TITLE:

Hyper-homocysteinemia Correlation With Coronary Artery Disease In Young Adults

ABSTRACT:
Objective: Coronary artery disease is major cause of mortality and morbidity.
Homocysteine has long been postulated as an underlying factor for atherosclerosis leading
to coronary artery disease, yet its role in young age group patients is uncertain This study is
therefore aimed to study the correlation between plasma homocysteine and cardiovascular
thrombosis in young adults in absence of conventional risk factors.
Methods: Case control study, carried out at Rehman Medical institute from 1st Oct, 2016
to 30th Sept, 2017 using universal sampling technique. Total of 158 subjects were included
among whom 30 subjects were in control group and 128 subjects in patient group, who had
moderate to severe stenosis in either single or multiple major coronary arteries on coronary
angiography having age less than 40 years.
Results: Cases and controls had similar characteristics but differed significantly in serum
homocysteine concentration. Mean age was 32.84(±2.14) years in control group and 33.81
(±2.74) years in cases group with mean plasma homocysteine concentration of
6.3(±2.05)μmol/L in control group and 44.5(±14.01)μmol/L in patients group. All the
h
.

IKeyWords: Hyper-homocysteinemia, Risk factor, atherosclerosis, cardiovascular,

n
thrombosis, Young Adults

p
INTRODUCTION:
a
Cardiovascular diseases are the leading cause of both mortality and morbidity. Globally
t
one third of all deaths occur due to cardiovascular events.1 In countries with lower and
i
middle income, mortality from cardiovascular diseases are at the fore-front and in
e
developed countries like United States, they contribute to more than 6 hundred thousand
n
deaths yearly.2 Ischemic heart disease is the biggest culprit contributing to major chunk of
t
mortality from heart diseases.3 Atherosclerosis is hallmark of ischemic heart disease which
s characterized by narrowing or blockage of coronary arteries supplying all the nutrients
is
and oxygen to the heart.4 Coronary angiography is gold standard for detecting coronary
g
artery disease.5
r Homocysteine accelerates atherosclerosis by increasing the pro-thrombotic factors
o
XII, V and decreasing the antithrombotic factors along with endothelial derived nitric
h
oxide. Hyper-homocysteinemia also inculcates direct endothelial cell damage, which leads
to smooth muscle cells hyperplasia, contributing to occlusion or narrowing of the vessels.
a
Increased plasma levels of homocysteine contributes to cardiac morbidities and it has
m
positive association with hypertension.6 Aspirin resistance is also increased with high
a
levels of homocysteine in blood.7 Therefore homocysteine has been thought of as an
j
independent risk factor contributing to coronary artery disease and Framingham Risk score
o been challenged.8
has
Low levels of Vitamin B12 and high serum concentration of homocysteine has
Hype-homocysteinemia
been associated with coronary
has positive
artery disease
correlation
in Asians
with cardiovascular
and studied by thrombosis
two different
instudies
young
9,10
adults
in Indian
even population.
in the absence of
Homocysteine
conventional risk
is afactors.
modifiable risk factor and folic acid
supplementation improves the endothelial dysfunction caused by high serum concentration
of homocysteine.11 This study is therefore aimed to study correlation of serum
homocysteine and cardiovascular thrombosis in young adults. As it’s a modifiable risk
factor, targeting it will decrease both the cardiovascular mortality and morbidity.

METHODS:
This study was carried out at Rehman Medical Institute (RMI), which is a specialized
tertiary care hospital providing modern state of the art facilities to the patients from all
across the province and neighboring countries. It was an observational case control study
carried out for a period of 12 months from 1st October, 2016 to 30th September, 2017.A total
of 158 subjects were included having 128 subject in patient group and 30 subjects in
control group. Control group was selected randomly from the general population having
age less than 40 years and more than 25 years having same features as patient group. In
patients group universal sampling technique was adopted and every patient who had
angiographic evidence of moderate to severe stenosis in single or more major coronary
artery on coronary angiography with age less than 40 years was included. Patients from
both genders, different ethnic backgrounds, multiple geographic locations and different
socioeconomic statuses were part of the study population in both cases and control groups.
All those subjects who had mild stenosis in coronary arteries, suffering from
diabetes, hypertension, were active smokers, having dyslipidemia, taking drugs like
anti-depressants (S-adenosyl methionine), methotrexate, phenytoin, carbamazepine,
6-azauridine triacetate, recently undergone general Anesthesia or were exposed to nitrous
oxide were excluded from the study population. Patients who did not give written consent
were also not included. Informed written consent was taken and confidentiality of the
patient was ensured. Data was collected using a printed Performa. Fasting sample was
collected using 8 hours fasting cutoff index. Under aseptic technique using alcohol swab
5ml of blood was collected in EDTA tube. Plasma homocysteine levels were assessed using
the quantitative measurement on AxSYM System - Fluorescence Polarization
Immunoassay (FPIA). This study was duly approved by Research and Ethics committee
(RnE) of Rehman Medical Institute.
Shapiro-wilk test was applied to check the distribution of data. Continuous
variables were determined as mean ± standard deviation test and ANOVA were used for
comparison of mean. Pearson test was used to compare the severity of
hyper-homocysteinemia with severity of coronary artery disease and number of coronary
artery disease. P value of less than 0.5 was considered significant.

