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 ad...

<ol>
	<li>Abiko, Y., Greenburg, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Metabolic Pathways. 7, 1-25 (1975).</li><li>Leonardi, R., Zhang, Y. -. M., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+A:+Back+in+action.">Coenzyme A: Back in action.</a> Progress in Lipid Research. 44, 125-153 (2005).</li><li>Jackowski, S., Rock, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+coenzyme+A+biosynthesis.">Regulation of coenzyme A biosynthesis.</a> Journal of Bacteriology. 148, 926-932 (1981).</li><li>Robishaw, J. D., Berkich, D. A., Neely, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate-limiting+step+and+control+of+coenzyme+A+synthesis+in+cardiac+muscle.">Rate-limiting step and control of coenzyme A synthesis in cardiac muscle.</a> Journal of Biological Chemistry. 257, 10967-10972 (1982).</li><li>Di Meo, I., Carecchio, M., Tiranti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+errors+of+coenzyme+A+metabolism+and+neurodegeneration.">Inborn errors of coenzyme A metabolism and neurodegeneration.</a> Journal of Inherited Metabolic Disease. 42, 49-56 (2019).</li><li>Zhou, B., 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=A+novel+pantothenate+kinase+gene+(PANK2)+is+defective+in+Hallervorden-Spatz+syndrome.">A novel pantothenate kinase gene (PANK2) is defective in Hallervorden-Spatz syndrome.</a> Nature Genetics. 28, 345-349 (2001).</li><li>Dusi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exome+sequence+reveals+mutations+in+CoA+synthase+as+a+cause+of+neurodegeneration+with+brain+iron+accumulation.">Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.</a> American Journal of Human Genetics. 94, 11-22 (2014).</li><li>Dansie, L. E., 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=Physiological+roles+of+the+pantothenate+kinases.">Physiological roles of the pantothenate kinases.</a> Biochemical Society Transactions. 42, 1033-1036 (2014).</li><li>Leonardi, R., Rehg, J. E., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pantothenate+kinase+1+is+required+to+support+the+metabolic+transition+from+the+fed+to+the+fasted+state.">Pantothenate kinase 1 is required to support the metabolic transition from the fed to the fasted state.</a> PLoS ONE. 5, 11107 (2015).</li><li>Leonardi, R., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pank1+deletion+in+leptin-deficient+mice+reduces+hyperglycaemia+and+hyperinsulinaemia+and+modifies+global+metabolism+without+affecting+insulin+resistance.">Pank1 deletion in leptin-deficient mice reduces hyperglycaemia and hyperinsulinaemia and modifies global metabolism without affecting insulin resistance.</a> Diabetologia. 57, 1466-1475 (2014).</li><li>Israel, B. C., Smith, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+and+chronic+ethanol+ingestion+on+pantothenate+and+CoA+status+of+rats.">Effects of acute and chronic ethanol ingestion on pantothenate and CoA status of rats.</a> The Journal of Nutrition. 117, 443-451 (1987).</li><li>Garcia, M., Leonardi, R., Zhang, Y. M., Rehg, J. E., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+deletion+of+pantothenate+kinases+1+and+2+reveals+the+key+roles+for+CoA+in+postnatal+metabolism.">Germline deletion of pantothenate kinases 1 and 2 reveals the key roles for CoA in postnatal metabolism.</a> PLoS One. 7, 40871 (2012).</li><li>Corbin, D. 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=Excess+coenzyme+A+reduces+skeletal+muscle+performance+and+strength+in+mice+overexpressing+human+PANK2.">Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.</a> Molecular Genetics and Metabolism. 120, 350-362 (2017).</li><li>Shumar, S. A., Kerr, E. W., Fagone, P., Infante, A. M., Leonardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Nudt7+decreases+bile+acid+levels+and+peroxisomal+fatty+acid+oxidation+in+the+liver.">Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver.</a> Journal of Lipid Research. 60, 1005-1019 (2019).</li><li>Shumar, S. A., 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=Induction+of+neuron-specific+degradation+of+Coenzyme+A+models+pantothenate+kinase-associated+neurodegeneration+by+reducing+motor+coordination+in+mice.">Induction of neuron-specific degradation of Coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.</a> PLoS ONE. 10, 0130013 (2015).</li><li>Zano, S. P., Pate, C., Frank, M., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+a+genetic+deficiency+in+pantothenate+kinase+1+using+phosphopantothenate+replacement+therapy.">Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.</a> Molecular Genetics and Metabolism. 16, 281-288 (2015).</li><li>Sharma, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+therapeutic+approach+to+pantothenate+kinase+associated+neurodegeneration.">A therapeutic approach to pantothenate kinase associated neurodegeneration.</a> Nature Communications. 