International Journal of Biological & Medical Research

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1 Int J Biol Med Res. 21; 1(4): Int J Biol Med Res Volume 3, Issue 4, Sep 21 ISS: 976:6685 BioMedSiDiret Publiations Contents lists available at BioMedSiDiret Publiations International Journal of Biologial & Medial Researh Journal homepage: International Journal of BIOLOGICAL AD MEDICAL RESEARCH Original Artiles Evaluation of endothelial and platelet funtions with intergenotypi variation of eos Glu298Asp gene polymorphism in relation to postmenopausal women a a b Pradeep Kumar Dabla *, Sarika Arora, Shubha Sagar Trivedi, ibhriti Das, a Jayashree Bhattaharjee a* Department of Biohemistry, Lady Hardinge Medial College, ew Delhi-111, India b Department of Obstetris & Gynaeology, Lady Hardinge Medial College, ew Delhi -111, India Department of Biohemistry, All India Institute of Medial Sienes, ew Delhi-1129, India A R T I C L E I F O Keywords: Postmenopausal Women Estrogen itri Oxide Platelet Fator-4 eos Glu298Asp gene polymorphism A B S T R A C T Menopause is assoiated with inreased Coronary Artery Disease (CAD) risk. This study was planned to eluidate the endothelial hanges and platelet dysfuntion and intergenotypi variation of O levels in eos Glu298Asp gene polymorphism in postmenopausal women. This study was done on 2 women in the age group 4-55 years were seleted from a tertiary are hospital. Of these, 1 postmenopausal women were inluded in study group and 1 premenopausal women served as ontrol. Fasting blood samples were evaluated for plasma estriol, itri Oxide (O) and Platelet Fator-4 (PF-4) levels. PCR-RFLP was done in remaining ell aggregate to study eos Glu298Asp gene polymorphism. Statistial evaluation was done on SPSS. Postmenopausal women had signifiantly lower estriol and O levels as ompared to premenopausal women (p<.1). Signifiant positive orrelation was found between age with PF4 in study group (p<.1) and O with estriol in both groups whereas negative orrelation was observed between O with PF4 in both groups. Distribution of genotype GG, GT and TT in postmenopausal and premenopausal women was found to be,, % and 9, 1, % respetively. All genotypes (GG+GT, GG and GT) showed signifiant derease (p<.1) in O level in postmenopausal women. Dereased estrogen levels in postmenopausal state lead to endothelial and platelet dysfuntion due to low O levels. Further presene of eos gene polymorphism may provide additional insult by dereasing O levels adding to the CAD risk. Hene, postmenopausal women those who presents with eos Glu298Asp polymorphi gene may have higher CAD risk. Copyright 21 BioMedSiDiret Publiations IJBMR - ISS: 976:6685. All rights reserved. 1. Introdution Menopause and impaired itri Oxide (O) release are assoiated with endothelial dysfuntion and are risk fators for Coronary Artery Disease (CAD). [1] CAD is rare in premenopausal women but beomes inreasingly prevalent after menopause. Premenopausal women have 66% lower risk of stroke and 33% lower risk of sudden ardia death than males of omparable age and the risk inreases and beomes equal to males after menopause. [2] After menopause, marked deline in estrogen level takes plae. Estrogen exerts its various ations by sex steroid reeptors present in arterial endothelium and inreases Low-density lipoprotein (LDL) atabolism resulting in lower LDL and higher High-density * Corresponding Author : Dr. Pradeep Kumar Dabla Department of Biohemistry, Lady Hardinge Medial College, ew Delhi-15, India. pradeep_dabla@yahoo.om Copyright 21 BioMedSiDiret Publiations. All rights reserved. lipoprotein (HDL) levels. [3] It is also known to inhibit lipoprotein oxidation. [4] The vasular endothelium helps to maintain vasular homeostasis by generating vasodilator and vasoonstritor substanes. itri oxide is one of most known versatile moleules and has a role in almost every biologial system. It is synthesized from L-arginine through atalyti ativity of endothelial itri Oxide Synthase (eos). [5] The O ats as double edged sword and its various effets result either diretly from reation between O and speifi biologial moleules through S-nitrosylation [6] or indiretly from formation of reative nitrogen speies through oxidation. [7] O regulates arterial tone by relaxation of vasular smooth musle and vasodilatation. [8] itri oxide inhibits various key steps in development of atheroslerosis and CAD via inhibition of platelet aggregation and adhesion, adhesion moleular and hemokine expression, inflammatory ell infiltration and smooth musle ell proliferation. [9,1]

