Determination of Coenzyme A and Acetyl-Coenzyme A in Biological Samples Using HPLC with UV Detection

Abstract

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) play essential roles in cell energy metabolism. Dysregulation of the biosynthesis and functioning of both compounds may contribute to various pathological conditions. We describe here a simple and sensitive HPLC-UV based method for simultaneous determination of CoA and acetyl-CoA in a variety of biological samples, including cells in culture, mouse cortex, and rat plasma, liver, kidney, and brain tissues. The limits of detection for CoA and acetyl-CoA are >10-fold lower than those obtained by previously described HPLC procedures, with coefficients of variation <1% for standard solutions, and 1-3% for deproteinized biological samples. Recovery is 95-97% for liver extracts spiked with Co-A and acetyl-CoA. Many factors may influence the tissue concentrations of CoA and acetyl-CoA (e.g., age, fed, or fasted state). Nevertheless, the values obtained by the present HPLC method for the concentration of CoA and acetyl-CoA in selected rodent tissues are in reasonable agreement with literature values. The concentrations of CoA and acetyl-CoA were found to be very low in rat plasma, but easily measurable by the present HPLC method. The method should be useful for studying cellular energy metabolism under normal and pathological conditions, and during targeted drug therapy treatment.

Keywords: Acetyl-coenzyme A; UV detection; coenzyme A; high performance liquid chromatography.

Figure 1

Representative chromatograms for (A) Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) standards 1.25–10 µM (the retention times (RTs) for CoA and acetyl-CoA are 3.8 min and 7.8 min, respectively); and calibration curves for (B) CoA and (C) acetyl-CoA standards measured over the physiologically relevant concentration range of 0–5 µM. AU, arbitrary units.

Figure 2

Confirmation of the identity of CoA and acetyl-CoA in the peaks eluting at 3.8 and 7.8 min, respectively, for neutralized deproteinized rat liver extracts. (A) Use of citrate synthase (CS) to confirm that the peak at 3.8 min is due to CoA. Brown trace: initial sample prior to treatment with CS and oxaloacetate; Orange trace: sample treated with CS and oxaloacetate for 5 min at 37 °C; Yellow trace: sample spiked with 10 µM CoA, 10 µM acetyl-CoA and oxaloacetate at zero time; Green trace: sample spiked with 10 µM CoA, 10 µM acetyl-CoA and oxaloacetate after incubation with CS for 5 min at 37 °C. Note that the magnitude of the CoA peak in the initial sample at the magnification shown is relatively small. The insert depicts an expanded scale shown for better resolution; (B) Use of phosphotransacetylase (PTA) to confirm that the peak at 7.8 min is due to acetyl-CoA. Brown trace: initial sample; Orange trace: sample after 5 min treatment at 37 °C with PTA and acetyl phosphate; Yellow trace: sample spiked with 10 µM CoA, 10 µM acetyl-CoA, PTA and acetyl phosphate at zero time; Green trace: spiked sample after incubation with PTA and acetyl phosphate for 5 min at 37 °C.

Figure 2

Confirmation of the identity of CoA and acetyl-CoA in the peaks eluting at 3.8 and 7.8 min, respectively, for neutralized deproteinized rat liver extracts. (A) Use of citrate synthase (CS) to confirm that the peak at 3.8 min is due to CoA. Brown trace: initial sample prior to treatment with CS and oxaloacetate; Orange trace: sample treated with CS and oxaloacetate for 5 min at 37 °C; Yellow trace: sample spiked with 10 µM CoA, 10 µM acetyl-CoA and oxaloacetate at zero time; Green trace: sample spiked with 10 µM CoA, 10 µM acetyl-CoA and oxaloacetate after incubation with CS for 5 min at 37 °C. Note that the magnitude of the CoA peak in the initial sample at the magnification shown is relatively small. The insert depicts an expanded scale shown for better resolution; (B) Use of phosphotransacetylase (PTA) to confirm that the peak at 7.8 min is due to acetyl-CoA. Brown trace: initial sample; Orange trace: sample after 5 min treatment at 37 °C with PTA and acetyl phosphate; Yellow trace: sample spiked with 10 µM CoA, 10 µM acetyl-CoA, PTA and acetyl phosphate at zero time; Green trace: spiked sample after incubation with PTA and acetyl phosphate for 5 min at 37 °C.