Metabolic Interaction Between Folate, Vitamin B12, and Polyunsaturated Fatty Acids in Pregnancy.

Fetal growth and development are influenced by maternal nutrition and gestational weight gain. Adequate intake of nutrients such as folate, vitamin B12, and docosahexaenoic acid (DHA) is essential for healthy fetal and placental development. Many countries have a national flour fortification program with folic acid (FA), together with pre-pregnancy supplementation of FA (400 µg/day) during the first trimester of pregnancy. The latter has been recommended by the WHO and adapted to local requirements by perinatal guidelines. On the other hand, in population studies, many women of childbearing age have vitamin B12 deficiency (<148 pmol/L), which can be additionally masked by high FA intake and maternal pregestational obesity. Under these conditions, these patients could be having pregnancies in a folate/vitamin B12 imbalance, which is associated with higher adiposity, insulin resistance, altered lipid metabolism, and low DHA levels in their offspring. However, if these neonatal consequences of maternal pregestational obesity and folate/vitamin B12 imbalance can be reverted by DHA supplementation during pregnancy has not been addressed. This chapter reviews the literature and exposes the current gaps in knowledge and challenges in maternal nutrition with a life-course perspective.

[Folate and Pregnancy, current concepts: It is required folic acid supplementation?] / Folatos y Embarazo, conceptos actuales: ¿Es necesaria una suplementación con Acido Fólico?

Folate intake during pregnancy is essential for an adequate fetal and placental development and for the long time health of the individual. Its deficiency may induce fetal pathologies, including neural tube disease (NTD). Therefore, several countries implemented public policies to fortify foods with folic acid (FA). Chile started the fortification of wheat flour with FA in the year 2000, decreasing a 43% the prevalence of NTD. However, despite the high consumption of bread (the main fortified food with FA) by our population, a high number of pregnant women consume FA supplements, thus, over passing the maximal recommended FA intake. Additionally, if the diet is reduced in vitamin B12, the optimal ratio folates/vit B12 may be altered, thus inducing changes in the methylation of specific genes and other metabolic pathways, affecting fetal development and the long-term health of the neonates. We think that, after 16 years of the initiation of the fortification of wheat flour with FA, it is necessary to evaluate the possible side effects of a high intake of FA in the pregnant population and their offspring. This article shows antecedents about mechanisms of folates and vit B12 at cellular level, and their possible consequences of an elevated FA maternal intake on the offspring.

Folates Induce Colorectal Carcinoma HT29 Cell Line Proliferation Through Notch1 Signaling.

Folic acid (FA) consumption at high levels has been associated with colon cancer risk. Several mechanisms have been proposed to explain this association. The Notch signal pathway has been implicated in the regulation of cellular proliferation. Our aim was to demonstrate that high concentrations of FA or its reduced form, 5-methyltetrahydrofolic acid (5-MTHF), increase colorectal carcinoma HT29 cell proliferation through an increase of Notch1 activation and to prove if the inhibition of Notch1 activation by gamma secretase inhibitor, reduce the effect of folic acid. HT29 cells were cultured in high (400 nM), low (20 nM), or 0 nM FA or 5-MTHF concentrations during 96 h with or without DAPT (gamma secretase inhibitor). Cell proliferation was determined by the methylthiazole tetrazolium method, and Notch1-intracellular domain (NICD) was analyzed by flow cytometry. HT29 cells exposed to 400 nM FA or 5-MTHF showed higher proliferation rate than those exposed to 20 nM of FA or 5-MTHF (P < 0.01) during 96 h. NICD expression increased at higher FA or 5-MTHF concentrations compared with lower concentrations (P < 0.01). This effect on proliferation was partially reversible when we blocked Notch1 activation with the inhibitor of γ-secretase (P < 0.05).These data suggest that high concentration of FA and 5-MTHF induce HT29 cell proliferation activating Notch1 pathway.

