Coenzyme A and Its Derivatives in Cellular Metabolism and Disease
Methods for measuring CoA and CoA derivatives in biological samples Yugo Tsuchiya*1 , Uyen Pham* and Ivan Gout*1 *Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract CoA (coenzyme A) is a ubiquitous and essential cofactor that acts as an acyl group carrier in biochemical reactions. Apart from participating in numerous metabolic pathways as substrates and intermediates, CoA and a number of its thioester derivatives, such as acetyl-CoA, can also directly regulate the activity of proteins by allosteric mechanisms and by affecting protein acetylation reactions. Cellular levels of CoA and CoA thioesters change under various physiological and pathological conditions. Defective CoA biosynthesis is implicated in NBIA (neurodegeneration with brain iron accumulation). However, the exact role of CoA in the pathogenesis of NBIA is not well understood. Accurate and reliable assays for measuring CoA species in biological samples are essential for studying the roles of CoA and CoA derivatives in health and disease. The present mini-review discusses methods that are commonly used to measure CoA species in biological samples.
Introduction
Extraction and sample handling
Since the discovery of CoA (coenzyme A) by Fritz Lipmann over 60 years ago, various methods have been developed to isolate and measure CoA and its thioester derivatives to study their biological functions. Early methods for measuring CoA included enzymatic assays, paper and thinlayer chromatography, and HPLC [1]. Many of these techniques are still used today. In addition, MS-based methods have been increasingly used recently for sensitive detection of CoA species. The present mini-review gives an overview of methods that are used for detecting and measuring CoA and CoA derivatives in biological samples.
CoA species have been measured in a wide range of biological samples such as isolated animal organs and tissues [2–5], human biopsies [6], plants [7], cultured cells [8– 11], single cell organisms [12,13] and isolated organelles [14–18]. Unesterified CoA and short-chain acyl-CoAs are most commonly quantified after acid deproteinization of samples, usually with ice-cold PCA (perchloric acid; HClO4 ; 3.6–6%) [5,19,20], TCA (trichloroacetic acid; 10%) [8] or SSA (5-sulfosalicylic acid; 5%) [3,4]. The soluble fraction (supernatant) contains CoASH and short-chain acyl-CoAs, whereas long-chain acyl-CoAs are found in the acid-insoluble fraction. The acid extracts are used for quantification of CoASH and short-chain esters without further treatment (e.g. for HPLC–MS analysis [3]) or after neutralization/removal of the acid. The pellet can be used to quantify the total levels of long-chain acyl-CoAs. This is accomplished by treating the pellet with an alkali (KOH) in the presence of a reducing agent and measuring the free CoA released [20,21]. Measurement of individual long-chain acyl-CoA levels usually involves extraction of samples in the presence of organic solvents and the use of solvent partitioning to separate long-chain CoA esters from more hydrophobic lipids [22–26]. Extracted longchain acyl-CoA are usually further clarified and concentrated using solid-phase purification (oligonucleotide purification cartridge, alumina or C18 cartridge) before analysis by HPLC, GC–MS or HPLC–MS [22–26]. As with other metabolites, it is important that enzymatic reactions in samples are quenched rapidly by deproteinization with an acid or organic solvent, or by snap-freezing to preserve the natural levels of CoA species. Cells or organelles in suspension may be quickly separated from media and quenched by the silicone oil layer centrifugation technique [12,27]. No appreciable degradation of CoASH and acetyl
CoA species grouping and nomenclature Unesterified CoA is often referred to as CoASH to distinguish it from CoA thioesters. Acyl-CoA esters are grouped into short-, medium- and long-chain esters based on the carbon-chain length of the group forming a thioester linkage with CoA. Short-chain esters include those with chain lengths of two to six carbons, such as malonyl-, acetyl-, 3hydroxy-3-methyl-glutaryl-, β-hydroxybutyryl-, succinyl-, propionyl- and isobutyryl-CoA. Medium-chain (C6 –C10 ) and long-chain acyl (C12 –C18 ) CoA esters include those derived from esterification of fatty acids of various chain lengths. The term acyl-CoA is sometimes used to specifically mean long-chain acyl-CoA derivatives.
Key words: coenzyme A, enzymatic assay, HPLC, mass spectrometry, unesterified coenzyme A (CoASH). Abbreviations: CoA, coenzyme A; CoASH, unesterified CoA; DTE, dithioerythritol; NEM, Nethylmaleimide; PCA, perchloric acid; RP-HPLC, reverse-phase HPLC; SSA, 5-sulfosalicylic acid. 1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
Biochem. Soc. Trans. (2014) 42, 1107–1111; doi:10.1042/BST20140123
C The
C 2014 Biochemical Society Authors Journal compilation
1107
1108
Biochemical Society Transactions (2014) Volume 42, part 4
Table 1 Examples of methods for measuring CoA and CoA derivatives Methods
Example
CoA species
Limit of detection
Enzymatic
CoA recycling assay (Allred and Guy 1969) [2]
CoASH and/or acetyl-CoA
20–40 pmol*
CoA cycling assay (Kato 1975) [31,32] Radiochemical malonyl-CoA assay (McGarry et al. 1978) [5,34]
CoASH and/or acetyl-CoA Malonyl-CoA
0.04 pmol [31] ∼10 pmol [5]
Malonyl-CoA:acetyl-CoA cycling assay (Takamura et al. 1985) [35,36] RP-HPLC analysis of short-chain CoA species [4,6,8,15,19,28,38] RP-HPLC analysis of long-chain CoA species [23,25,26]
Malonyl-CoA and/or acetyl-CoA Multiple Multiple
∼1 pmol [36] 3–12 pmol [19,28,38] 12 pmol [23]
GC–MS analysis of CoA esters [46-48] LC–ESI–MS analysis of short-chain CoA [3,45] LC–ESI–MS analysis of medium-chain CoA [49]
Multiple Multiple Multiple
0.3–0.5 pmol [46,47] 0.2 pmol [3] ∼0.5 pmol [49]
LC–ESI–MS/MS analysis long-chain CoA [50] OxiRed based colorimetric/fluorimetric assay (Abcam, Abnova and BioVision)
Multiple CoASH
10 fmol [50] 100 pmol (2.5μM)†
Europium chloride based fluorimetric assay (Cayman Chemical) AmpliteTM fluorimetric CoA assay (AAT Bioquest®) Acetyl-CoA fluorimetric assay (Sigma–Aldrich)
CoASH CoASH Acetyl-CoA
250 pmol (5 μM)‡ 4 pmol (40 nM)† 20 pmol (0.4 μM)†
ELISA (MyBioSource)
Malonyl-CoA
18.3fmol (183 pM)†
HPLC MS-based
Other
Y. Tsuchiya, unpublished results. †Based on the information provided by the suppliers. ‡Based on the standard curve provided by the supplier.
CoA occurs in snap-frozen liver pieces stored at − 80◦ C for at least 3 months [4]. However, approximately 40– 50% of CoASH and acetyl-CoA degrades in snap-frozen liver at − 20◦ C after a week [4]. To prevent oxidation of CoASH, a reducing agent is usually included during or after sample extraction. DTT and DTE (dithioerythritol) are commonly used for this purpose. However, DTT can interfere with HPLC detection of CoA compounds (Y. Tsuchiya, unpublished work and [28]). For quantification of CoA compounds by HPLC, tris-(2-carboxyethyl)phosphine (TCEP) can be used as an alternative. Stability of some CoA species in PCA extracts is dependent on the pH [19]. Succinyl-CoA is particularly known to be labile. In PCA extracts adjusted to pH 6, approximately 80% of succinylCoA originally present degrades after 12 h at 8◦ C [19]. Degradation is much slower at pH 3 with only 20% degraded in the same time period [19]. CoASH and short-chain acylCoAs extracted from liver in 5% SSA and 50 μM DTE are stable for at least 12 h at 4◦ C and for 55 days if stored at − 80◦ C [3].
