Determination of folate concentrations in diverse potato germplasm using a trienzyme extraction and a microbiological assay

Folate deficiency is a leading cause of birth defects and is implicated in several other diseases. We are interested in how much folate concentrations vary among potato germplasm. We determined total folate concentrations of potato tubers from 67 cultivars, advanced breeding lines, or wild species. Folates were extracted by a tri-enzyme treatment and analyzed by using a Lactobacillus rhamnosus microbiological assay. Folate concentrations varied from 521 +/- 96 to 1373 +/- 230 ng/g dry weight and were genotype and location dependent. The highest folate concentrations were mostly found in color-fleshed potatoes. Variations of folate concentrations within either color- or white-fleshed tubers were similar ( approximately 2-fold). Skin contained approximately 30% higher folate concentrations than flesh. Storage of tubers for 7 months generally led to an increase in folate contents. Semiquantitative RT-PCR analyses showed that higher folate contents were correlated with lower mRNA expression of some folate genes.     

Development and validation of a computer program to design and calculate ROPS

In Spain, there are more than 250,000 tractors built before 1980, when it became mandatory for all new tractors to be equipped with a rollover protective structure (ROPS). A similar situation is found in the European Union, but the situation is worse in the U.S. and in developing countries. Directive 2003/37/EEC establishes that tractors over 800 kg weight can be homologated by using the OECD standard code for the official testing of protective structures on agricultural and forestry tractors (static test), called Code 4. A ROPS attachable to the rear axle of different tractor models has been designed, and a computer program for the calculation of the ROPS design has been developed. The program, named ESTREMA, is available at: www.cfnavarra.es/insl. Using this program, it has been possible to design a ROPS for the Massey Ferguson model 178 tractor, one of the most common tractor models without a ROPS in Spain. After the tractor was equipped with the designed ROPS, it was tested at the Spanish Authorized Station for testing ROPS and passed the homologation test (OECD Code 4), the main results being a maximum distortion of 21.3 cm when the absorbed energy was 5437 N and the maximum force applied was 34 kN during loading from the side. The ROPS was improved, redesigned, and remounted on the tractor, the tractor was tested in a real overturn, and no part of the structure intruded on the driver's clearance zone during the test. In conclusion, the ESTREMA program worked correctly, and the designed ROPS was able to pass the authorized test and provide adequate protection to the operator during a real overturn.     

Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry

Tetracyclines are widely used in farm animals. This can cause drug residues in products of animal origin and, after excretion of these substances, in animal slurry and in soil fertilized with that slurry. In this paper, we present a method based on a microbiological assay coupled with HPLC for the detection of oxytetracycline, tetracycline, and chlortetracycline in eggs. After a simple liquid extraction of the samples and HPLC separation, fractions were collected on microtiter plates, and the tetracyclines were analyzed using the Staphylococcus aureus assay. This method was able to identify residues of tetracyclines in eggs at a level set by regulatory agencies (i.e., 200 microg/kg). In addition, it was shown that the described microbiological method can be used as a screening assay for the detection of tetracyclines and possible biologically active metabolites in animal slurry and soil samples. Employing the same extraction procedure, it was demonstrated that LC-MS-MS allowed the quantification of 20-400 microg/kg in eggs with recoveries ranging from 71 to 109% and RSDs of 3-15%.     

Degradation of primisulfuron by a combination of chemical and microbiological processes

Microbial degradation of the herbicide primisulfuron was investigated using enrichment cultures from contaminated soils and 20 axenic cultures. At neutral pH, no disappearance of the herbicide was detected either in the enrichment cultures or in the growth media of the axenic microbial cultures. During the growth of some of the microbial strains, however, the pH of the medium dropped below 6, resulting in the hydrolysis of primisulfuron. The rate of primisulfuron hydrolysis was clearly pH dependent; primisulfuron was more persistent in neutral or weakly basic solutions than in acidic solutions. After hydrolysis of the herbicide, four products were observed. These were identified as methyl 2-(aminosulfonyl)benzoate, 2-amino-4,6-(difluoromethoxy)pyrimidine, 2-N-[[[[[4, 6-bis(difluoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl ]be nzoic acid, and 2-(aminosulfonyl)benzoic acid. After hydrolysis, it was found that the fungus Phanerochaete chrysosporium mineralized 27 and 24% of (14)C-phenyl- and (14)C-pyrimidine-labeled products, respectively, after 24 days of incubation. Similarly, Trametes versicolor mineralized 13 and 11% of (14)C-phenyl- and (14)C-pyrimidine-labeled hydrolysis products, respectively. In addition, primisulfuron in a hydrolytically stable solution, at pH 7. 0, was rapidly decomposed after ultraviolet irradiation, and two photolysis products were isolated [methylbenzoate and 4, 6-(difluoromethoxy)pyrimidin-2-ylurea]. When (14)C-phenyl-labeled primisulfuron was exposed to photolysis for 24 h, 32% of the initial radioactivity was recovered as (14)CO(2), whereas no (14)CO(2) was detected if the herbicide was labeled at the (14)C-pyrimidine position. Mineralization of (14)C-pyrimidine-labeled products of photolyzed primisulfuron by P. chrysosporium was approximately 25% after 24 days. These results clearly indicate that hydrolysis and photolysis of primisulfuron facilitated microbial degradation.     

