OBJECTIVES: Perchlorate is an emerging contaminant that is found everywhere, including various foods. Perchlorate is known to disturb the production of thyroid hormones and leads to mental disorders in fetuses and infants, as well as metabolic problems in adults. In this study, we attempted to establish an LC-MS/MS method for measuring perchlorate in dairy products and used this developed method to investigate perchlorate levels in Korean milk and yogurt samples. METHODS: The developed method of perchlorate analysis requires a shaker and 1% acetic acid/acetonitrile as the extracting solvent. Briefly, the samples were extracted and then centrifuged (4000 rpm, 1hour), and the supernatant was then passed through a Envitrade mark Carb SPE cartridge that had been prewashed sequentially with 6 mL of acetonitrile and 6 mL of 1% acetic acid in water. The final volume of the sample extract was adjusted to 40 mL with reagent water and the final sample was filtered through a 0.20-microm pore size PTFE (Polytetrafluoroethylene) syringe filter prior to LC-MS/MS. RESULTS: The average levels of perchlorate in milk and yogurt samples were 5.63 +/- 3.49 microg/L and 3.65 +/- 2.42 microg/L, respectively. The perchlorate levels observed in milk samples in this study were similar to those reported from China, Japan, and the United States. CONCLUSIONS: The exposure of Koreans to perchlorate through the consumption of dairy products was calculated based on the results of this study. For all age groups, the calculated exposure to perchlorate was below the reference of dose (0.7 microg/kg-day) proposed by the National Academy of Science, USA, but the perchlorate exposure of children was higher than that of adults. Therefore, further investigation of perchlorate in other food samples is needed to enable a more exact assessment of exposure of children to perchlorate.
Acetic Acid ; Acetonitriles ; Adult ; Child ; China ; Dairy Products ; Fetus ; Humans ; Infant ; Japan ; Mental Disorders ; Milk ; Perchloric Acid ; Polytetrafluoroethylene ; Syringes ; Thyroid Hormones ; Water ; Yogurt

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method has been validated for determining concentrations of glutamate, glycine, and alanine in human plasma. Proteins in plasma were precipitated with perchloric acid, followed by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Simultaneous analysis of glutamate, glycine, and alanine is achieved using reversed-phase HPLC conditions and ultraviolet detection. Excellent linearity was observed for these three amino acids over their concentration ranges with correlation coefficients (r)>0.999. The intra- and inter-day precision were below 10%. This method utilizes quality control samples and demonstrates excellent plasma recovery and accuracy. The developed method has been successfully applied to measure plasma glutamate, glycine, and alanine in twenty volunteers.
Alanine ; Amino Acids ; Aminoquinolines ; Carbamates ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Glutamic Acid ; Glycine ; Humans ; Perchloric Acid ; Plasma ; Proteins ; Quality Control

The perchloric acid soluble, heat stable, and ethanol insoluble antigen of M. tuberculosis (TB-PBE) was prepared, and antigenicity of this antigen was studied in vivo and in vitro. TB-PBE showed a single band of 60 kDa by SDS-PAGE. Sera from the patients with active pulmonary tuberculosis did not react with this antigen by ELISA. A delayed hypersensitivity skin reaction was induced with this antigen and was correlated with the reaction with PPD. Skin biopsy was performed in this skin lesion induced by TB-PBE and stained by H-E and immunohistochemical methods. TB-PBE induced an inflammatory lesion similar to a lesion induced by PPD. Blastogenic activity of the peripheral blood mononuclear cells stimulated by TB-PBE increased, and showed a peak reaction at 7 days after stimulation. The blastogenic activity changed in a dose-dependent manner. After stimulation with TB-PBE, mononuclear cells were analyzed by FACS. DR+ T cells and CD4/CD8 ratio increased after stimulation by TB-PBE. These cells secreted IL-2, not IL-4 after stimulation with TB-PBE. In the immunofluorescence test, mouse antiserum against TB-PBE showed a positive reaction with M. tuberculosis and showed cross-reactivity with M. bovive and other atypical mycobacteria, but not with S. aureus. With these results, it is evident that TB-PBE is an antigen which can induce cell mediated immunity in vivo and in vitro.
Animal ; Antigens, Bacterial/immunology/*isolation & purification ; Cytokines/biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Human ; Hypersensitivity, Delayed ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis/*immunology ; Perchloric Acid ; Skin/pathology ; T-Lymphocytes/immunology

