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Analysis of N-nitroso folic acid, a nitrosamine impurity found in folic acid, is challenging due to the complex sample matrices. Many of such supplements contain not only a variety of vitamins, including vitamin A, Bs, C, D and E, but also other ingredients such as minerals, docosahexaenoic acid (DHA), glucose syrup, sugar, and herbs. On the other hand, the strength of folic acid is typically low, ranging from 50 µg to 5 mg per unit. In this study, a highly selective and sensitive LC-MS/MS method was developed to accurately quantify N-nitroso folic acid in supplements containing folic acid. The sample was extracted by 0.1% ammonia solution: MeOH (9:1, v/v) containing 5 ng/mL of N-nitroso folic acid-d 4 (Isotope internal standard). The quantification was performed by MRM in negative ionization mode. Mobile phases A and B were 0.1% formic acid in deionized water and methanol, respectively. The method was validated and found to have sufficient linearity (R 2  > 0.995), accuracy (recovery 83-110%), precision (RSD 3%) and low LOD, LOQ (4 and 10 µg/g respectively, with respect to folic acid). The method was applied to the determination of N-nitroso folic acid in 40 supplements containing folic acid with different strengths and formulation. The content of N-nitroso folic acid was found to be up to 898 ng/unit (1794 µg/g with respect to folic acid). It enabled regulatory actions, such as product recall, to safeguard public health from unsafe products.

This study explored whether using a coal or biomass stove for cooking was associated with a greater risk of red blood cell (RBC) folate insufficiency among pregnant women compared to using clean energy. A researcher-designed questionnaire was used to collect information on exposure-related factors and confounding factors. RBC folate concentrations were examined by microbiological assay. Binary logistic regression analysis was used to identify factors related to RBC folate insufficiency. The use of coal or firewood for cooking was associated with an increased risk of RBC folate insufficiency (<906 nmol/L) compared to gas. In subgroup analyses, associations between the use of polluting cooking fuels and folate insufficiency were positive for both urban and rural residents and statistically significant for rural women. Efforts to promote the use of clean energy and proper ventilation, especially in rural areas, are recommended to improve the health of pregnant women and their offspring.

Background: As folates are essential for embryonic development and growth, it is necessary to accurately determine the levels of folates in plasma and red blood cells (RBCs) for clinical intervention. The aims of this study were to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of folates in plasma and RBCs and to examine the association between plasma and RBC folate concentrations and gestational diabetes mellitus (GDM), gestational hypertension (GH) and preeclampsia (PE)., Methods: With the in-house developed LC-MS/MS, a retrospective cross-sectional study was conducted. The healthy pregnant women of first- ( n  = 147), second- ( n  = 84) and third-trimester ( n  = 141) or the women diagnosed with GDM ( n  = 84), GH ( n  = 58) or PE ( n  = 23), that were aged between 22 and 46 years old and registered at our institute, were subjected for measurement of folic acid (FA) and 5-methyltetrahydrofolate (5-MTHF), followed by appropriate statistical association analysis., Results: The assay for simultaneous quantitation of FA and 5-MTHF in plasma and RBCs was linear, stable, with imprecision less than 15% and recoveries within ±10%. The lower limits of quantification for FA and 5-MTHF measurement in whole blood were 0.57 and 1.09 nmol/L, and in plasma were 0.5 and 1 nmol/L, respectively. In the association analysis, the patients with lower RBC folate level (<906 nmol/L) presented higher risks of PE development (OR 4.861 [95% CI 1.411-16.505]) by logistic regression and restricted cubic spline (RCS) regression in a nonlinear fashion. In addition, higher level of plasma folates in pregnancy was significantly associated with GH risk but may be protective for the development of GDM., Conclusions: The in-house developed LC-MS/MS method for folates and metabolites in plasma or RBC showed satisfactory analytical performance for clinical application. Further, the levels of folates and metabolites were diversely associated with GDM, GH and PE development.

