The figures produced in this manuscript were created with BioRender.com. We are grateful to Amy Walker for providing feedback on this article. J.B.S. was supported by the Worcester Foundation for Biomedical Research Grant.

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

<ol>
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The authors have no conflicts of interest to report.

As emerging research demonstrates that mammalian mitochondria can function without consuming molecular oxygen, it is of the utmost importance for researchers to employ orthogonal assays, beyond OCR measurements, to accurately quantify mitochondrial function. Here, we compiled a series of assays that can be used to directly assess the activities of complex I, complex II, complex V, and DHODH by measuring the mitochondrial NAD+/NADH balance, the utilization of adaptive terminal electron acceptors, the production of ATP, de novo pyrimidine biosynthesis, and mitochondrial-derived ROS. Notably, these assays more directly measure mitochondrial function than OCR measurements. Furthermore, these assays provide researchers with tractable ways to quantify mitochondrial function during hypoxia, for which OCR measurements are largely irrelevant due to fumarate being used as the favored terminal electron acceptor. Finally, the proliferation-based methods described here are more cost-effective than classical respirometry experiments, thus providing a broadly accessible way to study mitochondrial function in mammalian systems.
There are key considerations when utilizing these assays to measure mitochondrial function in cultured cells. Regarding the proliferation assays, it is important to adjust the number of cells seeded for the doubling rate of each cell line. The cells should be seeded to at least 10% confluence and with enough space to allow for three to four doublings so that differences in proliferation can be quantified. Another consideration for each assay is the concentration of the small molecules used as controls for the activities of each ETC complex. As different cell lines may exhibit different sensitivities to these inhibitors, it is critical to test the dose of these small molecules to identify the optimal concentration.
A universal limitation of assays studying mitochondrial function in vitro, including OCR measurements and all the assays described here, is the metabolic composition of the culture medium. Standard cell culture medium tends to bias systems into superficially high levels of mitochondrial function. For example, supraphysiological glutamine levels increase its anaplerosis of the TCA cycle25, which fuels mitochondrial NADH synthesis and, consequently, increases oxidative phosphorylation. Similarly, the partial pressure of oxygen ranges between 3 mmHg and 100 mmHg (approximately 0.1%-13% O2) in mammalian tissues but is atmospheric (140 mmHg, approximately 21%) in vitro26,27. This excess O2 maximizes the mitochondrial respiratory capacity and superoxide production28. Recently, efforts have been made to design culture media to be more physiological29,30. Notably, culturing cells in human plasma-like media decreases mitochondrial respiration in some cancer cell lines30, mitochondrial ROS in T cells31, and mitochondrial adaptations to cancer therapeutics32. Thus, it is critical to be mindful of the composition of the culture media being used and understand how it may impact the mitochondrial function.
Another important and universal limitation in the interpretation of mitochondrial function is the potential for differences in the number of mitochondria. It is, therefore, critical to measure the mitochondrial content through either the quantification of mtDNA33, the measurement of mitochondrial mass with membrane potential-insensitive dyes34, or western blotting of mitochondrial markers. This is a critical control so that a decrease in the number of mitochondria is not mistaken for a decrease in mitochondrial function.
There are also specific limitations and troubleshooting that apply to the assays described here. First, given that differentiated cells do not proliferate, the proliferation-based assays will not be useful for assessing mitochondrial function in this context. A key limitation of the 13C4-aspartate tracing protocol to measure DHODH activity is that aspartate uptake in cells can be extremely inefficient35. To overcome this potential limitation, researchers can overexpress the aspartate transporter, SLC1A3, to facilitate 13C4-aspartate uptake35.
A limitation of the protocol using 13C5-glutamine tracing to measure SDH activity is that this assay requires cells to utilize the reductive carboxylation pathway to enrich the M+3 isotopologues in order to measure the reverse activity. Some cell lines are incapable of reductive carboxylation flux due to low ATP citrate lyase expression36, insufficient HIF stabilization37, or an &#945;-KG:citrate ratio that is too low38. To overcome this limitation, one could utilize 13C4-aspartate tracing to measure the SDH forward and reverse activities7. In this assay, the SDH forward activity can be measured by the ratio of fumarate M+2:succinate M+2 and the reverse reaction by succinate M+4:fumarate M+4. Notably, this tracing circumvents most of the enzymes in the reductive carboxylation pathway.
A limitation of the complex I activity assay using DCPIP reduction as the readout is that the mitochondria are not structurally intact. The process of freeze-thawing the mitochondria to enable their NADH uptake for the assay can certainly tarnish the structural integrity of the mitochondrial membrane39. This assay should be performed in parallel with assays such as the complex I proliferation assay to ensure that the changes in complex I activity observed are also true with intact&#160;cells.
In future studies, some of these techniques can be adapted for measuring mitochondrial functions in vivo using model organisms such as mice and Caenorhabditis elegans. The current methods used to measure mitochondrial function in vivo are centered on the organismal-level OCR, specifically the respiratory exchange rate when using mouse models. A clear limitation of this method is that oxygen serves many biochemical and signaling functions beyond its role as a ubiquitous terminal electron acceptor in the mitochondrial ETC. For example, oxygen is &#34;consumed&#34; by the catalytic activity of enzymes in the dioxygenase family. Although these enzymes contribute to the cellular oxygen consumption rate, they do not participate in, regulate, or reflect mitochondrial function. Classical respirometry experiments in vitro typically control for &#34;non-mitochondrial OCR&#34;, whereas organismal respiratory exchange ratio (RER) experiments cannot control for this, limiting the interpretation of RER as a metric for mitochondrial function in vivo. However, it is feasible to adapt the protocols to measure DHODH activity via&#160;13C4-aspartate tracing, complex II activity via&#160;13C5-glutamine tracing, complex I activity on mitochondria purified from tissues, and mitochondrial ROS using LC-MS friendly compounds such as MitoB in order to measure mitochondrial function in vivo. These direct assays to interrogate mitochondrial functions, in combination with classical respirometry experiments, provide researchers with a more comprehensive and accurate assessment of mitochondrial function in mammalian cells and tissues.

