2(1), 42-48, Feb 15, 2006
© 2005 Master Publishing Group
A Study of the Relationships between Serum Calcitonin Gene-Related Peptide, Sex Hormone, Homocysteine and
Coronary Heart Disease in Postmenopausal Women
Wang Zhen, Guo Jingxuan,
Wang Xian, Zhao Yiming,
Hou Lingfei
Wang Zhen
Department of Emergency
Peking University Third Hospital
Beijing 100083
P. R. of China.
Tel : 0086-010- 62017691-8229
Email: Wangzhen1369@Hotmail.com
Short title: Aminopeptidase A gene and hypertension
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ABSTRACT
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Objective. To investigate the relationships among serum calcitonin gene-related peptide (CGRP), homocysteine (Hcy), female hormone, and coronary arteriopathy in postmenopausal women. Method. In a cross-sectional study, serum CGRP, estrodiol (E2), progesterone（P）and Hcy levels of 144 postmenopausal women with coronary heart disease (CHD) and non-coronary heart disease (NCHD) were measured. Results. The mean serum CGRP level was significantly lower in CHD patients than in NCHD subjects. The mean serum E2 and P level were significantly lower in CHD patients than in NCHD subjects. The mean serum Hcy level was significantly higher in CHD patients than in NCHD subjects. By multivariate logistic regression, the OR of high Hcy level ≥1, p<0.01，which suggests that Hcy is an independent risk factor in the development of coronary arteriopathy. The OR of CGRP, E2 , and P are all≤1, indicating that CGRP , E2 and P are independent protective factor. Conclusion. The results of our study show that Hcy is an independent risk factor in the development of arteriopathy. CGRP and endogenous E2 are independent protective factors in the development of coronary arteriopathy. There are no relationships between Hcy, CGRP, and endogenous E2.
KEY WORDS:
Coronary heart disease
Calcitonin Gene-Related Peptide
Homocysteine Estradiol
postmenopausal women
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INTRODUCTION |
Numerous possible risk factors for coronary heart disease (CHD) have
been identified, of which include smoking, physical inactivity, gender, obesity,
age, hypertension, hyperlipemia and diabetes. In the 1990s many reports
associated elevated serum or plasma homocysteine (Hcy) levels with CHD,
suggesting that Hcy is a potent inducer of atherosclerosis [1-4]. The results of
our previous studies also confirm theses findings[5]. Calcitonin gene-related
peptide (CGRP) is a 37-amino acid neuropeptide mainly present in sensory nerve
fibers, which is present in almost all mammalian organs, including humans. It is
widely known that CGRP has various protective effects on the cardiovascular
system [6-7].
Hormonal decline is an endocrine characteristic of postmenopausal women, who
show a general decrease in female gonad hormones. Recent studies have found that
the decline of gonad hormone levels with age is accompanied by an increase risk
of cardiovascular diseases. Furthermore, some reports have shown that hormone
replacement therapy reduces Hcy levels [8-9], while other studies have shown no
correlation between hormone replacement therapy and Hcy[10-11] in postmenopausal
women. Some reports have demonstrated that E2 affects CGRP level in animals
[12-13].
Whether the decreases in endogenous female gonadal hormones are involved in the
age-related alterations of Hcy and CGRP remain unknown. There has also been
little reported on the relationships between serum Hcy, serum CGRP, and
endogenous female gonad hormone levels. The present study is designed to
determine the relationships among serum Hcy, serum CGRP, endogenous female gonad
hormone, and coronary arteriopathy in postmenopausal women.
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METHODS
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Case
ascertainment and control identification
One hundred and forty four postmenopausal women from a consecutive series of
subjects were enrolled in the study. The ages of the postmenopausal women ranged
from 51-73 years, with absence of menstrual bleedings for at least one year.
