Relation between the angiotensin-converting enzyme insertion/deletion polymorphism and blood pressure in Japanese male subjects

Abstract

Inconsistent results have been reported regarding the association of the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism and hypertension. Recent studies of population-based samples of three different areas in Japan presented conflicting results regarding this association. We, thus, investigated the relation between the ACE I/D polymorphism and blood pressure (BP), or the frequency of hypertension, respectively, in 706 Japanese male subjects who participated in the health check-up programme of our hospital. The ACE I/D polymorphism was determined by the polymerase chain reaction technique. Of 706 subjects, 203 were found to have hypertension and the other 503 were found to be normotensive. In all subjects, the frequencies of the DD, ID, and II genotypes were 0.123, 0.432, and 0.445, respectively, and the allelic frequency of the D allele was 0.339. In the younger subjects aged <50 years (n=264), neither systolic nor diastolic BP differed significantly among the genotypes. Conversely, in the older subjects aged50 years (n=442), the systolic BP was significantly higher by 5.9 mmHg in the subjects with the ID genotype than those with the II genotype (P<0.01), and the diastolic BP was significantly higher in the subjects with the DD and ID genotypes by 5.1 and 3.3 mmHg, respectively than those with the II genotype (P<0.05 for each), although age, BMI, percentage of smoking habits, drinking habits, or the use of antihypertensive drugs did not differ significantly among the genotypes. In addition, in the older subjects, the hypertensive subjects showed significantly higher frequencies of the DD and ID genotypes and the D allele than the normotensive subjects. These results demonstrated that there was no significant association of the ACE I/D polymorphism with BP or a prevalence of hypertension in younger Japanese men aged <50 years but there was in older Japanese men aged 50 years.

Introduction

Angiotensin-converting enzyme (ACE) catalyses the conversion of angiotensin I to angiotensin II (AII) and the degradation of bradykinin. Both AII and bradykinin are potent vasoactive peptides and have various acute and chronic effects on the cardiovascular system. ACE is considered to play an important role in cardiovascular hormonal regulatory systems. In humans, the ACE gene is located on chromosome 17, and exhibits an insertion/deletion (I/D) polymorphism characterized by either insertion (I) or deletion (D) of a 287-base pair (bp) fragment including the Alu-like sequence on intron 16, resulting in three genotypes (DD and II homozygotes and ID heterozygotes).1 It was reported that the ACE I/D polymorphism is associated with cardiovascular diseases such as myocardial infarction,2,3,4 although studies questioning the findings have also been reported.5

Regarding the association of the ACE I/D polymorphism and hypertension, inconsistent results have also been reported.6 Several studies reported evidence of a significant relation between the ACE I/D polymorphism and hypertension in male subjects but not in female subjects.7,8,9 However, it was also shown that this relation was not consistently seen in male subjects, but was changeable depending on other factors such as age.10 In addition, recent studies of population-based samples of three different areas in Japan presented conflicting results regarding the association of the ACE I/D polymorphism and hypertension.9,11,12 We, thus, performed the present study to determine the relation between the ACE I/D polymorphism and blood pressure (BP), or frequency of hypertension, respectively, in Japanese male subjects who participated in the health check-up programme of our hospital.

Methods

Subjects

A total of 706 Japanese male subjects aged 31–87 years who entered the 2-day hospitalized health check-up programme of our hospital between December, 1999 and November, 2000 were included in this study. Only male subjects were enrolled, because (1) nearly 70% of the participants in the health check-up programme of our hospital were males, and (2) the main purpose of the current study was to determine the relation between the ACE I/D polymorphism and hypertension in male subjects.

Methods

After >10 min of supine rest, systolic and diastolic BPs were measured twice in the right arm by a trained nurse using a sphygmomanometer. The diastolic BP was taken at the fifth phase Korotkoff sound. The mean value was used for the BP evaluation. Hypertension was defined as systolic BP140 mmHg and/or diastolic BP90 mmHg, or currently under antihypertensive medication.13,14 No subjects were found to have secondary hypertension by the comprehensive check-up. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m2). Information about current smoking and drinking habits and the use of antihypertensive drugs was obtained using a self-administered questionnaire.

Antecubical venous blood was collected into a plain tube and a tube containing EDTA (1 mg/ml) in the morning after the subjects had fasted overnight. Plasma was separated by centrifugation at 4°C and stored at −80°C until assayed. Routine laboratory tests including blood cell counts and blood chemistry were performed using automatic analyzers (HITACHI 7170, Hitachi Ltd. Instruments, Tokyo, Japan).

The study protocol was in accordance with the recommendations of the World Medical Association for biochemical research involving human subjects (Somerset West version, 1996). All subjects gave written informed consent for analysis of the ACE I/D polymorphism.

