Antibodies raised against trifluoroacetyl-protein adducts bind to N-trifluoroacetyl-phosphatidylethanolamine in hexagonal phase phospholipid micelles.
The delayed fulminant form of halothane hepatotoxicity is thought to be triggered by an immune response to haptenic adducts formed by a metabolite, trifluoroacetyl chloride. In this study we demonstrate that antibodies purified from the sera of rabbits sensitized to a trifluoroacetyl-protein adduct will cross-react with a trifluoroacetyl-phosphatidylethanolamine adduct. Trifluoroacetyl adducts of both rabbit serum albumin (TFA-RSA) and dioleoylphosphatidylethanolamine (TFA-DOPE) were prepared. The TFA-RSA was coupled to an Affigel-10 affinity column to purify hapten-selective immunoglobulin (Ig) G antibodies (anti-TFA-RSA IgG) from the sera of rabbits given i.m. injections of TFA-RSA. The TFA-DOPE was purified by high-performance liquid chromatography and the structure confirmed with direct chemical ionization mass spectrometry. Lamellar liposomes containing a mixture of 5% TFA-DOPE, 71% DOPE and 24% dioleoyl-phosphatidylcholine, as well as hexagonal phase micelles containing 5% TFA-DOPE and 95% DOPE, were prepared by sonication. Anti-TFA-RSA IgG antibodies were added to each of these lipid mixtures for 30 min, fluorescein-conjugated goat-antirabbit IgG antibodies were added next for an additional 30 min and then binding of anti-TFA-RSA IgG antibodies to TFA-DOPE was quantified by flow cytometry. Anti-TFA-RSA IgG antibodies bound to TFA-DOPE only when it was incorporated into hexagonal phase micelles. These findings suggest that TFA-phosphatidylethanolamine adducts that reside in nonlamellar domains on the hepatocyte surface could be recognition sites for anti-TFA-adduct antibodies and potentially participate in immune-mediated hepatotoxicity.
Trudell
J R
JR
Department of Anesthesia, Stanford University School of Medicine, California.
Ardies
C M
CM
Anderson
W R
WR
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000915:Antibody Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions / Q000276:immunology; D005434:Flow Cytometry; D005463:Fluoroacetates; D008081:Liposomes / Q000276:immunology / Q000378:metabolism; D008823:Micelles; D010713:Phosphatidylcholines / Q000276:immunology* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D012709:Serum Albumin / Q000276:immunology* / Q000378:metabolism; D014269:Trifluoroacetic Acid / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D005463:Fluoroacetates; D008081:Liposomes; D008823:Micelles; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D012709:Serum Albumin; C053344:trifluoroacetyl-serum albumin; C068825:N-trifluoroacetyl-1,2-dioleoylphosphatidylethanolamine; D014269:Trifluoroacetic Acid; C017251:1,2-oleoylphosphatidylcholine