Effect of eNOS polymorphisms on salbutamol evoked endothelium dependent vasodilation in South Indian healthy subjects.

BACKGROUND: The model of pulse plethysmograph using inhalational salbutamol 400 mcg is studied well to assess endothelium dependent vasodilation. Endothelial nitric oxide synthase (eNOS) gene polymorphism may influence the response to salbutamol in healthy subjects. AIM: To find the effect of polymorphisms 894G>T and -786T>C of eNOS gene on endothelium dependent vasodilation in healthy subjects.
MATERIALS AND METHODS: One hundred and two south Indian healthy subjects of either sex, aged between 18 to 35 years were recruited for the study. The digital volume pulse (DVP)was measured by pulse plethysmograph before and after salbutamol 400mcg inhalation. Three predose and five postdose recordings of DVP were measured. The average change in the DVP parameters namely reflection index (RI) and stiffness index (SI) were determined. The eNOS894G>T and -786T>C gene polymorphism were genotyped using polymerase chain reaction followed by restriction fragment length polymorphism. The percentage changes in RI and SI from predose baseline recordings were calculated and compared between the genotype groups.
RESULTS: The genotype and allele frequency of study subjects were in Hardy-Weinberg equilibrium. The changes in DVP parameters were not significantly different between the genotype groups.
CONCLUSION: eNOS polymorphism do not affect salbutamol evoked endothelium dependent vasodilation in the model of pulse plethysmograph in healthy subjects.

Introduction

Nitric oxide (NO) synthesized from L-arginine and molecular oxygen by the action of endothelial nitric oxide synthase (eNOS) enzyme, mediates endothelium dependent vasodilation (EDV). The enzyme eNOS is encoded by the eNOS gene which is mapped to chromosome 7q36 and consists of 26 exons. Many variants have been identified in the eNOS gene, among them 894G>T and -786T>C are the most characterized variants.[1] Carriers of variant allele for eNOS894G>T polymorphism have been shown to be associated with coronary heart disease and hypertension.[2-4] Similarly, the eNOS-786 T>Cin the promoter region has been shown to be associated with coronary artery disease.[56] Endothelial dysfunction has been regarded as an impending marker of atherosclerosis[7] which has been characterised by reduced availability of nitric oxide (NO) for maintenance of vascular tone in response to shear stress. Common risk factors of cardiovascular diseases namely diabetes and hypertension are also associated with endothelial dysfunction.

Endothelial function is clinically evaluated by measuring EDV with pulse plethysmograph (PPG).[89] Thedevice measures the changes in blood volume by emitting infrared light into the skin, which is transmitted through a capillary bed found in the finger tip. As arterial pulsations fill the capillary bed, changes in the volume of blood vessels modify the absorption, reflection and scattering of the light. Thus nonabsorbed light is measured and the signal is transduced to pulse plethysmographic recording in the form of digital volume pulse (DVP).[10-12] The DVP exhibits systolic and diastolic waves with a characteristic “notch” or point of inflection in its downslope. DVP and its parameters represent the vascular tone and stiffness of arteries. In an earlier study, endothelial function was assessed by measuring the effect of β2–adrenergic agonist salbutamol on inflection point (IP) of pulse plethysmographic DVP.[10] In humans, the vasodilating effect of salbutamol on the forearm resistance arteries is partially mediated through the L-arginine–NO pathway.[1314] The blunted response has been demonstrated in patients with hypertension, coronary heart disease and diabetes as compared to normal response in healthy subjects.[1015]

The role of eNOS894G>T and -786T>C polymorphism on response to salbutamol in model of pulse plethysmograph in healthy subjects has not been studied so far. This adds to the knowledge of genetic determinants of the pulse plethysmograph model of endothelial function. Thus, this study was designed to assess the effect of eNOS894G>T and -786T>Cpolymorphism on salbutamol evoked EDV as assessed by pulseplethysmograph in healthy subjects.

Materials and Methods

Subjects

Unrelated healthy subjects aged between 18 to 35 years of either gender, residing in South India for atleast more than three generations and speaking any of the South Indian language as their mother tongue were included in the study. Subjects with history of diabetes or hypertension, asthma, hypercholesterolemia, pregnancy and lactating were excluded. Chronic smokers, alcoholics and those on chronic medications or on any drugs in one week prior to the study were also excluded. The subjects were screened by general physical examination, biochemical analysis and electrocardiogram (ECG). Subjects were required to have normal biochemical profile and ECG to enter into study. The study was approved by the Institute Ethics Committee. All subjects gave written informed consent.

