Effect of ACE inhibitors and b-blockers on homocysteine ... - Nature

Journal of Human Hypertension (2008) 22, 289–294 & 2008 Nature Publishing Group All rights reserved 0950-9240/08 $30.00 www.nature.com/jhh

ORIGINAL ARTICLE

Effect of ACE inhibitors and b-blockers on homocysteine levels in essential hypertension A Poduri1, J Kaur1, JS Thakur2, S Kumari3, S Jain3 and M Khullar1 1

Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India; 2Department of Community Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India and 3Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Recent studies have shown that antihypertensive drugs like diuretics increase plasma homocysteine (Hcy) levels. However, the effect of other antihypertensive drugs on plasma Hcy levels has not been tested extensively. The aim of present study was to investigate the effect of antihypertensive therapy (AHT) on Hcy levels in essential hypertensive subjects. A case–control study of 273 patients with essential hypertension (EH) and 103 normotensive controls was undertaken. Plasma Hcy levels were measured before and after 6 weeks of AHT. The genotyping of MTHFR C677T polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism. Angiotensin-converting enzyme (ACE) inhibitors and b-blockers significantly

decreased and hydrochlorothiazides significantly increased the plasma Hcy levels in hypertensive patients (Po0.05). No significant association between MTHFR C677T genotypes and changes in Hcy levels in response to antihypertensive was observed in EH patients. The decrease in Hcy induced by b-blockers and ACE inhibitors observed in our study may be due to the improvement of endothelial function along with improved renal function. Thus, our results suggest that ACE inhibitors and b-blockers may provide additional beneficial therapeutic effects to the EH patients by decreasing Hcy levels. Journal of Human Hypertension (2008) 22, 289–294; doi:10.1038/sj.jhh.1002325; published online 17 January 2008

Keywords: essential hypertension; MTHFR C677T polymorphism; antihypertensive therapy; homocysteine; ACE

inhibitors; b-blockers

Introduction Recent studies have shown high incidence of hypertension in both urban and rural Indian population. The prevalence of hypertension in north Indian population has been reported to be 22–29% in the urban population in past one decade.1–3 Hyperhomocysteinaemia is an independent risk factor found to be associated with hypertension and other cardiovascular diseases.4,5 Several studies have shown that homocysteine (Hcy) is positively correlated with high blood pressure (BP),5–12 however, this association is not reported in other studies.13,14 Moreover, the subjects with higher quartile of Hcy levels have been shown to be more susceptible to hypertension.5,15–17 Recent studies have shown that the drug therapy for common

Correspondence: Professor M Khullar, Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, UT 160012, India. E-mail: [email protected] Received 3 October 2007; revised 16 November 2007; accepted 1 December 2007; published online 17 January 2008

medical conditions may have severe effects on plasma Hcy levels.18–22 Thiazide-type diuretics are recognized as the cornerstone of antihypertensive therapy (AHT) in elderly because of their extensive track record in preventing stroke and cardiovascular events and their low cost.23 Outcome benefits of diuretics as first-line agents have been demonstrated only at moderate doses (equivalent to X25 mg hydrochlorothiazide).24 A significant increase in plasma Hcy levels has been reported in hypertensive patients on a long-term diuretic therapy.4,5 Increased Hcy levels have been also observed in hypertensive patients on hydrochlorothiazide treatment.25 The effect of other antihypertensive drugs such as angiotensin-converting enzyme (ACE) inhibitors, b-blockers and calcium channel blockers on plasma Hcy levels has not been studied extensively. Atar et al.26 have reported that b-blocker treatment significantly decreases plasma Hcy levels in patients with hypertension, especially in women;26 however, another study did not find any change in plasma Hcy with b-blocker therapy.27 The increase in Hcy concentrations after AHT may be clinically relevant if it increases the risk of cardiovascular disease and

ACE inhibitors, b-blockers and homocysteine A Poduri et al 290

may counteract the desired cardiovascular protection conferred by lowering the BP. In the present study, we have examined the effect of various antihypertensive drugs on plasma Hcy levels in a cohort of essential hypertensive patients. We also studied the association of plasma Hcy levels with MTHFR C677T polymorphism in essential hypertension (EH) patients.

