• Title/Summary/Keyword: Liquid Chromatography-tandem Mass Spectrometry

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Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

  • Yoo, Byong Chul;Yeo, Seung-Gu
    • Radiation Oncology Journal
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    • v.35 no.3
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    • pp.281-288
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    • 2017
  • Purpose: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. Materials and Methods: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. Results: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. Conclusion: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

A Survey on Ochratoxin a Contamination of Commercial Medicinal Herbs in Incheon (인천지역 유통 생약 중 Ochratoxin A 오염실태 조사)

  • Lim, Soo-sun;Joo, Gwang-sig;Choi, Eun-Jeong;Lee, Soo-Yeon;Lim, Yun-sun;Eum, Sun-Ah;Jo, Nam-Kyu;Lee, Sung-Mo
    • Korean Journal of Pharmacognosy
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    • v.48 no.1
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    • pp.56-62
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    • 2017
  • The aim of this study was to screen the contamination by ochratoxin A of mycotoxins in various medicinal herbs. We conducted a survey of ochratoxin A in medicinal herb on the retail market in Incheon in 2016. 116 medicinal herb samples were evaluated for the ochratoxin A contamination. They were analyzed for ochratoxin A using immunoaffinity column and high-performance liquid chromatography(HPLC)-fluorescence detection and the positive samples were confirmed using HPLC-tandem mass spectrometry. Ochratoxin A was detected in 4 medicinal herb samples; the concentrations of ochratoxin A were containing between 20.11 and $372.90{\mu}g/kg$. This study shows that in general, this kind of commodity may be contaminated by mycotoxins. Also this contamination is not limited to only aflatoxin of mycotoxins.

Evolution of 49 Phenolic Compounds in Shortly-aged Red Wines Made from Cabernet Gernischt (Vitis vinifera L. cv.)

  • Li, Zheng;Pan, Qiu-Hong;Jin, Zan-Min;He, Jian-Jun;Liang, Na-Na;Duan, Chang-Qing
    • Food Science and Biotechnology
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    • v.18 no.4
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    • pp.1001-1012
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    • 2009
  • A total of 49 phenolic compounds were identified from the aged red wines made from Cabernet Gernischt (Vitis vinifera L. cv.) grapes, a Chinese characteristic variety, including 13 anthocyanins, 4 pryanocyanins, 4 flavan-3-ol monomers, 6 flavan-3-ol polymers, 7 flavonols, 6 hydroxybenzoic acids, 5 hydroxycinnamic acids, 3 stilbenes, and 1 polymeric pigment. Evolution of these compounds was investigated in wines aged 1 to 13 months. Variance analysis showed that the levels of most phenolics existed significant difference in between wines aged 3 and 9 months. Cluster analysis indicated that 2 groups could be distinguished, one corresponding to wines aged 1 to 3 months and the other to wines aged 4 to 13 months. It was thus suggested that there were 2 key-stages for the development of fine wine quality, at the aged 3 and 9 months, respectively. This work would provide some helpful information for quality control in wine production.

Bioequivalence of Mepiril Tablet to Amaryl Tablet (Glimepiride 2 mg) by Liquid Chromatography/Electrospray Tandem Mass Spectrometry

  • Lee, Heon-Woo;Cho, Sung-Hee;Park, Wan-Su;Im, Ho-Taek;Rew, Jae-Hwan;Lee, Kyung-Tae
    • Journal of Pharmaceutical Investigation
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    • v.35 no.4
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    • pp.287-293
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    • 2005
  • The purpose of the present study was to evaluate the bioequivalence of two glimepiride tablets, Amaryl tablet (Handok & Aventis Korea, reference drug) and Mepiril tablet (Myungmoon Pharm. Co., Ltd., Korea, test drug), according to the guidelines of Korea Food and Drug Administration (KFDA). After adding an internal standard (glibenclamide) to human plasma, plasma samples were extracted using 1mL of methyl tertiary butyl ether. Compounds extracted were analyzed by reverse-phase HPLC with multiple reaction monitoring (MRM) mode analyte detection. This method for determination glimepiride proved accurate and reproducible, with a limit of quantitation of 2 ng/mL in human plasma. Twenty-four healthy male Korean volunteers received each medicine at the glimepiride dose of 2 mg in a $2{\times}2$ crossover study. There was a one-week washout period between the doses. Plasma concentrations of glimepiride were monitored by a LC-MS/MS for over a period of 12 hr after the administration. $AUC_t$ (the area under the plasma concentration-time curve from time zero to 12 hr) was calculated by the linear trapezoidal rule method. $C_{max}$ (maximum plasma drug concentration) and $T_{max}$ (time to reach $C_{max}$) were compiled from the plasma concentration-time data. Analysis of variance was carried out using logarithmically transformed $AUC_t$ and $C_{max}$. No significant sequence effect was found for all of the bioavailability parameters indicating that the crossover design was properly performed. The 90% confidence intervals of the $AUC_t$ ratio and the $C_{max}$ ratio for Amaryl/Mepiril were log 0.9583-log 1.1357 and log 1.0570-log 1.2376, respectively. These values were within the acceptable bioequivalence intervals of log 0.80-log 1.25. Taken together, our study demonstrated the bioequivalence of Amaryl and Mepiril with respect to the rate and extent of absorption.

