Quantitative analysis of bisphenol A eluted into canned tomato and mackerel by matrix-assisted laser desorption ionization mass spectrometry using anthracene as matrix.

Quantitative analysis of bisphenol A (BPA) was performed by matrix-assisted laser desorption ionization mass spectrometry. It was found that BPA was ionized as deprotonated species when anthracene was used as the matrix. A peak of deprotonated BPA and a peak assignable to epoxy resin were observed on analysis of liquids in canned tomato and mackerel samples. In addition, many identical peaks were observed from the liquids in both cans, indicating that epoxy resin was degraded and BPA was eluted into the canned tomato and mackerel during the storage period. It was suggested that the mackerel heat-treatment process and the acidity of tomato were responsible for the elution of BPA. Using bisphenol B (BPB) as the internal standard, the concentrations of BPA were determined to be 0.55 ± 0.05 and 1.72 ± 0.13 ng/µL (µg/mL) for the canned tomato and mackerel samples, respectively. These canned products were imported goods, and their BPA levels exceeded the safe concentration recommended by The Can Manufacturers Institute of Japan. The results indicate that consumers should exercise caution when consuming canned products particularly those manufactured overseas, which have different safety standards.

Enhancement of Ionization Efficiency Using Zeolite in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Multiple Drugs in Cancer Cells (Mass Spectrometry of Multiple Drugs in Cells Using Zeolite).

Combined therapy using photodynamic therapy (PDT) and chemotherapy has been proposed for anticancer-drug-resistant cancer cells. To evaluate the efficacy of such a combined therapy, the uptakes of an anticancer drug and a photosensitizer in cancer cells must be assessed. Mass spectrometry using matrix-assisted laser desorption/ionization can detect multiple drugs simultaneously. Human prostate cancer cells PC-3 or docetaxel-resistant cancer cells PC-3-DR were incubated in a serum-free medium containing a photosensitizer, protoporphyrin IX (PpIX), and an anticancer drug, docetaxel. A zeolite matrix was created by mixing 6-aza-2-thiothymine and NaY5.6 zeolite, and dissolving in water with 50% acetone. Ions were obtained with a time-of-flight mass spectrometer using a Nd:YAG laser at a wavelength of 355 nm. The cell morphology was preserved by washing the cells with ammonium acetate and drying in a vacuum after drug administration. Protonated PpIX (m/z 563.3) and the sodium adduct ion of docetaxel (m/z 829.9) were obtained from PC-3 cells simultaneously using the zeolite matrix. On the other hand, PpIX was detected but ions originating from docetaxel were not detected from PC-3-DR cells. The result indicated the efficacy of PDT for docetaxel-resistant cancer cells.

MALDI Mass Spectrometry of Small Molecules Using Nanometer-sized Clay.

Nanometer-sized clay, allophane, was used as the matrix for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and applied to the ionization of small molecules. First, the laser desorption ionization mass spectrum of cation-exchanged allophane was measured, and it was found that the cation exchange proceeded smoothly with increasing atomic number of alkali metals in the periodic table. This phenomenon was explained by considering the size of the counter anion on the allophane surface. Then, fructose was measured as the analyte using each alkali-cation-exchanged allophane as the matrix. Contrary to the measurements using allophane itself, the peak intensity of fructose decreased with increasing atomic number of alkali metals in the periodic table. This phenomenon was clarified by considering the stability of alkali cation in the presence of a surface anion, the desorption energy, and the solvation enthalpy of each alkali cation. The applicability of allophane to high molecular weight compounds was also confirmed by measuring cyclodextrin, angiotensin II, and insulin. Finally, a combination of allophane and zeolite was examined by assuming proton relay among allophane, zeolite, and analyte. As a result of proton supply from zeolite to allophane, the peak intensity of the proton sponge (1,8-bis(dimethylamino)naphthalene) was enhanced by almost 2.2 times.

Chirality and structure-selective MALDI using mold matrix.

