Name: a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s
Uploaded: 2017-09-25T13:00:00.000+00:00
Duration: 7 min 38 s
Description: Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

이 연구 보조금에 의해 지원 되었다 아니. 캘리포니아-81301 국립 암 연구소에서. 우리 편집 지원에 대 한 칼 슨 밥, 박사 피터 Villalta 프리메이슨 암 센터의 분석 생화학 공유 리소스에서 질량 분석에 쉰 밍 박사 아담 T. Zarth 그리고 그들의 귀중 한 토론에 대 한 박사 안 나 공화국 미셸 감사 합니다. 그리고 입력. 분석 생화학 공유 리소스는 부분적으로 지원 하 여 국립 암 연구소 암 센터 지원 그랜트 CA-77598

1. 자료 및 일반 절차 <ol><li>종합 NNN 앞 15에서 설명한. Norcotinine, P450 2A6 Baculosomes, NADPH 재생 시스템, 0.5 x 반응 버퍼, 및 다른 모든 화학 시 약 급료에 상용 소스 로부터 용 매 또는.</li>
	<li>500 MHz 분석기에 기록 NMR 스펙트럼. 보고서 화학 당 부품으로 교대 백만 (ppm). 잔류 용 매 피크를 사용 하 여 내부 참조 1 H NMR (7.26 ppm CDCl 3)와 13 C NMR (77.2 ppm CDCl 3). 
	참고: 피크 분할은 다음과 같은 약어를 사용 하 고 있다: s 내의 d = 더블 릿, dd = = 남자, dt의 남자 용 상의 삼중 항, dq의 더블 릿 = 사중주, ddd의 더블 릿 = = 남자, 그리고 m의 남자 용 상의의 더블 릿 multiplet =.</li>
	<li>LTQ Orbitrap Velos와 m/z로 보고서 데이터에 선택 된 화합물에 대 한 고 분해능 질량 분석 (HRMS) 수행.</li>
	<li>사용 polygram 미리 254 nm 형광 표시기 얇은 층 크로마토그래피 (TLC)와 실리 카 젤 TLC 판 (40 m m x 80 m m, 두께 0.2 m m) 코팅. UV 램프 조사 하 여 TLC 번호판을 시각화.</li>
	<li>1.5 cm x 15 cm 유리 열을 사용 하 여 Å, 70-150 메쉬 실리 카 젤 60에 플래시 크로마토그래피를 수행.</li>
</ol> 2. Nitrosamide 긍정적인 통제의 준비 (N &#8242;-nitrosonorcotinine, NNC) <ol><li>건조, 하룻밤 오븐에서 (16 h, ~ 140 ° C), 25 mL, 둥근 바닥 플라스 크 자기 볶음 bar가 포함 된. 아침에 차가운 흐름의 N 2 N 2 흐름을 유지 하는 석유 bubbler를 사용 하는 동안 플라스 크 속도 초당 1 개 거품. 
	참고: 냉각 후, 아무 주의 N 2 플라스 크를 유지 하는 필요.</li>
	<li>연기 후드, norcotinine (31.8 mg, 0.196 mmol), 아세트산 무수 물 (5 mL), 및 초 산 (1 mL)을 추가 냉각된 플라스 크. 지속적으로 물이 얼음 욕조와 0 ° C에 냉각 하는 동안이 혼합물을 저 어. 