They were all with chest pain that suggest CHD or myocardial infarction (MI)
undergoing diagnostic coronary angiography for investigation of chest pain. CHD
indicates those subjects, the ages of postmenopausal women ranged from 56-72
years, with a mean ± standard deviation [SD] age of 63.9±6.5 years, whose scores
for severity of coronary arteriopathy≥1. NCHD indicates those subjects, the ages
of the postmenopausal women ranged from 50-71 years, with a mean ± standard
deviation [SD] age of 58.9±5.4 years, whose scores for severity of coronary
arteriopathy =0. All two groups had no history of renal, hepatic, endocrinologic,
gastrointestinal disease. They all did not take any estrogen and progestogen
therapy. All patients were evaluated for CHD risk factors. Clinical details,
including conventional risk factors for CHD (age, smoking, drinking, previous
MI, family history for premature CHD, hypertension, diabetes, hyperlipemia, drug
therapy, blood pressure, height and weight) were recorded for all patients by
interviews. Diagnostic coronary arteriography was carried out in all CHD and
NCHD patients. The World Health Organization criteria verified definite or
possible MI based on chest pain symptoms, electrocardiogram changes, and enzyme
determination were used to define previous MI [14]. Body mass index was
calculated (kg/m2).
Interpretation and scoring of coronary angiograms.
Angiographic scoring for each vessel segment was based on the severity and
extent of disease observed in the “most severe” projection. For the purpose of
analysis, the presence of angiographically detectable CHD was defined by an
extent score. All angiograms were reviewed by two experienced cardiologists who
had no knowledge of the patients clinical history and laboratory results.
Scoring of severity of coronary artery disease was performed with a modification
of the coronary atherosclerosis scoring system described previously [15]. The
percentage by which each lesion in the proximal coronary circulation narrowed
the artery was assessed according to the maximal narrowing of the diameter of
the artery in all projections. The extent and severity of the proximal coronary
disease was assessed by assigning points to each lesion as follows: less than
50% stenosis of the luminal diameter, 1 point; 50% to 74% stenosis, 2 points;
75% to 99% stenosis, 3 points; total obstruction, 4 points. The points for each
lesion in the proximal coronary circulation were summed and a score for severity
of coronary atherosclerosis was obtained.
Laboratory measurements
All venous blood specimens were drawn from patients at 8:00 a.m. after 12 hours
fast before angiography. Blood samples were centrifuged in low temperature
within one hour. Part of the serum was used to determine serum lipids, and the
remaining was stored at -30C for subsequent analysis for Hcy, estradiol (E2),
progesterone (P), and CGRP. Serum lipids were measured with Technicin
Autoanalyzer 7010A (Hitachi, Japan). High-density lipoprotein cholesterol (HDL-C),
low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and
triglyceride (TG) were measured by enzymatic method.
Hcy were measured using high–performance liquid chromatography (HPLC) with
420-AC fluorescence detection (Waters, U.S.A) and HPLC reagent kits (Bio-Rad,
U.S.A).
The radioimmunoassay (RIA) technique used for the measurement of CGRP levels in
the present study was similar to our previous reports [16]. Briefly, standards
of synthetic CGRP (rat amino acid sequence, Peninsula Laboratory, USA) ranging
from 2.5 to 1000 pg/assay tube or the dried samples were reconstituted in a
volume of 200 µl RIA buffer (0.1 M, pH 7.4, phosphate buffer containing 0.1% BSA,
0.01% NaN3, 50 mM NaCl, 0.1% Triton X-100) and incubated for 24 h at 4°C with
100 µl anti-CGRP antibody (Peninsula Laboratory, Belmont, CA, USA) diluted in
RIA buffer. The mixture was then incubated for another 24 h at 4°C with 100 µl
125I-labelled CGRP (125ICGRP [human sequence] was purchased from Amersham
(Arlington Heights, IL, USA).) in RIA buffer. Free and bound fractions were
separated by adding 100 µl goat anti-rabbit IgG and normal rabbit serum incubate
at room temperature for 2 h. An additional 0.5 ml RIA buffer was added, and the
RIA test tubes were centrifuged (2800 ×g, 4°C) for 20 min. After removal of the
supernatant, the RIA test tubes were counted by [gamma]-counter. The IC50 values
for the CGRP RIA were 30–40 pg/RIA tube. The intraassay coefficient of variation
was 5.1%. For the intraassay coefficients of variation, the corresponding values
4.5%.
