Coenzyme A and Coenzyme A‐Glutathione Mixed Disulfide Measurements by HPLC

Lynette K. Rogers1, Charles V. Smith1

1 The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.9
DOI:  10.1002/0471140856.tx0609s15
Online Posting Date:  May, 2003
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This unit describes a method for measuring coenzyme A and coenzyme A‐glutathione mixed disulfide in tissue homogenates obtained from tissues frozen in liquid nitrogen, homogenized in the presence of N‐ethylmaleimide, and then acidified. to avoid the oxidation, reduction, and thiol exchange reactions that can occur during tissue processing and isolation of subcellular fractions. This method may provide a way to assess changes in the oxidative state of mitochondria in intact tissues.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1:

  • Test animal
  • Liquid N 2
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A), 4°C
  • 1 M NEM (see recipe), 4°C
  • 4% perchloric acid (HClO 4): dilute 5.1 ml concentrated (68% to 70%) HClO 4 to 100 ml with H 2O; prechill to 4°C at time of use
  • Mobile phases A, B, and C for either tertiary/quaternary or binary HPLC solvent program (see recipe)
  • CoAS‐NEM and CoASSG standards (see reciperecipes)
  • Freeze clamps: 2 × 2 × 0.5–in. aluminum blocks and stainless steel towel clamps altered to hold them (see recipe)
  • Mortar and pestle designed for grinding under liquid nitrogen (Fisher Scientific)
  • 2‐ml dounce homogenizer with loose and tight pestles (Wheaton; VWR)
  • Refrigerated centrifuge
  • HPLC system with UV detector (capable of measurement at 254 nm)
  • Precolumn filter (0.062 × 0.250‐µm, 4 µm pore size; Western Analytical Inc.)
  • C 18 guard column (4.6 × 125 mm, Zorbax SB; Mac‐Mod Analytical, Inc.)
  • C 18 analytical HPLC column (4.6 × 150 mm, Zorbax SB; Mac‐Mod Analytical, Inc.)
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Literature Cited

Literature Cited
   Ansensi, M., Sastre, J., Pallardo, F.V., de la Asuncion, J.G., Estrela, J.M., and Vina, J. 1994. A high‐performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Anal. Biochem. 217:323‐328.
   Baker, F.C. and Schooley, D.A. 1981. Separation of S‐acyl‐CoA thioesters and related compounds by reversed‐phase ion‐pair chromatography. Methods Enzymol. 72:41‐52.
  Gilbert, H.F. 1982. Biological disulfides: The third messenger? J. Biol. Chem. 257:12086‐12091.
   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.
   Reed, D.J. and Savage, M.K. 1995. Influence of metabolic inhibitors on mitochondrial permeability transition and glutathione status. Biochim. Biophys. Acta. 1271:43‐50.
   Robishaw, J.D. and Neely, J.R. 1985. Coenzyme A metabolism. Am. J. Physiol. 248:E1‐E9.
   Rogers, L.K., Valentine, C.J., Szczpyka, M., and Smith, C.V. 2000. Effects of hepatotoxic doses of acetaminophen and furosemide on tissue concentrations of CoASH and CoASSG in vivo. Chem. Res. Tox. 13:873‐882.
   Sacchetta, P., Di Cola, D., and Federici, G. 1986. Alkaline hydrolysis of N‐ethylmaleimide allows a rapid assay of glutathione disulfide in biological samples. Anal. Biochem. 154:205‐208.
   Schluter, H., Meissner, M., van der Giet, M., Tepel, M., Bachmann, J., Grob, I., Nordhoff, E., Karas, M., Spieker, C., Witzel, H., and Zidek, W. 1995. Coenzyme A glutathione disulfide, a potent vasoconstrictor derived from the adrenal gland. Circ. Res. 76:675‐680.
   Tahiliani, A. G. and Neely, J. R. 1987. A transport system for coenzyme A in isolated rat heart mitochondria. J. Biol. Chem. 262:11607‐11610.
   van der Geit, M., Schmidt, A., Jankowski, J., Schluter, H., Zidek, W., and Tepel, M. 2001. Coenzyme A glutathione disulfide is a potent modulator of angiotensin II‐induced vasoconstriction. Am. J. Hyper. 14:164‐168.
   Wong, Y.L., Smith, C.V., McMicken, H.W., Rogers, L.K., and Welty, S.E. 2001. Mitochondrial thiol status in the liver is altered by exposure to hyperoxia. Toxicol. Lett. 123:179‐93.
Key References
   Rogers et al., 2000. See .
  Describes the method for sample preparation and HPLC analysis in this protocol and applies the protocol to a biological situation.
   Baker and Schooley, 1981. See .
  Describes the original HPLC separation of both short and long chain acyl‐CoA moieties and the ion‐pairing chromatography adapted for this protocol.
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