Covalent modification of hepatic microsomal lipids of rats by carbon tetrachloride

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Abstract

The present study was undertaken to isolate and identify various lipids bound to 14C label during hepatic microsomal metabolism of 14CCl4 in vitro under anaerobic conditions and in vivo in rats. The two major radioactive fractions identified by thin-layer chromatography each for neutral lipids and phospholipids from in vitro and in vivo experiments corresponded to fatty acids and triglycerides and to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Approximately 89% of the radioactivity associated with phospholipids was found in PC and PE fractions. Hydrolysis of PC and PE with phospholipase A2 (EC 3.1.1.4) released about 50% of the total radioactivity as lipid moieties corresponding to fatty acids. The radioactive neutral lipids and the lipid moieties hydrolyzed from PC and PE were methylated with boron trifluoride in methanol. These methylated lipids were separated by reversed-phase high-performance liquid chromatography (HPLC), and the elution profiles of 14C label found for the lipids obtained from in vitro experiments were similar to those from in vivo. The major radioactive fractions eluted immediately after methyl oleate were identified as trichloromethyloctadecenoic and trichloromethyleicosatrienoic acid methyl esters by chemical ionization mass spectrometry. The mass spectral analysis of these fractions also indicated the formation of dichlorocarbene adduct of oleic acid. However, similar mass spectrometric detection of trichloromethylated lipids was not evident in neutral lipids and phospholipids isolated from in vitro studies. The 14C-labeled lipids eluted as a nonpolar fraction exhibited a high molecular weight containing more than three chlorines. Dimerization and cross-linking of trichloromethylated lipids based on HPLC and mass spectral analysis are also discussed in this paper.

Original languageEnglish (US)
Pages (from-to)199-213
Number of pages15
JournalMolecular Toxicology
Volume2
Issue number3
StatePublished - 1989

Fingerprint

Carbon Tetrachloride
Rats
Lipids
Liver
Phosphatidylcholines
Phospholipids
Radioactivity
High performance liquid chromatography
Spectrum analysis
Labels
Fatty Acids
High Pressure Liquid Chromatography
Thin layer chromatography
Dimerization
Phospholipases A2
Chlorine
Reverse-Phase Chromatography
Oleic Acid
Thin Layer Chromatography
Metabolism

ASJC Scopus subject areas

  • Toxicology

Cite this

Covalent modification of hepatic microsomal lipids of rats by carbon tetrachloride. / Kaphalia, Bhupendra; Ansari, Ghulam.

In: Molecular Toxicology, Vol. 2, No. 3, 1989, p. 199-213.

Research output: Contribution to journalArticle

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abstract = "The present study was undertaken to isolate and identify various lipids bound to 14C label during hepatic microsomal metabolism of 14CCl4 in vitro under anaerobic conditions and in vivo in rats. The two major radioactive fractions identified by thin-layer chromatography each for neutral lipids and phospholipids from in vitro and in vivo experiments corresponded to fatty acids and triglycerides and to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), respectively. Approximately 89{\%} of the radioactivity associated with phospholipids was found in PC and PE fractions. Hydrolysis of PC and PE with phospholipase A2 (EC 3.1.1.4) released about 50{\%} of the total radioactivity as lipid moieties corresponding to fatty acids. The radioactive neutral lipids and the lipid moieties hydrolyzed from PC and PE were methylated with boron trifluoride in methanol. These methylated lipids were separated by reversed-phase high-performance liquid chromatography (HPLC), and the elution profiles of 14C label found for the lipids obtained from in vitro experiments were similar to those from in vivo. The major radioactive fractions eluted immediately after methyl oleate were identified as trichloromethyloctadecenoic and trichloromethyleicosatrienoic acid methyl esters by chemical ionization mass spectrometry. The mass spectral analysis of these fractions also indicated the formation of dichlorocarbene adduct of oleic acid. However, similar mass spectrometric detection of trichloromethylated lipids was not evident in neutral lipids and phospholipids isolated from in vitro studies. The 14C-labeled lipids eluted as a nonpolar fraction exhibited a high molecular weight containing more than three chlorines. Dimerization and cross-linking of trichloromethylated lipids based on HPLC and mass spectral analysis are also discussed in this paper.",
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