A matrix that enabled chirality and structure-selective detection in matrix assisted laser desorption ionization mass spectrometry (MALDI MS) has been developed. Molds of L- or D- alanine were made on a thermoreversible polymer (polyvinyl methyl ether) with 2,4,6-trihydroxyacetophenone, and this was used as a matrix. Separate detection of one optical isomer of alanine was realized in MALDI MS. This technique was also applied to the detection of trisaccharides having the same molecular weight but different structures. Separate detection of raffinose and maltotriose in MALDI MS were presented.

Detection and Quantification of 4-Methylimidazole in Cola by Matrix-assisted Laser Desorption Ionization Mass Spectrometry with Fe2O3 Nanoparticles on Zeolite.

Food additives generally used in carbonated drinks, such as 4-methylimidazole (4MI), caffeine (Caf?), citric acid (CA), and aspartame (Apm), were measured by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) using nanometer-sized particles of iron oxide (Fe2O3 NPs). The quantification of 4MI in Coca Cola (C-cola) was carried out. In order to improve the reproducibility of the peak intensities, Fe2O3 NPs loaded on ZSM5 zeolite were used as the matrix for quantification. By using 2-ethylimidazole (2EI) as the internal standard, the amount of 4MI in C-cola was determined to range from 88 to 65 µg/355 mL. The results agree with the published value (approx. 72 µg/355 mL). It was found that MALDI using Fe2O3 was applicable to the quantification of 4MI in C-cola.

Ammonium Ion Exchanged Zeolite for Laser Desorption/Ionization Mass Spectrometry of Phosphorylated Peptides.

α-Cyano-4-hydroxycinnamic acid (CHCA), an organic matrix molecule for matrix-assisted laser desorption/ionization mass spectrometry, was adsorbed to NH4 (+)-type zeolite surface, and this new matrix was used for the detection of low-molecular-weight compounds. It was found that this matrix could simplify the mass spectrum in the low-molecular-weight region and prevent interference from fragments and alkali metal ion adducted species. CHCA adsorbed to NH4 (+)-type ZSM5 zeolite (CHCA/NH4ZSM5) was used to measure atropine and aconitine, two toxic alkaloids in plants. In addition, CHCA/NH4ZSM5 enabled us to detect phosphorylated peptides; peaks of the protonated peptides had higher intensities than the peaks observed using CHCA only.

Analyte-Size-Dependent Ionization and Quantification of Monosaccharides in Human Plasma Using Cation-Exchanged Smectite Layers.

Smectite, a synthetic inorganic polymer with a saponite structure, was subjected to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Typical organic matrix molecules 2,4,6-trihydroxyacetophenone (THAP) and 2,5-dihydroxybenzoic acid (DHBA) were intercalated into the layer spacing of cation-exchanged smectite, and the complex was used as a new matrix for laser desorption/ionization mass spectrometry. Because of layer spacing limitations, only a small analyte that could enter the layer and bind to THAP or DHBA could be ionized. This was confirmed by examining different analyte/matrix preparation methods and by measuring saccharides with different molecular sizes. Because of the homogeneous distribution of THAP molecules in the smectite layer spacing, high reproducibility of the analyte peak intensity was achieved. By using isotope-labeled (13)C6-d-glucose as the internal standard, quantitative analysis of monosaccharides in pretreated human plasma sample was performed, and the value of 8.6 ± 0.3 µg/mg was estimated.

Quantitative analysis of free fatty acids in human serum using biexciton auger recombination in cadmium telluride nanoparticles loaded on zeolite.

Nanometer-sized semiconductor particles (CdTe) were used as an inorganic matrix for the laser desorption/ionization mass spectrometry of fatty acids. The excitation power dependence of the peak intensity of stearic acid (Ste), [Ste - H](-), was observed, and it was proportional to the square of the excitation power. The peak intensity of [Ste - H](-) decreased by addition of a hole scavenger (KSCN). It was understood that the ionization of fatty acids were due to the biexciton Auger recombination and electron ejection from CdTe. CdTe were then loaded on zeolite surface. The peak intensity enhancement of the deprotonated ion of fatty acid were observed. This phenomenon was explained by measuring the carrier lifetime for Auger recombination in CdTe. In addition, reproducibility of fatty acid ions was highly improved reflecting homogeneous distribution of CdTe on zeolite surface. CdTe/HM20 was successfully applied to the quantitative analysis of Ste in human serum by isotope dilution using (13)C18-Ste. The concentration of Ste in human serum samples was estimated to be 76.62 mg/kg with the standard deviation (SD) of 2.37 mg/kg.