Methods for measuring CoA and CoA derivatives Table 1 shows examples of methods that are used for measuring CoASH and CoA thioesters. Currently, CoASH and CoA thioesters are most commonly measured using enzymatic, HPLC- and MS-based methods. These methods are discussed in more detail below. Other methods of detection include TLC, which is useful for detecting the C The
C 2014 Biochemical Society Authors Journal compilation
formation of particular CoA species in cells after incubating cells with radiolabelled precursors [29].
Enzymatic methods Owing to the relatively low abundance of CoA compounds in biological samples, spectrophotometric end-point assays that measure stoichiometric conversion of CoA or CoA derivatives into a product are generally not sensitive enough for most applications, especially when the amount of materials is limited. The sensitivity of end-point assays can be improved by incorporating fluorometric or radiochemical detection methods [14,29,30]. Several recycling assays have been described for measuring CoASH and CoA esters. These assays are based on the CoA- or CoA esterdependent amplification of a specific reaction product that can be measured spectrometrically or fluorometrically. The recycling assay by Allred and Guy, first described in 1969 [2], allows quantification of CoASH and acetyl-CoA levels and has been widely used. This assay utilizes citrate synthase, which catalyses the transfer of the acetyl group from acetyl-CoA to oxaloacetate, generating free CoA and citrate. This reaction is linked to two other enzymatic reactions: phosphotransacetylase and malate dehydrogenase reactions. The former converts the free CoA released from citrate synthase back into acetyl-CoA, allowing continuous recycling of CoA. The malate dehydrogenase reaction replaces the oxaloacetate consumed by the citrate synthase reaction with accompanying generation of NADH. The recycling of CoA by phosphotransacetylase therefore results in continuous conversion of oxaloacetate into citrate and
Coenzyme A and Its Derivatives in Cellular Metabolism and Disease
the accumulation of NADH, which can be measured spectrophotometrically or fluorometrically. The rate of NADH generation is dependent on the amount of CoASH and acetyl-CoA originally present in the sample. The amount of acetyl-CoA alone can be measured by pre-treating samples with NEM (N-ethylmaleimide) to block CoASH. It is recommended that internal standards be used for the quantification of CoA and acetyl-CoA since the activity of the enzymes can be affected by the chemical environment of samples. Using the 96-well plate format and an absorbance at 340 nm, we found the detection limit of this method to be approximately 20–40 pmol (0.1–0.2 μM). The assay is sufficiently sensitive for reliably measuring CoA and acetylCoA in tissue samples, but may not be suitable for cultured cells or if the amount of samples is limited. A more sensitive method was described by Kato in 1975 [31] and later modified by Szutowicz and Bielarczyk in 1987 [32]. This method is also based on the cyclic reactions involving citrate synthase and phosphotransacetylase. However, instead of including malate dehydrogenase and measuring NADH, the method involves measuring citrate which is accumulated after many cycles of CoA recycling. An excess of oxaloacetate and acetyl phosphate are included in the reaction mixture. As for the recycling assay by Allred and Guy [2], the method can measure combined levels of CoASH and acetyl-CoA. AcetylCoA can be measured separately by inactivating CoASH with NEM. However, owing to the slow hydrolysis of CoA– NEM thioether, small amounts of free CoASH are released during the amplification step (which lasts for 1 h) [31,32]. More accurate measurement of acetyl-CoA can be achieved by blocking CoASH with maleic anhydride, which forms a more stable thioether bond with CoASH [32]. The detection limit of this method was reported to be as low as 40 fmol [31] and the method has been used to measure acetyl-CoA levels in synaptosomes isolated from rat brains [32]. Malonyl-CoA is an allosteric inhibitor of carnitine palmitoyltransferase 1 and acts as an important signalling molecule regulating β-oxidation of long-chain fatty acids [33]. Its levels in tissues can be as low as 0.2–3 pmol/mg of wet mass [3] and its quantification therefore requires particularly sensitive assays. A widely used enzymatic assay for malonyl-CoA is that developed by McGarry et al. [34], which measures malonyl-CoA-dependent incorporation of radiolabelled acetyl-CoA into fatty acid by fatty acid synthase. A cycling assay for malonyl-CoA and acetylCoA was described by Takamura et al. in 1985 [35]. This method is based on the amplification of acetate by malonate decarboxylase in the presence of malonyl-CoA and acetyl-CoA. The amplified acetate is first converted into acetyl phosphate and then to acetohydroxamate, which can be measured spectrophotometrically at 540 nm. The assay measures combined levels of acetyl-CoA and malonylCoA. Selective measurement of malonyl-CoA is achieved by enzymatically eliminating acetyl-CoA using citrate synthase and oxaloacetate. The detection limit of this method is approximately 1 pmol and the method has been used to quantify malonyl-CoA in mouse hypothalami [36].
HPLC Since the initial report of separation of CoA compounds using HPLC by Baker and Schooley [37], HPLC, particularly RPHPLC (reverse-phase HPLC), has been used extensively for measuring CoA species. Owing to the presence of adenosine in their structures, CoA compounds can be detected by UV absorbance at 254–260 nm. Separation of CoA from the bulk of other highly abundant nucleotides, such as ATP, by RPHPLC is usually straightforward due to the presence of the relatively hydrophobic pantetheine tail in CoA compounds, and can be achieved by increasing the salt concentration in the mobile phase. HPLC allows simultaneous detection of multiple CoA species and have been used to quantify CoASH as well as short- and long-chain acyl-CoAs in extracts from animal tissues [4,15,19,23,25,26,38], human needle biopsies [6], cultured cells [8] and bacteria [12]. The detection limit of this technique is approximately 3–12 pmol [19,28,38] and typical analysis time for each sample is 20–60 min. Recent advances in ultra-HPLC and HPLC analytical columns with smaller particle size are expected to help shorten analysis time and improve the quality of separation. A major disadvantage of this technique, if absorbance alone is used to detect CoA compounds, is uncertainty regarding the identity and purity of detected peaks. However, there are several ways to validate the identity and purity of CoASH/CoA thioester peaks. Alkaline hydrolysis of CoA thioesters and consequent disappearance of peaks corresponding to CoA thioesters is commonly used to demonstrate the purity of CoA thioester peaks [6,25,38]. More conclusive characterization of peaks can be achieved by using enzymes that specifically catalyse the conversion of specific CoA species. For example, the identity and purity of the acetyl-CoA peak can be demonstrated by the complete disappearance of the acetyl-CoA peak after treatment of the sample with citrate synthase and oxaloacetate. Phosphotransacetylase and an excess of acetyl phosphate may be used to verify the CoASH peak. Sensitivity and specificity of detection can be further improved by derivatization or radiolabelling of CoASH and/or derivatives before HPLC analysis. CoASH can be derivatized by thiol-reactive labels, such as monobromobimane and SBDF (ammonium 7-flurobenzo-2-oxa-1,3-doazole-4-sulfonate), which can be detected by a fluorescent detector [39–41]. Subpicomole amounts of CoASH can be detected by the latter method. However, these approaches are only applicable for quantification of CoASH or total CoA levels (after alkaline hydrolysis) and not thioesters. CoASH and CoA thioesters can be detected fluorometrically by converting them into fluorescent 1,N6-ethenoadenine derivatives using bromoacetaldehyde or chloroacetaldehyde before or after HPLC separation [7,12]. Wadler and Cronan [42] described a method for specifically labelling CoA and derivatives using dephospho-CoA kinase. In their approach, the 3 phosphate groups of CoASH and acyl-CoAs are first dephosphorylated by a phosphatase, and then the 3 hydroxy group is phosphorylated again using dephosphoCoA kinase and radioactive ATP. The radiolabelled CoA species are detected by HPLC connected to a flow-through C The
C 2014 Biochemical Society Authors Journal compilation
1109
1110
Biochemical Society Transactions (2014) Volume 42, part 4
beta-counter. This method can significantly improve the specificity of detection however the efficiency of enzymatic labelling of CoA species may be affected by the chemical environment in each sample.