	주의: 아세트산 무수 물과 아세트산은 부식성.</li>
	<li>추가 나노 2 (33.3 mg, 0.483 mmol) 한 부분을 지속적으로 2.5 h. TLC에 의해 모니터 반응 진행에 대 한 혼합물을 저 어 (100 %EtOAc, R f = 0.19, 단계 1.4 참조). 
	참고:이 시간이 지남에 혼합 될 것입니다 점점 노란색 가끔 버블링.</li>
	<li>(18 mL) 얼음 처럼 차가운 H 2 O로 혼합물을 붓는 의해 반응을 끄다. 바로 채널 2 Cl 2 100 mL separatory 깔때기에 18 mL와 용액을 추출 합니다. 추출 물의 레이어 채널 2 Cl 2 (각 9 mL)와 적어도 2 추가 시간.</li>
	<li>풀링된 생명체 2 분 필터 MgSO 4 이상 ~ 100 mg을 건조 하 고 원유, 노랑, 석유를 회전 증발에 의해 솔루션을 집중. 증발 잔류 초 산을 제거 하는 동안 30 ° C에 물 목욕 열. 
	참고: 프로토콜 일시 중지할 수 있습니다 여기 화합물 채널 2 Cl 2와 2-8 ° c.에 어둠 속에 저장 하는 솔루션에 용 해 하는 경우</li>
	<li>칼럼 크로마토그래피에 의해 원유 화합물 정화 (단계 1.5 참조) 실리 카 젤을 사용 하 여 고정 위상 및 100%로 EtOAc eluent 16. 
	주의: NNC와 관련된 nitrosamides 처리 주의 그들은 인간의 발암 물질로 간주 됩니다.</li>
	<li>CDCl 3 순수 화합물을 녹이 고 NMR을 사용 하 여 (단계 1.2 참조) 구조를 확인 하 고이 솔루션 17의 몰 확인 하. 2-8 ° C까지 필요에 어둠 속에서이 솔루션을 저장. 
	참고:이 솔루션 (3.5 단계)의 체 외에 분석 결과에 긍정적인 컨트롤로 사용 됩니다. NMR 스펙트럼 및 화학 교대 지원 정보에서 사용할 수 있습니다.</li>
	<li>순수한 NNC 솔루션으로는 정확한 확인 질량 부모 고 1.3 및 4.3 단계에서 설명 하는 매개 변수에서 고해상도 질량 분 서 계 (HRMS)에 직접 주입 하 여 제품 이온 대중의 결정. 
	참고: 제품 질량 체 외에 대사 분석 결과 (4.3 단계)에서 화합물 검출을 위해 사용 됩니다.</li>
</ol> 3. 예제 nitrosamine P450 생체 외에서 부 화 <ol><li>-80 ° C 냉동 고에서 P450 2A6 Baculosomes, 생생한 NADPH 재생 시스템, 그리고 10 mM NADP + 재고 솔루션을 제거 하 고 얼음에 녹여 그들. 일단 해 동, 희석 Baculosomes 및 NADPH 재생 시스템 1: 10과 1:50, 0.5 X 반응 버퍼 단일 1 mL 튜브에 각각. 마찬가지로, 추가 반응 버퍼 X + 재고 솔루션 0.5 97 μ를 NADP의 3 μ. 
	참고: NADP +은 빛에 민감한입니다. 이 솔루션의 컨테이너 알루미늄 호 일에 감싸서 보호 유지. 효소 솔루션은 민감한 freeze-thaw 주기; 이 2 개 이상의 주기를 제한 합니다. Aliquots 미래 실험을 위한 필요에 따라 준비.</li>
