Name: lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism
Uploaded: 2016-04-04T15:00:00
Description: Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

Reconocemos el apoyo de NIH subvenciones P30ES013508 y T32ES019851.

Ética Declaración: Todos los protocolos relativos al tratamiento de muestras humanas siguen las directrices de la Universidad de Pennsylvania comité de ética de investigación humana. 1. Preparación de tampones y 100x Soluciones madre <ol><li> Preparar 1 L de tampón de base de Tyrode. Combinar 8.123 g de NaCl, 1,428 g de NaHCO3, 0,466 g de CaCl2 ∙ 2 H2O, 0,224 g de KCl, y 0,095 g de MgCl 2. Ajustar el volumen total de 1 litro con ddH...

<ol>
	<li>Ault, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+utility+of+flow+cytometry+in+the+study+of+platelets.">The clinical utility of flow cytometry in the study of platelets.</a> Semin. Hematol. 38 (2), 160-168 (2001).</li><li>Avila, 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=Platelet+mitochondrial+dysfunction+is+evident+in+type+2+diabetes+in+association+with+modifications+of+mitochondrial+anti-oxidant+stress+proteins.">Platelet mitochondrial dysfunction is evident in type 2 diabetes in association with modifications of mitochondrial anti-oxidant stress proteins.</a> Exp. Clin. Endocrinol. Diabetes. 120 (4), 248-251 (2012).</li><li>Basu, S. S., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotenone-mediated+changes+in+intracellular+coenzyme+A+thioester+levels:+implications+for+mitochondrial+dysfunction.">Rotenone-mediated changes in intracellular coenzyme A thioester levels: implications for mitochondrial dysfunction.</a> Chem. Res. Toxicol. 24 (10), 1630-1632 (2011).</li><li>Basu, S. S., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SILEC:+a+protocol+for+generating+and+using+isotopically+labeled+coenzyme+A+mass+spectrometry+standards.">SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards.</a> Nat. Protoc. 7 (1), 1-12 (2012).</li><li>Basu, S. S., Deutsch, E. C., Schmaier, A. A., Lynch, D. R., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelets+as+a+platform+to+monitor+metabolic+biomarkers+using+stable+isotopes+and+LC-MS.">Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.</a> Bioanalysis. 5 (24), 3009-3021 (2013).</li><li>Berson, A., 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=Mechanisms+for+experimental+buprenorphine+hepatotoxicity:+major+role+of+mitochondrial+dysfunction+versus+metabolic+activation.">Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.</a> J. Hepatol. 34 (2), 261-269 (2001).</li><li>Castell, J. V., Jover, R., Martinez-Jimenez, C. P., Gomez-Lechon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+cell+lines:+their+use,+scope+and+limitations+in+drug+metabolism+studies.">Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies.</a> Expert. Opin. Drug Metab Toxicol. 2 (2), 183-212 (2006).</li><li>Ciccimaro, E., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable-isotope+dilution+LC-MS+for+quantitative+biomarker+analysis.">Stable-isotope dilution LC-MS for quantitative biomarker analysis.</a> Bioanalysis. 2 (2), 311-341 (2010).</li><li>Dang, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Links+between+metabolism+and+cancer.">Links between metabolism and cancer.</a> Genes Dev. 26 (9), 877-890 (2012).</li><li>Darnell, M., Weidolf, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+xenobiotic+carboxylic+acids:+focus+on+coenzyme+A+conjugation,+reactivity,+and+interference+with+lipid+metabolism.">Metabolism of xenobiotic carboxylic acids: focus on coenzyme A conjugation, reactivity, and interference with lipid metabolism.</a> Chem. Res. Toxicol. 26 (8), 1139-1155 (2013).</li><li>Des Rosiers, C., Fernandez, C. A., David, F., Brunengraber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversibility+of+the+mitochondrial+isocitrate+dehydrogenase+reaction+in+the+perfused+rat+liver.+Evidence+from+isotopomer+analysis+of+citric+acid+cycle+intermediates.">Reversibility of the mitochondrial isocitrate dehydrogenase reaction in the perfused rat liver. Evidence from isotopomer analysis of citric acid cycle intermediates.</a> J. Biol. Chem. 269 (44), 27179-27182 (1994).</li><li>Ellis, J. K., 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=Metabolic+profiling+detects+early+effects+of+environmental+and+lifestyle+exposure+to+cadmium+in+a+human+population.">Metabolic profiling detects early effects of environmental and lifestyle exposure to cadmium in a human population.</a> BMC. Med. 10, 61 (2012).</li><li>Grevengoed, T. J., Klett, E. L., Coleman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl-CoA+metabolism+and+partitioning.">Acyl-CoA metabolism and partitioning.</a> Annu. Rev. Nutr. 34, 1-30 (2014).</li><li>Haynes, C. A., 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=Quantitation+of+fatty+acyl-coenzyme+As+in+mammalian+cells+by+liquid+chromatography-electrospray+ionization+tandem+mass+spectrometry.">Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry.</a> J. Lipid Res. 49 (5), 1113-1125 (2008).</li><li>Ikegawa, S., 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=Characterization+of+cholyl-adenylate+in+rat+liver+microsomes+by+liquid+chromatography/electrospray+ionization-mass+spectrometry.">Characterization of cholyl-adenylate in rat liver microsomes by liquid chromatography/electrospray ionization-mass spectrometry.</a> Anal. Biochem. 266 (1), 125-132 (1999).</li><li>Kalghatgi, S., 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=Bactericidal+antibiotics+induce+mitochondrial+dysfunction+and+oxidative+damage+in+Mammalian+cells.">Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in Mammalian cells.