Mucosal-associated invariant T-cells (MAIT) can react to metabolites of the vitamins riboflavin and folate which are produced by the human gut microbiota. Since several studies showed that the pesticide chlorpyrifos (CPF) and glyphosate (GLP) can impair the gut microbiota, the present study was undertaken to investigate the impact of CPF and GLP treatment on the metabolism of gut microbiota and the resulting bacteria-mediated modulation of MAIT cell activity. Here, Bifidobacterium adolescentis ( B. adolescentis ), Lactobacillus reuteri ( L. reuteri ), and Escherichia coli ( E. coli ) were treated with CPF (50-200 µM) or GLP (75-300 mg/L) and then used in MAIT cell stimulation assays as well as in vitamin and proteome analyses. All three bacteria were nonpathogenic and chosen as representatives of a healthy human gut microflora. The results showed that E. coli activated MAIT cells whereas B. adolescentis and L. reuteri inhibited MAIT cell activation. CPF treatment significantly increased E. coli -mediated MAIT cell activation. Treatment of B. adolescentis and L. reuteri with CPF and GLP weakened the inhibition of MAIT cell activation. Riboflavin and folate production by the test bacteria was influenced by CPF treatment, whereas GLP had only minor effects. Proteomic analysis of CPF-treated E. coli revealed changes in the riboflavin and folate biosynthesis pathways. The findings here suggest that the metabolism of the analyzed bacteria could be altered by exposure to CPF and GLP, leading to an increased pro-inflammatory immune response.

Rice fortification offers great potential to deliver essential micronutrients to a large part of the world population. However, high temperatures required for cooking rice are deleterious to thermally labile micronutrients. This study evaluated the content and stability of thiamin and folic acid in fortified rice after different cooking methods (stir-frying, boiling, cooking in a microwave oven and boiling in a Food Service). The analyses were performed by High Performance Liquid Chromatography (HPLC). The fortified rice showed the highest thiamin content (0.97 mg/kg) and retention (65.4%) when cooked in microwave oven, and for folic acid the lowest content (0.17 mg/kg) and the highest retention (96.11%) when cooked in a Food Service and stir-fried, respectively. In conclusion, the stability of thiamin and folic acid varied according to the cooking method and the stability of folic acid presented higher percentages in relation to thiamin in the different methods. [Formula: see text].

A simple and efficient ultrasonic-assisted extraction (UAE) procedure has been proposed for the pre-concentration of (2S)-2-[(4-{[(2-amino-4-hydroxypteridin-yl)methyl]amino}phenyl)formamido]pentanedioic acid (folic acid) in vegetables, pharmaceuticals and foods prior to determination at 540 nm using UV-Vis spectrophotometry. The method is based on hydrophobic ternary complex formation of folic acid with silver ions in the presence of cetyltrimethylammonium bromide (CTAB) as a sensitivity enhancer counter ion at pH 7.0, and then extraction into a micellar phase of polyethylene glycol monoalkyl ether (Genapol X-080). The impacts on the extraction efficiency and complex formation of analytical parameters such as sample pH, concentration of silver, concentration of surfactants and extraction time, ultrasonic time and sample volume, were investigated and optimised in detail. The matrix effect on the pre-concentration and determination of folic acid was investigated, and it was observed that the proposed method was highly selective against possible matrix co-extractives. Under optimised conditions, a good linear relationship between the analytical signal and folic acid concentration was obtained in the range of 0.6-180 μg l(-1) with a detection limit of 0.19 μg l(-1) and quantification limit of 0.63 μg l(-1). The applicability was evaluated using samples fortified at different concentration levels, and recoveries higher than 94.1% were obtained. The precision as the percent relative standard deviation (RSD%) was in range of 2.5-3.8% (10 and 40 μg l(-1), n = 5). The proposed method was validated by analysis of two standard reference materials (SRMs) and various real samples, and satisfactory results were obtained.