MS-based methods MS-based quantification of CoA species has been increasingly used in recent years [43–45]. GC–MS has been used for measuring acyl-CoA esters after their hydrolysis and derivatization [46–48]. More recently, MS or tandem MS (MS/MS) coupled with HPLC and electrospray ionization (LC–ESI–MS and LC–ESI–MS/MS) have been used to directly detect short- and long-chain acyl-CoAs in biological extracts [3,13,45,49,50]. Femtomole amounts of CoA species can be quantified by these methods. Since a mass spectrometer detects compounds on the basis of mass charge ratios, baseline separation of compounds by HPLC is usually not required. However, identification of CoA compounds may be complicated by the fact that some CoA derivatives have almost identical masses. For instance, Gao et al. [3] were unable to distinguish malonyl-CoA and β-hydroxybutyrylCoA by their mass charge ratios or by unique product ions after MS/MS. Separate detection of these compounds was achieved by their prior separation by HPLC using dimethylbutyl amine as an ion-pairing agent [3].
Commercially available kits A number of companies supply kits for assaying CoA compounds. Examples of these are shown in Table 1. On the basis of information provided by the suppliers, some of these kits, including those based on OxiRed, do not appear to be sensitive enough for detecting CoA in small quantities of biological materials. The possible exceptions are the Amplite fluorometric Coenzyme A assay kit by AAT Bioquest® (detection limit 4 pmol) and the acetyl-CoA assay kit (detection limit 20 pmol) by Sigma–Aldrich. There is also an ELISA-based malonyl-CoA assay kit that can supposedly measure femtomole amounts of malonyl-CoA. However, as far as we are aware, these assay kits have not yet been widely used.
Conclusions There are currently a number of different methods for measuring CoA compounds. Different methods may be used depending on the aims and requirements of individual studies. MS-based methods are highly sensitive and specific, but its routine use may be limited by the availability of sensitive mass spectrometers. If routine analysis of a specific CoA species is required, a sensitive enzymatic assay may be used as an alternative. Many enzymatic assays can also be performed in microplate formats, allowing high-throughput analysis of multiple samples. HPLC is a useful and reliable technique for simultaneously measuring multiple CoA species, provided the identity and purity of peaks are validated. CoA levels differ considerably in different cell types and in different subcellular compartments. Changes in total CoA C The
C 2014 Biochemical Society Authors Journal compilation
levels in tissues or cells therefore may not reflect changes in CoA levels in specific cell types or compartments. Cytosolic and mitochondrial CoA levels are usually measured after their separation by subcellular fractionation or by using digitonin to selectively permeabilize the cell membrane [15– 18]. However, potential problems with these methods, if not carefully controlled, include leakage or enzymatic conversion of CoA species during the fractionation step. Measurement of CoA species in subcellular fractions also requires highly sensitive assays. Development of molecular probes for CoA compounds that can be used to detect CoA species in different subcellular compartments or different cell types would be highly useful for studying the consequences of altered CoA levels on cellular processes.
Funding This work was supported by UCLB (University College London Business) Proof of Concept funding [grant numbers UCLB PoC-11018 and UCLB PoC-13-014].
References 1 Bieber, L.L. (1992) Quantitation of CoASH and acyl CoA. Anal. Biochem. 204, 228–230 CrossRef PubMed 2 Allred, J.B. and Guy, D.G. (1969) Determination of coenzyme A and acetyl CoA in tissue extracts. Anal. Biochem 29, 293–299 CrossRef PubMed 3 Gao, L., Chiou, W., Tang, H., Cheng, X., Camp, H.S. and Burns, D.J. (2007) Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS. J. Chromatogr. B 853, 303–313 CrossRef 4 Shibata, K., Nakai, T. and Fukuwatari, T. (2012) Simultaneous high-performance liquid chromatography determination of coenzyme A, dephospho-coenzyme A, and acetyl-coenzyme A in normal and pantothenic acid-deficient rats. Anal. Biochem. 430, 151–155 CrossRef PubMed 5 Alam, N. and Saggerson, E.D. (1998) Malonyl-CoA and the regulation of fatty acid oxidation in soleus muscle. Biochem. J. 334, 233–241 PubMed 6 Corkey, B.E., Hale, D.E., Glennon, M.C., Kelley, R.I., Coates, P.M., Kilpatrick, L. and Stanley, C.A. (1988) Relationship between unusual hepatic acyl coenzyme A profiles and the pathogenesis of Reye syndrome. J. Clin. Invest. 82, 782–788 CrossRef PubMed 7 Larson, T.R. and Graham, I.A. (2001) Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues. Plant J. 25, 115–125 CrossRef PubMed 8 Corkey, B.E., Glennon, M.C., Chen, K.S., Deeney, J.T., Matschinsky, F.M. and Prentki, M. (1989) A role of malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic β-cells. J. Biol. Chem. 264, 21608–21612 PubMed 9 MacDonald, M.J., Smith, III, A.D., Hasan, N.M., Sabat, G. and Fahien, L.A. (2007) Feasibility of pathways for transfer of acyl groups from mitochondria to the cytosol to form short chain acy-CoAs in the pancreatic beta cell. J. Biol. Chem. 282, 30596–30606 CrossRef PubMed 10 Basu, S.S., Mesaros, C., Gelhaus, S.L. and Blair, I.A. (2011) Stable isotope labelling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters. Anal. Chem. 83, 1363–1369 CrossRef PubMed 11 Palekar, A (2000) Effect of pantothenic acid on hippurate formation in sodium benzoate-treated HepG2 cells. Pediatr. Res. 48, 357–359 CrossRef PubMed 12 Shimazu, M., Vetcher, L., Galazzo, J.L., Licari, P. and Santi, D.V. (2004) A sensitive and robust method for quantification of short-chain coenzyme A esters. Anal. Biochem. 328, 51–59 CrossRef PubMed 13 Seifer, R.M., Ras, C., Deshmukh, A.T., Bekers, K.M., Suarez-Mendez, C.A., de Cruz, A.L.B., van Gulik, W.M. and Heijnen, J.J. (2013) Quantitative analysis of intracellular coenzymes in Saccharomyces cerevisiae using ion pair reversed phase ultra high performance liquid chromatography tandem mass spectrometry. J. Chromatogr. A 1311, 115–120 CrossRef PubMed
Coenzyme A and Its Derivatives in Cellular Metabolism and Disease