Clinical Study

In this prospective study, subjects were interviewed and explained the purpose of the study. After obtaining written informed consent, five ml of blood was collected in polypropylene tubes containing 100 mL of 10% ethylene diamine tetra acetic acid (EDTA) from median cubital vein for biochemical analysis and genotyping. The DVP were measured during 8.00 am to 11.30 am after overnight fast. The room temperature was maintained at 25-27°C. Baseline DVP along with BP were recorded in supine every five for 15minutes. After baseline measurements, subjects were asked to inhale 400 micrograms of salbutamol (Cipla, India) by metered dose inhaler (MDI) via a spacer, while sitting. The subjects were properly instructed to inspire deeply synchronous with the release of each puff.After administration of salbutamol, the subjects were again asked to lie supine and DVP measurements were taken every five for 25minutes.Thus three predose and five postdose DVP measurements were obtained from each subject. The average of DVP parameters namely reflection index (RI) and stiffness index (SI) before and after salbutamol were estimated and the percentage difference between them were calculated for each subject.

Determination of Genotype

The sample was centrifuged at 2500 rpm for 10 minutes at 4°C. The plasma was separated and used for biochemical analysis for fasting glucose, urea, creatinine and lipid profile.Deoxyribonucleic acid (DNA) was extracted from the cellular part by standard phenol-chloroform method and genotyping was determined by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP).

After PCR, amplification check was carried out on 1% agarose. The amplified PCR products were digested using restriction enzymes MboI and NgoM-IV respectively for eNOS894G>T and -786T>C by incubating at 37°C overnight. The digested products were analysed on 8% and 12% polyacrylamide gel and identified by ethidium bromide staining. The gel was viewed and documented using gel documentationimaging system. Digestion of 206-bp amplicon for eNOS894G>T resulted in either retention of 206-bp amplicon or complete digestion to 119-bp and 87-bp fragments corresponded to individuals homozygous for the GG or TT genotypes, respectively. The presence of all three fragments at 206, 119 and 87 bp corresponded to GT heterozygous genotypes. Digestionof 223- bp amplicon for eNOS-786T>C resulted in either retention of 223-bp amplicon or complete digestion to 115-bp and 108-bp fragments corresponded to TT and CC genotyperespectively. The presence of all three fragments at 223, 115 and 108 bp corresponded to TC heterozygotes.

Statistical Analysis

Statistical analysis was done using statistical package for social sciences (SPSS) version 13.0 (SPSS, Chicago, IL) and p value <0.05 was considered significant. Subject characteristics and genotype frequencies were described using descriptive statistics. The parameters were expressed as the percentage change in reflection index, stiffness index from the baseline values of pre-salbutamol administration to that of the postdose.The homozygous mutants were combined with heterozygous mutant and compared with wild homozygous genotypes. Assessing for Hardy-Weinberg equilibrium and comparison of genotype, allele frequency with that of previous study was done by Chi-square test. Comparison of percentage changes in DVP parameters was done by Mann-Whitney test followed by application of analysis of covariance (ANCOVA) to adjust for the confounding variables. These confounding variables were age, body mass index (BMI), systolic blood pressure, diastolic blood pressure, total cholesterol, change in heart rate and plasma glucose.

Results

One hundred and twenty subjects were screened by detailed medical history, physical examination, ECG and biochemical analysis. One subject was excluded due to persistent high blood pressure. However, 109 subjects agreed to participate in the endothelial function test using salbutamol as a provocative challenge out of which seven samples were lost during processing for genotyping. Thus one hundred and two subjects had both phenotypic and genotypic data. None of the subjects were on any long term medications and had not taken any drug before one week of the study. The mean±SD age of the study subjects was 26.5 ± 4.5 years and BMI was 22.39 ± 3.26. Their SBP and DBP were 112 ± 8 and 67± 6.5 mmHg respectively [Table 1]. All the biochemical parameters were within the normal range.

Table 1

Subject characteristics

Genotype and Allele Frequency of the Study Subjects

The frequency distribution of the genotypes of eNOS 894G>T namely GG, GT and TT, were 60.7%, 34.3% and 4.9% respectively. The allele frequency of G and T were 0.78 and 0.22 respectively.The frequency distribution of the genotypes of our study was in accordance with Hardy-Weinberg equilibrium with p value of 0.98. The genotype frequency was also compared with the previous study[5] in South Indian population and found to be not significant (p = 0.15 for genotype frequency and p = 0.20 for allele frequency). The frequency distribution of the genotypes of eNOS-786T>C namely TT, TC,and CC, were 69.61, 27.45 and 2.94% respectively [Table 2]. The allele frequency of T and C were 0.83 and 0.17 respectively. The frequency distribution of the genotypes of our study was in accordance with Hardy-Weinberg equilibrium with p value of 0.90.