Mitochondrial function is a critical metric of cellular health, as it sustains key biosynthetic, bioenergetic, and signaling functions in mammalian cells1. The vast majority of mitochondrial functions require electron flow through the electron transport chain (ETC), and disruptions in electron flow in the ETC cause severe mitochondrial disease2. The ETC is comprised of a series of reduction and oxidation (redox) reactions that are embedded in the inner mitochondrial membrane, and these electron transfer reactions release free energy that can be harnessed to support ATP synthesis, physiological processes such as thermogenesis, biosynthetic pathways such as de novo pyrimidine biosynthesis, and the balance of the redox status of co-factors such as NADH. ETC complex I and&#160;III produce reactive oxygen species (ROS)3,4,5, which, in turn, regulate signaling key pathways such as HIF, PI3K, NRF2, NF&#954;B, and MAPK6. Consequently, metrics of electron flow in the ETC are classically used as a proxy for mitochondrial function in mammalian cells.
Respirometry experiments are frequently employed to measure mitochondrial function in mammalian cells. Since O2 is the most ubiquitous terminal electron acceptor for the mammalian ETC, its reduction is used as a proxy for mitochondrial function. However, emerging evidence demonstrates that mammalian mitochondria can employ fumarate as an electron acceptor to sustain mitochondrial functions that depend on the ETC, including de novo pyrimidine biosynthesis7, NADH oxidation7, and the detoxification of hydrogen sulfide8. Thus, in certain contexts, especially in hypoxic environments, measurements of the O2 consumption rate (OCR) do not provide a precise or accurate indication of mitochondrial function.
Here, we outline a series of assays that can be employed to measure mitochondrial function independently of the OCR. We provide assays to directly measure complex I-mediated NADH oxidation, dihydroorotate dehydrogenase-mediated de novo pyrimidine biosynthesis, complex V-dependent ATP synthesis, the net directionality of the succinate dehydrogenase (SDH) complex, and mitochondrial-derived ROS. These assays are meant to be performed on cultured mammalian cells, although many can be adapted to study mitochondrial functions in vivo. Notably, the assays described in this protocol are more direct measurements of mitochondrial functions than the OCR. Moreover, they enable the measurement of mitochondrial function in hypoxia, a context in which the OCR is not an indicative measurement. Taken together, these assays, in combination with classical respirometry experiments, will provide researchers with a more comprehensive assessment of mitochondrial function in mammalian cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;1.5 mL tube</td>
			<td>Cell Treat</td>
			<td>667443</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL tube</td>
			<td>Cell Treat</td>
			<td>229446</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Cell Treat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Cell Treat</td>
			<td>229112</td>
			<td></td>
		</tr>
		<tr>
			<td>13C4-aspartate</td>
			<td>Sigma-Aldrich</td>
			<td>604852</td>
			<td></td>
		</tr>
		<tr>
			<td>13C5-Glutamine</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>285978-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Cell Treat</td>
			<td>667411</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Cell Treat</td>
			<td>667421</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm tissue culture dish</td>
			<td>Cell Treat</td>
			<td>229651</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate-buffered saline</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-dichlorophenolindophenol</td>
			<td>Honeywell</td>
			<td>33125</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>37999</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascentis Express C18</td>
			<td>Sigma-Aldrich</td>