14 Garland, P.B., Shepherd, D. and Yates, D.W. (1965) Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate. Biochem. J. 97, 587–594 PubMed 15 Lysiak, W., Lilly, K., DiLisa, F., Toth, P. and Bieber, L.L. (1988) Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria. J. Biol. Chem. 236, 1151–1156 PubMed 16 Idell-Wenger, J.A., Grotyohann, L.W. and Neely, J.R. (1978) Coenzyme A and carnitine distribution in normal and ischemic hearts. J. Biol. Chem. 253, 4310–4318 PubMed 17 Brass, E.P. and Ruff, L.J. (1992) Rat hepatic coenzyme A is redistributed in response to mitochondrial acyl-coenzyme A accumulation. J. Nutr. 122, 2094–2100 PubMed 18 Siess, E.A., Brocks, D.G., Lattke, H.K. and Wieland, O.H. (1977) Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate. Biochem. J. 166, 225–235 PubMed 19 King, M.T. and Reiss, P.D. (1985) Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase-high-performance liquid chromatography. Anal. Biochem. 146, 173–179 CrossRef PubMed 20 Zammit, V.A. (1981) Regulation of hepatic fatty acid metabolism. Biochem. J. 198, 75–83 PubMed 21 Lopaschuk, G.D., Hansen, C.A. and Neely, J.R. (1986) Fatty acid metabolism in hearts containing elevated levels of CoA. Am. J. Physiol. 250, H351–H359 PubMed 22 Prasad, R.M., Sauter, J. and Lands, W.E.M. (1987) Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters. Anal. Biochem. 162, 202–212 CrossRef PubMed 23 Mangino, M.J., Zografakis, J., Murphy, M.K. and Anderson, C.B. (1992) Improved and simplified tissue extraction method for quantitating long-chain acyl-coenzyme A thioesters with picomolar detection using high-performance liquid chromatography. J. Chromatogr. 577, 157–162 CrossRef PubMed 24 Mancha, M., Stokes, G.B. and Stumpf, P.K. (1975) Fat metabolism in higher plants. The determination of acyl-acyl carrier protein and acyl coenzyme A in a complex lipid mixture. Anal. Biochem. 68, 600–608 CrossRef PubMed 25 Woldegiorgis, G., Spennetta, T., Corkey, B.E, Williamson, J.R. and Shrago, E. (1985) Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography. Anal. Biochem. 150, 8–12 CrossRef PubMed 26 Golovko, M.Y. and Murphy, E.J. (2004) An improved method for tissue long-chain acyl-CoA extraction and analysis. J. Lipid. Res. 45, 1777–1782 CrossRef PubMed 27 Hwang, C., Sinskey, A.J. and Lodish, H.F. (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257, 1496–1502 CrossRef PubMed 28 Corkey, B.E., Brand, M., Williams, R.J. and Williamson, J.R. (1981) Assay of short-chain acyl coenzyme A intermediates in tissue extracts by high-pressure liquid chromatography. Anal. Biochem. 118, 30–41 CrossRef PubMed 29 Zhang, Y., Chohnan, S., Virga, K.G., Stevens, R.D., Llkayeva, O.R., Wenner, B.R., Bain, J.R., Newgard, C.B., Lee, R.E., Rock, C.O. and Jackowski, S. (2007) Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis. Chem. Biol. 14, 291–302 CrossRef PubMed 30 Rabier, D., Briand, P., Petit, F., Kamoun, P. and Cathelineau, L. (1983) Radioisotopic assay of picomolar amounts of coenzyme A. Anal. Biochem. 134, 325–329 CrossRef PubMed 31 Kato, T. (1975) CoA cycling: an enzymatic amplification method for determination of CoASH and acetyl CoA. Anal. Biochem. 66, 373–392 32 Szutowicz, A. and Bielarczyk, H. (1987) Elimination of CoASH from acetyl-CoA cycling assay by maleic anhydride. Anal. Biochem. 164, 292–296 CrossRef PubMed 33 Saggerson, D. (2008) Malonyl-CoA, a key signaling molecule in mammalian cells. Annu. Rev. Nutr. 28, 253–272 CrossRef PubMed
34 McGarry, J.D., Stark, M.J. and Foster, D.W. (1978) Hepatic malonyl-CoA levels of fed, fasted and diabetic rats as measured using a simple radioisotopic assay. J. Biol. Chem. 253, 8291–8293 PubMed 35 Takamura, Y., Kitayama, Y., Arakawa, A., Yamanaka, S., Tosaki, M. and Ogawa, Y. (1985) Malonyl-CoA: acetyl-CoA cycling. A new micromethod for determination of acyl-CoAs with malonate decarboxylase. Biochim. Biophys. Acta 834, 1–7 CrossRef PubMed 36 Hu, Z., Cha, S.H., Chohnan, S. and Lane, M.D. (2003) Hypothalamic malonyl-CoA as a mediator of feeding behaviour. Proc. Natl. Acad. Sci. U.S.A. 100, 12624–12629 CrossRef PubMed 37 Baker, F.C. and Schooley, D.A. (1979) Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography. Anal. Biochem. 94, 417–424 CrossRef PubMed 38 Demoz, A., Garras, A., Asiedu, D.K., Netteland, B. and Berge, R.K. (1995) Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography. J. Chromatogr. B 667, 148–152 CrossRef 39 Newton, G.L., Dorian, R. and Fahey, R.C. (1981) Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography. Anal. Biochem. 114, 383–387 CrossRef PubMed 40 Siudeja, K., Srinivasan, B., Xu, L., Rana, A., de Jong, J., Nollen, E.A., Jackowski, S., Sanford, L., Hayflick, S. and Sibon, O.C. (2011) Impaired coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration. EMBO Mol. Med. 3, 755–766 CrossRef PubMed 41 Imai, K., Toyo’oka, T. and Watanabe, Y. (1983) A novel fluorogenic reagent for thiols: ammonium 7-fluorobenzo-2-oxa-1, 3-diazole-4-sulfonate. Anal. Biochem. 128, 471–473 CrossRef PubMed 42 Wadler, C. and Cronan, J.E. (2007) Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters. Anal. Biochem. 368, 17–23 CrossRef PubMed 43 McCoy, F., Darbandi, R., Lee, H.C., Bharatham, K., Moldoveanu, T., Grace, C.R., Dodd, K., Lin, W., Chen, S.I., Tangallapally, R.P. et al. (2013) Metabolic activation of CAMKII by coenzyme A. Mol. Cell 52, 325–339 CrossRef PubMed 44 Marino, ˜ G., Pietrocola, F., Eisenberg, T., Kong, Y., Malik, S.A., Andryushkova, A., Schroeder, S., Pendl, T., Harger, A., Niso-Santano, M. et al. (2014) Regulation of autophagy by cytosolic acetyl-coenzyme A. Mol. Cell 53, 710–725 CrossRef PubMed 45 MacDonald, M.J. (2007) Synergistic potent insulin release by combinations of weak secretagogues in pancreatic islets and INS-1 cells. J. Biol. Chem. 282, 6043–6052 CrossRef PubMed 46 Tamvakopoulos, C.S. and Anderson, V.E. (1992) Detection of acyl-coenzyme A thioester intermediates of fatty acid β-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography-mass spectrometry. Anal. Biochem. 200, 381–387 CrossRef PubMed 47 Kopka, J., Ohlrogge, J.B. and Jaworski, J.G. (1995) Analysis of in vivo levels of acyl-thioesters with gas chromatography/mass spectrometry of the butylamide derivative. Anal. Biochem. 224, 51–60 CrossRef PubMed 48 Kasumov, T., Martini, W.Z., Reszko, A.E., Bian, F., Pierce, B.A., David, F., Roe, C.R. and Brunengraber, H. (2002) Assay of the concentration and 13C isotopic enrichment of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA by gas chromatography-mass Spectrometry. Anal. Biochem. 305, 90–96 CrossRef PubMed 49 Kasuya, F., Oti, Y., Tatsuki, T. and Igarashi, K. (2004) Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography-electrospray ionization mass spectrometry. Anal. Biochem. 325, 196–205 CrossRef PubMed 50 Haynes, C.A., Allegood, J.C., Sims, K., Wang, E.W., Sullards, M.C. and Merrill, Jr, A.H. (2008) Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry. J. Lipid Res. 49, 1113–1125 CrossRef PubMed
Received 30 April 2014 doi:10.1042/BST20140123
C The
C 2014 Biochemical Society Authors Journal compilation
1111
Methods for measuring CoA and CoA derivatives in biological samples Yugo Tsuchiya*1 , Uyen Pham* and Ivan Gout*1 *Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.
