Challenges in the determination of unsubstituted food folates: impact of stabilities and conversions on analytical results.

Tetrahydrofolate is the parent molecule of the folate coenzymes required for one carbon metabolism. Together with other unsubstituted folates such as dihydrofolate and folic acid, tetrahydrofolate represents the third pool of dietary folates following 5-methyltetrahydrofolate and formyl folates. Low intake of dietary folates and poor folate status are common problems in many countries. There is a critical need for reliable methods to determine folate in foods to accurately estimate folate intakes in populations. However, current values for folates in foods in databanks are often underestimated due to the high instability of several folate forms, especially tetrahydrofolate. The present review highlights the occurrence of unsubstituted folates in foods and their oxidation mechanisms and chemical behavior as well as interconversion reaction between tetrahydrofolate and 5,10-methylenetetrahydrofolate. The review shows also the important role of antioxidants in protecting folates during analysis and describes strategies to stabilize unsubstituted folates throughout all steps of the analytical procedure.

Toxicity of 15 veterinary pharmaceuticals in zebrafish (Danio rerio) embryos.

Extensive use of veterinary pharmaceuticals may result in contamination of water bodies adjacent to pasture land or areas where animal manure has been applied. In order to evaluate the potential risk to fish embryos 15 veterinary pharmaceuticals were investigated by use of an extended zebrafish embryo toxicity test. Chemical analysis of the exposure medium was performed by solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for 11 of the compounds and potential metabolism by the embryos was studied for albendazole, febantel, fenbendazole and oxfendazole. Newly fertilized zebrafish eggs were exposed under static conditions in 96-well plates for 6 days to the pharmaceuticals: 5 antibacterials and 10 antiparasitics. Endpoints including mortality, malformations and other sublethal responses were recorded at 24, 48 and 144 h post fertilization (hpf). The pharmaceuticals causing the highest toxicity were antiparasitics whereas the tested antibacterials, danofloxacin, enrofloxacin, tylosine, trimethoprim and oxytetracyclin had a much lower toxic potency in zebrafish embryos. Most toxic were fenbendazole, albendazole and flumethrin with no observed effect concentrations (NOECs) around 0.02 mg/L. The overall NOEC was determined by lethality for the following pharmaceuticals: albendazole, fenbendazole and oxfendazole. Sublethal endpoints, including malformations, side-laying embryos, tremors, reduced movements and altered heart rate increased the sensitivity of the tests and determined the overall NOECs for febantel, doramectin, ivermectin, flumethrin and toltrazuril. Exposure to doramectin and ivermectin caused a decrease in movements at 24 hpf and a decrease in heart rate at 48 hpf. Flumethrin exposure resulted in decreased time to hatching, except at the highest concentrations, and caused an increase in heart rate at 48 hpf. In contrast, toltrazuril caused an increased time to hatching and a decrease in heart rate. Chemical analysis of the exposure medium after the tests revealed great differences between nominal and measured concentrations, emphasizing the need of including analysis of the actual exposure concentrations. The results indicated that metabolism of albendazole into its sulfoxide protected the embryos from toxicity. Albendazole was metabolized efficiently into albendazole sulfoxide at lower exposure concentrations, resulting in reduced toxicity. At higher concentrations, an increasing proportion of albendazole remained unmetabolized and embryo mortality occurred. Metabolism by the embryos of febantel into fenbendazole and oxfendazole and of fenbendazole into oxfendazole was demonstrated. It is suggested that the toxic effect of febantel in zebrafish embryos is due to metabolism into fenbendazole.

Folate content and composition of vegetables commonly consumed in China.

UNLABELLED: Folate deficiency increases the risk of chronic diseases, including neural tube defects (NTDs) in infants, megaloblastic anemia, cardiovascular disease, and some cancers in adults. China is the most NTDs prevalent area in the world. Folate deficiency in China can be reduced by proper supply of fresh leafy green vegetables but little is known about the folate content and vitamers in the vegetables commonly consumed by Chinese population. The purposes of this study were first to analyze most commonly consumed important vegetables that contribute to folate intake in the Chinese population and second to estimate the significance of selected vegetables as a source of dietary folate intake. Folate content and vitamers forms in vegetables were analyzed using a valid liquid chromatography method. Monoenzyme treatment was used for leafy green and some fruit vegetables, and dienzyme treatment for some root vegetables. Total folate content in commonly consumed vegetables ranged from 14.78 to 145.54 µg/100 g in edible portion with an average of 61.99 µg/100 g. The highest folate content (>140 µg/100 g) was found in pak choi and spinach. Total folate contents in leafy vegetables, fruit vegetables, and root vegetables were in the range of 17.22 to 145.54 µg/100g, 18.14 to 86.04 µg/100g, and 14.78 to 75.81 µg/100g, respectively. The considerable variations in folate content were found in different types of vegetables commonly consumed by Chinese population. Leafy vegetables are a better source of folate than fruit and root vegetables commonly consumed by Chinese population. PRACTICAL APPLICATION: Data from this research would facilitate to accurately establish the actual folate intake by Chinese population. Our folate composition data on vegetables can be incorporated into the national food databases. Availability of such data is essential for estimating folate intake and defining an optimal level for fortification program in China.

