Alcohol and Alcoholism Advance Access originally published online on March 22, 2006
Alcohol and Alcoholism 2006 41(3):240-246; doi:10.1093/alcalc/agl009
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METHOD TO ASSESS FATTY ACID ETHYL ESTER BINDING TO ALBUMIN
1 Department of Biomedical Sciences, Northeastern University, Boston, MA 02115, USA, 2 Division of Laboratory Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA, 3 Department of Pathology, Dartmouth-Hitchcock Medical Center and Dartmouth Medical School, Lebanon, NH 03756, USA
* Author to whom correspondence should be addressed at: Catherine A. Best, PhD, Room 235 Gray Building, 55 Fruit Street, Massachusetts General Hospital, Boston, MA 02114, USA. Tel.: +1 617 726 8174; Fax: +1 617 726 3256; E-mail: cbest1{at}partners.org
(Received 31 May 2005; first review notified 11 August 2005; in revised form 21 January 2006; accepted 23 January 2006)
Aims: To develop a method to assess the relative binding of fatty acid ethyl esters (FAEE) and free fatty acids (FFA) to albumin, and to determine if binding affinity is related to fatty acid chain length and or degree of saturation. Methods: Radiolabelled ethyl-[14C] oleate -bound to albumin was challenged with various ratios of FFA to FAEE. The displacement of ethyl-[14C] oleate by FFA was visualized and quantitated through a combination of native-PAGE, autoradiography, and liquid scintillation counting (LSC). Results: As the ratio of FFA to FAEE increased from 0:1 to 12:1, for all fatty acids tested (myristate, palmitate, stearate, oleate, linoleate, and arachidonate), ethyl-[14C] oleate displacement increased, when expressed as radioactivity (in DPM) as a percentage of control. In contrast, ethyl oleate did not displace stearate or oleate from albumin at molar ratios up to 5:1 (FAEE:FFA). Conclusions: The method developed gave reproducible visualization of noncovalent binding of radiolabelled FAEE to albumin. The combination of native-PAGE and autoradiography LSC works well in assessing the binding properties of albumin and radiolabelled FAEE. The data indicate preferential binding of FFA over FAEE to albumin with six different FFA displacing FAEE to an approximately equal extent.