Pearce R E, McIntyre C J, Madan A, Sanzgiri U, Draper A J, Bullock P L, Cook D C, Burton L A, Latham J, Nevins C, Parkinson A
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, 66160-7417, USA.
Arch Biochem Biophys. 1996 Jul 15;331(2):145-69. doi: 10.1006/abbi.1996.0294.
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 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 b5 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-de-methylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4solidus5 (testosterone 6beta-hydroxylation), and CYP4A9solidus11 (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 b5 and 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 degrees 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 degrees C, indicating there was no differential loss of cytochrome P450 activity, cytochrome b5 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.
在以下几种情况下检测了细胞色素P450酶、细胞色素b5和NADPH-细胞色素c还原酶的稳定性：(A) 液氮冷冻并储存在-80℃的人肝脏样本；(B) 悬浮于250 mM蔗糖中并储存在-80℃的人肝脏微粒体；(C) 经历多达10次冻融循环的人肝脏微粒体。在研究A中，从新鲜（未冷冻）组织以及储存在-80℃达1、2、4或6个月的冷冻组织中制备了来自5个人肝脏的微粒体。冷冻肝脏导致细胞色素P450和b5的表观浓度以及NADPH-细胞色素c还原酶的活性降低了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酶的活性方面也观察到了类似的降低，这些酶包括CYP1A2（7-乙氧基异吩唑酮O-脱烷基化和咖啡因N3-去甲基化）、CYP2A6（香豆素7-羟基化）、CYP2C9（甲苯磺丁脲甲基羟基化）、CYP2C19（S-美芬妥因4'-羟基化）、CYP2D6（右美沙芬O-去甲基化）、CYP2E1（氯唑沙宗6-羟基化）、CYP3A4/5（睾酮6β-羟基化）以及CYP4A9/11（月桂酸12-羟基化）。冷冻人肝脏并未使细胞色素P450转化为其无活性形式细胞色素P420，但增加了肝脏微粒体被血红蛋白或其他含血红素蛋白的污染，这导致细胞色素P450和b5的比活性以及所有P450酶的比活性均出现一致下降。在研究B中，测定了储存在-80℃约0、1或2年的10份人肝脏微粒体样本中细胞色素P450和b5的浓度、NADPH-细胞色素c还原酶的活性以及各个细胞色素P450酶的活性。细胞色素P450、细胞色素b5和NADPH-细胞色素c还原酶的浓度和活性在样本间的差异，在人肝脏微粒体于-80℃长期储存时受到的影响较小，表明细胞色素P450活性、细胞色素b5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，将来自一组人肝脏的微粒体在-80℃进行1、2、3、5、7或10次冷冻，随后在室温下解冻。对肝脏微粒体进行多达10次的冻融循环既未使细胞色素P450转化为P420，也未导致CYP1A2、CYP2A6、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A4/5或CYP4A9/11活性的显著损失。总体而言，这些结果表明我们目前储存和处理人肝脏的方法非常适合保存微粒体P450酶的活性。
