Name: high precision zinc isotopic measurements applied to mouse organs
Uploaded: 2015-05-22T13:00:00.000+00:00
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FM erkennt Finanzierung von der ANR durch eine chaire d&#39;Excellence IDEX Sorbonne Paris Cité, die INSU durch einen PNP-Zuschuss, der Institut Universitaire de France sowie die Labex UniverEarth Programm an der Sorbonne Paris Cité (ANR-10-LABX-0023 und ANR -11-IDEX-0005-02). Wir danken auch Mittel aus dem Europäischen Forschungsrat unter der Europäischen Gemeinschaft H2020 Rahmenprogramm / ERC Finanzhilfevereinbarung # 637503 (Pristine).

HINWEIS: Verfahren, die Tiere wurden von der Institutional Animal Care und Verwenden Committee (IACUC) an der Université Paris Diderot genehmigt. 1. Vorbereitung der Materialien <ol><li> Unter Kochen destillieren 1 L der Säuren (HNO 3, HBr), um sie von Unreinheit zu reinigen. </li><li> Reinigen Sie die Becher und Spitzenadapter in einem heißen (~ 100 ° C) konzentrierte HNO 3 Säurebad für mindestens zwei Tage. </li><li> Waschen Sie die Pipettenspitzen in einer kalten 3 N HNO 3 Bad für mehrere Tage und spülen einzeln dreimal mit entionisiertem Wasser. </li></ol> 2. Probenvorbereitung <ol><li> Betäuben die Mäuse durch intraperitoneale Injektion von Ketamin und Xylazin. Beurteilen Anästhesie durch den Zehen Prise Methode. </li><li> Das Blut durch eine kardiale Punktion in Gegenwart von Heparin in 1,5-ml-Röhrchen. </li><li> Trennen des Plasmas von Blutzellen durch Zentrifugation (10 min, 1.500 x g) und übertragen das Plasma auf Polypropylen cryogenic Fläschchen mit Polypropylen-Tipps. </li><li> Entfernen Sie das restliche Blut von Organen, die durch Schneiden der Lebervene und Einspritzen von DPBS durch das Herz. Beurteilen Tod der Maus durch zervikale Dislokation. </li><li> Ernte der Organe mit sterilen Instrumenten erreicht, befreien sie von umgebenden Fett wenn überhaupt, und Schnapp frieren sie in Polypropylen Kryoröhrchen. </li></ol> 3. Chemische Reinigung <ol><li> Zunächst lösen die Proben in einer Mischung von ~ 1 ml konzentrierter (30%) H 2 O 2 und ~ 1 ml konzentrierter (etwa 15 M) HNO 3. Haben alle diese Schritte innerhalb einer Abzugshaube. <ol><li> Legen Sie das ganze Organ von Interesse in einen 15 ml Teflonbecher. Fügen Sie dann die H 2 O 2 / HNO 3 in das Becherglas 5. Halten Sie das Becherglas offen für ein paar Minuten, um Spritzer auf die Reaktion der Oxidation des organischen Materials und die Freisetzung von CO 2 zu vermeiden. </li><li> Schließlich legte das Becherglas auf einer Heizplatte bei about 100 ° C für ein paar Stunden oder bis die Lösung völlig klar. </li></ol></li><li> Öffnen der Becher und trocknen der Lösung auf einer heißen Platte bei etwa 100 ° C. </li><li> Sobald die Probe trocken ist, fügen Sie 1 ml 1,5 N HBr zu den Proben; schließen Sie das Becherglas und lassen Sie es auf einer heißen Platte lösen sich bei 100 ° C zwei Stunden. </li><li> In der Zwischenzeit die 500 ul Spalten vorzubereiten. <ol><li> Fügen Sie 500 ul der AG1X8 200-400 Mesh-Harz auf die Säule und legte es auf die Säule Rack mit einem Papierkorb-Becher darunter. Waschen des Harzes mit Wechselstrom: 5 ml von 18,2 M &amp; Omega; cm ⋅ Wasser, 5 ml 0,5 N HNO 3, 5 ml Wasser, 5 ml 0,5 N