</a> Sci. Transl. Med. 5 (192), 192ra85 (2013).</li><li>Liu, X., 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=High-Resolution+Metabolomics+with+Acyl-CoA+Profiling+Reveals+Widespread+Remodeling+in+Response+to+Diet.">High-Resolution Metabolomics with Acyl-CoA Profiling Reveals Widespread Remodeling in Response to Diet.</a> Mol. Cell Proteomics. 14 (6), 1489-1500 (2015).</li><li>Lopez-Gallardo, E., Iceta, R., Iglesias, E., Montoya, J., Ruiz-Pesini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OXPHOS+toxicogenomics+and+Parkinson's+disease.">OXPHOS toxicogenomics and Parkinson's disease.</a> Mutat. Res. 728 (3), 98-106 (2011).</li><li>Magnes, C., Sinner, F. M., Regittnig, W., Pieber, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC/MS/MS+method+for+quantitative+determination+of+long-chain+fatty+acyl-CoAs.">LC/MS/MS method for quantitative determination of long-chain fatty acyl-CoAs.</a> Anal. Chem. 77 (9), 2889-2894 (2005).</li><li>Mauriala, T., Herzig, K. H., Heinonen, M., Idziak, J., Auriola, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+long-chain+fatty+acid+acyl-coenzyme+A+compounds+using+liquid+chromatography-electrospray+ionization+tandem+mass+spectrometry.">Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 808 (2), 263-268 (2004).</li><li>Murphy, T. A., Dang, C. V., Young, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopically+nonstationary+13C+flux+analysis+of+Myc-induced+metabolic+reprogramming+in+B-cells.">Isotopically nonstationary 13C flux analysis of Myc-induced metabolic reprogramming in B-cells.</a> Metab Eng. 15, 206-217 (2013).</li><li>Paglia, 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=Metabolomic+analysis+of+platelets+during+storage:+a+comparison+between+apheresis-+and+buffy+coat-derived+platelet+concentrates.">Metabolomic analysis of platelets during storage: a comparison between apheresis- and buffy coat-derived platelet concentrates.</a> Transfusion. 55 (2), 301-313 (2015).</li><li>Patti, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+strategies+for+untargeted+metabolomics.">Separation strategies for untargeted metabolomics.</a> J. Sep. Sci. 34 (24), 3460-3469 (2011).</li><li>Robishaw, J. D., Neely, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+A+metabolism.">Coenzyme A metabolism.</a> Am. J. Physiol. 248 (1 Pt 1), E1-E9 (1985).</li><li>Rodgers, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+platelet+physiology+and+laboratory+evaluation+of+platelet+function.">Overview of platelet physiology and laboratory evaluation of platelet function.</a> Clin. Obstet. Gynecol. 42 (2), 349-359 (1999).</li><li>Salles, I., 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=Development+of+a+high-throughput+ELISA+assay+for+platelet+function+testing+using+platelet-rich+plasma+or+whole+blood.">Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.</a> Thromb. Haemost. 104 (2), 392-401 (2010).</li><li>Snyder, N. W., Basu, S. S., Worth, A. J., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+propionic+acid+to+a+novel+acyl-coenzyme+A+thioester+by+mammalian+cell+lines+and+platelets.">Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.</a> J. Lipid Res. 56 (1), 142-150 (2015).</li><li>Snyder, N. W., Basu, S. S., Zhou, Z., Worth, A. J., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+dilution+liquid+chromatography/mass+spectrometry+analysis+of+cellular+and+tissue+medium-+and+long-chain+acyl-coenzyme+A+thioesters.">Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters.</a> Rapid Commun. Mass Spectrom. 28 (16), 1840-1848 (2014).</li><li>Snyder, N. W., 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=Production+of+stable+isotope-labeled+acyl-coenzyme+A+thioesters+by+yeast+stable+isotope+labeling+by+essential+nutrients+in+cell+culture.">Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.</a> Anal. Biochem. 474, 59-65 (2015).</li><li>Wallace, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+in+man+and+mouse.">Mitochondrial diseases in man and mouse.</a> Science. 283 (5407), 1482-1488 (1999).</li><li>Wojtovich, A. P., Brookes, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+II+inhibitor+atpenin+A5+protects+against+cardiac+ischemia-reperfusion+injury+via+activation+of+mitochondrial+KATP+channels.">The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial KATP channels.</a> Basic Res. Cardiol. 104 (2), 121-129 (2009).</li><li>Worth, A. J., 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=Stable+isotopes+and+LC-MS+for+monitoring+metabolic+disturbances+in+Friedreich's+ataxia+platelets.">Stable isotopes and LC-MS for monitoring metabolic disturbances in Friedreich's ataxia platelets.</a> Bioanalysis. 7 (15), 1843-1855 (2015).</li><li>Worth, A. J., Basu, S. S., Snyder, N. W., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neuronal+cell+mitochondrial+complex+I+with+rotenone+increases+lipid+beta-oxidation,+supporting+acetyl-coenzyme+A+levels.">Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid beta-oxidation, supporting acetyl-coenzyme A levels.</a> J. Biol. Chem. 289 (39), 26895-26903 (2014).</li><li>Zhou, L., Schmaier, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+testing+in+platelet-rich+plasma:+description+of+procedures+with+the+aim+to+develop+standards+in+the+field.">Platelet aggregation testing in platelet-rich plasma: description of procedures with the aim to develop standards in the field.</a> Am. J. Clin. Pathol. 123 (2), 172-183 (2005).</li></ol>

