Gender difference of serum angiotensin-converting enzyme (ACE) activity in DD genotype of ACE insertion/deletion polymorphism in elderly Chinese

Abstract

Introduction:

In this study we investigated the gender difference of serum angiotensin-converting enzyme (ACE) activity in a population of Hong Kong-dwelling elderly Chinese.

Materials and methods:

A total of 1767 (843 male, 924 female) Hong Kong-dwelling elderly Chinese were recruited. ACE I/D genotypes were identified by polymerase chain reaction amplification and serum ACE activity was determined using a commercially available kinetic kit. ACE I/D genotype distribution was compared by chi-square test, the correlation between ACE I/D polymorphism and serum ACE activity was analysed by ANOVA test and gender difference of serum ACE activity of different genotypes was compared by independent sample t-test.

Results:

No statistically significant difference of genotype distribution between male and female subjects was found. Serum ACE activity was significantly correlated with ACE genotype. Overall, there was no gender difference of serum ACE activity; however, when sub-grouping the subjects by ACE I/D genotype, male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype.

Conclusions:

No significant gender difference of genotype distribution was found in elderly Chinese. Serum ACE activity was significantly correlated with ACE I/D polymorphism in elderly Chinese. Male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype.

Keywords

Angiotensin-converting enzyme insertion/deletion polymorphism gender difference

Introduction

Angiotensin-converting enzyme (ACE) is a transmembrane zinc metallopeptidase which plays a major role in the metabolism of vasoactive peptides by converting decapeptide angiotensin I into vasoconstrictor octapeptide angiotensin II and inactivating bradykinin.^1,2 ACE is bound to endothelial surface membrane by an anchor peptide, which could be cleaved by ACE secretase to form a soluble enzyme called serum ACE.³

ACE plays an important role in the regulation of systemic blood pressure and renal development and function.⁴ ACE inhibitors are widely used in the treatment of cardiovascular diseases, such as hypertension, ischaemic heart disease and congestive heart failure.⁵ However, hypertension, ischaemic heart disease and congestive heart failure are male biased diseases and underlying mechanisms are still not well elucidated. In an animal study male mice were shown to have higher plasma ACE activity than females,⁴ which suggested that gender specific regulation of ACE may be a pathway to understand the male bias of cardiovascular diseases. So in this study, we first set out to clarify whether there is a gender difference in serum ACE activity in older subjects who may be prone to cardiovascular disease.⁶

It was reported that genetic factors had great impact on serum ACE activity.⁷ In 1990, Rigat et al. found that a polymorphism involving the presence (insertion, I) or absence (deletion, D) of a 287-bp sequence of DNA in intron 16 of ACE gene accounted for approximately half of the variant in serum ACE activity in Caucasians. Subjects with DD genotype had the highest serum ACE activity, subjects with II genotype had the lowest serum ACE activity and subjects with ID genotype had intermediate serum ACE activity.⁸ In Asian subjects, ACE I/D polymorphism also had great impact on serum ACE activity, with the same trend as in Caucasians.⁹ It was reported that ACE D allele was positively correlated with atherosclerosis.¹⁰ There is a gender difference of blood pressure response to hydrochlorothiazide, with declining diastolic blood pressure greater in male DD genotype holders than female DD genotype holders.¹¹ So there seems to be a sex dimorphism in the significance of ACE genotype in disease outcomes and mortality; therefore, secondly in this study, we set out to sub-group the subjects by ACE genotype to try to compare the serum ACE activity between male and female subjects in different ACE genotypes.

Materials and methods

Subjects

The subjects of this study were from two large cohort studies which investigated risk factors of osteoporotic fractures in Hong Kong-dwelling elderly Chinese aged 65 years and above. The details of including and excluding criteria and data collection methods were demonstrated elsewhere.^12,13 Briefly, 4000 community-dwelling Chinese (2000 of each gender) aged 65 years and above were recruited using a combination of private solicitation and public advertising in Hong Kong. Written informed consent was obtained from all the subjects, and the study protocol was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong.

A standardized structured face-to-face interview was performed by certificated research assistants to collect demographic information, lifestyle, personal medical history, including hypertension, diabetes mellitus, cardiac failure, respiratory diseases, etc. and medication history. Participants were asked to bring all the medications they were taking to the clinical centre, then trained research assistants recorded all drugs for each person.

