The authors acknowledge funding for sponsored research provided by CoA Therapeutics, Inc., a subsidiary of BridgeBio LLC, the National Institutes of Health grant GM34496, and the American Lebanese Syrian Associated Charities.

The animal procedure referred to in this protocol was performed according to protocols 323 and 556 and specifically approved by the St. Jude Children&#39;s Research Hospital Institutional Animal Care and Use Committee.
1. Preparation of solutions
NOTE: Use ultrapure water for all solutions and when stated in procedures.
<ol>
	<li>Prepare 1 mM potassium hydroxide (KOH) in water.</li>
	<li>Prepare 0.25 M KOH in water.</li>
	<li>Prepare 1 M Trizma-HCl in water and adjust to pH 8.0.</li>
	<li>Prepare 100 mM monobromobimane (mBBr) in acetonitrile (Optima Grade). Stock solutions of mBBr are stable at -20 &#176;C for many months provided they are kept in the dark as exposure to light can photolyze the monobromobimane to bimane26.</li>
	<li>Prepare Wash Buffer for solid phase extraction (SPE) column: 50% methanol (Optima Grade) + 2% acetic acid.</li>
	<li>Prepare Elution Buffer for SPE column: 95% ethanol (HPLC Grade) containing 50 mM ammonium formate.</li>
</ol>
2. Preparation of CoA-bimane standard
<ol>
	<li>Purchase a high-quality CoA standard from a reputable source (e.g., Avanti Polar Lipids, Inc.).</li>
	<li>Prepare a high concentration stock solution of CoA in 20 mM Tris, pH 8. The amount of CoA in the high concentration stock is determined or confirmed by spectrophotometric absorbance at &#955;260 nm (&#400; = 16,800 M-1&#8729;cm-1). Add 4-fold molar excess of mBBr; vortex on high for 10 s. For example, for 10 mM CoA in buffer, add 40 mM mBBr. The mBBr is added in excess to ensure that all the CoA is derivatized.</li>
	<li>Incubate at room temperature for 4 h in the dark without shaking to derivatize the CoA with mBBr. mBBr reacts with the thiol group of the CoA, and it is pH and temperature dependent27. Addition of the mBBr converts CoA to a water-soluble fluorescent (or ultraviolet absorbant) CoA-bimane (CoA-bimane; Figure 2).</li>
	<li>Add 100 &#956;L acetic acid and vortex on high for 10 s to stop the reaction.</li>
	<li>Centrifuge at 2,000 x g for 15 minutes to remove any precipitant.</li>
	<li>Clean-up the supernatant using an SPE-column to remove the unreacted mBBr (described below). Filtration and centrifugation of the eluate is not necessary after the SPE column clean-up (Section 5.8).</li>
	<li>Collect the eluate from the SPE column containing the CoA-bimane in a pre-weighed tube, dry under nitrogen gas, weigh and construct a standard calibration curve with known quantities of CoA-bimane.
	<ol>
		<li>Check the CoA-bimane standard for purity by HPLC (see below) with absorbance detection at both &#955;260 (adenine moiety) and &#955;393 (bimane moiety) and coincidence of both peaks in the chromatogram.</li>
		<li>Confirm the quantity of the CoA-bimane standard in the high concentration stock using &#955;260 nm (&#400; = 16,800 M-1&#8729;cm-1).</li>
	</ol>
	</li>
	<li>Register the HPLC retention time for identification of CoA-bimane in experimental samples using the CoA-bimane standard.</li>
	<li>Store aliquots of the high concentration stock of the CoA-bimane standard protected from light and stored at -20 &#176;C for up to 2 years. Avoid repeated thawing and re-freezing.</li>
</ol>
3. Extraction and derivatization of CoA in cultured cells
<ol>
	<li>Harvest adherent cells in culture when approaching late subconfluent densities. For example, human HepG2/C3A cells are grown to a density of 6-8 x 106, or HEK 293T cells are grown to a density of ~1.3 x 107 per 100 mm dish.
	<ol>
