Name: simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures
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Este trabalho foi apoiado pela União Europeia do Fundo Europeu de Desenvolvimento Regional no âmbito do Programa Operacional Desenvolvimento da Polônia Oriental 2007-2013 (POPW.01.03.00-06-003/09-00), pela Faculdade de Biotecnologia e Ciências Ambientais da Universidade Católica John Paul II de Lublin (Young Scientists grant for Ilona Sadok), Centro Nacional de Ciências Poloneses, OPUS13 (2017/25/B/NZ4/01198 para Magdalena Staniszewska, Pesquisadora-Princípio).  Os autores agradecem ao Prof. Agnieszka Ścibior, do Laboratório de Estresse Oxidativo, Centro de Pesquisa Interdisciplinar, JPII CUL por compartilhar os equipamentos para cultivo celular e quantificação de proteínas.

1. Preparação das soluções de estoque padrão 3NT, Kyn, 3HKyn, 3HAA, XA standard
<ol><li>Pesar os reagentes em um frasco com a maior precisão (0,3 mg cada). Para melhor precisão, dimensione os reagentes, ajustando o volume do solvente de acordo com a etapa 1.2.</li>
	<li>Dissolver os reagentes em 300 μL do solvente para obter uma solução de estoque de 1 g/L. Dissolver 3NT em 1% (v/v) ácido fórmico (FA) na água; Kyn, 3HAA e XA em sulfóxido de dimetil (DMSO); 3HKyn em água acidificada para pH 2.5 com ácido...

<ol>
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G., 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=Concurrent+quantification+of+tryptophan+and+its+major+metabolites.">Concurrent quantification of tryptophan and its major metabolites.</a> Analytical Biochemistry. 443 (2), 222-231 (2013).</li><li>Badawy, A. A. B., Morgan, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isocratic+liquid+chromatographic+separation+and+quantification+of+tryptophan+and+six+kynurenine+metabolites+in+biological+samples+with+ultraviolet+and+fluorimetric+detection.">Rapid isocratic liquid chromatographic separation and quantification of tryptophan and six kynurenine metabolites in biological samples with ultraviolet and fluorimetric detection.</a> International Journal of Tryptophan Research. 3, 175-186 (2010).</li><li>Vignau, J., Jacquemont, M. 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Este artigo apresenta um protocolo LC-SQ detalhado para a quantificação simultânea de quatro principais metabólitos Trp (3HKyn, Kyn, 3HAA, XA) que são medidos em um meio a partir de células cancerígenas humanas in vitro cultivadas. Atenção especial é dada à preparação da amostra, procedimento de espectrometria cromatográfica/de massa e interpretação dos dados, os pontos mais importantes dentro da análise.
Em geral, a análise da LC-MS, devido à alta sensibilidade, re...

