Quantification of Coenzyme A in Cells and Tissues

Summary

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.

Abstract

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.

Introduction

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 regulated^3,4; 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 functions^13,14,15. Preclinical development of potential therapies for PKAN or CoPAN includes cell or tissue CoA measurements as indicators of efficacy^{16,17,18,19,20}. 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 detection^23,24,25. 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.

Protocol

The animal procedure referred to in this protocol was performed according to protocols 323 and 556 and specifically approved by the St. Jude Children'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.

1. Prepare 1 mM potassium hydroxide (KOH) in water.
2. Prepare 0.25 M KOH in water.
3. Prepare 1 M Trizma-HCl in water and adjust to pH 8.0.
4. Prepare 100 mM monobromobimane (mBBr) in acetonitrile (Optima Grade). Stock solutions of mBBr are stable at -20 °C for many months provided they are kept in the dark as exposure to light can photolyze the monobromobimane to bimane²⁶.
5. Prepare Wash Buffer for solid phase extraction (SPE) column: 50% methanol (Optima Grade) + 2% acetic acid.
6. Prepare Elution Buffer for SPE column: 95% ethanol (HPLC Grade) containing 50 mM ammonium formate.

2. Preparation of CoA-bimane standard

1. Purchase a high-quality CoA standard from a reputable source (e.g., Avanti Polar Lipids, Inc.).
2. Prepare a high concentration stock solution of CoA in 20 mM Tris, pH 8. The amount of CoA in the high concentration stock is determined or confirmed by spectrophotometric absorbance at λ260 nm (Ɛ = 16,800 M^-1∙cm^-1). Add 4-fold molar excess of mBBr; vortex on high for 10 s. For example, for 10 mM CoA in buffer, add 40 mM mBBr. The mBBr is added in excess to ensure that all the CoA is derivatized.
3. Incubate at room temperature for 4 h in the dark without shaking to derivatize the CoA with mBBr. mBBr reacts with the thiol group of the CoA, and it is pH and temperature dependent²⁷. Addition of the mBBr converts CoA to a water-soluble fluorescent (or ultraviolet absorbant) CoA-bimane (CoA-bimane; Figure 2).
4. Add 100 μL acetic acid and vortex on high for 10 s to stop the reaction.
5. Centrifuge at 2,000 x g for 15 minutes to remove any precipitant.
6. Clean-up the supernatant using an SPE-column to remove the unreacted mBBr (described below). Filtration and centrifugation of the eluate is not necessary after the SPE column clean-up (Section 5.8).
7. Collect the eluate from the SPE column containing the CoA-bimane in a pre-weighed tube, dry under nitrogen gas, weigh and construct a standard calibration curve with known quantities of CoA-bimane.
   1. Check the CoA-bimane standard for purity by HPLC (see below) with absorbance detection at both λ260 (adenine moiety) and λ393 (bimane moiety) and coincidence of both peaks in the chromatogram.
   2. Confirm the quantity of the CoA-bimane standard in the high concentration stock using λ260 nm (Ɛ = 16,800 M^-1∙cm^-1).
8. Register the HPLC retention time for identification of CoA-bimane in experimental samples using the CoA-bimane standard.
9. Store aliquots of the high concentration stock of the CoA-bimane standard protected from light and stored at -20 °C for up to 2 years. Avoid repeated thawing and re-freezing.

3. Extraction and derivatization of CoA in cultured cells

1. Harvest adherent cells in culture when approaching late subconfluent densities. For example, human HepG2/C3A cells are grown to a density of 6-8 x 10⁶, or HEK 293T cells are grown to a density of ~1.3 x 10⁷ per 100 mm dish.
   1. Use duplicate or triplicate cultures routinely for CoA determinations. Incubate separate culture dishes in parallel with the sample cell cultures to determine viable cell number. Calculate the amount of CoA per 10⁶-10⁷ cells after HPLC purification.
2. Aspirate culture medium off the dish. Quickly wash cells on the dish with ice-cold phosphate-buffered saline (PBS) to remove any residual medium and aspirate the PBS off the dish. Quickly wash cells with ice-cold water to remove residual PBS and aspirate the water off the dish quickly. Avoid disturbing the adherent cells when adding the PBS or water.
3. Add 1 mL of ice-cold water to the culture dish. Scrape cells into the cold water in the dish and transfer the cell suspension to a glass test tube containing 400 μL of 0.25 M KOH and 1.5 mL of water.
   1. Mix the suspension vigorously by vortex on high for 10 s. Then cover tightly with paraffin film and incubate at 55 °C for 1 h without shaking in a water bath. The pH of the sample should be ≥ 12. The high pH is important to hydrolyze the CoA thioesters and can be adjusted with additional aliquots of KOH if necessary.
4. Harvest cultured cells growing in suspension by centrifugation at low speed: 136 x g for 6 min at 4 °C. Wash once with ice-cold PBS, then briefly wash again with cold water, and finally resuspend in 2.5 mL of cold water plus 400 µL 0.25 M KOH. Mix vigorously and incubate at 55 °C as described above.
5. Add 160 μL of 1 M Trizma-HCl and 10 μL of 100 mM mBBr and mix by vortex on high for 10 s. This brings the pH to approximately 8 to support the mBBr reaction with free CoA. Cover and incubate samples at room temperature for 2 h in the dark for the mBBr to react with the thiol of CoA.
6. Add 100 μL of acetic acid and mix by vortex on high for 10 s to stop the reaction
7. Centrifuge at 2,000 x g for 15 min to remove precipitated cell debris.
8. Remove and save supernatant in a glass test tube for the SPE column clean-up that is described below.

