Effects of Freezing, Thawing, and Storing Human Liver Microsomes on Cytochrome P450 Activity

Abstract

The stability of cytochrome P450 enzymes, cytochrome b5, and NADPH–cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at −80°C, (B) human liver microsomes suspended in 250 mMsucrose and stored at −80°C, and (C) human liver microsomes subjected to as many as 10 cycles of thawing and freezing. In study A, microsomes from five human livers were prepared from fresh (unfrozen) tissue and from tissue that was stored frozen at −80°C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b5and the activity of NADPH–cytochrome c reductase decreased 20–40% as a result of freezing the liver, regardless of whether the liver was stored for 1 or 6 months. Similar decreases were observed in the activities of cytochrome P450 enzymes belonging to several gene families, namely CYP1A2 (7-ethoxyresorufin O-dealkylation and caffeine N3-demethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide methylhydroxylation), CYP2C19 (S-mephenytoin 4′- hydroxylation), CYP2D6 (dextromethorphan O-de- methylation), CYP2E1 (chlorzoxazone 6-hydroxyla- tion), CYP3A4/5 (testosterone 6β-hydroxylation), and CYP4A9/11 (lauric acid 12-hydroxylation). Freezing human liver did not convert cytochrome P450 to its inactive form, cytochrome P420, but it increased the contamination of liver microsomes with hemoglobin or other heme-containing proteins, which resulted in a uniform decrease in the specific activity of cyto- chromes P450 and b5and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and b5, the activity of NADPH–cytochrome c reductase, and the activity of individual cytochrome P450 enzymes were determined in 10 samples of human liver microsomes stored at −80°C for approximately 0, 1, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome b5, and NADPH–cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at −80°C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5concentration, or NADPH–cytochrome c reductase activity. In study C, microsomes from a pool of human livers were subjected to 1, 2, 3, 5, 7, or 10 cycles of freezing at −80°C followed by thawing at room temperature. Freezing/thawing liver microsomes for up to 10 cycles did not convert cytochrome P450 to P420, nor did it cause significant loss of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, or CYP4A9/11 activity. Overall, these results suggest that our current methods for storing and processing human liver are well suited to preserving microsomal P450 enzyme activity.