			<td>53825-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filter 500 mL, 0.22 &#181;m, PES 9 9 mm membrane diameter</td>
			<td>Cell Treat</td>
			<td>229717</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Brequinar</td>
			<td>Sigma-Aldrich</td>
			<td>SML0113</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter, Double End Flat and Narrow Blade</td>
			<td>Cell Treat</td>
			<td>229305</td>
			<td></td>
		</tr>
		<tr>
			<td>CentriVap -105 Cold Trap</td>
			<td>Labconco</td>
			<td>7385020</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Tablets</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coulter Counter Cups</td>
			<td>Fisher Scientific</td>
			<td>07-000-694</td>
			<td></td>
		</tr>
		<tr>
			<td>Decylubiquinone</td>
			<td>Sigma-Aldrich</td>
			<td>D7911</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5425R</td>
			<td>Eppendorf</td>
			<td>2231000908</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5910 Ri</td>
			<td>Eppendorf</td>
			<td>5943000343</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G5388</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-free DMEM</td>
			<td>Gibco</td>
			<td>11966025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine-free DMEM</td>
			<td>Thermo Fisher</td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade 35% Ammonium hydroxide</td>
			<td>Thermo Scientific</td>
			<td>460801000</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>900667</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade formic acid</td>
			<td>Thermo Scientific</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>563935</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade MeOH</td>
			<td>Sigma-Aldrich</td>
			<td>900688</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Water</td>
			<td>Sigma-Aldrich</td>
			<td>270733</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Osteosarcome Cell Line 143B</td>
			<td>ATCC</td>
			<td>CRL-8303</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotone buffer</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P2222</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSox Red</td>
			<td>Invitrogen</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>75351-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potter-Elvehjem Tissue Grinder, Size 21</td>
			<td>Kimble</td>
			<td>885502-0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate-free DMEM media</td>
			<td>Gibco</td>
			<td>11965175</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>726030</td>
			<td></td>
		</tr>
		<tr>
			<td>ReCO2ver Incubator</td>
			<td>Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrivap Benchtop Vacuum Concentrator</td>
			<td>Labconco</td>
			<td>7310020</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Millipore Sigma</td>
			<td>20188</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>SeQuant ZIC-pHILIC 5&#956;m 150 x 2.1 mm analytical column</td>
			<td>Sigma-Aldrich</td>
			<td>1.50460.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>SeQuant ZIC-pHILIC guard kit</td>
			<td>Millipore Sigma</td>
			<td>1.50438.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, Pellets</td>
			<td>Millipore Sigma</td>
			<td>567530-250GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SW, TRACEFINDER 5.1 SP3</td>
			<td>Thermo Scientific</td>
			<td>OPTON-31001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tert-butyl hydroperoxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>458139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>93352</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25-200-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>VANQUISH HORIZON / FLEX HPLC</td>
			<td>Thermo Scientific</td>
			<td>VF-S01-A-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Z2 Coulter Particle count and size analyzer</td>
			<td>Beckman Coulter</td>
			<td>BZ10131270</td>
			<td></td>
		</tr>
	</tbody>
</table>