Biochemical Society Transactions
www.biochemsoctrans.org
Abstract CoA (coenzyme A) is a ubiquitous and essential cofactor that acts as an acyl group carrier in biochemical reactions. Apart from participating in numerous metabolic pathways as substrates and intermediates, CoA and a number of its thioester derivatives, such as acetyl-CoA, can also directly regulate the activity of proteins by allosteric mechanisms and by affecting protein acetylation reactions. Cellular levels of CoA and CoA thioesters change under various physiological and pathological conditions. Defective CoA biosynthesis is implicated in NBIA (neurodegeneration with brain iron accumulation). However, the exact role of CoA in the pathogenesis of NBIA is not well understood. Accurate and reliable assays for measuring CoA species in biological samples are essential for studying the roles of CoA and CoA derivatives in health and disease. The present mini-review discusses methods that are commonly used to measure CoA species in biological samples.
Introduction
Extraction and sample handling
Since the discovery of CoA (coenzyme A) by Fritz Lipmann over 60 years ago, various methods have been developed to isolate and measure CoA and its thioester derivatives to study their biological functions. Early methods for measuring CoA included enzymatic assays, paper and thinlayer chromatography, and HPLC [1]. Many of these techniques are still used today. In addition, MS-based methods have been increasingly used recently for sensitive detection of CoA species. The present mini-review gives an overview of methods that are used for detecting and measuring CoA and CoA derivatives in biological samples.
CoA species have been measured in a wide range of biological samples such as isolated animal organs and tissues [2–5], human biopsies [6], plants [7], cultured cells [8– 11], single cell organisms [12,13] and isolated organelles [14–18]. Unesterified CoA and short-chain acyl-CoAs are most commonly quantified after acid deproteinization of samples, usually with ice-cold PCA (perchloric acid; HClO4 ; 3.6–6%) [5,19,20], TCA (trichloroacetic acid; 10%) [8] or SSA (5-sulfosalicylic acid; 5%) [3,4]. The soluble fraction (supernatant) contains CoASH and short-chain acyl-CoAs, whereas long-chain acyl-CoAs are found in the acid-insoluble fraction. The acid extracts are used for quantification of CoASH and short-chain esters without further treatment (e.g. for HPLC–MS analysis [3]) or after neutralization/removal of the acid. The pellet can be used to quantify the total levels of long-chain acyl-CoAs. This is accomplished by treating the pellet with an alkali (KOH) in the presence of a reducing agent and measuring the free CoA released [20,21]. Measurement of individual long-chain acyl-CoA levels usually involves extraction of samples in the presence of organic solvents and the use of solvent partitioning to separate long-chain CoA esters from more hydrophobic lipids [22–26]. Extracted longchain acyl-CoA are usually further clarified and concentrated using solid-phase purification (oligonucleotide purification cartridge, alumina or C18 cartridge) before analysis by HPLC, GC–MS or HPLC–MS [22–26]. As with other metabolites, it is important that enzymatic reactions in samples are quenched rapidly by deproteinization with an acid or organic solvent, or by snap-freezing to preserve the natural levels of CoA species. Cells or organelles in suspension may be quickly separated from media and quenched by the silicone oil layer centrifugation technique [12,27]. No appreciable degradation of CoASH and acetyl
CoA species grouping and nomenclature Unesterified CoA is often referred to as CoASH to distinguish it from CoA thioesters. Acyl-CoA esters are grouped into short-, medium- and long-chain esters based on the carbon-chain length of the group forming a thioester linkage with CoA. Short-chain esters include those with chain lengths of two to six carbons, such as malonyl-, acetyl-, 3hydroxy-3-methyl-glutaryl-, β-hydroxybutyryl-, succinyl-, propionyl- and isobutyryl-CoA. Medium-chain (C6 –C10 ) and long-chain acyl (C12 –C18 ) CoA esters include those derived from esterification of fatty acids of various chain lengths. The term acyl-CoA is sometimes used to specifically mean long-chain acyl-CoA derivatives.
Key words: coenzyme A, enzymatic assay, HPLC, mass spectrometry, unesterified coenzyme A (CoASH). Abbreviations: CoA, coenzyme A; CoASH, unesterified CoA; DTE, dithioerythritol; NEM, Nethylmaleimide; PCA, perchloric acid; RP-HPLC, reverse-phase HPLC; SSA, 5-sulfosalicylic acid. 1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
Biochem. Soc. Trans. (2014) 42, 1107–1111; doi:10.1042/BST20140123
C The
C 2014 Biochemical Society Authors Journal compilation
1107
1108
Biochemical Society Transactions (2014) Volume 42, part 4
Table 1 Examples of methods for measuring CoA and CoA derivatives Methods
Example
CoA species
Limit of detection
Enzymatic
CoA recycling assay (Allred and Guy 1969) [2]
CoASH and/or acetyl-CoA
20–40 pmol*
CoA cycling assay (Kato 1975) [31,32] Radiochemical malonyl-CoA assay (McGarry et al. 1978) [5,34]
CoASH and/or acetyl-CoA Malonyl-CoA
0.04 pmol [31] ∼10 pmol [5]
Malonyl-CoA:acetyl-CoA cycling assay (Takamura et al. 1985) [35,36] RP-HPLC analysis of short-chain CoA species [4,6,8,15,19,28,38] RP-HPLC analysis of long-chain CoA species [23,25,26]
Malonyl-CoA and/or acetyl-CoA Multiple Multiple
∼1 pmol [36] 3–12 pmol [19,28,38] 12 pmol [23]
GC–MS analysis of CoA esters [46-48] LC–ESI–MS analysis of short-chain CoA [3,45] LC–ESI–MS analysis of medium-chain CoA [49]
Multiple Multiple Multiple
0.3–0.5 pmol [46,47] 0.2 pmol [3] ∼0.5 pmol [49]
LC–ESI–MS/MS analysis long-chain CoA [50] OxiRed based colorimetric/fluorimetric assay (Abcam, Abnova and BioVision)
Multiple CoASH
10 fmol [50] 100 pmol (2.5μM)†
Europium chloride based fluorimetric assay (Cayman Chemical) AmpliteTM fluorimetric CoA assay (AAT Bioquest®) Acetyl-CoA fluorimetric assay (Sigma–Aldrich)
CoASH CoASH Acetyl-CoA
250 pmol (5 μM)‡ 4 pmol (40 nM)† 20 pmol (0.4 μM)†
ELISA (MyBioSource)
Malonyl-CoA
18.3fmol (183 pM)†
HPLC MS-based
Other
Y. Tsuchiya, unpublished results. †Based on the information provided by the suppliers. ‡Based on the standard curve provided by the supplier.
CoA occurs in snap-frozen liver pieces stored at − 80◦ C for at least 3 months [4]. However, approximately 40– 50% of CoASH and acetyl-CoA degrades in snap-frozen liver at − 20◦ C after a week [4]. To prevent oxidation of CoASH, a reducing agent is usually included during or after sample extraction. DTT and DTE (dithioerythritol) are commonly used for this purpose. However, DTT can interfere with HPLC detection of CoA compounds (Y. Tsuchiya, unpublished work and [28]). For quantification of CoA compounds by HPLC, tris-(2-carboxyethyl)phosphine (TCEP) can be used as an alternative. Stability of some CoA species in PCA extracts is dependent on the pH [19]. Succinyl-CoA is particularly known to be labile. In PCA extracts adjusted to pH 6, approximately 80% of succinylCoA originally present degrades after 12 h at 8◦ C [19]. Degradation is much slower at pH 3 with only 20% degraded in the same time period [19]. CoASH and short-chain acylCoAs extracted from liver in 5% SSA and 50 μM DTE are stable for at least 12 h at 4◦ C and for 55 days if stored at − 80◦ C [3].
