The impact of serum homocysteine on intima-media thickness in normotensive, white-coat and sustained hypertensive adolescents

Abstract

Background and purpose. In recent decades, elevated levels of homocysteine (Hcy) have been found to be associated with an increased risk of vascular events. Additionally, in some case–control studies, hyperhomocysteinaemia has been found to be related to higher intima-media thickness (IMT), but the results are inconclusive. Therefore, in the present study we intended to assess the relationship between serum levels of Hcy and IMT in normotensive and hypertensive adolescents. Patients and methods. 59 normotensive 47 white coat hypertensive and 73 sustained hypertensive adolescents were included in our study. IMT of the common carotid arteries was measured by B-mode ultrasonography. Plasma NOx as well as homocysteine levels were measured in all cases. The relationship between IMT and Hcy and NOx were assessed by a pooled analysis. Additionally, serum levels of Hcy and NOx were compared between normotensives and hypertensive subgroups. Results. IMT was elevated in hypertensive adolescents (means ± SD: 0.055 ± 0.01 cm) compared with normotensives (0.048 ± 0.008 cm, p < 0.01). Higher serum concentrations of homocysteine were measured in hypertensive teenagers (11.9 ± 7.25 µmol/l for hypertensive and 9.85 ± 3.12 µmol/l for normotensives respectively, p < 0.01). In contrast to this, serum NOx was lower in patients (28.8 ± 14.9 µmol/l) compared with controls (38.8 ± 7.6 µmol/l, p < 0.01). The pooling of homocysteine and IMT data of hypertensive and normotensive adolescents revealed a significant positive relationship between the two parameters (r = 0.43, p < 0.001). Conclusions. We conclude that elevated serum levels of homocysteine may play a role in increased IMT in adolescent hypertension.

Key Words::

• adolescent hypertension
• intima-media thickness
• homocysteine

Introduction

In recent decades, hyperhomocysteinaemia has been extensively studied in relation to different atherosclerotic diseases. It has been postulated that mild or severely elevated levels of serum total homocysteine are positively associated with an increased risk of coronary, peripheral vascular and cerebral vascular disease (1–2). However, the question whether hyperhomocystainaemia is a marker of atherosclerosis or merely a marker of the atherosclerotic process remains a debated issue (3).

Intima-media thickness (IMT) of the carotid arteries has been considered by some authors a marker of subclinical atherosclerosis. Additionally, in young hypertensive subjects its seems to represent target organ damage (4,5). It is also clear that thickening of the intima-media layer in hypertensives is a continuous process; thus, it develops in a stepwise fashion during the course of hypertension, as demonstrated by Puato and co-workers (6).

Many authors have found that there is a relationship between total homocysteine levels and intima media thickness, although the relationship is not evidence-based and therefore remains a debated issue (3). In previous case–control studies, homocysteine was found to be an independent risk factor for carotid IMT (7,8) while others could not prove this relationship (9,10).

According to previous observations, there is a relationship between increased levels of homocysteine and endogenous inhibition of nitric oxide synthase (NOS) (11). In previous reports, the working group of the present material has presented data on increased IMT in hypertensive adolescents (12) and proved a relationship between increased IMT and decreased nitric oxide concentrations as well as elevated serum levels of endothelin-1 (13). However, no data are at present available as to whether serum homocysteine plays a role in the development of target-organ damage at different stages of adolescent hypertension. In view of this, we intended to answer the following study questions:

(i) Is there any difference between the serum levels of homocysteine and NOx measured in normotensive, white coat hypertensive and sustained hypertensive adolescents?

(ii) What is the relationship between serum homocysteine and IMT in normotensive and hypertensive teenagers?

Patients and methods

We included 59 healthy, non-hypertensive, 47 white coat hypertensive and 73 sustained hypertensive adolescents in our study. All subjects were selected from the Debrecen Hypertension Study (14). Students from all 26 secondary schools of Debrecen (230 000 inhabitants) were enrolled in the study. A subsample of 10 359 teenagers (14–20 years old) decided to participate, with 22 declining participation. Height and weight were measured before blood pressure was taken. Blood pressure was always recorded between 08:00 and 13:00 h at school. The study was approved by the local medical ethics committee of Debrecen University.

