Angiotensin-converting enzyme (ACE) converts angiotensin I to angiotensin II and thus has an important role in the control of blood and sodium balance. The gene encoding for ACE is subject to an insertion/deletion (I/D) polymorphism associated with different levels of the enzyme in serum (1). Subjects homozygous for the deletion allele (DD) have serum ACE levels 48% higher than those homozygous for the insertion allele (II), whereas heterozygotes (ID) have intermediate levels (1). This polymorphism accounts for 47% of the variability in serum ACE concentrations between subjects (1). The deletion allele may be a risk factor for myocardial infarction (2,3), ischaemic and dilated cardiomyopathy (4), and the development of left ventricular hypertrophy (5). The mechanism by which the D allele may exert such detrimental effects is unknown but could be through an enhanced rate of production of angiotensin II, resulting in increased pressor and trophic cardiovascular responses.
The physiologic significance of the ACE gene polymorphism has been studied little. ACE is distributed widely throughout the tissues, largely bound to cell surfaces, especially the vascular endothelium (6). The distribution of ACE levels in human T lymphocytes may be influenced by the I/D polymorphism (7), but to our knowledge, the expression of ACE at the vascular endothelium has not been examined. We studied this indirectly by examining the response to intravenous infusion of angiotensin I, which causes a dose-dependent pressor effect (8-12) with steady-state levels of blood pressure achieved within 3 min (9). The aim was to determine whether responses to angiotensin I infusion differ in healthy subjects homozygous for either the I or D allele. While this study was in progress, Ueda et al. (13) reported an increased pressor sensitivity to angiotensin I infusion in DD subjects by using similar methods.
Sixteen healthy white men aged a mean of 26 years (range, 19-36 years) were selected to provide equal numbers of the two genotypes DD (n = 8) and II (n = 8). Supine blood pressure was measured on three occasions over a 1-week period, and those with a mean diastolic pressure >80 mm Hg were excluded. All had normal serum creatinine, electrolyte levels, and electrocardiograph. Subjects gave written informed consent to the study, which was approved by the district ethics committee.
Subjects attended at 0900 h, having avoided added salt and foods with a high salt content for 3 days before the study and having refrained from food, smoking, caffeine, and strenuous exercise for 12 h. Urinary sodium excretion over a 24-h period was measured immediately before the infusion. Sodium chloride, 0.9%, was infused intravenously for a 30-min baseline period at 48 ml/h via an IVAC p1000 pump whilst subjects lay supine. Angiotensin I (Sigma-Aldrich Company Limited) was prepared as a sterile solution containing 50 μg/ml in 0.9% sodium chloride and then diluted to concentrations of 1.2 and 4 μg/ml. Angiotensin I was infused at an initial rate of 0.1 μg/min and increased to 0.3, 0.9, 1.8, 3.0, 4.0, 5.0, and 6.0 μg/min at 3-min intervals, or until an increase in diastolic pressure of 25 mm Hg was achieved. Blood pressure and heart rate were measured 30, 15, 10, 5, and 0 min before the angiotensin infusion, during the final 30 s of each 3-min infusion period, and at 10-min intervals after the infusion was discontinued. Basline blood pressure was defined as the average of the preinfusion pressures. Blood was taken for serum ACE activity and plasma renin, angiotensin II, and aldosterone concentrations before and at the end of the angiotensin I infusion. Identical syringes were used during the baseline and angiotensin I infusion periods so that subjects were unaware when the angiotensin infusion began. The investigators were blind to the genotype of the subject.
DNA was extracted from whole blood by a standard protocol, and the ACE genotype was determined by the polymerase chain reaction based on the method of Rigat et al. (14). Serum ACE activity was measured by a standard spectrophotometric method assessing the degradation of FAPGG by ACE (15). Plasma renin (16), angiotensin II (17), and aldosterone (Coat-acount Kit; Diagnostic Products Corporation) concentrations were measured by radioimmunoassay. Blood pressure and heart rate were measured by a Dinamap semiautomated recorder. The R(d)25 was the rate of angiotensin I infusion that caused a 25 mm Hg increase in diastolic blood pressure, and the R(s)25 was the rate of infusion at which a 25 mm Hg increase in systolic pressure occurred. Changes in heart rate (HR25) were analysed at the time the R(d)25 was attained.
By using a published standard deviation for the diastolic pressor response to angiotensin I infusion in white subjects (12), it was calculated that eight subjects were required in each group to detect a difference in R(d)25 between groups of 1.75 μg/min of angiotensin I with 80% power and alpha = 0.05. After logarithmic transformation of the rate of infusion of angiotensin I to approximate a linear dose response, the R(d)25 and R(s)25 were interpolated from the plotted dose responses for individual subjects. Fifteen subjects achieved a ≥25 mm Hg increase in diastolic blood pressure, but one achieved a increase of only 21 mm Hg and had the R(d)25 calculated by extrapolation. The R(d)25 and R(s)25 are expressed as geometric means of the responses for each genotype, and the difference between genotypes is expressed as the ratio of the geometric means with 95% confidence intervals for this ratio. Unpaired t tests were used to investigate differences in blood pressure, heart rate, and neurohormonal responses between the groups at baseline and after angiotensin I infusion. Paired t tests were used to examine neurohormonal responses within groups.
Comparability of groups
Age, weight, baseline blood pressure, heart rate, and 24-h urinary sodium excretion were similar in the two groups (Table 1). As anticipated, there was a significant relation between ACE genotype and serum ACE activity at baseline, with mean serum ACE 46.3 U/L in group DD and 12.3 U/L in group II (p < 0.001). There were no significant differences in baseline plasma renin and aldosterone levels between the genotypes. Baseline angiotensin II levels were higher in the DD subjects (7.0 pg/ml) than in the II subjects (4.7 pg/ml), and this difference approached significance (difference DD − II = 2.3 pg/ml; 95% CI, 0.0-+4.6; p = 0.07).