		<li>Use duplicate or triplicate cultures routinely for CoA determinations. Incubate separate culture dishes in parallel with the sample cell cultures to determine viable cell number. Calculate the amount of CoA per 106-107 cells after HPLC purification.</li>
	</ol>
	</li>
	<li>Aspirate culture medium off the dish. Quickly wash cells on the dish with ice-cold phosphate-buffered saline (PBS) to remove any residual medium and aspirate the PBS off the dish. Quickly wash cells with ice-cold water to remove residual PBS and aspirate the water off the dish quickly. Avoid disturbing the adherent cells when adding the PBS or water.</li>
	<li>Add 1 mL of ice-cold water to the culture dish. Scrape cells into the cold water in the dish and transfer the cell suspension to a glass test tube containing 400 &#956;L of 0.25 M KOH and 1.5 mL of water.
	<ol>
		<li>Mix the suspension vigorously by vortex on high for 10 s. Then cover tightly with paraffin film and incubate at 55 &#176;C for 1 h without shaking in a water bath. The pH of the sample should be &#8805; 12. The high pH is important to hydrolyze the CoA thioesters and can be adjusted with additional aliquots of KOH if necessary.</li>
	</ol>
	</li>
	<li>Harvest cultured cells growing in suspension by centrifugation at low speed: 136 x g for 6 min at 4 &#176;C. Wash once with ice-cold PBS, then briefly wash again with cold water, and finally resuspend in 2.5 mL of cold water plus 400 &#181;L 0.25 M KOH. Mix vigorously and incubate at 55 &#176;C as described above.</li>
	<li>Add 160 &#956;L of 1 M Trizma-HCl and 10 &#956;L of 100 mM mBBr and mix by vortex on high for 10 s. This brings the pH to approximately 8 to support the mBBr reaction with free CoA. Cover and incubate samples at room temperature for 2 h in the dark for the mBBr to react with the thiol of CoA.</li>
	<li>Add 100 &#956;L of acetic acid and mix by vortex on high for 10 s to stop the reaction</li>
	<li>Centrifuge at 2,000 x g for 15 min to remove precipitated cell debris.</li>
	<li>Remove and save supernatant in a glass test tube for the SPE column clean-up that is described below.</li>
</ol>
4. Extraction and derivatization of CoA in tissues
<ol>
	<li>Dissect animal tissue pieces, about 0.5 cm diameter or smaller, and blot briefly (1-2 s) on absorbent paper and then flash freeze in liquid nitrogen and store at -80 &#176;C until processing. CoA in tissues is hydrolyzed or converted to a thioester if not flash frozen. This step is important to obtain the maximum yield of CoA in tissues.</li>
	<li>Weigh frozen tissue pieces (5 &#8211; 60 mg) quickly prior to starting the analysis, and record the weight for each sample. This wet weight determination is used for calculation of the CoA content following HPLC purification. Avoid complete thaw of the tissue pieces.</li>
	<li>Add 2 mL of 1 mM KOH to a glass test tube (e.g., 13 mm x 100 mm disposable) destined for insertion of a probe and tissue disruption with a rotor-stator homogenizer. Keep the tube on ice until use. This is done to ensure that the samples are homogenized at a cold temperature and CoA is not destroyed due to the heat generated during homogenization.</li>
	<li>Transfer and homogenize tissue (30 &#8211; 40 mg) in the glass test tube (containing cold 1 mM KOH) for 30 s. Avoid complete thawing of the tissue.</li>
	<li>Add 500 &#956;L of 0.25 M KOH; vortex on high for 10 s and keep on ice until all samples are homogenized. This will bring the pH of the sample above 12 to hydrolyze the CoA thioesters to yield total CoA (free CoA + hydrolyzed CoA thioesters).</li>
	<li>Incubate samples at 55 &#176;C for 2 h in a water bath without shaking to support hydrolysis.</li>
