Voluntary fortification with folic acid in Spain: An updated food composition database.

Folic acid (FA) is a key vitamin in the prevention of many diseases including neural tube defects. In Spain, only voluntary FA food fortification is allowed and there is a lack of compositional data to assess the contribution of these products to population's dietary folate intakes. Since 2007, our group has been compiling and updating a FA fortified food composition database. FA levels were obtained from retailers in Madrid and information provided by manufacturers. FA was also quantified by an affinity chromatography-HPLC method. In the present study we recorded 375 products. Our results show a high variability in the declared FA levels amongst different products, and food groups, which is also dependant on the commercial brand. FA overages are commonly added by manufacturers to some fortified products. FA content label claims are missing in 64% of products. This database is a useful tool to manage FA fortified foods data but it is necessary to continuously update it for the sound evaluation and monitoring of population's FA dietary intakes.

Voluntary food fortification with folic acid in Spain: predicted contribution to children's dietary intakes as assessed with new food folate composition data.

The Spanish market offers a significant number of folic acid (FA) voluntarily fortified foods. We analysed FA and (6S)-5-methyltetrahydrofolic acid ((6S)-5-CH3-H4PteGlu) content in ready-to-eat cereals (RTEC) (n=68) and cow's milk (n=25) by a previously validated affinity chromatography-HPLC method. Contribution to potential FA intakes for children aged 2-13years, was assessed using food consumption data from a representative nationwide study, folate Recommended Dietary Intakes (RDI), and Upper Levels (UL). Results showed that at all food fortification levels obtained, fortified products provided more than tenfold FA than (6S)-5-CH3-H4PteGlu. For RTEC, the high fortification level provided 6-21%, per serving, of RDI and ⩽32% of ULs at 90th percentile of RTEC consumption (P90). Milk products fortified at the higher level reached on average 54-136% of RDI per serving and only exceeded UL at P90 of milk consumption in children aged 2-5years.

Moderate or supranormal folic acid supplementation does not exert a protective effect for homocysteinemia and methylation markers in growing rats.

BACKGROUND/AIMS: Folic acid (FA) deficiency/supplementation effects seem to be dependent on age group and/or physiological status. The aim was to evaluate changes associated with rapid growth in relation to methionine metabolism in rats. METHODS: Four groups (n = 10 each) of male Sprague Dawley rats (5 weeks old) were on diets that varied in their FA content: 0 mg FA/kg diet (deficient), 2 mg FA/kg diet (control), 8 mg FA/kg diet (moderate supplementation), 40 mg FA/kg diet (supranormal supplementation). Animals were fed ad libitum for 30 days. Biomarkers of methionine metabolism and antioxidant status were evaluated. RESULTS: Serum total homocysteine concentration increased (p < 0.01) in FA deficient animals, with no differences between the supplemented groups. The hepatic 'methylation ratio' (S-adenosylmethionine/S-adenosylhomocysteine) of the FA content groups reached similar values, which were significantly higher compared to the deficient group. The brain 'methylation ratio', however, remained unmodified independently of FA content in the diet. FA deficiency induced hepatic DNA hypomethylation, and supranormal FA supplementation exerted the most protective effect (p < 0.01). Serum folate levels increased according to FA dietary level, whereas no differences were seen for vitamin B(12) and vitamin B(6). CONCLUSIONS: FA deficiency compromises methionine metabolism whereas supplementation does not show an additional positive effect compared to the control diet in growing animals.

[Folic acid fortified foods available in Spain: types of products, level of fortification and target population groups]. / Alimentos fortificados con ácido fólico comercializados en España: tipo de productos, cantidad de ácido fólico que proporcionan y población a la que van dirigidos.

