This study is fully supported by the Division of Intramural Research of the National Institute of Nursing Research of the NIH, Bethesda, Maryland.

The current study (NCT00852111) was approved by the Institutional Review Board (IRB) of the National Institutes of Health (NIH), Bethesda, Maryland. Participants enrolled in this study were euthymic men, 18 years of age or older, who were diagnosed with non-metastatic prostate cancer with or without prior prostatectomy and were scheduled to receive external beam radiation therapy (EBRT). Potential participants were excluded if they had a progressive disease that could cause significant fatigue, had psychiatric disease within the past five years, had uncorrected hypothyroidism or anemia, or had a second malignancy. Individuals who used sedatives, steroids, or non-steroidal anti-inflammatory agents were also excluded. Healthy control blood samples were obtained at the NIH Department of Transfusion medicine from healthy donors under an IRB-approved protocol (NCT00001846). All participants are recruited at the Magnuson Clinical Research Center at the NIH. Signed written informed consents were obtained prior to study participation.
1. Mitochondrial Function Measurement Preparation (Day 1 of the Experiment)
<ol>
	<li>Hydrate Sensor Cartridge (see Table of Materials; approximate duration: 5 min).
	<ol>
		<li>Remove Sensor Cartridge from package. Add 200 &#956;L Calibrant solution into each well of the utility plate, then fill each moat with 400 &#956;L Calibrant solution.</li>
		<li>Return Sensor plate to the utility plate, which now has Calibrant solution in it. Hydrate the Cartridge in a non-CO2 37 &#176;C incubator overnight. 
		NOTE: Sensor Cartridge needs to be hydrated for a minimum of 4 h and a maximum of 72 h.</li>
	</ol>
	</li>
</ol>
2. Clinical Sample Preparation (Day 2 of the Experiment)
<ol>
	<li>Measure fatigue using the 13-item Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-F)2,23,24 at baseline (prior to EBRT initiation), midpoint and completion of EBRT, and 1-year post-EBRT.
	<ol>
		<li>Use a 0 - 4 scale for each item response, where a 0 represents &#34;not at all&#34; and a 4 indicates that the respondent relates to the corresponding statement &#34;very much.&#34; Total scores should range from 16 - 53, with lower scores reflecting high fatigue intensity.</li>
		<li>Define fatigue as a FACIT-F score lower than 43, with a FACIT-F score of &#8805;43 indicating absence of or not clinically-meaningful fatigue2. 
		NOTE: A FACIT-F score of 43 best divides fatigue scores of cancer patients and the general population2.</li>
		<li>Include the following 13 items in the FACIT Fatigue Scale: 1) I feel fatigued; 2) I feel weak all over; 3) I feel listless (&#34;washed out&#34;); 4) I feel tired; 5) I have trouble starting things because I&#39;m tired; 6) I have trouble finishing things because I am tired; 7) I have energy; 8) I am able to do my usual activities; 9) I need to sleep during the day; 10) I am too tired to eat; 11) I need help doing my usual activities; 12) I am frustrated by being too tired to do the things I want to do; and 13) I have to limit my social activity because I am tired.</li>
	</ol>
	</li>
	<li>Isolate PBMC from freshly collected blood samples (approximate duration: 1 h).
	<ol>
		<li>Collect 8 mL of blood into a Mononuclear Cells Preparation Tube (see Table of Materials). 
		NOTE: Blood samples should be processed within 2 h of collection. The quality of PBMCs may be compromised if blood samples are processed more than 2 h after sample collection.</li>
		<li>Centrifuge at 1,750 x g for 30 min at room temperature (18 - 25 &#176;C). Transfer the cloudy layer to a 15 mL conical tube. Add up to 15 mL of PBS and invert 5 times.</li>
		<li>Centrifuge at 300 x g for 15 minutes at 4 &#176;C. Carefully remove and discard the supernatant without disturbing pellet. Re-suspend the pellet by adding up to 10 mL PBS, and invert 5 times.</li>
		<li>Centrifuge at 300 x g for 10 min at 4 &#176;C. Discard liquid supernatant and re-suspend cells in 1 mL PBS. Transfer the PBMCs to a 1.5 mL microfuge tube.</li>
		<li>Centrifuge at 610 x g for 10 minutes. Carefully remove supernatant and resuspend pellet in complete RPMI-1640 (RPMI-1640 supplemented with 10% FBS, 10 mM Penicillin/Streptomycin).</li>
	</ol>
	</li>
	<li>Coat cell plates for non-adherent cells (approximate duration: 30 min).
	<ol>
		<li>Prepare cell and tissue adhesive solution (see Table of Materials) by diluting the stock solution in 0.1 M sodium bicarbonate pH 8.0. The working solution should be at 3.5 &#956;g/cm2 of surface area. 
		NOTE: This solution contains polyphenolic proteins extracted from the marine mussel, Mytilus edulis. These proteins are key components of the glue secreted by the mussel to anchor itself to surfaces. We find that Cell-Tak works the best with PBMCs.</li>
