea, glucose oxidase, total bilirubin, or uric acid. Routine histological staining as well as immunohistochemistry with proliferating cell nuclear antibody for dividing cells, and cytokeratin-8 against bile ductal cells, showed that aged, supplemented rats had the same number of hepatocytes as both control and supplemented weanling rats, and tended to have more (17%, P = 0.07) hepatocytes than aged, control rats. Moreover, the bile duct cells of aged, control rats proliferated and transformed into cholestatic rosettes at a higher frequency than in aged, supplemented rats. The morphology of the liver in weanling rats was similar in both diet groups, and comparable to the supplemented, aged rats, thus indicating that a high intake of FA improves normal liver morphology in livers of aged rats.     

Bioavailability of folate following ingestion of cholestyramine in the rat

The effect of cholestyramine ingestion on the intestinal deconjugation and absorption of folic acid (PGA) and brewers yeast folate was investigated using a rat bioassay and liver folate uptake as the response parameter. Male weanling Sprague Dawley rats were depleted on a low AIN-76A formulated basal diet for 21 days. During a 14 day repletion period folic acid (PGA) and brewers yeast were added to provide 0.25, 0.5 and 1.0 mg of folate per kg of diet. Cholestyramine was administered directly as part of the diet at 1.1%. All diets were made isonitrogenous and isocaloric. Based on a parallel line assay, the relative biological value of folate for PGA + cholestyramine (79) was significantly different from the standard diet (PGA = 100), while those for brewers yeast (88) and for brewers yeast + cholestyramine (88) did not differ from the standard diet. Ingestion of cholestyramine significantly reduced the bioavailability of PGA versus brewers yeast folate in rats.     

Plasma, liver and kidney folate and plasma homocysteine concentrations are poor response variables at very low dietary folate intakes, in a folate depletion/repletion rat model

Folate depletion/repletion rat models are popular protocols for assessing the bioavailability of folate. Much of the early work carried out on folate bioavailability concentrated on foods naturally high in folate. However, foods low in folate often contribute significantly to folate intake because of their high consumption in the general population. Therefore, the assessment of the bioavailability of foods low in folate is essential to properly estimate folate intake. The present study investigated plasma, liver and kidney folate and plasma homocysteine concentrations as appropriate response variables for measuring folate bioavailability in the rat at very low dietary folate intakes. One hundred and one weanling male rats (Wistar strain) were fed a folate-deficient diet containing 1% succinyl sulfathiazole for 28 d. Following depletion, six rats were randomly assigned to each of 16 repletion diets containing folic acid, fortified white bread, unfortified wholemeal bread or unfortified rye bread calculated to provide 6.25, 12.5, 18.75 and 25 micrograms folate/kg of each diet. After a further 28 d, plasma, liver and kidney folate concentrations were determined by microbiological assay. Plasma homocysteine was measured by HPLC as a functional indicator of folate status. Only a weak correlation was found between the response variables measured and dietary folate intake, indicating that this folate depletion/repletion rat model is not suitable for testing the response of rats fed diets containing very low levels of folate.     

Genomic DNA methylation decreases in response to moderate folate depletion in elderly women

BACKGROUND: Methylation of genomic DNA is dependent on an adequate supply of folate coenzymes. Previous data support the hypothesis that abnormal DNA methylation plays an integral role in carcinogenesis. To date, no studies assessing the effect of inadequate folate status on DNA methylation in older women (aged >63 y) have been reported. OBJECTIVE: The effect of moderate folate depletion followed by folate repletion on leukocyte genomic DNA methylation was investigated in elderly women (aged 60-85 y) to evaluate whether DNA methylation could be used as a functional indicator of folate status. DESIGN: Healthy, postmenopausal women (n = 33) consumed a moderately folate-depleted diet (118 microg folate/d) for 7 wk, followed by 7 wk of folate repletion with 200 or 415 microg/d, each provided as 2 different dietary treatments for a total of 4 treatment groups (n = 30). Leukocyte DNA methylation was determined on the basis of the ability of DNA to incorporate [(3)H]methyl groups from labeled S:-adenosylmethionine in an in vitro assay. RESULTS: Incorporation of [(3)H]methyl groups increased significantly (P: = 0.0025) in response to folate depletion, suggesting undermethylation of DNA. No significant changes were detected in [(3)H]methyl incorporation in any group over the 7-wk repletion period compared with postdepletion values. CONCLUSIONS: DNA methylation status may be used as a functional indicator of moderately depleted folate status. The slow response to the repletion diets observed suggests that normalization of DNA methylation after moderate folate depletion may be delayed in older women.     