In the Netherlands, vitamin intake occurs mainly via food and for some vitamins also via fortified food. In addition, some people take dietary supplements. Information on the bioavailability of vitamins is important for a good estimation of the actual exposure to vitamins. Furthermore, for a reliable intake estimation, it is important to know the accurateness of the claimed vitamin concentration on the product label. In the current study, the amount of vitamin A, vitamin C, and folic acid in different products and their maximum bioavailability (bioaccessibility) were investigated. In about half of the products, the amount of vitamins significantly deviated from the declared amounts. The vitamin bioaccessibility ranged from <1% to 100%. When assessing the dietary intake exposure of vitamins, it is important to take into account both the possible deviation from the declared level and (the variability of) the bioaccessibility of the vitamin in the products.

Objective: To investigate the frequencies of vitamin B12 and folate deficiencies in pregnant women in low socioeconomic group, the relation between the animal-source foods consumption and maternal vitamin B12-folate statuses, and their impacts on anthropometric measurements of the infants., Methods: A total of 208 pregnant women in the last trimester were included in the study. A questionnaire about socio-demographic status, consumption of meat, egg, milk-dairy products, multivitamin supplementation was used. Vitamin B(12) and folate concentrations were studied by chemiluminescence method. The babies of Vitamin B(12) deficient mothers were evaluated after birth., Results: The rate of vitamin B(12) deficiency was 47.6% and folate deficiency was 17.3% of pregnant women. Animal food consumption was inadequate about half of pregnant women and vitamin B(12) levels in these women were significantly low. There were no statistically significant relationships between the birth weight, birth length and head circumference measurements, and maternal vitamin B(12) and folate concentrations., Conclusion: The rate of vitamin B12 deficiency in pregnant women in low socioeconomic population is high. Although there were no significant effects of the vitamin B12 and folate deficiencies on birth size, additional studies are required to elucidate the subsequent effects.

Objective: To determine if maternal circulating red blood cell (RBC) folate concentration in early pregnancy is associated with late gestation pregnancy complications including small for gestational age (SGA) infants, preeclampsia and preterm birth (PTB) in a socioeconomically disadvantaged population., Method: This was a retrospective case control study, conducted at Lyell McEwin Health Service, South Australia, including 400 primiparous women. RBC folate and demographic data were collected at 10-12 weeks gestation. Pregnancy outcome data were obtained from patient case notes., Results: Patients who were folate deficient were more likely to develop pregnancy complications, specifically SGA (OR 6.9, 95% CI 2-24.3) and PTB (OR 5.4 95% CI 1.4-21.2). Those who were folate insufficient were also at increased risk of SGA (OR 3.0, 95% CI 1.3-7.7). No association between folate and preeclampsia was found. Women who were supplementing with folic acid delivered infants who were 179 g heavier (5.5% increased birth weight, P = 0.003) and 4.5 days later, compared to those who did not supplement. Furthermore, low RBC folate was associated with cigarette smoking (P < 0.001)., Conclusions: Maternal RBC folate concentration in early pregnancy is associated with SGA and PTB, but not with preeclampsia.

Objective: A prospective cohort study conducted in Italy on homocysteine and red cell folate levels in pregnancy and pregnancy outcome., Methods: A cohort of pregnant women was identified in 21 Italian obstetric centers. Women were eligible at 8th-10th week of gestation. All women were followed up to delivery in order to collect information on pregnancy outcome and birth weight (BW)., Results: Two hundred forty-four women were included in the analysis. In women not reporting current use of folate at study entry, the median homocysteine levels (micromol/l) were 7.9, 8.1, and 8.5 at the 8th-10th, 16th, and 22nd week of gestation, respectively; the median red cells folate levels (nmol/dl) were 3.9, 6.1, and 7.0. The mean BW tended to be lower in the 2nd and 3rd tertile of homocysteine levels in comparison with the 1st tertile (p<0.05 at the 8th-10th and 22nd week). The mean week of gestation at birth was higher in subjects in the 2nd and 3rd tertile of red cell folate at the 8th?10th and 16th week of gestation., Conclusion: This study estimated homocysteine and plasma folate levels in Italian pregnant women and suggested that homocysteine level was associated with BW.