INTRODUCTION: Folic acid is a potentially relevant factor in the prevention of a number of pathologies (congenital abnormalities, cardiovascular disease, colorectal cancer and neurocognitive decline). This has led to the introduction of different strategies in order to increase folate intake: nutritional education, pharmacological supplementation and mandatory or voluntary fortification of staple foods with folic acid. In Spain there is a growing number of folic acid fortified products on a voluntary basis, but there is also a lack of reliable data to assess their impact on the population's dietary folate intakes. OBJECTIVE: To gather a better knowledge of folic acid food fortification practices in Spain. METHODS: A Food Composition Database was developed using data from a market study. Also, previously published data of unfortified staple foods from Food Composition Tables was reviewed. RESULTS AND DISCUSSION: The Database included 260 folic acid fortified food items and it was periodically updated. Food groups included were primarily "Cereals and derivatives" (52%) followed by "Dairy products". Most of these foodstuffs lacked a target population for their consumption (37%) or were aimed at "Weight control" (28%) and "Children" (23%), but only 2% targeted women at a reproductive age. Number of unfortified foods included was 690. Fortification levels declared by manufacturers ranged between 15 and 430% of the Recommended Dietary Allowances (RDA) for folic acid per 100 g/ml, and simultaneous addition of B6 and B12 vitamins was observed in 75% of the products. Currently, Spain market offers a significant number of folic acid fortified products on a voluntary basis and at a level > or = 15% of the RDA per 100 g/ml or serving declared by manufacturers.

Alimentos fortificados con ácido fólico comercializados en España: tipo de productos, cantidad de ácido fólico que proporcionan y población a la que van dirigidos / Folic acid fortified foods available in Spain: Types of products, level of fortification and target population groups

Introducción: El ácido fólico presenta una relevancia potencial en la prevención de diversas patologías (malformaciones congénitas, enfermedad cardiovascular, cáncer, enfermedades neurodegenerativas), lo cual ha supuesto la implantación de estrategias para aumentar su ingesta: educación nutricional, suplementación farmacológica y fortificación obligatoria o voluntaria. En España se comercializan alimentos fortificados voluntariamente con ácido fólico, aunque se carece de datos específicos y no es posible evaluar su impacto en la ingesta de la población. Objetivos: Obtener un mayor conocimiento de las prácticas de fortificación de los alimentos con ácido fólico. Métodos: Se diseñó una Base de Datos de alimentos fortificados con ácido fólico a partir de un estudio de mercado y se recopilaron datos de alimentos no fortificados procedentes de Tablas de Composición de Alimentos. Resultados y discusión: La Base de Datos incluyó 260 alimentos fortificados. El grupo mayoritario fue el de "Cereales y derivados" (52%) seguidos por "Leche y derivados" (17%). La mayoría de productos carecía de población diana de consumo (37%) o iban dirigidos a población con "Sobrepeso" (28%) e "Infantil" (23%), siendo minoritarios los dirigidos a mujeres en edad fértil (2%). El número de alimentos no fortificados fue de 690. El nivel de fortificación declarado por los fabricantes se encontró entre 15 y 430% de la Cantidad Diaria Recomendada (CDR) de ácido fólico por 100 g/ml, y la adición conjunta de vitaminas B6 y B12 en un 75% de los productos. El mercado español ofrece ya una importante cantidad de alimentos fortificados con ácido fólico de forma voluntaria, a un nivel ≥ 15% de la CDR por 100 g/ml o ración (AU)

Introduction: Folic acid is a potentially relevant factor in the prevention of a number of pathologies (congenital abnormalities, cardiovascular disease, colorectal cancer and neurocognitive decline). This has led to the introduction of different strategies in order to increase folate intake: nutritional education, pharmacological supplementation and mandatory or voluntary fortification of staple foods with folic acid. In Spain there is a growing number of folic acid fortified products on a voluntary basis, but there is also a lack of reliable data to assess their impact on the population's dietary folate intakes. Objective: To gather a better knowledge of folic acid food fortification practices in Spain. Methods: A Food Composition Database was developed using data from a market study. Also, previously published data of unfortified staple foods from Food Composition Tables was reviewed. Results and discussion: The Database included 260 folic acid fortified food items and it was periodically updated. Food groups included were primarily "Cereals and derivatives" (52%) followed by "Dairy products". Most of these foodstuffs lacked a target population for their consumption (37%) or were aimed at "Weight control" (28%) and "Children" (23%), but only 2% targeted women at a reproductive age. Number of unfortified foods included was 690. Fortification levels declared by manufacturers ranged between 15 and 430% of the Recommended Dietary Allowances RDA) for folic acid per 100 g/ml, and simultaneous addition of B6 and B12 vitamins was observed in75% of the products. Currently, Spain market offers a significant number of folic acid fortified products on a voluntary basis and at a level ≥ 15% of the RDA per 100 g/ml or serving declared by manufacturers (AU)

Folate status and S-adenosylmethionine/S-adenosylhomocysteine ratio in colorectal adenocarcinoma in humans.

