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Angiotensin-converting enzyme (ACE) plays an important role in the renin-angiotensin system and ACE activity is usually assayed in vitro by monitoring the transformation from a substrate to the product catalyzed by ACE. A rapid and sensitive analysis method or ACE activity by quantifying simultaneously the substrate hippuryl-histidyl-leucine and its product hippuric acid using an ultra-performance liquid chromatography coupled with electrospray ionization-mass spectrometry (UPLC-MS) was first developed and applied to assay the inhibitory activities against ACE of several natural phenolic compounds. The established UPLC-MS method showed obvious advantages over the conventional HPLC analysis in shortened running time (3.5 min), lower limit of detection (5 pg) and limit of quantification (18 pg), and high selectivity aided by MS detection in selected ion monitoring (SIM) mode. Among the six natural products screened, five compounds, caffeic acid, caffeoyl acetate, ferulic acid, chlorogenic acid and resveratrol indicated potent in vitro ACE inhibitory activity with IC(50) values of 2.527 +/- 0.032, 3.129 +/- 0.016, 10.898 +/- 0.430, 15.076 +/- 1.211 and 6.359 +/- 0.086 mm, respectively. A structure-activity relationship estimation suggested that the number and the situation of the hydroxyls on the benzene rings and the acrylic acid groups may play the most predominant role in their ACE inhibitory activity., (2009 John Wiley & Sons, Ltd.)

A high-performance liquid chromatographic method for indirect determination of enalapril in human plasma, was developed and validated. An exogenous angiotensin converting enzyme after drug inhibition was determined by reacting with hippuryl-histidyl-leucine to produce hippuric acid (HA) which was inversely proportional to the amount of enalaprilat in plasma. The HPLC was carried out on a Lichrosphere 60RP-select B, C18, 5 microm (125 mm x 4.0 mm i.d.) column at flow rate of 1.0 ml/min. The analysis time per injection was within 6.5 min. The lowest concentration of enalaprilat to be quantitated was 3.0 ng/ml with the acceptable accuracy and precision. This successfully developed method was practically and accurately used for pharmacokinetics and bioequivalent study of enalapril.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.

A novel sensitive and specific method for the measurement of tissue angiotensin-converting enzyme (ACE) activity utilizing HPLC is described. ACE activity was determined in detergent-extracted canine hearts utilizing the synthetic ACE-specific substrate hippuryl histidyl leucine (HHL), both in the presence and the absence of the site-specific inhibitor captopril. Tissue ACE activity was quantitated from the moles of hippuric acid (HA) formed, in time-fixed assays, utilizing HPLC separation of HA from HHL and UV-spectrophotometry for quantitation of HA as in the standard Cushman and Cheung assay (Cushman DW and Cheung HS, Biochem Pharmacol 20: 1637-1648, 1971). Separation of HA from HHL was performed by reverse phase HPLC on a phenyl silica gel column with an eluent consisting of 20% acetonitrile in 0.1 M aqueous ammonium phosphate buffer, pH 6.8. After the standard liquid/liquid extraction procedure with ethyl acetate, HPLC analysis revealed the presence of unreacted substrate, HHL, in amounts comparable to the product of interest, HA, in the final assay; moreover, the amount of HA formed did not fall completely to zero in the presence of captopril. Regional studies of canine cardiac ACE activity utilizing the HPLC-based assay and the standard assay method showed a significantly higher ACE activity in the right ventricle compared with the left ventricle (2.37 +/- 0.7 vs 1.24 +/- 0.18 mU/g, P < 0.05 [N = 6], respectively) in the HPLC-based assay, but no difference in right and left ventricular ACE activities by the standard assay (0.25 +/- 0.08 vs 0.31 +/- 0.09 mU/g [N = 6], respectively). Kinetic studies utilizing the HPLC-based assay coupled with the use of captopril showed Km (1.34 +/- 0.08 mM) and Vmax (36.8 +/- 11.5 x 10(-10) M/min) values in agreement with those in the literature. Our results demonstrate that the application of HPLC to the standard Cushman and Cheung assay improves the sensitivity and specificity of the standard assay and enables the use of much smaller amounts (approximately 4 vs approximately 400 mg for the Cushman and Cheung assay) of tissue for ACE activity assay.