Maternal-child transfer of essential and toxic elements through breast milk in a mine-waste polluted area.

OBJECTIVE: To determine the daily intake of essential micronutrients and toxic elements through breast milk in exclusive and nonexclusive breastfed infants living in an area with major mine tailing deposition (n = 24), compared with a control area (n = 11). STUDY DESIGN: The milk volume ingested by 2 to 4 and 4 to 6 month infants was measured by a stable isotopic method. Elements in milk, maternal and infant urine, and drinking water were measured by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: Similar breast milk volume and essential micronutrients intake in groups of exclusively breastfed infants, but more cadmium, boron, and lithium through breastfeeding in experimental area was found. This exposure was even higher in the nonexclusively breastfed infants, who also ingested more arsenic, boron, and lithium than exclusive breastfed infants. CONCLUSION: The use of the deuterium and the ICP-MS methods made it possible to evaluate the exact amount of essential and toxic elements ingested by infants through breast milk demonstrating that lower amount of toxic elements are transferred to exclusive breastfed infants compared with those who additionally received nonmaternal milk.

[A role for the endocannabinoid system in hepatic steatosis]. / Sistema endocanabinoide y desarrollo de esteatosis hepática.

The endocannabinoid system (SEC) is an important modulator of several metabolic functions. This system is composed by cannabinoid receptors type 1 and 2 (RCB1 and RCB2), their endogenous ligands, known as endocannabinoids, and the enzymes involved in their synthesis and degradation. A deregulated SEC originates metabolic alterations in several tissues, resulting in the typical manifestations of the metabolic syndrome. Liver steatosis of different origins constitutes a physiopathological condition where an altered hepatic SEC is observed. In this condition, there is an increased expression of RCB1 and/or higher endocannabinoid levels in different hepatic cells, which may exert an autocrine/paracrine hyperstimulation of RCB1/RCB2. Activation of RCB1 stimulate the expression of several hepatocyte lipogenic factors, thus leading to increased de novo fatty acids synthesis and consequently to an abnormal accumulation of triglycerides. The effect of RCB2 activity on hepatic function is still controversial because, on one side its stimulation has an interesting protective effect on alcoholic liver disease while, on the other, it may enhance the development of hepatic steatosis in experimental models of diet-induced obesity. In this review we discuss the proposed mechanisms by which SEC is involved in the etiology of hepatic steatosis, as well as the therapeutic possibilities involving peripheral RCB1/RCB2 antagonism/agonism, for the treatment of this condition.

DHA Supplementation during Pregnancy in Women with Obesity Normalizes IGF2R Levels in the Placenta of Male Newborns.

Introduction: Insulin-like growth factor receptor 2 (IGF2R) regulates placental nutrient transport, and its soluble form is related to obesity in adults. If the placental expression of IGF2R is altered in women with obesity is unknown. Whether maternal supplementation with docosahexaenoic acid (DHA), a polyunsaturated fatty acid with anti-inflammatory properties, has a modulatory role in IGF2R's function has not been elucidated. We hypothesized that maternal obesity (Ob) would be associated with alterations in placental IGF2R expression, which may be prevented with DHA supplementation during pregnancy. Methods: At delivery, we obtained placentas from women with Ob (BMI ≥ 30 kg/m2, n = 17), Ob supplemented with 800 mg/day of DHA during pregnancy (Ob + DHA, n = 13), and normal-weight women (Nw, BMI ≥ 18.5 ≤ 24.9 kg/m2, n = 14). The IGF2R mRNA and protein were determined by RT-PCR and western blotting, respectively. Moreover, we quantified the gene expression of molecules that modulate the IGF2R function in the extracellular domain, such as TACE/ADAM17, PLAU, and IGF2. Mann-Whitney and Kruskal-Wallis nonparametric tests were used to compare results between two or three groups accordingly. Results: The IGF2R levels in the Ob placentas of the male offspring were higher than in the Nw group. The DHA supplementation prevented this effect, suggesting an unknown relationship between IGF2R-Ob-DHA in placental tissues. Conclusion: We report, for the first time, that DHA supplementation during pregnancy in women with obesity normalizes the increased IGF2R levels in male placentas, reducing the risk of adverse outcomes related to the IGF2/IGF2R system in male newborns.