	<li>각 보육에 대 한 추가 50 μ 효소 솔루션 (5 pmol P450 포함) 1 mL의 튜브 반응 버퍼와 4 μ M NNN 솔루션의 포함 40 μ. 미리 물을 욕조를 사용 하 여 37 ° C에서 2 분에 대 한이 새로운 혼합물을 품 어 고 희석된 NADP + 솔루션의 10 μ를 추가 합니다. 37에 1-30 분에 대 한 전체 시스템을 품 어 ° c. 
	참고: 보육 시간 5 분 간격을 사용 하 여 (예: 5, 10, 15 분, 등) 충분 한 nitrosamide 형성 시간 코스를 제공 합니다.</li>
	<li>원하는 보육 시간 후 3.0 N ZnSO 4의 첫 번째 추가 10 μ와 3.0 N Ba(OH)의 다음 10 μ 끄다 2. 소용돌이이 솔루션 및 백색 침전 물은 형성할 것 이다. 원심 침전 작은 4 분 8000 x g에서 샘플. 
	참고: 절차 해야 하지 일시 중지이 단계에서 수성 해결책에 있는 안정성을 제한 하는 구성 된 nitrosamides로 합니다. 허위 제외 어 긴 일시 중지 될 수 있습니다.</li>
	<li>즉시는 상쾌한 플라스틱 및 액체 크로마토그래피 긍정적인 nanoelectrospray-이온화 고해상도 탠덤 질량 분석에 의해 2 μ를 분석 (LC-NSI +-HRMS/MS, 4.1 4.3 단계).</li>
	<li>긍정적인 제어 외피에 대 한 흐름의 N 2 1 mL 튜브에 합성된 NNC 솔루션의 100 fmol을 포함 하는 약 수를 증발. 이 유리병에 3.2 단계에서 전체 효소 시스템을 추가 하 고 3.3-3.4 단계에 설명 된 대로 진행.</li>
	<li>부정적인 제어 외피 수행 실험 설명된 (3.2-3.4 단계)로 바꾸기는 50 μ 효소 반응 버퍼 X 0.5 솔루션 제외.</li>
</ol> 4. LC-NSI +에 의해 nitrosamide 탐지에 대 한 매개 변수 예제-HRMS/MS <ol><li>LC에 대 한 구성 요소를 사용 하는 상업, 손 포장 18 C18 UPLC 시스템 단계 4.2에서에서 다음과 같은 여러 단계로 그라데이션 적용 (5 μ m), 100 m m x 75 μ m, 15 μ m 구멍 모 세관 칼럼.</li>
	<li>용 매와 용 매 b MeCN로 사용 하 여 5 mM NH 4 OAc 1 μ/분 5 %B 실행 하 여 열에 샘플을 로드 0-5 분, 그리고 다음 이후에 0.3 μ/분 유량을 둔화. 다음, 실행 그라디언트 5에서 20% 이상 4 분, 55% B 10 분 이상 램프 및 다음 5%를 다시 평형 b.</li>
	<li>수행 LC NSI +-LTQ Orbitrap에 HRMS/MS. 전체 검사와 MS 2 조각화 NNC 위한 모니터. 60, 000의 해상도에서 전체 검사를 수행 하 고 5 ppm의 대량 허용에 정확한 부모 질량 (192.07670)를 추출 합니다. 격리 하 부모 이온 (2.0 amu) 및 조각 충돌 유도 된 분리 (CID) 충돌 에너지 25 eV, 15000의 해상도의 여 고 30 양의 시간을 스캔 사용 MS 2 조각화 추출 NNC (m/z의 제품 이온에 대 한 정확한 질량 192 m/z 134.04739와 162.07874) 5 ppm의 대량 허용 오차에서.</li>
</ol>