HNO 3, und dann mit 5 ml Wasser versetzt. Zustand des Harzes mit 5 ml 1,5 N HBr. </li></ol></li><li> Entfernen Sie die Bechergläser aus dem Kochplatte und setzen Sie sie in einem Ultraschallbad für etwa 30 min, und dann lassen Sie die Becher abkühlen auf RT. </li><li> Sobald der Becher wird abgekühlt und das Harz wird gewaschen, öffnen Sie das Becherglas. Setzen Sie die Spitze Adapter bis ter spritze, fügen Sie eine Pipettenspitze; Pipette die 1 ml Probe und laden Sie es auf dem Harz (sehr langsam, um nicht, um das Harz zu bewegen). </li><li> Nachdem die gesamte Flüssigkeit durch die Säule passiert, 5 ml 1,5 N HBr. </li><li> Sobald die 5 ml 1,5 N HBr durch die Säule, ersetzen Sie den Papierkorb-Becher mit einem sauberen 15 ml-Becher. </li><li> 5 ml von 0,5 N HNO 3 mit 2,5 ml auf einmal. In diesem Stadium der Zn von dem Harz eluiert. </li><li> Nachdem 5 ml HNO 3 durch die Säule passiert, nimmt das Becherglas und legen Sie sie auf einer heißen Platte bei 100 ° C bis getrocknet. </li><li> Entfernen Sie die Spalte aus der Säulenhalter; trash das Harz (verwenden Sie eine neue Harz für jede Probe). </li><li> Sobald die Probe trocken ist, wiederholen Sie das Protokoll mit dem gleichen Volumen von Säuren auf einer kleineren Kolonne (100 ul) und legen Sie sie auf einer heißen Platte bis getrocknet. Die Probe ist nun für die Massenspektrometrie. </li></ol> 4. Massenspektrometrie Measurement <ol><li> Analysieren Sie den Zn Isotopen composition auf einer Mehrkollektor-induktiv gekoppeltes Plasma-Massenspektrometer (MC-ICP-MS). <ol><li> Verwenden Sie die Maschinenparameter in Tabelle 1 zusammengefasst. </li></ol></li><li> Positionieren Sie die Faraday-Cups auf Masse (m / z) von 62 Ni, 63 Cu, Zn 64, 65 Cu, 66 Zn, 67 Zn und 68 Zn zu sammeln. </li><li> Bereiten Sie eine Lösung, die 500 ppb Zn in 0,1 M HNO 3 für die Isotopenanalyse. </li><li> Analysieren Sie die 500 ppb-Lösung von Zn mit Hilfe eines Spritzkammer in Verbindung mit einer 100 &amp; mgr; l / min Teflon Vernebler. Für jede Probe sind 30 Scans (1 Block von 30 Zyklen), in denen die Integrationszeit jeder Abtastzeile ist 8,389 sec. </li><li> Korrigieren Sie den Hintergrund durch Subtraktion der auf Spitze Null-Intensitäten aus einem Rohling-Lösung (0,1 M HNO 3 Lösung zur Wiederauflösung der Proben). </li><li> Kontrolle und korrekte möglich 64 Ni isobare Interferenzen durch Messung der Intensität der 62 Ni Höhepunkt.Angenommen, der 64 Ni / 62 Ni-Verhältnis ist natürlich (0,2548), korrigieren Sie diesen Wert aus der instrumentellen Massen bias, und entfernen Sie die 64 Ni auf die Masse 64, wie: 64 Zn Real = 64 Zn gemessen - 64 Ni = 64 Zn gemessen - (64 Ni / 62 Ni) Natur x 62 Ni gemessen. </li><li> Korrigieren Sie die instrumental Massen Bias von Belichtungsreihen jede der Proben mit 500 ppb Standardlösung des JMC Lyon Zn-Standard (oder einem anderen verfügbaren Standard wie IRMM-3702). Führen die Standardreihe durch Dividieren der 66 Zn / Zn 64 Verhältnis von der Probe durch den Mittelwert der 66 Zn / 64 Zn-Verhältnis der beiden Standards vor und nach der Probe minus 1 und multipliziert mit 1000 (siehe Gleichung 1). Typische externen Präzision auf der JMC Lyon Zn-Standard ist 0,05 permil / amu (2 Standardabweichung, 2 sd). </li></ol>

