Abstract
We examined three primary variables in the preparation of human liver microsomes. In three experiments, each using three livers, we manipulated 1) the force of the first centrifugation (9,000, 10,500, or 12,000g); 2) the presence of sucrose in the homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate dehydrogenase, NADPH cytochrome P450 reductase (reductase), and glutathione S-transferase in the resulting premicrosomal, microsomal, and cytosolic fractions suggest that enhanced purity of microsomes can be obtained by reducing force of centrifugation, including sucrose, and increasing the number of homogenization strokes. Each microsomal fraction was also assayed for protein content, cytochrome P450, NADH cytochromeb5 reductase, cytochromeb5, absorbance at 420,p-nitrophenol hydroxylation, tolbutamide hydroxylation, dextromethorphan N- and O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage ofp-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram of liver) and specific activity (per milligram of protein). The inclusion of sucrose improved activity specific to reductase activity. Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphanO-demethylation to dextrorphan and theN-demethylation of l-α-acetylmethadol (LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences were found in the concentration-dependent activities. Variation of the methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.
Footnotes
-
Send reprint requests to: David E. Moody, Ph.D., University of Utah, Center for Human Toxicology, 20 S. 2030 E. RM 490, Salt Lake City, UT 84112-9457. E-mail: dmoody{at}alanine.pharm.utah.edu
-
↵1 Current Address: Secor International, Inc., Salt Lake City, UT 84107.
-
This research was supported by U.S. Public Health Service Grant R01 DA10100. A preliminary report of these results was presented at the Experimental Biology 1999 meeting in Washington, D.C.
- Abbreviations used are::
- HLM
- human liver microsomes
- P450
- cytochrome P450
- LAAM
- l-α-acetylmethadol
- HPLC
- high-performance liquid chromatography
- Received November 9, 2000.
- Accepted December 5, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|