Dietary inulin suppresses azoxymethane-induced aberrant crypt foci and colon tumors at the promotion stage in young Fisher 344 rats

This study was designed to determine the effect of 10% dietary long-chain inulin on the azoxymethane (AOM)-induced colonic preneoplastic aberrant crypt foci (ACF) and small intestinal and colon tumors at the initiation (I), promotion (P) and I + P stages (20 rats per treatment) in Fisher 344 male weanling rats. After an acclimatization period of 1 wk, groups of Fisher 344 male weanling rats were assigned to consume AIN 93G diet (control) or AIN 93G diet containing 10% inulin. All the rats received 16 mg/kg body AOM dissolved in saline subcutaneously at 7 wk of age followed by a second injection at 8 wk of age. An additional group of five rats received only saline and consumed the control diet. The rats received the assigned diets until asphyxiation by CO(2) at 16 wk of age for the ACF experiment and 45 wk for the end-point tumor experiment. Feed intake, weight gain, diarrheal index, cecal weight, cecal pH, ACF and tumors in the colon were determined. Rats fed inulin had diarrhea after 2 wk of feeding and recovered by approximately 4 wk. Cecal weight was greater in rats fed inulin and cecal pH was lower. The inulin group had more than 66% fewer aberrant crypts and 60% fewer ACF compared with the control group. Tumor incidences in the small intestine and colon of rats in the control, I, P and I + P groups were: 78, 31, 0 and 11% and 90, 73, 69 and 50%, respectively. The corresponding values for the distal portion of the colon were 87, 63, 45 and 33%, respectively. Colon tumors per tumor-bearing rat were 4.2, 3.09, 1.36 and 1.2 for the control, I, P and I + P groups, respectively. All groups differed, P < 0.05. The results of this study indicate that dietary long-chain inulin suppresses AOM-induced ACF formation, an early preneoplastic marker of colon tumorigenesis in rats, and colon tumors, particularly at the promotion stage.     

Effect of nitrite ingestion on the bioavailability of folate in the rat.

The effect of nitrite ingestion on the intestinal deconjugation and absorption of folic acid (PGA) and brewers yeast folate was investigated using a rat bioassay and liver folate uptake as the response parameter. Male weanling Sprague Dawley rats were depleted on a low folate AIN-76A formulated basal diet for 21 days. During a 14 day repletion period, folic acid (PGA) and brewers yeast were added to provide 0.25, 0.5 and 1.0 mg of folate per kg of diet. Potassium nitrite was administered as part of the diet at 0.5%. All diets were made isonitrogenous and isocaloric. Based on a parallel line assay, the relative bioavailability of folate in the brewers yeast diet (109) was significantly higher than in the standard diet (PGA = 100). When combined with nitrite, the relative bioavailability of the PGA diet was not significantly different (101), while that of the brewers yeast diet was significantly lower than the standard diet. Concomitant ingestion of nitrite significantly reduced the bioavailability of brewers yeast folate but not that of PGA in rats. This appeared to be a direct effect of oxidation by nitrite on the more susceptible substituted folates in brewers yeast.     

Effects of parental folate deficiency on the folate content,global DNA methylation,and expressions of FRα, IGF-2 and IGF-1R in the postnatal rat liver