OBJECTIVES: This study reports the influence of colorectal neoplasia on methylation intermediates and folate concentrations in human colonic mucosa, as well as systemic measures of folate status, to examine biomarkers and possible mechanisms of folate-related carcinogenesis. SUBJECTS: A total of 47 patients were selected from those previously diagnosed with adenocarcinoma of the colorectum undergoing surgery. For each individual, we obtained a biopsy of the adenocarcinoma and a biopsy of normal appearing mucosa, to perform an intra-individual comparison. RESULTS: The 'methylation' ratio (S-adenosylmethionine/S-adenosylhomocysteine (SAH)) was lower in pathological tissue vs normal mucosa (P=0.08), mainly due to a much higher SAH concentration (P<0.005). Colonic folate concentration was significantly diminished in malignant tissue (P<0.0001). Plasma homocysteine concentration was within the normal to high range, and folate and vitamin B12 plasma concentrations were within the low to normal range as compared with normative values. CONCLUSION: Our results contribute to the hypothesis that altered DNA methylation and methyl metabolism is associated with colorectal neoplasia.

[Dietary food habits, nutritional status and lifestyle in menopausal women in Spain]. / Hábitos alimentarios, estado nutricional y estilos de vida en una población de mujeres menopaúsicas españolas.

AIM: To analyse the diet and the dietary food habits in a sample of 1,218 Spanish women, aged 40 to 77 years, in the menopausal or perimenopausal condition. Women were recruited nationwide and the diet was assessed using a food frequency questionnaire. RESULTS: Diet in our population group was close to the representative dietary habits of the Spanish population, except for dairy products. These were highly consumed (approximately 4 servings/day) by menopausal women. Energy, protein and cholesterol intakes, as well as most vitamins and mineral intakes, were very high, as compared to recommended dietary allowances. We only found marginal deficient intakes for folate and vitamin D. Women over 60 years consume less eggs, sweats, sugar and miscellany, and therefore less lipids and cholesterol, as well as vitamin D, than younger women. A high rate (61%) of overweight and obesity (BMI > 25) was present in our population group and was associated with a higher intake of meat, fish and eggs, as well as energy. A healthier diet (high intake of dairy products, fresh vegetables, fruits, legumes, cereals and fish) was associated with a healthier lifestyle (no smoking, physical exercise) and the use of fortified foods and vitamin/mineral supplements. A high percentage of women regularly consumed fortified food (52,7%) and micronutrient supplements (22,8%) although diets provided enough vitamins and minerals. CONCLUSION: Diet in our study group is in accordance to healthy dietary habits and physical activity recommendations. This is probably due to a greater concern for health in women close to the menopause condition.

[Homocysteine related vitamins and lifestyles in the elderly people: The SENECA study]. / Homocisteína, vitaminas relacionadas y estilos de vida en personas de edad avanzada: estudio SENECA.

