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

Figures

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  Figure 1. Separation of endogenous uACE inhibitors by molecular-weight filtration (a). uACE (>150 kDa in size) activity measured in native urine and in retentates from filtration using 5 kDa, 10 kDa, 30 kDa, and 50 kDa membrane filters. Adding back the <10 kDa filtrate inhibits uACE activity (b). The apparent presence of a 5–10 kDa inhibitor is only due to methodological reasons (c). Each bar represents the mean and standard deviation of three independent experiments. ACE activity values were compared to the activity of 10 kDa retentate. ACE activity values significantly different from uninhibited samples are indicated by asterisks (*).

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  Figure 2. Dilution-corrected uACE activity was measured in serial dilutions (2-fold to 256-fold) (a). Significant increase in activity was observed up to a 128-fold dilution, beyond which no further change occurred. ACE activity values significantly different from those measured at 128-fold dilution are indicated by asterisks (*). uACE activity can be completely inhibited with ACE-inhibitor drugs, lisinopril (b) and captopril (c). The IC50 values for lisinopril and captopril on uACE activity are 2.034 nM and 4.497 nM, respectively. Each symbol represents the mean ± SD of at least three independent measurements.

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  Figure 3. uACE activity was measured in the presence of various peptidase inhibitors: amastatin hydrochloride (aminopeptidase A and N inhibitor) (a), apstatin (Xaa-Pro aminopeptidase inhibitor) (b), bestatin hydrochloride (aminopeptidase B and N inhibitor) (c), MLN-4760 (ACE2 inhibitor) (d), sacubitrilate (neprilysin inhibitor) (e), Z-prolyl-prolinal (prolyl endopeptidase inhibitor) (f), and phosphoramidon disodium salt (neprilysin inhibitor) (g). The effect of phosphoramidon disodium salt on serum ACE activity is shown in subfigure (h). Each symbol represents the mean ± SD of at least three independent measurements, and activities are expressed as percentages of the initial inhibitor-free 10 kDa retentate. uACE activity values that are significantly different from the inhibitor-free values are indicated by asterisks (*). Subfigure (i) shows the residual uACE activity after immunodepletion, compared with the initial sample and the negative control containing only Protein G Sepharose resin (without antibody). Bars represent the mean ± SD of four independent measurements, and a statistically significant difference from the initial sample is indicated by an asterisk (*).

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  Figure 4. ACE activity was measured in purified urine samples (30 kDa retentate) combined with serial dilutions of urea (a), uric acid (b), and urobilinogen (c). Each symbol represents the mean and standard deviation of three independent measurements. IC50 values are indicated for each compound. The green zone indicates the physiological concentration range of each molecule in urine. Because of measurement-related limitations, the gray points serve illustrative purposes only. Significant differences from the initial value are indicated by an asterisk (*).

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  Figure 5. The effect of storage temperature on uACE activity was determined at +25 °C (a), +4 °C (b) and −20 °C (c). Each symbol represents the mean and standard deviation of three independent measurements. Significant differences from the initial value are indicated by an asterisk (*).

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  Figure 6. (a) Distribution of uACE activity and creatinine concentration in individuals not taking ACE inhibitors, shown with median values and interquartile ranges. (b) Correlation between uACE activity and creatinine concentration. Each point represents one individual data point.

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  Figure 7. uACE activity, normalized to creatinine concentration, is shown for patients not receiving ACE inhibitors (n = 20) and for those undergoing ACE inhibitor therapy (n = 33). Each point represents an individual value. Group medians and interquartile ranges are indicated. A significant difference between groups is denoted by the p value.