Maternal exposure to cadmium during gestation perturbs the vascular system of the adult rat offspring.

Several cardiovascular diseases (CVD) observed in adulthood have been associated with environmental influences during fetal growth. Here, we show that maternal exposure to cadmium, a ubiquitously distributed heavy metal and main component of cigarette smoke is able to induce cardiovascular morpho-functional changes in the offspring at adult age. Heart morphology and vascular reactivity were evaluated in the adult offspring of rats exposed to 30ppm of cadmium during pregnancy. Echocardiographic examination shows altered heart morphology characterized by a concentric left ventricular hypertrophy. Also, we observed a reduced endothelium-dependent reactivity in isolated aortic rings of adult offspring, while endothelium-independent reactivity remained unaltered. These effects were associated with an increase of hem-oxygenase 1 (HO-1) expression in the aortas of adult offspring. The expression of HO-1 was higher in females than males, a finding likely related to the sex-dependent expression of the vascular cell adhesion molecule 1 (VCAM-1), which was lower in the adult female. All these long-term consequences were observed along with normal birth weights and absence of detectable levels of cadmium in fetal and adult tissues of the offspring. In placental tissues however, cadmium levels were detected and correlated with increased NF-κB expression--a transcription factor sensitive to inflammation and oxidative stress--suggesting a placentary mechanism that affect genes related to the development of the cardiovascular system. Our results provide, for the first time, direct experimental evidence supporting that exposure to cadmium during pregnancy reprograms cardiovascular development of the offspring which in turn may conduce to a long term increased risk of CVD.

Fatty acid and lipid metabolism in liver of pregnant mice and their offspring is influenced by unbalanced folates/vitamin B12 diets.

Micronutrients (folates and vitamin B12) and long chain polyunsaturated fatty acids (LC-PUFAs) are linked through the one carbon cycle. We studied the effects of pre and postnatal high FA/low B12 diets (HFLB12) on hepatic fatty acid metabolism. Pregnant C57BL/6 mice were divided in two groups: control (2 mg folic acid: FA/25 µg vitamin B12/Kg food) and HFLB12 diets (8 mg FA/5 µg vitamin B12/Kg food). Offspring continued on the same diets until 60 days old. We determined hepatic fatty acid profile in dams and offspring and the expression of PPARα, Cpt-1, Acox-1 and Fas and the enzymatic activity of desaturases, all involved in lipid metabolism. In liver of dams, the HFHB12 diet decreased total fatty acids and desaturase activities; in offspring, effects were opposite, being more noticeable in females. Prenatal and postnatal unbalanced folic acid/B12 diets play a crucial role in regulating genes and enzymes involved in lipid metabolism in liver of dams and their offspring in adulthood.

Fetal growth restriction is related to placental levels of cadmium, lead and arsenic but not with antioxidant activities.

The objectives of this study were: to measure some essential metals and toxicants in placentas of mothers delivering neonates with fetal growth restriction, and to establish potential associations between environmental adverse stimulus and antioxidant protective mechanisms. Placentas of 20 mothers delivering neonates with low birth weight (<2500g) and normal birth weight (>3000g) at term were collected. Placental concentration of zinc, mercury, selenium and arsenic were measured by Instrumental Neutron Activation Analysis (INAA), and iron, copper, cadmium and lead by Atomic Absorption Spectrometry (AAS). Total glutathione, lipid peroxidation, total antioxidant activity and antioxidant enzyme activities (superoxide dismutase and glutathione peroxidase) were determined spectrophotometrically. Results showed reduced iron levels and increased concentrations of cadmium, lead and arsenic in placentas of mothers delivering low birth weight neonates, but not differences in oxidative stress parameters or antioxidant enzymatic activities, suggesting a relationship between low birth weight and placental concentration of cadmium, arsenic and lead.

