Bragat A C, Blumenfeld J, Sealey J E
Cardiovascular Center, Cornell University Medical College, New York 10021, USA.
J Hypertens. 1997 May;15(5):459-65.
Angiotensin II (Ang II) levels are normally very low in human plasma, approximately 5 pg/ml. They are usually measured by radioimmunoassay after extraction and concentration. An additional high-performance liquid chromatography (HPLC) step is reportedly necessary for accurate measurement but it is laborious and time-consuming, severely limiting the number of samples that can be assayed.
To investigate whether the HPLC step was necessary for measuring Ang II in human plasma samples in our laboratory using our own Ang II antiserum.
Human plasma Ang II levels, measured with and without the HPLC step, were compared in two different studies. Since the action of renin is the rate-limiting step in the production of Ang II in plasma, the relationships of plasma renin activity (PRA) to Ang II levels measured with and without HPLC were also evaluated. In the first study, 108 blood samples were collected from 29 hypertensive patients during placebo or treatment with the Ang II antagonist BMS-186295. In the second study blood samples were collected from 12 normal subjects before and during beta-adrenergic blockade.
In samples collected during angiotensin II antagonism, which predictably increased plasma Ang II levels, a highly significant relationship between the Ang II measurements with and without HPLC was found (y = 0.99x + 1.7; r = 0.97, P < 0.001). The y intercept of 1.7 pg/ml suggested that the nonspecific immunoreactivity was close to 2 pg/ml in samples assayed without the HPLC step. During beta-adrenergic blockade, which predictably suppressed plasma renin levels, highly significantly linear relationships between HPLC and non-HPLC Ang II measurements (y = 1.3x + 1.6; r = 0.93. P < 0.001, n = 16) and between non HPLC Ang II and PRA (y = 1.9x + 1.7; r = 0.73, P < 0.001, n = 108) were again found. The relationship between PRA and HPLC Ang II was also highly significant (y = 1.4x + 0.04; r = 0.92, P < 0.001, n = 16), but the y intercept was significantly lower (P < 0.001), approaching zero, indicating the removal of nonspecific immunoreactivity during the HPLC step.
These results demonstrate once more that, when using polyclonal antibody 182, the accuracy of the Ang II measurement in human plasma is improved by the inclusion of a HPLC step, especially for samples with Ang II levels in the normal-to-low range. They also show that plasma Ang II and PRA increase or decrease proportionally during treatment with Ang II antagonists or beta-adrenergic blockade, respectively.
人血浆中血管紧张素II（Ang II）水平通常非常低，约为5皮克/毫升。通常在提取和浓缩后通过放射免疫测定法进行测量。据报道，为了进行准确测量，还需要额外的高效液相色谱（HPLC）步骤，但这既费力又耗时，严重限制了可检测的样本数量。
使用我们自己的Ang II抗血清，研究在我们实验室中测量人血浆样本中的Ang II时HPLC步骤是否必要。
在两项不同的研究中比较了有或没有HPLC步骤时测量的人血浆Ang II水平。由于肾素的作用是血浆中Ang II产生的限速步骤，因此还评估了血浆肾素活性（PRA）与有或没有HPLC时测量的Ang II水平之间的关系。在第一项研究中，从29名高血压患者在安慰剂治疗期间或使用Ang II拮抗剂BMS - 186295治疗期间采集了108份血样。在第二项研究中，从12名正常受试者在β - 肾上腺素能阻滞剂治疗前和治疗期间采集了血样。
在血管紧张素II拮抗作用期间采集的样本中，血浆Ang II水平可预见地升高，发现有和没有HPLC时Ang II测量值之间存在高度显著的关系（y = 0.99x + 1.7；r = 0.97，P < 0.001）。1.7皮克/毫升的y轴截距表明，在没有HPLC步骤的样本中，非特异性免疫反应性接近2皮克/毫升。在β - 肾上腺素能阻滞剂治疗期间，血浆肾素水平可预见地受到抑制，再次发现HPLC和非HPLC Ang II测量值之间存在高度显著的线性关系（y = 1.3x + 1.6；r = 0.93，P < 0.001，n = 16）以及非HPLC Ang II和PRA之间存在高度显著的线性关系（y = 1.9x + 1.7；r = 0.73，P < 0.001，n = 108）。PRA与HPLC Ang II之间的关系也高度显著（y = 1.4x + 0.04；r = 0.92，P < 0.001，n = 16），但y轴截距显著更低（P < 0.001），接近零，表明在HPLC步骤中去除了非特异性免疫反应性。
这些结果再次证明，当使用多克隆抗体182时，通过加入HPLC步骤可提高人血浆中Ang II测量的准确性，特别是对于Ang II水平处于正常至低范围的样本。它们还表明，在使用Ang II拮抗剂或β - 肾上腺素能阻滞剂治疗期间，血浆Ang II和PRA分别成比例地增加或减少。