Folate concentrations in selected fruits were measured using the trienzyme extraction and microbiological assay with Lactobacillus casei (subsp. Rhamnosus) as an assay organism. Fruits were purchased from different retail outlets at Coimbatore, Tamilnadu, India and were analyzed for total folate content. The folate content in all fruits varied considerably on a fresh weight basis from 10 to 328 microg/100 g, with tropical fruits ranging between 10 and 211 microg/100 g, temperate fruits from 11 to 328 microg/100 g, and the subtropical fruits in the range of 9-237 microg/100 g. Amongst all fruits, plum had the highest content of folate (328 microg/100 g). Data analyzed will assist dietary studies to estimate and evaluate the adequacy of folate intakes of the population, to formulate experimental diets for folate bioavailability studies, and to revise dietary recommendations for the population. In addition, the data will assist the health authorities in planning and executing strategies for intervention programs.

The aim of the present work was to determine parameters of the corn and wheat flour matrix, such as protein, lipid, moisture, ash and carbohydrates, folic acid and iron contents. Three principal components explained 91% of the total variance. Wheat flours were characterized by high protein and moisture content. On the other hand, the corn flours had the greater carbohydrates, lipids and folic acid levels. The concentrations of folic acid were lower than the issued value for wheat flours. Nevertheless, corn flours presented extremely high values. The iron concentration was higher than that recommended in Brazilian legislation. Poor homogenization of folic acid and iron was observed in enriched flours. This study could be useful to help the governmental authorities in the enriched food programs evaluation.

The prevalence estimate of neural tube defects in India is restricted to some village clusters and has been reported to be 6.57-8.21 per 1,000 live births, one of the highest in the world. Dietary intake data for folate are scant and do not provide an accurate estimate because of the lack of analysed data in commonly consumed cooked/processed foods. A 24-h dietary recall survey of 200 respondents aged 17-24 years in one of the Southern states of India was used to prioritize the common foods consumed. Forty-three foods/preparations were analysed for their total folate content using the tri-enzyme extraction technique and microbiological assay using Lactobacillus casei, subsp. Rhamnosus. The mean dietary intake of the selected population was estimated to be 277 microg/day based on the analytical results obtained through this study. This value is relatively higher (2.8 times) than the calculated values reported on the intakes of the rural population (98 microg/day) reported by the National Nutrition Monitoring Bureau, India.

Objective: To compare the nutritional parameters of individuals with a previous diagnosis of coeliac disease (CD) with those of screen-detected patients in a large cohort of adults in primary care., Material and Methods: A cohort of 1900 adults (aged 18-64 years) was screened for tissue transglutaminase antibodies (TG2A) in primary care in the capital region of Finland. IgA-endomysium antibodies (EmA), HLADQ2/8 associated with CD were determined in positive individuals. Folate, iron, and transferrin receptor in sera were assessed in patients reporting a previous diagnosis of CD and patients positive for the above tests., Results: Twenty-two out of 1900 (1%) patients reported a previous diagnosis of CD (biopsy-based 16/22 cases; 6/22 cases diagnostic criteria unknown). Among the screen-detected cases with TG2A> or = the cut-off value, 14/32 cases were considered to have CD based on high levels of both TG2A and EmA, DQ2/8 genotype and/or biopsy results. The prevalence of CD was as high as 1:53 in the total study population (36/1900), and in women even 1:46 (2.2%). Nutritional deficiencies were rare among CD patients diagnosed earlier (low iron = 1; low folate n=1) but common among those who had an undiagnosed CD (low folate n=6; p<0.005; concomitant iron deficiency n=2). One-third of the screen-detected CD patients were obese. Screen-detected patients did not present more abdominal symptoms than those with no CD., Conclusions: CD is common, the proportion among women possibly being as high as 2.2%. Although the great majority of screen-detected patients do not present any gastrointestinal symptoms at primary care, nutritional deficiencies such as low folate levels and iron deficiency are common.