oxygen-independent assays to measure mitochondrial function in mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

The activities of DHODH, complex I, and complex V can all be assessed using proliferation assays. Upon deprivation of uridine from the culture medium, the cells become more dependent on the de novo pathway for pyrimidine biosynthesis. Thus, when cells were challenged to proliferate in a uridine-free medium, they were more sensitive to the inhibition of DHODH activity by brequinar than cells cultured in a medium containing uridine (Figure 6A). Similarly, the deprivation of pyruvate from the culture medium renders the cells more dependent on complex I activity for proliferation. Thus, when cells were challenged to proliferate in a pyruvate-free medium, they were more sensitive to the inhibition of complex I activity by rotenone than cells cultured in a pyruvate-containing medium (Figure 6B). Complex V activity can be assessed by challenging cells to proliferate in a medium containing galactose instead of glucose. As galactose yields net zero ATP in glycolysis, cells growing in this fuel are more reliant on mitochondrial ATP synthesis via complex V activity. Thus, cells proliferating in a galactose-containing medium were more sensitive to complex V inhibition by oligomycin than cells proliferating in a glucose-containing medium (Figure 6C).
SDH activity can be measured using 13C5-glutamine tracing and by monitoring its incorporation into fumarate and succinate isotopologues. In vehicle-treated conditions, the SDH complex favored the forward activity, and the incorporation of 13C4-succinate into 13C4-fumarate was higher than the incorporation of 13C3-fumarate into 13C3-succinate (Figure 7). In antimycin-treated conditions, the SDH complex favored the reverse activity, and the incorporation of 13C3-fumarate into 13C3-succinate was greater than the incorporation of 13C4-succinate into 13C4-fumarate (Figure 7).
Superoxide production inside the mitochondria can be measured using the fluorescent reporter MitoSox, which generates 2-hydroxy-mitoethidium upon reaction with superoxide. In this study, cells treated with MitoSox in the presence of tertbutyl hydrogen peroxide had higher levels of 2-hydroxy-mitoethidium in a manner that was suppressed by the addition of NAC, an antioxidant that quenches cellular ROS (Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65184/65184fig01.jpg" /> 
Figure 1: Mechanistic basis for the complex V proliferation assay. Oxidation of glucose and galactose via glycolysis. Glucose yields net two ATP from glycolysis, whereas galactose yields net zero ATP because UTP synthesis is required for UDP-galactose. Thus, cells grown in galactose are more dependent on mitochondrial ATP synthesis due to a lack of ATP produced from glycolysis. Abbreviations: GALK = galactokinase; GALT = galactose-1-phosphate uridylyltransferase; PGM1 = phosphoglucomutase 1; GPI = glucose-6-phosphate isomerase, UGP = UDP-glucose pyrophosphorylase; GALE = UDP-galactose-4-epimerase; NDK = nucleotide diphosphate kinase; UMPK = uridine monophosphate kinase; HK = hexokinase; PFK = phosphofructokinase; ALDO = aldolase; TPI = triosephosphate isomerase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; PGK = phosphoglycerate kinase; PGM = phosphoglucomutase; ENO = enolase; PK = pyruvate kinase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65184/65184fig02.jpg" /> 
Figure 2: Mechanistic basis for the complex I proliferation assay. Schematic of the metabolic pathways that are altered upon the inhibition of complex I activity. In high-pyruvate media, complex I inhibition is bypassed via LDH-mediated NADH oxidation. In low-pyruvate media, this adaptation is less feasible, making cells more reliant on complex I activity to reoxidize NADH. Abbreviations: LDH = lactate dehydrogenase; TCA cycle = tricarboxylic acid cycle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65184/65184fig03.jpg" /> 