<ol>
	<li>Rom, W. N., Markowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Environmental and Occupational Medicine. , 1226-1239 (2007).</li><li>Preussmann, R., Stewart, B. W., Searle, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Carcinogens, ACS Monograph 182. 2, 643-828 (1984).</li><li>Magee, P. N., Montesano, R., Preussmann, R., Searle, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical Carcinogens. ACS monograph 173. , 491-625 (1976).</li><li>Zhu, A. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alaska+Native+smokers+and+smokeless+tobacco+users+with+slower+CYP2A6+activity+have+lower+tobacco+consumption,+lower+tobacco-specific+nitrosamine+exposure+and+lower+tobacco-specific+nitrosamine+bioactivation.">Alaska Native smokers and smokeless tobacco users with slower CYP2A6 activity have lower tobacco consumption, lower tobacco-specific nitrosamine exposure and lower tobacco-specific nitrosamine bioactivation.</a> Carcinogenesis. 34 (1), 93-101 (2013).</li><li>Mesi&#263;, M., Revis, C., Fishbein, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+structure+on+the+reactivity+of+alpha-hydroxydialkynitrosamines+in+aqueous+solutions.">Effects of structure on the reactivity of alpha-hydroxydialkynitrosamines in aqueous solutions.</a> J. Am. Chem. Soc. 118, 7412-7413 (1996).</li><li>Mochizuki, M., Anjo, T., Okada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+N-alkyl-N-+(hydroxymethyl)nitrosamines+from+N-alkyl-N-+(hydroperoxymethyl)nitrosamines+by+deoxygenation.">Isolation and characterization of N-alkyl-N- (hydroxymethyl)nitrosamines from N-alkyl-N- (hydroperoxymethyl)nitrosamines by deoxygenation.</a> Tetrahedron Lett. 21, 3693-3696 (1980).</li><li>Guttenplan, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cytosol+on+mutagenesis+induced+by+N-nitrosodimethylamine,+N-nitrosomethylurea+and+&#224;-acetoxy-N-nitrosodimethylamine+in+different+strains+of+Salmonella:evidence+for+different+ultimate+mutagens+from+N-nitrosodimethylmine.">Effects of cytosol on mutagenesis induced by N-nitrosodimethylamine, N-nitrosomethylurea and &#224;-acetoxy-N-nitrosodimethylamine in different strains of Salmonella:evidence for different ultimate mutagens from N-nitrosodimethylmine.</a> Carcinogenesis. 14, 1013-1019 (1993).</li><li>Elespuru, R. K., Saavedra, J. E., Kovatch, R. M., Lijinsky, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a-carbonyl+derivatives+of+nitrosodimethylamine+in+ethylnitrosomethyamine+as+putative+proximate+carcinogens.">Examination of a-carbonyl derivatives of nitrosodimethylamine in ethylnitrosomethyamine as putative proximate carcinogens.</a> Carcinogenesis. 14, 1189-1193 (1993).</li><li>Chow, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ACS Symposium Series. 101, 13-37 (1979).</li><li>von Weymarn, L. B., Retzlaff, C., Murphy, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYP2A6-+and+CYP2A13-catalyzed+metabolism+of+the+nicotine+delta5'(1')iminium+ion.">CYP2A6- and CYP2A13-catalyzed metabolism of the nicotine delta5'(1')iminium ion.</a> J. Pharmacol. Exp. Ther. 343 (2), 307-315 (2012).</li><li>Bell-Parikh, L. C., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+cytochrome+P450+2E1-catalyzed+oxidation+of+ethanol+to+acetic+acid+via+acetaldehyde.">Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.</a> J Biol Chem. 274 (34), 23833-23840 (1999).</li><li>Chowdhury, G., Calcutt, M. W., Nagy, L. D., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+methyl+and+ethyl+nitrosamines+by+cytochrome+P450+2E1+and+2B1.">Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.