Availability of non-heme iron has been extensively discussed when meals comprise heme as well as non-heme iron, but seldom so for exclusively vegetarian meals. The present study aimed to develop a statistical model for predicting non-heme iron availability from a composite vegetarian meal. Radioisotopic measurements of in vitro iron dialyzability of 208 out of 274 meals representing vegetarian diets from Asia, Africa, Europe and Latin America and the meal contents of iron, zinc, copper, ascorbic acid, beta-carotene, riboflavin, thiamin, folic acid, tannic acid, fiber and degraded phytate forms (IP6-IP1) were used for development of the model. A multiple regression model weighted for calorie contents was developed for the percentage iron dialyzability with the possible predictors as meal contents along with plausible interaction terms. The model was validated with in vitro iron dialyzability of 66 meals and in vivo iron absorption in five ileostomized adults. Application of the model was demonstrated using data on the daily dietary intake of 215 young adults whose hemoglobin levels were estimated twice in 3 weeks. Weighted multiple regression model was: ln(% Fe dialyzability)=1.340-0.259xln(IP2 [mg])+0.188xln(IP3 [mg])-0.278xln(IP5 [mg])+0.0912xln(ascorbic acid [mg])+0.06693xln(tannins [mg])+0.09552xln(beta-carotene [microg])+0.137xln(hemicellulose [g]) (P<0.01, R2=0.51). Good agreement was seen between observed and predicted dialyzability (r=0.90) and human absorption (r=0.89). The model would be useful to estimate bioavailable iron intakes of vegetarian populations and to identify at-risk individuals.

Kinetics of folic acid degradation in cowpea (Vigna catjang L.) as well as in pure folic acid solutions at initial concentrations present in cowpea over a temperature range of 50-120 degrees C (isothermal conditions) followed first order reaction kinetics, where the rate constant increased with an increase in temperature. The temperature dependence of degradation was adequately modeled by Arrhenius equation. The degradation was also evaluated in normal open pan cooking, pressure-cooking and a newly developed and patented fuel-efficient Eco Cooker (non-isothermal heating process). A mathematical model was developed using the isothermal kinetic parameters obtained to predict the losses of folic acid from the time-temperature data of the non-isothermal heat processing method. The results obtained indicate the folic acid degradation is of a similar order of magnitude in all the methods of cooking.

Background: Folate deficiency occurs frequently and the related hyper-homocysteinaemia is considered a risk factor for thrombosis. We investigated folate status and homocysteine (Hcy) concentration in patients under 60 years on oral anticoagulant therapy (OAT) for previous venous or arterial thrombosis and in healthy blood donors., Patients and Method: Thirty-nine patients (mean age 35.2 years) on OAT for longer than 6 months and forty 44 healthy blood donors (mean age 36.0 years) were evaluated. Diet, serum folate (SF), red blood cell folate (RCF), homocysteinaemia, vitamin B12 levels and the mutation C677T of methylenetetrahydrofolate-reductase (MTHFR) gene were determined., Results: The mean SF and Hcy concentrations were significantly higher in patients compared with blood donors (SF = 17.7 versus 10.5 nmol/L, P < 0.0001; Hcy = 11.7 versus 8.9 micromol/L, P = 0.009). Twelve out of 39 patients and 7 out of 44 blood donors were homozygous for the mutation C677T of MTHFR gene. Among the remaining subjects, non-homozygous for the mutation, the patients (27) had mean SF and Hcy levels significantly higher than the (37) blood donors (SF = 18.1 versus 10.8 nmol/L, P < 0.0001; Hcy = 10.3 versus 7.9 micromol/L P < 0.0006)., Conclusion: Italian patients aged under 60 years on OAT and non-homozygous for the mutation C677T of MTHFR gene, had SF and Hcy concentrations significantly higher than the control group.