Blood sampling

About 5 ml of venous blood sample was drawn from overnight fasting subjects and placed in plain tubes as well as in tubes containing disodium EDTA for further analysis. After collection, samples were centrifuged 2000 g for 15 min and aliquots were stored at 20 1C. Biochemical investigations

Materials and methods Study population

A total of 273 hypertensive patients attending Hypertension Clinic of Nehru Hospital from January to December 2006 were enrolled in the present study. Patients were diagnosed with EH according to Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure VII (JNC VII) criteria (systolic blood pressure (SBP)X140 mm Hg and diastolic blood pressure (DBP)X90 mm Hg) or if they were administered at least one hypertensive medication. Mild hypertensive patients who had no associated systemic diseases, previous history of stroke, coronary artery disease, myocardial infarction, peripheral vascular disease, renal hypertension, diabetes mellitus and chronic liver disease were included in the present study. The research protocol was approved by Institute Research Ethics Committee, PGIMER, Chandigarh, India and informed consent was obtained from patients/caregivers and controls before the collection of blood samples. Patients and controls were of north Indian origin and were ascertained to have at least parents and grandparents born in northern India to ensure ethnicity. A total of 103 unrelated, age- and sex-matched healthy normotensive (SBP/DBPo130/80 mm Hg) individuals who had no family history of hypertension served as controls. All the subjects underwent a detailed physical examination and their baseline BP was measured. A standardized protocol in which the appropriate cuff size was used depending on the arm circumference was followed. Three measurements were taken 5 min apart in the right arm in the sitting position after 5 min of rest. The average of the second and third measurements was taken as the participant’s BP.28 The patients on antihypertensive drugs were taken off their drug(s) for 2 weeks to ensure washout of the drug and their baseline BP was measured and a fasting blood sample was taken. These patients were re-started on antihypertensive medication which they were taking earlier (ramipril (5 mg per day), atenolol (50 mg per day), amlodipine (5 mg per day) and hydrochlorothiazide (25 mg per day). All untreated hypertensives were prescribed ramipril (5 mg per day). Each individual’s BP was re-evaluated after 2 and 6 weeks of treatment. Journal of Human Hypertension

Plasma total Hcy concentrations, folate, vitamin B12, total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride were measured in the patients before starting antihypertensive treatment and after 6 weeks of antihypertensive treatment. Plasma total Hcy was measured in overnight fasting plasma samples by enzyme-linked immunosorbent assay-based kit (Axis Shield Diagnostics, UK). A fasting blood sample was collected from each patient to measure baseline levels of Hcy, folate, vitamin B12, total cholesterol, LDL, HDL and triglyceride before starting the antihypertensive medication. Folic acid and vitamin B12 were measured using AIA-Pack-Folate Assay Kit and AIA-Pack B12 assay kit (Tosoh Biosciences NV, Tessenderlo, Belgium). Genotyping of MTHFR

Genomic DNA was isolated from the whole blood by the method of Lahiri et al.29 Genotyping of MTHFR C677T polymorphism was analysed by polymerase chain reaction using the primers described by Frosst et al.30 followed by restriction digestion with Hinf I enzyme (Bangalore Genei, India). The restriction fragments were observed by using 12% polyacrylamide gel followed by silver staining. Statistical analysis

Statistical analyses were performed using the SPSS (Version 10; SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as mean±s.d.. Paired t-test was performed to compare the Hcy, SBP and DBP levels, folate, vitamin B12, total cholesterol, LDL, HDL and triglyceride levels before and after antihypertensive treatment. Unpaired Student’s t-test was carried out to analyse the data between control and patient groups. We calculated the genotype and allelic frequency for the MTHFR C677T polymorphism in the study population. Pearson’s w2-square test was used for statistical comparisons between the groups. The multiple logistic regression analysis was performed to estimate the main effects of the antihypertensive drugs on Hcy levels. Risk magnitudes were estimated by calculating odds ratios with 95% confidence intervals. All P-values were two tailed. The conventional P-value of 0.05 was specified as the significance threshold.