		<li>Add 100 &#956;L of the diluted adhesive solution to each well and incubate for at least 20 min at room temperature. Wash three times with DI water and air dry.</li>
	</ol>
	</li>
</ol>
3. Mitochondrial Function Measurement
<ol>
	<li>Preparation of Assay Media (approximate duration: 10 min). 
	NOTE: Assay media must be freshly prepared on the day of the experiment.
	<ol>
		<li>Add L-glutamine, pyruvate, and glucose to base media (the same constituents as Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), but without any sodium bicarbonate, glucose, glutamine, or sodium pyruvate) to make assay media. Warm media up to 37 &#176;C, then adjust pH to 7.4. 
		NOTE: Concentrations of L-glutamine, pyruvate, and glucose are usually the same as concentrations in normal growth media, but can be adjusted based on the assay.</li>
	</ol>
	</li>
	<li>Prepare PBMCs for the Mito Stress test (approximate duration: 2 h).
	<ol>
		<li>Plate enough PBMCs from Step 2 into each well to reach 80 - 90% confluency. In our experience, 1.5 x 105 cells/well yielded the most consistent results. 
		NOTE: Wells A and H are background wells and should only contain assay media with no cells. In wells B to G, we recommend plating at least 3 - 6 wells per patient sample in order to account for outliers.</li>
		<li>Spin plates down at 200 x g for 2 min to allow cells to adhere to the bottom of the wells. Wash the cells once with Assay media. This step removes serum and sodium bicarbonate from the growth media. 
		NOTE: In our experience, the washing step typically removes 20 - 30% of cells.</li>
		<li>Add 180 &#956;L Assay media into each well, including background wells A and H. Incubate in a non-CO2 37 &#176;C incubator for 45 - 60 min.</li>
	</ol>
	</li>
	<li>Running the Mito Stress Test
	<ol>
		<li>Reconstitute drugs in the Mito Stress Kit with assay media as follows:
		<ol>
			<li>Oligomycin: Add 252 &#956;L assay media into the vial to generate a stock solution at 50 &#956;M.</li>
			<li>FCCP: Add 288 &#956;L of assay media into the vial to generate a stock solution at 50 &#956;M.</li>
			<li>Antimycin A/Rotenone: Add 216 &#956;L of assay media into the vial to generate a stock solution at 25 &#956;M.</li>
		</ol>
		</li>
		<li>Vortex reconstituted drugs for approximately 1 minute. Dilute into working solutions. 
		NOTE: Concentrations of the working solutions should be titrated and pre-determined prior to the experiment depending on the cell type. For PBMCs, we find that 1 &#956;M of Oligomycin, 1 &#956;M FCCP, and 0.5 &#956;M antimycin A/Rotenone work the best.</li>
		<li>Pipette the drugs into each port in the sensor cartridge sequentially:
		<ol>
			<li>Port A: Pipette 20 &#956;L of 10 &#956;M Oligomycin, for a final concentration in each well of 1 &#956;M.</li>
			<li>Port B: Pipette 22 &#956;L of 10 &#956;M FCCP, for a final concentration in each well of 1 &#956;M.</li>
			<li>Port C: Pipette 25 &#956;L of 5 &#956;M antimycin A/rotenone for a final concentration in each well of 0.5 &#956;M.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select &#34;Mito Stress Test&#34; on an extracellular flux instrument. Follow instrument prompt and insert the sensor cartridge. The instrument will automatically perform sensor calibration. Insert cell plate after sensor calibration, and the instrument will finish the rest of the assay. Oxygen dynamics is measured at 530 nm (excitation)/650nm (emission), and proton concentration is measured at 470 nm (excitation)/ 530 nm (emission).</li>
</ol>
4. Normalization of Mitochondrial Function Data
<ol>
	<li>Prepare Cell Proliferation Assay solution (approximate duration: 5 min).
	<ol>
		<li>Add 48 &#956;L nucleic acid stain (500x) and 240 &#956;L background suppressor to 11.7 mL PBS (2x). The nucleic acid stain is a cell-permeant DNA-binding dye, and the background suppressor is a masking dye which blocks dead cells or cells with compromised cell membrane integrity from being stained. The combination of the DNA-binding dye and the background suppressor ensures that only live cells are stained.</li>
		<li>Add equal volume of 2x&#160;working solution (180 &#956;L) directly into the medium in each well after the Mito Stress Test has completed.</li>
	</ol>
	</li>
	<li>Incubate the cells in the presence of cell proliferation assay solution in a 37 &#176;C incubator for 45 - 60 min. Quantify the number of live cells (fluorescent) on a plate reader at 508 nm (excitation)/527 nm (emission) and normalize OCR data (approximate duration: 10 min). 
	NOTE: in addition to the number live cells, users may also choose to normalize their data with the total number of cells, amount of nucleic acid, protein concentrations, etc.</li>
</ol>