2 Pradeep Kumar Dabla et al. / Int J Biol Med Res. 21; 1(4): The funtional and morphologial alteration in endothelial ells is ritial to atherosleroti proess. Therefore, eos is a potential andidate gene for atheroslerosis. The endothelial itri Oxide Synthase (eos or OS 3: hromosome 7q) is found primarily in the endothelium and at low levels in platelets and is almodulin dependent. Of the several sequene variants in humans, only G894T is found to be in exon 7. [11] Interation of blood platelets with arterial endothelium plays an important role in pathogenesis of CAD. [12] Platelet fator-4 (PF4) released from á-granules of platelets an serve as a useful marker for platelets in serum or plasma beause of its platelet speifiity due to synthesis in megakaryoytes. [13] However, so far no data is available in relation to menopausal CAD risk and eos gene polymorphism. Hene this study was planned to analyze the endothelial dysfuntion and intergenotypi variation of O level in eos gene polymorphism in relation to inreasing CAD risk in postmenopausal women for prevention and early intervention. 2. Materials and Methods 2.1. Study population The 2 women were seleted from gynaeology linis and wards of a tertiary are hospital in India with informed onsent. The women were divided into study group onsisting of 1 postmenopausal women and ontrol group onsisting of 1 premenopausal women in age group of 4-55 years. The serum FSH levels were measured in postmenopausal women, as it is an established indiret marker of folliular ativity. The study was approved by the Institutional Ethial Committee. 2.2.Seletion Criteria Inlusion riteria were: 1. Women in age group of 4-55 years with either natural or surgial menopause (hysteretomy with b i l a t e r a l salpingo-oopheretomy). 2.Premenopause in age group of 4-55 years without any gynaeology omplaints (tumors, dysfuntional uterine bleeding). 3.FSH levels 3 miu/ml. Exlusion riteria were: 1. Women assoiated with any malignany. 2. Women who have had HRT (hormone replaement therapy). 3. Women with diagnosed CVD Sample olletion and proessing Brief history and linial examination was arried out with onsent before examination. Abdominal ultrasound for pelvi region was arried out in all women at the time of enrolment to rule out any organi illness. Venous blood was olleted from the subjets under sterile onditions after overnight fasting. Seven milliliters of blood was olleted in sterile srew apped vials ontaining 5% antioagulant (1 unit of 5% EDTA for eah 9 units of blood). Platelet rih plasma (PRP) was prepared by entrifugation of olleted blood at 1 rpm for 15min at 37 for O and estrogen estimation and aliquot. Remaining PRP was entrifuged at 3 rpm for 15 min at 37 C to get platelet poor plasma (PPP) for measuring PF4 levels. The plasma obtained was transferred to sterile srew apped vial and stored at -2 C till bath analyzed. The remaining ell aggregate was transferred to polyethylene ontainer and stored at -2C till analyzed for PCR and RFLP itri Oxide estimation itri oxide was estimated in platelet rih plasma and plasma by modified Griess reation. [14] Griess reation involves the formation of a hromophore during the reation of nitrite (O -) 2 with sulfanilamide and heteroyli amine of (1-napthyl) ethylenediamine (Griess reagent) under onditions of low ph. In an oxygenated solution, O deomposes to form nitrite (O -) 2 and nitrate (O -) as shown below: 3 2 O + O 2 2 O 2 2 O + 2 O2 2 2 O3 2 2 O3 + 2 H2O 4 O H 2.5.Determination of estriol Serum estriol was measured by ompetitive EIA (enzymati immunoassay). [15] The estriol ELISA is based on ompetitive interation of estriol and the hormone-enzyme onjugate for a limited number of immobilized anti estriol antibodies (rabbit). Thus, the amount of bound hormone-enzyme onjugate in the well, unbound onjugate is removed by washing. When substrate solution is added, a blue olor develops hanging to yellow after stopping the reation. The intensity of olor is inversely proportional to the amount of estriol in the speimen. 2.6.Estimation of PF4 It is based on the priniple of sandwih ELISA in whih the PF4 to be measured reats with speifi antihuman PF4 antibodies oated on the wells. [16] A rabbit anti PF4 antibody oupled with peroxidase reats with another epitope of the PF4. Upon addition of substrate OPD (orthophenylenediamine) in the presene of H2O 2, the intensity of olor reation bears a diret relationship with the PF4 onentration. 2.7.Identifiation of genotype and genotyping Genomi DA isolation was done from stored ell aggregate. RBC were lysed using RBC lysis buffer (Tris, MgCl2, acl, ph-7.6) and DA extration was done by salting out method. [17] The DA was amplified for eos gene polymorphism by PCR using flanking intron primers 5'-TCC CTG AGG AGG GCA TGA GGCT (sense) and 5'-TGA GGG TCA CAC AGG TTC CT (antisense), whih was later sreened for RFLP using BanII restrition endonulease digestion for 2hrs at 37º C. [] The restrited fragments were resolved on 2% agarose gel and visualized by ethidium bromide staining. 2.8.Statistial Analysis Statistial analysis was arried out using SPSS for windows 12. software (SPSS In., Chiago, IL, USA). Data are expressed in Mean ± Standard Error of Mean. The differene between groups was ompared by independent sample t test or Mann-Whitney test for ontinuous variables. Spearman's rank orrelation was applied to test for assoiation between ontinuous variables. A two-tailed p value<.5 was onsidered statistially signifiant. Endothelial OS gene G894T polymorphism genotypes (GG, GT, TT) and alleles (G, T) frequeny was analyzed by using Fisher's exat and hi-square tests. 3.Results The study group mean age was 51.76±.697 years and among the ontrols were 45.6±.622 years. Among the ases mean systoli blood pressure was ±2.77 mm Hg and diastoli blood pressure was 78.1±1.15mm Hg. The mean systoli blood pressure was 13.7±2.65 mm Hg among the ontrols and diastoli blood pressure was 78.3±1.55 mm Hg.