<ol>
	<li>Marechal, C. N., Telouk, P., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+analysis+of+copper+and+zinc+isotopic+compositions+by+plasma-source+mass+spectrometry.">Precise analysis of copper and zinc isotopic compositions by plasma-source mass spectrometry.</a> Chemical Geology. 156 (1), 251-273 (1999).</li><li>Marechal, C. N., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+fractionation+of+copper+and+zinc+isotopes.">Ion-exchange fractionation of copper and zinc isotopes.</a> Geochim. Cosmochim. Acta. 66 (9), 1499-1509 (2001).</li><li>Weiss, D. J., Mason, T. F. D., Zhao, F. J., Kirk, G. J. D., Coles, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+discrimination+of+zinc+in+higher+plants.">Isotopic discrimination of zinc in higher plants.</a> New Phytologist. 165 (3), 703-710 (2005).</li><li>Jouvin, D., Louvat, P., N, M. F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+isotopic+fractionation:+why+organic+matters.">Zinc isotopic fractionation: why organic matters.</a> Environ Sci Technol. 43 (15), 5747-5754 (2009).</li><li>Moynier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+fractionation+and+transport+mechanisms+of+Zn+in+plants.">Isotopic fractionation and transport mechanisms of Zn in plants.</a> Chemical Geology. 267 (3-4), 125-130 (2009).</li><li>Moynier, F., Herzog, G., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+composition+of+zinc,+copper,+and+iron+in+lunar+samples.">Isotopic composition of zinc, copper, and iron in lunar samples.</a> Geochim. Cosmochim. Acta. 70 (24), 6103-6117 (2006).</li><li>Moynier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+fractionation+of+zinc+in+tektites.">Isotopic fractionation of zinc in tektites.</a> Earth Planet. Sci. Lett. 277 (3-4), 482-489 (2009).</li><li>Chen, H., Savage, P., Teng, F. Z., Helz, R., Moynier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+isotope+fractionation+during+magmatic+differentiation+and+the+isotopic+composition+of+the+bulk+Earth.">Zinc isotope fractionation during magmatic differentiation and the isotopic composition of the bulk Earth.</a> Earth Planet. Sci. Lett. 369-370, 34-42 (2013).</li><li>Moeller, 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=Calibration+of+the+new+certified+materials+ERM-AE633+and+ERM-AE6447+for+copper+and+IRMM+3702+for+zinc+isotope+amount+ratio+determination.">Calibration of the new certified materials ERM-AE633 and ERM-AE6447 for copper and IRMM 3702 for zinc isotope amount ratio determination.</a> Geostd. Geoan. Res. 36 (2), 177-199 (2012).</li><li>Bigeleisen, J., Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+equilibrium+constants+for+isotopic+exchange+reactions.">Calculation of equilibrium constants for isotopic exchange reactions.</a> J. Chem. Phys. 15, 261-267 (1947).</li><li>Luck, J. M., Ben Othman, D., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zn+and+Cu+isotopic+variations+in+chondrites+and+iron+meteorites:+Early+solar+nebula+reservoirs+and+parent-body+processes.">Zn and Cu isotopic variations in chondrites and iron meteorites: Early solar nebula reservoirs and parent-body processes.</a> Geochim. Cosmochim. Acta. 69 (22), 5351-5363 (2005).</li><li>Moynier, F., Dauphas, N., Podosek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Search+for+70Zn+Anomalies+in+Meteorites.">A Search for 70Zn Anomalies in Meteorites.</a> Astrophys. J. 700 (2), L92-L95 (2009).</li><li>Paniello, R., Day, J., Moynier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zn+isotope+evidence+for+the+origin+of+the+Moon.">Zn isotope evidence for the origin of the Moon.</a> Nature. 490 (7420), 376-380 (2012).</li><li>Pichat, S., Douchet, C., Albarede, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+isotope+variations+in+deep-sea+carbonates+from+the+eastern+equatorial+Pacific+over+the+last+175+ka.">Zinc isotope variations in deep-sea carbonates from the eastern equatorial Pacific over the last 175 ka.</a> Earth and Planetary Science Letters. 210 (1-2), 167-178 (2003).</li><li>Moynier, F., Fujii, T., Shaw, A., Le Borgne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+of+natural+Zn+isotopes+in+mice.">Heterogeneous of natural Zn isotopes in mice.</a> Metallomics. 5 (6), 693-699 (2013).</li><li>Balter, V., 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=Bodily+variability+of+zinc+natural+isotope+abundance+in+sheep.">Bodily variability of zinc natural isotope abundance in sheep.</a> Rapid Com. Mass. Spec. 24, 605-612 (2010).</li><li>Balter, V., 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=Contrasting+Cu,+Fe,+and+Zn+isotopic+patterns+in+organs+and+body+fluids+of+mice+and+sheep,+with+emphasis+on+cellular+fractionation.">Contrasting Cu, Fe, and Zn isotopic patterns in organs and body fluids of mice and sheep, with emphasis on cellular fractionation.</a> Metallomics. 5 (11), 1470-1482 (2010).</li><li>Urgast, D. 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=Zinc+isotope+ratio+imaging+of+rat+brain+thin+sections+from+stable+isotope+tracer+by+LA-MC-ICP-MS.">Zinc isotope ratio imaging of rat brain thin sections from stable isotope tracer by LA-MC-ICP-MS.</a> Metallomics. 4, 1057-1063 (2012).</li><li>Marin-Carbonne, J., Rollion-Bard, C., Luais, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+measurements+of+iron+isotopes+by+SIMS:+MC-ICP-MS+intercalibration+and+application+to+a+magnetite+crystal+from+the+Gunflint+chert.">In-situ measurements of iron isotopes by SIMS: MC-ICP-MS intercalibration and application to a magnetite crystal from the Gunflint chert.</a> Chem. Geol. 285 (1-4), 50-61 (2011).</li><li>Fietzke, 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=Boron+isotope+ratio+determination+in+carbonates+via+LA-MC-ICP-MS+using+soda-lime+glass+standards+as+reference+material.">Boron isotope ratio determination in carbonates via LA-MC-ICP-MS using soda-lime glass standards as reference material.</a> J. Anal. Atom. Spec. 25, 1953-1957 (2010).</li></ol>

