Bessho M, Ohsuzu F, Yanagida S, Sakata N, Aosaki N, Tajima T, Nakamura H
Eisai Research Laboratories, Eisai Company Ltd., Tokyo, Japan.
Anal Biochem. 1991 Jan;192(1):117-24. doi: 10.1016/0003-2697(91)90195-y.
To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 M HClO4. Powdered samples (6-15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 M HClO4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO3. The same powdered samples were directly homogenized with 2 ml of 0.4 M HClO4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.
为了通过乙醇萃取比较磷酸肌酸与三磷酸腺苷（ATP）的可萃取性，使用不同浓度梯度的乙醇和0.4M高氯酸从正常灌注的大鼠心脏组织中提取这两种化合物。将来自冷冻钳夹组织的粉末状样品（湿重6 - 15mg）在2ml乙醇溶液中匀浆。离心后，去除上清液；将每个离心后的沉淀物再用2ml 0.4M高氯酸匀浆并离心。上清液用0.4m碳酸氢钾中和。将相同的粉末状样品直接用2ml 0.4M高氯酸匀浆并以相同方式处理。组织中只有少量ATP可被85%或更高浓度的乙醇萃取。此外，约13%的组织ATP不能被随后的高氯酸萃取。与ATP相反，组织中的磷酸肌酸可被95%乙醇部分萃取，几乎所有组织中的磷酸肌酸可被70%乙醇萃取。通过70%乙醇和随后的高氯酸萃取获得的总磷酸肌酸明显高于直接高氯酸萃取获得的量。从这些结果得出结论，乙醇萃取法对组织中磷酸肌酸的可萃取性与ATP明显不同。此外，逐步萃取法被推荐为一种用于萃取灌注大鼠心脏组织中能量代谢物的有用方法。
