Effect of Enalapril on Exhaled Nitric Oxide in Normotensive and Hypertensive Subjects

Subjects

Normotensive subjects in both study 1 and study 2 underwent physical examinations to rule out the presence of disease. None of the subjects had evidence of active infection or respiratory disease. None was a smoker. None of the subjects had taken any prior medication. Subjects were also excluded if they had had any clinically relevant illness in the 3 weeks preceding the study or if any clinically relevant abnormality was identified during the screening examination.

Hypertensive subjects in study 2 also underwent a physical examination (Table 1⇓). We also assessed the following laboratory parameters: blood cell count, urinalysis, and serum levels of electrolytes, creatinine, fasting blood sugar, cholesterol, triglycerides, and liver enzymes. Blood pressure was measured on at least 3 different occasions. Hypertension was defined as a systolic or diastolic blood pressure consistently >140 mm Hg or 90 mm Hg, respectively, or both. An ECG, echocardiogram, and chest radiograph were also obtained. Echocardiographic and ECG findings suggestive of left ventricular hypertrophy were present in 2 patients. Mild proteinuria was present in 1 patient and hypercholesterolemia in 3 patients. None of the hypertensive patients had diabetes mellitus or liver dysfunction. No patients were taking antihypertensive medications before the study. None of them was a smoker.

Alcohol and caffeine were prohibited for 12 hours before and during the study. Antihypertensive drugs were administered after fasting overnight. Subjects were told to maintain a limited-nitrate diet (which excluded foodstuffs containing a high concentration of nitrate such as cured meat, fruit, and, in particular, leafy green vegetables) from 3 days to 14 hours before the administration of antihypertensive drugs. Because tap water contains variable amounts of nitrate, distilled water was provided during the study. Subjects maintained their usual exercise habits throughout the study. All subjects underwent pulmonary function testing (Chestac-55V; Chest M.I.) before beginning the study protocol. The forced vital capacity is expressed as a percentage of the reference value (%VC). The forced expiratory volume in 1 second is expressed as a percentage of the forced expiratory volume (FEV₁%). The study protocol was approved by the Institutional Committee on Human Research of Maebashi-Jonan Hospital. Informed consent for participation was obtained from all subjects.

Protocol

Collection of Exhaled Air

Samples of exhaled air were obtained and NO gas was analyzed and the rate of NO release determined as previously described.⁶ In brief, after application of a nose clip, air inspired through the mouth was exhaled through the mouth into a 6-L bag made of polyvinylfluoride film (Tedlar bag, Iuchi). A Teflon column (15×50 mm) was inserted between the mouthpiece and the Tedlar bag. The column was packed with 6.7 g of silica gel (particle size, 1.7 to 4.0 mm, Kanto Chemical). This column was used to dry the exhaled air sufficiently to prevent the condensation of vapor on the walls of the Tedlar bag. NO is not adsorbed by the silica gel and is stable in the Tedlar bag for several hours if the sample is dry and is not exposed to light. The study was performed only when the concentration of NO in the room air was <3 parts per billion (ppb). The room air concentration of NO was 1.2±0.6 ppb (0.6 to 2.8 ppb) immediately before each measurement.

Analysis of NO and Determination of NO Release Rate in Exhaled Air

The NO concentration was measured with a chemiluminescence analyzer (model GLN-32; Denki-Kagaku-Keiki). The flow rate for sampling was 800 mL/min. The limit of detection for NO was 0.2 ppb. The coefficient of variation was 2% at 20 ppb of NO.

Subjects were instructed to exhale air after making a full inspiratory effort at 6 flow rates that were determined arbitrarily by each subject. The flow rates ranged from very fast to very slow. Air volume in the 6 efforts corresponded to each person’s vital capacity. Samples were collected in separate bags. The time from the start to the end of each sampling was measured with a stopwatch. Sample volumes were determined on a chart recorder (model 056; Hitachi).

The concentration of NO in exhaled air was positively correlated with the duration of the exhalation. The rate of NO release for each subject was calculated on the basis of the slope of a simple linear regression model of the relation between the concentration of gas and the duration of exhalation as follows: Release rate of NO (pmol/s)=slope (ppb/s)×average of 6 sample volumes (L)×1/V(L⁻¹)×10³, where V corresponds to the volume of 1 mole of dry air at ambient temperature (24.5 L at 25°C at 760 mm Hg). The release rate was constant (36±4 pmol/s) in 5 control subjects who repeated the protocol 10 times over a 2-week period. In addition, the NO release rate at 0, 2, 4, and 8 hours (37±5 pmol/s, 36±5 pmol/s, 37±3 pmol/s, 36±4 pmol/s, respectively) was not changed for 8 hours in 5 control subjects.

Measurement of Serum Level of NOx

Samples of venous blood were centrifuged at 500g for 10 minutes at 4°C. The resulting serum samples were stored at −70°C until assayed. For NOx measurements, 0.125 mL of thawed serum was added to 0.5 mL of 75 mmol/L ZnSO₄ solution, to which 0.625 mL of 55 mmol/L NaOH was then added. The final pH was maintained between 7.0 and 7.5. Treated samples were mixed by vortexing, allowed to stand for 10 minutes, and then centrifuged at 5000g for 10 minutes. The resulting supernatant was applied to a copperized cadmium reduction column to reduce nitrate to nitrite and subsequently reacted with the Griess reagent with the use of an autoanalyzer (TCI-NOX 1000; Tokyo Kasei Kogyo). The absorbance of the reaction mixture at 540 nm was measured by spectrophotometry. The limit of nitrite detection was 0.2 μmol/L.