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R., 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=Clinical+Predictors+of+Fatigue+in+Men+With+Non-Metastatic+Prostate+Cancer+Receiving+External+Beam+Radiation+Therapy.">Clinical Predictors of Fatigue in Men With Non-Metastatic Prostate Cancer Receiving External Beam Radiation Therapy.</a> Clinical Journal of Oncology Nursing. 19 (6), 744-750 (2015).</li><li>Feng, L. R., Wolff, B. S., Lukkahatai, N., Espina, A., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploratory+Investigation+of+Early+Biomarkers+for+Chronic+Fatigue+in+Prostate+Cancer+Patients+Following+Radiation+Therapy.">Exploratory Investigation of Early Biomarkers for Chronic Fatigue in Prostate Cancer Patients Following Radiation Therapy.</a> Cancer Nursing. 40 (3), 184-193 (2017).</li><li>Myhill, S., Booth, N. 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The authors have nothing to disclose.

Fatigue in cancer patients is a debilitating condition that is not well defined or characterized1. Diagnosis of fatigue entirely relies on subjective reporting and there is no current diagnostic standard or treatment for this condition, largely due to a lack of understanding in its pathobiology2. Of the proposed mechanisms underlying fatigue in cancer patients, impairment in mitochondrial function is one of the most therapeutically targetable pathways. Therefore, we develop...

