Lazzarino G, Nuutinen M, Tavazzi B, Di Pierro D, Giardina B
Department of Experimental Medicine and Biochemical Sciences, II University of Rome, Italy.
Anal Biochem. 1989 Sep;181(2):239-41. doi: 10.1016/0003-2697(89)90236-4.
A rapid and simple method for preparing freeze-clamped tissue samples for metabolite determinations is described. Freeze-clamped rat heart tissue samples weighing from 0.8 to 1.0 g were homogenized directly in an Ultra-Turrax homogenizer for 60 s in 3.5 ml of ice-cold 0.6 M HClO4 without pulverizing them in liquid nitrogen. After centrifugation, the pellet was rehomogenized in the Ultra-Turrax homogenizer for 30 s in 1.5 ml of HClO4. Following a further centrifugation the extracts were combined and the pH was adjusted to 7.0 by adding 5 M K2CO3. The neutralized supernatant was used for the desired assays. The analyses of the tissue extracts obtained from isolated perfused rat hearts by the present method give similar results for different kinds of metabolites than those processed according to the previous classical method. Moreover, the values of the various parameters determined from the tissue extracts prepared according to the method described here are similar to the data reported in literature. The method can be readily applied to any other freeze-clamped tissue. The greatest improvement obtained is that the homogenization procedure can be accomplished easily and conveniently in about one-tenth of the time required for the earlier classical method without the time-consuming and unpleasant tissue grinding in liquid nitrogen.
本文描述了一种快速简便的制备用于代谢物测定的冷冻钳夹组织样品的方法。将重0.8至1.0 g的冷冻钳夹大鼠心脏组织样品直接在Ultra-Turrax匀浆器中于3.5 ml冰冷的0.6 M高氯酸中匀浆60秒，无需在液氮中研磨。离心后，沉淀在1.5 ml高氯酸中于Ultra-Turrax匀浆器中再匀浆30秒。再次离心后，合并提取物，并通过加入5 M碳酸钾将pH调至7.0。中和后的上清液用于所需的测定。用本方法从离体灌注大鼠心脏获得的组织提取物分析得出的不同种类代谢物结果与按照先前经典方法处理的结果相似。此外，根据本文所述方法制备的组织提取物测定的各种参数值与文献报道的数据相似。该方法可轻松应用于任何其他冷冻钳夹组织。最大的改进是，匀浆过程可以在比早期经典方法所需时间约十分之一的时间内轻松便捷地完成，无需在液氮中进行耗时且麻烦的组织研磨。
