Estimated daily intake and seasonal food sources of quercetin in Japan.

Quercetin is a promising food component, which can prevent lifestyle related diseases. To understand the dietary intake of quercetin in the subjects of a population-based cohort study and in the Japanese population, we first determined the quercetin content in foods available in the market during June and July in or near a town in Hokkaido, Japan. Red leaf lettuce, asparagus, and onions contained high amounts of quercetin derivatives. We then estimated the daily quercetin intake by 570 residents aged 20-92 years old in the town using a food frequency questionnaire (FFQ). The average and median quercetin intakes were 16.2 and 15.5 mg day(-1), respectively. The quercetin intakes by men were lower than those by women; the quercetin intakes showed a low correlation with age in both men and women. The estimated quercetin intake was similar during summer and winter. Quercetin was mainly ingested from onions and green tea, both in summer and in winter. Vegetables, such as asparagus, green pepper, tomatoes, and red leaf lettuce, were good sources of quercetin in summer. Our results will help to elucidate the association between quercetin intake and risks of lifestyle-related diseases by further prospective cohort study and establish healthy dietary requirements with the consumption of more physiologically useful components from foods.

Effects of polishing, cooking, and storing on total arsenic and arsenic species concentrations in rice cultivated in Japan.

The effects of polishing, cooking, and storing on total arsenic (As) and As species concentrations in rice were studied adopting typical Japanese conditions. Total and inorganic As levels in three white rice samples polished by removing 10% of bran by weight were reduced to 61-66% and 51-70% of those in brown rice. The As levels in the white rice after three washings with deionized water were reduced to 81-84% and 71-83% of those in raw rice. Rinse-free rice, which requires no washing before cooking because bran remaining on the surface of the rice was removed previously, yielded an effect similar to that of reducing As in rice by washing. Low-volume cooking (water:rice 1.4-2.0:1) rice to dryness did not remove As. The As content of brown rice stored in grain form for one year was stable.

Development of a reference material of a single DNA molecule for the quality control of PCR testing.

We developed a reference material of a single DNA molecule with a specific nucleotide sequence. The double-strand linear DNA which has PCR target sequences at the both ends was prepared as a reference DNA molecule, and we named the PCR targets on each side as confirmation sequence and standard sequence. The highly diluted solution of the reference molecule was dispensed into 96 wells of a plastic PCR plate to make the average number of molecules in a well below one. Subsequently, the presence or absence of the reference molecule in each well was checked by real-time PCR targeting for the confirmation sequence. After an enzymatic treatment of the reaction mixture in the positive wells for the digestion of PCR products, the resultant solution was used as the reference material of a single DNA molecule with the standard sequence. PCR analyses revealed that the prepared samples included only one reference molecule with high probability. The single-molecule reference material developed in this study will be useful for the absolute evaluation of a detection limit of PCR-based testing methods, the quality control of PCR analyses, performance evaluations of PCR reagents and instruments, and the preparation of an accurate calibration curve for real-time PCR quantitation.

Interlaboratory study of immunochromatography for the rapid determination of cadmium concentrations in cereals and soybeans.

Cadmium (Cd) is one of the most toxic heavy metals to humans. To prevent the distribution of Cd-contaminated food, a simple and quick on-site test for measuring Cd concentrations in agricultural products is needed. Recently, an immunochromatography kit developed for determining Cd in rice was reported to be useful for determining Cd in many other crops. We conducted an interlaboratory study to evaluate the kit for determining Cd in cereals (wheat and rice) and soybeans. Ten test materials were used, and 12 test samples including two sets of blind duplicates were distributed to 12 laboratories in Japan. The Cd recoveries (relative to certified values or values determined by inductively coupled plasma-MS) from all test materials were 84.6-125.1%. Repeatability RSD values of the test materials ranged from 8.8 to 14.8%. Reproducibility RSD values ranged from 13.4 to 27.6%, averaging 21.3%. The Horwitz ratio ranged from 0.61 to 1.36. The reproducibility was within the range of ELISA results for measuring toxins and allergens in food. Our results indicated that the kit was an inexpensive, reliable tool for quick and easy on-site determination of Cd in cereals and soybeans.