Concentration of E2 and P were measured by using Immulite Automated Immunoassay
System (DPC Inc. American). The intra-assay and inter-assay variability was less
than 5%.
Statistical analyses. Statistical analysis was performed with SPSS
10.0(Statistical computer package for the Social Science; SPSS Inc., Chicago,
IL.) Data of continuous variables are expressed as mean (±standard deviation
[SD]). Data were analyzed by one-way analysis of variance (ANOVA). Student’t
test for means between two groups. P<0.05 indicates significant difference. Data
were further analyzed by logistic regression for multiple comparisons. A
logistic analysis remains an effective statistical tool for investigating the
role of a series of measured independent variables. That is, the relationship
among variables is modeled by an additive logistic equation. Then coefficient of
a sequential analysis is given with forward method to identify if the factor is
independent or not. In logistic regression model, if OR of a variable>1, means
this factor is a risk factor, otherwise, OR<1, a protect factor [17].
Results
Descriptive characteristics of subjects in the study are listed in table 1.
Subjects of CHD group have high rates of suffering diabetes and hypertension,
there were significant difference compared with NCHD group. P<0.05. Ages of CHD
group were higher than NCHD group, P<0.05. Subjects of CHD group have higher
serum Hcy level than NCHD group. Serum CGRP level, E2 level and P level in two
groups showed an opposite trend compared with Hcy level. CHD group have lower
serum CGRP level, E2 level and P level than NCHD group. Serum TG level was
higher in CHD group compared with NCHD group, P<0.05. There were no difference
in serum TCHO, LDL-C, HDL-C and BMI between CHD and NCHD group.
Single factor analysis showed elevated Hcy level, higher ages, hypertension,
diabetes, lower CGRP level, E2 level and P level having relationship with CHD.
That means all these factors may have influence on the generation of
arteriopathy. But which one is independent risk factor? Which one is not? These
factors contribute unequally to the outcome (CHD). Therefore, single factor
analysis could not answer this question. An extremely useful approach to
identifying the relationship among multivariate measurements involves the
application of logistic regression model. It gives a comprehensive picture of
the application of logistic model to explore the relationships with
multivariable data. In order to understand the effects of elevated Hcy level and
other factors, the data were further analyzed by multivariate logistic
regression model (Table 2). We can found that when Hcy alone entering the
regression model, the OR of Hcy≥1, the difference is very small, p<0.01. We can
identify that elevated Hcy level was an independent risk factor in the
development of women CHD. Similarly, when old age, hypertension, diabetes, TG
alone entering the regression model, the OR of old age, hypertension, diabetes,
TG were all ≥1, the difference is very small, p<0.01. We can identify that old
age, hypertension, diabetes, TG were all independent risk factor in the
development of women CHD. This is similar with the results of single factor
analysis. Oppositely, when CGRP alone entering the regression model, the OR of
CGRP ≤1, the difference is very small, p<0.01. In accordance with CGRP, when E2
and P alone entering the regression model, the OR of CGRP ≤1, the difference is
very small, p<0.01. We can identify that CGRP, E2 and P were all independent
protective factor in the development of women CHD. This is similar with the
results of single factor analysis also.
This study aimed to find the relationship among Hcy, CGRP, and E2. Therefore
with forward method, from table 3-5, we can found that when hypertension
entering regression model, the OR of hypertension ≥1, the difference is very
small, p<0.01. But when CGRP entering the regression model, the OR of
hypertension changed greatly, P>0.05, showing hypertension has the similar
effects compared with lower CGRP level. That means lower CGRP level and
hypertension were all independent risk factor in the development of women CHD.
Similarly, when age, TG and diabetes entering the regression model, the OR of
TG, age and diabetes were all≥1, p<0.01. But when CGRP entering the regression
model, the OR of age, TG and diabetes changed greatly, P>0.05. That means lower
CGRP level, TG, high age and diabetes were all independent risk factor in the
development of women CHD. Above analysis showed that age, diabetes,
hypertension, TG and lower CGRP level have similar effects.
