ENALAPRIL ACTS THROUGH RELEASE OF NITRIC OXIDE IN PATIENTS WITH ESSENTIAL HYPERTENSION^*

Abstract

Background: Endothelial dysfunction has been proposed as an etiological agent in the pathogenesis of essential hypertension. Amongst the various antihypertensive drugs, angiotensin converting enzyme inhibitors (ACEI) have been implicated in modifying the vascular endothelium by the release of mediators that include bradykinin, nitric oxide, prostaglandins and thromboxane A2. Materials and Methods: To study the mechanism of action of enalapril, an ACEI, serum reactive nitrite intermediates (RNI) and citrulline, by products of nitric oxide metabolism were measured before and after treatment with enalapril in 25 consecutive patients of essential hypertension. Results: Following treatment serum RNI intermediate increased from a pretreatment value of 164.5 ± 20.2 nmol/mL to a post treatment value of 266.9 ± 47.3 nmol/mL (p < 0.05), however there was no significant change in the levels of citrulline (p > 0.1). There was no significant correlation between the severity of hypertension and serum RNI. Serum RNI levels were lower in the postmenopausal women but did not reach statistical significance. Conclusions: It is postulated that enalapril exhibits its antihypertensive property through release of nitric oxide.

• Angiotensin converting enzyme
• Hypertension
• Nitric oxide

INTRODUCTION

Although the etiology of essential hypertension is not known, various mechanisms have been proposed for its pathogenesis. These include salt retention, increase in the plasma renin activity and endothelial factors. The vascular endothelium is recognized to have profound influence in maintaining the vascular tone and proliferation of vascular smooth muscles. It acts through the release of various mediators – endothelin and nitric oxide (NO) being the most important ones [1-2]. Many studies have shown that endothelial dysfunction is responsible for the hypertension and a deficiency of NO plays a major role in its pathogenesis [3-4]. Amongst the numerous antihypertensives used in the treatment of hypertension, angiotensin enzyme inhibitors (ACEI) have been implicated in modifying the vascular endothelium by the release of nitric oxide. In animal models and isolated cell cultures ACEI have been shown to release mediators like bradykinin, nitric oxide, prostaglandins and thromboxane A₂[5-10]. Release of NO may be an important mechanism of action of ACEI contributing to its vasodilator and antitrophic action. This study was conducted to find the effect of enalapril, ACEI on serum reactive nitrite and citrulline levels, byproducts of NO synthesis, in patients with essential hypertension.

Materials and Methods

Twenty-five consecutive patients of essential hypertension who were not on any medication were drawn from a special hypertension clinic of PGIMER, Chandigarh, India. The patients in the age group of 15–70 years were enrolled in the study. Patients with associated diseases, pregnancy or having secondary cause of hypertension, recent or current infection or on oral contraceptives and steroids were excluded from the study. An informed consent was obtained from each patient prior to his or her inclusion and the ethical committee of the institute approved the study. All patients underwent a thorough clinical evaluation: laboratory investigations including a urinalysis, blood urea, serum creatinine, complete hemogram, electrocardiogram and a chest skiagram were done in all the patients. Ultrasound abdomen, captopril renography and urinary vanillyl mandelic acid levels were estimated as and when required to exclude secondary hypertension. These patients were advised oral enalapril to control the hypertension. 10 mL of heparinized blood was taken in a plastic vial, centrifuged and plasma was separated and stored at −20°C. The samples were drawn, coded and analyzed in a blind fashion, before and after 4 weeks of therapy.

Plasma Nitrite Estimation

Nitrite, the stable end product of nitric oxide metabolism, was measured in plasma as reactive nitrogen intermediates. This procedure is based on the method described by Green [[11]]. In brief the procedure followed was as follows: 100 μL of plasma was diluted with distilled water and reacted with 500 μL of Griess reagent (1 : 1 v/v 0.1% n-1-naphthylethylendiamine in distilled water and 1% sulfanilamide in 5% o-phosphoric acid) and was kept at room temperature for 10 minutes. Simultaneously five different concentrations of the standard (100 μL of 10 mM sodium nitrite diluted to 10 mL) were taken. Absorbance at 546 mm was measured using a Uvicon spectrometer. The test was carried out in duplicate and the average values were taken. The nitrite concentration was calculated from the standard sodium nitrite curve.

Estimation of Citrulline

It was done by the method described by Boyde and Rahmatulla [[12]]. The procedure was as follows: 200 μL of plasma was deproteinized with zinc sulfate and centrifuged. 20 μL of supernatant was added to 480 μL of 0.1M HCI. This was reacted with 1.5 mL of a reagent comprising of two parts of solution A and one part of solution B. Solution A was prepared by mixing 137.5 mL of water, 62.5 mL of 95% sulphuric acid and 50 mL of 85% phosphoric acid and cooled to room temperature. 82.5 mg of Ferric chloride was then added to make a total volume of 250 mL of solution A. Solution B contained 2-Diacetyl monoxamine (0.5% w/v) and thiosemicarbazide (0.01%w/v) in water. After a thorough mixing, the tubes were put in a boiling water bath for 5 minutes. The tubes were cooled to room temperature and the absorbency was measured at 530 nm in Uvicon Spectrophotometer. L-citrulline concentration was calculated from the standard curve drawn by using different concentration of the citrulline standard.

Statistical Analysis

Paired ‘t’ test was applied to analyze the nitrite and citrulline levels before and after treatment. The coefficient of variation ‘r’ was derived between the severity of hypertension and the plasma nitrite and citrulline levels.