The SENECA study started in 1988 and consisted of a random age- and sex-stratified sample of inhabitants of 19 European towns. A total of 2.100 elderly people were finally able to be included in the study. The present study includes results for total plasma homocysteine (tHcy) and the related vitamins folate, B12 and B6. Other style factors as alcohol consumption or smoking have been also evaluated. The lowest values for tHcy corresponded to Mediterranean countries (Portugal, Spain, and Greece), compared to central or northern european countries (Netherland or Belgium (differences higher than 4 micromol/l). In addition, an interesting north-south gradient is observed, with the lowest values for tHcy corresponding to Betanzos (Spain), 12.38 micromol/l followed by both centers in Portugal, whereas the highest concentrations are found in Maki (Poland), 21.92 pmol/I and Culemborg (Netherlands), 20.41 mircromol/l. The mean tHcy concentration for all the European centers was 15.98 micromol/l. Effect of sex has been also evaluated: those countries with the lowest tHcy concentration (i.e. Spain or Portugal) show significant (p < 0.01) higher tHcy concentration in men vs women, whereas these differences by sex are not observed in countries with the highest tHcy values. The effect of "aging" within the same individuals after ten years of follow up was also evaluated: a significant difference was observed for the same individuals in the 10-years period. Plasma folic acid was compared to tHcy values, resulting also in marked differences between north and southern countries. Plasma vitamin B12 also shows a close pattern. Either plasma folate or vitamin B12 were shown as strong predictors of tHcy. This effect was not observed for plasma vitamin B6. Total alcohol intake was positively and significantly (p < 0.01) correlated with tHcy ("no" intake corresponded with the lowest tHcy, 14.3 micromol/l vs "high" intake-over 30 g/d-with the highest tHcy, 17 micromol/l). The type of alcoholic beverage was also evaluated: wine and spirits drinkers showed positively significant (p < 0.005) correlation whereas beer intake was not significantly associated. Smoking was also analysed: "never" smokers had the lowest tHcy concentration (13.82 +/- 0.20 micromol/1) vs "current" smokers (16.64 +/- 0.35 pmol/1), a significant difference (p < 0.05).

Hábitos alimentarios, estado nutricional y estilos de vida en una población de mujeres menopáusicas españolas / Dietary food habits, nutritional status and lifestyle in menopausal women in Spain

Objetivo: Conocer la dieta y los hábitos alimentarios, a partir de un cuestionario de frecuencia de consumo de alimentos, de una muestra de 1.218 mujeres españolas en estado de menopausia-perimenopausia y edades comprendidas entre 40 y 77 años, procedentes de todas las Comunidades Autónomas. Resultados: La dieta de la muestra de estudio es equivalente a la dieta media de la población española, destacando los productos lácteos, que son consumidos en mayor cantidad por las mujeres participantes (aproximadamente 4 raciones/ día). La ingesta energética, de proteínas y de colesterol es elevada, así como de la mayoría de vitaminas y minerales. Únicamente se han encontrado ingestas deficitarias moderadas para el ácido fólico y la vitamina D. Las mujeres de mayor edad (> 60 años) consumen menos alimentos del grupo de huevos, dulces y azúcar y varios y, por tanto, de lípidos y colesterol, aunque también de vitamina D. Un IMC elevado (IMC > 25; 61% de la población) está asociado con un mayor consumo de carnes, pescados y huevos y, por tanto, de energía. Una dieta más adecuada (mayor consumo de lácteos, frutas, verduras, legumbres, cereales y pescados) está asociada a un estilo de vida más saludable (no fumar, practicar ejercicio físico) y a la ingesta de alimentos fortificados (52,7% de la muestra total) y suplementos vitamínicos y minerales (22,8% de la muestra total), aun cuando el aporte de los micronutrientes es suficiente a través de la dieta habitual. Conclusiones: En conjunto, la dieta de nuestra población de estudio se adecúa a los patrones de dieta saludable y actividad física, reflejo de la preocupación por la salud de la mujer en la etapa de la menopausia