Determination of folate concentrations in pulses by a microbiological method employing trienzyme extraction

Over the past two decades, the role of folate in human nutrition has been of much interest because of its relationship to diseases such as neural tube defects and heart disease. Since 1998, the U.S. Food and Drug Administration has mandated that cereal products be fortified with 140 microg of folic acid/100 g. It is important, therefore, to be able to determine accurately the folate concentrations in cereals and other grains to ensure proper dietary intake of folate. In this study, a microbiological method employing a trienzyme extraction procedure was applied to the analysis of folate in several starchy grain legumes (pulses). Differences in the folate content of dry bean were observed among some market classes but not between cultivars in the same market class. Location had a significant effect on the folate content of lentil and dry pea; cultivar did not. The significant effect of market class, cultivar, and growth environment on the levels of folate in pulses is of particular importance to pulse processors and pulse breeders.     

Quantification of the mycotoxin patulin by a stable isotope dilution assay.

Two stable isotope dilution assays for the quantification of patulin [4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one] in foods were developed using (13)C-labeled patulin as the internal standard. One method was performed by means of LC/MS in negative electrospray ionization mode without derivatization; the other used HRGC/HRMS after trimethylsilylation of the patulin isotopomers. In comparison with previously reported methods based on high-performance liquid chromatography with UV detection, HRGC/HRMS of the derivatized samples showed better repeatability, higher recovery rates (96% at a spike level of 200 ng/L), and a 100 times lower detection limit (12 ng/L). In contrast, LC/MS showed a much lower performance as compared to HPLC/UV or HRGC/HRMS. Using HRGC/HRMS, the mycotoxin was quantified in many different fruit products and in molded wheat bread.     

Inhibition of uropathogenic Escherichia coli by cranberry juice: a new antiadherence assay

A combination of microplate technology and turbidity assessment for testing the adherence of P-fimbriated Escherichia coli to human uroepithelial cell line T24, validated with the addition of the known inhibitor 4-O-alpha-D-galactopyranosyl-alpha-D-galactopyranose (galabiose), resulted in a high-throughput, biologically relevant assessment of cranberry (Vaccinium macrocarpon). P-fimbriated ATCC E. coli strains 25922, 29194, and 49161 were inhibited by galabiose. ATCC 29194, a representative urine isolate containing the papGII allele (Class II fimbrial adhesin) and demonstrating the most significant inhibition in the presence of galabiose, was chosen for further testing. In this assay, a low-polarity fraction of cranberry juice cocktail demonstrated dose-dependent inhibition of E. coli adherence. Reported here, for the first time in V. macrocarpon, are 1-O-methylgalactose, prunin, and phlorizin, identified in an active fraction of cranberry juice concentrate. This in vitro assay will be useful for the standardization of cranberry dietary supplements and is currently being used for bioassay-guided fractionation of cranberry juice concentrate.     

Reduction of acrylamide level in french fries by employing a temperature program during frying

In this study, the effect of employing an oil temperature program during frying on the acrylamide content of French fries was investigated. The frying conditions that could lead to lower acrylamide levels in French fries were first simulated by means of an experimentally validated frying model. Then, experiments were conducted to test the simulated conditions in real frying process. Different time/temperature combinations (4 min at 170 degrees C, 2 min at 170 degrees C + 2 min at 150 degrees C, 1 min at 170 degrees C + 3 min at 150 degrees C, 1 min at 190 degrees C + 3 min at 150 degrees C) were employed for frying potato strips (8.5 x 8.5 x 70 mm), and the resultant acrylamide levels were determined with a gas chromatography-mass spectrometry (GC-MS) method. The results indicated that acrylamide levels in French fries can be reduced by half if the final stage of the frying process employs a lower oil temperature. Therefore, the method appears to be an effective way of controlling the acrylamide level in the final product.     