Methods for measuring CoA and CoA derivatives Table 1 shows examples of methods that are used for measuring CoASH and CoA thioesters. Currently, CoASH and CoA thioesters are most commonly measured using enzymatic, HPLC- and MS-based methods. These methods are discussed in more detail below. Other methods of detection include TLC, which is useful for detecting the C The
C 2014 Biochemical Society Authors Journal compilation
formation of particular CoA species in cells after incubating cells with radiolabelled precursors [29].
Enzymatic methods Owing to the relatively low abundance of CoA compounds in biological samples, spectrophotometric end-point assays that measure stoichiometric conversion of CoA or CoA derivatives into a product are generally not sensitive enough for most applications, especially when the amount of materials is limited. The sensitivity of end-point assays can be improved by incorporating fluorometric or radiochemical detection methods [14,29,30]. Several recycling assays have been described for measuring CoASH and CoA esters. These assays are based on the CoA- or CoA esterdependent amplification of a specific reaction product that can be measured spectrometrically or fluorometrically. The recycling assay by Allred and Guy, first described in 1969 [2], allows quantification of CoASH and acetyl-CoA levels and has been widely used. This assay utilizes citrate synthase, which catalyses the transfer of the acetyl group from acetyl-CoA to oxaloacetate, generating free CoA and citrate. This reaction is linked to two other enzymatic reactions: phosphotransacetylase and malate dehydrogenase reactions. The former converts the free CoA released from citrate synthase back into acetyl-CoA, allowing continuous recycling of CoA. The malate dehydrogenase reaction replaces the oxaloacetate consumed by the citrate synthase reaction with accompanying generation of NADH. The recycling of CoA by phosphotransacetylase therefore results in continuous conversion of oxaloacetate into citrate and
Coenzyme A and Its Derivatives in Cellular Metabolism and Disease
the accumulation of NADH, which can be measured spectrophotometrically or fluorometrically. The rate of NADH generation is dependent on the amount of CoASH and acetyl-CoA originally present in the sample. The amount of acetyl-CoA alone can be measured by pre-treating samples with NEM (N-ethylmaleimide) to block CoASH. It is recommended that internal standards be used for the quantification of CoA and acetyl-CoA since the activity of the enzymes can be affected by the chemical environment of samples. Using the 96-well plate format and an absorbance at 340 nm, we found the detection limit of this method to be approximately 20–40 pmol (0.1–0.2 μM). The assay is sufficiently sensitive for reliably measuring CoA and acetylCoA in tissue samples, but may not be suitable for cultured cells or if the amount of samples is limited. A more sensitive method was described by Kato in 1975 [31] and later modified by Szutowicz and Bielarczyk in 1987 [32]. This method is also based on the cyclic reactions involving citrate synthase and phosphotransacetylase. However, instead of including malate dehydrogenase and measuring NADH, the method involves measuring citrate which is accumulated after many cycles of CoA recycling. An excess of oxaloacetate and acetyl phosphate are included in the reaction mixture. As for the recycling assay by Allred and Guy [2], the method can measure combined levels of CoASH and acetyl-CoA. AcetylCoA can be measured separately by inactivating CoASH with NEM. However, owing to the slow hydrolysis of CoA– NEM thioether, small amounts of free CoASH are released during the amplification step (which lasts for 1 h) [31,32]. More accurate measurement of acetyl-CoA can be achieved by blocking CoASH with maleic anhydride, which forms a more stable thioether bond with CoASH [32]. The detection limit of this method was reported to be as low as 40 fmol [31] and the method has been used to measure acetyl-CoA levels in synaptosomes isolated from rat brains [32]. Malonyl-CoA is an allosteric inhibitor of carnitine palmitoyltransferase 1 and acts as an important signalling molecule regulating β-oxidation of long-chain fatty acids [33]. Its levels in tissues can be as low as 0.2–3 pmol/mg of wet mass [3] and its quantification therefore requires particularly sensitive assays. A widely used enzymatic assay for malonyl-CoA is that developed by McGarry et al. [34], which measures malonyl-CoA-dependent incorporation of radiolabelled acetyl-CoA into fatty acid by fatty acid synthase. A cycling assay for malonyl-CoA and acetylCoA was described by Takamura et al. in 1985 [35]. This method is based on the amplification of acetate by malonate decarboxylase in the presence of malonyl-CoA and acetyl-CoA. The amplified acetate is first converted into acetyl phosphate and then to acetohydroxamate, which can be measured spectrophotometrically at 540 nm. The assay measures combined levels of acetyl-CoA and malonylCoA. Selective measurement of malonyl-CoA is achieved by enzymatically eliminating acetyl-CoA using citrate synthase and oxaloacetate. The detection limit of this method is approximately 1 pmol and the method has been used to quantify malonyl-CoA in mouse hypothalami [36].
HPLC Since the initial report of separation of CoA compounds using HPLC by Baker and Schooley [37], HPLC, particularly RPHPLC (reverse-phase HPLC), has been used extensively for measuring CoA species. Owing to the presence of adenosine in their structures, CoA compounds can be detected by UV absorbance at 254–260 nm. Separation of CoA from the bulk of other highly abundant nucleotides, such as ATP, by RPHPLC is usually straightforward due to the presence of the relatively hydrophobic pantetheine tail in CoA compounds, and can be achieved by increasing the salt concentration in the mobile phase. HPLC allows simultaneous detection of multiple CoA species and have been used to quantify CoASH as well as short- and long-chain acyl-CoAs in extracts from animal tissues [4,15,19,23,25,26,38], human needle biopsies [6], cultured cells [8] and bacteria [12]. The detection limit of this technique is approximately 3–12 pmol [19,28,38] and typical analysis time for each sample is 20–60 min. Recent advances in ultra-HPLC and HPLC analytical columns with smaller particle size are expected to help shorten analysis time and improve the quality of separation. A major disadvantage of this technique, if absorbance alone is used to detect CoA compounds, is uncertainty regarding the identity and purity of detected peaks. However, there are several ways to validate the identity and purity of CoASH/CoA thioester peaks. Alkaline hydrolysis of CoA thioesters and consequent disappearance of peaks corresponding to CoA thioesters is commonly used to demonstrate the purity of CoA thioester peaks [6,25,38]. More conclusive characterization of peaks can be achieved by using enzymes that specifically catalyse the conversion of specific CoA species. For example, the identity and purity of the acetyl-CoA peak can be demonstrated by the complete disappearance of the acetyl-CoA peak after treatment of the sample with citrate synthase and oxaloacetate. Phosphotransacetylase and an excess of acetyl phosphate may be used to verify the CoASH peak. Sensitivity and specificity of detection can be further improved by derivatization or radiolabelling of CoASH and/or derivatives before HPLC analysis. CoASH can be derivatized by thiol-reactive labels, such as monobromobimane and SBDF (ammonium 7-flurobenzo-2-oxa-1,3-doazole-4-sulfonate), which can be detected by a fluorescent detector [39–41]. Subpicomole amounts of CoASH can be detected by the latter method. However, these approaches are only applicable for quantification of CoASH or total CoA levels (after alkaline hydrolysis) and not thioesters. CoASH and CoA thioesters can be detected fluorometrically by converting them into fluorescent 1,N6-ethenoadenine derivatives using bromoacetaldehyde or chloroacetaldehyde before or after HPLC separation [7,12]. Wadler and Cronan [42] described a method for specifically labelling CoA and derivatives using dephospho-CoA kinase. In their approach, the 3 phosphate groups of CoASH and acyl-CoAs are first dephosphorylated by a phosphatase, and then the 3 hydroxy group is phosphorylated again using dephosphoCoA kinase and radioactive ATP. The radiolabelled CoA species are detected by HPLC connected to a flow-through C The
C 2014 Biochemical Society Authors Journal compilation
1109
1110
Biochemical Society Transactions (2014) Volume 42, part 4
beta-counter. This method can significantly improve the specificity of detection however the efficiency of enzymatic labelling of CoA species may be affected by the chemical environment in each sample.