The perchloric acid soluble, heat stable, and ethanol insoluble antigen of M. tuberculosis (TB-PBE) was prepared, and antigenicity of this antigen was studied in vivo and in vitro. TB-PBE showed a single band of 60 kDa by SDS-PAGE. Sera from the patients with active pulmonary tuberculosis did not react with this antigen by ELISA. A delayed hypersensitivity skin reaction was induced with this antigen and was correlated with the reaction with PPD. Skin biopsy was performed in this skin lesion induced by TB-PBE and stained by H-E and immunohistochemical methods. TB-PBE induced an inflammatory lesion similar to a lesion induced by PPD. Blastogenic activity of the peripheral blood mononuclear cells stimulated by TB-PBE increased, and showed a peak reaction at 7 days after stimulation. The blastogenic activity changed in a dose-dependent manner. After stimulation with TB-PBE, mononuclear cells were analyzed by FACS. DR+ T cells and CD4/CD8 ratio increased after stimulation by TB-PBE. These cells secreted IL-2, not IL-4 after stimulation with TB-PBE. In the immunofluorescence test, mouse antiserum against TB-PBE showed a positive reaction with M. tuberculosis and showed cross-reactivity with M. bovive and other atypical mycobacteria, but not with S. aureus. With these results, it is evident that TB-PBE is an antigen which can induce cell mediated immunity in vivo and in vitro.
Animal ; Antigens, Bacterial/immunology/*isolation & purification ; Cytokines/biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Human ; Hypersensitivity, Delayed ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis/*immunology ; Perchloric Acid ; Skin/pathology ; T-Lymphocytes/immunology

PURPOSE: Cancer specific killing can be achieved by therapeutic gene activated by cancer specific promotor. Expression of sodium iodide symporter (NIS) gene causes transportation and concentration ofiodide into the cell, therefore radioiodine treatment after NIS gene transfer to cancer cell could be a form of radionuclide gene therapy. luciferase (Luc) gene transfected cancer cell can be monitored by in vivo optical imaging after D-luciferin injection. Aims of the study are to make vector with both therapeutic NIS gene driven by AFP promoter and reporter Luc gene driven by CMV promoter, to perform hepatocellular carcinoma specific radiodiodine gene therapy by the vector, and assessment of the therapy effect by optical imaging using luciferase expression. MATERIALS AND METHODS: A Vector with AFP promoter driven NIS gene and CMV promoter driven Luc gene (AFP-NIS-CMV-Luc) was constructed. Liver cancer cell (HepG2, Huh-7) and non liver cancer cell (HCT-15) were transfected with the vector using liposome. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Radioiodide uptake, perchlorate blockade, and washout tests were performed and bioluminescence also measured by luminometer in these cells. In vitro clonogenic assay with I-131 was performed. In vivo nuclear imaging was obtained with gamma camera after I-131 intraperitoneal injection. RESULTS: A Vector with AFP-NIS-CMV-Luc was constructed and successfully transfected into HepG2, Huh-7 and HCT-15 cells. HepG2 and Huh-7 cells with AFP-NIS-CMV-Luc gene showed higher iodide uptake than non transfected cells and the higher iodide uptake was totally blocked by addition of perchlorate. HCT-15 cell did not showed any change of iodide uptake by the gene transfection. Transfected cells had higher light output than control cells. In vitro clonogenic assay, transfected HepG2 and Huh-7 cells showed lower colony count than non transfected HepG2 and Huh-7 cells, but transfected HCT-15 cell did not showed any difference than non transfected HCT-15 cell. Number of Huh-7 cells with AFP-NIS-CMV-Luc gene transfection was positively correlated with radioidine accumulation and luciferase activity. In vivo nuclear imaging with I-131 was successful in AFP-NIS-CMV-Luc gene transfected Huh-7 cell xenograft on nude mouse. CONCLUSION: A Vector with AFP promoter driven NIS and CMV promoter driven Luc gene was constructed. Transfection of the vector showed liver cancer cell specific enhancement of I-131 cytotoxicity by AFP promoter, and the effect of the radioiodine therapy can be successfully assessed by non-invasive luminescence measurement.
Animals ; Benzothiazoles ; Carcinoma, Hepatocellular ; Gamma Cameras ; Genetic Therapy ; Homicide ; Injections, Intraperitoneal ; Ion Transport ; Light ; Liposomes ; Liver ; Liver Neoplasms ; Luciferases ; Luminescence ; Mice ; Mice, Nude ; Molecular Imaging ; Optical Imaging ; Perchloric Acid ; RNA, Messenger ; Sodium ; Sodium Iodide ; Symporters ; Transfection ; Transplantation, Heterologous ; Transportation

Bone and Bones ; Carbocyanines ; Cell Culture Techniques ; Coculture Techniques ; Collagen ; Collagenases ; Drug Combinations ; Durapatite ; Endothelial Cells ; Laminin ; Lipoproteins ; Magnetics ; Magnets ; Mandible ; Mesenchymal Stromal Cells ; Molar, Third ; Nylons ; Osteoblasts ; Osteogenesis ; Perchloric Acid ; Periosteum ; Proteoglycans