</a> Biochemistry. 51 (50), 9995-10007 (2012).</li><li>Chowdhury, G., Calcutt, M. W., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+N-nitrosoalkylamines+by+human+cytochrome+P450+2A6:+sequential+oxidation+to+aldehydes+and+carboxylic+acids+and+analysis+of+reaction+steps.">Oxidation of N-nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.</a> J Biol Chem. 285 (11), 8031-8044 (2010).</li><li>Carlson, E. S., Upadhyaya, P., Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+nitrosamide+formation+in+the+cytochrome+P450-mediated+metabolism+of+tobacco-specific+nitrosamines.">Evaluation of nitrosamide formation in the cytochrome P450-mediated metabolism of tobacco-specific nitrosamines.</a> Chem Res Toxicol. 29 (12), 2194-2205 (2016).</li><li>Amin, S., Desai, D., Hecht, S. S., Hoffmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+tobacco-specific+N-nitrosamines+and+their+metabolites+and+results+of+related+bioassays.">Synthesis of tobacco-specific N-nitrosamines and their metabolites and results of related bioassays.</a> Crit. Rev. Toxicol. 26, 139-147 (1996).</li><li>Clark, A. G., Wong, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+chromatographic+technique+for+the+detection+of+dye-binding.">A rapid chromatographic technique for the detection of dye-binding.</a> Anal Biochem. 89 (2), 317-323 (1978).</li><li>Pauli, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+purity+evaluation+and+the+potential+of+quantitative+(1)H+NMR+as+a+purity+assay.">Importance of purity evaluation and the potential of quantitative (1)H NMR as a purity assay.</a> J Med Chem. 57 (22), 9220-9231 (2014).</li><li>van der Heeft, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microcapillary+column+switching+HPLC-electrospray+ionization+MS+system+for+the+direct+identification+of+peptides+presented+by+major+histocompatibility+complex+class+I+molecules.">A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules.</a> Anal Chem. 70 (18), 3742-3751 (1998).</li><li>White, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Chemistry+of+the+N-Alkyl-N-nitrosoamides.+I.+Methods+of+Preparation.">The Chemistry of the N-Alkyl-N-nitrosoamides. I. Methods of Preparation.</a> J. Am. Chem. Soc. 77, 6008-6010 (1955).</li><li>Patten, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+cytochrome+P450+2A6+and+3A4+as+major+catalysts+for+N'-nitrosonornicotine+alpha-hydroxylation+by+human+liver+microsomes.">Evidence for cytochrome P450 2A6 and 3A4 as major catalysts for N'-nitrosonornicotine alpha-hydroxylation by human liver microsomes.</a> Carcinogenesis. 18, 1623-1630 (1997).</li><li>Wong, H. L., Murphy, S. E., Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+2A-catalyzed+metabolic+activation+of+structurally+similar+carcinogenic+nitrosamines:+N'-nitrosonornicotine+enantiomers,+N-nitrosopiperidine,+and+N-nitrosopyrrolidine.">Cytochrome P450 2A-catalyzed metabolic activation of structurally similar carcinogenic nitrosamines: N'-nitrosonornicotine enantiomers, N-nitrosopiperidine, and N-nitrosopyrrolidine.</a> Chem. Res. Toxicol. 18, 61-69 (2004).</li><li>Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry,+biology,+and+carcinogenicity+of+tobacco-specific+N-nitrosamines.">Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines.</a> Chem. Res. Toxicol. 11, 559-603 (1998).</li><li>von Weymarn, L. B., Zhang, Q. Y., Ding, X., Hollenberg, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+8-methoxypsoralen+on+cytochrome+P450+2A13.">Effects of 8-methoxypsoralen on cytochrome P450 2A13.</a> Carcinogenesis. 26 (3), 621-629 (2005).</li></ol>

