Name: methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart
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This work was supported by National Institutes of Health Grants R00 HL112952 (to R. H.), R01 HL108618 (to J.P.G.), P01 HL051971, and P20 GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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The authors declare that they have no competing financial interests.

önceki protokol aynı zamanda izole çalışma sıçan kalbinde glukoz oksidasyonu ve yağ asidi oksidasyon yoluyla substratın akı ölçmek için yöntemler göstermektedir. Ölçümler daha sonra (... vb iş yükü değişimi, iskemi-reperfüzyon) taban ve stres koşullarında yüzeyler metabolizma ve kalp çalışma arasındaki ilişkiyi belirlemek için kaydedilen kardiyak fonksiyonel parametrelere yerleştirilebilir. Metabolizma / daralma ilişkisi, kalp yetmezliği ve diyabet gibi önceden var olan koş...

klinik Alaka Memeli kalbinde, oksidatif metabolik yollar, ATP üretimi ve kalp işi 1 ile substratların akı arasında güçlü bir pozitif ilişki vardır. Son yirmi yılda, kalp metabolizması ve fonksiyon arasındaki karmaşık bağlantı soruşturma kalp metabolizmasında değişiklikler kalp hastalığının 2-4 farklı türde ortamında kasılma disfonksiyon ve patolojik yapısal yeniden bir nedeni olduğunu fark yol açmıştır. nedenle, stresli kalp metabolik biçimlenme düzenleyen mekanizmaların anlaşılması, kalp yetmezliği, 5-7 önlenmesi ya da tedavisi için bir terapötik hedeflerin tanımlanmasına yol açacağı beklenmektedir. &quot;Kardiyak Metabolizma Değerlendirilmesi&quot; Amerikan Kalp Derneği bilimsel deyimi son yayın t bilimsel topluluğun artan ilgi vurgularAraştırmanın 8 onun alanı. Kardiyak görüntülemede teknolojik gelişmeler artık kardiyak morfoloji ve fonksiyon hızlı ve doğru bir değerlendirme için izin verirken Ancak, kalp metabolizması in vivo çalışma sınırlı ve külfetli kalır: Nükleer Manyetik Rezonans (NMR) spektroskopisi ve Pozitron Emisyon Tomografisi (PET) görüntüleme can ex vivo olarak bu tarihten .To kararlı durum koşullarında oksidatif metabolizma çeşitli yüzeylerde katkısını 9 belirlemek için kalp yüksek enerji fosfat metabolizması ve Krebs döngüsü aktivitesini izlemek için kullanılabilir, ancak bu teknikler yüksek işletme maliyetleri ile ve kendi yetersizlik boğulmuş kalp hazırlık çalışma eş zamanlı ve gerçek zamanlı, oksidatif metabolik yollar 7,9 içine kasılma fonksiyonu ve substratların akı, çalışmak için tek ve benzersiz tekniği kullanılabilir temsil eder. Aşağıdaki protokol sıçan belirlemek için kullanılan reaktiflerin hazırlanması ve kullanımı yönergeleri sağlamayı amaçlarİzole çalışma sıçan kalbinde substratlar kullanım es. İzole Çalışma Kemirgen Kalp Aparatı teknik neredeyse yarım asırlık olmasına rağmen, izole çalışma sıçan kalp hazırlık kardiyovasküler araştırma için seçim yöntemi olmaya devam etmektedir. Langendorff kalp hazırlanması gibi, çalışma kemirgen kalp nörohormonal diğer organların ve diğer dolaşım faktörlerin karıştırıcı etkilerinden bağımsız olarak kalp parametreleri geniş bir yelpazede ölçmek için nispeten basit, güvenilir ve ucuz bir yol sağlar. Ancak Langendorff perfüze kalp aksine, çalışma kalp yakın fizyolojik kalp işi, in vivo koşullarında alakalı seviyelere oksidatif metabolik akışın üretilmesi için bir ön koşul yerine devam eder. Bu, sol atrium bağlı bir kanül aracılığı ile sol ventrikül (LV) perfüzyon tampon elde ederek ve AG, doldurur ve sözleşmeler gibidirtampon kararlı afterload hidrostatik