	<li>Add 150 &#956;L of 1 M Trizma-HCl and 10 &#956;L of 100 mM mBBr; vortex on high for 10 seconds. This brings the pH to about 8 to support the mBBr reaction with free CoA.</li>
	<li>Incubate samples at room temperature for 2 h in the dark to ensure all the CoA is derivatized with mBBr.</li>
	<li>Add 100 &#956;L of acetic acid and vortex on high for 10 s to stop the reaction.</li>
	<li>Centrifuge at 2,000 x g for 15 min to pellet precipitated cell debris.</li>
	<li>Remove and save supernatant in a glass test tube for the SPE column clean-up (described below).</li>
</ol>
5. Sample clean-up with solid phase extraction (SPE) column
<ol>
	<li>At room temperature, equilibrate each disposable 2-(2-pyridyl)-ethyl silica gel column (1 mL size) with 1 mL of Wash Buffer to ensure that the pyridyl functional group is protonated and will function as an anion-exchanger. 2-(2-pyridyl)-ethyl silica gel is a weak anion exchanger which is ideal for CoA-bimane at a basic pH. 2-(2-pyridyl)-ethyl silica gel has a pKa of 6 and the elution is done pH &#8805; 7.</li>
	<li>Add sample supernatant (step 4.11) to column and collect eluate.</li>
	<li>Wash column twice with 1 mL of Wash Buffer to remove any unretained species.</li>
	<li>Wash column once with 1 mL of water.</li>
	<li>Wash column twice with 1 mL of Elution Buffer into a separate glass test tube (12 mm x 75 mm) to collect the CoA-bimane.</li>
	<li>Dry CoA-bimane sample in tube under nitrogen gas to dryness. The dried sample is stable at room temperature until further use. Seal and completely cover the tube and store.</li>
	<li>When ready for HPLC analysis, resuspend sample in 300 &#956;L of water and mix vigorously by vortex on high for 10 s.</li>
	<li>Transfer resuspended sample to a centrifuge tube filter (0.22 &#181;m cellulose acetate, 2 mL size) and centrifuge at 5,000 x g for 10 min to remove any precipitant.</li>
	<li>Transfer filtered sample to a glass vial suitable for HPLC injection.</li>
</ol>
6. HPLC purification and measurement of CoA-bimane
<ol>
	<li>Prepare buffers. Buffer A: 50 mM KH2PO4, pH 4.6; Buffer B: Acetonitrile (Optima Grade).</li>
	<li>Power up the HPLC system. 
	NOTE: The HPLC system in our laboratory is a Waters e2695 Separations Module equipped with a 2489 Ultraviolet-Visible (UV-Vis) absorbance detector and a 2475 fluorescence detector controlled with Empower 3 software. The system also has an automated sample injector.</li>
	<li>Inject each sample (e.g., 20 &#956;L) for separation on a Gemini C18, 3 &#956;m 100 &#197;, 4.6 mm x 150 mm column using the program in Table 1 with a flow rate of 0.5 mL/min. The column temperature is 25 &#176;C. Measure the UV/Visible detector absorbance at &#955;393 nm, and fluorescent detection at &#955;ex = 393 nm, &#955;em = 470 nm. The retention time is determined by running a CoA-bimane standard curve before each set of samples.</li>
	<li>Record the area under the CoA-bimane peak for each sample and compare with the standard curve to calculate pmol of CoA-bimane injected onto the HPLC column. The CoA-bimane absorbance standard curve is used for tissue samples, and the CoA-bimane fluorescence standard curve is used for tissue culture samples.</li>
	<li>As the CoA-bimane values represent total CoA for each sample, normalize to the number of viable cells for culture samples, or mg wet weight for tissue samples (Figure 6). Alternately, calculate the total CoA values following normalization to DNA or protein content for each sample. DNA or protein content can be determined separately using sample aliquots that are removed during sample preparation prior to KOH addition.</li>
</ol>

