Effects of Extraction with Isooctane upon the Properties of Liver Microsomes.
Leibman KC; Estabrook RW
Mol Pharmacol 1971 Jan; 7(1):26-32
The effects of extraction with isooctane (540841) on substrate hemoprotein spectrophotometric binding properties of liver microsomes were studied in-vitro. Liver microsomes obtained from male Holtzman-rats, pretreated with 50mg/kg sodium-phenobarbital daily for 3 days were used. Twenty milliliters (ml) microsomal suspension were extracted with 13.4ml isooctane or unextracted and assayed for protein, cytochrome-b5, cytochrome-P450, and the extent of aniline/microsomal binding. The effect on the hexobarbital binding spectrum of the preparations was investigated. The effects on aminopyrine-demethylase and acetanilide-hydroxylase activity were evaluated. The extracts were examined by thin layer chromatography (TLC). Extraction with isooctane reduced microsomal protein content by about 80 percent. Microsomal cytochrome-b5 and cytochrome-P450 concentrations were reduced to a similar extent, about 80 percent, so that the relative proportions per milligram protein were not significantly altered. The apparent aniline/microsomal dissociation constants were not significantly affected. The type-I difference spectrum seen with hexobarbital, peak at 390 nanometers (nm) and trough at 425nm, disappeared at concentrations of 0.8 or 4.9 millimolar (mM). At hexobarbital concentrations of 12.5mM or higher a type-II binding spectrum was obtained. Isooctane did not inhibit aminopyrine-demethylase or acetanilide-hydroxylase activity. TLC spots corresponding to phosphatidylcholine and phosphatidylethanolamine were found in the isooctane extracts. The authors conclude that after isooctane extraction rat liver microsomes cannot bind hexobarbital and produce a type-I difference spectrum. Isooctane apparently destroys or modifies the normal hexobarbital binding form of cytochrome-P450 in liver microsomes.
NIOSH-Publication; NIOSH-Grant; Grants-other; Organic-solvents; Solvent-extraction; Laboratory-techniques; Chemical-binding; Physiological-chemistry; Liver-microsomes; Absorption-spectrophotometry;
Pharmacology and Therapeutics Univ of Florida Coll of Med Dept of Pharma & Therapeutics Gainesville, Fla 32601
Other Occupational Concerns; Grants-other;
University of Florida Gainesville, Gainesville, Florida