We examined the effect of parental folate deficiency on the folate content, global DNA methylation, folate receptor-alpha (FRα), insulin-like-growth factor-2 (IGF-2) and -1 receptor (IGF-1R) in the liver and plasma homocysteine in the postnatal rat. Male and female rats were randomly fed a folic acid-deficient (paternal folate-deficient, PD and maternal folate-deficient, MD), or folic acid-supplemented diet (paternal folate-supplemented, PS and maternal-folate-supplemented, MS) for four weeks. They were mated and grouped accordingly: PSxMS, PSxMD, PDxMS, and PDxMD. Pups were killed on day 21 of lactation. The hepatic folate content was markedly reduced in the PDxMD and PSxMD and PDxMS as compared with the PSxMS group. The hepatic global DNA methylation was decreased in the PDxMS and PSxMD groups as much as in the PDxMD group, and all the three groups were significantly lower as compared to the PSxMS group. There were no significant differences in the hepatic FRα, IGF-2 and IGF-1R expressions among the groups. Positive correlations were found between the hepatic folate content and global DNA methylation and protein expressions of FRα, IGF-2 and IGF-1R, whereas an inverse correlation was found between hepatic folate content and plasma homocysteine level in the 3-week-old rat pup. The results of this study show that both paternal and maternal folate deficiency at mating can influence the folate content and global DNA methylation in the postnatal rat liver.     

Folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers

This study was designed to determine whether nutritional folate depletion exerts hepatic oxidative stress in relation to elevated plasma homocysteine. To mimic various extents of folate depletion status in vivo, male Wistar rats were fed an amino acid-defined diet containing either 8 (control), 2, 0.5, or 0 mg folic acid/kg diet. After a 4-wk feeding period, the plasma and hepatic folate concentrations of the rats decreased significantly with each decrement of dietary folate. Folate depletion did not significantly affect two major liver antioxidants: reduced glutathione and alpha-tocopherol. Conversely, folate depletion decreased Cu-Zn superoxide dismutase and glutathione peroxidase activities, but had no effect on catalase activity in liver homogenates. Lipid peroxidation products, as measured by thiobarbituric acid-reactive substances, were significantly higher in livers of folate-depleted rats than in those of the controls. This occurrence of hepatic oxidative stress in folate-depleted rats was confirmed by demonstrating an increased susceptibility of livers of folate-depleted rats to lipid peroxidation induced by additional H2O2 or Fe(2+) treatments compared with the controls. Decreasing dietary folate intake resulted in graded increases in plasma homocysteine concentrations of folate-depleted rats. Elevated plasma homocysteine and decreased plasma and hepatic folate concentrations in folate-depleted rats were all strongly and significantly correlated with increased liver lipid peroxidation (/r/ > or = 0.58, P < 0.0003). These data demonstrate that folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers.     

Dietary folate and selenium affect dimethylhydrazine-induced aberrant crypt formation,global DNA methylation and one-carbon metabolism in rats

Several observations suggest a role for DNA methylation in cancer pathogenesis. Although both selenium and folate deficiency have been shown to cause global DNA hypomethylation and increased cancer susceptibility, the nutrients have different effects on one-carbon metabolism. Thus, the purpose of this study was to investigate the interactive effects of dietary selenium and folate. Weanling, Fischer-344 rats (n = 23/diet) were fed diets containing 0 or 2.0 mg selenium (as selenite)/kg and 0 or 2.0 mg folate/kg in a 2 x 2 factorial design. After 3 and 4 wk of a 12-wk experiment, 19 rats/diet were injected intraperitoneally with dimethylhydrazine (DMH, 25 mg/kg) and 4 rats/diet were administered saline. Selenium deficiency decreased (P < 0.05) colonic DNA methylation and the activities of liver DNA methyltransferase and betaine homocysteine methyltransferase and increased plasma glutathione concentrations. Folate deficiency increased (P < 0.05) the number of aberrant crypts per aberrant crypt foci, the concentration of colonic S-adenosylhomocysteine and the activity of liver cystathionine synthase. Selenium and folate interacted (P < 0.0001) to influence one-carbon metabolism and cancer susceptibility such that the number of aberrant crypts and the concentrations of plasma homocysteine and liver S-adenosylhomocysteine were the highest and the concentrations of plasma folate and liver S-adenosylmethionine and the activity of liver methionine synthase were the lowest in rats fed folate-deficient diets and supplemental selenium. These results suggest that selenium deprivation ameliorates some of the effects of folate deficiency, probably by shunting the buildup of homocysteine (as a result of folate deficiency) to glutathione.     

