J. Lipid Res.
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A more recent version of this article appeared on September 1, 2004

Papers In Press, published online ahead of print June 21, 2004
J. Lipid Res., doi:10.1194/jlr.D400004-JLR200
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Submitted on April 19, 2004
Revised on June 2, 2004
Accepted on June 3, 2004

An improved method for tissue long chain acyl-CoA extraction and analysis

Mikhail Y. Golovko and Eric J. Murphy

Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, ND 58202-9037

Corresponding Author: emurphy{at}medicine.nodak.edu

We report an extensively modified method for the extraction, solid phase purification, and HPLC analysis of long-chain acyl-CoA from tissues. Tissue samples were homogenized in glass homogenizer in KH2PO4 buffer (100 mM, pH 4.9) and again after addition of 2-propanol. Acyl-CoA were then extracted from the homogenate with acetonitrile. The acyl-CoA in the extract were bound to an oligonucleotide purification column and eluted using 2-propanol. This eluent was concentrated and then loaded onto a C-18 column and eluted using a binary gradient system where solvent A was KH2PO4 (75 mM, pH 4.9) and solvent B was acetonitrile containing 600 mM glacial acetic acid. Initial flow rate was 0.5 ml/min or 0.25 ml/min depending upon the tissue used. The HPLC eluent was monitoring at 260 nm. Our modifications increased the recovery of extraction procedure to 70-80%, depending upon tissue, with high reproducibility and significantly improved separation of the most common unsaturated and saturated acyl-CoA. We also report, for the first time, the mass (nmole/g ww) of the most common polyunsaturated acyl-CoA in rat heart, kidney, and muscle tissues. The modifications and high recovery permit use of tissue samples less than 100 mg, making this method useful for analysis of small tissue amounts associated with mice.


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