Evaluation of plasma chemerin levels in patients with non-dipper blood pressure patterns

Background

Hypertension remains a major public health problem despite continuous research and development in cardiovascular medicine [1,2]. Hypertension is one of the leading causes of cardiovascular mortality [3]. Besides being one of the most important risk factors of ischemic heart disease, hypertension has other major adverse effects on target organs, including the heart, kidneys, and eyes.

Blood pressure (BP) and heart rate have long been known to show circadian changes depending on sleep and wakefulness. Ambulatory blood pressure monitoring allows for the evaluation of daily rhythms of blood pressure and a determination of various parameters demonstrated to be associated with adverse cardiovascular prognoses. In many studies, a non-dipper condition, which is one of the most important abnormalities in circadian BP rhythm, was associated with target organ damage in both hypertensive (HT) and normotensive subjects [4]. Patients demonstrating a drop of 10% or more in blood pressure values when measured during the night as compared to daytime values were defined as “dippers”, whereas patients demonstrating a drop of less than 10% were defined as “non-dippers” [5]. In prospective studies, lack of nighttime blood pressure drop, or a blood pressure higher at night than during the day, were identified as independent risk factors for cardiovascular disease [6–9].

Adipose tissue has been identified as an important organ affecting energy regulation and metabolism. It acts through bioactive mediators called adipokines, among which there are many hormones and cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, angiotensinogen, adiponectin, leptin, and resistin [10–12]. Chemerin is an adipokine and is also known as tazarotene-induced gene 2 protein (TIG2) or a retinoid acid receptor responder 2 (RARRES2). However, chemerin has been defined as a new adipokine that plays a role in acquired and natural immunity [13,14]. Chemerin is secreted as a ligand for “orphan” G protein-coupled receptor chemokine-like receptor (CMKLR) 1, chemokine (C-C motif) receptor-like (CCRL) 2, and G protein-coupled receptor (GPR) 1 [13].

Chronic inflammation has been demonstrated to be a risk factor for the development of arterial hypertension. Inflammation markers such as C-reactive protein (CRP), TNF-α, and IL-6, were shown to increase in patients with essential hypertension. Therefore, the chemerin/CMKLR1 signal, the secretion of which is controlled by inflammatory cytokines, was found to contribute to pathogenesis of hypertension [15–17].

In our study, we aimed to determine whether chemerin levels are elevated in non-dipper patients, compared with dippers and healthy controls. We also tried to determine if chemerin levels are related to circadian blood pressure patterns in essential hypertensive patients.

Material and Methods

STUDY POPULATION:

This study included a group of 90 patients: 60 hypertensive patients and 30 healthy control subjects (18 females, 12 males, mean age =53.2±15.4 years). Ambulatory BP monitoring devices (ABPM) were connected to all patients. Through the use of data from the ABPMs, hypertensive patients were divided into 2 groups: 30 dipper patients (18 females, 12 males, mean age =52.5±15.1 years) and 30 non-dipper patients (19 females, 11 males, mean age =54.6±13.0 years). The ABPM also confirmed that the subjects in the control group were normotensive and have a dipper profile.

Hypertension was defined as systolic BP (SBP) ≥140 mmHg or a diastolic BP (DBP) ≥90 mmHg and/or using anti-hypertensive drug therapy [18]. After the diagnosis of hypertension, ambulatory blood pressure monitoring (ABPM) was performed for all patients. Exclusion criteria included secondary hypertension, chronic heart failure, diabetes mellitus, renal or hepatic dysfunction, clinical evidence of cancer, systemic inflammatory disease, chronic or acute infectious disease, auto-immune disease, serious valvular heart disease, cardiomyopathy, atrial fibrillation, hematological disease, and known coronary artery or cerebrovascular disease.

Clinical BP measurements were conducted in the morning using a sphygmomanometer, and the average of 3 measurements was used. Prior to BP measurements, the subject was allowed to rest for a minimum of 5 minutes. Blood pressure was measured when the subject had not consumed tea/coffee within the last hour and had not smoked within the last 30 min. BP was measured while the subject’s arm was supported at the heart level in a sitting position. The measurement was made on both arms and the higher value was used.

The patients’ clinical and demographic characteristics were noted and included age, sex, smoking habits, and antihypertensive drug use. Additionally, fasting blood glucose levels, creatinine levels, and fasting serum lipid status, including total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride levels were recorded. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m²).

This study complied with the Declaration of Helsinki, was approved by the local ethics committee, and each patient gave written informed consent.

AMBULATORY BLOOD PRESSURE MONITORING AND DIPPING STATUS:

Ambulatory BP monitoring studies were carried out using a Tracker NIBP2 (Del Mar Reynolds Medical Ltd, Hertford, UK) monitoring device. Blood pressure was measured and recorded for 24 h with 15-min daytime intervals and 30-min nighttime intervals using this device, which performs oscillometric measurements. The cuff of the ABPM device was placed on the non-dominant arm if the BP difference between the 2 arms during clinical BP measurement was less than 10 mmHg. However, if the BP difference was greater than 10 mmHg, the cuff was placed on the arm that had the higher reading. The recordings were analyzed with interactive software. The subjects were given information about the procedure and asked to perform daily routine activities, avoid excessive activities, and hold their arms still at heart level during measurements.