9, 4399 (2018).</li><li>Alvarez-Cordoba, M., 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=Pantothenate+Rescues+Iron+Accumulation+in+Pantothenate+Kinase-Associated+Neurodegeneration+Depending+on+the+Type+of+Mutation.">Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation.</a> Molecular Neurobiology. 56, 3638-3656 (2019).</li><li>Arber, C., 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=iPSC-derived+neuronal+models+of+PANK2-associated+neurodegeneration+reveal+mitochondrial+dysfunction+contributing+to+early+disease.">iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease.</a> PLoS One. 12, 0184104 (2017).</li><li>Di Meo, I., 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=Acetyl-4'-phosphopantetheine+is+stable+in+serum+and+prevents+phenotypes+induced+by+pantothenate+kinase+deficiency.">Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency.</a> Scientific Reports. 7, 11260 (2017).</li><li>Nishikawa, T., Edelstein, D., Brownlee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+missing+link:+a+single+unifying+mechanism+for+diabetic+complications.">The missing link: a single unifying mechanism for diabetic complications.</a> Kidney International Supplements. 77, 26-30 (2000).</li><li>Tsuchiya, Y., Pham, U., Gout, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+CoA+and+CoA+derivatives+in+biological+samples.">Methods for measuring CoA and CoA derivatives in biological samples.</a> Biochemical Society Transactions. 42, 1107-1111 (2014).</li><li>Demoz, A., Netteland, B., Svardal, A., Mansoor, M. A., Berge, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+Detection+of+Tissue+Coash+and+Long-Chain+Acyl-Coa+by+Reversed-Phase+High-Performance+Liquid-Chromatography+after+Precolumn+Derivatization+with+Monobromobimane.">Separation and Detection of Tissue Coash and Long-Chain Acyl-Coa by Reversed-Phase High-Performance Liquid-Chromatography after Precolumn Derivatization with Monobromobimane.</a> Journal of Chromatography. 635, 251-256 (1993).</li><li>Shimada, K., Mitamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Thiol-Containing+Compounds.">Derivatization of Thiol-Containing Compounds.</a> Journal of Chromatography B. 659, 227-241 (1994).</li><li>Minkler, P. E., Kerner, J., Ingalls, S. T., Hoppel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+isolation+procedure+for+short-,+medium-,+and+long-chain+acyl-coenzyme+A+esters+from+tissue.">Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue.</a> Analytical Biochemistry. 376, 275-276 (2008).</li><li>Newton, G. L., Fahey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+biothiols+by+bromobimane+labeling+and+high-performance+liquid+chromatography.">Determination of biothiols by bromobimane labeling and high-performance liquid chromatography.</a> Methods in Enzymology. 251, 148-166 (1995).</li><li>Radkowsky, A. E., Kosower, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimanes+.17.+(Haloalkyl)-1,5-Diazabicyclo[3.3.0]Octadienediones+(Halo-9,10-Dioxabimanes)+-+Reactivity+toward+the+Tripeptide+Thiol,+Glutathione.">Bimanes .17. (Haloalkyl)-1,5-Diazabicyclo[3.3.0]Octadienediones (Halo-9,10-Dioxabimanes) - Reactivity toward the Tripeptide Thiol, Glutathione.</a> Journal of the American Chemical Society. 108, 4527-4531 (1986).</li><li>Zhang, Y. M., 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=Chemical+knockout+of+pantothenate+kinase+reveals+the+metabolic+and+genetic+program+responsible+for+hepatic+coenzyme+A+homeostasis.">Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis.</a> Chemistry &amp; Biology. 14, 291-302 (2007).</li><li>Tokutake, Y., Onizawa, N., Katoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toyoda+A,Chohnan+S.+Coenzyme+A+and+its+thioester+pools+in+fasted+and+fed+rat+tissues.">Toyoda A,Chohnan S. Coenzyme A and its thioester pools in fasted and fed rat tissues.</a> Biochemical and Biophysical Research Communications. 402, 158-162 (2010).</li><li>Shibata, K., Nakai, T., Fukuwatari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-performance+liquid+chromatography+determination+of+coenzyme+A,+dephospho-coenzyme+A,+and+acetyl-coenzyme+A+in+normal+and+pantothenic+acid-deficient+rats.">Simultaneous high-performance liquid chromatography determination of coenzyme A, dephospho-coenzyme A, and acetyl-coenzyme A in normal and pantothenic acid-deficient rats.</a> Analytical Biochemistry. 430, 151-155 (2012).</li><li>Chohnan, S., Takamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+micromethod+for+measurement+of+CoASH+and+its+use+in+measuring+the+intracellular+levles+of+CoASH+and+short+chain+acyl-CoAs+in+Escherichia+coli+K12+cells.">A simple micromethod for measurement of CoASH and its use in measuring the intracellular levles of CoASH and short chain acyl-CoAs in Escherichia coli K12 cells.</a> Agricultural and Biological Chemistry. 55, 87-94 (1991).</li></ol>