Blood pressure measurement procedure

Systolic and diastolic blood pressures were measured in the sitting position on the right arm using OMRON M4 digital oscillometry manometer (OMRON Healthcare GmbH, Hamburg, Germany). If the forearm circumference was 34 cm or over, an obesity cuff was used. Three measurements were performed in each case with 5-min intervals and the average values of the systolic and diastolic blood pressure were used for further analyses.

For the study, the guidelines of “The Fourth Task Force on Blood Pressure in Children and Adolescents” (15) were applied. Based on age, gender and height, subjects were divided into 32 subgroups. Repeated measurements (three consecutive measurements on two further occasions) were performed in adolescents whose systolic and/or diastolic BP exceeded the 90th percentile value for age, gender and height specific subgroups, as suggested by the guidelines. Thus, 3 × 3 = 9 BP measurements were performed in this group in order to prove the subjects’ hypertension. The selection of hypertensive adolescents and the assessment of hypertension prevalence were based on the average of the nine BP measurements, considering the initial 95th percentile value of BP measurements obtained for the entire population.

Subsequently, 1461 teenagers underwent a second and third assessment of three BP recordings. Each occasion was separated by 3 months and totalled nine measurements. Thus, finally 3 × 3 = 9 blood pressure measurements were performed in this group of 1461 adolescents in order to prove their hypertension. Finally, 216 young secondary school students (2.01%) were considered to have hypertension. They were asked to participate in further investigations (e.g. blood tests, transthoracal echocardiography, 24-h ambulatory blood pressure monitoring and carotid ultrasonography). After having the investigation procedure explained to them, approximately half of the hypertensive adolescents (120 persons, 55%) decided to continue the study and the remaining 96 hypertensive adolescents refused further participation. Thus, 120 patients and 59 healthy normotensive adolescent controls (29 girls and 30 boys) as volunteers completed the study.

Twenty-four-hour ambulatory blood pressure monitoring (ABPM)

Ambulatory blood pressure monitoring was performed using an ABPM-04 (Meditech Ltd. Budapest, Hungary) oscillometry device, which was validated earlier according to the protocol of the British Hypertension Society (16) and the Association for the Advancement of Medical Instrumentation (17). In every case, ABPM devices were installed between 08:00 and 09:00 h and were removed 24 h later. Before starting the 24-h monitoring, blood pressures measured by the ABPM device were controlled by a standard mercury sphygmomanometer. Blood pressure was measured by the ABPM between the period of 06:00 and 22:00 h every 15 min, while between 22:00 and 06:00 h, every 30 min. Blood pressures were analysed off-line. Based on the results of ABPM measurements, hypertensive adolescents were grouped into white coat and sustained hypertension groups as follows: in cases where either systolic or diastolic average of the ABPM-measured blood pressure reading exceeded the 95th percentile value of the gender- and height-matched control values, the patients were considered to have sustained hypertension. All other cases of hypertensive adolescents were classified as white-coat hypertensives.

Measurement of IMT

This was done in the common carotid artery was performed using a 7-MHz linear array probe (Hewlett-Packard Sonos 2000, USA). During scanning, the probe was placed just behind the sternocleidomastoid muscle. The bifurcation of the carotid artery was found first, followed by the flow divider between the internal and external carotid arteries. Measurements were performed 2 cm proximal from the flow divider at the far wall of the vessel. IMT was defined as the distance between the media-adventitia layer and the lumen-intima interface and was expressed in centimetres. Three measures from three different images were performed in each carotid artery; thereafter IMT values were averaged and these averaged values were used for further statistical analysis. IMT was assessed by the same investigator, who was unaware of the patients grouping status (hypertensive or not).

Laboratory analysis

Routine laboratory parameters [serum cholesterol, triglyceride high-density lipoprotein (HDL) and low-density lipoprotein (LDL)-cholesterol] were determined by methods used by routine automated clinical chemistry laboratory systems. Plasma NOx level (NOx = measured as nitrite and nitrate) was determined immediately after blood withdrawal using the method of Green et al. (18). Plasma NOx levels were expressed in μmol/l. Serum homocysteine was measured using colorimetric enzyme assays on routine clinical chemistry analyser based on enzymatic release of hydrogen sulphide, which reacts to form a chromogen.