To study the relationship among CGRP, Hcy and female gonad hormone, with forward
method, they were also analyzed by logistic regression model (table 6). The last
main logistic regression model involves CGRP, Hcy and E2. We can find that when
CGRP and Hcy separately entering the regression model, the coefficients of E2
changed to –0.052→-0.052→-0.058. When Hcy entering the regression model, the
coefficients of CGRP changed from -0.022 to -0.018, the coefficients changed
little (table 7). This showed the three factors are independent.
 DISCUSSIONS
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Numerous possible risk factors for coronary heart disease (CHD) have been
identified. In the present study, we found that old ages, diabetes TG and
hypertension are all independent risk factors for CHD in postmenopausal women,
which is consistent with prior reports [18]. Hcy is a sulfur-containing amino
acid, formed from methionine as a product of numerous S-adenosylmethionine-dependent
transmethylation reactions. Two enzymes utilize Hcy, including
tathionineβ-synthase(CBS) and methionine synthase(MS)[19]. A very wide range of
effects has been attributed to Hcy. These include direct damage to endothelial
cells, flawed platelet activity, elevated procoagulant activity, increased
collagen synthesis, and enhanced proliferation of smooth muscle cells.
Biochemically, it has been proposed that Hcy modifies eicosanoid metabolism,
promotes translocation of protein kinase C from cell nuclei and cyto-plasm to
the cell membranes, and induces c-fos and c-myb activity [20-24]. In this study
we found that an elevated increase in Hcy level is associated positively with
higher scores for severity of coronary atherosclerosis, which is also consistent
with other case control studies [1-5]. Hcy levels increase with age in women,
but the mechanism is not clear [25-26]. In the present study, the mean serum Hcy
level was significantly higher in CHD postmenopausal women than in NCHD
postmenopausal women and young subjects. However, a multivariate logistic
regression analysis showed no relationship between Hcy and E2 levels.
CGRP is a sensory neuropeptide with potent vasodilatory and cardiotonic actions.
It is a more potent vasodilator than other endogenous or synthetic compounds,
which include prostacyclin, adrenaline, histamine, vasoactive intestinal
polypeptide, and sodium nitroprusside. Furthermore, CGRP is present in the
adventitial nerves surrounding most peripheral blood vessels, including coronary
and cerebral vessels [6-7]. In the present study, the mean serum CGRP level is
significantly lower in CHD postmenopausal women than in NCHD postmenopausal
women. In the multivariate logistic regression analysis, CGRP was an independent
protective factor in the development of coronary arteriopathy in women, but
there was no relationship between CGRP and E2 level.
In recent years people have begun to pay much more attention to the relationship
between CHD and hormone replacement therapy, as well as health benefits and
risks of hormone therapy. HERS II study indicated that after 6.8 years, hormone
therapy did not reduce risk of cardiovascular events in women with CHD. Results
from WHI study showed that overall health risks exceeded benefits from the use
of combined estrogen and progestin for an average 5.2-year follow-up among
healthy postmenopausal US women [27-28]. In the present study, we found that
there is no relationship between CGRP and endogenous female gonad hormone (E2)
in postmenopausal women without hormone therapy. That is, endogenous female
gonad hormone (E2) neither increases serum CGRP levels (which is a protective
factor to CHD) nor drops serum Hcy levels (which is a risk factor to CHD) in
postmenopausal women with CHD. However, the fact is that postmenopausal women do
have higher rates of CHD than young women, of which there is no simple
explanation. The decline of endogenous female gonad hormone levels with age is
accompanied by other pathophysiologic changes, such serum lipids, etc. The
relationship between female gonad hormone and CHD will continue to remain an
important topic.
Furthermore, serum lipids are risk factors for CHD. In the present study, we
found no differences in serum lipid levels (TCHO, HDL-C, LDL-C) between the CHD
and NCHD groups. This may be due to the limits in choosing patients from the
patient population. All of the patients that underwent diagnostic coronary
angiography have been treated for the secondary prevention of cardiovascular
disease (including lipid-therapy).
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CONCLUSION |
The results of the present study show that Hcy is an independent risk
factor in the development of arteriopathy in postmenopausal women. CGRP and
endogenous E2 are independent protective factors in the development of coronary
arteriopathy. There are no relationships between Hcy, CGRP and endogenous E2
levels.
