Measurement of rates of cholesterol synthesis using tritiated water.
Measurement of rates of cholesterol synthesis using tritiated water.
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DOI：
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发表时间：
1984-12
影响因子：
6.5
通讯作者：
J. Dietschy;D. Spady
中科院分区：
文献类型：
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作者：
J. Dietschy;D. Spady
通常通过测定分离的微粒体上 3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 还原酶的水平或通过测量体外全细胞制剂或体内整个动物组织将各种 14C 标记底物或 [3H] 水掺入胆固醇的速率来评估各种组织中甾醇的合成速率。虽然测量 HMG-CoA 还原酶的活性或 14C 标记底物掺入胆固醇的速率给出了有用的甾醇产生的相对速率，但这两种方法都不能产生胆固醇合成的绝对速率。 [3H]水的使用避免了使用 14C 标记底物时遇到的前体池比活性可变和未知稀释的问题，并且只要已知特定组织的 3H/C 掺入比，确实会产生胆固醇合成的绝对速率。在12个不同的实验情况下，发现在几种动物的不同组织中，有21至27微克的3H原子从[3H]水中掺入胆固醇中，因此3H/C掺入比率在几乎所有实验条件下都相似，变化范围为0.78至1.00。当体内施用时，[3H]水迅速与细胞内水平衡，并以相对于时间呈线性的速率并入各个器官内的甾醇中。从这些数据可以获得整个动物和动物各个器官中胆固醇合成的绝对速率。因此，目前的数据表明，使用[3H]水可以在体外和体内产生最准确的胆固醇合成率。
Rates of sterol synthesis in various tissues commonly are assessed by assaying levels of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase on isolated microsomes or by measuring the rates of incorporation of various 14C-labeled substrates or [3H]water into cholesterol by whole cell preparations in vitro or by the tissues of the whole animal in vivo. While measurement of activities of HMG-CoA reductase or rates of incorporation of 14C-labeled substrates into cholesterol give useful relative rates of sterol production, neither method yields absolute rates of cholesterol synthesis. The use of [3H]water circumvents the problem of variable and unknown dilution of the specific activity of the precursor pool encountered when 14C-labeled substrates are used and does yield absolute rates of cholesterol synthesis provided that the 3H/C incorporation ratio is known for a particular tissue. In 12 different experimental situations it has been found that from 21 to 27 micrograms atoms of 3H are incorporated into cholesterol from [3H]water in different tissues of several animal species, so that the 3H/C incorporation ratio is similar under nearly all experimental conditions and varies from 0.78 to 1.00. When administered in vivo, [3H]water rapidly equilibrates with intracellular water and is incorporated into sterols within the various organs at rates that are linear with respect to time. From such data it is possible to obtain absolute rates of cholesterol synthesis in the whole animal and in the various organs of the animal. Current data suggest, therefore, that use of [3H]water yields the most accurate rates of cholesterol synthesis both in vitro and in vivo.