O caminho de kynurenine (KP) é a principal rota do catabolismo triptofano (Trp) em células humanas. Indoleamina-2,3-dioxygenase (IDO-1) em células extrahêspticas é a primeira enzima limitante de KP e converte Trp em N-formylkynurenine1. Outros passos dentro do KP geram outros metabólitos secundários, ou seja, kynurenines que exibem várias propriedades biológicas. Kynurenine (Kyn) é o primeiro catabólito Trp estável que mostra propriedades tóxicas e regula eventos celulares após a ligação ao receptor de hidrocarbonetos aryl (AhR)2. Posteriormente, Kyn é transformado em várias moléculas espontaneamente ou nos processos mediados por enzimas, gerando metabólitos como 3-hidroxikynurenine (3HKyn), ácido antoraniano (AA), ácido 3-hidroxyanthranilic (3-HAA), ácido kynurênico (Kyna) e ácido xanturênico (XA). Outro metabólito a jusante, 2-amino-3-carboxymuconic acid-6-semialdeído (ACMS), sofre ciclização não enzimática para ácido quinolínico (QA) ou ácido picolínico (PA)1. Finalmente, a QA é ainda transformada em dinucleotídeo de nicotinamida-adenina (NAD+)3, o metabólito de ponto final KP que é um importante cofator de enzimas. Algumas kynurenines têm propriedades neuroprotetoras como Kyna e PA, enquanto as outras, ou seja, 3HAA e 3HKyn, são tóxicas4. O ácido xanthurenic, formado a partir de 3HKyn, apresenta propriedades antioxidantes e vasorelaxação5. XA se acumula em lentes de envelhecimento e leva à apoptose das células epiteliais6. KP, descrito em meados do séculoXX, ganhou mais atenção quando seu envolvimento em vários transtornos foi demonstrado. O aumento da atividade dessa rota metabólica e o acúmulo de algumas kynurenines modulam a resposta imune e estão associados a diferentes condições patológicas como depressão, esquizofrenia, encefalopatia, HIV, demência, esclerose lateral amiotrófica, malária, Alzheimer, doença de Huntington e câncer4,7. Algumas alterações no metabolismo Trp são observadas em microambientes tumorais e células cancerosas2,8. Além disso, as kynurenines são consideradas como marcadores promissores da doença9. Na pesquisa sobre câncer, os modelos de cultura in vitro são bem estabelecidos e amplamente utilizados para avaliação pré-clínica de respostas a medicamentos anticancerígenos10. Os metabólitos trp são secretados pelas células no meio da cultura e podem ser medidos para avaliar o status da via kynurenina. Portanto, é necessário desenvolver métodos apropriados para a detecção simultânea de tantos metabólitos KP quanto possível em uma variedade de espécimes biológicos com um protocolo fácil, flexível e confiável.
Neste artigo, descrevemos um protocolo para a determinação simultânea de quatro metabólitos de via kynurenina: Kyn, 3HKyn, 3HAA e XA pela LC-SQ em um meio pós-cultura coletado de células cancerosas. Em uma abordagem analítica moderna, a cromatografia líquida13,14,15,16 é preferida para a detecção e quantificação simultâneas dos catabólitos triptofanos individuais, em contraste com ensaios bioquímicos inespecíficos utilizando o reagente Ehrlich11,12. Atualmente, existem muitos métodos disponíveis para a determinação de kynurenines em espécimes humanos, principalmente baseados na cromatografia líquida com detectores de ultravioleta ou fluorescência13,17,18,19. A cromatografia líquida aliada a um detector de espectrometria de massa (LC-MS) parece mais adequada para este tipo de análise, devido à sua maior sensibilidade (limites mais baixos de detecção), seletividade e repetibilidade.
Metabólitos trp já foram determinados em soro humano, plasma e urina13,20,21,22,23, no entanto, os métodos para outros espécimes biológicos, como o meio de cultura celular também são desejados. Anteriormente, o LC-MS era utilizado para compostos derivados de TRP em um meio coletado após acultura de células de glioma humanos, monócitos, células dendríticas ou astrócitos tratados com gama de interferon (IFN-γ)24,25,26. Atualmente, há a necessidade de novos protocolos validados que possam permitir uma avaliação de vários metabólitos em diferentes mídias culturais, células e tratamentos usados em modelos de câncer.
O objetivo do método desenvolvido é quantificar (dentro de uma corrida analítica) quatro kynurenines principais que podem indicar anormalidades em KP. Apresentados aqui estão etapas críticas do nosso protocolo recentemente publicado para análise quantitativa lc-SQ de kynurenines significativas selecionadas usando um padrão interno (3-nitrotyrosina, 3NT) no meio coletado de células cancerígenas humanas in vitro cultivadas 27. Para nosso melhor conhecimento, é o primeiro protocolo LC-SQ para quantificação simultânea de 3HKyn, 3HAA, Kyn e XA em um meio de cultura obtido a partir das células cultivadas in vitro. Após algumas modificações, o método pode ser aplicado ainda mais para estudar as mudanças no metabolismo Trp em uma gama mais ampla de modelos de cultura celular.