Aim: To analyse the diet and the dietary food habits in a sample of 1,218 Spanish women, aged 40 to 77 years, in the menopausal or perimenopausal condition. Women were recruited nationwide and the diet was assessed using a food frequency questionnaire. Results: Diet in our population group was close to the representative dietary habits of the Spanish population, except for dairy products. These were highly consumed (approximately 4 servings/day) by menopausal women. Energy, protein and cholesterol intakes, as well as most vitamins and mineral intakes, were very high, as compared to recommended dietary allowances. We only found marginal deficient intakes for folate and vitamin D. Women over 60 years consume less eggs, sweats, sugar and miscellany, and therefore less lipids and cholesterol, as well as vitamin D, than younger women. A high rate (61%) of overweight and obesity (BMI > 25) was present in our population group and was associated with a higher intake of meat, fish and eggs, as well as energy. A healthier diet (high intake of dairy products, fresh vegetables, fruits, legumes, cereals and fish) was associated with a healthier lifestyle (no smoking, physical exercise) and the use of fortified foods and vitamin/mineral supplements. A high percentage of women regularly consumed fortified food (52,7%) and micronutrient supplements (22,8%) although diets provided enough vitamins and minerals. Conclusion: Diet in our study group is in accordance to healthy dietary habits and physical activity recommendations. This is probably due to a greater concern for health in women close to the menopause condition

Homocisteína, vitaminas relacionadas y estilos de vida en personas de edad avanzada: estudio SÉNECA / Homocysteine related vitamins and lifestyles in the elderly people: SENECA study

El Estudio SÉNECA es un estudio longitudinal prospectivo, con un seguimiento de diez años, en personas de edad avanzada europeas (19 ciudades, 2.100 hombres y mujeres, nacidos entre 1913 y 1918). Se han evaluado en el presente trabajo la homocisteinemia en los diferentes países participantes, su relación con las vitaminas ácido fólico, B12 y B6, así como otros factores y estilos de vida que potencialmente pueden modificar la concentración de tHcy: alcohol y tabaco. La homocisteinemia media de todos los centros participantes es de 15,98 μmol/l, aunque con importantes diferencias geográficas: se observa un marcado patrón norte-sur en el que las concentraciones más bajas corresponden a los países mediterráneos, obteniéndose diferencias por encima de 4 μmol/l al comparar con países del centro y norte europeo. Los hombres presentan generalmente valores más elevados de tHcy que las mujeres, aunque esta diferencia no se observa en los países donde la homocisteinemia media es más elevada. Para estudiar el efecto del envejecimiento per se, se compararon los resultados de tHcy con los obtenidos diez años antes, encontrándose diferencias significativas para los mismos individuos. Los valores sanguíneos de folato y vitamina B12 también difieren significativamente según los centros, mostrando el mismo patrón norte sur y se correlacionan de forma inversa con los valores de homocisteinemia. Tanto la concentración plasmática de folato como la de vitamina B12, pero no la de vitamina B6, se comportan como predictores potentes de la tHcy. La ingesta absoluta de alcohol se correlaciona positiva y significativamente con la tHcy: los no bebedores presentan la homocisteinemia más baja mientras que los consumidores habituales de más de 30 g diarios de etanol presentan la homocisteinemia más elevada, encontrándose diferencias significativas entre ambos grupos. No obstante, el efecto difiere según el tipo de bebida más frecuente: el consumo de vino y bebidas de alta graduación alcohólica se correlaciona positivamente con los valores de Hcy, mientras que no se observa asociación en el caso de la cerveza. El tabaquismo influye en la tHcy. Así, los fumadores, pero también los ex fumadores, presentan concentraciones más elevadas que los no fumadores

