Method to assess fatty acid ethyl ester binding to albumin.
OBJECTIVE
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.
Best
Catherine A
CA
Department of Biomedical Sciences, Northeastern University, Boston, MA 02115, USA. cbest1@partners.org
Laposata
Michael
M
Proios
Veronia G
VG
Szczepiorkowski
Zbigniew M
ZM
D000418:Albumins / Q000378:metabolism*; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism*; D005230:Fatty Acids, Nonesterified / Q000097:blood; D006801:Humans; D007553:Isotope Labeling; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000138:chemical synthesis; D012394:Rosaniline Dyes; D012588:Scintillation Counting
D000418:Albumins; D005227:Fatty Acids; D005230:Fatty Acids, Nonesterified; D019275:Radiopharmaceuticals; D012394:Rosaniline Dyes; C048139:Coomassie blue