Subjects taking ACE inhibitors and angiotensin II type I receptors (ARBs) were excluded from this study, and subjects with hypertension, diabetes mellitus and respiratory diseases, including chronic obstructive lung disease, chronic bronchitis, asthma and emphysema, were also excluded. Finally, 1767 Hong Kong-dwelling elderly Chinese entered into this study (843 male: 71.8±4.9 years; 924 female: 72.4±5.4 years).

DNA extraction and ACE I/D genotyping

Peripheral venous blood was taken after an overnight fast for serum isolation and DNA extraction. DNA was later extracted using the standard phenol/chloroform extraction method. I/D genotypes of ACE were determined by polymerase chain reaction (PCR) amplification. PCR mixtures (25 µl) were set up which contained 1X reaction buffer (Fermentas Life Sciences), 2 mM MgCl2, 1 µM of each forward and reverse primer, 0.2 mM each dNTP, 0.6 U Taq polymerase (Fermentas Life Sciences) and 50 ng DNA. The sequences of forward and reverse primer were 5’-AGAGAGACTCAAGCACGCCC-3’ and 5’-ACCCAAGTGCCAGTGATGTT-3’, respectively. The thermal cycling profile began with initial denaturation at 96°C for 5 min, followed by 35 cycles at 96°C for 30 s, 63.8°C for 45 s, 72°C for 30 s and a final extension at 72°C for 10 min. Amplification products yielded were of 439 bp for the D allele and 727 bp for the I allele. The products were then separated by electrophoresis in 2% agarose gels with ethidium bromide and were visualized under UV transillumination.

Serum ACE activity measurement

Serum ACE activity was determined photometrically by a commercially available kinetic kit purchased from Bühlmann Laboratories AG (Allschwil, Switzerland). Testing was performed according to the manufacturer’s instructions. ACE catalyses the hydrolysation of the synthetic substance N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine. This hydrolysis results in a decrease in absorbance at 340 nm. The measurement was automatically done by Roche COBAS MIRA Plus Chemistry Analyser.

Statistical methods

Continuous variables were presented as mean ± standard deviation (SD) and categorical variables as number (frequency). The gender difference of genotype distribution was analysed by chi-square test. The correlation between ACE genotype and serum ACE activity was analysed using the ANOVA test. Independent sample t test was used for comparing serum ACE activity between genders. All analyses were performed with SPSS software (version 11.0, Chicago, USA). All statistical tests were two-sided and a p-value of less than 0.05 was considered to indicate a statistically significant difference.

Results

General characteristics of study populations

The general characteristics of study populations are presented in Table 1. Generally, there was no statistically significant difference of general characteristics between different genotypes.

Table 1.

General characteristics of study populations.

Male	II (n=378)	ID (n=386)	DD (n=79)	p-value
Age (years)	71.90±4.71	71.77±5.00	71.73±5.16	0.920
Weight (kg)	61.70±9.67	60.55±8.53	61.54±10.62	0.215
BMI	23.01±3.20	22.73±2.84	22.88±3.47	0.449
Smoking	66 (17.4%)	53 (13.7%)	17 (21.5%)	0.149
SBP (mmHg)	137.41±19.62	138.83±18.98	138.44±19.94	0.591
DBP (mmHg)	77.48±9.22	77.65±9.06	78.49±7.68	0.663

Female	II (n=437)	ID (n=382)	DD (n=105)	p-value

Age (years)	71.84±5.22	72.58±5.44	73.43±5.92	0.012
Weight (kg)	53.59±8.38	53.49±8.81	52.53±8.43	0.512
BMI	23.45±3.38	23.54±3.70	23.16±3.38	0.622
Smoking	5 (1.1%)	15 (3.9%)	2 (1.9%)	0.118
SBP (mmHg)	139.88±18.89	141.43±18.74	142.96±16.04	0.232
DBP (mmHg)	76.89±9.17	77.75±8.92	76.40±9.21	0.256

Data were presented as mean ± standard deviation or number (frequency). There was no statistically significant difference between different genotypes.

BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure.

ACE I/D genotype distribution of the subjects

In total in this study, the homozygous genotype II was present in 46.1% subjects, heterozygous genotype ID was present in 43.5% subjects, and 10.4% of subjects held the homozygous DD genotype. When sub-grouping the subjects by gender, the frequency of II, ID and DD genotype was 44.8%, 45.8% and 9.4%, respectively, in male subjects, and 47.3%, 41.3%, and 11.4%, respectively, in female subjects. There was no statistically significant gender difference of genotype frequency (details are presented in Table 2).

Table 2.

ACE I/D genotype frequency in male and female subjects.

