Plasma Homocysteine Levels in Indian Patients with Essential Hypertension and Their Siblings

Abstract

Background. An association between plasma homocysteine and essential hypertension and their non-hypertensive siblings is controversial. Methods and Results. Plasma homocysteine levels were measured in subjects with essential hypertension (n = 50), their normotensive siblings (n = 50) and normotensive controls (n = 50). All the subjects were non-diabetic, had no past history of myocardial infarction, stroke or peripheral vascular disease and had normal renal functions. The mean homocysteine values were 18.96 ± 8.08 µmol/L in patients, 14.84 ± 5.55 µmol/L in siblings and 10.50 ± 4.92 µmol/L in controls. Plasma homocysteine level were found to be significantly higher in patients with hypertension (p < 0.0001) and their normotensive siblings (p<0.0001) when compared to controls. Also patients with hypertension had their higher plasma homocysteine levels compared to their siblings (p<0.0036). Conclusion. Plasma homocysteine levels are significantly elevated in Indian patients with essential hypertension and their normotensive siblings. Thus, plasma homocysteine may serve as a marker for the development of essential hypertension.

• Essential hypertension
• Homocysteine
• Siblings
• Risk factors
• Indians

Introduction

High blood pressure is a major health problem in the world. The JNC V[[1]] estimates that hypertension affects more than 50 million Americans and contributes to more than 1/2 million strokes and 1.25 million coronary attacks per year. Obesity, smoking, excessive alcohol consumption, hypercholesterolemia and diabetes are some of the important risk factors contributing to coronary artery disease and hypertension.[[2]] Homocysteine is one of the newer risk factors found to be associated with hypertension.

McCully first linked hyperhomocystinemia to precocious atherosclerosis, in children with inborn errors of metabolism.[[3]] Patients with hyperhomocystinemia have been observed to show a high incidence of cardiovascular disease in early adolescence and even in childhood. Studies have associated mild to moderate elevation in plasma homocysteine levels in patients with coronary,[[4]], [[5]], [[6]] cerebral[[7]], [[8]], [[9]]and peripheral vascular disease.[[10]] Recently an increased plasma homocysteine levels have also been reported in patients with hypertension, indicating that hyperhomocystinemia in hypertension may be an important factor contributing to cardiovascular risk in these subjects.[[11]] The mechanisms of homocysteine causing hypertension are not very clear.

A higher prevalence of hypertension has been found among the Indian population in both urban and rural areas.[[12]] However, homocysteine levels in Indian population are not clearly defined. The present study aims to determine the plasma homocysteine levels hypertensive subjects, their normotensive siblings and to compare them with plasma homocysteine levels in normotensive controls.

Methods

The target subjects were 50 consecutive patients of essential hypertension (as defined by JNC VI[[13]] criteria (BP>140/90 mm of Hg), 50 normotensive siblings of the above patients and 50 normotensive subjects without any family history of hypertension. Subjects between 18 and 65 years of both sexes were included.

Subjects with secondary hypertension, a past history of stroke, coronary artery disease, myocardial infarction, peripheral vascular disease with evidence of tissue injury or loss, transient ischemic attacks, diabetes mellitus or renal insufficiency were excluded from the study.

A fully informed consent was obtained from all the subjects prior to participation in the study. All the subjects underwent a detailed clinical examination. The blood pressure measurement was carried out as per JNC VI recommendations. Each subject underwent baseline investigations including a complete hemogram, blood urea, serum creatinine, lipid profile, blood sugar (fasting), urine routine examination, electrocardiogram and a chest X-ray. Two milliliters of fasting blood sample was collected in EDTA vial, was immediately centrifuged and the plasma was separated and stored at −70°C.

Homocysteine was measured by a modified method of Araki and Sako.[[14]] Homocysteine is dissociated from non-relevant proteins and other disulfides by reduction with dithiothreital. In brief, the procedure that was followed is as follows. The proteins were precipitated by using 0.6 M perchloric acid. The sulfhydryl amino acids so obtained were made to react with fluorescent reagent 7-fluro, benzo, 2-oxa, 1,3-diazole–4 sulfonic acid (SBDF) (sigma laboratories). Homocysteine was quantified by using reverse phase high performance liquid chromatography (Waters India Ltd.) and fluorescence detection was done (emission wave length—515 nm, excitation wave length—385 nm).

Statistical Analysis

Descriptive values were expressed as Mean ± SD. One way ANOVA test was used for statistical analysis among the three groups. Further analysis was done using students t-test. Chi square test was used for testing proportions. Pearsons correlation coefficient was calculated to study the relationship between homocysteine and other variables. A p value of <0.05 was considered statistically significant.