4. Extraction and derivatization of CoA in tissues

1. Dissect animal tissue pieces, about 0.5 cm diameter or smaller, and blot briefly (1-2 s) on absorbent paper and then flash freeze in liquid nitrogen and store at -80 °C until processing. CoA in tissues is hydrolyzed or converted to a thioester if not flash frozen. This step is important to obtain the maximum yield of CoA in tissues.
2. Weigh frozen tissue pieces (5 – 60 mg) quickly prior to starting the analysis, and record the weight for each sample. This wet weight determination is used for calculation of the CoA content following HPLC purification. Avoid complete thaw of the tissue pieces.
3. Add 2 mL of 1 mM KOH to a glass test tube (e.g., 13 mm x 100 mm disposable) destined for insertion of a probe and tissue disruption with a rotor-stator homogenizer. Keep the tube on ice until use. This is done to ensure that the samples are homogenized at a cold temperature and CoA is not destroyed due to the heat generated during homogenization.
4. Transfer and homogenize tissue (30 – 40 mg) in the glass test tube (containing cold 1 mM KOH) for 30 s. Avoid complete thawing of the tissue.
5. Add 500 μL of 0.25 M KOH; vortex on high for 10 s and keep on ice until all samples are homogenized. This will bring the pH of the sample above 12 to hydrolyze the CoA thioesters to yield total CoA (free CoA + hydrolyzed CoA thioesters).
6. Incubate samples at 55 °C for 2 h in a water bath without shaking to support hydrolysis.
7. Add 150 μL of 1 M Trizma-HCl and 10 μL of 100 mM mBBr; vortex on high for 10 seconds. This brings the pH to about 8 to support the mBBr reaction with free CoA.
8. Incubate samples at room temperature for 2 h in the dark to ensure all the CoA is derivatized with mBBr.
9. Add 100 μL of acetic acid and vortex on high for 10 s to stop the reaction.
10. Centrifuge at 2,000 x g for 15 min to pellet precipitated cell debris.
11. Remove and save supernatant in a glass test tube for the SPE column clean-up (described below).

5. Sample clean-up with solid phase extraction (SPE) column

1. At room temperature, equilibrate each disposable 2-(2-pyridyl)-ethyl silica gel column (1 mL size) with 1 mL of Wash Buffer to ensure that the pyridyl functional group is protonated and will function as an anion-exchanger. 2-(2-pyridyl)-ethyl silica gel is a weak anion exchanger which is ideal for CoA-bimane at a basic pH. 2-(2-pyridyl)-ethyl silica gel has a pKa of 6 and the elution is done pH ≥ 7.
2. Add sample supernatant (step 4.11) to column and collect eluate.
3. Wash column twice with 1 mL of Wash Buffer to remove any unretained species.
4. Wash column once with 1 mL of water.
5. Wash column twice with 1 mL of Elution Buffer into a separate glass test tube (12 mm x 75 mm) to collect the CoA-bimane.
6. Dry CoA-bimane sample in tube under nitrogen gas to dryness. The dried sample is stable at room temperature until further use. Seal and completely cover the tube and store.
7. When ready for HPLC analysis, resuspend sample in 300 μL of water and mix vigorously by vortex on high for 10 s.
8. Transfer resuspended sample to a centrifuge tube filter (0.22 µm cellulose acetate, 2 mL size) and centrifuge at 5,000 x g for 10 min to remove any precipitant.
9. Transfer filtered sample to a glass vial suitable for HPLC injection.

6. HPLC purification and measurement of CoA-bimane

1. Prepare buffers. Buffer A: 50 mM KH₂PO₄, pH 4.6; Buffer B: Acetonitrile (Optima Grade).
2. Power up the HPLC system.
   NOTE: The HPLC system in our laboratory is a Waters e2695 Separations Module equipped with a 2489 Ultraviolet-Visible (UV-Vis) absorbance detector and a 2475 fluorescence detector controlled with Empower 3 software. The system also has an automated sample injector.
3. Inject each sample (e.g., 20 μL) for separation on a Gemini C18, 3 μm 100 Å, 4.6 mm x 150 mm column using the program in Table 1 with a flow rate of 0.5 mL/min. The column temperature is 25 °C. Measure the UV/Visible detector absorbance at λ393 nm, and fluorescent detection at λex = 393 nm, λem = 470 nm. The retention time is determined by running a CoA-bimane standard curve before each set of samples.
4. Record the area under the CoA-bimane peak for each sample and compare with the standard curve to calculate pmol of CoA-bimane injected onto the HPLC column. The CoA-bimane absorbance standard curve is used for tissue samples, and the CoA-bimane fluorescence standard curve is used for tissue culture samples.
5. As the CoA-bimane values represent total CoA for each sample, normalize to the number of viable cells for culture samples, or mg wet weight for tissue samples (Figure 6). Alternately, calculate the total CoA values following normalization to DNA or protein content for each sample. DNA or protein content can be determined separately using sample aliquots that are removed during sample preparation prior to KOH addition.

Results

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<...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-(2-pyridyl)-ethyl silica gel SPE column	Millipore-Sigma	54127-U
coenzyme A	Avanti Polar Lipids	870700
Gemini C18 3 μm 100 Å HPLC column	Phenomenex	00F-4439-E0
monobromobimane	ThermoFisher Scientific	M-1378
Omni-Tip probe tissue disrupter	Omni International	32750H
Parafilm	Fisher	S37440
PowerGen 125 motorized rotor stator homogenizer	ThermoFisher Scientific	NC0530997
Spin-X centrifuge tube filter	CoStar	8161
Trizma-HCl	Fisher	T395-1
Waters 2475 fluorescence detector	Waters	2475
Waters 2489 UV-Vis detector	Waters	2489
Waters e2695 separations module	Waters	e2695