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MWF, CS and SJ wrote the paper, MWF and CS provided the data and figures, COR designed the experiments and critically reviewed the manuscript. SJ and COR are inventors on a pending patent application (PCT/US17/39037) &#34;Small molecule modulators of pantothenate kinases&#34;&#160;held by St. Jude Children&#39;s Research Hospital that covers the PZ-2891 compound referenced in this article.

Here we demonstrate a reliable, step-by-step procedure for quantifying total CoA in cells and animal tissues with a wide range of linear detection that is accessible in many laboratories that have an HPLC with either an absorbance or fluorescence output detector. Alternatively, mass spectrometry is a common technique for evaluating CoA and CoA thioesters, but is not widely available due to the cost of the instrumentation and the specialized knowledge required for development of methodology and interpretation of data. Iso...

Coenzyme A (CoA) is an essential cofactor in all living organisms and is synthesized from pantothenic acid, also called pantothenate (the salt of pantothenic acid) or vitamin B5. CoA is the major intracellular carrier of organic acids, including short-chain acids such as acetate and succinate, branch-chain acids such as propionate and methylmalonate, long-chain fatty acids such as palmitate and oleate, very long-chain fatty acids such as polyunsaturated fatty acids, and xenobiotics such as valproic acid. The organic acid forms a thioester linkage enzymatically with CoA to enable its use as a substrate in over 100 reactions in intermediary metabolism1. CoA thioesters are also allosteric regulators and transcriptional activators. It is now appreciated2 that the cellular total CoA supply is regulated3,4; thus, CoA availability can be limiting, and that CoA deficiencies can be catastrophic, as exemplified by inherited genetic disorders that impact CoA biosynthesis5. Pantothenate kinase catalyzes the first step in CoA biosynthesis (Figure 1) and Pantothenate Kinase Associated Neurodegeneration, called PKAN, is caused by mutations in the PANK2 gene6. CoA synthase, encoded by the COASYN gene, catalyzes the last two steps in CoA biosynthesis (Figure 1) and COASY Protein-Associated Neurodegeneration, called CoPAN, is caused by a mutation in the COASYN gene7. Both PKAN and CoPAN are inherited neurodegenerative diseases associated with iron accumulation in the brain and CoA deficiencies underly the disease pathologies.
Cellular levels of total CoA vary among tissues8 and total CoA can increase or decrease under a variety of physiological, pathological and pharmacological states. Liver CoA increases during fuel switching from the fed to the fasted state9, and liver CoA levels are abnormally high in leptin-deficient obese mice10. Liver CoA decreases in response to chronic ethanol ingestion11. Brain CoA levels in the Pank2 knockout mouse model are depressed during the perinatal period, but later in the adult stage brain CoA content is equivalent to wild-type levels, indicating an adaptive CoA response during development12. Manipulation of tissue CoA content by transgenesis or gene delivery methods impacts metabolic and neural functions13,14,15. Preclinical development of potential therapies for PKAN or CoPAN includes cell or tissue CoA measurements as indicators of efficacy16,17,18,19,20. Evaluation of all of these conditions and their metabolic or functional consequences requires a quantitative method for measurement of total CoA.
An accurate, reliable assay for measuring CoA in biological samples is a technical challenge in many labs. Unfortunately, there are no probes available to evaluate or quantify CoA or CoA thioesters in intact cells, although analogs of natural CoA thioesters have been widely used as mechanistic probes in studies of CoA ester utilizing enzymes21. The conversion of CoA, with a free sulfhydryl (-SH) moiety, to a CoA thioester (or vice versa) is rapid in cells or animal tissues during transfer to a different environment and during cell lysis. Numerous acyl-CoA synthetases and acyl-CoA thioesterases in cells mediate the interconversions within the CoA pool, and additional enzymes that utilize CoA thioesters as substrates remain active in biological samples until quenched by chemical or physical means. The off-loading of acyl-groups from CoA to carnitine by acyl-transferases is one example within the network of reactions that can alter the CoA/CoA thioester distribution. Radioactive tracers can be used to measure rates of CoA synthesis in cells. Current methods for measuring CoA and CoA derivatives in biological samples have been reviewed22 and include coupled enzymatic spectrophotometric assays, high-pressure liquid chromatography and mass spectrometry-based procedures. However, these methods are often focused on particular CoA molecular species and are blind to variation of the total CoA pool. The coupled enzymatic assays generally require larger amounts of input material due to low detection sensitivities and have a limited range of linearity.
Our laboratory has developed a reliable procedure for quantification of total CoA in cultured cells and animal tissues. The strategy includes hydrolysis of all CoA thioesters to yield only free CoA during sample preparation, rather than making efforts to maintain and analyze the entire spectrum of CoA species. The procedure is a compilation of individual published methods for sample preparation, CoA derivatization, purification and identification following high-pressure liquid chromatography (HPLC), and quantification of the derivatized CoA by absorbance or fluorescence detection23,24,25. The CoA determinations obtained using this procedure have enabled our understanding of CoA regulation and the development of a therapeutic approach for treatment of CoA deficiencies.