MS-based methods MS-based quantification of CoA species has been increasingly used in recent years [43–45]. GC–MS has been used for measuring acyl-CoA esters after their hydrolysis and derivatization [46–48]. More recently, MS or tandem MS (MS/MS) coupled with HPLC and electrospray ionization (LC–ESI–MS and LC–ESI–MS/MS) have been used to directly detect short- and long-chain acyl-CoAs in biological extracts [3,13,45,49,50]. Femtomole amounts of CoA species can be quantified by these methods. Since a mass spectrometer detects compounds on the basis of mass charge ratios, baseline separation of compounds by HPLC is usually not required. However, identification of CoA compounds may be complicated by the fact that some CoA derivatives have almost identical masses. For instance, Gao et al. [3] were unable to distinguish malonyl-CoA and β-hydroxybutyrylCoA by their mass charge ratios or by unique product ions after MS/MS. Separate detection of these compounds was achieved by their prior separation by HPLC using dimethylbutyl amine as an ion-pairing agent [3].
Commercially available kits A number of companies supply kits for assaying CoA compounds. Examples of these are shown in Table 1. On the basis of information provided by the suppliers, some of these kits, including those based on OxiRed, do not appear to be sensitive enough for detecting CoA in small quantities of biological materials. The possible exceptions are the Amplite fluorometric Coenzyme A assay kit by AAT Bioquest® (detection limit 4 pmol) and the acetyl-CoA assay kit (detection limit 20 pmol) by Sigma–Aldrich. There is also an ELISA-based malonyl-CoA assay kit that can supposedly measure femtomole amounts of malonyl-CoA. However, as far as we are aware, these assay kits have not yet been widely used.
Conclusions There are currently a number of different methods for measuring CoA compounds. Different methods may be used depending on the aims and requirements of individual studies. MS-based methods are highly sensitive and specific, but its routine use may be limited by the availability of sensitive mass spectrometers. If routine analysis of a specific CoA species is required, a sensitive enzymatic assay may be used as an alternative. Many enzymatic assays can also be performed in microplate formats, allowing high-throughput analysis of multiple samples. HPLC is a useful and reliable technique for simultaneously measuring multiple CoA species, provided the identity and purity of peaks are validated. CoA levels differ considerably in different cell types and in different subcellular compartments. Changes in total CoA C The
C 2014 Biochemical Society Authors Journal compilation
levels in tissues or cells therefore may not reflect changes in CoA levels in specific cell types or compartments. Cytosolic and mitochondrial CoA levels are usually measured after their separation by subcellular fractionation or by using digitonin to selectively permeabilize the cell membrane [15– 18]. However, potential problems with these methods, if not carefully controlled, include leakage or enzymatic conversion of CoA species during the fractionation step. Measurement of CoA species in subcellular fractions also requires highly sensitive assays. Development of molecular probes for CoA compounds that can be used to detect CoA species in different subcellular compartments or different cell types would be highly useful for studying the consequences of altered CoA levels on cellular processes.
Funding This work was supported by UCLB (University College London Business) Proof of Concept funding [grant numbers UCLB PoC-11018 and UCLB PoC-13-014].
References 1 Bieber, L.L. (1992) Quantitation of CoASH and acyl CoA. Anal. Biochem. 204, 228–230 CrossRef PubMed 2 Allred, J.B. and Guy, D.G. (1969) Determination of coenzyme A and acetyl CoA in tissue extracts. Anal. Biochem 29, 293–299 CrossRef PubMed 3 Gao, L., Chiou, W., Tang, H., Cheng, X., Camp, H.S. and Burns, D.J. (2007) Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS. J. Chromatogr. B 853, 303–313 CrossRef 4 Shibata, K., Nakai, T. and Fukuwatari, T. (2012) Simultaneous high-performance liquid chromatography determination of coenzyme A, dephospho-coenzyme A, and acetyl-coenzyme A in normal and pantothenic acid-deficient rats. Anal. Biochem. 430, 151–155 CrossRef PubMed 5 Alam, N. and Saggerson, E.D. (1998) Malonyl-CoA and the regulation of fatty acid oxidation in soleus muscle. Biochem. J. 334, 233–241 PubMed 6 Corkey, B.E., Hale, D.E., Glennon, M.C., Kelley, R.I., Coates, P.M., Kilpatrick, L. and Stanley, C.A. (1988) Relationship between unusual hepatic acyl coenzyme A profiles and the pathogenesis of Reye syndrome. J. Clin. Invest. 82, 782–788 CrossRef PubMed 7 Larson, T.R. and Graham, I.A. (2001) Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues. Plant J. 25, 115–125 CrossRef PubMed 8 Corkey, B.E., Glennon, M.C., Chen, K.S., Deeney, J.T., Matschinsky, F.M. and Prentki, M. (1989) A role of malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic β-cells. J. Biol. Chem. 264, 21608–21612 PubMed 9 MacDonald, M.J., Smith, III, A.D., Hasan, N.M., Sabat, G. and Fahien, L.A. (2007) Feasibility of pathways for transfer of acyl groups from mitochondria to the cytosol to form short chain acy-CoAs in the pancreatic beta cell. J. Biol. Chem. 282, 30596–30606 CrossRef PubMed 10 Basu, S.S., Mesaros, C., Gelhaus, S.L. and Blair, I.A. (2011) Stable isotope labelling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters. Anal. Chem. 83, 1363–1369 CrossRef PubMed 11 Palekar, A (2000) Effect of pantothenic acid on hippurate formation in sodium benzoate-treated HepG2 cells. Pediatr. Res. 48, 357–359 CrossRef PubMed 12 Shimazu, M., Vetcher, L., Galazzo, J.L., Licari, P. and Santi, D.V. (2004) A sensitive and robust method for quantification of short-chain coenzyme A esters. Anal. Biochem. 328, 51–59 CrossRef PubMed 13 Seifer, R.M., Ras, C., Deshmukh, A.T., Bekers, K.M., Suarez-Mendez, C.A., de Cruz, A.L.B., van Gulik, W.M. and Heijnen, J.J. (2013) Quantitative analysis of intracellular coenzymes in Saccharomyces cerevisiae using ion pair reversed phase ultra high performance liquid chromatography tandem mass spectrometry. J. Chromatogr. A 1311, 115–120 CrossRef PubMed
Coenzyme A and Its Derivatives in Cellular Metabolism and Disease
