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 cytochrome b(5)
reductase,
cytochrome b(5), absorbance at 420,
p-nitrophenol hydroxylation,
tolbutamide hydroxylation,
dextromethorphan N- and O-demethylation, glucuronidation of
morphine and 1-
naphthol, and
ester cleavage of p-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
dextromethorphan O-demethylation to
dextrorphan and the N-demethylation of l-alpha-
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.