De Silva P E, Husain A, Smeby R R, Khairallah P A
Research Institute, Cleveland Clinic Foundation, Ohio 44195-5071.
Anal Biochem. 1988 Oct;174(1):80-7. doi: 10.1016/0003-2697(88)90521-0.
Angiotensin II, the major effector peptide of the renin-angiotensin system, is an endocrine and paracrine regulator of tissue function. To determine its physiological role, it is important to quantify angiotensin II and related fragment peptides in tissues and plasma as a first step toward understanding angiotensin II metabolism within tissues. A fully characterized, sensitive, and reproducible immunochemical assay has been developed for quantitating angiotensin II immunoreactivity in tissues and plasma. We identified two methodological events of critical importance, incompletely addressed in previously reported studies. First, the nonspecific interference resulting from Sep-Pak processing was found to be due to hydrophobic impurities in the octade-casilane absorbent which were eliminated by washing the Sep-Pak with tetrahydrofuran and hexane before use. Second, a significant discrepancy was observed in the recoveries of angiotensin II and 125I-angiotensin II added to tissue extracts following high-pressure liquid chromatography. Angiotensin II immunoreactivity extracted from decapitated rat adrenal gland, brain, and kidney (target organs for angiotensin II), ovary and uterus (potential target organs for angiotensin II), and plasma has been characterized. The predominant component of the angiotensin II immunoreactivity was the biologically active octapeptide angiotensin II. However, in the brain, the ratio of angiotensin II to C-terminal angiotensin II immunoreactive fragments was lower than observed in other tissues studied. Other angiotensin II C-terminal immunoreactive peptide fragments-the biologically active heptapeptide and the biologically inactive angiotensin(3-8) and angiotensin(4-8)--were also detected in variable quantities in the various tissues.
血管紧张素II是肾素-血管紧张素系统的主要效应肽，是一种组织功能的内分泌和旁分泌调节因子。为了确定其生理作用，作为了解组织内血管紧张素II代谢的第一步，对组织和血浆中的血管紧张素II及相关片段肽进行定量非常重要。已经开发出一种完全表征的、灵敏且可重复的免疫化学测定法，用于定量组织和血浆中的血管紧张素II免疫反应性。我们确定了两个至关重要的方法学事件，而先前报道的研究并未完全解决这些问题。首先，发现Sep-Pak处理产生的非特异性干扰是由于十八烷基硅烷吸附剂中的疏水杂质所致，在用四氢呋喃和己烷在使用前洗涤Sep-Pak可消除这些杂质。其次，在高压液相色谱后添加到组织提取物中的血管紧张素II和125I-血管紧张素II的回收率中观察到显著差异。已对从断头大鼠的肾上腺、脑、肾（血管紧张素II的靶器官）、卵巢和子宫（血管紧张素II的潜在靶器官）以及血浆中提取的血管紧张素II免疫反应性进行了表征。血管紧张素II免疫反应性的主要成分是具有生物活性的八肽血管紧张素II。然而，在脑中，血管紧张素II与C末端血管紧张素II免疫反应性片段的比率低于在其他研究组织中观察到的比率。在各种组织中还检测到了其他血管紧张素II C末端免疫反应性肽片段——具有生物活性的七肽以及无生物活性的血管紧张素(3-8)和血管紧张素(4-8)，其含量各不相同。
