Naik G O, Moe G W, Armstrong P W
St. Michael's Hospital and the Department of Medicine, Division of Cardiology, University of Toronto, 8F 30 Bond Street, Toronto, ON, Canada M5B 1W8.
J Pharm Biomed Anal. 2001 Mar;24(5-6):947-55. doi: 10.1016/s0731-7085(00)00565-3.
Although angiotensin II (ANG II) has been the focal regulatory peptide of the renin-angiotensin system, its proteolytic fragments have recently been demonstrated to have biological effects. Conventional measurement of angiotensins involves radioimmunoassay (RIA), which is a sensitive binding technique capable of measuring low physiological concentrations. However, ANG II antibody cross-reacts with ANG II and its fragments (ANG II cascade), rendering RIA measurement alone to be a non-specific measure of immunoreactive ANG II (ir-ANG II). On the other hand, high-performance liquid chromatography (HPLC) is capable of separating immunoreactive ANG II cascade members, but may not be sensitive enough to detect these low peptide concentrations often present in biological samples. Consequently, a reverse-phase HPLC method, with triethylammoniun formate as an ion-pair reagent, was developed to separate ANG II and its fragments, ANG III, ANG IV and ANG V. This HPLC separation was applied to extracts from normal canine hearts and ANG II cascade immunoreactive fractions were collected. Collected fractions were quantified by RIA, with the use of separate standard curves. The isocratic HPLC separation of ANG II, ANG III, ANG IV and ANG V was achieved in less than 5 min with adjacent peaks having baseline resolution. Measured cardiac left ventricle ANG III, ANG IV and ANG V concentrations (mean+/-SD) were 5.3+/-2.2,4.0+/-1.0 and 3.1+/-1.0 fmol/g (n=9), respectively. There was a significant difference (P=0.003, n=9) between left ventricular immunoreactive ANG II and 'true' ANG II, corrected for recovery rates of 86.2+/-22.5 and 53.5+/-16.2 fmol/g, respectively. We conclude that the combination of HPLC with RIA ensures the specific measurement of the ANG II cascade family members while non-chromatographic processing of tissue renders ANG II measurement non-specific. In addition, the use of triethylammonium formate as mobile phase additive is superior in the HPLC separation of the angiotensins.
尽管血管紧张素II（ANG II）一直是肾素-血管紧张素系统的核心调节肽，但其蛋白水解片段最近已被证明具有生物学效应。传统的血管紧张素测量方法包括放射免疫测定法（RIA），这是一种能够测量低生理浓度的灵敏结合技术。然而，ANG II抗体与ANG II及其片段（ANG II级联反应）发生交叉反应，使得仅用RIA测量成为免疫反应性ANG II（ir-ANG II）的非特异性测量方法。另一方面，高效液相色谱法（HPLC）能够分离免疫反应性ANG II级联反应成员，但可能不够灵敏，无法检测生物样品中经常存在的这些低肽浓度。因此，开发了一种以甲酸三乙铵作为离子对试剂的反相HPLC方法，用于分离ANG II及其片段ANG III、ANG IV和ANG V。这种HPLC分离方法应用于正常犬心脏的提取物，并收集ANG II级联反应免疫反应性组分。收集的组分通过RIA进行定量，使用单独的标准曲线。ANG II、ANG III、ANG IV和ANG V的等度HPLC分离在不到5分钟内完成，相邻峰具有基线分辨率。测量的心脏左心室ANG III、ANG IV和ANG V浓度（平均值±标准差）分别为5.3±2.2、4.0±1.0和3.1±1.0 fmol/g（n = 9）。左心室免疫反应性ANG II与“真实”ANG II之间存在显著差异（P = 0.003，n = 9），分别校正回收率为86.2±22.5和53.5±16.2 fmol/g。我们得出结论，HPLC与RIA相结合可确保特异性测量ANG II级联反应家族成员，而组织的非色谱处理会使ANG II测量具有非特异性。此外，在血管紧张素的HPLC分离中，使用甲酸三乙铵作为流动相添加剂更具优势。