Development of laser ionization techniques for evaluation of the effect of cancer drugs using imaging mass spectrometry.

Recently, combined therapy using chemotherapy and photodynamic therapy (PDT) has been proposed as a means of improving treatment outcomes. In order to evaluate the efficacy of combined therapy, it is necessary to determine the distribution of the anticancer drug and the photosensitizer. We investigated the use of imaging mass spectrometry (IMS) to simultaneously observe the distributions of an anticancer drug and photosensitizer administered to cancer cells. In particular, we sought to increase the sensitivity of detection of the anticancer drug docetaxel and the photosensitizer protoporphyrin IX (PpIX) by optimizing the ionization-assisting reagents. When we used a matrix consisting of equal weights of a zeolite (NaY5.6) and a conventional organic matrix (6-aza-2-thiothymine) in matrix-assisted laser desorption/ionization, the signal intensity of the sodium-adducted ion of docetaxel (administered at 100 µM) increased about 13-fold. Moreover, we detected docetaxel with the zeolite matrix using the droplet method, and detected PpIX by fluorescence and IMS with α-cyano-4-hydroxycinnamic acid (CHCA) using the spray method.

Laser desorption ionization mass spectrometry of bioactive substances by using 2,4,6-trihydroxyacetophenone on cation-substituted zeolite.

By using 2,4,6-trihydroxyacetophenone adsorbed to cation-substituted zeolite, laser desorption/ionization mass spectrometry for six kinds of bioactive substances was carried out. The compounds, which were usually difficult to observe by conventional matrix-assisted laser desorption/ionization mass spectrometry, were ionized by cation adduction from the zeolite surface.

Association of ITPA polymorphism with outcomes of peginterferon-α plus ribavirin combination therapy.

AIM: To analyzed the association between inosine triphosphatase (ITPA) (rs1127354) genotypes and sustained virological response (SVR) rates in peginterferon (Peg-IFN)α + ribavirin (RBV) treatment. METHODS: Patients who underwent Peg-IFNα + RBV combination therapy were enrolled (n = 120) and they had no history of other IFN-based treatments. Variation in hemoglobin levels during therapy, cumulative reduction of RBV dose, frequency of treatment withdrawal, and SVR rates were investigated in each ITPA genotype. RESULTS: In patients with ITPA CC genotype, hemoglobin decline was significantly greater and the percentage of patients in whom total RBV dose was < 60% of standard and/or treatment was withdrawn was significantly higher compared with CA/AA genotype. However, SVR rates were equivalent between CC and CA/AA genotypes, and within a subset of patients with Interleukin 28B (IL28B) (rs8099917) TT genotype, SVR rates tended to be higher in patients with ITPA CC genotype, although the difference was not significant. CONCLUSION: ITPA CC genotype was a disadvantageous factor for Peg-IFNα + RBV treatment in relation to completion rates and RBV dose. However, CC genotype was not inferior to CA/AA genotype for SVR rates. When full-length treatment is accomplished, it is plausible that more SVR is achieved in patients with ITPA CC variant, especially in a background of IL28B TT genotype.

Add-on therapy of pitavastatin and eicosapentaenoic acid improves outcome of peginterferon plus ribavirin treatment for chronic hepatitis C.