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 &#956;m 100 &#197; 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. ...

Coenzyme A, called CoA for short, is an important cofactor in cellular metabolism. Quantitative measurements reveal changes that happen with nutritional and hormonal states in animal models. This method quantitates the total amount of cellular CoA, including CoA derivatives and free CoA, in a simple, reliable method.Several types of neurodegeneration with brain iron accumulation are associated with mutations in the genes of the CoA biosynthetic pathway. This method can be applied to any biological system since all organisms require CoA for survival, growth, and function. A first-time researcher may struggle with the first steps in sample preparation, where it's important to keep the samples absolutely cold and then quickly transfer them to potassium hydroxide.Limit the number of samples in a batch before transfer to potassium hydroxide, and practice with the transfer before working with the experimental samples. Many researchers are not acquainted with solid-phase extraction in the preparation of biological samples for later biochemical analysis. To begin, incubate triplicate culture dishes in parallel, two for biochemical analysis and one for determination of viable cell number.When the culture approaches the late subconfluent densities, for example, human HepG2/C3A cells grown to a density of six to eight times 10 to the six or HEK 293T cells grown to a density of about 1.3 times 10 to the seven per 100-millimeter dish, harvest the adherent cells in the culture. Then, add one milliliter of ice-cold water to the same culture dish. Scrape cells into the cold water in the dish, and transfer the cell suspension into a glass test tube containing 400 microliters of 0.25-molar potassium hydroxide and 1.5 milliliters of water to bring the pH above 12.Mix the suspension vigorously by vortexing on high for 10 seconds. Then cover tightly with paraffin film, and incubate at 55 degrees Celsius for one hour without shaking in a water bath. Then, add 160 microliters of one-molar Trizma-hydrochloride solution and 10 microliters of 100-millimolar mBBr.Mix by vortexing on high for 10 seconds. This brings the pH to approximately eight to support the mBBr reaction with free CoA. Cover the tube with paraffin film, and incubate the samples at room temperature for two hours in the dark for the mBBr to react with the thiol of CoA.After two hours, add 100 microliters of acetic acid, and mix by vortexing for 10 seconds to stop the reaction. A precipitation forms in the tube. Next, centrifuge at 2, 000 times g for 15 minutes.To remove the precipitated cell debris, transfer and save the supernatant in a glass test tube for the solid-phase extraction column clean-up. Prior to starting the analysis, add two milliliters of one-millimolar potassium hydroxide to a glass test tube designed for insertion of a probe and tissue disruption with a rotor-stator homogenizer. Keep the tube on ice until use.Weigh the frozen tissue pieces quickly, and record the wet weight for each sample. Transfer the tissue into the glass tube, and homogenize the tissue for 30 seconds. Avoid complete thawing of the tissue.Add 500 microliters of 0.25-molar potassium hydroxide. Vortex on high for 10 seconds, and keep on ice. This brings the pH of the sample above 12 to hydrolyze the CoA thioesters to yield total CoA.Cover the tube with paraffin film. The preparation of cultured cells and the preparation of tissues are the most critical steps in this procedure. It is important to add high potassium hydroxide quickly to prevent CoA degradation.Incubate the samples at 55 degrees Celsius for two hours in a water bath without