The SENECA study started in 1988 and consisted of a random age- and sex-stratified sample of inhabitants of 19 European towns. A total of 2.100 elderly people were finally able to be included in the study. The present study includes results for total plasma homocysteine (tHcy) and the related vitamins folate, B12 and B6. Other style factors as alcohol consumption or smoking have been also evaluated. The lowest values for tHcy corresponded to Mediterranean countries (Portugal, Spain, and Greece), compared to central or northern european countries (Netherland or Belgium (differences higher than 4 μmol/l). In addition, an interesting north-south gradient is observed, with the lowest values for tHcy corresponding to Betanzos (Spain), 12.38 μmol/l followed by both centers in Portugal, whereas the highest concentrations are found in Maki (Poland), 21.92 μmol/l and Culemborg (Netherlands), 20.41 μmol/l. The mean tHcy concentration for all the European centers was 15.98 μmol/l. Effect of sex has been also evaluated: those countries with the lowest tHcy concentration (i.e. Spain or Portugal) show significant (p < 0.01) higher tHcy concentration in men vs women, whereas these differences by sex are not observed in countries with the highest tHcy values. The effect of “aging” within the same individuals after ten years of follow up was also evaluated: a significant difference was observed for the same individuals in the 10-years period. Plasma folic acid was compared to tHcy values, resulting also in marked differences between north and southern countries. Plasma vitamin B12 also shows a close pattern. Either plasma folate or vitamin B12 were shown as strong predictors of tHcy. This effect was not observed for plasma vitamin B6. Total alcohol intake was positively and significantly (p < 0.01) correlated with tHcy (“no” intake corresponded with the lowest tHcy, 14.3 μmol/l vs “high” intake-over 30 g/d-with the highest tHcy, 17 μmol/l). The type of alcoholic beverage was also evaluated: wine and spirits drinkers showed positively significant (p < 0.005) correlation whereas beer intake was not significantly associated. Smoking was also analysed: “never” smokers had the lowest tHcy concentration (13.82 ± 0.20 μmol/l) vs “current” smokers (16.64 ± 0.35 μmol/l), a significant difference (p < 0.05)

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.

Drugs-nutrient interactions: a potential problem during adolescence.

The concept of drug-nutrient interactions is not new, but it has only recently gained currency in medicine. Although the elderly are normally considered to be at particular risk, other groups may also be at risk: infants, adolescents, pregnant women, alcohol and tobacco users, etc. In infants and adolescents there are several factors that may influence the possible interactions: firstly, nutrient needs are usually higher, mainly micronutrients; systems for detoxification of anutrients are not complete; the tendency to restricted diets (especially girls) that are unable to cover the actual recommended intakes for a number of micronutrients (i.e. vitamins); and the dangerous increase in alcohol consumption either in males or females. Administration of drugs in population with adequate vitamin intake is usually not a problem, but administration of drugs in those with borderline intake of vitamins or in patients with low nutritional status can result in symptomatic vitamin deficiency states. The groups at risk of poor vitamin status are smokers (a high proportion of adolescents are active smokers); dieters (skipping meals and dieting to lose weight frequently compromise micronutrient intake, and it should be considered that it is extremely difficult to meet all the requirements at intakes of less than 1,200 calories per day), oral contraceptive users, and pregnant and lactating women, excessive alcohol users, etc. The chapter also focuses on the case of folate: rapidly dividing tissues during the adolescent growth spurt increase requirements for folate. Because of this increased need, folate status appears to be of concern during the age of this rapid growth. A variety of drugs are known to interfere with vitamin utilization by blocking or altering transformation of the vitamin to its metabolically active form. Serum folate levels are known to be low in a high percentage of patients with rheumatoid arthritis, suggesting that aspirin alters the transport of folate by competition for binding sites on serum proteins. Methotrexate, a drug commonly used at low doses for the treatment of psoriasis, rheumatoid arthritis and certain liver disorders, limits the availability of methyl groups derived from one-carbon metabolism by inhibiting competitively a key enzyme in the intracellular folate metabolism. In humans, the antiepileptic drug valproic acid (VPA) is associated with two major adverse effects: teratogenicity and folate deficiency. The mechanisms by which VPA exerts the teratogenic or antifolate effect remain unclear, but an alteration in the methionine cycle is the strongest hypothesis proposed.

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.

High dietary folate supplementation affects gestational development and dietary protein utilization in rats.