From the hourly averages of ambulatory BP recordings, daytime, nighttime, and 24-h averages of SBP, DBP, and mean BP were calculated for each patient. Patients with a BP drop of 10% or more during nighttime were accepted as dipper hypertensives, whereas patients with a BP drop of less than 10% were accepted as non-dipper hypertensives, according to the criteria of Verdecchia et al. [9].

COLLECTION OF BLOOD SAMPLES AND BIOCHEMICAL ANALYSES:

Blood samples were taken in the morning after a 30-min rest following a 12-h fasting period. Samples were centrifuged (Shimadzu UV160A, S. No: 28006648, Japan) at 3000 rpm for 10 min and the serum was stored at −80°C. Serum glucose, creatinine, total cholesterol, HDL cholesterol, LDL cholesterol, and triglyceride concentrations were analyzed by a Roche Hitachi Cobas 8000 device using Roche Diagnostics GmbH kits. Hemoglobin and leukocyte concentrations were determined by using a Siemens Advia 2120i Hematology System device. The concentrations of chemerin in the serum were measured using commercially available ELISA kits (Human Chemerin Elisa kit, BioVendor-Laboratorni Medicina AS, Cat No.RD191136200R, Brno, Czech Republic). The enzymatic reactions were quantified in an automatic microplate photometer and the chemerin levels were expressed as ng/ml. The mean interassay coefficient of variation (CV) percent and intraassay CV percent for chemerin were 8.3% and 5.1%, respectively. All assays were conducted according to the manufacturer’s instructions. The samples that showed higher concentrations were diluted and measured in duplicate.

STATISTICAL ANALYSIS:

Statistical analyses were conducted using the Statistical Package for the Social Sciences for Windows 21.0 (SPSS, Chicago, IL). Descriptive statistics are given as mean, standard deviation, frequency, and percentage. The Kolmogorov-Smirnov test and graphical methods were used to test the normality of continuous variables. One-way analysis of variance (ANOVA) (for data with normal distribution) or Kruskal-Wallis test (for data without normal distribution) was used for the comparison of the 3 groups with regard to baseline clinical characteristics. Tukey HSD was used as the post hoc test after ANOVA and built-in pairwise comparison test of the software was used after Kruskal-Wallis test. For the comparison of dippers and non-dippers with regard to blood pressure measurements, either Student’s t test for independent variables or Mann-Whitney U test was used, depending on the normality of data. Comparisons of categorical values were carried out by the chi-squared test. Correlations between blood pressure and chemerin levels were tested using Spearman correlation analysis because chemerin data did not have normal distribution. Additionally, we performed a receiver operating characteristic (ROC) analysis to identify the most sensitive chemerin cut-off level for identifying patients with non-dipper hypertension. A probability value <0.05 was considered the minimum level of statistical significance. A 2-sided p-value was considered for all comparisons.

Results

Demographic characteristics, laboratory data, and medication usage of patient and control groups are given in Table 1. There was no significant difference between the groups in terms of age, sex, creatinine, fasting glucose, fasting lipid status, hemoglobin levels, white blood cell, body mass index, smoking, or antihypertensive drug use.

Table 2 shows the comparisons of clinical blood pressure and chemerin levels of the groups at baseline. As expected, clinical BP values were significantly higher in dipper and non-dipper patients compared to normotensive patients (SBP 144.5±15.9 and 148.2±21.8 vs. 126.3±8.8 mmHg, p<0.001 for both; DBP 83.4±12.3 and 83.7±13.3 vs. 75.5±9.1 mmHg, p=0.029 and 0.022, respectively). However, clinical BP values were similar between dipper and non-dipper patients (Table 2).

Nocturnal SBP, DBP, and mean BP were significantly higher in non-dippers compared with dippers (Table 3). While daytime BP measurements were similar between non-dippers and dippers, there was a significant difference between these groups during nighttime measurements (nighttime SBP 132.4±20.1 vs. 114.2±11.2 mmHg, p<0.001; nighttime DBP 73.9±12.6 vs. 64.8±7.7 mmHg, p=0.001).

Chemerin levels were significantly higher in non-dipper patients compared to dippers and normotensives. As reported in Table 2 and Figure 1, non-dipper patients demonstrated higher levels of chemerin compared to dippers and normotensives (219.7±16.3 vs. 182.4±21.4 ng/ml; 219.7±16.3 vs. 85.4±38.1 ng/ml, p<0.001 for both, respectively).

In hypertensive patients, ROC curves discovered the correlation between the non-dipping status and chemerin, for which ROC analysis showed an optimum cut-off of 201.4 (area under the curve 0.972, p<0.001). According to the cut-off, we calculated the sensitivity and specificity as 90% and 90%, respectively (Figure 2).

In dipper and non-dipper hypertensive patients, we found a positive correlation between chemerin and all ambulatory BP values (daytime DBP and SBP, nighttime SBP and DBP, and 24-h SBP and DBP) (Table 4).

There was no significant difference for antihypertensive medications taken by the dipper and non-dipper groups. Normotensives did not take any medicine (Table 1).

Discussion

LIMITATIONS OF THE STUDY:

Our sample size was small, thus the statistical power of the study was limited. Other inflammation markers were not studied.

Conclusions

Chemerin, which plays a role in inflammation and atherosclerosis, was found to be higher in non-dippers compared to dippers and normotensives. Chemerin levels also show a positive correlation with blood pressure. These results are important because they show the likely role of inflammation in the pathogenesis of hypertension.