basınca karşı aort hattı üzerinden çıkarılır. Başlangıçta 10 daha sonra Taegtmeyer, hems ve Krebs 11 tarafından geliştirilen, fakat Neely ve arkadaşları tarafından tarif edilen perfüzyon aparatı tasarımı zamandan beri çok az değişmiştir. Orijinal cihaz tarif edildiği gibi, kontraktil fonksiyon aort ölçmek için herhangi bir daha fazla dereceli silindirler ve bir kronometre kullanılarak kalp çıkışının saptanması yoluyla tespit edilebilir ve koroner 10,11 akar. Çeşitli satıcılar artık tam çalışma kemirgen kalp perfüzyon sistemleri sunuyoruz. Bu ticari olarak temin edilebilen bir düzen flowprobes, basınç gücü çeviricisi, bir basınç-hacim kateter ve kalp işlevsel veri alımı ve analizi için gerekli tüm ekipman ile elde edilebilir. satıcıları, kendi ekipman ile yeni bir kullanıcı tanımak için kapsamlı dokümantasyon ve eğitim oturumları sağlamak. çalışma kalp instrum birkaç da inceleme makaleleri detay protokollerientation ve kateter kullanımı ile ilgili kemirgenler 12-15 kardiyak fonksiyonunu ölçmek için. Bu nedenle, sadece kısa bir süre perfüzyon aparatı ve kayıt donanımları set-up değineceğim. Mevcut protokol yerine eş zamanlı olarak glikoz ve uzun zincirli yağ asidi oksidasyon oranları, normal kalpte iki büyük enerji sağlayan yüzeyler ölçmek için uygulanabilecek yöntemler bir açıklama ile zaten mevcut bilgiyi tamamlamayı amaçlamaktadır. Veri analizi, örneklerin kurtarma ve işleme reaktif ve tampon hazırlanmasından, miyokard oksidatif metabolizma değerlendirilmesi için radyoaktif işaretli enerji substratların kullanımı dahil burada tüm adımları açıklar. Yönteminin İlkeleri Kardiyomiyositlerde (glikoz yağ asidi (özellikle uzun zincirli yağ asidi) ve karbonhidrat oksidatif fosforilasyon ile ilgili daralma enerjilerinin bir kısmını oluştururE ve laktat). Kalp çok enerjik rezervleri sınırlı ve dolaşımdan bu enerji sağlayan substratların sürekli bir tedarik dayanmaktadır etti. glikolitik bir yol üzerinden glikoz katabolizması daha sonra iç mitokondriyal zarın piruvat dehidrojenaz kompleksi tarafından dekarboksile edilir piruvat elde edilir. albümin ya da lipoprotein trigliseritleri dolaşımdaki ekstre uzun zincirli yağlı asitler, ilk sitoplazmada asil-CoA moleküllere aktif ve daha sonra p-oksidasyon yolu girmek için karnitin transfer yoluyla mitokondrial matris içinde taşınmaktadır. Glukoz ve yağ asitlerinin katabolizması ile üretilen asetil CoA moleküller iç mitokondriyal membran boyunca proton motiv güç oluşturmak için elektron taşıma zinciri tarafından kullanılan indirgeyici eşdeğerleri (NADH ve FADH 2) oluşturmak için Krebs döngüsü yakıt ve ATP sentaz aktivitesi ile ATP üretir. Su ve karbondioksit sonu yan ürünleridirKrebs döngüsü içinde yer alan enzimatik reaksiyonlar. 14 C- ve 3H-radyo-etiketli substratlar tedarik çalışmakta olan kalp (örneğin, 14, C-radyo-etiketli glukoz ve 3-radyo işaretli oleik asit gibi), bunun sonucu olarak 14 CO 2 ve 3 H2O üretimine yol açacaktır olabilir kantitatif koroner atık geri kazanılabilir. 14 CO 2 koleksiyonu kapalı bir bölmeye izole perfüze kalbi tutarak ve kalbi çıkarken hemen koroner atık kurtarma tarafından yürütülmektedir. Küçük bir anyon değiştirme sütunu ayırmak ve koroner çıkış maddesinden 3 H2O kurtarmak için kullanılır. işlenmiş numuneler arasında radyoaktivite bir sıvı sintilasyon sayacı vasıtasıyla ölçüldü ve ikinci radyo-etiketli substratlar spesifik aktivitenin bilgisi ile birlikte, tek tek glukoz ve yağlı asit akışı ölçmek mümkün olur olanoksidasyon 16,17 yolaklarıyla.