Genotyping for the ACE I/D polymorphism

Genomic DNA was extracted from white blood cells obtained from the buffy coat layer that remained in the sample tube using a Genomic DNA extraction kit (Talent, Italy). Template genomic DNA (0.5 μg) was amplified by polymerase chain reaction (PCR) with a thermal cycler (Astec, Fukuoka, Japan), as described previously.15,16 The ACE I/D polymorphism was determined by agarose gel electrophoresis with ethidium bromide staining, and the DD genotype was reconfirmed by insertion allele-specific amplification.

Statistical analysis

All values are expressed as means±s.e.m. Statistical analyses were performed using the unpaired t-test, the χ2 test, or an analysis of variance (ANOVA) where appropriate. When a significant overall effect was detected by ANOVA, Scheffe's F-test was used for the comparison of two variables. Multiple regression techniques were conducted to determine the association of systolic and diastolic BPs with age, BMI, with or without smoking habits, with or without drinking habits, and the ACE I/D polymorphism (the DD, ID, and II genotypes). Values of P<0.05 were considered significant. Agreement with the Hardy–Weinberg equilibrium for the ACE I/D polymorphism was checked by the χ2 test.

Results

Of 706 subjects, 203 (28.8%) were found to have hypertension and the other 503 (71.2%) were found to be normotensive. Table 1 shows the clinical characteristics of all subjects, and hypertensive and normotensive subjects, respectively. Age, weight, BMI, percentages of drinking habits and the use of antihypertensive drugs, systolic and diastolic BPs, haematocrit, uric acid levels, and plasma glucose levels 2 h after oral administration of 75 g of glucose were significantly higher in the hypertensive subjects than in the normotensive subjects. In contrast, the percentage of smoking habits was significantly lower in the hypertensive subjects than in the normotensive subjects.

Table 1 Clinical characteristics of the study subjects

By multiple regression analysis, age (r=0.164, P<0.0001), BMI (r=0.232, P<0.0001), and with or without drinking habits (r=0.192, P<0.0001) were positive explanatory variables of systolic BP, while with or without smoking habits (r=−0.080, P=0.026) was a negative explanatory variable of systolic BP. In addition, by multiple regression analysis, BMI (r=0.289, P<0.0001), with or without drinking habits (r=0.166, P<0.0001), and the ACE I/D polymorphism (r=0.076, P=0.032) were positive explanatory variables of diastolic BP, while with or without smoking habits (r=−0.119, P=0.001) was a negative explanatory variable of diastolic BP.

The frequencies of the DD, ID, and II genotypes were 0.123, 0.432, and 0.445, respectively (Table 2). Thus, the allelic frequency of the D allele was 0.339, which was lower than the reported frequency in Caucasians, but similar to the frequency reported previously in Japanese.5,11,12 The observed genotype frequencies were in agreement with the frequencies predicted by the Hardy–Weinberg equilibrium. As shown in Table 2, in all subjects, age, BMI, the percentage of smoking habits, drinking habits, or the use of antihypertensive drugs, or systolic or diastolic BP did not differ significantly among the genotypes.

Table 2 Age, body mass index, percentages of smoking and drinking habits, and the use of antihypertensive drugs, and systolic and diastolic blood pressures among the ACE genotypes in all subjects, the subjects aged <50 years, and the subjects aged 50 years

In the present study, additional analyses were performed in the younger subjects aged<50 years (n=264; mean age: 44.0±0.3 years: range: 31–49 years) and the older subjects aged50 years (n=442; mean age: 57.0±0.3 years; range: 50–87 years), separately. As shown in Table 2, in the younger subjects, age, BMI, the percentage of smoking habits, drinking habits, or the use of antihypertensive drugs, or systolic or diastolic BP did not differ significantly among the genotypes. In contrast, in the older subjects, systolic BP was significantly higher by 5.9 mmHg in the subjects with the ID genotype than those with the II genotype (P<0.01), and diastolic BP was significantly higher in the subjects with the DD and ID genotypes by 5.1 and 3.3 mmHg, respectively, than those with the II genotype (P<0.05 for each), although age, BMI, or the percentage of smoking habits, drinking habits, or the use of antihypertensive drugs did not differ significantly among the genotypes. Also in the older subjects aged50 years who were not under antihypertensive medication (n=362; mean age: 56.7±0.3 years; range: 50–77 years), systolic BP was significantly higher by 5.2 mmHg in the subjects with the ID genotype than those with the II genotype (P<0.05), and diastolic BP was significantly higher in the subjects with the DD and ID genotypes by 5.2 and 2.9 mmHg, respectively, than those with the II genotype (P<0.05 for each).