Methylation status in healthy subjects with normal and high serum folate concentration.

OBJECTIVE: We assessed the impact of high serum folate concentration on erythrocyte S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) concentrations, SAM/SAH ratio, CpG methylation levels across the promoter region of the extracellular superoxide dismutase (ec-SOD) gene, and ec-SOD activity in healthy men. METHODS: Serum folate levels were measured in 111 subjects who were categorized in quintiles according to their folate status. Subjects located at the lowest, middle, and upper quintiles were selected for assessment of SAM and SAH by high-performance liquid chromatography, C677T genotype of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, ec-SOD methylation of CpG sites in lymphocytes genomic DNA by bisulfate treatment, and ec-SOD activity by a chemical assay. RESULTS: Sixteen subjects were in the lowest serum folate quintile (<23.6 nmol/L), 17 in the middle (>34-<42 nmol/L), and 14 in the highest (>45nmol/L). SAM concentration was higher in the upper than in the middle and lowest quintiles (5.57 +/- 1.58, 2.52 +/- 0.97, 2.29 +/- 1.2 micromol/L; P < 0.0001). SAH concentration was higher in the upper compared with the lowest quintile (0.76 +/- 0.24 versus 0.52 +/- 0.23 micromol/L, P < 0.001). There were no differences in the SAM/SAH ratio, ec-SOD activity, methylation status of CpG sites of the ec-SOD gene, and TMTHFR C677T genotype between groups. CONCLUSION: Serum folate concentrations in the highest quintile among healthy humans are associated with increased erythrocyte SAM and SAH concentrations, but not with SAM/SAH ratio or with methylation levels of CpG sites across the promoter region of the ec-SOD gene. Further research is required to determine if these findings are beneficial or harmful.

Fraude alimentario: Pasado, presente y futuro

La adulteración y el fraude de productos alimenticios son tan antiguos como los sistemas de procesamiento y producción de alimentos, sin embargo, en la actualidad son cada vez más frecuentes. Con la globalización y los sistemas de producción y distribución de alimentos cada día más complejos, la adulteración puede ocurrir en distintos puntos de la cadena alimentaria y tener un impacto de gran alcance, e incluso, consecuencias adversas para la salud de las personas. Los enfoques regulatorios de la comunidad internacional para enfrentar y resolver el fraude alimentario están dispersos y en constantes ajustes. Se necesita un enfoque colectivo y coordinado para identificar a todos los interesados en la cadena de suministro de alimentos, calificarlos y certificarlos, excluir a aquellos que no cumplan con los estándares adecuados y rastrear los alimentos en tiempo real. Este artículo de actualización revisará conceptos claves asociados a la integridad alimentaria, historia del fraude alimentario y episodios de fraude alimentario de connotación pública en el mundo y en Chile, herramientas analíticas y alimentos más vulnerables al fraude, reglamentos y nuevas acciones en el mundo y en Chile para enfrentar la inocuidad y el riesgo de fraude alimentario.

Food adulteration and food fraud is as old as food production and processing however, it is increasingly prevalent today. With globalization and increasingly complex food production and distribution systems, adulteration can occur at different points in the food chain and may have far-reaching impacts and even adverse consequences for human health. The international community's regulatory approaches to confronting and resolving food fraud are scattered and in constant adjustment. A collective and coordinated approach is needed to identify all stakeholders in the food supply chain, certify and qualify them, exclude those who do not meet applicable standards, and trace food in real time. This update provides definitions and background on key concepts associated with food integrity, episodes of food fraud in Chile and the world, main foods vulnerable to food fraud, common fraud practices and analytical techniques, regulations and new actions in Chile and the world to face food safety and the risk of food fraud.