Results

Discussion

The demographic characteristics of the subjects are shown in Table 1. Plasma Hcy levels were significantly higher in patients than in controls (Po0.05) (Table 1). No significant difference in other biochemical parameters was observed. We did not observe any correlation between age, body mass index (BMI), smoking and Hcy levels. There was a significant decrease in SBP and DBP levels with AHT (Po0.05). No significant change was observed in total cholesterol, HDL-C and LDL-C levels but there was a significant increase in the triglyceride levels after AHT therapy (Po0.05). No significant change was observed in folate and vitamin B12 levels with AHT. Plasma Hcy levels were significantly increased in patients taking diuretics (Po0.05); a significant decrease in plasma Hcy levels was observed in patients on b-blockers and on ACE inhibitors (Po0.05); no significant change in plasma Hcy levels was observed with calcium channel blockers (Table 2).

Recent studies suggest that drugs used for the prevention or treatment of hypertension may modulate plasma Hcy levels.18,19 In the present study, we analysed the effect of commonly used antihypertensive drugs such as ACE inhibitors, calcium channel blockers, b-blockers and hydrochlorothiazide on plasma Hcy levels in essential hypertensive subjects. We observed that ACE inhibitor and b-blocker resulted in significantly reduced plasma Hcy levels in hypertensive patients. The effect of ACE inhibitors on plasma Hcy is not well known. ACE inhibitors are first-choice drugs for lowering elevated BP and hence reducing risk of cardiovascular disease. The vascular mechanism of ACE inhibitors is to block the angiotensin II formation,31 to stimulate the release of endothelin-1 within endothelial32 and smooth muscle cells,33 to remedy the endothelial dysfunction and to restore endothelium-dependent vasodilatation.34,35 Impaired endothelium-dependent vasodilatation indicating endothelial dysfunction has been observed in patients with high plasma Hcy level.36,37 It has been suggested that elevated Hcy level can act synergistically with angiotensin II and endothelin in enhancing the cardiac and carotid rings contraction.37,38 The decrease in Hcy induced by ACE inhibitors observed in our study may be due to the improvement of endothelial function along with improved renal function following ACE inhibitors therapy.39 There are few studies on the effect of b-blockers on plasma Hcy levels. A decrease in Hcy levels has been reported in hypertensive patients on b-blockers by Sharabi et al.40 In an experimental study conducted by Do et al.,41 b-adrenergic-receptor stimulation of astrocyte cultures resulted in enhanced secretion of homocysteinic acid, and atenolol reduced this effect. Thus, our study further confirms the reduction of plasma Hcy levels by b-blocker therapy in hypertensive patients. The reduction of elevated homocysteinaemia by ACE inhibitors seen in our study suggests that these drugs thus could provide additional beneficial therapeutic effects. However, association of Hcy with cardiovascular risk remains the subject of ongoing debate, and more studies are needed to confirm the beneficial effects of such treatments on cardiovascular risk.

Genotype analysis

There was no significant difference in MTHFR 677CC and heterozygous 677CT genotype frequencies between patients (65.20 and 34.79%) and controls (71.84 and 28.15%). The frequency of the mutant T allele was 0.18 in patient group and 0.14 in controls (P40.05). No significant correlations were seen in MTHFR C677T polymorphism and Hcy levels before or after the treatment in EH patients (Table 3).

Table 1 Demographic profile of controls and patients Characteristics Age (years) Sex Males Females BMI (kg m2) SBP (mm Hg) DBP (mm Hg) Homocysteine (mmol l1)

Controls (n ¼ 103) 43.25±6.70 61 (59.22%) 42 (40.77%) 23.25±3.41 117.33±8.28 78.13±6.16 11.95±5.67

Patients (n ¼ 273) 48.03±8.98 174 (63.73%) 99 (36.26%) 24.94±3.23 143.05±17.89** 92.74±10.63** 17.68±7.90*

Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; SBP, systolic blood pressure. Values are given as mean±s.d.; **Po0.001, *Po0.05.