Observation and Results

Of the 25 patients studied, 16 were female and 9 were male patients with a range from 23 to 60 years (mean 43.7 ± 10.73). The common symptoms were headache (11 patients), palpitation (5 patients), dyspnea and chest pain (3 patients each). Four patients had pedal edema on presentation and these patients did not have any renal, liver or cardiac disease on clinical evaluation or investigations. Six patients were asymptomatic. Three patients had cardiomegaly on chest x-ray while ECG abnormalities (left ventricular hypertrophy, right bundle branch block and ST-T wave changes) were seen in 4 patients. Fundus examination revealed hypertensive changes (grade I and II) in 9 patients. The mean height and weight of the patients were 161 ± 7.9cm and 66.7 ± 9.9 kg respectively. The body mass index ranged from 19.8 to 36.4 kg/m² (mean 25.9 ± 4.8 kg/m²). The waist hip circumference ratio varied between 0.7 and 1.05 (mean 0.89 ± 0.07). The biochemical blood parameters were normal. The risk factors of the development of hypertension included a positive family history of hypertension in 11 patients (27.5%), postmenopausal status in 10 patients (25%), obesity (Waist/Hip ratio of more than 0.9 in males and 0.95 in females) in 9 patients (22.5%), body mass index of more than 30 in 5 patients (12.5%), age more than 60 years in 3 patients (7.5%), smoking in one patient (2.5%), serum LDL cholesterol more than 160 mg% in 7 patients (17.5%), serum HDL cholesterol less than 35 mg% in 9 patients (22.5%) and serum triglycerides more than 160 mg% in 19 patients (47.5%).

At the beginning of the study, the systolic blood pressure of the study group was 157.8 ± 20.0 mm Hg (range 130–230 mmHg) while the diastolic blood pressure was 100.1 ± 7.3 mmHg (range 90–116 mmHg). After therapy with enalapril, systolic blood pressure fell to 126.4 ± 10.4 mm Hg while a mean diastolic pressure of 81.2 ± 4.4 mm Hg could be achieved. The diastolic fall in blood pressure was more than the systolic blood pressure. Following treatment, serum RNI intermediate increased from a pretreatment value of 164.5 ± 20.2 nmol/mL to a post treatment value of 266.9±47.3 nmol/mL (p < 0.05) while the citrulline levels changed from 2197.5 ± 229.1 nmol/mL pretreatment to 1867.9 ± 162.4 nmol/mL posttreatment (p > 0.1). There was no significant correlation of serum RNI with the severity of hypertension as judged by their levels (r = 0.08). It was observed that serum RNI levels were lower in the postmenopausal women (131.2 ± 21.2 nmol/mL) than premenopausal women (186.6 ± 47.3 nmol/mL) but did not reach statistical significance (p > 0.05).

Discussion

The pathophysiology of essential hypertension is complex and involves numerous neurohumoral factors. Of recent interest is the role of nitric oxide in its pathogenesis [1-2]. There is a paucity of studies in humans involving direct measurement of nitric oxide. Most of the studies have shown a rise in systemic vascular resistance following infusion of L-NMMA, a competitive inhibitor of L-arginine, and a prerequisite for the synthesis of NO [13-14]. In our study, we estimated the RNI and citrulline levels that are stable byproducts of nitric oxide breakdown and showed that RNI increases with ACEI treatment. In a study, an increase in plasma nitrite and a reduction of free radicals occurred after control of hypertension with β blockers or calcium channel blockers [[15]]. It has also been shown that endothelium dependent vasodilatation improves with various angiotensin converting enzyme inhibitors – captopril, enalapril, ramipril, perinodopril and trandalopril [[6]], [16-17]. It has been proposed that the improvement seen could be due to the intrinsic property of the antihypertensives and not due to the control of blood pressure. The plasma nitrite levels also would depend on the etiology of hypertension. Gooneshekhar et al observed that plasma nitrite levels and their urinary excretion were elevated in hypertensive children with predominantly renovascular or renal parenchymal hypertension [[18]]. It is possible that basic vascular pathology results in a reduction of nitric oxide production in essential hypertension whereas a compensatory increase in NO production occurs in secondary hypertension.

We studied the correlation between the severity of hypertension with the RNI levels and found that there was no significant correlation. In contrast, a multiple regression analysis has revealed that serum nitrite correlates with the systolic blood pressure but not with diastolic blood pressure [[19]]. In our study the number of subjects is too small to draw a valid conclusion. Also, plasma nitrite levels depend on the dietary nitrate intake, renal function, smoking, hypercholesterolemia, estrogens and acute illnesses like infection. In our study the dietary intake was more or less constant. Therefore, the changes in the plasma nitrite are expected to be minimal as observed by Forte et al [[20]].

We observed that in females RNI were lower even though it was statistically insignificant. In contrast, Takahashi et al [[19]] have proposed that estradiol induces constitutive nitric oxide synthesis, which is responsible for a high plasma level in females. The difference from our study may be contributed by the fact that more than half of our female subjects were post menopausal and not on hormone replacement therapy and the mean age of the female patients was higher than the males.

We found no significant difference in the L-citrulline levels following therapy with enalapril. Hecker et al [[21]] have demonstrated and noted that L-citrulline does not accumulate despite considerable formation of nitric oxide by cultured endothelial cells. They demonstrated that L-citrulline is recycled to L-arginine, the nitric oxide precursor. Thus, in individuals with increased nitric oxide production, L-citrulline may not be a sensitive marker for nitric oxide production due to its recycling to L-arginine. This may be the reason that L-citrulline did not rise in parallel with plasma nitrite levels in our study. We found that there was a wide variation of plasma nitrite and citrulline levels that were similar to a previous study. As we compared the plasma levels before and after treatment in the same individual, it may not interfere with the ultimate outcome. In conclusion, serum reactive nitrogen intermediates increase in patients with essential hypertension following therapy with enalapril and one of the mechanisms of the action of enalapril is through an increased NO production.