Interference-Free Measurement of Urinary Angiotensin-Converting Enzyme (ACE) Activity: Diagnostic and Therapeutic Monitoring Implications

<b>Background/Objectives</b>: Urinary angiotensin-converting enzyme (uACE) activity has long been regarded as a promising biomarker for kidney and cardiovascular diseases; however, its clinical applicability has been limited by the presence of endogenous urinary inhibitors and technically demanding assay protocols. We aimed to establish a fast and reproducible method for measuring uACE activity to identify the inhibitory compounds responsible for previous assay failures and to define practical preanalytical conditions suitable for routine laboratory implementation. <b>Methods</b>: A fluorescence-based kinetic assay was optimized for urine samples. Endogenous inhibitors were isolated by membrane filtration and chemically characterized, while the effect of sample dilution was evaluated as a simplified alternative for eliminating inhibitory interference. We assessed the stability of ACE activity under various storage conditions to support reliable measurement. <b>Results</b>: Urea (IC₅₀ = 1.18 M), uric acid (IC₅₀ = 3.61 × 10⁻³ M), and urobilinogen (IC₅₀ = 2.98 × 10⁻⁴ M) were identified as the principal reversible inhibitors, jointly accounting for up to 90% suppression of uACE activity. Their inhibitory effect was effectively eliminated by a 128-fold dilution. ACE activity remained stable for 24 h at 25 °C but was completely lost after freezing. A strong positive correlation between uACE activity and creatinine concentration (r = 0.76, <i>p</i> < 0.0001) justified normalization. ACE activity-to-creatinine ratio turned out to be significantly lower in ACE inhibitor-treated patients than in untreated controls (6.49 vs. 36.69 U/mol, <i>p</i> < 0.0001). <b>Conclusions</b>: Our findings demonstrate that accurate measurement of uACE activity is feasible using a rapid dilution-based protocol. The normalized ACE activity can serve as a practical biomarker for detecting pharmacological ACE inhibition and monitoring therapy adherence in cardiovascular care and may also provide insight into renal pathophysiology such as tubular injury or local RAAS-related processes.