Table 2 Effect of various antihypertensive drugs on plasma homocysteine levels Antihypertensive drug ACE inhibitors Diuretics b-Blockers CCB

Patients (N)

Homocysteine levels (before AHT)

Homocysteine levels (after AHT)

P-value

94 52 103 24

19.12±6.94 18.81±7.92 18.35±9.23 19.83±9.80

14.39±5.75* 22.87±6.89* 14.09±7.07* 17.55±7.69

o0.05 o0.05 o0.05 0.71

Abbreviations: ACE, angiotensin-converting enzyme; AHT, antihypertensive therapy; CCB, calcium channel blockers. Values are given in mmol l1, mean±s.d; *Po0.05. Journal of Human Hypertension


Table 3 Association of MTHFR C677T genotypes with change in homocysteine levels after antihypertensive therapy Treatment

677 CC Baseline

ACE Int. (N) Diuretics (N) BB (N) CCB (N)

18.78±7.46 (54) 19.15±8.05 (31) 17.55±7.77 (60) 22.38±11.51 (16)

677 CT After AHT 15.22±6.18 22.63±9.89 14.85±6.05 20.83±9.18

(54) (31) (60) (16)

Baseline 17.19±6.18 20.42±8.91 18.20±9.11 14.47±2.91

After AHT (40) (21) (43) (8)

14.79±5.56 23.46±9.73 15.28±6.73 13.31±1.95

(40) (21) (43) (8)

Abbreviations: ACE, angiotensin-converting enzyme; AHT, antihypertensive therapy; BB, b-blockers; CCB, calcium channel blockers. Values are given in mmol l1, mean±s.d.

Several factors such as dietary intake of folate levels, niacin,42 vitamin B6 as well as vitamin B12 plasma levels43 and physical activity44,45 have been found to affect plasma total Hcy levels. Increased Hcy levels have been associated with low folate and vitamin B12 levels. Asian Indians have low folate levels, probably due to their dietary habits;46,47 however, we did not observe any correlation between folate, B12 and change in Hcy levels in our subjects. We did not observe any change in folate or vitamin B12 levels after AHT, thus the reduction in Hcy levels with ACE inhibitors or b-blockers in the present study does not appear to be influenced by these factors. As reported in earlier studies,48,49 we too found that plasma Hcy levels increased significantly following treatment by hydrochlorothiazide. This increase in Hcy levels has been proposed to be due to alternation of renal function.50 Decrease in the concentration of folate in red blood cells is as a result of a depletion of water-soluble vitamins. This elevation of Hcy after hydrochlorothiazide use may be of clinical importance, thus, increase in Hcy may now be added to the unfavourable adverse effects of hydrochlorothiazide as also suggested by others.51 There is limited data on the effect of calcium channel blocker (CCB) therapy on plasma Hcy level. In a recent study, Muda et al.52 reported no significant change in plasma Hcy levels in hypertensive patients on either candesartan or amlodipine treatment, however they observed at least 2 mmol l1 increase in Hcy concentration in 12 of 49 patients. We too did not find any significant change in Hcy levels in hypertensive patients on amlodipine treatment, but the number of patients (n ¼ 24) is too small to make any definite conclusions regarding this. A recent study indicated that MTHFR C677T polymorphism may modulate BP responsiveness to short-term treatment of ACE inhibitor in Chinese essential hypertensive patients.39 A correlation between MTHFR polymorphisms and hypertension has been observed in several studies.53,54 MTHFR C677T and A1298C polymorphisms have been also found to be associated with plasma Hcy concentrations.48 Thus, we examined if there was any association between change in plasma Hcy concentrations and MTHFR C677T polymorphism in our Journal of Human Hypertension

cohort. We did not find any correlation between MTHFR C677T genotypes and change in plasma Hcy levels following any AHT. Thus, our data suggest that the observed changes in Hcy concentrations with antihypertensive drugs are independent of MTHFR polymorphisms. However, these findings need to be replicated and expanded to a large population of hypertensive individuals to understand the effect of antihypertensive treatment on Hcy levels. What is known about the topic K Several recent studies have revealed that elevated homocysteine (Hcy) levels are positively correlated with hypertension and various cardiovascular diseases. K A significant increase in plasma Hcy levels has been reported in hypertensive patients on long-term diuretic therapy. K The effects of other antihypertensive drugs such as ACE inhibitors, b-blockers and calcium channel blockers on plasma Hcy levels are not studied widely in hypertensive subjects. What this paper adds K Our results suggest that ACE inhibitors and b-blockers decrease the plasma Hcy levels in hypertensive patients. K The present study has shown that the observed changes in Hcy concentrations with antihypertensive drugs are independent of MTHFR C677T polymorphism. Abbreviation: ACE, angiotensin-converting enzyme.

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