<table><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>BP366</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>P284</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M63</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>AG 1-X8 resin, chloride form, 100 - 200 dry mesh size, 500 g</td><td>Bio-Rad</td><td>1401441</td><td>This item can be replaced by purchasing directly the hydoxide form&amp;nbsp; (see reference below), but this will cost almost 8 times more</td></tr><tr><td>AG 1-X8 resin, hydroxide form, 100 - 200 dry mesh size, 100 g</td><td>Bio-Rad</td><td>1432445</td><td>Purchasing this item allows to bypass the conversion of the anion exchange resin from the chloride form to the hydroxide form (See section 1.2 of protocol)</td></tr><tr><td>Glass Microanalysis Vacuum Filter Holder</td><td>Fisher Scientific</td><td>09-753-2</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318</td><td>Corrosive. Consult the product MSDS for appropriate handling and storage.</td></tr><tr><td>Gas Dispersion Tube with Fritted Cylinder</td><td>Fisher Scientific</td><td>11-138B</td><td></td></tr><tr><td>Probumin Bovine Serum Albumin Fatty Acid Free, Powder</td><td>EMD Millipore</td><td>820027</td><td>We recommend the use of a charcoal-defatted BSA, as other purification process such as cold ethanol fractionation may leave residues toxic for the heart.</td></tr><tr><td>Sodium Oleate</td><td>Sigma-Aldrich</td><td>O7501</td><td></td></tr><tr><td>Oleic Acid, [9,10-&lt;sup&gt;3&lt;/sup&gt;H(N)]-</td><td>PerkinElmer</td><td>NET289005MC</td><td>Radioactive material. Follow your Institution&#039;s radiation safety office guidelines for ordering and handling.</td></tr><tr><td>Dialysis Membrane Tubing, 29 mm diameter</td><td>Fisher Scientific</td><td>08-667E</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glucose, D-[14C(U)]-</td><td>PerkinElmer</td><td>NEC042B005MC</td><td>Radioactive material. Follow your Institution&#039;s radiation safety office guidelines for ordering and handling.</td></tr><tr><td>Humulin R U-100</td><td>Eli Lilly and Company</td><td>NDC 0002-8215-01 (HI-210)</td><td></td></tr><tr><td>Inactin Hydrate</td><td>Sigma-Aldrich</td><td>T133</td><td>Controlled substance on USDEA Schedule III</td></tr><tr><td>3-0 Silk Black Braid</td><td>Roboz Surgical</td><td>SUT-15-3</td><td></td></tr><tr><td>10x Hyamine Hydroxide</td><td>PerkinElmer</td><td>6003005</td><td>Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage</td></tr><tr><td>20 ml Glass Scintillation Vials</td><td>Fisher Scientific</td><td>03-341-25E</td><td>Use glass vials for quantitative recovery of &lt;sup&gt;14&lt;/sup&gt;CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>20 ml HDPE Scintillation Vials</td><td>Fisher Scientific</td><td>03-337-23B</td><td>Use HDPE vials for quantitative recovery of &lt;sup&gt;3&lt;/sup&gt;H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Red Rubber Sleeve Stoppers</td><td>Fisher Scientific</td><td>14-126DD</td><td>Fit 20 mL scintillation vials; Reusable</td></tr><tr><td>BD PrecisionGlide Needle 23G x 40 mm</td><td>BD</td><td>305194</td><td>Use to inject perchloric acid through the rubber sleeve stopper of the CO&lt;sub&gt;2&lt;/sub&gt; trap</td></tr><tr><td>Perchloric Acid, 60%</td><td>Fisher Scientific</td><td>A228</td><td>Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage</td></tr><tr><td>Ultima Gold, Scintillation Cocktail</td><td>PerkinElmer</td><td>6013327</td><td></td></tr><tr><td>Glass Wool</td><td>Fisher Scientific</td><td>AC38606</td><td></td></tr><tr><td>Decon Dri-Clean Detergent Powder</td><td>Fisher Scientific</td><td>04-355</td><td>For cleaning of glassware, plastic parts, and tubing</td></tr><tr><td>Alconox Tergazyme Enzyme-Active Powered Detergent</td><td>Fisher Scientific</td><td>16-000-115</td><td>For cleaning of &quot;hard to reach&quot; surfaces (tubing, glassware) contaminated by fatty acid-BSA residue</td></tr></tbody></table>

methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart

Memeli kalp ATP önemli bir tüketici ve daralma enerji substratların sabit bir kaynağı gerektirir. Şaşırtıcı olmayan bir şekilde, miyokard metabolizması değişiklikleri kasılma disfonksiyonu ve kalp yetmezliği gelişimine bağlantılı olmuştur. Bu nedenle, metabolizma ve daralma arasındaki bağlantıyı çözülüyor hastalık durumlarında kardiyak adaptasyon veya davranışsal sorunlar düzenleyen mekanizmaların bazıları da ışık tutmalıdır. çalışmakta olan sıçan kalbi hazırlığı aynı anda ve gerçek zamanda, kalp kasılma fonksiyonunun ve oksidatif metabolik yollar içinde alt-tabakaların temin enerji akı takip etmek için kullanılabilir. Mevcut protokol glikoz ve yağ asitleri, kalp substratlar sağlayan ana enerji oksidasyon oranları kantitatif ölçümü için tamponların hazırlanması ve kullanımında kullanılan yöntemlerin ayrıntılı bir açıklamasını sunmayı amaçlamaktadır. Örnek analizi ve veri yorumlama için kullanılan yöntemler de ele alınmıştır.Kısaca, bu teknik normotermik kristalloid perfüzyon yoluyla bir ex vivo dayak kalbe 14 arz C radyo-etiketli glukoz ve 3H-radyo-etiketli, uzun zincirli yağ asidi dayanır. 14 CO2 ile 3 H2O, son yan ürünleri bu enerji sağlayan alt-tabakaların kullanımına katılan enzimatik reaksiyonlar, ardından nicel koroner çıkış maddesinden geri kazanılır. kullanılan radyoaktif işaretli substratlar spesifik aktivitesi bilgisi ile, tek tek oksidasyon yolaklarında glükoz ve yağlı asit akışı ölçmek için mümkündür. izole edilmiş kalbin kasılma işlevi uygun kayıt cihazının paralel olarak belirlenir ve doğrudan metabolik akı değerleri ile ilişkili olabilir. teknik, öncesi ve yük ve iskemi, bir ilaç veya bir dolaşım sistemlerini sonra değişiklikler gibi çeşitli stres koşullarına cevaben metabolizma / daralma ilişkisini incelemek için son derece yararlıdırbir gen ürününün ekspresyonu değiştirilmesinden ting faktörü veya.

İki Örnek deneyler aşağıdaki şekillerde tarif edilmektedir. Her iki durumda da, 16 haftalık erkek Sprague Dawley sıçan kalbi izole edilmiştir ve önceki protokole göre hazırlandı KH tamponu ile çalışma modunda perfüze. Her deneyde, kalp, kalp çalışma değiştirmek için bir stres durumu tabi tutulmuştur. Kalp kasılma fonksiyonu aort hattına bir basınç transdüktörünün yerleştirilmesi ile ve kalp gücünün belirlenmesi ile nabız basıncı sürekli kayıt ile...

Watch this Scientific Journal Video about Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart at JoVE.com

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

İzole Çalışma Sıçan Kalp Glukoz ve Yağ Asidi Oksidasyonu Oranlarının Belirlenmesine Yöntemleri

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

methods-for-determination-rates-glucose-fatty-acid-oxidation-isolated

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.6K Görüntüleme.  University of Mississippi Medical Center. The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Video: Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Yüksek verim Mikroplate Solunum Ölçümler Fare İskelet Kası Minimal Miktarları gelen kondriyayı İzole kullanma

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Gelişim Biyolojisi

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

İzole Biventriküler ve NADH Floresans Görüntüleme Tavşan Kalpler Çalışma

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Tıp

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Yeni İzole Edilmiş Fare Hepatositlerinin Süspansiyonunda Yağ Asidi β-Oksidasyonunun Ölçülmesi

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

Biyokimya

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Perkütan İğne Biyopsisi ile Elde Edilen İskelet Kası Doku İzole Mitokondri hazırlanması ve Respirometrik Değerlendirmesi

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

Bir Optimize Protokol Lenfositlerindeki Glikoliz ve Mitokondriyal Solunum Analiz

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

İmmünoloji ve Enfeksiyon

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Perfkullanılmış Mammalin kalpleri transmural absorbance spektroskopisi kullanarak hücresel metabolizmayı Izleme için kalp içi yan ateş ışık kateter

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Biyomühendislik

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Mitokondri İzole Fare Heart Geçirgenliği Geçiş Gözenek Açma Multi-parametre ölçümü

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Biyoloji

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Oksijen tüketim oranını bir hücre dışı akı Analyzer kullanarak birincil kültürlü fare yenidoğan Cardiomyocytes içinde analiz

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Ex Vivo Retinal Doku Örneklerinde Mitokondriyal Solunum ve Glikoliz Tayini

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Nörobilim

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...