Fatigue is a prevalent and distressing condition that has a negative impact on the quality of life of cancer patients1. To this date, cancer fatigue remains poorly defined and relies only on subjective reporting by patients2. Therefore, there is an urgent need to identify an easily adaptable diagnostic laboratory test to objectively characterize fatigue in the clinical setting3,4.
Multiple underlying mechanisms, including mitochondria dysfunction, have been proposed to cause fatigue5. Mitochondria are the powerhouse organelles, providing 95% of cellular energy needs via oxidative phosphorylation, and play an important role in calcium signaling, apoptosis, immune signaling, and regulation of other intracellular signaling events6. Accordingly, impaired mitochondrial bioenergetics and defects in energy production may contribute to fatigue. Supporting this hypothesis, previous studies have observed mutations in mitochondrial DNA in patients with chronic fatigue syndrome7. While it remains unclear whether the pathophysiological origin of fatigue lies within the central nervous system or peripheral tissues, such as skeletal muscles8,9, there is currently no direct method to accurately assess mitochondrial dysfunction related to fatigue in live, respiring cells.
Using peripheral blood mononuclear cells (PBMCs) to study mitochondrial function offers several advantages. First, PBMCs are readily available in the clinical setting using standard phlebotomy and can be isolated quickly using basic laboratory techniques. Second, blood collection is less invasive than collecting other tissues such as a muscle biopsy. Thus, blood samples can be collected from the same patient repeatedly over time, which facilitates longitudinal assessment of treatment effects. Interestingly, mitochondrial function in PBMCs appeared to be well correlated with kidney mitochondrial status in an animal model10. Furthermore, immune cell mitochondria have been used as a proxy for detecting systemic changes under different disease conditions11,12. Mitochondria in circulating immune cells are particularly sensitive to changes in immune functions and immune signaling molecules such as cytokines13,14,15. For example, it has been observed that PBMCs from patients with acute rheumatic inflammatory diseases exhibit high baseline oxygen consumption14. In contrast, oxygen consumption was reduced in PBMCs isolated from patients with systemic inflammatory conditions including sepsis16. Under inflammatory conditions, free radicals produced by dysfunctional mitochondria may further contribute to elevated oxidative stress and prolonged inflammation17. The central role of mitochondria in energy production as well as in oxidative stress suggests the potential utility of using mitochondrial function as a proxy for studying fatigue in cancer patients 13.
Previous studies examining mitochondrial function utilized biochemical techniques, mitochondrial membrane potential measurement, or isolation of specific cell populations that may not be readily adaptable in the clinical setting5,14,18. In recent years, the development of extracellular flux assays has allowed researchers to easily and accurately examine changes in oxygen consumption rate (OCR) in response to automated injections of respiratory inhibitors19,20,21,22. However, most of these studies are designed for specific cell types and the large high-throughput format may not be applicable in a clinical setting. In this manuscript, we describe an optimized protocol for examining mitochondrial function for clinical use.

<table><tbody><tr><td>CPT Mononuclear Cells Preparation Tube&amp;nbsp;</td><td>BD Biosciences</td><td>362761</td><td>For isolating PBMCs following phlebotomy</td></tr><tr><td>RPMI-1640&amp;nbsp;</td><td>Corning</td><td>10-040</td><td>For making growth media for PBMCs</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td>For making growth media for PBMCs</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisher</td><td>15140122</td><td>For making growth media for PBMCs</td></tr><tr><td>Cell-Tak</td><td>Corning</td><td>354240</td><td>Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface</td></tr><tr><td>Seahorse XF Calibrant Solution</td><td>Agilent</td><td>103059-000</td><td>For hydrating cartridges</td></tr><tr><td>XFp Fluxpak (miniplates and sensor cartridges)</td><td>Agilent</td><td>103022-100</td><td>Contains XFp cell culture miniplates and sensor cartridges</td></tr><tr><td>XF base media</td><td>Agilent</td><td>103335-100</td><td>For making XF assay media</td></tr><tr><td>45% cell culture D-(+)-Glucose solution</td><td>Corning</td><td>25-037-CI</td><td>For making XF assay media</td></tr><tr><td>Sodium pyruvate solution</td><td>Corning&amp;nbsp;</td><td>25-000-CI</td><td>For making XF assay media</td></tr><tr><td>L-glutamine solution</td><td>ThermoFisher</td><td>25030081</td><td>For making XF assay media</td></tr><tr><td>Seahorse XFp Mito Stress Test Kit</td><td>Agilent</td><td>103010-100</td><td>Contains oligomycin, FCCP, antimycin A/rotenone</td></tr><tr><td>CyQUANT Direct Cell Proliferation Assay</td><td>ThermoFisher</td><td>C35011</td><td>For quantification of live cells and data normalization</td></tr><tr><td>Seahorse XFp Analyzer</td><td>Agilent</td><td>S7802AEA</td><td>For measuring mitochondrial function in live cells</td></tr><tr><td>Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate)</td><td>BioTek</td><td>BTCYT5PV</td><td>For quantification of live cells and data normalization</td></tr></tbody></table>

evaluating the role of mitochondrial function in cancer-related fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

The Mito Stress Test relies on measuring oxygen consumption rate (OCR) after sequential injection of various respiratory inhibitors to map a complete mitochondrial profile. OCR measurements after each drug injection can be used to calculate the following parameters related to mitochondrial health: Basal OCR is first measured before any drug injection to assess oxygen consumption needed to meet resting level ATP demand. Basal respiration is calculated by subtracting non-mi...