Results

The baseline characteristics of the subjects are shown in Table 1. Hypertensive patients had higher body mass index, LDL cholesterol, total cholesterol and triglyceride levels as compared to normotensive controls. Mean plasma homocysteine levels in different groups are given in Table 2. The hypertensive patients had significantly higher homocysteine levels when compared to controls and their normotensive siblings (p<0.0001). Normotensive siblings of hypertensive patients also had higher plasma total homocysteine levels as compared to controls (p<0.0001). The mean plasma homocysteine levels in male patients was higher than male siblings (p<0.0001), and male controls (p<0.0001), also siblings has a higher level than (p<0.0001) controls. Though female patients had a higher mean plasma homocysteine levels than female siblings and female controls the difference was significant only between patients and controls (p<0.0006).

Table 1. Baseline characteristics of the subjects

Groups	Patients (n = 50)	Siblings (n = 50)	Controls (n = 50)
Age (in years)	46.4±8.6	42.0±8.1	45.1±8.4
Sex (M/F)	26/24	37/13	27/23
BMI (kg/m^2)	26.00±4.58	23.67±3.79	22.82±3.96
W/Ha Ratio	0.95±0.08	0.98±0.06	0.95±0.09
HDL (mg/dL)	44.2±8.9	46.8±1.2	45.6±1.37
LDL (mg/dL)	91.1±37.7	88.0±42.7	75.4±34.2
Total cholesterol (mg/dL)	169.6±37.6	166.3±42.6	146.8±39.3
Triglycerides (mg/dL)	170.9±78.2	161.2±71.7	135.4 + 53.7

All values showing mean ± SD.

^aWaist:hip ratio.

Table 2. Mean homocysteine values of the subjects (µmol/L)

Groups	Patients	Siblings	Controls
Males	20.08 ± 8.41	15.10 ± 5.9	10.16 ± 5.44
Females	17.76 ± 7.18	14.09 ± 4.52	11.05 ± 4.00
Total	18.96 ± 8.08	14.84 ± 5.55	10.50 ± 4.92

All values showing mean ± SD.

When plasma homocysteine levels of 16 µmol/L was taken as a cut off to define hyperhomocystinemia, 15.52% of patients had hyperhomocystinemia compared to 40% in siblings and 12% in controls and the difference was statistically significant (p<0.0001). No significant relation was found between homocysteine and BMI, age, sex, waist hip ratio, lipid profile and smoking.

Discussion

The present study shows a very strong association between plasma homocysteine levels and hypertension. Tyrell et al.,[[11]] also have shown this association in patients with isolated systolic hypertension. The Hordaland homocysteine study[[16]] also showed a positive association between homocysteine and both diastolic and systolic blood pressure. This relation was confined to the younger age group. Malinow et al.[[17]] showed a significant positive correlation between concentration of plasma homocysteine and systolic blood pressure that remained after adjusting for diastolic blood pressure and pulse wave velocity (an index of arterial rigidity). Another study[[18]] in patients with peripheral arterial disease showed higher homocysteine levels in patients with associated hypertension than that in those without hypertension. Araki et al.[[19]] in their study found a mean value of homocysteine to be 9.9 µmol/L for hypertensive patients and 7.3 µmol/L for controls and the difference was significant. However various other studies[[20]], [[21]], [[22]] did not find any significant association between plasma homocysteine levels and hypertension. Lack of association between coronary artery disease and homocysteine has been shown in various Indian studies.[[23]], [[24]], [[25]]

In our study we found homocysteine levels in patients to be higher when compared to their siblings. Higher levels of homocysteine in family members were also found in number of studies previously. A high prevalence of hyperhomocystinemia and asymptomatic vascular disease was found in siblings of young patients with vascular disease and hyperhomocystinemia.[[26]] Higher plasma homocysteine levels were observed among children with a positive family history of coronary artery disease.[[27]] Homocysteine lowering treatment with folic acid and Vitamin B6 in healthy siblings of patients with premature atherothrombotic disease was associated with a decreased occurrence of abnormal exercise electrocardiography test, which was consistent with a decreased risk of atherosclerotic coronary events.[[28]] Tonstad[[29]] found a higher level of plasma homocysteine in family members of coronary artery disease and suggested that a moderately elevated homocysteine level may partly account for the contribution of the family history to risk of cardiovascular disease.

The mechanism of hyperhomocystinemia causing hypertension is not clear. Hyperhomocystinemia induces an elastolytic process in arterial wall, by inducing synthesis and secretion of serine elastases.[[30]] The analysis of elastin in subrenal aortic wall in minipigs showed significantly lower level of elastin in hyperhomocystinemia minipigs compared to control animals.[[31]] The loss of elastin may lead to stiffening of arterial wall resulting in hypertension.[[32]] It was also found that elevated homocysteine was related to impaired nitric oxide mediated relaxation of vessels.[[33]]

To summarize, elevated levels of plasma homocysteine were found in patients with essential hypertension. Siblings of patients with essential hypertension also had higher levels of homocysteine, putting them at a greater risk of developing hypertension in future. Further studies need to be done in Indian population on the effect of folic acid on homocysteine levels and whether any reduction in homocysteine levels result in lowering of blood pressure or prevents development of hypertension in the siblings of patients with hypertension.

Acknowledgment

We acknowledge the help of Dr. Veena Dhawan for performing the HPLC on the samples.