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

Vi erkjenner støtte fra NIH tilskudd P30ES013508 og T32ES019851.

Etikk Uttalelse: Alle protokoller vedrørende behandling av humane prøver følge retningslinjene i The University of Pennsylvania menneskelige forskningsetisk komité. 1. Utarbeidelse av buffere og 100x Stock Solutions <ol><li> Forbered en L base Tyrode buffer. Kombiner 8,123 g NaCl, 1,428 g NaHCO3, 0,466 g CaCl 2 ∙ 2 H 2 O, 0,224 g KCl, og 0,095 g MgCl 2. Juster totale volumet til en L med DDH 2 O. Filter sterilisere basen Tyrode b...

<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>

Her har vi vist nytten av isolerte blodplater som en plattform for å studere opprørt mitokondrie metabolisme. Nærmere bestemt har vi karakterisert metabolsk tilpasning i respons til kompleks I hemming av rotenon. Denne studien har forlenget tidligere rapporterte funnene på seg rollen som kompleks I hemming av rotenon i cellelinjer til humane blodplater. Viktigere, og dette viste at rotenon også hemmet blodplate-succinyl-CoA formasjon, stimuleres en økning i blodplate βHB-CoA, og hadde...

Dysfunksjonell mitokondrielle metabolismen har vært implisert i en rekke sykdommer, inkludert nevrodegenerasjon, kreft, kardiovaskulær sykdom og 30. Som sådan har stor innsats er lagt vekt på å karakterisere metabolske defekter som bidrar til sykdom patogenesen. Væskekromatografi-tandem massespektrometri (LC-MS / MS) er regnet som gullstandarden for kvantifisering av analytter fra komplekse biologiske matriser og er ofte brukt for metabolske studier 8. Imidlertid, som det ofte er tilfelle med biomedisinske studier, å oppnå en tilgjengelig og godt definert modell som er relevante for human sykdom er en utfordring. Mange studier ansetter transformert cellemodeller for undersøkelser av virkningen av xenobiotics eller genetiske avvik på cellenes stoffskifte 7,9. Den metabolske omprogrammering som oppstår i kreftceller kan innføre forstyrrende faktorer 21 og er derfor ikke ideelt. Disse problemene kan være circumvented med primære cellemodeller, selv om å skaffe tilstrekkelig biomasse til metabolske analyser kan være utfordrende. Videre er effekten av store mengder antibiotika som brukes i kulturen blitt markert som potensielt konfunderende mitokondrielle studier 16. Humane blodplater, hvilket gir mulighet til å utnytte en primær celle modell med tilstrekkelig mitokondrie-innhold for metaboliske studier 5,22,27,32. Først kan blodplater være lett kjøpt gjennom blod trekker fra individuelle givere, eller i store volumer fra blodbanker, og derfor gi en modell hvor ytre faktorer kan lett kontrolleres. For det andre, på grunn av sin lille størrelse, blodplater kan lett isoleres fra andre blodkomponenter med minimal forberedende arbeid i og med minimalt utstyrte laboratorier 5. Av notatet, blodplater ikke inneholde kjerner og kan derfor brukes til å studere endringer i metabolismen uavhengig av transkripsjonsregulering. Her viser vi ati tillegg til relativ kvantifisering av acyl-koenzym A (CoA) tioestere, kan det isolerte blodplate-systemet bli brukt til å undersøke karbon metabolisme. Spesielt rapporterer vi bruk av metabolsk merking med stabile isotoper (ikke-radioaktive) merket [13 C 6] -glukose og [13 C 16] -palmitate å sondere inkorporering av [13 C] label inn den viktige metabolitten acetyl CoA via glykolyse eller fettsyre oksidasjon. Dette gir en kraftig, generaliseres, og allsidig plattform på grunn av den omfattende involvering av acyl-CoA arter i biokjemiske pathways 13,24 og tractability av dette systemet til å teste andre variabler, for eksempel hemming av komplekse jeg med rotenon 3,33. I tillegg til informasjon gitt i protokollen under kan en omfattende beskrivelse av metodene som brukes for isotop merking og for LC-MS-baserte analyser finnes i Basu og 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.

For å demonstrere nytten av denne metodikken vi har gjengitt enes allmenn tidligere beskrevet kompenserende metabolske tilpasninger som følge av eksponering for rotenon. Dette funnet var tidligere identifisert i cellekultur modeller og denne undersøkelsen var rettet for å teste om dette metabolske forskyvning også forekommer i blodplater, som er anuclear og ikke er utsatt for de samme eksperimentelle gjenstander som cellekultur. Dette arbeidet ble utført med seks dager gamle blodpl...

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

Her viser vi isolerte humane blodplater kan anvendes som en rullestol ex vivo modell for å studere metabolske tilpasninger som reaksjon på det komplekse I-inhibitor rotenon. Denne tilnærmingen benytter isotopisk sporing og relativ kvantifisering ved væske-kromatografi-massespektrometri og kan brukes på en rekke forskjellige studiedesign.

LC-MS analyse av humane blodplater som en plattform for å studere mitokondrie Metabolism

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

Teknikk for å studere Arthropod og mikrobielle samfunn innen Tre Vev

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 ...

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

Forbigående Gene Expression i Tobacco bruker Gibson forsamling og Gene Gun

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

Bruk av Chironomidae (Diptera) Surface-Floa pupal Exuviae som Rapid Bioassessment protokoll for vannforekomster

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

Modifikasjon og anvendelse av en løvblåser-vac for Feltet Prøvetaking av Leddyr

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

Termisk skanning Conductometry (TSC) som en generell metode for å studere og kontrollere fase virkemåten til ledende fysisk Gels

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

Kvantifisere anlegget løselig Protein og fordøyelige karbohydrater innhold, bruke korn (Zea mays) som en Exemplar

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

Studere neurobehavioral effekter av miljøforurensende stoffer på sebrafisk larver

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

Effekten av konstruksjon og riving avfall plast fraksjoner på trepolymer kompositt egenskaper

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

Et gnotobiotisk system for å studere mikrobiomemontering i phyllosphere og i vegetabilsk gjæring

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

Microplot Design og plante og jord prøve forberedelse for 15Nitrogen analyse

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. ...