As shown in Table 3, in all subjects, neither the frequency of the ACE I/D polymorphism nor the D allele differed significantly between the hypertensive and normotensive subjects. In addition, in the younger subjects aged<50 years, neither the frequency of the ACE I/D polymorphism nor the D allele differed significantly between the hypertensive and normotensive subjects. In contrast, in the subjects aged50 years, the hypertensive subjects showed significantly higher frequencies of the DD and ID genotypes and the D allele than the normotensive subjects. In the present study, the frequency of the ACE I/D polymorphism did not differ significantly between the younger and older subjects (χ2=4.04, P=0.13).

Table 3 Genotype and allele distribution of the ACE I/D polymorphism in hypertensive and normotensive subjects

Discussion

In the present study, we demonstrated that Japanese male subjects with the D allele showed elevated BP levels in addition to an increased frequency of having hypertension. To date, convincing evidence of the association of the ACE I/D polymorphism with serum ACE levels has been reported.17 Previously, we also reported that serum ACE levels were significantly higher in the DD and ID genotypes than in the II genotype in subjects who underwent cardiac catheterization15 or renal biopsy16 in our hospital. Recently, three studies, two in Caucasians7,8 and one in Japanese,9 independently reported a significant association between the ACE I/D polymorphism and hypertension in male subjects. The current study confirmed these earlier observations in our study population.

A large number of studies have been undertaken to investigate the association between the ACE I/D polymorphism and BP, or the prevalence of hypertension, respectively. In their meta-analysis of 23 studies comprising 6923 subjects, Staessen et al3 found no association of the ACE I/D polymorphism and hypertension. Similarly, Agerholm-Larsen et al5 found no evidence for an association of the ACE I/D polymorphism with BP in their meta-analysis of 19 studies comprising 15 942 subjects. However, Staessen et al3 only included two studies from Japan in their meta-analysis; Agerholm-Larsen et al5 included none.

To date, there have been three population-based studies that were undertaken independently in different areas of Japan to determine the association of the ACE I/D polymorphism with BP or the prevalence of hypertension.9,11,12 First, Higaki et al9 reported that the D allele significantly increased the risk of essential hypertension in Japanese male subjects, using 5014 subjects (2340 males and 2674 females), who were randomly selected from the general population (the Suita Study). They reported that the estimated odds prevelence for hypertension (DD vs II) was 1.75 (95% confidental interval: 1.21–2.53). In contrast, recently, Zaman et al11 reported that there was no convincing evidence that the ACE I/D polymorphism was associated with hypertension, using 1340 subjects (464 males and 876 females) who were selected from the 1990s health examination data of four rural communities (the Shibata Study). More recently, Matsubara et al12 also reported that there was little association between the ACE I/D polymorphism and hypertension, using 1245 subjects (450 males and 795 females) who participated in the home or ambulatory BP measurement project in rural communities of Japan (the Ohasama Study). The results of the present study were similar to the findings of Higaki et al.9 We would like to emphasize that male subjects were enrolled more in the present study (n=706) than in the study of Zaman et al (n=464)11 or Matsubara et al (n=450).12 In addition, Zaman et al11 found a tendency of higher diastolic BP in male subjects with the DD genotype (P=0.06). Matsubara et al12 also observed a high prevalence of hypertension, specifically in elderly male subjects with the DD genotype, although no statistical significance was achieved.

In the present study, there was no significant association of the ACE I/D polymorphism with BP or a prevalence of hypertension in younger men aged <50 years, but there was in older men aged 50 years. In contrast, Higaki et al9 reported that the influence of the ACE I/D polymorphism on hypertension was greater in younger male subjects aged 60 years rather than in older male subjects aged >60 years. The reasons for these discrepancies are uncertain. However, a phenomenon of BP tracking is well recognized.18,19 We assume that the magnitude of the effects of the ACE I/D polymorphism on BP is amplified with ageing, although this hypothesis should be confirmed by further research.

In summary, we investigated the relation between the ACE I/D polymorphism and BP, or the frequency of hypertension, respectively, in 706 Japanese male subjects who participated in the health check-up programme of our hospital. We demonstrated that there was no significant association of the ACE I/D polymorphism with BP or a prevalence of hypertension in younger men aged <50 years, but there was in older men aged 50 years.

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Todoroki, M., Minami, J., Ishimitsu, T. et al. Relation between the angiotensin-converting enzyme insertion/deletion polymorphism and blood pressure in Japanese male subjects. J Hum Hypertens 17, 713–718 (2003). https://doi.org/10.1038/sj.jhh.1001601

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Keywords

  • angiotensin-converting enzyme
  • gene polymorphism
  • D allele
  • males
  • essential hypertension

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