There is new evidence that good folate status may play a critical role in the prevention of neural tube defects and in the maintenance of adequate homocysteine levels, an amino acid recently identified as a risk factor for cardiovascular disease. This has led to different folate recommendations, all of them much higher than the present dietary recommendations. Folic acid is a water-soluble vitamin with a low potential toxicity. However, the possible consequences of long-term, high folate intakes are unknown. Therefore, the present study was undertaken to determine the effects of long-term, high dietary folate supplementation on gestational and nutritional markers in pregnant and virgin rats. Four groups of Wistar rats were classified on the basis of physiological status (virgin or pregnant) and the experimental diets administered (folic acid supplemented, 40 mg/kg diet; or control diet, 2 mg folic acid/kg diet). Rats were fed their respective diets for 3 wk. Two critical periods were used for metabolic balance studies (experimental d 1-5 and 17-21), which involved the determination of fat and protein digestibilities as well as metabolic protein utilization (MPU) and net protein utilization (NPU). Gestational development (number of live fetuses) was adequate in both diet groups regardless of folate supplementation. However, body weight and vertex-coccyx length in fetuses from supplemented dams were less than (P < 0.0001) in fetuses of control dams. Fat and nitrogen digestibilities were not affected by supplementation, but MPU and NPU coefficients were significantly lower (P < 0.05) in the folic acid-supplemented groups, irrespective of physiological status, compared to control rats. These new findings of macro-micronutrient interactions caused by high folate supplementation are discussed on the basis that the vitamin may act as a xenobiotic more than as a nutrient.

Impaired methionine synthesis and hypomethylation in rats exposed to valproate during gestation.

BACKGROUND: The antiepileptic drug valproic acid (VPA) may be teratogenic. The mechanism of teratogenicity remains unclear, but it has been hypothesized that VPA interferes with folate metabolism. OBJECTIVE: To study the effect of VPA on the methionine cycle and transmethylation reactions in pregnant rats. METHODS: Wistar rats were treated with VPA (300 mg/kg/day) on gestation days 8, 9, and 10, alone or in combination with folinic acid (FOL, 4 mg/kg/day) on gestation days 8, 9, and 10 or S-adenosylmethionine (SAM, 10 mg/kg/day) throughout gestation days 1 to 10. RESULTS: VPA induced a reduction in maternal methionine serum concentration (p < 0.05) caused by a 24% reduction of methionine synthase activity in liver. This provoked hepatic DNA hypomethylation, although the methylation ratio (S-adenosylmethionine/S-adenosylhomocysteine) was not altered. Homocysteine, folate, and vitamin B12 serum concentrations, as well as methionine adenosyltransferase and betaine homocysteine methyltransferase hepatic activities, did not change. In fetuses exposed to VPA, no effect was observed in hepatic methionine content, but the methylation ratio was reduced (p < 0.01), leading again to hepatic DNA hypomethylation. Coadministration of FOL prevented VPA-induced alterations in methionine synthesis and corrected fetal DNA hypomethylation. By contrast, SAM did not exert a protective effect on fetal DNA methylation. CONCLUSION: Impaired methionine synthesis and DNA hypomethylation may be involved in VPA-induced teratogenesis.

[Morphological changes induced by valproate and its administration concomitant with folinic acid or S-adenosylmethionine in pregnant rats]. / Alteraciones morfológicas en rata gestante, inducidas por el valproato y su administración conjunta con ácido folínico o S-adenosilmetionina.

Neural tube defects (NTD) are serious congenital abnormalities that have a multifactorial etiology, including both genetic and environmental effectors (for example, diet and/or drugs). Valproic acid (VPA) is a frequently used anti-epileptic drug that has a potentially teratogenic character, as well as the capacity for inducing NTD and other less serious malformations. However, the mechanism of action of VPA has not been clearly established, and it has been suggested that it interferes in the folate cycle and therefore, with the methionine/methylation, possibly through a metabolic blocking of some biomarker that is a key of the cycle, such as for example S-adenosylmethionine (SAM) and folic acid (FA). The objective of the present study is to analyze the morphological and histological changes, which can occur in a high risk experimental model after the administration of VPA as well as for the induction of NTD and other malformations. In addition, the protective roles of the administration of folic acid, 5-formyltetrahydrofolate (FOL) and S-adenosylmethionine (SAM) are assessed. For this pregnant "Wistar" rats classified according to the following treatments: 1) VPA (300 mg/kg/day on days 8, 9, and 10 of the pregnancy); II) VPA (300 mg/kg/day on days 8, 9, and 10 of the pregnancy) and FOL (4 mg/kg/day i.p. on days 8, 9, and 10 of the pregnancy); III) VPA (300 mg/kg/day on days 8, 9, and 10 of the pregnancy) + SAM (10 mg/kg/day, on days 1-10 of the pregnancy); IV) CONTROL (no treatment). VPA decreases the fertility index by 25% compared to the control group, it increases the number of reabsorptions by mother (1.3 +/- 0.5 vs 1.0 +/- 0.5), and decreases the number of fetuses compared to the control (9.0 +/- 1.4 vs. 12.6 +/- 0.9). In the VPA + FOL group, the numbers for these parameters approach those of the control group and the VPA + SAM group is no different from the VPA group, showing no protective factors. With respect to the bone alterations observed, when these are grouped according to whether they affect the skull, trunk, and extremities, it is seen that there are no significant differences between the groups. The histological study and the immunohistochemical analysis show liver alterations in all groups treated, and a lower number of lymphocytes in the VPA group, and a greater number of Kupffer cells, The results are discussed in relation to, first, the effect of VPA per se in the interference of the methionine/methylation cycle, and secondly, with regard to how folic acid and/or S-adenosylmethionine can improve or not some of the harmful effects induced by VPA.