Measurement of Plasma Level of Bradykinin

For measurement of bradykinin, samples were placed in siliconized vacuum tubes containing aprotinin, soybean trypsin inhibitor, protamine sulfate, and disodium EDTA. All tubes were placed in a box filled with ice until they were centrifuged at 500g for 10 minutes at 4°C. Supernatants were stored in sealed polypropylene tubes at −80°C until analysis. The plasma bradykinin level was measured by radioimmunoassay.¹⁰

Measurement of Plasma Level of Renin Activity

Venous blood samples from the contralateral arm were collected in tubes containing EDTA-2Na (1 mg/mL) and promptly chilled in an ice bath. After samples of venous blood were centrifuged at 500g for 10 minutes at 4°C and plasma was removed, aliquots of the plasma sample were stored at −20°C until use. Plasma renin activity (PRA) was determined by radioimmunoassay with radioimmunoassay kits obtained from SRL Radioisotope Laboratories, Tachikawa.

Drugs

Enalapril and nitrendipine were provided by Banyu Pharmaceutical Co and Yoshitomi Pharmaceutical Co, respectively.

Statistical Analysis

Two-way ANOVA was used to compare measurements of blood pressure, the NO release rate, the heart rate, the serum level of NOx, and the plasma levels of bradykinin and renin activity at baseline and 2, 4, and 8 hours after administration of enalapril or nitrendipine in normotensive subjects in study 1 and at baseline and 2 and 4 hours after administration of enalapril between normotensive and hypertensive subjects in study 2. When ANOVA yielded a significant result, the location of difference was determined by the unpaired or paired t test. All values are expressed as mean±SEM. A value of P<0.05 was accepted as statistically significant.

Study 1

Before treatment, there were no differences in blood pressure, heart rate, PRA, plasma bradykinin, and the serum NOx concentration between subjects treated with the ACE inhibitor or the calcium antagonist (Tables 2⇓ and 3⇓). Enalapril significantly increased heart rate (P<0.05) and PRA (P<0.01) at 4 hours compared with baseline levels. Blood pressure, plasma bradykinin, and serum NOx showed no significant changes 8 hours after drug administration. Nitrendipine significantly increased heart rate at 2 hours compared with the baseline level (P<0.05). Blood pressure, PRA, bradykinin, and serum NOx showed no significant changes 8 hours after administration of nitrendipine.

In enalapril-treated subjects, the NO release rate from the lung was significantly higher at 4 hours (58.3±7.3 pmol/s) than at baseline (36.9±5.1 pmol/s) (P<0.01) (Figure 1⇓). The NO release rate from the lung was not significantly changed 8 hours after administration of nitrendipine (Figure 1⇓).

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Figure 1.

Time course of NO release from lung before and after administration of enalapril or nitrendipine in normotensive subjects (n=8). NO release rate from lung at 4 hours was significantly increased by enalapril administration (•, n=8) (*P<0.01) but was not changed by nitrendipine (○, n=8).

Study 2

Before treatment, both systolic (P<0.001) and diastolic (P<0.001) blood pressures were significantly higher in hypertensive subjects than in normotensive subjects (Table 4⇓). There were no differences in heart rate, PRA, plasma bradykinin, and the serum NOx concentration between hypertensive and normotensive subjects before treatment (Tables 4⇓ and 5⇓). In normotensive subjects, the systolic blood pressure was significantly decreased at 2 hours (P<0.05) and at 4 hours (P<0.01) and diastolic blood pressure was significantly decreased at 4 hours (P<0.05) compared with the baseline level. The PRA was significantly increased at 4 hours compared with the baseline level (P<0.01). There were no changes in diastolic blood pressure, plasma bradykinin, and serum NOx 8 hours after administration of enalapril. In hypertensive subjects, systolic blood pressure was significantly decreased at 4 hours (P<0.01) and diastolic blood pressure was significantly decreased at 2 hours (P<0.05) and at 4 hours (P<0.01) compared with the baseline level, but PRA was significantly increased at 4 hours compared with the baseline level (P<0.01). Heart rate, plasma bradykinin, and serum NOx were not significantly changed 8 hours after administration of enalapril.

Before treatment, the NO release rate from the lung in hypertensive subjects was significantly lower than in normotensive subjects (P<0.05). The NO release rate from the lung was significantly increased in normotensive subjects at 4 hours (70.3±11.4 pmol/s) compared with baseline (40.4±6.0 pmol/s) (P<0.01) (Figure 2⇓). The NO release rate from the lung was not significantly changed 4 hours after treatment in hypertensive subjects (Figure 2⇓). The NO release rate was significantly higher in normotensive subjects than in hypertensive subjects at 2 and 4 hours (P<0.01 and P<0.01, respectively).

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Figure 2.

Time course of NO release from lung before and after enalapril administration in normotensive subjects (n=10) and hypertensive subjects (n=10). NO release rate from lung at 4 hours was significantly increased by enalapril administration in normotensive subjects (•, n=10) (*P<0.01) but was not changed (▪, n=10). NO release rate at baseline, 2 hours, and 4 hours was significantly higher in normotensive subjects than in hypertensive subjects (†P<0.05, †P<0.01, and †P<0.01, respectively).