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

Alexandra Csongrádi; Attila Enyedi; István Takács; Tamás Végh; Ivetta S. Mányiné; Zsófia Pólik; István Tibor Altorjay; József Balla; György Balla; István Édes; János Kappelmayer; Attila Tóth; Zoltán Papp; Miklós Fagyas

doi:10.1515/cclm-2017-0837

Article

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

Alexandra Csongrádi , Attila Enyedi , István Takács , Tamás Végh , Ivetta S. Mányiné , Zsófia Pólik , István Tibor Altorjay , József Balla , György Balla , István Édes , János Kappelmayer , Attila Tóth , Zoltán Papp and Miklós Fagyas

Published/Copyright: February 9, 2018

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information

From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 56 Issue 7

Abstract

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.

Keywords: angiotensin-converting enzyme (ACE); genotype; reference interval; sarcoidosis

Corresponding author: Miklós Fagyas, MD, PhD, Specialist in Laboratory Medicine, Assistant Professor, Division of Clinical Physiology, Department of Cardiology, Faculty of Medicine, University of Debrecen, 22 Moricz Zsigmond Str., 4032 Debrecen, Hungary, Phone: +3652-411717; extension: 55404

aAlexandra Csongrádi and Attila Enyedi contributed equally to this work.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: The work was supported by the National Research, Development and Innovation Office – NKFIH, PD 116212 and K 116940 (Miklós Fagyas and Attila Tóth), ÚNKP-17-4-I-DE-40 the New National Excellence Program of the Ministry of Human Capacities (Hungary, to Miklós Fagyas) and the GINOP-2.3.2-15-2016-00043 project. The project is cofinanced by the European Union and the European Regional Development Fund.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Haimovic A, Sanchez M, Judson MA, Prystowsky S. Sarcoidosis: a comprehensive review and update for the dermatologist. J Am Acad Dermatol 2012;66:719.e1–10.10.1016/j.jaad.2012.02.003Search in Google Scholar PubMed

2. Costabel U, Hunninghake GW. ATS/ERS/WASOG statement on sarcoidosis. Sarcoidosis Statement Committee. American Thoracic Society. European Respiratory Society. World Association for sarcoidosis and other granulomatous disorders. Eur Respir J 1999;14:735–7.10.1034/j.1399-3003.1999.14d02.xSearch in Google Scholar PubMed

3. Grutters JC, Fellrath J-M, Mulder L, Janssen R, van den Bosch JM, van Velzen-Blad H. Serum soluble interleukin-2 receptor measurement in patients with sarcoidosis: a clinical evaluation. Chest 2003;124:186–95.10.1378/chest.124.1.186Search in Google Scholar PubMed

4. Grosso S, Margollicci MA, Bargagli E, Buccoliero QR, Perrone A, Galimberti D, et al. Serum levels of chitotriosidase as a marker of disease activity and clinical stage in sarcoidosis. Scand J Clin Lab Invest 2004;64:57–62.10.1080/00365510410004092Search in Google Scholar PubMed

5. Casanova N, Zhou T, Knox KS, Garcia JG. Identifying novel biomarkers in sarcoidosis using genome-based approaches. Clin Chest Med 2015;36:621–30.10.1016/j.ccm.2015.08.005Search in Google Scholar PubMed PubMed Central

6. Chopra A, Kalkanis A, Judson MA. Biomarkers in sarcoidosis. Expert Rev Clin Immunol 2016;12:1191–208.10.1080/1744666X.2016.1196135Search in Google Scholar PubMed

7. Pfeffer MA, Braunwald E, Moyé LA, Basta L, Brown EJ, Cuddy TE, et al. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators. N Engl J Med 1992;327:669–77.10.1056/NEJM199209033271001Search in Google Scholar PubMed

8. Oppong SY, Hooper NM. Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane. Biochem J 1993;292(Pt 2):597–603.10.1042/bj2920597Search in Google Scholar PubMed PubMed Central

9. Metzger R, Franke FE, Bohle RM, Alhenc-Gelas F, Danilov SM. Heterogeneous distribution of angiotensin I-converting enzyme (CD143) in the human and rat vascular systems: vessel, organ and species specificity. Microvasc Res 2011;81:206–15.10.1016/j.mvr.2010.12.003Search in Google Scholar PubMed

10. Fagyas M, Úri K, Siket IM, Daragó A, Boczán J, Bányai E, et al. New perspectives in the renin-angiotensin-aldosterone system (RAAS) III: endogenous inhibition of angiotensin converting enzyme (ACE) provides protection against cardiovascular diseases. PLoS One 2014;9:e93719.10.1371/journal.pone.0093719Search in Google Scholar PubMed PubMed Central

11. Sheffield EA. Pathology of sarcoidosis. Clin Chest Med 1997;18:741–54.10.1016/S0272-5231(05)70416-0Search in Google Scholar

12. Lynch JP, Kazerooni EA, Gay SE. Pulmonary sarcoidosis. Clin Chest Med 1997;18:755–85.10.1016/S0272-5231(05)70417-2Search in Google Scholar

13. Fagyas M, Úri K, Siket IM, Daragó A, Boczán J, Bányai E, et al. New perspectives in the renin-angiotensin-aldosterone system (RAAS) I: endogenous angiotensin converting enzyme (ACE) inhibition. PLoS One 2014;9:e87843.10.1371/journal.pone.0087843Search in Google Scholar

