Development and validation of an analysis method for pesticide residues by gas chromatography-tandem mass spectrometry in Daikenchuto.

Daikenchuto (DKT) is one of the most widely used "Kampo" in Japan as a representative of herbal medicine. Because DKT is made from a natural product like food, it requires the management of pesticides; therefore, an analysis of residual pesticides in Kampo is required. The World Health Organization (WHO) indicates that pesticide residue analysis by the U.S. Pharmacopeia (USP) is required. USP defines 107 compounds containing organochlorine pesticides and organophosphorus pesticides and their metabolites, which have a high residual risk. Accordingly, to guarantee the safety of herbal medicines according to global standards is a very important issue. In this study, we developed an analytical method for 91 compounds, which are listed in USP, using DKT as the subject. The method could extract pesticides from DKT with acetone, elute pesticides with acetonitrile using a SepPak C18 column (5 g) and with ethyl acetate using a DSC-NH2 column (2 g), and perform simultaneous analyses by gas chromatography-tandem mass spectrometry (GC-MS/MS). This method, which could quantify 88 compounds, was validated according to USP. A pesticide residue analysis method that meets USP requirements enables the analysis of pesticide residues with a high residue risk and contributes to improving the safety of "Kampo" and other herbal medicines.

Nickel-Catalyzed Hydroarylation of in Situ Generated 1,3-Dienes with Arylboronic Acids Using a Secondary Homoallyl Carbonate as a Surrogate for the 1,3-Diene and Hydride Source.

The nickel-catalyzed hydroarylation of 1,3-dienes with arylboronic acids using a secondary homoallyl carbonate as a surrogate for the 1,3-diene and hydride source has been developed. The synthetic strategy allowed an efficient access to a wide array of hydroarylation products in high yields with high functional group compatibility without the use of an external hydride source. Mechanistic experiments indicated that the alkene-directed oxidative addition and subsequent ß-hydride elimination would be a critical process in this transformation.

Quantitative analysis of hepatic fat fraction by single-breath-holding MR spectroscopy with T2 correction: phantom and clinical study with histologic assessment.

The focus of this study was on the investigation of the accuracy of the fat fraction of the liver by use of single-breath-holding magnetic resonance spectroscopy (MRS) with T (2) correction. Single-voxel proton MRS was performed with several TE values, and the fat fraction was determined with and without T (2) correction. MRS was also performed with use of the point-resolved spectroscopy sequence in single breath holding. The T (2) values of both water and fat were determined separately at the same time, and the effect of T (2) on the fat fraction was corrected. In addition, MRS-based fat fractions were compared with the degree of hepatic steatosis (HS) by liver biopsy in human subjects. With T (2) correction, the MRI-derived fat fractions were in good agreement with the fat fractions in all phantoms, but the fat fractions were overestimated without T (2) correction. R (2) values were in good agreement with the preset iron concentrations in the phantoms. The MRI-derived fat fraction was well correlated with the degree of HS. Iron deposited in the liver affects the signal strength when proton MRS is used for detection of the fat signal in the liver. However, the fat signal can be evaluated more accurately when the T (2) correction is applied. Breath-holding MRS minimizes the respiratory motion, and it can be more accurate in the quantification of the hepatic fat fraction.

[Influence of b value on the measurement of contrast and apparent diffusion coefficient in 3.0 Tesla breast magnetic resonance imaging].

Diffusion-weighted imaging (DWI) has been used to characterize not only the brain, but also the breast by implementation of faster imaging techniques and higher magnetic field strengths. However, the optimum b value, which is an important scan parameter for DW images contrast on 3 T breast magnetic resonance imaging (MRI) has not been established. The purpose of this study was to investigate the influence of different b value combinations on the image contrast and apparent diffusion coefficient (ADC) in patients with known invasive carcinoma, ductal carcinoma in situ (DCIS), and normal mammary gland in breast DWI. The analysis procedure consisted of the following methods: 1) T(2) correction of DW images with echo-planar imaging (EPI) T(2)-weighted images; 2) contrast measurement between normal mammary gland and tumor tissues; 3) ADC measurement of normal mammary gland and tumor tissues. In many cases, the highest contrast between normal mammary gland and tumor tissues was obtained using a b value of 1500 s/mm(2). Our results indicated that when only one b value is used, the b value in which signal intensities of normal mammary gland decreases down to noise level, and the contrast between normal mammary gland and tumor tissues is recommended. ADC value decreased with increasing b value. Therefore, when determining the ADC threshold level, it is important to perform the evaluation using ADC values calculated from DW images with the same b value in clinical studies.

[Quantitative evaluation of Gd-EOB-DTPA uptake in phantom study for liver MRI].