저자는 공개 없다.

니트로의 신진 대사를 elucidating 그들의 성에 이해 하는 중요 한 구성 요소입니다. 이기 때문에 관련 된 시 토 크롬 P450s와 다른 대사 효소 다형, 더욱이 지식을 응용 프로그램 식별할 수 있는 잠재적으로 위험이 높은 개인1,4. 새로운 데이터의 α-hydroxynitrosamines, DNA 바인딩에 관련 된 니트로의 추정된 주요 대사 산물을 더 산화, nitrosamides 하는 것은 가능 합?...

N-니트로 사 민 다이어트, 담배 제품 및 일반 환경;에 있는 발암 물질의 큰 클래스는 1인체에서를 endogenously 형성 수 있습니다 또한. 이상의 300 N-nitroso 화합물 테스트 및 &#62; 90%에서 발암 성 평가 했다 동물 모델2,3. 그들의 성에 전시, 먼저이 화합물 시 토 크롬 P450s1,,23에 의해 작동 한다. 연구는 시 토 크롬 P450s 쉽게 산화 α-hydroxynitrosamines (그림 1)는 ~ 5의 반감기와 반응성이 매우 높은 화합물에 니트로 자발적으로 alkyldiazohydroxides를 분해 하기 전에 s. 후자는 H2O N2의 손실 후에 DNA을 알 수 있습니다. 결과 DNA adducts unrepaired, 암 개발1이어질 중요 onco-또는 종양 억제기 유전자는 돌연변이 일으킬 수 있습니다. 이러한 이유로, 많은 노력 변화 통로의 완전 한 이해를 얻으려고 소비 되어, DNA adducts, 그리고 발암 성 니트로의 시 토 크롬 P450 산화의 다운스트림 대사 산물. 이 지식에 개별 암 위험 평가4잠재적인 응용 프로그램 합니다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg"> 
그림 1: 일반 및 니트로의 제안 된 대사. (1) 니트로 P450s α-hydroxynitrosamines (2) 자발적으로 alkyldiazohydroxides (3)을 분해 하는에 산화 됩니다. 이러한 화합물 형태로 DNA adducts DNA에 바인딩할 수 있습니다. 그것을 가정 했다 그 2 는 nitrosamides 4P450s에 의해 더 산화 된다. 이러한 형태로 소설 DNA adducts 또는 3 알려진된 DNA adducts 형태로 분해 DNA에 직접 바인딩할 수 있습니다. R1 과 R2 알 킬 그룹을 나타냅니다. <a href="//ecsource.jove.com/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
Α-hydroxynitrosamine 가설을 단단하게 광범위 한 데이터에 의해 지원 됩니다, 하지만 몇 가지 불일치;는 주요 것은 α-hydroxynitrosamines5,6의 짧은 반감기 이다. 이러한 화합물 바인딩과 그물 막에 생산 고 나중 핵 DNA를 알 알려져 있다. 몇 초 그들의 일생을 감안할 때, 그것은 어떻게 이러한 중간체 살아남을 필요한 여행 하지만 cytosol 수수께끼 이다. 하나의 가설은 이다 α-hydroxynitrosamines의 부분 산화 processively는 nitrosamides7,8, 비교9에서 매우 안정적입니다. 이것은 아마도 시 토 크롬 P450 활성 사이트에서 α-hydroxynitrosamines의 보존을 통해 발생 합니다. 산화의이 유형에 대 한 전례가 니코틴10, 알콜11, 그리고 간단한 alkylnitrosamines12,13보아 왔다. 또한, nitrosamides는 직접 연기 발암 물질2,3. 9그들의 반응 따라, 이러한 화합물 생산 DNA adducts 함께 새로운 α-hydroxynitrosamines에서 인 동일 하, 미개척된 DNA adducts (그림 1) 추정 된다. 따라서,이 가설 뿐만 아니라 설명 한다 cytosol, 통해 전송 하지만 또한 DNA의 형성 제품 손상.
이 문서에는 생체 외에서 시 토 크롬 P450 중재 변환 니트로의 nitrosamides 평가 하기 위한 일반 프로토콜을 설명 합니다. N ′-nitrosonorcotinine (NNC) 시 토 크롬 P450 2A6에 의해 n ′-nitrosonornicotine (NNN)의 이전에 보고 된 변환 예제14로 전시 됩니다. 다양 한 기질-효소 시스템에이 프로토콜의 응용 프로그램 전반적인 nitrosamine 대사 nitrosamides의 중요성을 결정 하는 데 도움이 됩니다.