Statistical analysis

We used the Statistica for Windows (Statsoft, Tulsa, USA) program for data analysis. Means and standard deviations were reported for all values. Parametric values were first analysed by normality tests in order to check whether they show normal or non-normal distribution. Normally distributed parameters were compared using t-test, and those with non-normal distribution with the appropriate Kruskal–Wallis tests. Bonferroni correction was performed when indicated. Spearman correlation was applied for the assessment of the relationship between homocysteine and IMT. A p < 0.05 value was accepted as statistically significant.

Results

We included 59 normotensive, 47 white coat hypertensive and 73 sustained hypertensive adolescents. The main confounding factors and results of the routine laboratory tests are summarised in Table I. Hypertensive patients had higher body mass index (BMI) values and blood pressure values, but the routine lipid parameters were similar in the three groups. Before analysing data, multiple regression analysis of variance (ANOVA) was performed in order to adjust potential confounders of IMT. IMT was independent of BMI, total cholesterol, HDL-, LDL-cholesterol and triglycerides (p = 0.202, multiple regression ANOVA).

Table I. The most important confounding factors and results of laboratory tests.

	Normotensive (n = 59)	White coat hypertensives (n = 47)	Sustained hypertensives (n = 73)
Female/male	29/30	28/19	29/44
Age (years)	15.8 ± 0.6	16.3 ± 1.1	16.5 ± 1.0
Body weight (kg)	58.3 ± 11.5	64.06 ± 13.8	71.6 ± 15.5
Height (cm)	169.0 ± 9.2	170.5 ± 9.9	174.2 ± 8.3
BMI (kg/m^2)	20.2 ± 2.7	21.8 ± 3.5	23.4 ± 4.2
Systolic BP average	114.9 ± 10	138.3 ± 11.4	144.8 ± 11.1
MAP average	83.0 ± 8.4	101.1 ± 7.9	103.2 ± 6.4
Diastolic BP average	66.8 ± .9	82.5 ± 11.0	82.4 ± 8.3
Serum glucose (mmol/l)	5.3 ± 0.7	5.4 ± 0.7	5.6 ± 1.5
Serum cholesterol (mmol/l)	4.2 ± 0.7	4.3 ± 0.7	4.2 ± 0.8
HDL-cholesterol (mmol/l)	1.5 ± 0.2	1.5 ± 0.2	1.4 ± 0.3
LDL-cholesterol (mmol/l)	2.3 ± 0.5	2.4 ± 0.6	2.4 ± 0.7
Serum triglyceride (mmol/l)	0.86 ± 0.44	0.93 ± 0.45	1.08 ± 0.65

BMI, body mass index; BP, blood pressure; MAP, mean arterial pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Analysis of IMT, homocysteine and serum NOx levels among normotensive and hypertensive adolescents

During the first analysis, we intended to compare IMT, homocysteine and NOx levels only between normotensives and hypertensives independent of the ABPM results. IMT was elevated in hypertensive adolescents (means ± SD: 0.055 ± 0.01 cm) compared with normotensives (0.048 ± 0.008 cm, p < 0.01). We also measured higher serum concentrations of homocysteine in hypertensive teenagers (11.9 ± 7.25 µmol/l for hypertensive and 9.85 ± 3.12 µmol/l for normotensives respectively, p < 0.01). In contrast to this, serum NOx was lower in patients (28.8 ± 14.9 µmol/l) compared with controls (38.8 ± 7.6 µmol/l, p < 0.01).

Relationship between serum homocysteine and IMT

The pooling of homocysteine and IMT data of hypertensive and normotensive adolescents revealed a significant positive relationship between the two parameters, e.g. the higher the serum concentration of homocysteine the higher the IMT was (Figure 1). During subgroup analysis this positive relationship was maintained in hypertensive adolescents (r = 0.43, p < 0.01), but not in controls (r = 0.25, p = 0.06).

Figure 1. Relationship between serum homocysteine (µmol/l) and intima-media thickness (cm).

