Van Der Gugten J Grace, Holmes Daniel T
Department of Pathology and Laboratory Medicine, University of British Columbia, Rm. G227 - 2211 Wesbrook Mall, Vancouver, BC, V6T 2B5, Canada.
Department of Pathology and Laboratory Medicine, St. Paul's Hospital, 1081 Burrard Street, Vancouver, BC, V6Z 1Y6, Canada.
Methods Mol Biol. 2016;1378:243-53. doi: 10.1007/978-1-4939-3182-8_26.
Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison's disease, congenital adrenal hyperplasia, Bartter and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semi-automated and simple method for the accurate and precise measurement of PRA from 500 μL of plasma (250 μL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 °C in buffering conditions at pH 6, the reaction is quenched with 10 % formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34-30.0 ng/mL which corresponds to PRA values of 0.11-10.0 ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8 to 5.0 % for Bio-Rad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43 ng/mL/h. At AngI concentrations of 0.06 ng/L (corresponding to a PRA of 0.02 ng/mL/h), signal to noise ratios are 50:1 indicating that the limit of quantitation is well below the level required for clinical use.
准确测定血浆肾素活性（PRA）对于原发性醛固酮增多症（PA）有效筛查方案的制定和维持至关重要。PRA测定在肾动脉狭窄、盐皮质激素过多综合征、艾迪生病、先天性肾上腺增生、巴特综合征和吉特曼综合征的调查中也很有用，并且可用于肾素 - 血管紧张素 - 醛固酮系统（RAAS）的遗传性缺陷研究。我们描述了一种半自动且简单的方法，用于使用液相色谱和串联质谱（LC-MS/MS）方法以96孔板形式从500 μL血浆（如讨论的那样，如果省略空白扣除则为250 μL）中准确精确地测量PRA。在pH 6的缓冲条件下于37 °C进行3小时的血管紧张素I（AngI）生成步骤后，用含有AngI内标的10%甲酸淬灭反应。然后进行离线固相萃取、两步洗涤步骤和甲醇洗脱，随后注入LC-MS/MS系统。根据在缓冲液中制备的7点校准线性曲线进行定量。该测定的校准范围为0.34 - 30.0 ng/mL，对应于PRA值为0.11 - 10.0 ng/mL/h：比使用传统基于竞争抗体的方法所能达到的范围宽得多。对于标称PRA值范围为1.73至12.43 ng/mL/h的Bio-Rad高血压对照材料，临床生产中的总精密度已观察到为5.8%至5.0%。在AngI浓度为0.06 ng/L（对应于PRA为0.02 ng/mL/h）时，信噪比为50:1，表明定量限远低于临床使用所需水平。