Despite the use of pegylated-interferon (peg-IFN) plus ribavirin combination therapy, many patients infected with hepatitis C virus (HCV)-1b remain HCV-positive. To determine whether addition of pitavastatin and eicosapentaenoic acid (EPA) is beneficial, the "add-on" therapy option (add-on group) was compared retrospectively with unmodified peg-IFN/ribavirin therapy (standard group). Association of host- or virus-related factors with sustained virological response was assessed. In HCV replicon cells, the effects of pitavastatin and/or EPA on HCV replication and expression of innate-immunity- and lipid-metabolism-associated genes were investigated. In patients infected with HCV-1b, sustained virological response rates were significantly higher in the add-on than standard group. In both groups, sustained virological response rates were significantly higher in patients with genotype TT of IL-28B (rs8099917) than in those with non-TT genotype. Among the patients with non-TT genotype, sustained virological response rates were markedly higher in the add-on than standard group. By multivariate analysis, genome variation of IL28B but not add-on therapy remained as a predictive factor of sustained virological response. In replicon cells, pitavastatin and EPA suppressed HCV replication. Activation of innate immunity was obvious in pitavastatin-treated cells and EPA suppressed the expression of sterol regulatory element binding protein-1c and low-density lipoprotein receptor. Addition of pitavastatin and EPA to peg-IFN/ribavirin treatment improved sustained virological response in patients infected with HCV-1b. Genotype variation of IL-28B is a strong predictive factor in add-on therapy.

Matrix-assisted laser desorption ionization mass spectrometry of maltohexaose and acetylsalicylic acid using alkali metal cation-substituted zeolites.

Using alkali-metal cation-substituted zeolites and 2,4,6-trihydroxyacetophenone (THAP), which is a typical organic matrix molecule for matrix-assisted laser desorption ionization (MALDI), mass spectrometry has been performed for maltohexaose and acetylsalicylic acid, and the cation-selective ionization of these analytes was achieved. It is found that a complex of cation-substituted zeolite and THAP can be applicable to a compound that is hard to be ionized by a proton adduction in conventional MALDI.

Assessing current nutritional status of patients with HCV-related liver cirrhosis in the compensated stage.

BACKGROUND/AIM: Nutritional states of Japanese patients with liver cirrhosis have recently shown great diversity, some show protein energy malnutrition and others excessive nutrition and obesity. For there to be adequate guidance regarding dietary treatment, it is important that a patient's current nutritional state be clarified. METHODS: We assessed nutritive intake in Japanese cirrhotic patients and determined their nutritional problems. Subjects were non-hospitalized patients with hepatitis C virus (HCV)-related cirrhosis in the compensated stage (n=47), chronic hepatitis C (n=46) or healthy volunteers (n=32). A brief self-administered diet history questionnaire was conducted with assistance from a registered dietitian. RESULTS: We categorized patients with cirrhosis according to daily intake of energy and protein; 10.6% had an energy and protein intake within a normal range, 72.4% showed excessive intake, and 17.0% showed insufficient intake of energy or protein. In cirrhotic patients with diabetic complications, the intake levels of energy, proteins, fat and carbohydrates were significantly higher than in patients without diabetes. Moreover, cirrhotic patients had significantly higher intake levels of energy, protein and fat than did chronic hepatitis C patients and healthy individuals. In patients with HCV-related liver cirrhosis, insufficient intake of energy and protein was shown in some, while many, especially those with diabetes, showed excessive intake. CONCLUSION: For nutritive management of cirrhotic patients, the intake of various nutrients should be appropriately assessed and effective nutritional education systems established.

Early dynamics of viremia in patients with genotype 1b chronic hepatitis C: Peg-IFNalpha2a shows earlier viral decline than peg-IFNalpha2b in combination therapy with ribavirin.

BACKGROUND: We aimed to assess differences in early viral dynamics following treatment with either peg-IFNalpha2a or peg-IFNalpha2b in combination with ribavirin in patients with chronic genotype 1b HCV infection. MATERIAL/METHODS: Sixty-one patients in the peg-IFNalpha2a + ribavirin treatment (group alpha2a) and 88 patients in the peg-IFNalpha2b + ribavirin treatment (group alpha2b) were retrospectively analyzed. The early dynamics of HCV RNA over 12 weeks were evaluated. Sustained virological response (SVR) was defined as undetectable HCV RNA at week 24 after end of therapy. First- (day 0-1) and second-phase (day 1-28) viral decline rates were calculated in accordance with theoretical formulae. RESULTS: Baseline HCV RNA concentrations were almost similar between the 2 groups. In group alpha2a, viral decline was significantly greater than in group alpha2b at weeks 4, 8, and 12. In group alpha2a, viral decline was significantly greater in SVR patients than in non-SVR patients at week 2, whereas significantly greater viral decline in SVR patients was found during weeks 1-12 in group alpha2b. The first-phase viral decline rate was significantly larger in group alpha2a than in group alpha2b (1.31 ± 0.84 vs. 0.70 ± 0.97 log IU/mL/day; p < 0.0001). Within SVR patients, first-phase viral decline rate was significantly larger in group α2a compared with group alpha2b (1.45 ± 0.85 vs. 0.78 ± 1.0 log IU/mL/day; p < 0.0001). Second-phase viral decline rate was comparable between the groups. CONCLUSIONS: Peg-IFNalpha2a showed earlier viral decline than peg-IFNalpha2b and the difference was obvious, especially in the first-phase viral decline.