14. Fagyas M, Úri K, Mányiné IS, Fülöp GÁ, Csató V, Daragó A, et al. New perspectives in the renin-angiotensin-aldosterone system (RAAS) II: albumin suppresses angiotensin converting enzyme (ACE) activity in human. PLoS One 2014;9:e87844.10.1371/journal.pone.0087844Search in Google Scholar

15. Carmona AK, Schwager SL, Juliano MA, Juliano L, Sturrock ED. A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay. Nat Protoc 2006;1:1971–6.10.1038/nprot.2006.306Search in Google Scholar

16. Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res 1992;20:1433.10.1093/nar/20.6.1433-aSearch in Google Scholar

17. Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer MJ, Grodstein F, LaMotte F, et al. A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 1995;332:706–11.10.1056/NEJM199503163321103Search in Google Scholar

18. Rodriguez S, Gaunt TR, Day IN. Hardy-Weinberg equilibrium testing of biological ascertainment for Mendelian randomization studies. Am J Epidemiol 2009;169:505–14.10.1093/aje/kwn359Search in Google Scholar

19. Bunting PS, Szalai JP, Katic M. Diagnostic aspects of angiotensin converting enzyme in pulmonary sarcoidosis. Clin Biochem 1987;20:213–9.10.1016/S0009-9120(87)80123-6Search in Google Scholar

20. Lieberman J, Beutler E. Elevation of serum angiotensin-converting enzyme in Gaucher’s disease. N Engl J Med 1976;294:1442–4.10.1056/NEJM197606242942609Search in Google Scholar PubMed

21. Lieberman J, Rea TH. Serum angiotensin-converting enzyme in leprosy and coccidioidomycosis. Ann Intern Med 1977;87:423–5.10.7326/0003-4819-87-4-422Search in Google Scholar

22. Alves MF, Araujo MC, Juliano MA, Oliveira EM, Krieger JE, Casarini DE, et al. A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity. Braz J Med Biol Res 2005;38:861–8.10.1590/S0100-879X2005000600007Search in Google Scholar

23. Lieberman J, Sastre A. An angiotensin-converting enzyme (ACE) inhibitor in human serum. Increased sensitivity of the serum ACE assay for detecting active sarcoidosis. Chest 1986;90:869–75.10.1378/chest.90.6.869Search in Google Scholar

24. Saxe AW, Hollinger MA, Essam T. Effect of bilirubin on the spectrophotometric and radionuclide assay for serum angiotensin-converting enzyme. Res Commun Chem Pathol Pharmacol 1986;51:129–36.Search in Google Scholar

25. Danilov SM, Lünsdorf H, Akinbi HT, Nesterovitch AB, Epshtein Y, Letsiou E, et al. Lysozyme and bilirubin bind to ACE and regulate its conformation and shedding. Sci Rep 2016;6:34913.10.1038/srep34913Search in Google Scholar

26. Dimeski G. Interference testing. Clin Biochem Rev 2008;29(Suppl 1):S43–8.Search in Google Scholar

27. Clinical and Laboratory Standards Institute (CLSI). Defining, establishing, and verifying reference intervals in the Clinical Laboratory, Wayne, PA, USA: CLSI document C28-A3c, 3rd ed. 2010:72.Search in Google Scholar

28. Henny J, Vassault A, Boursier G, Vukasovic I, Mesko Brguljan P, Lohmander M, et al. Recommendation for the review of biological reference intervals in medical laboratories. Clin Chem Lab Med 2016;54:1893–900.10.1515/cclm-2016-0793Search in Google Scholar

29. Biller H, Zissel G, Ruprecht B, Nauck M, Busse Grawitz A, Muller-Quernheim J. Genotype-corrected reference values for serum angiotensin-converting enzyme. Eur Respir J 2006;28:1085–91.10.1183/09031936.00050106Search in Google Scholar

30. Sharma P, Smith I, Maguire G, Stewart S, Shneerson J, Brown MJ. Clinical value of ACE genotyping in diagnosis of sarcoidosis. Lancet 1997;349:1602–3.10.1016/S0140-6736(05)61631-5Search in Google Scholar

31. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86:1343–6.10.1172/JCI114844Search in Google Scholar PubMed PubMed Central

32. Camós S, Cruz MJ, Morell F, Solé E. Genetic-based reference values for angiotensin-converting enzyme (ACE) according to I/D polymorphism in a Spanish population sample. Clin Chem Lab Med 2012;50:1749–53.10.1515/cclm-2012-0042Search in Google Scholar PubMed

33. Nissen PH, Campbell NB, Højskov CS, Fløe A, Hoffmann HJ, Hilberg O, et al. Development of a high-resolution melting genotyping assay for the angiotensin I converting enzyme insertion/deletion polymorphism and establishment of genotype-specific reference intervals in a Danish population. Ann Clin Biochem 2015;52:105–12.10.1177/0004563214529261Search in Google Scholar PubMed

Received: 2017-9-15

Accepted: 2018-1-7

Published Online: 2018-2-9

Published in Print: 2018-6-27

You are currently not able to access this content.

Articles in the same Issue

https://doi.org/10.1515/cclm-2017-0837

Keywords for this article

angiotensin-converting enzyme (ACE); genotype; reference interval; sarcoidosis