OPERATIONAL DEFINITIONS:
Coronary artery disease (CAD) was defined as stenosis on coronary angiography using the
QCA(quantification of coronary atherosclerosis) scoring system as follow;
a) Mild CAD was defined as <30% stenosis in coronary arteries.
b) Moderate CAD was defined as 30- 70% stenosis in coronary arteries.
c) Severe CAD was defined as >70% stenosis in coronary arteries.
Patients having moderate to severe stenosis in single vessel were labelled as single vessel
coronary artery disease (SVCAD)
Patients having moderate to severe stenosis in 2 major vessels were labelled as double
vessel coronary artery disease (DVCAD)
Patients having moderate to severe stenosis in 3 major vessels were labelled as triple vessel
coronary artery disease (TVCAD)
Mild Hyper-homocysteinemia was defined as Plasma Homocysteine level of >12µmol/L to
14.9µmol/L
Moderate Hyper-homocysteinemia was defined as Plasma Homocysteine level of
>15µmol/L to 29.9µmol/L
Intermediate Hyper-homocysteinemia was defined as Plasma Homocysteine level of
>30µmol/L to 100µmol/L
Severe Hyper-homocysteinemia was defined as Plasma Homocysteine level of
>100µmol/L.

RESULTS:
The total number of study population was 158 with 128 of those being patients and 30
being controls were included in the study. Mean age of 32.84(±2.14) years was observed
among controls and 33.81(±2.74) in patient group. Among controls 68.1% of subjects were
males and 31.9% females while in patients group total males were 67.2% and females
32.8%. A total of 13.5% subjects among control group and 14.3% in patients group were
smokers, while diabetes was present in 6.4% subjects of control group and 7.1% of patients
group. Hypertension was present in 9.4% subjects of control group and 10.2% of patients
group. Body Mass Index(BMI) was calculated as 22.95(±2.65) in control group while
23.28((±2.08) among the patients group. Mean plasma homocysteine concentration of
6.3(±2.05) µmol/L was observed in control group while patient group had plasma
concentration of 44.5(±14.01) µmol/L as shown in table 1.

Table 1: Basic characteristics of controls and cases

Controls Cases
Age 32.84(±2.14) 33.81(±2.74)
Gender Males 68.1 % 67.2 %

Females 31.9 % 32.8 %

Smoking 13.5 % 14.3 %

Diabetes Mellitus 6.4 % 7.1%
Hypertension 9.4 % 10.2%
BMI 22.95(±2.65) 23.28((±2.08)
Homocysteine (µmol/L) 6.3(±2.05) 44.5 (±14.01)
Figure 1.1 comparison of plasma homocysteine concentration between cases and
controls.
There was a significant difference between the plasma concentration of homocysteine between cases
and controls the average plasma concentration of homocysteine being 44.5 µmol/L in the cases and
6.3 µmol/L in controls

None of the patient was suffering from mild hyper-homocysteinemia with 11.7% patients
having moderate increase in plasma homocysteine level among whom 8.6% were males
and 3.1% were females. Intermediate increase was observed in 85.2% patients having
56.3% males and 28.9% females while 3.1% patients were having severe
hyper-homocysteinemia with 2.3% males and 0.8% females as shown in table # 2.
Table 2 The intensity of hyper-homocysteinemia in the patients

Intensity Of Hyper-homocysteinemia in Patients Group

Mild Moderate Intermediate Severe Increase
Gender Increase Increase Increase (>100µmol/L) Total
(12-14.9 (15-29.9 (30-100
µmol/L) µmol/L) µmol/L)

0 11(8.6%) 72(56.3%) 3(2.3%) 86(67.2%)

Male
0 4(3.1%) 37(28.9%) 1(0.8%) 42(32.8%)
Female
Total 0 15(11.7%) 109(85.2%) 4(3.1%) 128
Out of 15(11.7%) patients suffering from moderate hyper-homocystenemia 10 were having
moderate stenosis while 5 had severe stenosis in Single Vessel coronary artery disease
(SVCAD). Among 109(85.2%) patients suffering from intermediate increase in plasma
homocysteine levels 103 patients were having severe stenosis in SVCAD and 6 patients
had severe stenosis in more than one coronary artery disease. A total of 4(3.1%) patients
were suffering from severe hyper-homocysteinemia all of them having severe stenosis in
more than one major coronary artery as shown in table # 3.