5-methyltetrahydrofolic acid stimulates endothelin-1 production in low density lipoprotein-treated human endothelial cells.

BACKGROUND AND AIMS: Several studies have shown the beneficial effects of folate treatment in improving cardiovascular function. However, the mechanisms involved have not been clearly identified. The aim of this study is to determine the effect of folates and vitamin B12 on endothelial vasoconstriction/vasodilatation parameters in cultured human endothelial cells incubated with human low density lipoproteins (LDL). METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) were extracted from recently delivered umbilical cords, cultured until confluence was achieved, and then incubated for 24h with folic acid (FA), 5-methyltetrahydrofolic acid (5-MTHF) or vitamin B12 (B12) in the presence or absence of LDL that was isolated from healthy volunteers. Total nitrites (as a measure of nitric oxide production), thiobarbituric acid reactive species (TBARS, a parameter of lipid peroxidation), and endothelin-1 (ET-1) were determined in the incubation media. None of the vitamins, either in the presence or absence of LDL, was able to modify nitric oxide production by HUVEC. A significant reduction of ET-1 production was observed in LDL-treated cells. This effect was not modified by FA or B12; however, 5-MTHF caused a concentration-dependent increase on ET-1 production, an effect coincidental with reduced TBARS production. CONCLUSIONS: This study demonstrates for the first time that 5-MTHF, but not FA or B12, increases ET-1 production in LDL-treated endothelial cells. Although this effect was associated with the antioxidant properties of this folate, our results show that additional specific mechanisms involving 5-MTHF-LDL interactions may be operating to regulate endothelial function.

Folate Transporters in Placentas from Preterm Newborns and Their Relation to Cord Blood Folate and Vitamin B12 Levels.

Folate deficiency during pregnancy has been related to low birth weight, preterm (PT) birth and other health risks in the offspring; however, it is unknown whether prematurity is related to low folate transport through the placenta due to altered expression of specific folate transporters. We determined placental expression (mRNA and protein concentrations by RT-qPCR and WB respectively) of specific folate transporters: RFC, PCFT/HCP1 and FOLR1 in chorionic (fetal) and basal (maternal) plates of placentas of PT pregnancies (PT, 32-36 weeks, n = 51). Term placentas were used as controls (T, 37-41 weeks, n = 47). Folates and vitamin B12 levels were measured by electrochemiluminescence in umbilical cord blood of newborns. FOLR1 mRNA expression was lower and protein concentration higher in PT placentas (both plates) relative to the control group (p <0.05). In addition, gestational age was positively correlated with mRNA expression (Rho = 0.7), and negatively with protein concentration (Rho = -0.7 for chorionic and -0.43 for basal plate). PCFT/HCP1 mRNA was lower in PT placentas, without changes in protein levels. RFC did not differ in PT placentas compared to controls. PT newborns presented higher cord blood folate level (p = 0.049) along with lower vitamin B12 concentration compared to controls (p = 0.037).In conclusion, placental FOLR1 mRNA was positively associated with gestational age. Conversely, FOLR1 protein concentrations along with folate/vitamin B12 ratio in cord blood were negatively associated with gestational age. Placental FOLR1 is likely the main placental folate transporter to the fetus in newborns.

Smoking specifically induces metallothionein-2 isoform in human placenta at term.