<table border="0" cellpadding="0" cellspacing="0" style="width:1360px;" width="1359">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-hydroksy-DL-kynurenina</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">H1771</td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-hydroxyanthranilic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">148776</td>
			<td>97%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-nitro-L-tyrosine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">N7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Acetonitrile</td>
			<td style="width:175px;">Supelco</td>
			<td>1.00029</td>
			<td>hypergrade for LC-MS LiChrosolv</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Activated charcoal</td>
			<td style="width:175px;">Supelco</td>
			<td>05105</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Analytical balance</td>
			<td style="width:175px;">Ohaus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:411px;">Analytical column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>959764-902</td>
			<td>Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium formate</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">7022</td>
			<td>eluent additive for LC-MS, LiChropur, &#8805;99.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td style="width:175px;">Sigma</td>
			<td>1001887398</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caps for chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5185-5820</td>
			<td>blue screw caps,PTFE/red sil sepa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dish</td>
			<td style="width:175px;">Nest</td>
			<td>704004</td>
			<td>polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plate</td>
			<td style="width:175px;">Biologix</td>
			<td>07-6012</td>
			<td>12-well, non-pyrogenic, non-cytoxic, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>HERAcell 150i Cu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5415R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-2278</td>
			<td>Eppendorf type, 1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-3693</td>
			<td>Falcon type, 50 mL, PP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic data acqusition and analysis software</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td></td>
			<td>LC/MSD ChemStation (B.04.03-S92)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic insert vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-1387</td>
			<td>100 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5182-0714</td>
			<td>2 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dimethyl sulfoxide</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">1.02950</td>
			<td>Uvasol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dubelcco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:175px;">PAN Biotech</td>
			<td>P04-41450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dual meter pH/conductivity</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td style="width:261px;"></td>
			<td>SevenMulti</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evaporator</td>
			<td style="width:175px;">Genevac</td>
			<td style="width:261px;"></td>
			<td>model EZ-2.3 Elite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Formic acid</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">5.33002</td>
			<td>for LC-MS LiChropur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glass bottle for reagents storage</td>
			<td style="width:175px;">Bionovo</td>
			<td>S-2070</td>
			<td>50 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Guard column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">959757-902</td>
			<td>Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hydrochloric acid</td>
			<td style="width:175px;">Merck</td>
			<td>1.00317.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">L-kynurenine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">K8625</td>
			<td>&#8805;98% (HPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Magnetic stirrer</td>
			<td style="width:175px;">Wigo</td>
			<td style="width:261px;"></td>
			<td>ES 21 H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microbalance</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td></td>
			<td>model XP6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Milli-Q system</td>
			<td style="width:175px;">Millipore</td>
			<td>ZRQSVPO30</td>
			<td>Direct-Q 3 UV with Pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quadrupole mass spectrometer</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>G1948B</td>
			<td>model 6120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:175px;">Sigma-Adrich</td>
			<td>048M4874V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td style="width:175px;">Bio Tek</td>
			<td></td>
			<td>Synergy 2 operated by Gen5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td style="width:175px;">Merck</td>
			<td>1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium dihydrogen phosphate</td>
			<td style="width:175px;">Merck</td>
			<td>1.05108.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Assay Dye Reagent Concentrate</td>
			<td style="width:175px;">BioRad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">See-saw rocker</td>
			<td style="width:175px;">Stuart</td>
			<td>SSL4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipette</td>
			<td style="width:175px;">Nest</td>
			<td>326001</td>
			<td>5 mL, polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td style="width:175px;">Merck</td>
			<td>1.06346.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent inlet filter</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5041-2168</td>
			<td>glass filter, 20 &#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6524</td>
			<td>1 L, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6526</td>
			<td>1 L, amber glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spreadsheet program</td>
			<td style="width:175px;">Microsoft Office</td>
			<td></td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stir bar</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-2003</td>
			<td>teflon coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for culture medium filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>7-8803</td>
			<td>regenerated cellulose, &#216; 30 mm, 0,45 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for mobile phase components filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-0018</td>
			<td>nylon, &#216; 30 mm, 0,22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td style="width:175px;">VWR</td>
			<td>10062-900</td>
			<td>96-wells, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA Solution</td>
			<td style="width:175px;">Sigma-Aldrih</td>
			<td>T4049-100 mL</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:411px;">Ultra-High Performance Liquid Chromatography system</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">G1367D, G1379B, G1312B, G1316C</td>
			<td style="width:513px;">1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound bath</td>
			<td>Polsonic</td>
			<td>104533</td>
			<td>model 6D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>Biosan</td>
			<td></td>
			<td>model V-1 plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xanthurenic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">D120804</td>
			<td>96%</td>
		</tr>
	</tbody>
</table>

simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures

O caminho da kynurenine e os catabólitos do triptofano chamados kynurenines têm recebido maior atenção por seu envolvimento na regulação imunológica e biologia do câncer. Um ensaio de cultura in vitro é frequentemente usado para aprender sobre a contribuição de diferentes catabólitos triptofanos em um mecanismo de doença e para testar estratégias terapêuticas. O meio de cultura celular rico em metabólitos secretos e moléculas de sinalização reflete o status do metabolismo triptofano e outros eventos celulares. Novos protocolos para a quantificação confiável de múltiplas kynureninas no complexo meio de cultura celular são desejados para permitir uma análise confiável e rápida de múltiplas amostras. Isso pode ser realizado com cromatografia líquida aliada à espectrometria de massa. Esta poderosa técnica é empregada em muitos laboratórios clínicos e de pesquisa para quantificação de metabólitos e pode ser usada para medir kynurenines.
Apresentado aqui é o uso de cromatografia líquida juntamente com espectrometria de massa quadrupole única (LC-SQ) para a determinação simultânea de quatro kynurenines, ou seja, kynurenine, 3-hidroxikynurenine, 3-hidroxyanthranilic, e ácido xanturnic no meio coletado de células cancerígenas in vitro cultivadas. O detector SQ é simples de usar e menos caro em comparação com outros espectrômetros de massa. Na análise SQ-MS, vários íons da amostra são gerados e separados de acordo com sua relação massa-carga específica(m/z),seguida pela detecção usando um modo de Monitoramento de Íons Únicos (SIM).
Este artigo chama a atenção sobre as vantagens do método relatado e indica alguns pontos fracos. É focado em elementos críticos da análise LC-SQ, incluindo preparação de amostras, juntamente com cromatografia e análise de espectrometria de massa. Também são discutidas as condições de controle de qualidade, as condições de calibração do método e os problemas de efeito matricial. Descrevemos uma simples aplicação de 3-nitrotyrosina como um padrão analógico para todos os analitos alvo. Como confirmado por experimentos com ovário humano e células cancerígenas de mama, o método LC-SQ proposto gera resultados confiáveis e pode ser ainda mais aplicado a outros modelos celulares in vitro.

A LC-MS apresenta vantagens indiscutíveis na quantificação de moléculas biologicamente ativas, embora alguns componentes de espécimes complexos causem os chamados efeitos matriciais e comprometam a ionização dos analitos. Leva à supressão de íons ou ao aprimoramento de íons, diminuindo fortemente a precisão e afetando um limite de detecção/quantificação de LC-MS, que é considerado como um ponto ''fraco' do método. Em nosso protocolo, os íons são gerados pela ionização eletrospray (ESI) no modo posit...

Watch this Scientific Journal Video about Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures at JoVE.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

Descrito aqui é um protocolo para a determinação de quatro metabólitos triptofanos diferentes gerados na via kynurenine (kynurenine, 3-hidroxikynurenine, ácido xanthurenic, ácido 3-hidroxyanthranilic) no meio coletado de culturas de células cancerosas analisadas por cromatografia líquida acoplada com uma única espectrometria de massa quadrupole.

Quantificação simultânea de kynureninas selecionadas analisadas por espectrometria de cromatografia líquida-massa em média coletada de culturas celulares cancerosas

