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This study investigated whether patients receiving renin-angiotensin system inhibitors (RASIs) can undergo reliable screening for primary aldosteronism (PA) without discontinuation of therapy. Consecutive patients with hypertension who underwent PA screening at our hospital between 2016 and 2024 while on RASIs were recruited. Plasma aldosterone concentration (PAC) and direct renin concentration (DRC) were measured at three time points: pre-washout, post-washout, and post-captopril-challenge test (CCT). Subsequently, the aldosterone-to-renin ratio (ARR) was calculated, and PAC, DRC, and ARR were compared across time points. Receiver operating characteristic (ROC) curve analysis was performed to determine optimal diagnostic cutpoints. A total of 412 patients on RASIs, with or without calcium-channel blockers (CCBs) or α1-receptor antagonists, were analyzed. Among these, 175 had PA and 237 had essential hypertension (EH). PAC, DRC, and ARR were significantly different between the PA and EH groups across all three time points. Within the PA cohort, PAC (p < 0.001) and ARR (p = 0.016) differed significantly between the pre-washout and post-CCT measurements, whereas DRC did not (p = 0.456). The optimal pre-washout ARR cutpoint of 2.69 demonstrated a sensitivity of 83.3%, specificity of 87.2%, positive predictive value (PPV) of 82.2%, and negative predictive value (NPV) of 88.1% for diagnosing PA. These findings indicate that pre-washout PAC, DRC, and ARR retain high diagnostic performance for PA in patients treated with RASIs, provided that other agents affecting the renin-angiotensin-aldosterone system are not co-administered.