<table><tbody><tr><td>2-(2-pyridyl)-ethyl silica gel SPE column</td><td>Millipore-Sigma</td><td>54127-U</td><td></td></tr><tr><td>coenzyme A</td><td>Avanti Polar Lipids</td><td>870700</td><td></td></tr><tr><td>Gemini C18 3 &amp;mu;m 100 &amp;Aring; HPLC column</td><td>Phenomenex</td><td>00F-4439-E0</td><td></td></tr><tr><td>monobromobimane</td><td>ThermoFisher Scientific</td><td>M-1378</td><td></td></tr><tr><td>Omni-Tip probe tissue disrupter</td><td>Omni International</td><td>32750H</td><td></td></tr><tr><td>Parafilm</td><td>Fisher</td><td>S37440</td><td></td></tr><tr><td>PowerGen 125 motorized rotor stator homogenizer</td><td>ThermoFisher Scientific</td><td>NC0530997</td><td></td></tr><tr><td>Spin-X centrifuge tube filter</td><td>CoStar</td><td>8161</td><td></td></tr><tr><td>Trizma-HCl</td><td>Fisher</td><td>T395-1</td><td></td></tr><tr><td>Waters 2475 fluorescence detector</td><td>Waters</td><td>2475</td><td></td></tr><tr><td>Waters 2489 UV-Vis detector</td><td>Waters</td><td>2489</td><td></td></tr><tr><td>Waters e2695 separations module</td><td>Waters</td><td>e2695</td><td></td></tr></tbody></table>

quantification of coenzyme a in cells and tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

A relatively fast and reliable method for the detection of total CoA in cultured cells and tissues has been developed by derivatizing the thiol of CoA to a fluorescent agent using mBBr, and then purifying the derivatized CoA-bimane using reverse phase HPLC. A standard curve is first generated, where known and increasing amounts of the CoA-bimane standard are injected individually and the areas under the peaks in the CoA-bimane chromatograms are plotted as a function of the input CoA-bimane (Figure 4<...

Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

Quantification of Coenzyme A in Cells and Tissues

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

quantification-of-coenzyme-a-in-cells-and-tissues

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.2K Views.  St. Jude Children's Research Hospital. This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Video: Quantification of Coenzyme A in Cells and Tissues

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

Cancer Research

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.

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

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Biology

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.

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

Neuroscience

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Substrate Oxidation Assay in Cultured Cells: An In Vitro Assay to Quantify Substrate Oxidation in Cells by Measuring Radioactive Signals from Trapped CO2

Source: Song, C. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/63568">Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping.</a> J. Vis. Exp. (2022)
This video demonstrates a quantification technique for CO2 released during substrate oxidation using 14C-radiolabeled substrates. The released 14CO2 is trapped in an alkaline solution and quantified by a scintillation counter. The oxidation of different substrates varies between tissues and reflects the pathophysiological condition of the tissue.

Source: Song, C. et al., Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping. J. Vis. Exp. (2022)
This video demonstrates a quantification ...

Quantification of Coenzyme A in Cells and Tissues

Summary

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Chapters in this video

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

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

Measurement of Heme Synthesis Levels in Mammalian Cells

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

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Substrate Oxidation Assay in Cultured Cells: An In Vitro Assay to Quantify Substrate Oxidation in Cells by Measuring Radioactive Signals from Trapped CO₂