ABSTRACT years. Fasting serum lipid profile and homocysteine

ABSTRACT

Background: Cytochrome P450 (CYP)
epoxygenase metabolise arachidonic acid (AA) into four epoxyeicosatrienoic
acids (EETs) 5,6- EETs, 8,9 EETs, 11,12-EETs and 14,15-EETs.  Since, EETs are unstable eicosanoids they
rapidly get converted into dihydroxyeicosanoid trienoicacids (DHETs) by soluble
hydrolase. These eicosanoids promote defence mechanism against inflammatory
atherosclerosis process. However, 11,12-EETs are more potent eicosanoids in
maintaining anti-atherosclerotic  activity.
Endothelial dysfunction is the key step in the pathogenesis of atherosclerosis.
Polymorphism in CYP epoxygenase can alter individual’s risk for events in
coronary artery disease (CAD) patients. Therefore, we examined the impact of
CYP epoxygenase polymorphism indirectly through CYP metabolites on endothelial
dysfunction to predict the risk of events in CAD patients.

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Methods:
It
is a prospective case-control study consisting of 84 acute coronary syndrome (ACS)
patients and 84 healthy controls of either gender aged above 18 years. Fasting
serum lipid profile and homocysteine levels were measured in all subjects. We
measured plasma 11,12-dihydroxyeicosatrienoic acid (11,12-DHET) as indicative
of 11,12-EETs. Genotyping of CYP putative exons of CYP2C9, CYP2C19 and CYP2J2
epoxygenase were carried out by Polymerase Chain Reaction–Single Strand
Conformation Polymorphism (PCR-SSCP) method. Sanger’s sequencing chain
termination method was carried out for the SSCP subtle samples. All the data
obtained were analysed by using Ms-Excel, 2007 and SPSS, version 24.Software,
IBM, USA.

Results:
We
observed significantly higher levels of  homocysteine
in CAD group (35.1 ± 13.8 µmol/L) indicating higher inflammatory condition in
patients compared to control group (8.1 ± 2.9 µmol/L, p T (Case 47, Figure 3-A) and novel base
substitutions in other two patients (Case 58 and Case 73). CYP2C19 gene SNP base
substitutions were found in 5 patients of which 3 patients were found to report
CYP2C19*2, c.681G>A
(Figure 3-B) and two patients reported novel base substitutions (Case 5 and
Case 10). CYP2J2 gene
polymorphism constituting novel base substitutions (Figure 3-C) were found in 4
patients and with Cytosine base insertions in Case 31. In this connection, we
also noticed comparatively reduced levels of 11,12-DHETs in the patients
reporting CYP gene polymorphisms (Table 4).

Further,
data analysis revealed significant differences in 11,12-DHET levels between patients
reporting CYP polymorphism  and  patients without CYP polymorphism compared
with the control individuals (p T, R144C) was found only in one patient
and accounted for 1.2% which is less compared to the study by Jose R et al in
2004 reporting 4%.24 clinical
Importance  of base substitution.

CYP2C19 is also an important
epoxygenase and any variations in the gene encoding this enzyme may lead to
reduced epoxygenase activity. CYP2C19*2 allele has clinical importance especially
in acute coronary syndrome patients.12,25 
Akasava T et al. in 2016 also reported that patients with
CYP2C19*2 allele showed reduced levels of plasma 11,12- DHETs compared to the
patients without the mutant allele.19  Three of 84 CAD patients (case 8, Case 58
& case 68) were found to report CYP2C19*2 allele (c.681G>A, p.G228R) and accounted
for 3.6% and it was comparatively low with the studies reporting 12% and 10% by
Shuldiner AR et al 26 and Tantray JA et al 27
respectively in south Indian population. Genetic polymorphism
in CYP2C19 gene was reported to be independent risk factor for cardiovascular
events irrespective of clopidogrel resistance.28 Thus,
reduced levels of DHETs in CAD patients result in decline of DHET-mediated
defence activity against vascular inflammation that can cause endothelial dysfunction.

CYP2J2 is cardiac specific epoxygenase expressed
predominately in vascular endothelial cells. Presence of CYP2J2*4 allele in
exon 4 significantly decreases the CYP2J2 epoxygenase activity, especially
results in reduced expression of 11,12 DHETs compared to the wild type. However,
Asians are rare to this allele accounting for 0-2%29 and it was consistent with our study none was found
to report CYP2J2*4 allele. Variations in gene sequences may lead to altered
enzyme activity and have effect on cardiovascular homeostasis and disease
outcomes. We observed CYP2J2 novel base substitutions and C
insertions in the patients of case
23 and case 40 reporting single base substitution c.646G>A
and in patients of case 31 & case 73 reporting with C insertions at c.
664_665insC and c.673_674insC
respectively. We noticed comparatively reduced levels of 11,12- DHETs in the
patients reporting C base insertions in case 
31,  and case 73,  depicting reduced enzyme activity due to frame
shift mutation. A study by Indrayan A
in 2013 reported that  gene variations in
these epoxygenases influence their activity that can act as important modifiers
of cardiovascular risk in CAD patients.30

            Interestingly,
we observed negative correlation between 11,12-DHETs and  homocysteine levels in 11 patients reporting CY2C9,
CYP2C19 and CYP2J2 polymorphisms depicting the reduced enzyme activity. Thus,
our study findings showed that presence of CYP polymorphisms result in reduced
levels of 11,12-DHETs and decline of DHET mediated vasodilation that can cause
endothelial dysfunction and risk of events.

Conclusions:

We observed
decreased levels of 11,12- DHETs in these patients  compared to the patients without
polymorphism. Presence of lower levels of 11,12- DHETs is a reflection of poor
reserve defence mechanism in CAD patients that might result in endothelial
dysfunction and lead to cardiac events.

Therefore,
in acute coronary syndromes patients showing lower 11,12-DHET levels,
genotyping of CYP2C9, CYP2C19 and CYP2J2 genes may serve as a prognostic marker
for  future events.

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