LOW-DENSITY-LIPOPROTEIN RECONSTITUTED WITH FATTY-ACID ETHYL-ESTERS ASA PHYSIOLOGICAL VEHICLE FOR ETHYL-ESTER DELIVERY TO INTACT-CELLS

Fatty acid ethyl esters (FAEEs), esterification products of ethanol an d fatty acids, have been found selectively in the organs damaged by et hanol abuse, and on that basis have been implicated as contributors to ethanol-induced organ damage. To directly assess the cytotoxic potent ial of FAEEs with intact cells in a physiological system, solubility m ust be achieved for these highly nonpolar lipids in aqueous medium. Af ter ethanol ingestion, FAEEs can be found within low-density lipoprote ins (LDLs). Therefore, to achieve solubility with FAEEs bound to a nat urally occurring lipid carrier, we developed a method for FAEE solubil ization and delivery to cells in culture. We synthesized radiolabeled FAEEs and incorporated them into human LDL particles that bind to LDL receptors and deliver FAEEs to intact cells. Ethyl palmitate acid ethy l oleate were incorporated into LDLs yielding molar ratios of FAEEs to LDLs of 2,153 +/- 249 and 4,208 +/- 403, respectively. LDL reconstitu ted with FAEE had the same electrophoretic mobility on agarose gel ele ctrophoresis as native LDL, indicating that the reconstituted LDL (rLD L) was not oxidatively modified. Quantitative analysis of the solubili zation of FAEEs in aqueous medium was investigated by adding FAEEs to tissue culture medium either directly or reconstituted in LDL at a con centration of 27 mu M. The percentage of FAEE quantitated was 40.0 +/- 2.5% and 89.3 +/- 0.6% for FAEEs added directly and in rLDLs, respect ively. After sterile filtration of these two media, the percentage of FAEE that remained was 11.8 +/- 1.3% (direct addition) and 74.9 +/- 1. 3% (addition within rLDL), further demonstrating that the LDL particle did solubilize the FAEE. There was a linear relationship between the amount of rLDL in the medium and the amount of FAEE incorporated into the cells. Finally, native LDL (1:21 for rLDL:native LDL) was shown to decrease the uptake of ethyl oleate rLDL by HepG2 cells by 23.1 +/- 3 .1%, indicating the involvement of a receptor in rLDL uptake. Use of L DL reconstituted with FAEE as a solubilized farm of the ester will per mit in vivo and in vitro studies assessing the physiological and patho logical roles of these potentially toxic ethanol metabolites.