Urinary ACE Phenotyping as a Research and Diagnostic Tool: Identification of Sex-Dependent ACE Immunoreactivity

Figures

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  Figure 1. ACE activity in human concentrated urine. ACE activity in 12 samples of concentrated (30X) and dialyzed urine was quantified using a spectrofluorometric assay with Z-Phe-His-Leu (1 mM) (A) and Hip-His-Leu (2.5 mM) (B) as substrates (C). Ratio of the rate of hydrolysis of the two substrates (ZPHL/HHL ratio) in the tested samples. Data expressed as % of individual urinary ACE activity from the designated “gold standard” (1A-urine of healthy 22 y.o. male taken as 100%-green bar). Bars with significant changes in % of control ACE activity are colored as follows: increase of more than 20%—orange, >50%—brown, more than 2-fold—red, decrease of more than 20%—yellow, and more than 50%—light blue. Mean values (±SD) from 2 to 5 experiments (each made in triplicates).

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  Figure 2. Sex-dependence in urinary ACE immunoreactivity. ACE activity was precipitated from 30X concentrated urine samples of 4 apparently healthy females (A) and 4 apparently healthy males (B) by 25 mAbs to different epitopes of human somatic ACE. (C). Ratio of ACE activity precipitation from citrated plasma of females to that from males by 25 tested mAbs. Data are expressed as % from ACE activity precipitated from sample #1A (urine from 22 y.o. healthy male) used as a control sample and normalized by binding by mAb 9B9 (mAbs/9B9 ratios). Mean values (±SD) from 2 to 5 experiments (each made in triplicates). Bars are colored as in Figure 1.

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  Figure 3. The 2H9/2D1 binding ratio for ACE derived from several human tissues. ACE activity in the indicated tissue homogenates, or sera from male and female patients, was approximately equilibrated according to their ACE activity. Then, ACE activity from these samples was precipitated using mAbs 2H9 and 2D1 (as in Figure 2). The 2H9/2D1 binding ratio for each tissue was expressed in absolute values (A), or as a % of mean from the female sample values. Shown are mean values (±SD) from 4 to 6 experiments (each made in triplicates). M—male; F—female. Bars colored in (B)—as in Figure 1. Results from 10 male sera and 12 female sera are shown in (C). This experiment was performed in triplicates. The SD is <10% and was not shown for clarity.* Values in colored bars of Figure 3B for males are statistically significant (p < 0.05) from females.

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  Figure 4. Immunoreactivity of urinary ACEs and ACE purified from human lung. ACE activity was precipitated from 30x concentrated urine (A) of 22 y.o. healthy male-sample #1A and ACE preparation purified from male human lung (B) by 25 mAbs to different epitopes of human somatic ACE. Data are expressed as % of ACE activity precipitated by tested mAbs to that by mAb 9B9 used as a control mAb (mean ± SD). (C) Ratio of ACE activity precipitation from male urine to that from ACE purified from male human lung. Values of standard deviations for ratios, which were not exceeded 10%, were not shown for clarity. Bars are colored as in Figure 1. In addition, mAbs in which binding to ACE was less than 10% of that for mAb 9B9 were marked with light green.

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  Figure 5. Model of albumin docking to ACE dimer. A model of human somatic ACE presented as a dimer [26] was used for modeling human albumin docking to ACE. The surfaces of two ACE monomers in a dimer are colored beige and grey, respectively. Albumin structure (PDB 1E78) is shown in pink. Two molecules of albumin can bind to the ACE dimer, but only one albumin molecule is shown to demonstrate the ACE epitopes interacting with albumin. The epitopes for mAbs on the N and C domains of ACE are shown in different colors. Asparagine (N) residues of the putative glycosylation sites are highlighted in lime green.

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  Figure 6. ACE activity and the amount of ACE immunoreactive protein. Urine (200 mL) was collected from two subjects (#1A and #9Q) on each of 4 consecutive days and stored at −20C. On the day of the experiment, urine samples were concentrated 30-fold (using filters with 3 kD pores). A total of 0.5 mL from each sample was dialyzed (using 10 kD pores), and ACE activity was determined spectrofluorometrically using substrate ZPHL (A). The remainder of the concentrated urine samples were used for precipitation of ACE activity by three mAbs (B–D). Correlation of ACE activity with the amount of immunoreactive ACE protein, determined with these three mAbs, was calculated for each subject.