3 274 Pradeep Kumar Dabla et al. / Int J Biol Med Res. 21; 1(4): Plasma O, estriol and PF4 levels in study and ontrol groups The mean plasma O level in study group (14.9 µmol/l ± 1.8) was signifiantly lower (p<.1) as ompared to ontrol group (24.91 µmol/l± 1.75). Signifiant lower mean plasma estriol level (p<.1) was observed in study group (2.56 ng/ml ±.14) than in ontrols (5.58 ng/ml ±.27). Though the levels of PF4 were higher in the study group as ompared to the ontrol group but the differene was statistially insignifiant [Table1]. Table 1. Plasma O, Estrogen and PF4 levels in study and ontrol groups: Fig 1. Ethidium bromide-stained agarose gel used for genotyping PCR produts (457 bp) with primers flanking Glu298 Asp polymorphi region of e OS gene digested with Ban II. Homozygotes with G at with position (G/G) showed 2 bands at 32 bp and 137 bp. Heterozygotes for this mutation (G/T) showed 3 bands at 457 bp, 32bp, and 137 bp. M= moleular wt. marker (Promega USA). M GG GT GT GT Parameters (=1) Estriol(ng/ml) 2.56±.14 (=1) 5.58±.27 P value <.1 itri Oxide(µmol/L) 14.9± ±1.75 <.1 PF4(IU/ml) 1.31± ± Correlation between age, O, estriol and PF4 in study and ontrol groups In both groups, age has not shown signifiant orrelation with O and estriol whereas signifiant positive orrelation was found with PF4 in study group (r=+.37, p<.1). Plasma O demonstrated signifiant positive orrelation with estriol and signifiant negative orrelation with PF4 in both study group (r=+.35, p<.5 and r= -.41, p<.1) and ontrol group (r=+.46, p<.1 and r= -.29, p<.5) respetively. Both estriol and PF4 was found to be negatively orrelated with eah other insignifiantly [Table 2]. Table 2. Correlation of various parametres evaluated in study and ontrol group with their signifiane: Correlation Age with O Age with estriol (=1) (=1) R P R P Table 3. Distribution of genotypes and alleles of eos Glu298Asp polymorphism in postmenopausal and premenopausal women: Genotype Postmenopausal women Frequeny Postmenopausal women Frequeny Levelof Signifiane Age with PF < GG =1.329 O with Estriol +.35 < <.1 GT 1 1 df=1 O with PF < <.5 TT p Estriol with PF G Allele ot Signifiant 3.3.Distribution of genotypes and alleles of eos Glu298Asp polymorphism in the study and the ontrol group [Fig 1]. A total of 2 partiipants were genotyped for eos Glu298Asp polymorphism. GG genotype was found in subjets (%) of study group and 9 subjets (9%) of ontrol group whereas, subjets (%) of study group and 1 subjets (1%) of ontrol group have shown GT genotype. o TT was found in any group. The genotype frequenies were as predited by Hardy- Weinberg equilibrium (hi-square=1.329, df=1, P=.5, ot Signifiant). Study group has shown alleli frequenies of G and T allele as.91 and.9 respetively. The frequeny of G and T alleles in ontrol group was.95 and.5 [Table 3]. T Allele Intergenotypi variation of plasma O levels in study group and ontrol group [Fig 2] The highly signifiant differene (p<.1) was seen between O level of GG+GT genotype in study group (14.92±1.87 µmol/l) and ontrol group (24.916±1.758 µmol/l). The O level of GG genotype also showed highly signifiant differene (p<.1) between study group was (16.256±1.2 µmol/l) and ontrol group (26.6±1.874 µmol/l). Similarly, GT genotype plasma O level showed signifiant differene (p<.1) in both study group (8.733±.789 µmol/l) and ontrol group (15.1±2.45 µmol/l) [Table 4].