Expression profile of lipid metabolism-associated genes in hepatitis C virus-infected human liver.

AIM: Recent studies have shown that lipid metabolic pathways are required for the entry, replication and secretion of hepatitis C virus (HCV). Although little is known about the life cycle of HCV in humans, the activation of cholesterol and fatty acid biosynthesis may be critical for HCV proliferation. METHODS: We assessed the transcription levels of genes essential for cholesterol and fatty acid biosynthesis in liver samples obtained from patients with chronic hepatitis C and determined their correlations. The serum levels of low-density lipoprotein (LDL) cholesterol and HCV core antigen were also measured. RESULTS: The gene expression of the LDL receptor (LDLR) was suppressed, whereas that of SREBP1c, liver X receptor-α (LXRα), fatty acid synthase (FASN), and HMG-CoA reductase and synthase (HMGR and HMGS) was significantly increased, and SREBP2 transcription was comparable in HCV-infected liver compared with normal liver. Positive correlations were found for LDLR versus HMGR, HMGR versus SREBP1c, and LDLR versus SREBP2 in the HCV-infected and control liver. Although the LXRα-SREBP1c-FASN pathway was upregulated, proteasome activator 28γ (PA28γ) was downregulated at the transcriptional level in HCV-infected liver, and was not significantly correlated with the other genes examined. The serum LDL cholesterol level was negatively correlated with LDLR and HMGR expression. CONCLUSION: These results suggest that, in HCV-infected liver, the cholesterol load increases and cholesterol uptake is controlled, while de novo cholesterol synthesis is upregulated compared with the normal physiological state. The positive correlations in the expression levels of some cholesterol metabolism-associated genes indicate that not all of the metabolic pathways are dysregulated in HCV-infected liver.

MALDI mass spectrometry using 2,4,6-trihydroxyacetophenone and 2,4-dihydroxyacetophenone with cyclodextrins: suppression of matrix-related ions in low-molecular-weight region.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of a model peptide, Substance P (SubP), was carried out using 2,4,6-trihydroxyacetophenone (THAP) and 2,4-dihydroxyacetophenone (DHAP) with cyclodextrins (cyclodextrin-supported matrix). It was found that the use of a cyclodextrin-supported matrix simplified the mass spectrum in the low-molecular-weight region. The interaction between THAP/DHAP and cyclodextrin (CD) was studied by UV-vis absorption spectroscopy and the incorporation of matrix molecules into the cyclodextrin cavity was confirmed by (1)H-NMR spectroscopy. DHAP showed tight incorporation with betaCD (betaCD(DHAP)) rather than THAP and it was found that the matrix-related peaks could be weakened by less than one third of the peak intensity of a protonated analyte. The betaCD(DHAP) matrix was applied to the measurements of two low-molecular-weight compounds; adenosine and adrenaline. It became clear that the cyclic structure of the CD and the host-guest interaction between betaCD and the matrix molecule were important to reduce the matrix-related peaks of THAP and DHAP.

[Multisite-based approach to assure inter-assay system compatibility among different exclusive-typed immunoassay systems through determining exchanged calibrators].