<table><tbody><tr><td>Norcotinine</td><td>AKoS GmbH (Steinen, Germany)</td><td>CAS 17708-87-1, AKoS AK0S006278969</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma-Aldrich</td><td>242845</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>431311</td><td></td></tr><tr><td>Barium Hydroxide</td><td>Sigma-Aldrich</td><td>433373</td><td></td></tr><tr><td>D-Chloroform</td><td>Sigma-Aldrich</td><td>151823</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Sigma-Aldrich</td><td>34998</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methylene Chloride</td><td>Sigma-Aldrich</td><td>34856</td><td></td></tr><tr><td>Sodium Nitrite</td><td>Sigma-Aldrich</td><td>237213</td><td></td></tr><tr><td>ViVid CYP2A6 Blue Screening Kit</td><td>Life Technologies</td><td>PV6140</td><td></td></tr><tr><td>Zinc Sulfate</td><td>Sigma-Aldrich</td><td>221376</td><td></td></tr><tr><td>0.5 mL tubes</td><td>Fisher</td><td>AB0533</td><td></td></tr><tr><td>100 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z510424</td><td></td></tr><tr><td>125 mL Erlenmeyer flask</td><td>Sigma-Aldrich</td><td>CLS4980125</td><td></td></tr><tr><td>125 mL Separatory Funnel</td><td>Sigma-Aldrich</td><td>Z261017</td><td></td></tr><tr><td>25 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z278262</td><td></td></tr><tr><td>500 MHz NMR Spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Allegra X-22R Centrifuge</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>LC vials</td><td>ChromTech</td><td>CTC&amp;ndash;0957&amp;ndash;BOND</td><td></td></tr><tr><td>LTQ Orbitrap Velos</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Magnetic Stir bar</td><td>Sigma-Aldrich</td><td>Z127035</td><td></td></tr><tr><td>NMR tube</td><td>Sigma-Aldrich</td><td>Z274682</td><td></td></tr><tr><td>P1000, P200, and P10 pipettes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Rotary evaporator</td><td>Sigma-Aldrich</td><td>Z691410</td><td></td></tr><tr><td>RSLCnano UPLC system</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Shaking Water Bath</td><td>Fisher</td><td>FSSWB15</td><td></td></tr><tr><td>Stir plate</td><td>Sigma-Aldrich</td><td>CLS6795420</td><td></td></tr><tr><td>PicoFrit Column</td><td>New Objective</td><td>PF3607515N5</td><td></td></tr><tr><td>Luna C18, 5 um</td><td>Phenomenex</td><td>535913-1</td><td></td></tr></tbody></table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

N-니트로 시 토 크롬 P450 산화 전시 활동을 요구 하는 환경 발암 물질의 기초가 튼튼한 그룹. 대사 활성화의 허용된 메커니즘 저절로 DNA alkylating 에이전트를 분해 하는 α-hydroxynitrosamines의 대형을 포함 한다. DNA 손상의 결과 돌연변이 축적 궁극적으로 암으로 이어질 수 있습니다. 새로운 증거는 α-hydroxynitrosamines 수 수 추가 산화 nitrosamides에 processively 시 토 크롬 P450s에 의해 나타냅니다. Nitrosamides α-hydroxynitrosamines 보다 일반적으로 더 안정 하 고 또한 DNA를 알 수 있습니다, 때문에 nitrosamides 발암에 역할을 재생할 수 있습니다. 이 보고서에서 우리는 니트로의 생체 외에서 시 토 크롬 P450 촉매 물질 대사에서 nitrosamide 생산을 평가 하기 위한 일반 프로토콜을 설명 합니다. 이 프로토콜 관련 nitrosamides 및 생체 외에서 시 토 크롬 P450 물질 대사 분석 결과 검색에 대 한 액체 착 색 인쇄기-nanospray 이온화 높은 해상도 탠덤 질량 분석을 사용 하 여 합성 하는 일반적인 접근 방식을 활용 합니다. 이 방법은 n ′-예제 연구에서 nitrosonornicotine의 작은 대사 산물으로 n ′-nitrosonorcotinine를 감지. 메서드는 높은 감도 및 선택적으로 인해 정확한 대량 검출. 다양 한 nitrosamine-시 토 크롬 P450 시스템에이 방법의 응용이이 변환의 일반 성을 결정 하는 데 도움이 됩니다. 시 토 크롬 P450s 다형성을 활동에서 변화 하기 때문에 개별 암 위험 평가에 도움이 수 nitrosamide 대형의 더 나은 이해.