Gd-EOB-DTPA is a new liver specific MRI contrast media. In the hepatobiliary phase, contrast media is trapped in normal liver tissue, a normal liver shows high intensity, tumor/liver contrast becomes high, and diagnostic ability improves. In order to indicate the degree of uptake of the contrast media, the enhancement ratio (ER) is calculated. The ER is obtained by calculating (signal intensity (SI) after injection-SI before injection) / SI before injection. However, because there is no linearity between contrast media concentration and SI, ER is not correctly estimated by this method. We discuss a method of measuring ER based on SI and T(1) values using the phantom. We used a column phantom, with an internal diameter of 3 cm, that was filled with Gd-EOB-DTPA diluted solution. Moreover, measurement of the T(1) value by the IR method was also performed. The ER measuring method of this technique consists of the following three components: 1) Measurement of ER based on differences in 1/T(1) values using the variable flip angle (FA) method, 2) Measurement of differences in SI, and 3) Measurement of differences in 1/T(1) values using the IR method. ER values calculated by these three methods were compared. In measurement made using the variable FA method and the IR method, linearity was found between contrast media concentration and ER. On the other hand, linearity was not found between contrast media concentration and SI. For calculation of ER using Gd-EOB-DTPA, a more correct ER is obtained by measuring the T(1) value using the variable FA method.

Glycolysis module activated by hypoxia-inducible factor 1alpha is related to the aggressive phenotype of hepatocellular carcinoma.

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.

Decreased ID2 promotes metastatic potentials of hepatocellular carcinoma by altering secretion of vascular endothelial growth factor.

PURPOSE: We aimed to explore the molecular and biological functions of Inhibitor of DNA binding/differentiation 2 (ID2), which was found to be responsible for portal vein invasion of hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: We measured ID2 mRNA levels in 92 HCC patients by real-time reverse transcription-PCR and examined the relation to clinicopathologic features. To clarify the precise roles of ID2, we did in vitro analysis with expression vectors and small interfering RNAs. Effects of ID2 on cell invasive potential and expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha were analyzed by Matrigel-coated invasion chamber, ELISA, and Western blot analysis, respectively. RESULTS: ID2 mRNA level correlated inversely with portal vein invasion (P < 0.001), tumor-node-metastasis stage (P < 0.001), tumor size (P < 0.001), and early intrahepatic recurrence (P < 0.05). When limited to a cohort of hepatitis C virus-related HCCs, patients with low levels of ID2 had significantly shorter disease-free survival time than those with high levels of ID2. Invasive potential of cells transfected with ID2 expression vector was lower than that of empty vector-transfected cells. Cells overexpressing ID2 also showed decreased VEGF secretion and hypoxia-inducible factor-1alpha protein levels. The results of ID2-knockdown experiments were opposite to those of ID2 overexpression experiments. CONCLUSIONS: On the basis of our clinical and in vitro data, we suggest that ID2 plays a significant role in the metastatic process during progression of HCC. This action might be explained, at least in part, by altered cell mobility due to decreased secretion of VEGF.

A three-gene predictor for early intrahepatic recurrence of hepatocellular carcinoma after curative hepatectomy.

We previously developed a DNA microarray-based system that out-performs traditionally used clinical parameters for prediction of early intrahepatic recurrence (IHR) of hepatocellular carcinoma (HCC). Because DNA microarray is too expensive for daily clinical use, we used a quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) to develop a lower-cost predictor for early IHR. From the 12 early IHR-related genes integrated in the previous predictor, we selected 6 genes whose levels showed the strongest association between data from the 2 distinct DNA microarray platforms with the same sample set. Expression of these 6 genes relative to that of GAPDH was measured by QRT-PCR in 82 HCCs. Of the 82 HCCs, 39 and 43 were assigned to training and independent test sets, respectively. By searching all combinations (n=2-6) of the 6 genes, we found an optimal combination of 3 genes (HLADRA, DDX17 and LAPTM5) that minimized the leave-one-out error for prediction of early IHR in the training set. The 3-gene predictor constructed with the Fisher linear classifier correctly predicted early IHR or non-recurrence in 35 (81.4%) of 43 HCCs in the independent test set and had a high positive predictive value of 72.7% and a high negative predictive value of 84.4%. Multivariate analysis with the stepwise logistic regression showed that the 3-gene predictor [F(x)<0] was an independent risk factor for early IHR (risk ratio, 13.6; p=0.006), indicating its potential as an easy-to-use predictor for accurate prediction of early IHR of HCC.

Involvement of c-myc-regulated genes in hepatocellular carcinoma related to genotype-C hepatitis B virus.

PURPOSE: The purpose of this study was to elucidate the molecular basis of hepatocellular carcinoma (HCC) caused by genotype-C hepatitis B virus (HBV). METHODS: We compared molecular profiles of 15 HCCs and five non-tumorous livers, all of which were associated with genotype-C HBV infection, using DNA microarray technology. RESULTS: Our supervised learning identified 237 genes whose expression differed between HCCs and non-tumorous livers. This result was validated by a false discovery rate of 0%. Levels of expression of 35 and 202 genes were higher and lower, respectively, in HCCs than in non-tumorous livers. Among the 237 genes, we highlighted the top 35 upregulated and top 35 down regulated genes in tumor. Interestingly, when overlapping genes were excluded, 12 (e.g., NM23-H2, MCM7, PARP1, YWHAH, HSPB1, and MSH2) of the top 34 upregulated genes and five (e.g., MT1A and MT3) of the top 33 downregulated genes were c-myc-regulated genes. The microarray data for five randomly selected genes (MCM7, UBE2L3, PPIA, CXCL12, and ASS) were confirmed by quantitative real-time reverse transcription-polymerase chain reaction. CONCLUSIONS: Our results indicate that many c-myc-regulated genes are involved in genotype-C-HBV-related HCC, suggesting that c-myc is related to the hepatocarcinogenic activity of genotype-C HBV.