Determination of the total antioxidant activity of fruits and vegetables by a liposome assay

The effects of mixtures of antioxidants on the oxidation of phospholipids have been investigated in large unilamellar liposomes following initiation by 2,2'-azobis(2-aminopropane) dihydrochloride. The lag phase increased linearly with antioxidant concentration. The lag phases of mixtures containing alpha-tocopherol with ascorbic acid showed synergy between the antioxidants, but mixtures of beta-carotene with alpha-tocopherol or ascorbic acid were not synergistic. The liposome system was used to investigate the total antioxidant activity of lipid- and water-soluble extracts from 16 samples of fruits, vegetables, and related food products. The water-soluble extracts caused greater increases in lag phase than the lipid-soluble extracts. The lag phase of liposomes containing the water-soluble extracts from fruits and vegetables increased linearly with the total phenolic concentration, with the continental salad extract having the longest lag phase. The lipid-soluble extract from apples caused the largest increase in lag phase of the lipid-soluble extracts. The lag phases of the lipid-soluble and water-soluble extracts of all fruits and vegetables studied were additive, but no synergy was detected. The lag phase of the liposomes containing both the water-soluble and lipid-soluble extracts varied from 611.5 min for the continental salad extracts to 47.5 min for the cauliflower extracts.     

Quantification of free and bound pantothenic acid in foods and blood plasma by a stable isotope dilution assay

A stable isotope dilution assay for quantification of pantothenic acid in food and blood plasma uses a 4-fold labeled isotopomer of the vitamin as an internal standard. Pantothenic acid and its labeled analogue were detected as trimethylsilyl derivatives by gas chromatography-mass spectrometry, showing a minimized spectral overlap. In starch a detection limit of 44 microg/kg, an intrasample relative standard deviation of 6.7%, and recovery values ranging between 97.5 and 99.4% were determined. Total pantothenic acid content was determined in rice, milk powder, apple juice, and blood plasma after enzymatic hydrolysis of the vitamin's conjugates; free pantothenic acid was quantified prior to enzyme treatment. Almost all results were found to be in good agreement with literature data.     

Determination and distribution of 6-methoxymellein in fresh and processed carrot puree by a rapid spectrophotometric assay.

Isocoumarin or 6-methoxymellein (6-MM) was extracted from carrot tissue using alkali saponification to solubilize the lactone portion of its structure into an aqueous phase. Acidification and subsequent organic solvent extraction allowed isolates to be quantified and verified as 6-MM by spectrophotometric determination. 6-Methoxymellein was analyzed in carrot cross sections, as a function of depth and before and after thermal processing. A natural propensity for 6-MM accumulation was observed in root tip sections exposed to ethylene, and levels increased as a result of wounding. Consecutive layer peeling demonstrated that small-diameter roots accumulated greater amounts of 6-MM in periderm tissue compared to large roots. Processing carrots into a puree resulted in 10-25% greater extraction of 6-MM than grinding fresh carrot samples, whereas steam-cooked and thermally processed purees had 15% greater extraction than unheated purees. This analytical technique will allow carrot processors to accurately estimate raw and processed products for the bitter compound 6-MM.     

Quantitative analysis of N-phenylpropenoyl-L-amino acids in roasted coffee and cocoa powder by means of a stable isotope dilution assay

Since recent reports on the role of N-phenylpropenoyl-L-amino acids as powerful antioxidants and key contributors to the astringent taste of cocoa nibs, there is an increasing interest in the concentrations of these phytochemicals in plant-derived foods. A versatile analytical method for the accurate quantitative analysis of N-phenylpropenoyl-L-amino acids in plant-derived foods by means of HPLC-MS/MS and synthetic stable isotope labeled N-phenylpropenoyl-L-amino acids as internal standards was developed. By means of the developed stable isotope dilution assay (SIDA), showing recovery rates of 95-102%, 14 N-phenylpropenoyl-L-amino acids were quantified for the first time in cocoa and coffee samples. On the basis of the results of LC-MS/MS experiments as well as cochromatography with the synthetic reference compounds N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4'-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine, respectively, were detected for the first time in cocoa powder, and (-)-N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine, (-)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tyrosine, N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine, (+)-N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-aspartic acid, (+)-N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid, N-[3',4'-dihydroxy-(E)-cinnamoyl]-L-tryptophan, N-[4'-hydroxy-(E)-cinnamoyl]-L-tryptophan, and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tryptophan, respectively, were detected for the first time in coffee beverages.     

Curcuminoids-cellular uptake by human primary colon cancer cells as quantitated by a sensitive HPLC assay and its relation with the inhibition of proliferation and apoptosis