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Kynurenines are associated with immune response regulation in several diseases, including cancer. Reliable and validated methods for identifying multiple kynurenines aid in the development of more effective therapies. Our protocol uses liquid chromatography coupled with mass spectrometry to identify the different tryptophan metabolites generated by the kynurenine pathway in medium collected from cancer cell cultures.Unexperienced users may have some difficulty during the sample preparation or data acquisition and interpretation steps. By following our protocol and recommendations, one can avoid mistakes and obtain reliable data. To prepare stock solutions of the reagents, weigh out 0.3 milligrams of each reagent in a vial to the highest accuracy and dissolve each reagent in 300 microliters of the appropriate solvent to obtain one gram per liter stock solutions of each reagent.To prepare charcoal-treated culture medium, weigh out 280 milligrams of activated charcoal in a conical tube and add five milliliters of the liquid medium prepared for culturing the cells of interest. Shake the tube with the medium and charcoal on a seesaw rocker for two hours at room temperature and 50 oscillations per minute. At the end of the incubation, sediment the charcoal by centrifugation and carefully collect the supernatant without disturbing the charcoal, then filter the medium using a 0.45 micrometer syringe filter.To prepare the calibration solution, spike the charcoal-treated culture medium with 0.75 microliters of 0.1 gram per liter 3NT solution and add 3-H kynurenine, 3-HAA kynurenine, and xanthurenic acid to at least six different concentrations to cover all of the calibration ranges to a final volume of 150 microliters per sample. After vortexing, add 150 microliters of minus 20 degrees Celsius cold methanol supplemented with 1%formic acid to each tube for sample deproteinization and tightly cap and vortex the tubes again. After a 40-minute incubation at minus 20 degrees Celsius, centrifuge the samples and use an automatic pipette to transfer 270 microliters of each supernatant into individual flat bottom glass vials.Place the vials into an evaporator and use the appropriate program for water methanol fractions to gently evaporate the volatile components for about 30 minutes. When the tubes are dry, reconstitute each sample in 60 microliters of 0.1%formic acid in water and vortex the samples before transferring each solution into individual 1.5 milliliter tubes for centrifugation. Without disturbing the sediment, transfer the supernatants into chromatographic vials with conical glass inserts and place the vials into an LC-MS auto-sampler.Before running the samples, purge the LC system with the mobile phase to remove bubbles and to prime all of the solvent channels. Next, flush the guard and analytical column with 100%acetonitrile for about 30 minutes before flushing the system with 100%solvent A until a stable pressure is observed in the column, then set the appropriate LC parameters in the data acquisition software and construct a work list for running the samples on the LC system. To construct the calibration curve, in the data acquisition software, add the calibration standards into the work list and run the standards in triplicates, then integrate and measure the peak area corresponding to 3-hydroxykynurenine, kynurenine, 3-hydroxyanthranilic acid, 3-nitrotyrosine, and xanthurenic acid at the retention time of about 4.4, 10, 16, 21, and 30 minutes respectively.To collect supernatant samples from an in vitro cell culture, after 48 hours of standard cell culture of the cells of interest, transfer 500 microliters of supernatant from each well into individual 1.5 milliliter tubes and remove the cell debris by centrifugation, then transfer the medium supernatants into new tubes for minus 80 degrees Celsius storage until the analysis and maintain the cell pellets at minus 20 degrees Celsius until protein estimation analysis. To prepare samples for LC-MS analysis, transfer 149.25 microliters of each thawed culture medium sample into a new 1.5 microliter tube and add 0.75 microliters of 0.1 gram per liter 3NT solution to each tube. After preparing the samples as demonstrated, add 150 microliters of minus 20 degrees Celsius methanol supplemented with 1%formic acid to each tube and prepare the sample for LC-MS analysis as demonstrated.When the work list is complete, measure the peak area of each analyte and use an individual linear calibration equation dedicated for each analyte to calculate concentrations of the analytes present within each experimental sample. To assess cellular protein content corresponding to the sample of culture medium, resuspend each cell pellet in 100 microliters of PBS and freeze the samples at minus 20 degrees Celsius for one hour before thawing them on ice. After freeze-thawing the samples three times, collect the cell lysates by centrifugation and transfer the supernatants into new tubes without disturbing the pellets.Dilute the sample 10 times with fresh PBS and add the appropriate volumes of bovine serum albumin standard solution in duplicate to the appropriate wells of a 96-well plate. Load 10 to 15 microliters of each cell lysate sample supernatant to the appropriate wells of the 96-well plate and fill both the standard and sample wells to a final volume of 50 microliters of PBS per well. Next, add 200 microliters of a Bradford reagent diluted five times with ultra pure water to each well and incubate the plate for at least five minutes at room temperature.At the end of the incubation, load the plate onto a microplate reader and measure the absorbance at 595 nanometers, then construct a calibration curve to allow calculation of the amount of protein in each sample and divide the concentration of each kynurenine by the total protein content to normalize the kynurenine amount in each sample per one microgram of protein. Here are single quadrupole mass spectrometry results obtained during the analysis of medium containing 4.5 grams per liter of D-glucose and 10%fetal bovine serum. Note the small peak indicating the presence of kynurenine within the sample.Run a quality control sample before requiring the actual sample data to confirm the appropriate retention times of the analytes as a shift in LC signals or mismatches can be observed. Co-eluting matrix components can generate ions with the master charge ratio selected for the target analytes. For example, in this analysis, the peak from the unknown compound appeared in the scan period during which the ion of master charge ratio 206 was monitored.Due to the incompatibility of the retention time, the signal did not correspond to the analyte. Although the tryptophan isotope shared the same ion as master charge ratio 206, chromatographic analysis revealed that the tryptophan signal was well separated from the xanthurenic acid signal, indicating that the level of tryptophan present in the tested cancer cell medium did not impact xanthurenic acid quantification. This analysis of the culture medium from two types of cancer cell lines demonstrates that the evaluated human epithelial breast cancer cells released different amounts of tryptophan metabolites while the tested human ovarian cancer cells produced significantly higher levels of kynurenine.Using an internal standard during the sample preparation helps with reliable data acquisition. Normalization of the data to the protein content corrects for cell number variabilities within individual wells. Our protocol can be further expanded to determine additional analytes, but may accumulate in the culture medium under different experimental conditions.

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