Albendazole causes stage-dependent developmental toxicity and is deactivated by a mammalian metabolization system in a modified zebrafish embryotoxicity test.

The zebrafish embryotoxicity test has previously been combined with an external metabolic activation system (MAS) to assess developmental toxicity of metabolites produced by maternal metabolism. Due to toxicity of MAS the exposure was limited to one early and short period. We have modified the method and included additional testing time points with extended exposure durations. Using the anthelmintic drug albendazole as a model substance, we demonstrated stage-dependent toxic effects at three windows of zebrafish embryo development, i.e. 2-3, 12-14 and 24-28h post fertilization, and showed that MAS, by metabolic deactivation, reduced the toxicity of albendazole at all time points. Chemical analysis confirmed that albendazole was efficiently metabolized by MAS to the corresponding sulfoxide and sulfone, which are non-toxic to zebrafish embryos. To conclude, the modified zebrafish embryotoxicity test with MAS can be expanded for assessment of metabolites at different developmental stages.

Natural variation of folate content and composition in spinach (Spinacia oleracea) germplasm.

Breeding to increase folate levels in edible parts of plants, termed folate biofortification, is an economical approach to fight against folate deficiency in humans, especially in the developing world. Germplasm with elevated folates are a useful genetic source for both breeding and direct use. Spinach is one of the well-know vegetables that contains a relatively high amount of folate. Currently, little is known about how much folate, and their composition varies in different spinach accessions. The aim of this study was to investigate natural variation in the folate content and composition of spinach genotypes grown under controlled environmental conditions. The folate content and composition in 67 spinach accessions were collected from the United States Department of Agriculture (USDA) and Asian Vegetable Research and Development Center (AVRDC) germplasm collections according to their origin, grown under control conditions to screen for natural diversity. Folates were extracted by a monoenzyme treatment and analyzed by a validated liquid chromatography (LC) method. The total folate content ranged from 54.1 to 173.2 µg/100 g of fresh weight, with 3.2-fold variation, and was accession-dependent. Four spinach accessions (PI 499372, NSL 6095, PI 261787, and TOT7337-B) have been identified as enriched folate content over 150 µg/100 g of fresh weight. The folate forms found were H(4)-folate, 5-CH(3)-H(4)-folate, and 5-HCO-H(4)-folate, and 10-CHO-folic acid also varied among different accessions and was responsible for variation in the total folate content. The major folate vitamer was represented by 5-CH(3)-H(4)-folate, which on average accounted for up to 52% of the total folate pool. The large variation in the total folate content and composition in diverse spinach accessions demonstrates the great genetic potential of diverse genotypes to be exploited by plant breeders.

Developmental toxicity of albendazole and its three main metabolites in zebrafish embryos.

Albendazole (ABZ) is used as an anthelmintic drug in humans and animals. ABZ has been shown to cause developmental toxicity in experimental animals, however it is not clear if this is caused by the parent compound or a metabolite. Zebrafish embryos were exposed from 1 to 144hpf (hours post fertilization) to investigate the developmental toxicity of ABZ, the first metabolite albendazole sulphoxide and the subsequent metabolites albendazole sulphone (ABZSO(2)) and albendazole-2-aminosulphone (ABZSO(2)NH(2)). The results showed that ABZ caused malformations of head and tail and embryonic lethality from 0.3µM. In contrast, the metabolites did not display developmental toxicity at any tested concentration. Dechorionation did not influence the developmental toxic potential of ABZ and ABZSO, indicating that bioavailability was not a limiting factor. Chemical analysis showed that at sublethal concentrations, most of ABZ was metabolized to ABZSO. The results demonstrate that in zebrafish embryos ABZ rather than ABZSO displays developmental toxicity.

Biofortification of folates in white wheat bread by selection of yeast strain and process.

We here demonstrate that folate content in yeast fermented food can be dramatically increased by using a proper (i) yeast strain and (ii) cultivation procedure for the selected strain prior to food fermentation. Folate levels were 3 to 5-fold higher in white wheat bread leavened with a Saccharomyces cerevisiae strain CBS7764, cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation (135-139 microg/100 dry matter), compared to white wheat bread leavened with commercial Baker's yeast (27-43 microg/100 g). The commercial Baker's yeast strain had been industrially produced, using a fed-batch process, thereafter compressed and stored in the refrigerator until bakings were initiated. This strategy is an attractive alternative to fortification of bread with synthetically produced folic acid. By using a high folate producing strain cultured a suitable way folate levels obtained were in accordance with folic acid content in fortified cereal products.

Growth rate and medium composition strongly affect folate content in Saccharomyces cerevisiae.