We compared the effects of acute and chronic ACE inhibition (ACEi) on the 123I-hippurate in the stenotic kidney of two 2-kidney, 1-clip hypertensive dogs. In the period after clip implantation poststenotic renograms without ACEi of both dogs were normal. Acute ACEi always resulted in delayed hippurate handling. Chronic ACEi, however, induced abnormal poststenotic renograms in only 36% of the cases. Withdrawal of chronic ACEi restored the phenomenon of acute ACE-induced delayed hippurate handling within 5 months in both dogs. These data indicate that chronic ACEi or recent ACEi medication reduces the effectiveness of ACEi renography in diagnosing hypertension due to a moderate renal-artery stenosis. This phenomenon may explain why the sensitivity of ACEi renography in human studies varies more than in animal studies.

This assay of angiotensin-converting enzyme is based on release of hippuric acid from a synthetic tripeptide, hippuryl-L-histidyl-L-leucine. Hippuric acid is separated and quantitated on a reversed-phase column under conditions similar to those used for assay of theophylline and acetaminophen. The method is rapid and free from interferences.

The activity of serum angiotensin converting enzyme (ACE), assayed fluorometrically with the substrate hippuryl-L-histidyl-L-leucine was significantly higher in 116 patients with sarcoidosis than in 415 patients with other diseases--pulmonary and nonpulmonary--and in 58 normal subjects. Serum ACE was elevated in 59% of 46 recently diagnosed, untreated patients with sarcoidosis and was significantly reduced following steroid therapy in three patients with sarcoidosis, but in only one of four patients treated with placebos. The results suggest that elevated serum ACE activity is a valuable, although not absolutely specific, diagnostic indicator of sarcoidosis. It should be stressed that a normal value does not rule out the disease, and that steroid therapy tends to reduce the serum ACE activity.

This optimized two-point kinetic assay for serum angiotensin-converting enzyme is based on the colorimetric determination of hippurate with cyanuric chloride/dioxan reagent. The hippurate is released from hippuryl-L-histidyl-L-leucine by angiotensin-converting enzyme in the presence of chloride ion. CVs for the method (within-run and between-run) ranged from 2.1 to 3.2%. Linearity extends up to 200 U/L. Results are unaffected by lipemia and icterus. Hemoglobin in concentrations greater than 1.5 g/L causes a slight negative interference. Reference intervals for men and women are 22-82 U/L and 25-69 U/L, respectively.

The angiotensin I-coverting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) was isolated from both guinea pig lung and serum; Km and V values were determined using both angiotensin I and hippurylhistidylleucine as substrates. Km values for the lung enzyme were 3.1 mM for hippurylhistidylleucine hippurylhistidylleucine and 0.076 mM for angiotensin I. Inhibition studies were performed and I50 values were obtained with the following inhibitors: angiotensin II (lung, 1.9 - 10(-5) M; serum, 1.7 - 10(-5) M), bradykinin (lung, 2.6 - 10(-6) M; serum, 2.1 - 10(-6) M), and pyrrolidone-Lys-Trp-Ala-Pro (lung, 7.9 - 10(-8) M; serum, 5.6 - 10(-8) M). Both enzymes were glycoproteins and were inhibited by concanavalin A. A maximum inhibition of 35% initial enzymatic activity was observed for both enzymes at a concanavalin A concentration of 4 - 10(-4) M suggesting that the sugar moieties of each enzyme are similar. Both enzymes required NaCl for activity and were inhibited by EDTA. A comparison of kinetic and inhibition properties indicates that both enzymes are quite similar.