Die Autoren haben nichts zu offenbaren.

Die Reproduzierbarkeit der Messungen wird durch repliziert Analysen der gleichen Proben während verschiedenen analytischen Sitzungen durchgeführt bewertet. Zum Beispiel 6 haben wir die gleichen terrestrischen rock 7 Mal wiederholt, und wir erhalten die Ergebnisse in der Tabelle 2 angegeben. Wie aus der Theorie der Isotopenfraktionierung 10 erwartet und nach einem Sonnensystems Material bisher gemessen (zB Meteoriten 11-13, pflanzen...

Die Messung hoher Genauigkeit (besser als 100 ppm / atomare Masseneinheit) Zink stabilen Isotopenzusammensetzung erst seit etwa 15 Jahren durch die Entwicklung von Multi-Kollektor-Plasmaquelle Massenspektrometer möglich und wurde seitdem hauptsächlich in Erde angewendet und Planetenwissenschaften. Die Anwendungen auf dem Gebiet der Medizin, sind neu und haben ein starkes Potential als Biomarker für Krankheiten, die den Metabolismus von Zink (zB Alzheimer-Krankheit) zu ändern. Dieses Papier berichtet über eine Methode, um mit hoher Präzision in verschiedenen Organen der Maus messen die natürlichen stabilen Isotopen-Verhältnisse von Zink. Das gleiche würde für menschlichen Proben. Das Verfahren besteht in der Auflösung der Organe, die chemische Reinigung von Zink aus dem Rest der Atome, und die Analyse der Isotopenverhältnis auf einem Massenspektrometer. Die Qualität der Zn Isotopenmessungen ist abhängig von der Qualität der chemischen Reinigung (Reinheit von Zn, niedrigen leere Datei herunterladenared auf die Menge an Zn in der Probe vorhanden, hohe chemische Ausbeute des Verfahrens) und die Steuerung der Instrumental Bias. Die hohe Reinheit des Endproduktes Zn Fraktion benötigt wird, um sowohl isobare Interferenzen und nicht isobare Interferenzen, die eine Matrix Effekt entfernen. Isobar Nuklide erstellen direkten Eingriffe (zB 64 Ni). Non-isobare Interferenzen erzeugen die sogenannten &quot;Matrixeffekt&quot; und verändern die analytische Genauigkeit der Messungen durch Ändern des Zustands der Ionisation gegenüber dem reinen Zink Standards, nach dem die Proben 1 verglichen. Eine niedrige Leer (&lt;10 ng) zeigt an, daß es keine Verunreinigung der Proben durch externe Zn das würde den gemessenen Biasisotopenzusammensetzung. Zn Isotope während Ionenaustauschchromatographie 2 fraktioniert werden, die Sammlung aller Zn-Atome sichergestellt, dass keine isotopischen Fraktionierung auftritt, was bedeutet, daß der chemische Vorgang ein erfülltes Ertrag haben. Schließlich wird die Korrektur der instrument Isotopenfraktionierung während der Massenspektrometrie Messung über die &quot;Standard-Belichtungsreihe&quot; Methode. Daher sind die Hauptschwierigkeiten zu erhalten präzise Messungen sind die Steuerung der externen