Human Tissue Angiotensin Converting Enzyme (ACE) Activity Is Regulated by Genetic Polymorphisms, Posttranslational Modifications, Endogenous Inhibitors and Secretion in the Serum, Lungs and Heart.

Objective: Inhibitors of the angiotensin converting enzyme (ACE) are the primarily chosen drugs to treat heart failure and hypertension. Moreover, an imbalance in tissue ACE/ACE2 activity is implicated in COVID-19. In the present study, we tested the relationships between circulating and tissue (lung and heart) ACE levels in men. Methods: Serum, lung (n = 91) and heart (n = 72) tissue samples were collected from Caucasian patients undergoing lung surgery or heart transplantation. ACE I/D genotype, ACE concentration and ACE activity were determined from serum and tissue samples. Clinical parameters were also recorded. Results: A protocol for ACE extraction was developed for tissue ACE measurements. Extraction of tissue-localized ACE was optimal in a 0.3% Triton-X-100 containing buffer, resulting in 260 ± 12% higher ACE activity over detergent-free conditions. SDS or higher Triton-X-100 concentrations inhibited the ACE activity. Serum ACE concentration correlated with ACE I/D genotype (II: 166 ± 143 ng/mL, n = 19, ID: 198 ± 113 ng/mL, n = 44 and DD: 258 ± 109 ng/mL, n = 28, p < 0.05) as expected. In contrast, ACE expression levels in the lung tissue were approximately the same irrespective of the ACE I/D genotype (II: 1423 ± 1276 ng/mg, ID: 1040 ± 712 ng/mg and DD: 930 ± 1273 ng/mg, p > 0.05) in the same patients (values are in median ± IQR). Moreover, no correlations were found between circulating and lung tissue ACE concentrations and activities (Spearman's p > 0.05). In contrast, a significant correlation was identified between ACE activities in serum and heart tissues (Spearman's Rho = 0.32, p < 0.01). Finally, ACE activities in lung and the serum were endogenously inhibited to similar degrees (i.e., to 69 ± 1% and 53 ± 2%, respectively). Conclusion: Our data suggest that circulating ACE activity correlates with left ventricular ACE, but not with lung ACE in human. More specifically, ACE activity is tightly coordinated by genotype-dependent expression, endogenous inhibition and secretion mechanisms.

Chitotriosidase gene polymorphisms and mutations limit the determination of chitotriosidase expression in sarcoidosis.

Serum chitotriosidase (CTO) activity was proposed as a biomarker in sarcoidosis being potentially useful in diagnostics. Nevertheless, a common duplication polymorphism (c.1049_1072dup24, Dup24) of the CTO gene influences CTO activity and thereby compromises its use in sarcoidosis. Here we aimed to substitute CTO activity with CTO concentration to prevent the confounding effect of Dup24. CTO activity, concentration and genetic backgrounds were determined in 80 histopathology proven sarcoidosis patients and 133 healthy individuals. CTO activities were lower in healthy individuals and sarcoidosis patients heterozygous for Dup24 mutation (472 ± 367 mU/L, n = 49; 2300 ± 2105 mU/L, n = 29) than in homozygous wild types (838 ± 856 mU/L, n = 81; 5125 ± 4802 mU/L, n = 48; p < 0.001, respectively). Sera of Dup24 homozygous individuals had no CTO activity. CTO concentrations were also lower in healthy individuals and sarcoidosis patients heterozygous for Dup24 mutation (7.2 ± 1.9 µg/L, n = 11; 63.16 ± 56.5 µg/L, n = 29) than in homozygous wild types (18.9 ± 13.0 µg/L, n = 36; 157.1 ± 132.4 µg/L, n = 47, p < 0.001, respectively) suggestive for an interaction between Dup24 mutation and CTO concentration determinations. We also identified a healthy Hungarian male subject without CTO activity carrying a rare mutation (c.(965_993)del), which mutation has been considered unique for Cypriot population to date. Taken together, CTO concentration determination does not add to the CTO activity measurement when CTO is used as a biomarker in sarcoidosis. Therefore, genotyping of CTO gene should be involved in the interpretation of laboratory findings.

Angiotensin Converting Enzyme Activity in Anti-TNF-Treated Rheumatoid Arthritis and Ankylosing Spondylitis Patients.