It is well known that most exclusive-typed immunoassay systems are highly precise but are poor in compatibility of their determinations. Thus, it is difficult to compare the determinations among different systems, posing problems when a patient is transferred to different hospitals or when a laboratory intends to change the system currently used. In the study, we tried to approach how to assure inter-immunoassay compatibility among four different systems through determination of the exchanged calibrators. First, determinations of total protein and albumin, and electrophoretic fractionation demonstrated marked differences among calibrators in their protein constituent. Some calibrators were prepared with human sera, but others were with inorganic or non-human albumin-based solution. Regression analysis of calibrators between the indicated concentrations by manufacturers and those actually determined by the different immunoassay systems revealed that; most slopes were closed to 1.0 for alpha-fetoprotein and prostate-specific antigen, but widely dissociated from 0.28 to 4.71 for CA19-9. In evaluation of clinical serum samples, determinations by one immunoassay system were compared with those converted based on a linear regression equation that was obtained by determination of the exchanged calibrators. However, this procedure could not improve compatibility, and positive effects of conversion varied by immunoassay systems combined, and also by test parameters. With these, we concluded that simple conversion of determinations by using the exchanged calibrators and a statistical linear regression could not provide us with the expected compatibility. Thus, standardization of target molecules or probes, and of calibrator constituent were urgent issue to assure inter-immunoassay compatibility.

Changes in the expression of cholesterol metabolism-associated genes in HCV-infected liver: a novel target for therapy?

Recent investigations indicate that hepatitis C virus (HCV) infection is closely associated with hepatocytic lipid metabolism and induces hepatic steatosis. However, the actual lipid metabolism in HCV-infected liver has not been extensively investigated in humans. In this study, we evaluated the expression of lipid metabolism-associated genes in patients with HCV infection by real-time PCR. Sterol regulatory element-binding protein (SREBP)-2 expression was unchanged and low density lipoprotein receptor expression was markedly reduced by 90% in HCV-infected liver. The expression of apolipoprotein B100, microsomal triglyceride transfer protein and ATP-binding cassette G5 was significantly increased. Up-regulation of cholesterol synthesis-associated genes, including HMG-CoA reductase, HMG-CoA synthase, farnesyl-diphosphate synthase and squalene synthase, confirmed enhanced de novo cholesterol synthesis. The expression of cholesterol 7alpha-hydroxylase and farnesoid X receptor was enhanced, while bile salt export pump expression was unchanged. Fatty acid synthase expression was increased which was accompanied by increased expression of liver X receptor alpha and SREBP-1c. In summary, the regulation of lipid metabolism was impaired and cholesterol and fatty acid synthesis continued to increase without negative feedback in HCV-infected liver. These changes may be beneficial for HCV replication.

The state of cholesterol metabolism in the liver of patients with primary biliary cirrhosis: the role of MDR3 expression.

AIM: Because dyslipidemia, such as hypercholesterolemia, is a characteristic of primary biliary cirrhosis (PBC), hepatic lipid metabolism may be disturbed in PBC patients. We examined the expression of lipid metabolism-associated genes in PBC liver. METHODS: All of the patients examined were in stage I or II PBC and without medication. RNA was isolated from liver specimens by needle biopsies of PBC patients and controls. The expression levels of various genes were measured by real-time RT-PCR. Multidrug resistance 3 (MDR3) expression was examined immunohistochemically. Statistical correlations between the gene expression levels and indices of blood testing were calculated. RESULTS: The expression levels of sterol regulatory element-binding protein (SREBP) 2 and LDL receptor were significantly lower, and those of apolipoprotein B, microsomal triglyceride transfer protein, ATP-binding cassette G5, and liver X receptor α (LXRα) were significantly higher in the PBC liver than in the normal control liver. The expression levels of bile acid synthesis- and excretion-associated genes did not change, and those of farnesoid X receptor, peroxisome proliferator-activated receptor α, and SREBP-1c were similar between the PBC and normal liver. MDR3 gene expression levels in the PBC liver were more than 4-fold higher than those in the control liver. Immunohistochemically, strong canalicular staining for MDR3 was observed in the PBC liver. LXRα expression was positively correlated with MDR3 levels. Serum levels of γ-glutamyl transpeptidase (GGT) and IgM were negatively correlated with MDR3 levels. CONCLUSIONS: Hepatocellular cholesterol metabolism was at least partially disturbed, even in the early stage of PBC. The most characteristic finding was a distinct elevation of MDR3 expression, and the MDR3 levels were negatively correlated with GGT and IgM levels.