Folate content in a Saccharomyces cerevisiae strain was monitored during aerobic batch fermentation in synthetic growth medium, yeast peptone dextrose medium, and a molasses based medium. During growth in the synthetic medium large differences in intracellular folate content was observed at different phases. Specific folate levels, expressed per unit biomass, were highest during respiro-fermentative growth (120 microg/g) and decreased during the respiratory and stationary phases. Thus, the physiological state of the cells clearly affects the folate content. This was confirmed in chemostat cultures where total intracellular folate content increased linearly with increasing growth rate (r(2)=0.998), indicating high growth rate i.e. respiro-fermentative growth to be most favourable to obtain high specific folate content. In complex media however, much lower folate content (15-40 microg/g) was found throughout the batch growth. Only minor growth-phase related differences were detected. This shows the impact of cultivation medium on folate content in yeast. To further investigate which components that influence folate content, batch experiments in synthetic medium with addition of specific components were performed. Adding a raw mixture of peptides and amino acids (peptone) decreased folate levels extensively (90%) whereas adding amino acids one-by-one only had minor effects on the intracellular folate content. Furthermore, supplementing synthetic medium with pABA, folate or nucleotides did not change the intracellular folate content. This work constitutes the first steps towards an optimised process for production of natural folates for fortification purposes, as well as an effort to gain fundamental understanding of folate requirements in yeast in relation to environmental conditions.

Application of liquid chromatography-electrospray ionisation mass spectrometry for determination of dietary folates: effects of buffer nature and mobile phase composition on sensitivity and selectivity.

A sensitive and reliable liquid chromatography-mass spectrometry (LC-MS) method to determine dietary folates was developed and validated. Folates were detected and quantified using positive electrospray ionisation (ESI) with selective ion monitoring of protonated ions [M+H]+. The effects of buffer nature and mobile phase composition on separation, peak shape and intensity of MS signal were investigated. The acidic-basic properties of folates were successfully used to predict possible ionisation patterns, but they were not sufficient to predict the intensity of MS signal and the proportion of different ionisation products, which indicated that other parameters, such as gas phase acidity/basicity of analytes and ion evaporation mechanisms might be important. The use of aqueous acetic acid as volatile buffer was found to be preferable compared to formic acid due to considerable gain in intensity of MS signal for all folate forms studied. Limits of quantifications were 0.3 ng/mL for 5-methyltetrahydrofolate and 0.6 ng/mL for tetrahydrofolate, 10-formylfolic acid, 5-formyltetrahydrofolate and folic acid when using 20 microL injection. For 10-formylfolic acid, 5-formyltetrahydrofolate and folic acid the MS detection was found to be superior over commonly used fluorescence and UV detection in terms of selectivity and sensitivity. The method was successfully applied to analysis of folates in baker's yeast.

Applicability of alkyl-bonded ultra-pure silica stationary phases for gradient reversed-phase HPLC of folates with conventional and volatile buffers under highly aqueous conditions.

Applicability of several alkyl-bonded silica stationary phases was tested for gradient RP-HPLC of folates under highly aqueous conditions. High retention of folates was achieved on alternative phases with enhanced polarity and classical phases with higher carbon content. Phases exhibiting polar secondary interactions were found to provide better selectivity for late-eluting folates, whereas selectivity for early-eluting folates was mostly dependent on hydrophobic interactions. Best selectivity in phosphate buffered mobile phase was achieved on polar-endcapped silica phases (Aquasil C18 and HyPurity Aquastar) followed by alternative Atlantis dC18. Classical phases exhibited poorer separation of 10-formyl-folic acid and 5-formyl-tetrahydrofolate, but it could be considerably improved by increasing the buffer pH. Strong secondary interactions of ion-exchange character on polar-embedded phases resulted in marked peak deterioration, loss of recovery and dramatic changes in retention behaviour for early- and late-eluting folates when changing the mobile phase composition and pH. Therefore, polar-embedded phases such as HyPurity Advance were found to be unsuitable for separating folates. Stationary phases exhibited peak deterioration when using volatile buffer of low ionic strength. Better results were obtained with classical phases, whereas alternative phases showed not only peak deterioration but also a decrease in recovery and poorer selectivity due to increased secondary interactions in volatile buffer.

Development of a simplified method for the determination of folates in baker's yeast by HPLC with ultraviolet and fluorescence detection.

A simplified HPLC method for rapid determination of folates in yeast with ultraviolet and fluorescence detection without sample purification has been developed. By use of the column Aquasil C(18), specially designed for polar analytes, and gradient elution, it was possible to separate and determine five folate derivatives: tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate with fluorescence detection, and 10-formylfolic acid and folic acid with ultraviolet detection. The sample preparation required only a small amount of dry yeast (25-50 mg) and included an extraction of folates by heat treatment and deconjugation of folate polyglutamates to monoglutamates with the use of rat serum conjugase. Validation involved investigation of matrix effects, determination of recovery by standard addition method, repeatability, and stability tests. The dominating folate forms in commercial dry baker's yeast were found to be tetrahydrafolate and 5-methyltetrahydrofolate with a total folate content of 2890 microg/100 g (63.4 nmol/g). The simplicity of the method makes it suitable for folate screening studies of different yeast strains.