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 metabolism^1....

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 ....

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