Reproducibility of plasma angiotensin-converting enzyme activity in human subjects determined by fluorimetry with Z-phenylalanine-histidyl-leucine as substrate

Abstract

Despite the major physiologic role of angiotensin-converting enzyme (ACE), few studies have evaluated the ideal conditions for measuring human plasma ACE activity, specifically when using Z-phenylalanine-histidyl-leucine as substrate. This study, performed in volunteer patients, assessed the reproducibility of human plasma ACE activity measured by fluorimetry with Z-phenyl-histidyl-leucine as the substrate. After blood centrifugation, plasma was stored under different conditions until processing. The following sources of variability were evaluated: (1) the interval to centrifugation of blood after collection, (2) the temperature and (3) safe time for storing the plasma after cold centrifugation, (4) the effect of fasting. Plasma ACE activity was 20.6 ± 7.7 U/mL, 20.9 ± 8 U/mL, and 20.5 ± 7.9 U/mL (n = 25) when samples were centrifuged immediately, after 1 hour of blood sampling, and after 3 hours of blood sampling, respectively (not significant). In plasma kept at −20°C, ACE activity was not different after 1 week (17.4 ± 4.3 U/mL) nor after 1 month (17.9 ± 4 U/mL), whereas baseline ACE was 16.7 ± 4.3 U/mL (n = 10). In plasma stored at −80°C, ACE activity was 15.5 ± 5.7 U/mL after 1 month (baseline 15 ± 5.3 U/mL; not significant; n = 12). No evidence for hydrolysis of the reaction product of ACE (hisleu dipeptide) was observed in plasma samples kept for 1 month at −20°C or at −80°C (by high-performance liquid chromatography analysis). In plasma obtained before breakfast, ACE activity was 12.8 ± 7.1 U/mL, and it was 12.3 ± 7.5 U/mL 2 hours afterwards (not significant; n = 12). Thus, to determine human plasma ACE activity by fluorimetry with reliability, with Z-phenylalanine-histidyl-leucine used as a substrate, there is a safe interval of at least 3 hours before blood centrifugation at −4°C. Plasma may be kept at −20°C or at −80°C for at least 4 weeks before final processing. Fasting does not influence its enzymatic activity.