Abstract

Effects of freezing, thawing, and storage at room temperature of human liver samples on the contents and catalytic activities of individual forms of cytochrome P450 (P450 or CYP) were examined. The stability of liver genomic DNA was also investigated. There were no significant decreases in microsomal levels or catalytic activities of P450 enzymes by storage at −80°C for 5 years. We then examined the effects of freezing/thawing of liver samples. Levels of P450 forms were determined immunochemically and by the activities of typical drug oxidation reactions in liver microsomes of human samples, which were divided into two groups. One group of samples was thawed and kept at 25°C for 6 hr and then frozen again and kept for 1 week at −80°C. In the other group, liver microsomes were prepared at the same time (as those from the other group), but not thawed and refrozen. Thawing the liver samples and storage for 6 hr at 25°C decreased contents of total P450 by about 90% and activities of both NADH-ferricyanide and NADPH-cytochrome c reductases by about 80%. However, the decrease in b₅ levels was only about 30%. Spectral studies of P450 suggested that thawing the liver samples and holding them at 25°C produced inactive form P420. P450 proteins were detected by immunoblot analysis with or without thawing, but catalytic activities for individual P450s were decreased drastically by thawing and holding samples for 6 hr at 25°C. Only 10% of tolbutamide methyl hydroxylation activity was present, and there was no detectable ethoxyresorufin O-deethylation activity in such microsomes after thawing and holding samples for 6 hr at 25°C. Genomic DNA from human livers was also found to be degraded after the samples were thawing. These results suggested that thawing and holding the liver samples at 25°C decreased the levels and activities of P450s in microsomes and that there are differences in stabilities in individual forms of P450 proteins. P450 proteins determined immunochemically do not always reflect P450-catalytic functions in human liver microsomes because of difficulties in obtaining fresh liver samples.