ß-Cryptoxanthin alleviates diet-induced nonalcoholic steatohepatitis by suppressing inflammatory gene expression in mice.

Recent nutritional epidemiological surveys showed that serum ß-cryptoxanthin inversely associates with the risks for insulin resistance and liver dysfunction. Consumption of ß-cryptoxanthin possibly prevents nonalcoholic steatohepatitis (NASH), which is suggested to be caused by insulin resistance and oxidative stress from nonalcoholic fatty liver disease. To evaluate the effect of ß-cryptoxanthin on diet-induced NASH, we fed a high-cholesterol and high-fat diet (CL diet) with or without 0.003% ß-cryptoxanthin to C56BL/6J mice for 12 weeks. After feeding, ß-cryptoxanthin attenuated fat accumulation, increases in Kupffer and activated stellate cells, and fibrosis in CL diet-induced NASH in the mice. Comprehensive gene expression analysis showed that although ß-cryptoxanthin histochemically reduced steatosis, it was more effective in inhibiting inflammatory gene expression change in NASH. ß-Cryptoxanthin reduced the alteration of expression of genes associated with cell death, inflammatory responses, infiltration and activation of macrophages and other leukocytes, quantity of T cells, and free radical scavenging. However, it showed little effect on the expression of genes related to cholesterol and other lipid metabolism. The expression of markers of M1 and M2 macrophages, T helper cells, and cytotoxic T cells was significantly induced in NASH and reduced by ß-cryptoxanthin. ß-Cryptoxanthin suppressed the expression of lipopolysaccharide (LPS)-inducible and/or TNFα-inducible genes in NASH. Increased levels of the oxidative stress marker thiobarbituric acid reactive substances (TBARS) were reduced by ß-cryptoxanthin in NASH. Thus, ß-cryptoxanthin suppresses inflammation and the resulting fibrosis probably by primarily suppressing the increase and activation of macrophages and other immune cells. Reducing oxidative stress is likely to be a major mechanism of inflammation and injury suppression in the livers of mice with NASH.

Inter-laboratory study of an LC-MS/MS method for simultaneous determination of fumonisin B1, B2 and B3 in corn.

To validate an LC-MS/MS method using a strong anion exchange cartridge for simultaneous determination of fumonisin B1, B2 and B3 in corn, an inter-laboratory study was performed in 9 laboratories using one fumonisin-negative corn sample, three spiked corn samples (FB1: 100-1,000 µg/kg, FB2 and FB3: 10-100 µg/kg) and two naturally contaminated corn samples. The recoveries were in the ranges of 79.7-87.2% for FB1, 78.6-103.2% for FB2 and 80.1-92.8% for FB3. The relative standard deviations for repeatability (RSDr) ranged from 3.7 to 8.0% for FB1, from 2.6 to 15.3% for FB2 and from 4.3 to 9.7% for FB3. The relative standard deviations for reproducibility (RSDR) for FB1, FB2 and FB3 were in the ranges of 6.3-10.1%, 5.9-18.7% and 9.3-16.0%, respectively. The HorRat values for all analytes ranged from 0.2 to 0.9. The difference of the trueness between the two kinds of commercially available anion exchange cartridges used in this study was not significant (p>0.05). Surveillance for fumonisins in corn grits was performed using the validated method. All of the samples were contaminated with fumonisins and the mean concentrations for FB1, FB2 and FB3 were 118.1, 37.3 and 17.9 µg/kg, respectively. These results indicated that the method for simultaneous determination of FB1, FB2 and FB3 in corn was successfully developed and validated.

Identification of possible technical problems in determination of the major inorganic constituents of brown-rice flour by evaluating proficiency test results.