Recently, we reported the presence of higher levels of metallothionein (MT) in placentas of smokers compared to non-smokers. In the present study, we designed experiments to separate and evaluate two isoforms of MT (MT-1 and MT-2) in placentas of smokers and non-smokers. Metallothionein was extracted and separated by ion-exchange high performance liquid chromatography (HPLC), previous saturation with cadmium chloride. Two peaks eluting at 6 and 12.5 min, corresponding to MT-1 and MT-2, respectively, were obtained. Metallothionein present in both peaks was identified by Western blot analysis using a monoclonal antibody directed against MT-1 and MT-2. Each isoform concentration was calculated after measuring its cadmium content by atomic absorption spectrometry with inductively coupled-plasma. In placentas of smokers, MT-2 levels increased by seven-fold compared to non-smokers, whereas MT-1 was not changed. Total placental cadmium and zinc concentrations, determined by atomic absorption spectrometry and neutron activation analysis, respectively, were higher in smokers. Metallothioneins levels were clearly in excess to bind all cadmium ions present in placentas. However, most of placental zinc remains unbound to MTs, although as much as twice zinc ions could be bound to MT in smokers. In conclusion, MT-2 is the main isoform induced by smoking, suggesting that this isoform could be involved in placental cadmium and zinc retention. This fact, which could contribute to reduce the transference of zinc to the fetus, may be associated to detrimental effects on fetal growth and development.

Acrilamida en los alimentos: Valores de referencia, recomendaciones y acciones de mitigación

RESUMEN Diversas agencias internacionales han considerado que la acrilamida puede producir efectos dañinos en la salud de la población debido a una serie de estudios toxicológicos realizados en modelos animales, en los cuales se observan efectos cancerígenos, genotóxicos, neurotóxicos, inmunológicos y en la salud reproductiva. A pesar de la creciente preocupación en diversos países sobre los potenciales efectos en salud humana, los organismos encargados de determinar límites toxicológicos no han definido aún los límites máximos de acrilamida que pueden estar presente en los diferentes tipos de alimentos para que sean inocuos para la población. El objetivo de esta actualización es revisar las regulaciones existentes sobre la acrilamida y enfatizar la necesidad de establecer límites que la industria alimentaria pueda aplicar efectivamente, además de la necesidad de contar con valores máximos diarios tolerables para prevenir los efectos nocivos para la salud de la población.

ABSTRACT Several international agencies have considered that acrylamide can induce deleterious effects in human health due to a series of toxicological studies conducted in animal models, in which carcinogenic, genotoxic, neurotoxic, immunological and reproductive effects have been observed. Despite a growing concern about these effects on human health, agencies responsible for determining toxicological limits in various countries have not yet defined the maximum levels of acrylamide that may be present in the different types of food to be safe for the population. The objective of this updated review is to evaluate the existing regulations on acrylamide and emphasize the need to establish limits that the food industry can effectively apply, in addition to the need to have tolerable daily maximum values to prevent harmful effects on the population health.

Relationship between folate transporters expression in human placentas at term and birth weights.

BACKGROUND: Adequate folate levels are essential for successful pregnancy outcomes. We aimed to study the relationship between placental mRNA and protein levels of folate transporters to birth weight. METHODS: Placental folate transporters (FOLR1, RFC1 and HCP1/PCFT) mRNA and protein levels in basal (BP) and chorionic plate (CP) of small (SGA), appropriate (AGA) and large (LGA) for gestational age term infants (≥37 weeks gestation, n = 111) were determined by real-time PCR and Western blot respectively. RESULTS: FOLR1 and HCP1/PCFT mRNA were lower in both plates of SGA and LGA placentas compared to AGA (p < 0.01) and RFC1 mRNA was lower only in CP (p < 0.02). RFC1 protein levels were lower in BP of SGA (p < 0.05) and LGA (p < 0.01), and FOLR1 protein levels were lower in CP of SGA (p < 0.02) and LGA (p < 0.01) groups compared to AGA. HCP1/PCFT protein levels remained unchanged in all groups. CONCLUSION: Placentas of SGA and LGA groups showed a reduced mRNA expression and protein levels of folate transporters, with some differences depending on the location within the placenta (BP or CP). This suggests the presence of specific placental regulation mechanisms in gene expression that may be associated to birth weight.

Increased levels of metallothionein in placenta of smokers.

