Yamazaki H, Inoue K, Turvy C G, Guengerich F P, Shimada T
Osaka Prefectural Institute of Public Health, Higashinari-ku, Japan.
Drug Metab Dispos. 1997 Feb;25(2):168-74.
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 degrees 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 degrees C for 6 hr and then frozen again and kept for 1 week at -80 degrees 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 degrees 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 degrees 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 degrees 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 degrees 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 degrees 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.
研究了人肝脏样本的冷冻、解冻及室温保存对细胞色素P450（P450或CYP）各形式的含量及催化活性的影响。还研究了肝脏基因组DNA的稳定性。在-80℃保存5年，微粒体中P450酶的水平及催化活性没有显著降低。然后我们研究了肝脏样本冷冻/解冻的影响。通过免疫化学方法以及人样本肝脏微粒体中典型药物氧化反应的活性来测定P450形式的水平，样本分为两组。一组样本解冻后在25℃保存6小时，然后再次冷冻并在-80℃保存1周。另一组在同一时间制备肝脏微粒体（与另一组同时），但不解冻再冷冻。解冻肝脏样本并在25℃保存6小时使总P450含量降低约90%，NADH-铁氰化物和NADPH-细胞色素c还原酶的活性降低约80%。然而，b5水平的降低仅约30%。P450的光谱研究表明，解冻肝脏样本并在25℃保存会产生无活性形式的P420。通过免疫印迹分析检测有无解冻时的P450蛋白，但解冻并在25℃保存样本6小时后，各P450的催化活性急剧下降。解冻并在25℃保存样本6小时后，此类微粒体中仅存在10%的甲苯磺丁脲甲基羟化活性，且未检测到乙氧基异吩恶唑酮O-脱乙基活性。还发现人肝脏样本解冻后基因组DNA会降解。这些结果表明，解冻肝脏样本并在25℃保存会降低微粒体中P450的水平和活性，且P450蛋白各形式的稳定性存在差异。由于难以获得新鲜肝脏样本，通过免疫化学方法测定的P450蛋白并不总是反映人肝脏微粒体中P450的催化功能。
