Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity
Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity
复制标题
DOI：
10.1006/abbi.1996.0294
复制
发表时间：
1996-07-15
影响因子：
3.9
通讯作者：
Parkinson, A
中科院分区：
文献类型：
--
作者：
Pearce, RE;McIntyre, CJ;Parkinson, A
在（A）冷冻于液氮中并储存于-80 ℃的人肝样品、（B）悬浮于250 mM蔗糖中并储存于-80 ℃的人肝微粒体和（C）经历多达10次解冻和冷冻循环的人肝微粒体中检查了细胞色素P450酶、细胞色素bg和NADPH-细胞色素c还原酶的稳定性。在研究A中，从新鲜（未冷冻）组织和在-80 ℃冷冻储存1、2、4或6个月的组织中制备了5个人肝脏的微粒体。细胞色素P450和B（5）的表观浓度以及NADPH-细胞色素c还原酶的活性由于冷冻肝脏而降低20-40%，无论肝脏储存1个月还是6个月。在属于几个基因家族的细胞色素P450酶（即CYP 1A 2）的活性中观察到类似的降低（7-乙氧基试卤灵O-脱烷基化和咖啡因N3-脱甲基化），CYP 2A 6（香豆素7-羟基化），CYP 2C 9（甲苯磺丁脲甲基羟基化），CYP 2C 19（S-美芬妥英4 '-羟基化），CYP 2D 6（美沙芬O-去甲基化），CYP 2 E1（氯唑沙宗6-羟基化）、CYP 3A 4/5（睾酮6 β-羟基化）和CYP 4A 9/11（月桂酸12-羟基化）。冷冻人肝脏不会将细胞色素P450转化为非活性形式细胞色素P420，但会增加血红蛋白或其他含血红素蛋白质对肝微粒体的污染，导致细胞色素P450和B（5）的比活性以及所有P450酶的比活性均匀降低。在研究B中，测定了在-80 ℃下储存约0、1或2年的10份人肝微粒体样品中细胞色素P450和bg的浓度、NADPH-细胞色素c还原酶的活性以及单个细胞色素P450酶的活性。细胞色素P450、细胞色素bg和NADPH-细胞色素c还原酶的浓度和活性的样品间变化在名义上受到人肝微粒体在-80 ℃下长期储存的影响，表明细胞色素P450活性、细胞色素bg浓度或NADPH-细胞色素c还原酶活性没有差异损失。在研究C中，将来自人肝脏池的微粒体在-80 ℃下进行1、2、3、5、7或10个循环的冷冻，然后在室温下解冻。冻融肝微粒体长达10个循环不会将细胞色素P450转化为P420，也不会导致CYP 1A 2、CYP 2A 6、CYP 2C 9、CYP 2C 19、CYP 2D 6、CYP 2 E1、CYP 3A 4/5或CYP 4A 9/11活性显著丧失。总的来说，这些结果表明，我们目前用于储存和处理人肝脏的方法非常适合于保存微粒体P450酶活性。(C)出版社：Academic Press，Inc.
The stability of cytochrome P450 enzymes, cytochrome bg, and NADPH-cytochrome c reductase was examined in (A) human liver samples frozen in liquid nitrogen and stored at -80 degrees C, (B) human liver microsomes suspended in 250 mM sucrose and stored at -80 degrees 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 degrees C for 1, 2, 4, or 6 months. The apparent concentration of cytochromes P450 and b(5) and 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-demethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4/5 (testosterone 6 beta-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 cytochromes P450 and b(5) and in the specific activity of all P450 enzymes. In study B, the concentration of cytochromes P450 and bg, 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 degrees C for approximately 0, I, or 2 years. The sample-to-sample variation in the concentration and activity of cytochrome P450, cytochrome bg, and NADPH-cytochrome c reductase was nominally affected by long-term storage of human liver microsomes at -80 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome bg concentration, 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 degrees 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. (C) 1996 Academic Press, Inc.