INTRODUCTION: Angiotensin-converting enzyme (ACE) and ACE2 have been implicated in the regulation of vascular physiology. Elevated synovial and decreased or normal ACE or ACE2 levels have been found in rheumatoid arthritis (RA). Very little is known about the effects of tumor necrosis factor α (TNF-α) inhibition on ACE or ACE2 homeostasis. In this study, we assessed the effects of one-year anti-TNF therapy on ACE and ACE2 production in RA and ankylosing spondylitis (AS) in association with other biomarkers. PATIENTS AND METHODS: Forty patients including 24 RA patients treated with either etanercept (ETN) or certolizumab pegol (CZP) and 16 AS patients treated with ETN were included in a 12-month follow-up study. Serum ACE levels were determined by commercial ELISA, while serum ACE2 activity was assessed using a specific quenched fluorescent substrate. Ultrasonography was performed to determine flow-mediated vasodilation (FMD), common carotid intima-media thickness (ccIMT) and arterial pulse-wave velocity (PWV) in all patients. In addition, CRP, rheumatoid factor (RF) and ACPA were also measured. All assessments were performed at baseline and 6 and 12 months after treatment initiation. RESULTS: Anti-TNF therapy increased ACE levels in the full cohort, as well as in the RA and AS subsets. ACE2 activity increased in the full cohort, while the ACE/ACE2 ratio increased in the full cohort and in the RA subset (p < 0.05). Uni- and multivariable regression analyses determined associations between ACE or ACE/ACE2 ratios at different time points and disease duration, CRP, RF, FMD and IMT (p < 0.05). ACE2 activity correlated with CRP. The changes of ACE or ACE2 over 12 months were determined by treatment together with either RF or FMD (p < 0.05). CONCLUSIONS: Anti-TNF treatment may increase ACE and ACE2 in the sera of RA and AS patients. ACE and ACE2 may be associated with disease duration, markers of inflammation and vascular pathophysiology. The effects of TNF inhibition on ACE and ACE2 may reflect, in part, the effects of these biologics on the cardiovascular system.

Combined application of angiotensin converting enzyme and chitotriosidase analysis improves the laboratory diagnosis of sarcoidosis.

Establishing the diagnosis of sarcoidosis most often requires biopsy and histopathologic evaluation, since there is no single marker with sufficient specificity and sensitivity for the disease. Our aims were to determine and compare the diagnostic accuracies of several potential biomarkers and to develop a combined biomarker analysis tool for the diagnosis of sarcoidosis. 133 healthy individuals and 104 patients with suspected sarcoidosis and diagnostic thoracic surgery were enrolled into this study. Histopathologic results were contrasted to biomarker levels of chitotriosidase (CTO), serum amyloid-A (SAA), soluble interleukin-2 receptor (sIL-2R), lysozyme (LZM) or angiotensin converting enzyme (ACE). Sarcoidosis was confirmed by histopathology in 69 patients. CTO activity, sIL-2R concentration and ACE activity could discriminate between sarcoidosis and control patients, while SAA and LZM concentrations could not. A new combined parameter, which was derived from the multiplication of ACE by CTO activities (double product) showed the best diagnostic accuracy in this clinical study: (AUC = 0.898, sensitivity: 90.5%, specificity: 79.3%, positive and negative predictive values: 90.5% and 79.3%, respectively). Sarcoidosis can be diagnosed with the combined analysis of ACE and CTO activities more accurately than with single serum biomarkers in the absence of invasive biopsy in the majority of cases with pulmonary manifestation of sarcoidosis.

Optimized angiotensin-converting enzyme activity assay for the accurate diagnosis of sarcoidosis.

BACKGROUND: Serum angiotensin-converting enzyme (ACE) activity determination can aid the early diagnosis of sarcoidosis. We aimed to optimize a fluorescent kinetic assay for ACE activity by screening the confounding effects of endogenous ACE inhibitors and interfering factors. Genotype-dependent and genotype-independent reference values of ACE activity were established, and their diagnostic accuracies were validated in a clinical study. METHODS: Internally quenched fluorescent substrate, Abz-FRK(Dnp)P-OH was used for ACE-activity measurements. A total of 201 healthy individuals and 59 presumably sarcoidotic patients were enrolled into this study. ACE activity and insertion/deletion (I/D) genotype of the ACE gene were determined. RESULTS: Here we report that serum samples should be diluted at least 35-fold to eliminate the endogenous inhibitor effect of albumin. No significant interferences were detected: up to a triglyceride concentration of 16 mM, a hemoglobin concentration of 0.71 g/L and a bilirubin concentration of 150 µM. Genotype-dependent reference intervals were considered as 3.76-11.25 U/L, 5.22-11.59 U/L, 7.19-14.84 U/L for II, ID and DD genotypes, respectively. I/D genotype-independent reference interval was established as 4.85-13.79 U/L. An ACE activity value was considered positive for sarcoidosis when it exceeded the upper limit of the reference interval. The optimized assay with genotype-dependent reference ranges resulted in 42.5% sensitivity, 100% specificity, 100% positive predictive value and 32.4% negative predictive value in the clinical study, whereas the genotype-independent reference range proved to have inferior diagnostic efficiency. CONCLUSIONS: An optimized fluorescent kinetic assay of serum ACE activity combined with ACE I/D genotype determination is an alternative to invasive biopsy for confirming the diagnosis of sarcoidosis in a significant percentage of patients.