Serum angiotensin-converting enzyme (ACE) was studied in 50 patients with sarcoidosis (39 active, 11 inactive) as well as in 50 control patients (34 with chronic nonspecific lung disease, 9 with Hodgkin and 7 with rheumatoid arthritis). There was a significant difference of ACE activity between sarcoidosis patients (28.6 +/- 11.4) and controls (14.8 +/- 4), and also between active (32.8 +/- 11) and the inactive (21.9 +/- 5.1) sarcoidosis (p less than 0.001). Coricosteroid treatment seems to lower ACE activity in patients with sarcoidosis without offering a clue for clinical improvement. Increased ACE activity in other granulomatous disorders is being discussed. ACE activity thus proves to be a valuable test especially in differentiating active from inactive sarcoidosis.

Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purfied using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1 mg/200 mg protein) for 2 h could be divided into three components: (i) an enzyme of molecular weight 290 000 (peak I), (ii) an enzyme of molecular weight 180 000 (peak II) and (iii) an enzyme of molecular weight 98 000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1 mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.

Cultured endothelial cells provide a model for the study of interactions of vasoactive peptides with endothelium. Endothelial cell cultured from veins of human umbilical cords contain both angiotensin I converting enzyme (kininase II) and angiotensinase activities. Intact monolayers of cells can both activate angiotensin I and inactivate bradykinin when the peptides are added to culture flasks in protein-free medium. Intact suspended cells or lysed cells convert angiotensin I to angiotensin II, inactivate bradykinin, and hydrolyze hippuryldiglycine to hippuric acid and diglycine. These actions are inhibited by SQ 20881, the specific inhibitor of converting enzyme. The kininase activity of endothelial cells was partially inhibited by antibody to human lung converting enzyme. Endothelial cells also inactivate longer analogs of bradykinin, such as kallidin, methionyl-lysyl bradykinin, and bradykinin coupled covalently to 500,000 mol wt dextran. The endothelial cells retained converting enzyme activity through four successive subcultures, indicating that the enzyme is synthesized by the cells surface, and it is apparently a marker for endothelial cells, since cultured human fibroblasts, smooth muscle cells, and baby hamster kidney cells do not have it. Endothelial cells also contain an aminopheptidase which hydrolyzes both angiotensin II and the synthetic substrate, alpha-L-aspartyl beta-naphthylamide. The angiotensinase activity increased when the cells were lysed, which suggests that the enzyme is localized within the cells, Hydrolysis of both alpha-L-aspartyl beta-naphthylamide and angiotensin II was inhibited by omicron-phenanthroline, indicating that the enzyme is an A-tipe anigotensinase.

Using hippuryl-L-histidyl-L-leucine as a substrate analogue, serum angiotensin converting enzyme (ACE) was spectrophotometrically estimated in patients with bronchial asthma. The mean level of asthmatic patients was significantly lower than that of the control subjects. The reduced serum ACE activities did not change during an acute asthmatic attack. Significantly lower levels of serum ACE occurred in patients with chronic asthma than in those who only suffered with occasional asthma. Serum ACE activity was not reduced when the patients were taking steroids. Serum ACE activity could not be correlated with either the systolic blood pressure or the diastolic blood pressure of our asthmatic patients. However, serum ACE activity was correlated with the serum beta-globulin fraction in asthmatic patients.

Angiotensin-converting enzyme (ACE) catalyzes rapid hydrolytic cleavage of angiotensin I to form angiotensin II (AII). Inasmuch as converting enzyme activity is present at birth and increases postnatally to adult values it was of interest to determine the prenatal development of ACE. Converting enzyme activity was determined in the 20,000 x g supernatant fraction of lung homogenates using hippuryl-L-histidyl-L-leucine (HHL) as substrate. Hippuric acid liberated by the hydrolysis of HHL was quantified spectrophotometrically. ACE activity was first detectable at 18 days of gestation and increased fourfold prior to birth (21 days gestation). Pulmonary ACE activity of 1-day-old animals was twice that of fetuses at day 20 of gestation; however, this increase did not appear to result from ventilation alone. The Michaelis-Menten constant for fetal ACE (2.0 mM HHL) was not different from that calculated for ACE of adult rat lungs (2.6 mM). These data were interpreted to indicate that the age-related increase in ACE activity was due to greater ACE content as opposed to further activation of preexisting enzyme. This increase in fetal ACE activity may play an important role in preparing the renin-angiotensin system for postnatal function.