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Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

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Research

JoVE Journal

Cancer Research

9.1K Views.  National Institutes of Health. Ons doel was het ontwikkelen van een praktische protocol om te evalueren van de mitochondriale dysfunctie vermoeidheid bij kankerpatiënten is gekoppeld. Dit innovatieve protocol is geoptimaliseerd voor klinisch gebruik waarbij alleen standaard phlebotomy en basic Labor procedures.

Video: Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Behavior

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Biochemistry

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Medicine

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Immunology and Infection

Use of Minimally Invasive Methods to Assess Fuel Utilization and Circadian Rhythms in Older Adults

Aging is associated with multiple physiological changes that contribute synergistically and independently to physical disability and the risk of chronic disease. Although the etiology of age-related physical disability is complex and multifactorial, the decline in mitochondrial function appears to coincide with the progression of functional decline in many older adults. The reason why there is a decrease in mitochondrial function with aging remains elusive, but emerging science indicates that both fuel metabolism and circadian rhythms can influence mitochondrial function.
Recent studies have established that circadian rhythms become disturbed with aging, and that disrupted circadian rhythms have pathological consequences that impact mitochondrial function and overlap with many age-associated chronic diseases. Current quantitative methods for direct assessment of mitochondrial function are invasive and typically require a muscle biopsy, which can pose difficulties with participant recruitment and study adherence, given the perceived levels of potential pain and risk. Thus, an innovative and relatively noninvasive protocol to assess changes in mitochondrial function at the cellular level and circadian patterns in older adults was adapted. Specifically, a real-time metabolic flux analyzer is used to assess the mitochondrial bioenergetic function of white blood cells under differential substrate availability.
The expression of circadian clock genes in white blood cells to cross-correlate with the mitochondrial bioenergetics and circadian rhythm outcomes are also analyzed. It is believed that these innovative methodological approaches will aid future clinical trials by providing minimally invasive methods for studying mitochondrial substrate preference and circadian rhythms in older adults.

A novel and minimally invasive protocol is presented to evaluate the synergistic impact of fuel utilization and circadian rhythms on aging individuals, utilizing peripheral blood mononuclear cells.

Aging is associated with multiple physiological changes that contribute synergistically and independently to physical disability and the risk of chronic ...

Biology

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

Mitochondria

Mitochondria are eukaryotic cellular organelles that are known to produce energy through a process called oxidative phosphorylation. Besides their primary function, mitochondria are involved in various cellular processes, including cell growth, differentiation, signaling, metabolism, and senescence. Age-related changes cause a decline in mitochondrial quality and integrity due to increased mitochondrial mutations and oxidative damage. Thus, aging can severely impact mitochondrial functions, leading to abnormal cell processes.
Mitochondria are known for their structural plasticity and undergo fission or fusion depending on specific cellular processes. For instance, mitochondrial fission is associated with mitophagy, a regulatory process that specifically removes damaged mitochondria, thus maintaining tissue homeostasis. However, aging can cause loss or mutation in proteins involved in mitochondrial fission. This eventually impairs mitophagy, a condition often correlated with several age-related diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, cardiomyopathies, and cancer.
In another critical function, mitochondria associates with the cytoskeleton to facilitate their own mobility. It is a crucial factor that enables the distribution of mitochondria across cytoplasms in cells with a complex structure such as neurons. However, in aging cells, the cytoskeleton can become unstable, decreasing the mitochondrial movement and leading to abnormal neuronal functions.
Energy production through respiration is the fundamental function of the mitochondria. The mitochondrial respiratory chains generate superoxide radicals as a toxic byproduct. The mitochondrial antioxidant system typically neutralizes these radicals. However, the aging mitochondria have decreased antioxidant capacity and cannot combat the oxidative stress from the superoxide radicals. This results in the accumulation of reactive oxygen species in the cell that eventually cause cell death.

Mitochondria are eukaryotic cellular organelles that are known to produce energy through a process called oxidative phosphorylation. Besides their primary ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

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