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.
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.