4 Pradeep Kumar Dabla et al. / Int J Biol Med Res. 21; 1(4): Intergenotypi variation of plasma O levels in study group and ontrol group [Fig 2] Figure 2. Intergenotypi variation of plasma O levels in study group and ontrol group. The highly signifiant differene (p<.1) was seen between O level of GG+GT genotype in study group (14.92±1.87 µmol/l) and ontrol group (24.916±1.758 µmol/l). The O level of GG genotype also showed highly signifiant differene (p<.1) between study group was (16.256±1.2 µmol/l) and ontrol group (26.6±1.874 µmol/l). Similarly, GT genotype plasma O level showed signifiant differene (p<.1) in both study group (8.733±.789 µmol/l) and ontrol group (15.1±2.45 µmol/l) [Table 4]. Table 4. Intergenotypi variation of plasma O levels in study group and ontrol group: Genotype GG+GT GG GT µmol/l Disussion n 1 GG+GT GG GT =1 Levels of O(µmol/L) 14.92± ± ± = ± ± ±2.45 p value <.1 <.1 <.1 The present study was designed to eluidate the role of O, estrogen, PF4 and their interrelationship along with theirsignifiane of intergenotypi variation of O levels in eos Glu298Asp gene polymorphism in relation to postmenopausal endothelial dysfuntion. In the present study, a signifiantly lowelevels of plasma O and estriol were observed in study group as ompared to the ontrol group whereas PF4 did not show any signifiant differene whih may be due to the riteria of exlusion of diagnosed CAD and malignany ases. Menopause is assoiated with higher prevalene and inidene of ardiovasular disease and death ompared to premenopause, whih ould be attributed to the loss of ovarian funtion with subsequent defiieny of estrogen and O. [1,2] In the earlier study, we have shown that menopause tends to downregulate O-GMP pathway whih may be diretly through estrogen reeptors or indiretly through potentiation of dyslipidemia resulting in endothelial dysfuntion. [19] Similarly, Majumdar et al [2] have observed that menopause and male gender are assoiated with redued arterial O ativity whih orrelates well with our finding of derease O level in study group. Arteries from postmenopausal women demonstrated impaired endothelium dilatation to flow stimulus, as ompared to premenopausal women, whih is due to defet in O synthesis at the level of endothelial ell. [21] The loal vasodilating and antiplatelet ations of estrogen are a onsequene of endothelial response mediated by release of EDRF's (endothelial dependent relaxing fator), whih is O. [22] In the present study, plasma O level showed a signifiant positive orrelation with plasma estriol level in both groups. The vasuloprotetive effets of estradiol are suggested by Amant et al[23] by regulating Fasligand expression at the transitional level via an estrogen reeptor mediated O dependent mehanism. Chroni exposure to estradiol has been shown to upregulate gene expression of eos and thereby inreases O prodution. [24] Plasma O, estrogen and PF4 showed a non-signifiant negative orrelation with age whih may due to the small group size, riteria of agegrouping (4-55 years) and ardiovasular disease exlusion, whih shows insignifiant hanges over the limited time as the mean age of postmenopausal women is 51.3 years. Platelet fator-4 levels reflet the platelet ativity in vivo and hene serve as useful marker for platelets in serum or plasma. [13] The plasma O and PF4 showed a signifiant negative orrelation whereas estriol levels showed an insignifiant negative orrelation with PF4 levels. Interation of endothelium with blood platelets plays an important role in pathogenesis of ardiovasular diseases. [25] Bassenge E et al[26] have shown that shear fores auses the release of O from endothelial ells whih diffuses into platelets and inhibits their ativation. Aggregating platelets in CAD patients produe less O, [27] that thus explain the signifiant negative orrelation of PF4 with O observed in postmenopausal women in the present study. The non-signifiant negative orrelation between estriol and PF4 ould be explained by the indiret effet of estriol on PF4, probably mediated by O. 5.Conlusion Thus authors onlude that menopause is a period of inreasing risk that is related to both age and to estrogen defiieny. Estrogen defiieny dereases O levels by its negative influene on O--GMP pathway. The dereased O level auses platelet aggregation through various interrelated mehanisms. Thus, arriage of eos Glu298Asp gene polymorphism during menopause may ause low eos expression whih may manifest with ardiovasular risk fators and low estrogen levels. It points that development of CAD risk in postmenopausal women may be a multifatorial phenomenon and not just related to hyperlipidemia and other known risk fators. With CAD being a major health and eonomi burden, it is imperative to treat it at the earliest and avert its ourse. Hene, women with eos Glu298Asp gene polymorphism and low O level should be periodially monitored for development of Coronary Artery Disease. 6.Referenes [1] Kannel WB. Metaboli risk fators for oronary heart disease in women: perspetive from the Framingham Study. Am Heart J. 1987; 114(2):

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