During recent years importance of B complex vitamins, beta-carotene and vitamin C has been realised in terms of their antioxidative and anticarcinogenic properties. Fruits and vegetables are the rich sources of these vitamins. However, there are considerable cooking losses of vitamins, and information on vitamin contents of cooked foods is essential for assessing the adequacy of vitamin intakes. Secondly, there is a growing trend to consume ready-to-eat foods such as stuffed pancakes (samosa, patties), pastries, French fries; replacing traditional foods for lunch or dinner like roti, vegetable curry, bread, non-vegetarian items. Ready-to-eat foods are considered to give empty calories rather than a balanced diet. A study was undertaken to estimate ascorbic acid, folic acid, riboflavin, thiamine and beta-carotene of 263 cooked food samples and 260 meals representing dietary patterns of Asia, Africa, Europe, USA and Latin America by spectrophotometry and photoflurometry. A broad range of beta-carotene (84-2038 mcg%), riboflavin (0.01-0.48 mg%), thiamine (0.04-0.36 mg%), vitamin C (1-28 mg%) and folate (26-111 mcg%) was observed in individual foods. Bakery products and sweets were found to be poor sources and green leafy vegetables and fruits were good sources of these five vitamins. The differences between ready-to-eat foods and meals consumed during lunch or dinner were prominent for beta-carotene, ascorbic acid, riboflavin and folic acid (P < 0.05). The cooking losses were 34.6, 30, 52.2, 45.9 and 32.2% in case of ascorbic acid, thiamine, riboflavin, beta-carotene and folic acid respectively. Irrespective of whether it is ready-to-eat or a lunch/dinner food item, the contribution of vegetables in the preparations was found to make a marked impact on the vitamin profile. While results justify the concept of a food pyramid, emphasis needs to be given to types of fruits and vegetables rich in vitamins; preferably in their uncooked form, rather than considering their total consumption.

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.

The main objective of this study was to investigate the effects of pasteurisation, UHT processing and fermentation on the concentration of folate-binding proteins (FBP) and their folate binding capacity in comparison with the retention of the most predominant folate from, 5-CH3THF. The amount of folate-binding protein (FBP) was analysed using enzyme-linked immunosorbent assay (ELISA). Unprocessed milk and pasteurised milk were found to contain similar amounts, 211 and 168 nmol/l, of FBP, respectively. UHT-processed milk and Yoghurt naturelle, both processed at temperatures above 90 degrees C, contained only 5.2 and 0.2 nmol/l FBP, respectively. As an indication of the protein-binding capacity free and protein-bound folates were analysed after charcoal treatment using the radio-protein binding assay method (RPBA). These results indicated that all folates in unprocessed milk and pasteurised milk were protein-bound, while folates in UHT-processed milk and Yoghurt naturelle occurred freely which is supported by our findings on FBP. High-performance liquid chromatography analysis indicated that unprocessed milk, pasteurised milk, UHT-processed milk and Yoghurt naturelle contained 44.8 +/- 2.1 (n = 10), 41.1 +/- 0.9 (n = 10), 36.1 +/- 1.8 (n = 10) and 35.6 +/- 9.1 micrograms/l (n = 10) 5-methyltetrahydrofolates (5-CH3THF), respectively, after deconjugation. Corresponding values for total milk folates analysed using radio-protein binding assay were 80.4 +/- 0.9 (n = 10), 64.2 +/- 2.7 (n = 10), 48.2 +/- 1.8 (n = 10) and 54.0 +/- 8.2 micrograms/l (n = 10), respectively. Hence, both methods indicated significant (P < 0.05) losses of 5-CH3THF as a result of pasteurisation, UHT processing and fermentation, compared with unprocessed milk. In spite of apparent discrepancies in folate concentrations obtained using the two different methods, these results support the equimolar ratio of FBP and folates in unprocessed and pasteurised milk when data on 5-CH3THF, obtained using HPLC were corrected for differences in recovery. Thus, heat processing of milk not only reduced the amount of 5-CH3 THF significantly, but also changed the concentration of FBP and the folate-binding capacity of FBP, which may have implications on the bioavailability of milk folates.