Changes in blood pressure
The geometric mean rate of infusion of angiotensin I required to achieve the R(d)25 was 2.53 μg/min in II subjects and 2.67 μg/min in DD subjects [ratio of doses (II to DD) = 0.95; 95% CI, 0.44-2.02; p > 0.05; Table 2; Fig. 1]. The equivalent rates of infusion for systolic blood pressure (R(s)25) were 4.47 μg/min in II subjects and 3.39 μg/min in DD subjects [ratio of doses (II to DD) = 1.32; 95% CI for ratio, 0.49-3.56; p > 0.05].
Changes in heart rate
There was a significant difference between the groups in the chronotropic response to angiotensin I infusion (Table 2; Fig. 2). At the time of the R(d)25, the mean change from baseline heart rate was +1.2 beats/min for DD subjects and −9.5 beats/min for II subjects (Diff II − DD = 10.7 beats/min; 95% CI, 6.7-14.8; p = 0.01).
Changes in serum ACE activity and plasma renin, angiotensin II, and aldosterone concentrations
There was a small increase in serum ACE activity after angiotensin I infusion in all subjects (+2.0 U/L; p = 0.03), but there was no difference in this response between subjects of differing genotypes (Table 2). There was no change in the mean plasma renin concentration in response to angiotensin I infusion. Serum aldosterone and angiotensin II levels increased as expected, but there was no difference in responses between groups. There were also no differences between the genotypes when these measurements were calculated as the unit change per microgram of angiotensin I infused (i.e., the sensitivity to angiotensin I).
The strong relation between ACE genotype and serum ACE activity (1) was confirmed in this study. As expected, angiotensin I caused a pressor response, increased plasma angiotensin II, and aldosterone concentrations, but caused no change in plasma renin concentration. However, neither the pressor nor the hormonal responses differed significantly between the two genotypes despite a near fourfold higher mean serum ACE level in the DD subjects. The only significant difference in response between the genotypes was in heart rate.
Overall these results show no evidence that the sensitivity of the renin-angiotensin-aldosterone system to angiotensin I varies with serum ACE level or I/D genotype. However, although the geometric means for the R(d)25 were similar, with a ratio of 0.95, the 95% confidence intervals show that the study had insufficient power to exclude ratios between 0.44 (DD > II) and 2.02 (II > DD). This relatively low power was not expected and was the result of much greater variability in the pressor response to angiotensin I infusion by using this protocol (Fig. 1) than was anticipated from the data of Joubert and Brandt (12). The reasons for this are not clear, but as shown in Fig. 1, there was one outlying subject in the II group. However, even if this subject was excluded, the ratio of geometric means (II to DD) was 0.78, with 95% confidence intervals of 0.39-1.55, so that there was still no significant difference in the genotypes. While our study was in progress, a similar study of the pressor sensitivity to angiotensin I infusion in relation to ACE genotype was reported (13). Ueda et al. (13) showed a significant increase in pressor sensitivity to angiotensin I infusion in 10 healthy normotensive DD men compared with 10 II men. The II to DD ratio for the rates of infusion of angiotensin I required to achieve a 20 mm Hg increase in mean arterial pressure in their study was 1.68. We have recalculated our data in an analysis similar to that of Ueda et al., correcting for body weight and using as the end point a 20 mm Hg increase in mean arterial pressure. Analysed thus, the ratio of infusion rates (II to DD) was 1.12, with 95% CI, 0.65-1.92. Thus our study does not confirm their positive finding, although it had insufficient power to exclude a difference of the magnitude reported. The only significant difference between the two genotypes was in the chronotropic response to angiotensin I infusion. Subjects of II genotype exhibited bradycardia averaging nine beats/min with a 25 mm Hg increase in diastolic pressure, whereas DD subjects showed no change in heart rate. That subjects of DD genotype exhibited less heart rate slowing than did the II subjects at a similar pressor response could imply altered baroreceptor sensitivity between the genotypes. Angiotensin II is known to inhibit the reflex slowing of heart rate to a increase in arterial pressure (18). A previous study found no difference in the chronotropic response to a single oral dose of enalapril in healthy subjects of differing ACE genotype (19). As we found no evidence of other differences in haemodynamic responses in homeostasis or in the rate of production of angiotensin II between II and DD subjects, the difference between genotypes in chronotropic response to angiotensin I may be a chance observation. However, further study of cardiovascular reflexes in relation to ACE genotype may be of interest.
In conclusion, we showed no difference in the blood pressure or endocrine renin-angiotensin-aldosterone system responses to infusion of angiotensin I between healthy subjects homozygous for either the deletion or insertion allele of the ACE gene I/D polymorphism. The conversion of angiotensin I to angiotensin II is not the rate-limiting step in the renin-angiotensin cascade (20), and marked interindividual differences in serum ACE levels may be of little or no importance. However, because of the variability of pressor responses to angiotensin I infusion, our study cannot exclude with certainty important differences related to the ACE genotype. The link between the ACE genotype and cardiovascular disease (21) would be strengthened by showing that the ACE genotype has a consistent association with some physiologic activity. Until then, this link remains tenuous, as illustrated by recent studies showing an absence of association or genetic linkage between the ACE gene and cardiovascular disease (22,23).
Acknowledgment: This work was supported by a grant from Glaxo Group Research (IGC).
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