Kontamination (dh niedrige blank), wodurch eine vollständige Ausbeute chemische Reinigung, die sauber von anderen Atomen oder Molekülen ist, und die Korrektur der instrument Isotopenfraktionierung auf dem Massenspektrometer. In diesem Papier werden wir unsere analytischen Protokolls beschreiben Zn aus dem Maus-Organen sowie die massenspektrometrische Messung zu trennen. Die Extraktion unter Verwendung einer geringen Menge an verdünnten Säuren (HBr / HNO 3 Medien) auf Mikro-Säulen (0,5 &amp; mgr; l und 0,1 &amp; mgr; l) des Anionenaustauscherharzes durchgeführt. Es hat einen vollen Ertrag und die Messungen eine externe Reproduzierbarkeit besser als 50 ppm auf der 66 Zn / 64 Zn-Verhältnis. Ein weiterer Vorteil der Method ist, dass es sehr schnell ist. Das Verfahren ist daher sehr gut an den medizinischen Wissenschaften angepasst, wobei man braucht, um eine große Anzahl von Proben verglichen, um geowissenschaftlichen gegebenen wurden die analytischen Methoden entwickelt analysieren.

<table><tbody><tr><td>Multi-collection inductively-coupled-plasma mass-spectromter</td><td>Thermo-Fisher</td><td></td><td></td></tr><tr><td>Anion-exchange resin AG1 X8 200-400</td><td>Bio-Rad</td><td>140-1443-MSDS</td><td></td></tr><tr><td>Teflon beakers</td><td>Savillex&amp;nbsp;</td><td>200-015-12</td><td></td></tr><tr><td>In-house-made teflon colunms made with shrinkable teflon</td><td></td><td></td><td></td></tr></tbody></table>

high precision zinc isotopic measurements applied to mouse organs

Wir präsentieren ein Verfahren mit hoher Präzision Zink Isotopenverhältnisse in der Maus Organe messen. Zink wird von 5 stabile Isotope (64 Zn, 66 Zn, 67 Zn, 68 Zn und 70 Zn), die natürlicherweise zwischen Mausorgane fraktioniert werden zusammengesetzt. Wir zeigen zunächst, wie die verschiedenen Organe, um die Zn-Atome frei zu lösen; Dieser Schritt wird durch eine Mischung von HNO 3 und H 2 O 2 umgesetzt. Reinigen wir dann die Zinkatome von allen anderen Elementen, insbesondere von isobare Interferenzen (beispielsweise Ni), durch Anionenaustausch-Chromatographie in einer verdünnten HBr / HNO 3 -Medium. Diese ersten beiden Schritte in einem Reinlabor mit hochreinen Chemikalien durchgeführt. Schließlich werden die Isotopenverhältnisse durch die Verwendung eines Multikollektor-induktiv gekoppeltes Plasma-Massenspektrometer, mit einer niedrigen Auflösung gemessen. Die Proben werden mit einer Spritzkammer und der isotopischen Fraktionierung-Massenspektrometers induzierte correc spritztted durch Vergleich des Verhältnisses der Proben an das Seitenverhältnis eines herkömmlichen (Standardbelichtungstechnik). Das vollständige typische Vorgehensweise erzeugt ein Isotopenverhältnis mit einer 50 ppm (2 sd) Reproduzierbarkeit.