Vitamin B-12 deficiency induces anomalies of base substitution and methylation in the DNA of rat colonic epithelium

Derangements of one-carbon metabolism can directly affect the integrity of the genome by producing inappropriate uracil insertion into DNA and by altering patterns of DNA methylation. Vitamin B-12, a one-carbon nutrient, serves as a cofactor in the synthesis of precursors of biological methylation and in nucleotide synthesis. We therefore examined whether vitamin B-12 deficiency can induce these molecular anomalies in the colonic mucosa of rats. Weanling male Sprague-Dawley rats (n = 30) were divided into 2 groups and fed either a vitamin B-12-deficient diet or a similar diet containing adequate amounts of the vitamin. Rats from each group were killed at 6 and 10 wk. Uracil misincorporation into DNA was measured by GC/MS and genomic DNA methylation was measured by LC/MS. Plasma vitamin B-12 concentrations in deficient rats were below detectable limits at 6 and 10 wk; in control rats, concentrations were 0.46 +/- 0.07 and 0.42 +/- 0.10 nmol/L at those times. Although the colon total folate concentration did not differ between the groups, the proportion that was methylfolate was marginally greater in the deficient rats at 10 wk (P = 0.05) compared with control, consistent with the "methylfolate trap" that develops during vitamin B-12 deficiency. After 10 wk, the colonic DNA of the deficient rats displayed a 35% decrease in genomic methylation and a 105% increase in uracil incorporation (P < 0.05). This vitamin B-12-deficient diet, which was of insufficient severity to cause anemia or illness, created aberrations in both base substitution and methylation of colonic DNA, which might increase susceptibility to carcinogenesis.     

Phenytoin treatment and folate supplementation affect folate concentrations and methylation capacity in rats.

Phenytoin (PHT) has long been known to cause folate depletion with chronic use. In animal models PHT has been shown to interfere with folate-dependent one-carbon metabolism. Folic acid supplementation in humans has been shown to restore blood levels of folates to normal, but the effects of folic acid supplementation on the PHT-induced effects on one-carbon metabolism have not been addressed. In the present study rats were treated for 8 wk with 1) PHT, 2) folic acid, 3) PHT plus folic acid or 4) vehicle (propylene glycol). Phenytoin treatment caused a decrease in weight gain over the 8 wk of treatment. This effect on weight gain was reversed by folic acid supplementation, but the decrease in brain folate concentration caused by PHT was not reversed by folic acid supplementation, which by itself apparently caused a decrease in brain folate concentration. Phenytoin treatment tended to increase methylation capacity (S-adenosylmethionine:S-adenosylhomocysteine ratio) in the brain and decrease methylation capacity in the liver. Folate supplementation by itself increased methylation capacity in the liver but had no effect in the brain. Folic acid and PHT apparently had independent but opposite effects in the liver, leading to a normalization of methylation capacity. These data suggest that folic acid supplementation in PHT therapy may be effective in reversing the peripheral effects of chronic PHT treatment on one-carbon metabolism but not the central effects.     

Rat bioassay of wheat bran folate and effects of intestinal bacteria

This study estimates the folate endogenous to a food material (wheat bran) and examines the role of intestinal bacteria in the rat bioassay for folate. After a 4-wk folate depletion period, rats were fed for an additional 4 wk basal diets with or without 0.5% phthalylsulfacetamide and with 100, 200 or 300 g of wheat bran; or 50, 100 or 150 g of xylan; or 0, 0.25, 0.50 or 0.75 mg of folic acid added per kg of basal diet. Xylan increased both liver and fecal folate, and this effect was nearly eliminated by phthalylsulfacetamide. Wheat bran contributed 1.6 micrograms of available folate per g of wheat bran without phthalylsulfacetamide in an apparently valid slope-ratio analysis. With the addition of phthalylsulfacetamide, liver folate increased in rats fed wheat bran diets and decreased in rats fed folic acid diets. The slope-ratio analysis for wheat bran folate with phthalylsulfacetamide became invalid due to a lack of intersection. Phthalylsulfacetamide had no effect on fecal folate excretion from rats fed the wheat bran diets. Further studies are needed on a variety of foods with and without phthalylsulfacetamide to evaluate the effect and importance of intestinal folate synthesis in the rat.     