Relationship between Cholesterol and Oxidative Potential from Meat Cooking (고기구이 초미세먼지 내 콜레스테롤 및 산화 잠재력과의 관계)

  • Lee, Yongmin;Kim, Eunyoung;Ryu, Chunho;Oh, Sea-Ho;Joo, Hungsoo;Bae, Min-Suk
    • Journal of Korean Society for Atmospheric Environment
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    • v.34 no.5
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    • pp.639-650
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    • 2018
  • Identification of the major sources contributing to PM is of importance in order to understand their quantitative contributions to atmosphere. In the viewpoint of the meat cooking in Korea, only a few analyses of organic molecular markers have been conducted due to analytical difficulties. In this study, ten different parts of meat (i.e., blade shoulder, belly, and arm shoulder of pork; ribeye roll, top blade muscle, and short plate of beef; leg quarter, breast, and wing of chicken; duck; mackerel) were pyrolyzed to generate the cooked PM using an electronic heating plate. Generated PM were collected by the pyrolysis sampling system to identify total carbon (TC) using a carbon analyzer and cholesterol using a Liquid chromatography tandem-mass spectrometry (LC-MSMS) based on fragmentor voltage (FV), precursor ion, collision energy, product ion. In addition, oxydative potential (OP) analysis using dithiothreitol (DTT) method were discussed to investigate the toxicity relates. Highly correlated pairwise scatterplots between the cholesterol and TC indicate that oxydative potential was highly associated with different parts of meat. This study provides insight into the meat cooking components of PM, which could be drivers of the oxidative potential relates.

In Vitro Assessment of Cytochrome P450 Inhibition by Ambroxol and Cetirizine (암브록솔과 세티리진의 Cytochrome P450 저해 활성 평가)

  • Kim, Bong-Hee;Ryu, Chang Seon;Jang, Him Chan;Lee, Sang Yoon;Lee, Ji-Yoon;Chae, Jung-Woo;Kwon, Kwang-Il;Kim, Sang Kyum
    • YAKHAK HOEJI
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    • v.57 no.3
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    • pp.194-198
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    • 2013
  • In the present study we evaluated drug-drug interaction potential of ambroxol and cetirizine mediated by inhibition of CYP isoforms including CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 using pooled human liver microsomes (HLMs). As measured by liquid chromatography-electrospray ionization tandem mass spectrometry, cetirizine and ambroxol inhibited significantly CYP2E1 but the maximal inhibition was approximately 36% at 10 ${\mu}M$ cetirizine and 28% at 3 ${\mu}M$ ambroxol. In addition, CYP2D6 activity was decreased to approximately 83% of control activity in pooled HLM incubated with 3 ${\mu}M$ ambroxol. Activities of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 were not significantly inhibited by cetirizine and ambroxol. Considering their maximal plasma concentration in human ($C_{max}$ of cetirizine is approximately 0.67 ${\mu}M$ and $C_{max}$ of ambroxol is 0.044 ${\mu}M$), these two drugs have very low possibility in drug-drug interaction by CYP inhibition in clinical situations.

Anti-Inflammatory Properties of Flavone di-C-Glycosides as Active Principles of Camellia Mistletoe, Korthalsella japonica

  • Kim, Min Kyoung;Yun, Kwang Jun;Lim, Da Hae;Kim, Jinju;Jang, Young Pyo
    • Biomolecules & Therapeutics
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    • v.24 no.6
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    • pp.630-637
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    • 2016
  • The chemical components and biological activity of Camellia mistletoe, Korthalsella japonica (Loranthaceae) are relatively unknown compared to other mistletoe species. Therefore, we investigated the phytochemical properties and biological activity of this parasitic plant to provide essential preliminary scientific evidence to support and encourage its further pharmaceutical research and development. The major plant components were chromatographically isolated using high-performance liquid chromatography and their structures were elucidated using tandem mass spectrometry and nuclear magnetic resonance anlysis. Furthermore, the anti-inflammatory activity of the 70% ethanol extract of K. japonica (KJ) and its isolated components was evaluated using a nitric oxide (NO) assay and western blot analysis for inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. Three flavone di-C-glycosides, lucenin-2, vicenin-2, and stellarin-2 were identified as major components of KJ, for the first time. KJ significantly inhibited NO production and reduced iNOS and COX-2 expression in lipopolysaccharide-stimulated RAW 264.7 cells at $100{\mu}g/mL$ while similar activity were observed with isolated flavone C-glycosides. In conclusion, KJ has a simple secondary metabolite profiles including flavone di-C-glycosides as major components and has a strong potential for further research and development as a source of therapeutic anti-inflammatory agents.