Comparison of IMT, serum homocysteine and NOx in normotensive, white coat hypertensive and sustained hypertensive adolescents

IMT was higher in both white-coat (means ± SD: 0.056 ± 0.001 cm) and sustained hypertensive adolescents (means ± SD 0.054 ± 0.001 cm) compared with controls (means ± SD:0.048 ± 0.001 cm). Similarly, serum homocysteine concentrations appeared to be elevated among white coat and sustained hypertensive teenagers compared with controls (Figure 2). Serum levels of NOx were significantly lower in patients compared with controls, as shown in Figure 3.

Figure 2. Comparison of serum homocysteine (µmol/l) in normotensive, white coat and sustained hypertensive adolescents. Means and standard deviations are shown. p-values after Bonferroni correction are presented.

Figure 3. Comparison of serum NOx concentrations (µmol/l) in normotensive, white coat hypertensive and sustained hypertensive adolescents. Means and standard deviations are shown. p-values after Bonferroni correction are presented.

Because these data suggested the impact of both homocysteine and NOx in the intima-media thickening procedure, we also assessed the relationship between homocysteine and NOx serum levels. When pooling normotensive and hypertensive data together, no relationship was found between the two parameters (r = 0.01, p = 0.88), suggesting no relationship. Similarly, no significant relationship was found between the two parameters during subgroup analysis of normotensive, white coat hypertensive and sustained hypertensive patients.

Discussion

In the present study, we have found that serum homocysteine is elevated in hypertensive adolescents and this elevation is already present in the white coat form of adolescent hypertension. Additionally, we detected a relationship between serum levels of homocysteine and IMT among hypertensive teenagers. To our knowledge, this is the first study to assess the relationship between serum homocysteine and IMT among hypertensive adolescents.

Homocysteine is believed to influence the vascular processes in two ways: according to the oxidative stress hypothesis, elevated levels of homocysteine inhibit the nitric oxide synthesis and bioavailability, contribute to vascular smooth muscle proliferation and intimal hyperplasia. The other proposed mechanism of homocysteine is related to the generation of asymmetric dimethylarginine, an endogenous inhibitor of the NOS system (3). Whichever mechanism is dominant, elevated levels of homocysteine may lead to the alteration of the physiological balance between the NO/endothelin system, which is responsible for vasodilation/vasoconstriction and remodelling processes of the vasculature (19). In a previous study among hypertensive adolescents, we were able to demonstrate a significant positive relationship between serum endothelin-1 levels and IMT as well as left ventricular mass index of the heart. Additionally, a negative relationship was found between decreased NOx and IMT (13). Despite our inability to demonstrate a relationship between NOx and homocysteine levels in the present study, elevated levels of homocysteine among hypertensive adolescents may indicate a role of homocysteine in the development of IMT among adolescent hypertensives. One reason why a direct relationship could not be detected between Hcy and NOx may be that homocysteine is an intermediate product of the methionine metabolism, which influences the vascular endothelium through several pathways and the NOS activity is just one of these mechanisms (3).

In previous studies that assessed the relationship between Hcy and IMT, some authors found Hcy was an independent risk factor for IMT (7,8), while others could not prove this relationship (9,10). It has to be noted that the majority of the previous studies were performed on different aged adults with multiple vascular risk factors that may also influence IMT (such as diabetes, lipid disorders) through pathways other than elevated homocysteine. In the present study, however, we included young hypertensive teenagers aged between 14 and 18 years with no or modest other vascular risk factors. In this context, the present results may underline the possible role of elevated homocysteine levels in the development of elevated IMT.

Another important observation of the present study is that even white coat hypertension may result in increased IMT among adolescents and homocysteine is elevated in teenagers with white coat hypertension. White coat hypertension has long been considered a benign condition but recently published follow-up studies have indicated that white coat hypertensives may be at an increased risk for cardiovascular events (20,21). In agreement with our observations, several authors have reported on elevated levels of homocysteine in patients with white coat hypertension (22,23).

We conclude that elevated serum levels of homocysteine may play a role in increased IMT in adolescent hypertension. It is conceivable that inconclusive results on the impact of homocysteine in influencing vascular endothelial function in adults may be related to multiple risk factors (such as diabetes mellitus and hypercholesterolaemia) that also play a role in arterial wall thickening. Further, prospective studies are encouraged to show whether hyperhomocysteinaemia is an independent risk factor for IMT and long-term vascular events in adolescent hypertensives.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.