Figure 3: Schematic of the DHODH reaction upon 13C4-aspartate tracing. Brequinar inhibits dihydroorotate oxidation to orotate, thus preventing the downstream synthesis of UMP. 13C4-aspartate is incorporated into 13C3-UMP via DHODH activity. Abbreviations: OMM = outer mitochondrial membrane; IMM = inner mitochondrial membrane; DHODH = dihydroorotate dehydrogenase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65184/65184fig04.jpg" /> 
Figure 4: Measuring forward and reverse complex II activity via&#160;13C5-glutamine tracing. To measure forward complex II activity (left), the incorporation of 13C5-glutamine into 13C4-succinate and 13C4-fumarate is monitored. To measure reverse complex II activity (right), the incorporation of 13C5-glutamine into 13C3-succinate and 13C3-fumarate is monitored. Abbreviations: SDH = succinate dehydrogenase; CytC = cytochrome C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65184/65184fig05.jpg" /> 
Figure 5: MitoSox Red reaction with superoxide. The reaction of MitoSox with mitochondrial superoxides to form 2-OH-MitoE2+. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65184/65184fig06.jpg" /> 
Figure 6: Proliferation-based assays to measure mitochondrial function (A) Proliferation of 143B osteosarcoma cells treated with 5 uM brequinar, a DHODH inhibitor, in medium with &#177;100 ug/mL uridine. Data are mean &#177; SEM; N = 3 per condition. (B) Proliferation of 143B osteosarcoma cells treated with the 2 uM rotenone, a complex I inhibitor, in medium with &#177;5 mM pyruvate. Data are mean &#177; SEM; N = 3 per condition. (C) Proliferation of 143B osteosarcoma cells treated with the 5 uM oligomycin, a complex V inhibitor, in medium with either 10 mM glucose or 10 mM galactose as the sole central carbon source. Data are mean &#177; SEM; N = 3 per condition. * indicates p &#60; 0.05 using a one-way ANOVA test in Graphpad Prism. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65184/65184fig07.jpg" /> 
Figure 7: 13C5-glutamine tracing to measure complex II activity. Succinate oxidation and fumarate reduction (SDH reverse) in DMSO and 500 nM antimycin A-treated Caki1 and DLD1 cells. Data represent mean &#177; SEM; N = 3 per condition. *** indicates p &#60; 0.05 using an unpaired t-test in GraphPad Prism. Abbreviations: SDH = succinate oxidation; SDH reverse = fumarate reduction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65184/65184fig08v2.jpg" /> 
Figure 8: LCMS-based MitoSox assay to detect superoxide. Extracted ion chromatogram of 2-OH-Mito E2+ isolated from Caki1 cells treated with MitoSox for 30 min in the presence of tBuOOH &#177; NAC. Abbreviations: LCMS = liquid chromatography-mass spectrometry; NAC = N-acetyl cysteine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of reagents, buffers, and media used in this protocol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65184/65184_Table 1 Reaction Compositions_Revision (2).xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals at JoVE.com

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

oxygen-independent-assays-to-measure-mitochondrial-function-in-mammals

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.4K Views.  University of Massachusetts Chan Medical School. Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Video: Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

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.

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

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 ...

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

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Quantitative Immunofluorescence to Measure Global Localized Translation

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Inner Mitochondrial Membrane Sensitivity to Na⁺ Reveals Partially Segmented Functional CoQ Pools

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

An Improved Method to Isolate Mitochondrial Contact Sites