To support skill upgrading in analysis of inorganic constituents of environmental and food samples, the National Metrology Institute of Japan (NMIJ) and the National Food Research Institute (NFRI) have organized a proficiency test (PT) of determination of Mn, Fe, Cu, Zn, As, and Cd in brown-rice flour based on the international standard (ISO/IEC 17043:2010). One hundred and thirty-three sets of reports were assessed by use of the E(n)-number and z-score approaches in accordance with ISO/IEC 17043 and the international harmonized protocol for PT. The PT results and analytical procedures, reported in detail, were reviewed, and possible technical reasons for questionable or unsatisfactory results are discussed.

Simulation of collaborative studies for real-time PCR-based quantitation methods for genetically modified crops.

To study impacts of various random effects and parameters of collaborative studies on the precision of quantitation methods of genetically modified (GM) crops, we developed a set of random effects models for cycle time values of a standard curve-based relative real-time PCR that makes use of an endogenous gene sequence as the internal standard. The models and data from a published collaborative study for six GM lines at four concentration levels were used to simulate collaborative studies under various conditions. Results suggested that by reducing the numbers of well replications from three to two, and standard levels of endogenous sequence from five to three, the number of unknown samples analyzable on a 96-well PCR plate in routine analyses could be almost doubled, and still the acceptable repeatability RSD (RSDr < or = 25%) and the reproducibility RSD (RSDR < 35%) of the collaborative study could be met. Further, RSDr and RSD(R) were found most sensitive to random effects attributable to inhomogeneity among blind replicates, but they were little influenced by those attributable to DNA extractions. The proposed models are expected to be useful for optimizing standard curve-based relative quantitation methods for GM crops by real-time PCR and their collaborative studies.

Inter-laboratory study of an LC-MS/MS method for simultaneous determination of deoxynivalenol and its acetylated derivatives, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in wheat.

To validate an LC-MS/MS method for simultaneous determination of deoxynivalenol (DON) and its acetylated derivatives, 3-acetyl-deoxynivalenol (3ADON) and 15-acetyl-deoxynivalenol (15ADON), in wheat using a multifunctional column, an inter-laboratory study was performed in 9 laboratories using one blank wheat sample, three spiked wheat samples (10, 50, 150 µg/kg) and one naturally contaminated wheat sample. The recoveries ranged from 98.8 to 102.6% for DON, 89.3 to 98.7% for 3ADON, and from 84.9 to 90.0% for 15ADON. The relative standard deviations for repeatability (RSDR) and reproducibility (RSDR) of DON were in the ranges of 7.2-11.3% and 9.5-22.6%, respectively. For 3ADON, the RSDR ranged from 5.3 to 9.5% and the RSDR ranged from 16.1 to 18.0%, while for 15ADON, the RSDR ranged from 6.2 to 11.2% and the RSDR ranged from 17.0 to 27.2%. The HorRat values for the three analytes ranged from 0.4 to 1.2. These results validate this method for the simultaneous determination of DON and its acetylated derivatives, 3ADON and 15ADON.

Interlaboratory study of LC-UV and LC-MS methods for the simultaneous determination of deoxynivalenol and nivalenol in wheat.

To evaluate LC methods with UV or MS detection for simultaneous analysis of deoxynivalenol (DON) and nivalenol (NIV) in wheat, an interlaboratory study was conducted in 11 laboratories. DON and NIV were purified using a multifunctional column, and their concentrations were determined using LC-UV or LC-MS(/MS). No internal standards were used. Three fortified wheat samples (0.1, 0.5 and 1 mg/kg), one naturally contaminated wheat sample, and one blank wheat sample were used. The recoveries ranged from 90% to 110% for DON and from 76% to 83% for NIV. For DON, the relative standard deviations for repeatability (RSDr) ranged from 1.1% to 7.6%. The relative standard deviations for reproducibility (RSDr) ranged from 7.2% to 25.2%. For NIV, the RSDr ranged from 2.0% to 10.7%, and the RSDr ranged from 7.0% to 31.4%. Regardless of sample and detector, the HorRat values for DON and NIV ranged from 0.4 to 1.4. Both LC-UV and LC-MS(/MS) methods were considered to be suitable for application as an official method.