Aquí hemos demostrado la utilidad de plaquetas aisladas como una plataforma para el estudio del metabolismo mitocondrial perturbado. En concreto, hemos caracterizado adaptación metabólica en respuesta a la inhibición del complejo I de la rotenona. El presente estudio se ha extendido hallazgos presentados con anterioridad sobre el papel de la inhibición del complejo I de la rotenona en líneas celulares a las plaquetas humanas. Es importante destacar que esto se ha puesto de manifiesto q...

Metabolismo mitocondrial disfuncional se ha implicado en una amplia gama de enfermedades, incluyendo neurodegeneración, el cáncer y la enfermedad cardiovascular 30. Como tal, un gran esfuerzo se ha colocado en la caracterización de defectos metabólicos que contribuyen a la patogénesis de la enfermedad. Cromatografía líquida-espectrometría de masas (LC-MS / MS) se considera el estándar de oro para la cuantificación de analitos de matrices biológicas complejas y con frecuencia se emplea para estudios metabólicos 8. Sin embargo, como es a menudo el caso con los estudios biomédicos, la consecución de un modelo accesible y bien definida correspondiente a la enfermedad humana es un desafío. Muchos estudios emplean modelos celulares transformadas para sondear el impacto de xenobióticos o anomalías genéticas en el metabolismo celular 7,9. La reprogramación metabólica que se produce en las células cancerosas pueden introducir factores de confusión 21 y por lo tanto no son ideales. Estos problemas pueden ser circumvented con modelos celulares primarios, aunque la obtención de biomasa suficiente para los análisis metabólicos puede ser un reto. Por otra parte, el impacto de altas cantidades de antibióticos que se utilizan en la cultura se ha destacado como estudios mitocondriales 16 de confusión potenciales. Las plaquetas humanas ofrecen la oportunidad de utilizar un modelo celular primario con suficiente contenido mitocondrial para estudios metabólicos 5,22,27,32. En primer lugar, las plaquetas se pueden adquirir fácilmente, a través de la extracción de sangre de donantes individuales, o en grandes volúmenes de bancos de sangre, y por lo tanto proporcionar un modelo en el que factores externos pueden ser controlados fácilmente. En segundo lugar, debido a su pequeño tamaño, las plaquetas pueden ser fácilmente aislados de otros componentes de la sangre con un mínimo de trabajos de preparación en laboratorios incluso mínimamente equipados 5. Es de destacar que las plaquetas no contienen núcleos y por lo tanto se pueden utilizar para estudiar alteraciones en el metabolismo de manera independiente de la regulación transcripcional. Aquí nos muestran queAdemás de la cuantificación relativa de tioésteres de acil-coenzima A (CoA), el sistema de plaquetas aislado se puede utilizar para examinar el metabolismo del carbono. En concreto, el informe de uso de marcaje metabólico con isótopo estable (no radiactivo) marcado con [13 C 6] -glucosa y [13 C 16] palmitato para sondear la incorporación de [13C] -label en el metabolito importante acetil- CoA a través de la glicólisis o de la oxidación de ácidos grasos. Esto proporciona una plataforma de gran alcance, generalizable, y versátil debido a la amplia participación de especies acil-CoA reductasa en las vías bioquímicas 13,24 y la tratabilidad de este sistema para probar otras variables, tales como la inhibición del complejo I con rotenona 3,33. Además de la información proporcionada en el Protocolo a continuación, una descripción detallada de los métodos utilizados para el etiquetado de isótopos y para los análisis basados ​​en CL-EM se puede encontrar en Basu y Blair 4.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Reagent</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Chloride (NaCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">746398</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Bicarbonate (NaHCO3)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">S5761</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Calcium Chloride Dihydrate (CaCl2 * H2O)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">223506</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Potassium Chloride (KCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">P9541</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">208337</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">G8270</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C6-Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td>389374</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Palmitic acid</td>
			<td style="width:92px;">Cayman</td>
			<td style="width:68px;">10006627</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C16-Palmitic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">605573</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Rotenone</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">R8875</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Trichloro Acetic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">T6399</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">5-Sulfosalicylic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">390275</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Acetonitirle</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A996-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Water (H2O)</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">W7-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Formic acid</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">85171</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Dimethyl Sulfoxide</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">472301</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ethanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">04-355-222</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Methanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A454-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ammonium Acetate</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A639-500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">2 mL Eppendorf Tubes</td>