Brain folates and DNA methylation in rats fed a choline deficient diet or treated with low doses of methotrexate.

In a recent study we have demonstrated that induced changes in several parameters of one-carbon metabolism in livers of rats fed either a choline deficient diet or treated with low doses of methotrexate may be reversed through restoration of an adequate choline diet and discontinuing of methotrexate (MTX) administration. The present study was aimed at comparing these two treatments for their effect on brain: total folate was 0.69 +/- 0.35 nmol/g in the control group and was significantly lower in the other two groups (0.25 +/- 0.06 in the MTX and 0.45 +/- 0.24 nmol/g in the choline deficient groups, p < 0.01). Both treatments were associated with elongations of the glutamate chains of the folate molecules: the proportion of hepta- and octaglutamyl folates increased from 16% to 31% in the MTX treated and to 33% in the choline deficient group, accompanied with concomitant decreases in the concentrations of tetra- and pentaglutamyl folates. MTX could not be detected in brain tissue in contrast to previous observations of significant amounts found in liver. DNA was significantly (p < 0.05) undermethylated in both treated groups compared to controls. Results are discussed on the basis of the possibility that choline deficiency and MTX treatment appear to impair the capacity of tissues, either a peripheral one as liver or a central one as brain, to incorporate folate.

Carbon tetrachloride-induced hepatic injury is associated with global DNA hypomethylation and homocysteinemia: effect of S-adenosylmethionine treatment.

Carbon tetrachloride (CCl4) administration to rats produces hepatic cirrhosis and supplementation with S-adenosylmethionine (SAM) can partially prevent CCl4-induced liver injury. These effects are thought to be caused by oxidative stress and the subsequent formation of free radicals, but the mechanism whereby this occurs and the accurate nature of the mechanisms by which SAM exerts its protective action are not well understood. The effect of short-term administration of CCl4 on hepatic DNA methylation and on SAM and S-adenosylhomocysteine (SAH) were assessed. CCl4 administration to rats for 3 weeks resulted in hypomethylation of liver DNA, determined by comparing the extent to which DNA from livers of control or treated animals could be methylated in vitro using [3H-methyl] SAM as methyl donor. This CCl4 effect on DNA methylation was corrected by the administration of SAM (10 mg/kg/d, intramuscularly), with values of methyl groups incorporation comparable with those observed in the control animals. hepatic SAM was decreased by CCl4 (65.3 +/- 5.27 vs. 102.2 +/- 4.89 nmol/g; P < .05) and SAH was increased (69.5 +/- 14.6 vs. 29.4 +/- 3.83 nmol/g; P < .05). This led to a marked reduction of the SAM/SAH ratio (the methylation ratio) from 3.47 in control rats to 0.94 in CCl4-treated animals (P < .05). SAM treatment partially prevented (P < .05) the reduction of the ratio SAM/SAH induced by CCl4. CCl4 also induced a marked elevation of serum homocysteine levels (more than 20-fold; P < .001), which was partially prevented by SAM administration.(ABSTRACT TRUNCATED AT 250 WORDS)