Practicable group testing method to evaluate weight/weight GMO content in maize grains.

Because of the increasing use of maize hybrids with genetically modified (GM) stacked events, the established and commonly used bulk sample methods for PCR quantification of GM maize in non-GM maize are prone to overestimate the GM organism (GMO) content, compared to the actual weight/weight percentage of GM maize in the grain sample. As an alternative method, we designed and assessed a group testing strategy in which the GMO content is statistically evaluated based on qualitative analyses of multiple small pools, consisting of 20 maize kernels each. This approach enables the GMO content evaluation on a weight/weight basis, irrespective of the presence of stacked-event kernels. To enhance the method's user-friendliness in routine application, we devised an easy-to-use PCR-based qualitative analytical method comprising a sample preparation step in which 20 maize kernels are ground in a lysis buffer and a subsequent PCR assay in which the lysate is directly used as a DNA template. This method was validated in a multilaboratory collaborative trial.

Tendency for interlaboratory precision in the GMO analysis method based on real-time PCR.

The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.

Role of seed coat in imbibing soybean seeds observed by micro-magnetic resonance imaging.

BACKGROUND AND AIMS: Imbibition of Japanese soybean (Glycine max) cultivars was studied using micro-magnetic resonance imaging (MRI) in order to elucidate the mechanism of soaking injury and the protective role of the seed coat. METHODS: Time-lapse images during water uptake were acquired by the single-point imaging (SPI) method at 15-min intervals, for 20 h in the dry seed with seed coat, and for 2 h in seeds with the seed coat removed. The technique visualized water migration within the testa and demonstrated the distortion associated with cotyledon swelling during the very early stages of water uptake. KEY RESULTS: Water soon appeared in the testa and went around the dorsal surface of the seed from near the raphe, then migrated to the hilum region. An obvious protrusion was noted when water reached the hypocotyl and the radicle, followed by swelling of the cotyledons. A convex area was observed around the raphe with the enlargement of the seed. Water was always incorporated into the cotyledons from the abaxial surfaces, leading to swelling and generating a large air space between the adaxial surfaces. Water uptake greatly slowed, and the internal structures, veins and oil-accumulating tissues in the cotyledons developed after the seed stopped expanding. When the testa was removed from the dry seeds before imbibition, the cotyledons were severely damaged within 1.5 h of water uptake. CONCLUSIONS: The activation of the water channel seemed unnecessary for water entry into soybean seeds, and the testa rapidly swelled with steeping in water. However, the testa did not regulate the water incorporation in itself, but rather the rate at which water encountered the hypocotyl, the radicle, and the cotyledons through the inner layer of the seed coat, and thus prevented the destruction of the seed tissues at the beginning of imbibition.

Thawing of frozen vegetables observed by a small dedicated MRI for food research.

The thawing process for boiled and frozen edible vegetables was traced by a dedicated MRI for food research. The MRI system is small, with a 1.0-T static magnetic field, and can be placed in an ordinary research room with a light air conditioner. Images of green soybeans, broad beans, okra, asparagus and taro were measured by the spin-echo method (echo time=7 ms) with 0.1 or 0.2 s and 1 s repetition times. The images appeared along with the thawing time, and signals uniformly covered the sliced plane of the samples in the thawed condition. Information about the thawing process and tissue structures of the materials was obtained during transit thawing conditions. The thawing kinetics were examined with increased signal intensity, which were divided into two types. The signal increased linearly and saturated for okra and asparagus but exhibited convex curves for soybeans, broad beans and taro. The small MRI was stable, its handling was simple, and the internal structures of food materials could be accurately identified, although the grey-scale of the images was insufficient for determining precise textural fluctuations of tissue organization. We conclude that the devised MRI is useful for examining the quality of frozen foods and for developmental research into frozen foods.

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing.

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.

Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers.

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.