Table 3 severity of the hyper-homocysteinemia with no of vessels and severity of CAD

Severity Of No Of Vessels Involved Severity of CAD

Hyper-homocysteinemia SVCAD DVCAD TVCAD Moderate Severe
Stenosis Stenosis
Mild 0 0 0 0 0
Hyper-homocysteinemia
(12-14.9µmol/L)
Moderate 15 0 0 10 5
Hyper-homocysteinemia
(15-29.9µmol/L)
Intermediate 103 5 1 0 109
Hyper-homocysteinemia
(30-100µmol/L)
Severe 0 3 1 0 4
Hyper-homocysteinemia
(>100µmol/L)
There was statistically positive correlation between mean homocysteine concentration of
cases and control with p value of 0.003 on T test. The correlation between homocysteine
level and severity of coronary artery disease (moderate or severe stenosis in one or more
major coronary arteries) was statistically significant on Pearson’s test with r value of 0.85
and p < 0.01. Homocysteine level also had statistically significant correlation with number
of major coronary arteries having moderate to severe stenosis with r value of 0.22 and
p<0.01on pearson's test.

DISCUSSION:
A recent study conducted in Indian population showed that plasma homocysteine levels are
more in men as compared to female which is congruent to our result in which 67.2% of the
patients suffering from hyper-homocysteinemia were males as compared to 32.8% of
females.12 Homocysteine has pro-thrombotic effect by enhancing platelet aggregation via
the hydrogen sulfide pathway, thus contributing to intravascular thrombosis as reported by
different studies.13,14 It is hence identified and reported as an independent risk factor for
atherosclerosis.15,16 Ganguly P et al also identified detrimental effect of high homocysteine
levels on cardiovascular system mediated by catecholamine and Zang S et al showed its
deleterious effect in causing arterial stiffness.17,18 All these studies support our results
which showed that all the patients with moderate to severe stenosis in one or more major
coronary arteries had hyper-homocysteinemia. Amongst whom, 92.2 % had intermediate,
4.7% had moderate and 3.1% had severe increase in plasma homocysteine levels. None of
the patient who had moderate or severe stenosis in one or more major coronary arteries was
having a normal homocysteine level. Different geographic locations and different ethnic
backgrounds have different genetic responses and to evaluate hyper-homocysteinemia
effect in our population we compared our results to similar studies from Asian population.
Mahalle, Kulkarni, et al conducted a similar study in Asia where 216 patients with
coronary artery disease were studied, among whom 95.3% had hyper-homocysteinemia in
Indian population. 19 Hyper-homocysteinemia was thus identified as risk factor for
cardiovascular thrombosis in Asian population which strengthens our results. Mendis S, et
al conducted a case control study to look for association between the
hyper-homocysteinemia and coronary artery disease in Sirilankan patients and established
that there was a statistically significant association between raised level of homocysteine
and coronary artery disease which again was in coherence with the results of our study.20
Schaffer et al ran a large prospective study from March 2007 to October 2013 to
look for the relationship between homocysteine and coronary artery disease which
included 3056 patients, and found a positive correlation between raised homocysteine
levels and coronary artery disease and concluded that homocysteine was an independent
risk factor for coronary artery disease.21
The impact of homocysteine as an isolated and very important risk factor was
hallmarked in a meta-analysis of observational studies published in JAMA correlating
homocysteine levels with risk of ischemic heart disease, stroke and found that low levels of
homocysteine was associated with lower risk of ischemic heart disease and stroke which
was again in support of our results which showed that raised level of homocysteine
increases cardiovascular thrombosis.22 We also compared our target population age group
with other studies. Raised level of homocysteine was reported as cardiovascular risk factor
in young patients in another case report which supported our results and hypothesis.23
Our results showed that all the young patients with coronary artery disease had a
raised homocysteine level with no young patient suffering from moderate to severe
stenosis in single or more major coronary arteries having a normal serum homocysteine
concentration. As it’s a modifiable risk factor it needs urgent attention. Targeting it will
decrease both cardiovascular morbidity and mortality both in primary and secondary
prevention.
CONCLUSION: In absence of conventional risk factors hyper-homocysteinemia alone
can be major risk factor for cardiovascular thrombosis in young patients. Further studies
are required to elaborate such results.
LIMITATIONS OF THE STUDY: Having been a case control study our results can’t
truly depict general population but it will provide nidus for further population based
epidemiological studies and bring into limelight the grave impact hyper-homocysteinemia
is projecting as cardiovascular risk factor.
Sample size was relatively small due to low incidence of hyper-homocysteinemia
in general population and stringent inclusion exclusion criteria.

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