백인 외의 작업을 기반으로 합니다. 19, norcotinine 되었고 NNC에 니트로 소화 깔끔하게 높은 수율 (80-92%)에서 생체 외에서 실험에 대 한 표준 생산 하. 성공적인 반응에 대 한 구조적 증거 1H NMR, 13C NMR, 아늑한, 그리고 부모 질량 [M + H] 확인 HRMS 함께 HSQC (지원 정보)를 포함 하 여 분 광 분석에서 얻은+ 의 5 ppm 이내는 이론적...

Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

시 토 크롬 P450s에 의해 발암 성 니트로의 α-hydroxylation는 돌연변이 일으키는 원인이 되는 DNA 손상 중간체 생산 허용된 신진 대사 통로. 그러나, 새 데이터 나타냅니다 nitrosamides 산화 추가 발생할 수 있습니다. 우리는 일반 설명 nitrosamides을 탐지 하기 위한 방법 체 외에서 시 토 크롬 P450 촉매의 물질 대사 니트로에서 생산.

체 외에 대사 시 토 크롬 P450s에 의해 니트로의 Nitrosamide 형성을 탐지 하기 위한 일반적인 방법

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

a-general-method-for-detecting-nitrosamide-formation-vitro-metabolism

Research

JoVE Journal

Cancer Research

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

9.9K 조회수.  University of Minnesota. 시 토 크롬 P450s에 의해 발암 성 니트로의 α-hydroxylation는 돌연변이 일으키는 원인이 되는 DNA 손상 중간체 생산 허용된 신진 대사 통로. 그러나, 새 데이터 나타냅니다 nitrosamides 산화 추가 발생할 수 있습니다. 우리는 일반 설명 nitrosamides을 탐지 하기 위한 방법 체 외에서 시 토 크롬 P450 촉매의 물질 대사 니트로에서 생산.

비디오: A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

그만큼<em&gt; 초파리</em&gt; 가상적인 디스크 종양 모델 : 유전 모자이크를 사용하여 유전자 발현 및 종양 침윤의 시각화 및 정량화

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

연구

암 연구학

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

질량 분석 및 Luminogenic 기반의 접근 방법은 단계 I 대사 역량의의 특성을<em&gt; 체외</em&gt; 세포 배양

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

생화학

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

형성 화학식 DNA Adducts 효소 활성화 된 발암 물질 및 생체 외에서 마약 및 32P postlabeling에 의해 그들의 결정에 의해

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

질소 산화물 Bioactivity을 시금을위한 분석 기법

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

의학

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

3-Nitrotyrosine 대기 환경을 통해 고성능 액체 착 색 인쇄기-전기 화학 검출기 시스템에서의 탐지

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

환경과학

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

임상 응용을위한 인간의 소변에서 3- 니트로 타이로신의 소형 고체 상 추출 및 LC-MS / MS 검출의 통합

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

화학

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

14NH4+/15NH4+ N2O로순차적변환을 통한 분석 에 따른 암모늄에 대한 질산염 감소의 잠재적 비율 측정

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

안지오텐신 II에 의해 설명 니트로 펩티드 프로파일링 및 식별

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

산화 질소 및 그 유도체의 검출을위한 화학 발광 기반 분석 및 자동 산화 및 니트로화 화합물

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

화학 발광 법을 사용하여 생체 물질에서 산화 질소 통로에서 아질산염과 질산염, 대사 산물을 측정

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

체 외에 대사 시 토 크롬 P450s에 의해 니트로의 Nitrosamide 형성을 탐지 하기 위한 일반적인 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

그만큼

질량 분석 및 Luminogenic 기반의 접근 방법은 단계 I 대사 역량의의 특성을

형성 화학식 DNA Adducts 효소 활성화 된 발암 물질 및 생체 외에서 마약 및 ³²P postlabeling에 의해 그들의 결정에 의해