Curcumin, which is a bright orange-yellow pigment of turmeric with antioxidant properties, has been shown to produce a potent preventative action against several types of cancers in recent studies. It has also been reported to protect the development of colon tumor in animals being fed with carcinogen. In the colon cancer cells, curcumin was illustrated to inhibit cell proliferation and induce apoptosis. As an antioxidant, it acts as an anti-inflammatory as well as an antitumor agent. Curcumin has been detected to exist in nature in the form of curcuminoids, a mixture of curcumin, the major component, with two of its related demethoxy compounds (demethoxycurcumin and bisdemethoxycurcumin). In the present study, we have investigated the antiproliferation and induced apoptosis effects of curcuminoids on colon cancer, using the primary cancer cells isolated from Taiwanese colon cancer patients as the model for colorectal cancer. Results showed that curcuminoids inhibited cell proliferation and induced apoptosis of these human primary colon cancer cells. The effects were observed in a dose-dependent manner as dose increased from 12.5 to 100 microM. With the aim of furthering the fundamental understanding of the mechanisms underlying the antiproliferation and induced apoptosis effects of curcuminoids on these human colon cancer cells, we developed a sensitive, rapid, and reproducible assay method based on high-performance liquid chromatography (HPLC). This HPLC technique developed was found to successfully determine, in a quantitative manner, the cellular uptake of curcuminoids. The uptake of these curcuminoids by the colon cancer cells was shown to increase as the dose of curcuminoids was increased. The observations of inhibited proliferation and increased apoptosis in the colon cancer cells appeared to be associated with the cellular uptake of curcuminoids.     

Determination of selenium bioavailability from wheat mill fractions in rats by using the slope-ratio assay and a modified torula yeast-based diet

Selenium is an essential mineral micronutrient for animals, and significant evidence supports an association between supranutritional Se intake and a reduction in the incidence of some forms of cancer. Thus, supplemental Se intake may provide an avenue for reducing cancer incidence. However, an important issue to consider is the form of Se that should be provided in such a supplement, because the bioavailability and bioactivity of Se can vary dramatically depending on the chemical form in which it is delivered. Because wheat products are the largest source of Se in U.S. diets, the absorption of Se was evaluated in different fractions of milled wheat that exhibits very high Se levels, owing to its production on naturally Se-rich soils. An experiment was conducted to determine the bioavailability of Se from three milled fractions of high-Se wheat. The method used was the slope-ratio assay, which measures the ability of Se from the wheat fractions to regenerate Se-dependent enzyme activities and tissue Se concentrations in Se-deficient rats. The responses generated from wheat Se were compared to a standard response curve generated by feeding graded amounts of Se as sodium selenite (Na2SeO3; NaSelenite) or selenomethionine (SeMet) in an AIN-93G-Torula yeast-based diet. Results showed that Se from wheat flour ( approximately 75% extraction) was nearly 100% available by a number of measures including plasma, liver, kidney, and muscle Se concentrations and liver and erythrocyte Se-dependent enzyme activities when compared with similar measures in rats fed NaSelenite or SeMet. However, on the basis of similar criteria, Se from wheat shorts was only about 85% available and that from wheat bran was about 60% available for absorption. These results indicate that high-Se wheat products, mainly those made from refined flour alone, might be particularly well suited for use as dietary Se supplements.     

Increased electron donor and electron acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay

The adhesion of methyl ricinoleate droplets to cells of the yeast Yarrowia lipolytica was investigated. A new cytometric method, relying on the double staining of fatty globules with Nile Red and of cells with Calcofluor, enabled us to quantify methyl ricinoleate droplet adhesion to cells precultured on a hydrophilic or on a hydrophobic carbon source. In this last case, droplet adsorption was enhanced and a MATS (microbial adhesion to solvents) test revealed that this increase was due to Lewis acid-base interactions and not to an increase in the hydrophobic properties of the cell surface. These preliminary results demonstrate that the developed cytometric method is promising for various applications concerning the study of interactions between microorganisms and an emulsified hydrophobic substrates.     

Development of a quality assurance program for determination of ultratrace background levels of lead and cadmium in raw agricultural crops by differential pulse anodic stripping voltammetry

Data on the background levels of lead and cadmium in the food supply are essential in order to establish a baseline from which to evaluate the extent of contamination in transport, processing, industrial atmospheric particulate fallout, and soil treatment (e.g., fertilizers, sewage sludge, etc.). This requires the establishment of site selection and sampling criteria as well as the development of a rigorous analytical method capable of performing routine analyses of Pb and Cd at ultratrace levels. The method used in this study, which was published previously, was designed to provide high sample throughput with minimal contamination. This involved control and measurement of blank levels and the establishment of quality control procedures to maintain confidence in the accuracy and precision of the method.     

Estimation of nitrogen fixation by Trifoliumsubterraneum L. and Medicago truncatula gaertn. grown in pots using a non-destructive acetylene reduction assay

Non-destructive acetylene reduction assays were successfully performed using small (1.21) incubation chambers and a 1 h incubation at 20°C. The concentration of C₂H₂ substrate used in the assays reached saturation at a partial pressure of 10 kPa for nodulated subterranean clover and barrel medic grown in a sandy loam. The optimum rates of C₂H₂ reduction associated with both species occurred within the range of 25–50% of the soil moisture content at field capacity (33 kPa). The ratio of moles N₂ fixed to moles C₂H₂ reduced was calculated to be 1:2.9 for the subterranean clover—Rhizobium symbiosis and 1:3.3 for the barrel medic—Rhizobium symbiosis.