Table 2

Genotype and allele frequency of the study subjects (n = 102)

Comparison of DVP Parameters between Genotype Groups

DVP parameters, RI and SI were compared between the genotype groups. [Table 3] None of the DVP parameters showed any statistical difference between the two genotype groups. Further, diplotypic analysis also didnot find any statistical difference between the genotype groups (data not shown)

Table 3

Comparison of change in DVP parameters between genotype groups

Discussion

We assessed the effect of eNOS polymorphism on salbutamol evoked EDV using pulse plethysmograph in healthy subjects. The present study showed lack of association between eNOS and EDV. To the best of our knowledge, this is the first study investigating the effect of eNOS polymorphism on EDV as assessed by non-invasive pulse plethysmograph using inhalational salbutamol in healthy subjects. It has been reported that the T allele of eNOS 894G>T reduces the activity of the eNOS enzyme.[16] Many studies have shown association of eNOS 894G>T polymorphism with coronary heart disease and hypertension.[2-4] However, there are also studies which showed no association.[17-19]

The negative finding obtained in our study is in accordance with a functional genetic study done in patients with hypertension and coronary heart disease.[20] The eNOS894G>T genotype did not show any biological effect on EDV. However this study used strain gauze plethysmography for measuring forearm blood flow in response to intra-arterial acetylcholine and nitroprusside for studying endothelium dependent and independent vasodilations respectively in coronary heart disease patients. The non-invasive model of assessing endothelial function using inhalational salbutamol has been well studied.[1015] The dose of 400 mcg of inhalational salbutamol has been reported to sufficiently cause EDV as shown by the previous study.[12] This model was shown to have good sensitivity and specificity compared with FMD.[21] However, the subjective differences are more likely to affect this model of endothelial function using salbutamol.The subjective differences in inhalation of salbutamol between subjects were taken care by instructing the subjects priorly about proper inhalation; also the spacer was used to minimise the erratic absorption of salbutamol. Earlier study has shown that the measured plasma concentration of salbutamol is stable between 5 to 20 minutes after inhalation.[9] Therefore we measured the DVP every 5 mins upto 25 mins postdose.

Healthy subjects with T allele for eNOS894G>T aged between 18 to 44 years were shown to have blunted EDV in response to intra-arterial acetylcholine.[22] Our study subjects were aged 18 to 35 years and we used salbutamol as stimulus to evoke EDV.

Another study evaluated the effect of eNOS894G>T with environment and dietary factors on endothelial function using flow mediated dilatation (FMD) in healthy subjects.[23] Endothelial function was not related to genotype in the group as a whole or within both genders. However, among males, smoking was associated with lower FMD. In the present study although majority of the participants were males, the lack of association may be due to the non-smoking status of the subjects. Further, carriers of T allele for 894G>T with atherogenic lipid levels showed impaired EDV in a model of FMD.[24] The subjects of our study had normal lipid levels.

Thus positive association of eNOS with EDV depends not only on associated risk factors namely age, gender, smoking, atherogenic lipids of subjects but also on stimulus and model of testing endothelial function used. Possibly, the genetic determinants become more apparent in the presence of risk factors.

The confounding variables were minimised by choosing a homogenous group following strict inclusion and exclusion criteria. The important confounding factor which can potentially influence our study is the distribution of β2 receptor polymorphisms between the eNOS genotype groups. Recently, the study in healthy subjects concluded that endothelial function using PWA (pulse wave analysis) and salbutamol was not influenced by β2 receptor polymorphisms.[25] Even other confounding factors such as the amount of salbutamol reaching the circulation may be more influential than the genetic factors in this model of endothelial function with salbutamol as challenge in healthy subjects. The major limitation of our study is that the serum salbutamol concentrations were not measured and correlated due to practical reasons. Another limitation of the study is that the endothelium independent vasodilation (EIV) of the subjects by using nitroglycerin was not assessed, though our study subjects were homogenous and only the percentage change of DVP parameters from baseline measurements were used for analysis.

Thus, our study results indicate that in healthy subjects, eNOS894G>T and -786T>C polymorphisms do not affect the EDV in the pulse plethysmograph model using inhalational salbutamol as a challenge.