Ledwith B J, Cahill M K, Losse L S, Satiritz S M, Eydelloth R S, Dallob A L, Tanaka W K, Galloway S M, Nichols W W
Department of Safety Assessment, Merck Research Laboratories, West Point, Pennsylvania 19486.
Anal Biochem. 1993 Sep;213(2):349-55. doi: 10.1006/abio.1993.1431.
Measurement of plasma angiotensin II (AII) by radioimmunoassay (RIA) usually requires prior purification of the plasma to remove substances that cross-react in the RIA, most notably angiotensin III (AIII). Purification of AII is generally accomplished by solid-phase extraction (SPE) followed by reverse-phase HPLC, with tedious evaporation and resuspension steps in between, and requires collection of many HPLC fractions per sample for RIA. In this report, we describe a rapid two-step SPE procedure for the purification of plasma AII, including an improved protease inhibitor cocktail for preventing the formation or degradation of AII in vitro. Plasma is first extracted on an S-Sepharose cation-exchange column, in which AII is separated from AIII by virtue of their difference in net charge, and then extracted on a C8 SPE column, without need for intermediate sample handling. The two-step SPE method is fast, results in only a single fraction for RIA per sample, and yields consistently high recoveries (77-86%) of AII, reducing the volume of plasma needed from 2 to 0.5 ml. Rat plasma was used in the present study, but the complete conservation of angiotensin peptide sequences (except angiotensinogen) in mammals suggests that this method will be applicable for other species including humans. In summary, the two-step SPE method offers the speed and simplicity of solid phase extraction while achieving a purity in AII (i.e., free of AIII) previously only obtained by laborious procedures involving HPLC.
通过放射免疫测定法（RIA）测量血浆血管紧张素II（AII）通常需要事先纯化血浆，以去除在RIA中发生交叉反应的物质，最显著的是血管紧张素III（AIII）。AII的纯化一般通过固相萃取（SPE），然后进行反相高效液相色谱法（HPLC）来完成，中间还需要繁琐的蒸发和重悬步骤，并且每个样品进行RIA时需要收集多个HPLC馏分。在本报告中，我们描述了一种快速的两步SPE程序用于纯化血浆AII，包括一种改进的蛋白酶抑制剂混合物，以防止AII在体外形成或降解。血浆首先在S-Sepharose阳离子交换柱上萃取，其中AII因其净电荷差异与AIII分离，然后在C8 SPE柱上萃取，无需中间样品处理。两步SPE方法快速，每个样品进行RIA时仅产生单一馏分，并且AII的回收率始终很高（77 - 86%），将所需血浆体积从2 ml减少到0.5 ml。本研究使用大鼠血浆，但哺乳动物中血管紧张素肽序列（除血管紧张素原外）的完全保守性表明该方法适用于包括人类在内的其他物种。总之，两步SPE方法兼具固相萃取的速度和简便性，同时能达到此前只有通过涉及HPLC的繁琐程序才能获得的AII纯度（即不含AIII）。