	II	ID	DD
General (n=1767)	815 (46.1%)	768 (43.5%)	184 (10.4%)
Male (n=843)	378 (44.8%)	386 (45.8%)	79 (9.4%)
Female (n=924)	437 (47.3%)	382 (41.3%)	105 (11.4%)

Data were presented as number (frequency). There is no statistically significant difference of genotype frequency between male and female subjects (p=0.119).

Correlation between serum ACE activity and ACE I/D polymorphism

Serum ACE activity was significantly correlated with ACE I/D polymorphism both in male and in female subjects, subjects with DD genotype had the highest serum ACE activity (65.0±22.6 U/l), followed by subjects with ID genotype (53.1±18.0 U/l), and subjects with II genotype had the lowest serum ACE activity (37.9±13.5 U/l).

Gender difference of serum ACE activity in different ACE I/D genotype

The overall serum ACE activity was 47.3±19.1 U/l. It was 47.9±20.0 U/l and 46.8±18.3 U/l for male and female subjects, respectively. There was no statistically significant difference of serum ACE activity between male and female subjects. When sub-grouping the serum ACE activity by ACE I/D genotype, there was no gender difference of serum ACE activity in II genotype (male: 38.6±14.2 U/l; female: 37.2±12.8 U/l; p=0.144) and ID genotype (male: 52.5±18.4 U/l; female: 53.7±17.7 U/l; p=0.379); however, male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype (male: 69.8±26.1 U/l; female: 61.4±18.9 U/l; p=0.017) (details are presented in Table 3).

Table 3.

Comparison of serum ACE activity between gender and different ACE I/D genotype.

	Serum ACE activity (U/l)				p-value
	General	II	ID	DD
General	47.3±19.1	37.9±13.5	53.1±18.0	65.0±22.6	<0.005
Male	47.9±20.0	38.6±14.2	52.5±18.4	69.8±26.1	<0.005
Female	46.8±18.3	37.2±12.8	53.7±17.7	61.4±18.9	<0.005
p-value	0.211	0.144	0.379	0.017

Data were presented as mean ± standard deviation. Serum ACE activity was significantly correlated with ACE I/D genotype both in male and in female subjects (p<0.05). Totally, there is no gender difference of serum ACE activity. By sub-grouping analysis, the gender difference showed only in DD genotype (p=0.017).

Discussion

In this study we found no gender difference of ACE I/D genotype distribution. Serum ACE activity was significantly corrected with ACE I/D polymorphism both in male and in female subjects. Overall, there was no gender difference of serum ACE activity. However, when sub-grouping the subjects by ACE I/D genotype, male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype.

The ACE genotype frequency of our study was similar to other published studies of the same ethnics.^14,15 However, it was different from Caucasians: the frequency of DD genotype was lower and II genotype was higher in Asian subjects than in Caucasians.^16,17 No statistically significant difference of genotype distribution was found between male and female subjects in our study, which was similar to that of Caucasians.¹⁷ It was reported that ACE I/D polymorphism was related to many clinical outcomes. Some published papers reported that ACE I/D polymorphism was associated with blood pressure,^18,19 but these were not consistent with other published papers.^20,21 A meta-analysis listed 26 association studies, of which 12 published positive and 14 published negative results,²² so the effects of ACE I/D polymorphism on blood pressure, if any, may be very small. ACE D allele was also reported connected with atherosclerosis, particularly in those who carry other cardiovascular risk factors.²³ The relationship between ACE I/D polymorphism and coronary heart disease has also been investigated.^24,25 Interestingly, in large sample size studies, no positive relationship was found; however, in some small sample size studies, DD genotype was positively correlated with coronary heart disease, which may because of sample size bias. Small sample size studies usually select hospitalized or high risk patients,²⁶ so, generally, D allele was not clinically important in the general population, but may play an important role in certain groups of patients.²⁷ Our results showed there was no significant difference of ACE genotype frequency between male and female subjects. So factors other than ACE I/D polymorphism may be the underlying mechanism of the gender bias of some cardiovascular diseases.

In an animal study it was reported that plasma ACE activity but not tissue ACE activity was sexually dimorphic in mice, with male mice having a higher plasma ACE activity than female mice.²⁸ Plasma ACE activity decreased after gonadectomy both in male and in female mice, but the decrease was more severe in male mice.²⁸ It has been demonstrated that orchiectomy reduced blood pressure, while ovariectomy had no effect on blood pressure in spontaneously hypertensive rats.²⁹ Therefore, serum ACE activity may be important in understanding the gender bias in cardiovascular disease.