			<td style="width:92px;">BioExpress</td>
			<td style="width:68px;">C-3229-1</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">LC vials (plastic)</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">186002640</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">10 mL Glass Centrifuge Tubes</td>
			<td style="width:92px;">Kimble Chase</td>
			<td style="width:68px;">73785-10</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Oasis Solid Phase Extraxtion (SPE) Columns</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">WAT094225</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Pastuer Pipets</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">13-678-200</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Name</td>
			<td style="width:92px;">Company</td>
			<td style="width:68px;">Catalog Number</td>
			<td style="width:85px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Equipment</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:161px;">CO2 Water-Jacketed Incubator</td>
			<td style="width:92px;">Nuaire</td>
			<td style="width:68px;">AutoFlow NU-8500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">Triple Quadropole Mass Spectrometer</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Finnigan TSQ Quantum</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">HPLC</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Dionex Ultimate 3000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Source</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">HESI II</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:161px;">HPLC Column</td>
			<td style="width:92px;">Phenomenex</td>
			<td style="width:68px;">Luna C18</td>
			<td style="width:85px;">3 &#956;m particle size, 200 mm x 2 mm</td>
		</tr>
	</tbody>
</table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Para demostrar la utilidad de esta metodología hemos reproducido la generalización de la adaptación metabólica compensatoria se ha descrito previamente como resultado de la exposición a la rotenona. Este hallazgo fue identificado previamente en modelos de cultivo celular y esta investigación tenía por objeto poner a prueba si este cambio metabólico también se produce en las plaquetas, que son anuclear y no es propenso a los mismos artefactos experimentales como cultivo celular. ...

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Aquí mostramos plaquetas humanas aisladas se pueden utilizar como un accesible ex vivo modelo para estudiar adaptaciones metabólicas en respuesta al complejo I inhibidor de la rotenona. Este enfoque emplea trazado isotópico y la cuantificación relativa mediante espectrometría de cromatografía de masa líquida y se puede aplicar a una variedad de diseños de estudio.

LC-MS El análisis de las plaquetas humanas como una plataforma para el estudio del metabolismo mitocondrial

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Education

Research

JoVE Core

JoVE Journal

Statistics

Environment

Unit 1

Measures of Variation

Measures of Relative Standing

Probability Distributions

Estimates

Distributions

Hypothesis Testing

Analysis of Variance

Correlation and Regression

Statistics in Practice

SCIENTISTS IN ACTION

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Técnica para el estudio de los artrópodos y las comunidades microbianas dentro de los tejidos del árbol

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Investigación

Medio ambiente

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Expresión génica transitoria en tabaco utilizando Asamblea Gibson y la pistola de genes

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

El uso de Chironomidae (Diptera) en superficie flotante pupal Exuviae como Protocolo Bioassessment rápida para las Masas de Agua

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Modificación y aplicación de un soplador de hojas-vac de campo Muestreo de artrópodos

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

Exploración Conductometría (TSC) como un método General para estudiar y controlar el comportamiento de fase de conductor físico geles térmicos

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

Cuantificación de proteínas solubles de la planta y Digestible contenido de hidratos de carbono, utilizando el maíz (Zea mays) como un ejemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Estudio de los efectos neuroconductuales de los contaminantes ambientales en las larvas de peces cebra

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

El efecto de las fracciones de plástico de desecho de construcción y demolición en las propiedades compuestas de polímero de madera

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

Un sistema gnotobiótico para el estudio del ensamblaje del microbioma en la filosfera y en la fermentación vegetal

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Diseño de microparplots y preparación de muestras de plantas y suelos para 15 análisisde nitrógeno

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...