Supranormal dietary folic acid supplementation: effects on methionine metabolism in weanling rats

There are nationwide folic acid (FA) fortification programmes of staple foods established or under consideration in order to prevent neural tube defects. Universal FA fortification still remains controversial because of the concern that additional FA in the diets of population groups (e.g. children and elderly) not initially targeted for fortification may suffer adverse effects. However, dietary surveys regarding folate generally deal with adults and little is known about the consumption and long-term effects of fortified food and supplements in growing individuals. Recent reports from our laboratory show several effects of high-dose folate supplementation in rats. In the present work, we studied the effect of FA on the methionine cycle in weanling (3-week-old) male rats after 4 weeks of supplementation with 40 mg FA/kg diet v. control (1 mg FA/kg diet). FA supplementation resulted in a reduction of homocysteine and creatinine concentrations v. control group. FA supplementation did not alter S-adenosylmethionine/S-adenosylhomocysteine ratio, DNA methylation, enzymatic activities or concentrations of vitamins involved in the nutritional regulation of the methionine cycle, except for folate. FA supplementation of 40 mg/kg did not lead to hepatic or renal damage. In conclusion, there were no apparent adverse effects on one-carbon metabolism after FA supplementation in the studied conditions.     

Effect of dietary methyl group deficiency on folate metabolism in rats

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.     

High-dose folic acid supplementation in rats: effects on gestation and the methionine cycle

There is new evidence that a good folate status may play a critical role in the prevention of neural-tube defects and in lowering elevated homocysteine concentrations. This adequate folate status may be achieved through folic acid dietary supplementation. Folate is a water-soluble vitamin with a low potential toxicity. However, the possible consequences of long-term high-dose folic acid supplementation are unknown, especially those related to the methionine cycle, where folate participates as a substrate. With the aim of evaluating such possible effects, four groups of Wistar rats were classified on the basis of physiological status (virgin v. pregnant) and the experimental diet administered (folic-acid-supplemented, 40 mg/kg diet v. control, 2 mg folic acid/kg diet). Animals were fed on the diets for 3 weeks. Results showed that gestation outcome was adequate in both groups regardless of the dietary supplementation. However, there were reductions (P < 0.001) in body weight and vertex-coccyx length in fetuses from supplemented dams v. control animals. Folic acid administration also induced a higher (P < 0.01) S-adenosylmethionine: S-adenosylhomocysteine value due to increased S-adenosylmethionine synthesis (P < 0.01). However, hepatic DNA methylation and serum methionine concentrations remained unchanged. Serum homocysteine levels were reduced in supplemented dams (P < 0.05). Finally, pregnancy caused lower serum folate, vitamin B6 and vitamin B12 levels (P < 0.05). Folic acid administration prevented the effect of pregnancy and raised folate levels in dams, but did not change levels of vitamins B12 and B6. These new findings are discussed on the basis of potential benefits and risks of dietary folic acid supplementation.     

A postweaning iron-adequate diet following neonatal iron deficiency affects iron homeostasis and growth in young rats

Iron deficiency is among the most prevalent of nutrient-related diseases worldwide, but the long-term consequences of maternal and neonatal iron deficiency on offspring are not well characterized. We investigated the effects of a postweaning iron-adequate diet following neonatal iron deficiency on the expression of genes involved in iron acquisition and homeostasis. Pregnant rats were fed an iron-adequate diet (0.08 g iron/kg diet) until gestational d 15, at which time they were divided into 2 groups: 1) a control group fed an iron-adequate diet, and 2) an iron-deficient group fed an iron-deficient diet (0.005 g iron/kg diet) through postnatal d (P) 23 (weaning). After weaning, pups from both dietary treatment groups were fed an iron-adequate diet until adulthood (P75). Rat pups that were iron deficient during the neonatal period (IDIA) had reduced weight gain and hemoglobin concentrations and decreased levels of serum, liver, and spleen iron on P75 compared with rats that were iron sufficient throughout early life (IA). IDIA rats developed erythrocytosis during postweaning development. Further, hepatic expression of hepcidin in IDIA rats was 1.4-fold greater than in IA rats, which paralleled an upregulation of IL-1 expression in the serum. Our data suggest that an iron-adequate diet following neonatal iron deficiency induced an inflammatory milieu that affected iron homeostasis and early growth and development.