shaking to support hydrolysis. Then, add 150 microliters of one-molar Trizma-hydrochloride and 10 microliters of 100-millimolar mBBr to bring the pH to about eight for supporting the mBBr reaction with free CoA. Vortex on high for 10 seconds.Cover the tube with paraffin film, and incubate the samples at room temperature for two hours in the dark to ensure all the CoA is derivatized with mBBr. After that, add 100 microliters of acetic acid, and vortex on high for 10 seconds to stop the reaction. Centrifuge at 2, 000 times g for 15 minutes to pellet precipitated cell debris, and transfer the supernatant into a glass test tube for the solid-phase extraction column clean-up.At room temperature, equilibrate each one-milliliter disposable 2-2-pyridyl-ethyl silica gel column with one milliliter of wash buffer to ensure that the pyridyl functional group is protonated and will function as an anion exchanger. Then, pipette the sample supernatant onto the column, and collect the eluate. Wash the column twice with one milliliter of wash buffer and once with one milliliter of water to remove any unretained species.Next, into a separate glass test tube, wash the column twice with one milliliter of elution buffer to collect the CoA-bimane. Dry the CoA-bimane sample in the tube under nitrogen gas. Seal, completely cover the tube, and store at minus 20 degrees Celsius.When ready for HPLC analysis, resuspend the sample in 300 microliters of water, and mix vigorously by vortexing on high for 10 seconds. Transfer the resuspended sample to a centrifuge tube filter, and centrifuge at 5, 000 times g for 10 minutes to remove any precipitant. Then, transfer the filtered sample to a glass vial suitable for HPLC injection.In this study, a representative HPLC profile shows the retention time of the CoA-bimane standard on the C18 HPLC column. Increasing amounts of the CoA-bimane standard were injected individually, and the areas under the peaks in the CoA-bimane chromatograms were plotted as a function of the input CoA-bimane. The standard curve reflected the magnitude of absorbance of CoA-bimane at a wavelength of 393 nanometers.The fluorescence of CoA-bimane was also reflected at the excitation wavelength of 393 nanometers and emission wavelength of 470 nanometers. Subsequent HPLC separation and typical detection profiles for mouse liver or human cultured C3A cells are indicated here by red peaks, with a retention time between 11 and 12 minutes using the elution program. If the reaction with mBBr does not yield detectable results, the amount of Trizma-HCl may need to be adjusted to lower the pH to approximately eight.It is possible to detect additional cellular molecules that contain sulfhydryl or thioester moieties as bimane derivatives. For example, glutathione-bimane can be detected with the HPLC method. This technique will allow other researchers to investigate the potential role of CoA dysfunction associated with metabolic imbalance, such as animal models of type 2 diabetes.Researchers should use personal protective equipment when working with organic solvents used for the solid-phase extraction.

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

Research

JoVE Journal

Biochemistry

8.1K visualizações.  St. Jude Children's Research Hospital. A coenzima A, chamada CoA para abreviar, é um importante cofator no metabolismo celular. Medições quantitativas revelam alterações que acontecem com estados nutricionais e hormonais em modelos animais. Este método quantifica a quantidade total de CoA celular, incluindo derivados de CoA e CoA gratuito, em um método simples e confiável.Vários tipos de neurodegeneração com acúmulo de ferro cerebral estão associados a mutações nos genes da via biossintética coa. Este métod...