Serum ACE activity was significantly correlated with ACE genotype both in male and in female subjects, II genotype had the lowest serum ACE activity, DD genotype had the highest serum ACE activity and ID genotype had intermediate serum ACE activity. However, our results showed that, generally, there was no significant difference of serum ACE activity between male and female subjects. Since ACE I/D polymorphism had great impact on serum ACE activity, and ACE I/D polymorphism was correlated to some diseases and treatment outcomes,^10,11,23,26 we then sub-grouped the serum ACE activity by ACE I/D genotype, compared the serum ACE activity of different genotypes between male and female subjects and found that male subjects with DD genotype had higher serum ACE activity than female subjects with DD genotype.

Actually, the gender difference of serum ACE activity had been reported in a previous study, in a group of 159 healthy adult Caucasians (53 male and 106 female) from northern Germany. A male–female difference was apparent only in individuals with ID genotype; however, due to the sample size, female subjects were over-represented in the study and after correcting the value for sex ratio no significant gender difference existed.³⁰ Serum ACE activity was correlated to many diseases; it was evaluated in patients with untreated active sarcoidosis, and spontaneous or corticosteroid-induced remission of sarcoidosis could be heralded by a decrease in serum ACE level,^31,32 so measurement of serum ACE activity can be used as a tool in the monitoring of sarcoidosis. Since ACE I/D polymorphism had a great impact on serum ACE activity, a genotype-corrected reference value of serum ACE activity was quite important for clinical monitoring of sarcoidosis.³⁰ Our results indicated that, in DD genotype, a truer picture will be obtained if different reference values are used for male and female subjects, respectively. However, it is unknown whether a gender difference exists regarding the treatment effect of sarcoidosis. Further studies are required.

Serum ACE activity was also correlated with some other clinical outcomes, for example, coronary stent restenosis. It was reported that plasma ACE level determination was more predictive than ACE I/D genotype for risk of restenosis after coronary stent, which may due to the large variation of serum ACE activity in different ACE I/D genotypes.³³ Although serum ACE activity was significantly affected by ACE I/D genotype and the serum ACE activity of DD genotype was nearly twice as high as that of II genotype, the variation of serum ACE activity in DD genotype was still very large. As a categorical variable, ACE I/D genotype may be less precise than serum ACE activity, since serum ACE activity was a continuous variable. Serum ACE activity provided additional information for the correlation analysis of ACE I/D polymorphism and coronary stent and further studies should use serum ACE activity instead of ACE I/D polymorphism as a marker for this analysis. We are interested to know whether the gender difference of serum ACE activity in DD genotype will affect the incidence of coronary stent restenosis. Further studies need to be done.

In elder subjects, ACE II genotype was associated with insulin resistance.³⁴ Since genotype distribution was similar between male and female subjects, the frequency of hypoglycaemia may have no gender difference, which was consistent with data from the Diabetes Control and Complication Trial. In that study, it was demonstrated that the prevalence of hypoglycaemia in type I diabetes was gender neutral.³⁵ However, it was reported that plasma glucose levels fall to a lower level in women compared to men during short to moderate fasting,^36,37 which suggested a sexual dimorphism may exist in hypoglycaemic counter-regulation. Interestingly, it was demonstrated that high serum ACE activity is strongly associated with an increased risk of severe hypoglycaemia in type I diabetes.³⁸ Our results showed serum ACE activity of male subjects is higher than female subjects in DD genotype, which may be one of the possible reasons neutralizing the sexual dimorphism. Further studies need not be done to clarify the exact underlying mechanism.

In conclusion, in this study we found a gender difference of serum ACE activity in ACE DD genotype, which provided some additional information in explaining the correlation between ACE I/D genotype and some diseases or clinical outcomes. More research works need to be done to demonstrate the significance of this gender difference.

Footnotes

Acknowledgements

The authors would like to thank Dr Tanya Chu, Ms Emily Poon and Mr Martin Li from the Department of Medicine and Therapeutics, The Chinese University of Hong Kong, for their kindly help in serum ACE activity measurement.

Conflict of interest

We have no conflict of interest.

Funding

This research was supported by the Research Grants Council (Earmarked Grant CUHK4101/02M), the National Institute of Health (R01 grant AR049439-01A1), the Natural Science Fund of Jiangsu Province, PR China (ref. BK2010285) and the Collegiate Natural Science Fund of Jiangsu Province, PR China (ref. 10KJB320015).

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