Low-density lipoprotein reconstituted with fatty acid ethyl esters as a physiological vehicle for ethyl ester delivery to intact cells

D. A. Bird, Z. M. Szczepiorkowski, V. C. Trace, Michael Laposata

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been found selectively in the organs damaged by ethanol abuse, and on that basis have been implicated as contributors to ethanol- induced organ damage. To directly assess the cytotoxic potential of FAEEs with intact cells in a physiological system, solubility must be achieved for these highly nonpolar lipids in aqueous medium. After ethanol ingestion, FAEEs can be found within low-density lipoproteins (LDLs). Therefore, to achieve solubility with FAEEs bound to a naturally occurring lipid carrier, we developed a method for FAEE solubilization 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 and ethyl oleate were incorporated into LDLs yielding molar ratios of FAEEs to LDLs of 2,153 ± 249 and 4,208 ± 403, respectively. LDL reconstituted with FAEE had the same electrophoretic mobility on agarose gel electrophoresis as native LDL, indicating that the reconstituted LDL (rLDL) was not oxidatively modified. Quantitative analysis of the solubilization of FAEEs in aqueous medium was investigated by adding FAEEs to tissue culture medium either directly or reconstituted in LDL at a concentration of 27 μM. The percentage of FAEE quantitated was 40.0 ± 2.5% and 89.3 ± 0.6% for FAEEs added directly and in rLDLs, respectively. 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 LDL reconstituted with FAEE as a solubilized form of the ester will permit in vivo and in vitro studies assessing the physiological and pathological roles of these potentially toxic ethanol metabolites.

Original languageEnglish (US)
Pages (from-to)1265-1270
Number of pages6
JournalAlcoholism: Clinical and Experimental Research
Volume19
Issue number5
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

LDL Lipoproteins
Esters
Fatty Acids
Ethanol
Solubility
Lipids
Tissue culture
Electrophoretic mobility
Agar Gel Electrophoresis
LDL Receptors
Esterification
Poisons
Hep G2 Cells
Metabolites
Electrophoresis
Sepharose

Keywords

  • Ethanol
  • Fatty Acids
  • HepG2 Cells
  • Lipids
  • Lipoproteins

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Toxicology

Cite this

Low-density lipoprotein reconstituted with fatty acid ethyl esters as a physiological vehicle for ethyl ester delivery to intact cells. / Bird, D. A.; Szczepiorkowski, Z. M.; Trace, V. C.; Laposata, Michael.

In: Alcoholism: Clinical and Experimental Research, Vol. 19, No. 5, 1995, p. 1265-1270.

Research output: Contribution to journalArticle

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abstract = "Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been found selectively in the organs damaged by ethanol abuse, and on that basis have been implicated as contributors to ethanol- induced organ damage. To directly assess the cytotoxic potential of FAEEs with intact cells in a physiological system, solubility must be achieved for these highly nonpolar lipids in aqueous medium. After ethanol ingestion, FAEEs can be found within low-density lipoproteins (LDLs). Therefore, to achieve solubility with FAEEs bound to a naturally occurring lipid carrier, we developed a method for FAEE solubilization 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 and ethyl oleate were incorporated into LDLs yielding molar ratios of FAEEs to LDLs of 2,153 ± 249 and 4,208 ± 403, respectively. LDL reconstituted with FAEE had the same electrophoretic mobility on agarose gel electrophoresis as native LDL, indicating that the reconstituted LDL (rLDL) was not oxidatively modified. Quantitative analysis of the solubilization of FAEEs in aqueous medium was investigated by adding FAEEs to tissue culture medium either directly or reconstituted in LDL at a concentration of 27 μM. The percentage of FAEE quantitated was 40.0 ± 2.5{\%} and 89.3 ± 0.6{\%} for FAEEs added directly and in rLDLs, respectively. 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 LDL reconstituted with FAEE as a solubilized form of the ester will permit in vivo and in vitro studies assessing the physiological and pathological roles of these potentially toxic ethanol metabolites.",
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T1 - Low-density lipoprotein reconstituted with fatty acid ethyl esters as a physiological vehicle for ethyl ester delivery to intact cells

AU - Bird, D. A.

AU - Szczepiorkowski, Z. M.

AU - Trace, V. C.

AU - Laposata, Michael

PY - 1995

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N2 - Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been found selectively in the organs damaged by ethanol abuse, and on that basis have been implicated as contributors to ethanol- induced organ damage. To directly assess the cytotoxic potential of FAEEs with intact cells in a physiological system, solubility must be achieved for these highly nonpolar lipids in aqueous medium. After ethanol ingestion, FAEEs can be found within low-density lipoproteins (LDLs). Therefore, to achieve solubility with FAEEs bound to a naturally occurring lipid carrier, we developed a method for FAEE solubilization 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 and ethyl oleate were incorporated into LDLs yielding molar ratios of FAEEs to LDLs of 2,153 ± 249 and 4,208 ± 403, respectively. LDL reconstituted with FAEE had the same electrophoretic mobility on agarose gel electrophoresis as native LDL, indicating that the reconstituted LDL (rLDL) was not oxidatively modified. Quantitative analysis of the solubilization of FAEEs in aqueous medium was investigated by adding FAEEs to tissue culture medium either directly or reconstituted in LDL at a concentration of 27 μM. The percentage of FAEE quantitated was 40.0 ± 2.5% and 89.3 ± 0.6% for FAEEs added directly and in rLDLs, respectively. 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 LDL reconstituted with FAEE as a solubilized form of the ester will permit in vivo and in vitro studies assessing the physiological and pathological roles of these potentially toxic ethanol metabolites.

AB - Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been found selectively in the organs damaged by ethanol abuse, and on that basis have been implicated as contributors to ethanol- induced organ damage. To directly assess the cytotoxic potential of FAEEs with intact cells in a physiological system, solubility must be achieved for these highly nonpolar lipids in aqueous medium. After ethanol ingestion, FAEEs can be found within low-density lipoproteins (LDLs). Therefore, to achieve solubility with FAEEs bound to a naturally occurring lipid carrier, we developed a method for FAEE solubilization 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 and ethyl oleate were incorporated into LDLs yielding molar ratios of FAEEs to LDLs of 2,153 ± 249 and 4,208 ± 403, respectively. LDL reconstituted with FAEE had the same electrophoretic mobility on agarose gel electrophoresis as native LDL, indicating that the reconstituted LDL (rLDL) was not oxidatively modified. Quantitative analysis of the solubilization of FAEEs in aqueous medium was investigated by adding FAEEs to tissue culture medium either directly or reconstituted in LDL at a concentration of 27 μM. The percentage of FAEE quantitated was 40.0 ± 2.5% and 89.3 ± 0.6% for FAEEs added directly and in rLDLs, respectively. 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 LDL reconstituted with FAEE as a solubilized form of the ester will permit in vivo and in vitro studies assessing the physiological and pathological roles of these potentially toxic ethanol metabolites.

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KW - Fatty Acids

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KW - Lipids

KW - Lipoproteins

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