14 Garland, P.B., Shepherd, D. and Yates, D.W. (1965) Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate. Biochem. J. 97, 587–594 PubMed 15 Lysiak, W., Lilly, K., DiLisa, F., Toth, P. and Bieber, L.L. (1988) Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria. J. Biol. Chem. 236, 1151–1156 PubMed 16 Idell-Wenger, J.A., Grotyohann, L.W. and Neely, J.R. (1978) Coenzyme A and carnitine distribution in normal and ischemic hearts. J. Biol. Chem. 253, 4310–4318 PubMed 17 Brass, E.P. and Ruff, L.J. (1992) Rat hepatic coenzyme A is redistributed in response to mitochondrial acyl-coenzyme A accumulation. J. Nutr. 122, 2094–2100 PubMed 18 Siess, E.A., Brocks, D.G., Lattke, H.K. and Wieland, O.H. (1977) Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate. Biochem. J. 166, 225–235 PubMed 19 King, M.T. and Reiss, P.D. (1985) Separation and measurement of short-chain coenzyme-A compounds in rat liver by reversed-phase-high-performance liquid chromatography. Anal. Biochem. 146, 173–179 CrossRef PubMed 20 Zammit, V.A. (1981) Regulation of hepatic fatty acid metabolism. Biochem. J. 198, 75–83 PubMed 21 Lopaschuk, G.D., Hansen, C.A. and Neely, J.R. (1986) Fatty acid metabolism in hearts containing elevated levels of CoA. Am. J. Physiol. 250, H351–H359 PubMed 22 Prasad, R.M., Sauter, J. and Lands, W.E.M. (1987) Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters. Anal. Biochem. 162, 202–212 CrossRef PubMed 23 Mangino, M.J., Zografakis, J., Murphy, M.K. and Anderson, C.B. (1992) Improved and simplified tissue extraction method for quantitating long-chain acyl-coenzyme A thioesters with picomolar detection using high-performance liquid chromatography. J. Chromatogr. 577, 157–162 CrossRef PubMed 24 Mancha, M., Stokes, G.B. and Stumpf, P.K. (1975) Fat metabolism in higher plants. The determination of acyl-acyl carrier protein and acyl coenzyme A in a complex lipid mixture. Anal. Biochem. 68, 600–608 CrossRef PubMed 25 Woldegiorgis, G., Spennetta, T., Corkey, B.E, Williamson, J.R. and Shrago, E. (1985) Extraction of tissue long-chain acyl-CoA esters and measurement by reverse-phase high-performance liquid chromatography. Anal. Biochem. 150, 8–12 CrossRef PubMed 26 Golovko, M.Y. and Murphy, E.J. (2004) An improved method for tissue long-chain acyl-CoA extraction and analysis. J. Lipid. Res. 45, 1777–1782 CrossRef PubMed 27 Hwang, C., Sinskey, A.J. and Lodish, H.F. (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257, 1496–1502 CrossRef PubMed 28 Corkey, B.E., Brand, M., Williams, R.J. and Williamson, J.R. (1981) Assay of short-chain acyl coenzyme A intermediates in tissue extracts by high-pressure liquid chromatography. Anal. Biochem. 118, 30–41 CrossRef PubMed 29 Zhang, Y., Chohnan, S., Virga, K.G., Stevens, R.D., Llkayeva, O.R., Wenner, B.R., Bain, J.R., Newgard, C.B., Lee, R.E., Rock, C.O. and Jackowski, S. (2007) Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis. Chem. Biol. 14, 291–302 CrossRef PubMed 30 Rabier, D., Briand, P., Petit, F., Kamoun, P. and Cathelineau, L. (1983) Radioisotopic assay of picomolar amounts of coenzyme A. Anal. Biochem. 134, 325–329 CrossRef PubMed 31 Kato, T. (1975) CoA cycling: an enzymatic amplification method for determination of CoASH and acetyl CoA. Anal. Biochem. 66, 373–392 32 Szutowicz, A. and Bielarczyk, H. (1987) Elimination of CoASH from acetyl-CoA cycling assay by maleic anhydride. Anal. Biochem. 164, 292–296 CrossRef PubMed 33 Saggerson, D. (2008) Malonyl-CoA, a key signaling molecule in mammalian cells. Annu. Rev. Nutr. 28, 253–272 CrossRef PubMed
34 McGarry, J.D., Stark, M.J. and Foster, D.W. (1978) Hepatic malonyl-CoA levels of fed, fasted and diabetic rats as measured using a simple radioisotopic assay. J. Biol. Chem. 253, 8291–8293 PubMed 35 Takamura, Y., Kitayama, Y., Arakawa, A., Yamanaka, S., Tosaki, M. and Ogawa, Y. (1985) Malonyl-CoA: acetyl-CoA cycling. A new micromethod for determination of acyl-CoAs with malonate decarboxylase. Biochim. Biophys. Acta 834, 1–7 CrossRef PubMed 36 Hu, Z., Cha, S.H., Chohnan, S. and Lane, M.D. (2003) Hypothalamic malonyl-CoA as a mediator of feeding behaviour. Proc. Natl. Acad. Sci. U.S.A. 100, 12624–12629 CrossRef PubMed 37 Baker, F.C. and Schooley, D.A. (1979) Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography. Anal. Biochem. 94, 417–424 CrossRef PubMed 38 Demoz, A., Garras, A., Asiedu, D.K., Netteland, B. and Berge, R.K. (1995) Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography. J. Chromatogr. B 667, 148–152 CrossRef 39 Newton, G.L., Dorian, R. and Fahey, R.C. (1981) Analysis of biological thiols: derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography. Anal. Biochem. 114, 383–387 CrossRef PubMed 40 Siudeja, K., Srinivasan, B., Xu, L., Rana, A., de Jong, J., Nollen, E.A., Jackowski, S., Sanford, L., Hayflick, S. and Sibon, O.C. (2011) Impaired coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration. EMBO Mol. Med. 3, 755–766 CrossRef PubMed 41 Imai, K., Toyo’oka, T. and Watanabe, Y. (1983) A novel fluorogenic reagent for thiols: ammonium 7-fluorobenzo-2-oxa-1, 3-diazole-4-sulfonate. Anal. Biochem. 128, 471–473 CrossRef PubMed 42 Wadler, C. and Cronan, J.E. (2007) Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters. Anal. Biochem. 368, 17–23 CrossRef PubMed 43 McCoy, F., Darbandi, R., Lee, H.C., Bharatham, K., Moldoveanu, T., Grace, C.R., Dodd, K., Lin, W., Chen, S.I., Tangallapally, R.P. et al. (2013) Metabolic activation of CAMKII by coenzyme A. Mol. Cell 52, 325–339 CrossRef PubMed 44 Marino, ˜ G., Pietrocola, F., Eisenberg, T., Kong, Y., Malik, S.A., Andryushkova, A., Schroeder, S., Pendl, T., Harger, A., Niso-Santano, M. et al. (2014) Regulation of autophagy by cytosolic acetyl-coenzyme A. Mol. Cell 53, 710–725 CrossRef PubMed 45 MacDonald, M.J. (2007) Synergistic potent insulin release by combinations of weak secretagogues in pancreatic islets and INS-1 cells. J. Biol. Chem. 282, 6043–6052 CrossRef PubMed 46 Tamvakopoulos, C.S. and Anderson, V.E. (1992) Detection of acyl-coenzyme A thioester intermediates of fatty acid β-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography-mass spectrometry. Anal. Biochem. 200, 381–387 CrossRef PubMed 47 Kopka, J., Ohlrogge, J.B. and Jaworski, J.G. (1995) Analysis of in vivo levels of acyl-thioesters with gas chromatography/mass spectrometry of the butylamide derivative. Anal. Biochem. 224, 51–60 CrossRef PubMed 48 Kasumov, T., Martini, W.Z., Reszko, A.E., Bian, F., Pierce, B.A., David, F., Roe, C.R. and Brunengraber, H. (2002) Assay of the concentration and 13C isotopic enrichment of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA by gas chromatography-mass Spectrometry. Anal. Biochem. 305, 90–96 CrossRef PubMed 49 Kasuya, F., Oti, Y., Tatsuki, T. and Igarashi, K. (2004) Analysis of medium-chain acyl-coenzyme A esters in mouse tissues by liquid chromatography-electrospray ionization mass spectrometry. Anal. Biochem. 325, 196–205 CrossRef PubMed 50 Haynes, C.A., Allegood, J.C., Sims, K., Wang, E.W., Sullards, M.C. and Merrill, Jr, A.H. (2008) Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry. J. Lipid Res. 49, 1113–1125 CrossRef PubMed
Received 30 April 2014 doi:10.1042/BST20140123
C The
C 2014 Biochemical Society Authors Journal compilation
1111