In 1,5 N HBr, die Hauptzinkspezies (ZnBr3-) bildet sehr starke Komplexe mit dem Anionenaustauscherharz, während die meisten anderen Elemente nicht mit dem Harz in Wechselwirkung treten. Zink wird dann durch Ändern des Mediums verdünnt HNO 3, die Änderung der Speziation von Zn zu Zn 2+, die von dem Harz 6,7 freigesetzt wird, zurückgewonnen. Isotopenverhältnisse sind typischerweise als Teile pro 1.000 Abweichungen relativ zu einem Standard ausgedrückt: <...

Watch this Scientific Journal Video about High Precision Zinc Isotopic Measurements Applied to Mouse Organs at JoVE.com

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

High Precision Zink Isotopenmessungen an Maus-Orgeln Angewandte

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

high-precision-zinc-isotopic-measurements-applied-to-mouse-organs

Universidad de Cartagena UDEC

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7.4K Aufrufe.  Université Paris Diderot. We present the technique to measure with high precision zinc isotope ratios in mouse organs.

Serie: High Precision Zinc Isotopic Measurements Applied to Mouse Organs

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Ein umfassendes Verfahren zur Bewertung der<em&gt; In Vivo</em&gt; Performance von Krebs Nanomedizin

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Forschung

Krebsforschung

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the inverse electron demand Diels-Alder (IEDDA) cycloaddition between a radiolabeled tetrazines and trans-cyclooctene (TCO) linked to a biomolecule has proven to be a highly effective bioorthogonal approach to imaging specific biological targets. Despite the fact that technetium-99m remains the most widely used isotope in diagnostic nuclear medicine, there is a scarcity of methods for preparing 99mTc-labeled tetrazines. Herein we report the preparation of a family of tridentate-chelate-tetrazine derivatives and their Tc(I) complexes. These hitherto unknown compounds were radiolabeled with 99mTc using a microwave-assisted method in 31% to 83% radiochemical yield. The products are stable in saline and PBS and react rapidly with TCO derivatives in vitro. Their in vivo pre-targeting abilities were demonstrated using a TCO-bisphosphonate (TCO-BP) derivative that localizes to regions of active bone metabolism or injury. In murine studies, the 99mTc-tetrazines showed high activity concentrations in knees and shoulder joints, which was not observed when experiments were performed in the absence of TCO-BP. The overall uptake in non-target organs and pharmacokinetics varied greatly depending on the nature of the linker and polarity of the chelate.

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Here, we describe a protocol for radiolabeling and in vivo testing of tridentate 99mTc(I) chelate-tetrazine derivatives for pre-targeting and bioorthogonal chemistry.

Herstellung und Auswertung der<sup&gt; 99m</sup&gt; Tc-markierte Dreizähnige Chelate für Pre-Targeting Bioorthogonale Chemie

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...

Chemie

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Atomische Absorbance-Spektroskopie zur Messung von intrazellulären Zink-Pools in den Mamänarchalzellen

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Biochemie

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Schrotflinten-Lipidomik von Nagetiergewebe

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

Isolierung und Quantifizierung des Zika-Virus aus mehreren Organen in einer Maus

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

Immunologie und Infektion

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Körperzusammensetzung und festsetzende Stoffwechselanalyse bei hohen Fett Fed Mäusen

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Medizin

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Quantitative Analyse der zellulären Zusammensetzung in fortgeschrittene atherosklerotische Läsionen der glatten Muskulatur Zelle Abstammung-Tracing Mäuse

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Bildgebung von CD19+ B-Zellen in einem experimentellen autoimmunen Enzephalomyelitis-Mausmodell mit Positronen-Emissions-Tomographie

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

High Precision Zink Isotopenmessungen an Maus-Orgeln Angewandte

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