Identification and Characterization of a Novel Antioxidant Peptide from Bovine Skim Milk Fermented by Lactococcus lactis SL6

  • Kim, Sang Hoon;Lee, Ji Yoon;Balolong, Marilen P.;Kim, Jin-Eung;Paik, Hyun-Dong;Kang, Dae-Kyung
    • Food Science of Animal Resources
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    • v.37 no.3
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    • pp.402-409
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    • 2017
  • A novel peptide having free radical scavenging activity was separated, using an on-line high-performance liquid chromatography (HPLC) - ABTS screening method, from bovine skim milk fermented by Lactococcus lactis SL6 (KCTC 11865BP). It was further purified using reverse phase-HPLC (RP-HPLC) and sequenced by RP-HPLC-tandem mass spectrometry. The amino acid sequence of the identified peptide was determined to be Phe-Ser-Asp-Ile-Pro-Asn-Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu-Trp (2,362 Da), which is corresponding to the C-terminal fragment of bovine ${\alpha}_{s1}$-casein (f179-199). The hydroxyl radicals scavenging activity ($IC_{50}$ $28.25{\pm}0.96{\mu}M$) of the peptide chemically synthesized based on the MS/MS data showed a slightly lower than that of the natural antioxidant Trolox ($IC_{50}$ $15.37{\pm}0.52{\mu}M$). Furthermore, derivatives of the antioxidant peptide were synthesized. The antioxidative activity of the derivatives whose all three proline residues replaced by alanine significantly decreased, whereas replacement of two proline residues in N-terminal region did not affect its antioxidative activity, indicating that $3^{rd}$ proline in C-terminal region is critical for the antioxidative activity of the peptide identified in this study. In addition, N-terminal region of the antioxidant peptide did not show its activity, whereas C-terminal region maintained antioxidative activity, suggesting that C-terminal region of the peptide is important for antioxidative activity.

Development and Application of a Method for Rapid and Simultaneous Determination of Three β-agonists (Clenbuterol, Ractopamine, and Zilpaterol) using Liquid Chromatography-tandem Mass Spectrometry

  • Sung, In Kyung;Park, Seo Jung;Kang, Kyutae;Kim, Min Young;Cho, Seongbeom
    • Food Science of Animal Resources
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    • v.35 no.1
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    • pp.121-129
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    • 2015
  • ${\beta}$-agonists are anabolic compounds that promote fat loss and muscle gain, and their administration to livestock may provide economic benefits by increasing growth rate and feed efficiency. For these reasons, ${\beta}$-agonists are also commonly added to livestock feed as growth promoters. This can introduce a significant risk of secondary human poisoning through intake of contaminated meat. A new method for the simultaneous determination of three ${\beta}$-agonists (clenbuterol, ractopamine, and zilpaterol) was developed in this study and applied to various meat samples. The limits of quantification, derived through a validation test following Codex guidelines, were $0.2{\mu}g/kg$ for clenbuterol and zilpaterol, and $0.4{\mu}g/kg$ for ractopamine. The average recoveries for clenbuterol, ractopamine, and zilpaterol ranged from 109.1% to 118.3%, 95.3% to 109.0%, and 94.1% to 120.0%, respectively. The recovery and coefficient of variation (CV) values fell within the acceptable range according to the Codex guidelines. This method reduced the analysis time without decreasing detection efficiency by modifying the pretreatment steps. This method could be utilized to manage the safety of imported meat products from countries where zilpaterol use is still permitted, thereby improving public health and preventing ${\beta}$-agonist poisoning due to secondary contamination.

Effect of Pioglitazone on Perihematomal Edema in Intracerebral Hemorrhage Mouse Model by Regulating NLRP3 Expression and Energy Metabolism

  • Kim, Hoon;Lee, Jung Eun;Yoo, Hyun Ju;Sung, Jae Hoon;Yang, Seung Ho
    • Journal of Korean Neurosurgical Society
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    • v.63 no.6
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    • pp.689-697
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    • 2020
  • Objective : Cerebral edema is the predominant mechanism of secondary inflammation after intracerebral hemorrhage (ICH). Pioglitazone, peroxisome proliferator-activated receptor gamma agonist has been shown to play a role in regulation of central nervous system inflammation. Here, we examined the pharmacological effects of pioglitazone in an ICH mouse model and investigated its regulation on NLRP3 inflammasome and glucose metabolism. Methods : The ICH model was established in C57 BL/6 mice by the stereotactical inoculation of blood (30 µL) into the right frontal lobe. The treatment group was administered i.p. pioglitazone (20 mg/kg) for 1, 3, and 6 days. The control group was administered i.p. phosphate-buffered saline for 1, 3, and 6 days. We investigated brain water contents, NLRP3 expression, and changes in the metabolites in the ICH model using liquid chromatography-tandem mass spectrometry. Results : On day 3, brain edema in the mice treated with pioglitazone was decreased more than that in the control group. Expression levels of NLRP3 in the ICH model treated with pioglitazone were decreased more than those of the control mice on days 3 and 7. The pioglitazone group showed higher levels of glycolytic metabolites than those in the ICH mice. Lactate production was increased in the ICH mice treated with pioglitazone. Conclusion : Our results demonstrated less brain swelling following ICH in mice treated with pioglitazone. Pioglitazone decreased NLRP3-related brain edema and increased anaerobic glycolysis, resulting in the production of lactate in the ICH mice model. NLRP3 might be a therapeutic target for ICH recovery.