Experiments were designed to evaluate and compare metallothionein (MT), zinc and cadmium levels in human placentas of smoking and non-smoking women. Smoking was assessed by self-reported cigarette consumption and urine cotinine levels before delivery. Smoking pregnant women with urine cotinine levels higher than 130 ng/ml were included in the smoking group. Determination of placental MT was performed by western blot analysis after tissue homogenization and saturation with cadmium chloride (1000 ppm). Metallothionein was analyzed with a monoclonal antibody raised against MT-1 and MT-2 and with a second anti mouse antibody conjugated to alkaline phosphatase. Zinc and cadmium were determined by neutron activation analysis and atomic absorption spectrometry respectively. Smokers showed higher placental MT and cadmium levels, together with decreased newborn birth weights, as compared to non-smokers. The semi-quantitative analysis of western blots by band densitometry indicated that darker bands corresponded to MT present in smokers' samples. This study confirms that cigarette smoking increases cadmium accumulation in placental tissue and suggests that this element has a stimulatory effect on placental MT production.

Effect of homocysteine, folates, and cobalamin on endothelial cell- and copper-induced LDL oxidation.

Oxidation of LDL contributes to endothelial dysfunction and atherosclerosis. This process could be associated with hyperhomocysteinemia, a condition that can be reduced after folic acid treatment. Because a reduction in LDL oxidation may improve endothelial function, we studied the effect of some vitamins (folic acid, 5-methyltetrahydrofolic acid, and vitamin B-12) on LDL oxidation, either in the presence or absence of homocysteine. For this purpose, two in vitro systems were used: an endothelial cell-catalyzed LDL oxidation system and a cell-free copper-initiated LDL oxidation system. The kinetics of copper-catalyzed LDL oxidation was determined by continuous monitoring of the production of conjugated dienes in the reaction medium. TBARS production, a parameter of lipid peroxidation, was also evaluated. In both in vitro systems, only 5-methyltetrahydrofolic acid was able to decrease TBARS production in a concentration-dependent manner, independently of the presence or absence of homocysteine. In the copper-induced LDL oxidation system, vitamin B-12 and 5-methyltetrahydrofolic acid increased the lag time of conjugated diene production by 25 and 47%, respectively, suggesting that both vitamins in this system had antioxidant properties. Folic acid was unable to show antioxidant properties when included in either in vitro system. The results demonstrate that 5-methyltetrahydrofolic acid and vitamin B-12 are important protective agents against LDL oxidative modifications.

Folic acid supplementation during pregnancy induces sex-specific changes in methylation and expression of placental 11ß-hydroxysteroid dehydrogenase 2 in rats.

In the placenta, 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) limits fetal glucocorticoid exposure and its inhibition has been associated to low birth weight. Its expression, encoded by the HSD11B2 gene is regulated by DNA methylation. We hypothesized that maternal diets supplemented with folic acid (FA) during pregnancy modify the expression of placental HSD11B2 through gene methylation. Wistar rats were fed with high (8 mg/kg) or normal low (1mg/kg, control) levels of FA during pregnancy. Concentrations of mRNA and protein in placentas were determined by qRT-PCR and Western blot respectively. Methylation in five CpG sites of the placental HSD11B2 promoter (-378 to -275) was analyzed by bacterial cloning and subsequent sequencing. In the FA-supplemented group, mRNA and protein levels of 11ß-HSD2 decreased by 58% and increased by 89%, respectively, only in placentas attached to males. In controls, most CpG sites were not methylated except for the CpG2 site which was 80% methylated. CpG2 methylation level increased under the FA treatment; however, only in placentas attached to females was this increase significant (113%). This change was not related to HSD11B2 expression. Fetal weight of females from FA- supplemented mothers was 6% higher than females from control mothers. In conclusion, this is the first study reporting that FA over supplementation during pregnancy modifies the placental HSD11B2 gene expression and methylation in a sex-dependent manner, suggesting that maternal diets with high content of FA can induce early sex-specific responses, which may lead to long-term consequences for the offspring.