Office-blood pressure (BP) measurements alone overlook a significant number of individuals with masked-hypertension (office-BP: 120/80–139/89mmHg and 24-h ambulatory BP monitoring (ABPM) daytime ≥135/85mmHg or night-time ≥120/70mmHg). Diminished endothelial function contributes to the pathogenesis of hypertension. To better understand the pathophysiology involved in the increased cardiovascular (CV) disease risk associated with masked-hypertension, we estimated the occurrence, assessed the endothelial function, compared plasma levels of inflammatory markers, white blood cell count (WBC count), tumor necrosis factor-α (TNF-α), and high sensitivity C-reactive protein (hsCRP) and examined the possible relationship between endothelial function and inflammatory markers in apparently healthy prehypertensive (office-BP: 120/80–139/89mmHg) African Americans.
Fifty African Americans who were sedentary, nondiabetic, nonsmoking, devoid of CV disease were recruited. Office-BP was measured according to JNC-7 guidelines to identify prehypertensives in whom ABPM was then assessed. Fasting plasma samples were assayed for inflammatory markers. Brachial artery flow-mediated dilation (FMD) at rest and during reactive hyperemia was measured in a subset of prehypertensives.
Subjects in the masked-hypertension sub-group had a higher hsCRP (P = 0.04) and diminished endothelial function (P = 0.03) compared to the true-prehypertensive sub-group (office-BP: 120/80–139/89mmHg and ABPM: daytime <135/85mmHg or night-time <120/70mmHg). Regression analysis showed that endothelial function was inversely related to hsCRP amongst the masked-hypertensive sub-group (R2 = 0.160; P = 0.04).
Masked-hypertension was identified in 58% of African Americans which suggests that a masking phenomenon may exist in a sub-group of prehypertensives who also seem to have a diminished endothelial function that could be mediated by an elevated subclinical inflammation leading to the increased CV disease.
American Journal of Hypertension advance online publication 16 June 2011; doi:10.1038/ajh.2011.103
It has been recognized that 24-h ambulatory blood pressure monitoring (ABPM) is an optimal tool for the diagnosis of different phenotypes of hypertension including masked-hypertension.1,2 Mounting evidence suggests that masked-hypertension must be considered in the assessment of blood pressure (BP) as masked-hypertension has been recognized to have adverse prognostic consequences in terms of cardiovascular (CV) morbidity3,4,,–7 which is comparable to sustained-hypertension.7 Diagnosis of masked-hypertension is vital in that very little is known about its clinical detection or management and it is likely to be prevailing in a large number of individuals of various ages3,8 and in a wide array of BP values including prehypertension.4,9,–11
Prevalence of prehypertension is considerably higher amongst African Americans compared to whites and is a key factor in the racial disparity in CV disease.12,13 Previously, we have shown that BP levels in the prehypertension range were related to circulating inflammatory markers such as high sensitivity C-reactive protein (hsCRP) indicating that BP levels below the hypertensive range may potentially be a proinflammatory condition.14 Prior studies have also provided compelling evidence that African Americans have an early onset of diminished endothelial function. 15,16
Presently, to the best of our knowledge, there are no data available on the assessment of endothelial function in subjects with masked-hypertension. Moreover, research investigating masked-hypertension amongst African Americans is lacking. Thus, data are needed to better understand the underlying pathophysiologic mechanisms of masked-hypertension especially in African Americans in whom the ABPM data itself are sparse. Therefore, our aim was to determine the occurrence, assess the endothelial function via flow-mediated dilation (FMD), compare plasma levels of inflammatory markers, white blood cell count (WBC count), tumor necrosis factor-α (TNF-α), and hsCRP, and examine the possible relationship between endothelial function and inflammatory markers in a cohort of apparently healthy prehypertensive African Americans.
Methods
Subjects. We recruited 50 African-American men (N = 10) and women (N = 40) between the ages 40–75 years from the community within the city of Philadelphia in Pennsylvania through advertisements in the media. Subjects were diagnosed to be normotensives (N = 9; office-BP: <120/80mmHg), prehypertensives (N = 41; office-BP: 120/80–139/89mmHg). Only subjects diagnosed with prehypertension were included in statistical analyses as we wanted to identify at-risk prehypertensives.
For inclusion in the study, subjects were required to be sedentary (aerobic exercise <2 days/week, <20min/session or sedentary occupation), nonsmoking, nonmorbidly obese (body mass index (BMI) <40kg/m2), and not on any medication. Based on medical history, subjects were excluded if they had a history of coronary artery disease, congestive heart failure, renal insufficiency, diabetes, hypercholesterolemia, liver disease, lung disease, or any inflammatory disorders. Subjects were also excluded if they had an inter-current infection within the past 3 months. At the commencement of the study, subjects were not on antihistamine, anti-inflammatory or anticholesterol medications that would influence vascular function. The protocol was approved by the Temple University, institutional review board.
Screening and dietary stabilization. To ensure the eligibility of all qualified subjects, three screening visits were completed prior to inclusion in the study. Screening visit one consisted of a 12-h overnight fasting blood sample to measure blood chemistry, complete blood count, and lipid profile. Any subject who had total cholesterol >240mg/dl, fasting blood glucose >126mg/dl and BMI >40kg/m2 was excluded from the study. Screening visits two and three required all qualified subjects to undergo a physician administered physical examination and echocardiogram bicycle stress test to confirm that subjects displayed no evidence of CV, pulmonary or other chronic conditions. Then, subjects underwent dietary stabilization for 6-weeks. They were instructed by a registered dietitian on the American Heart Association Dietary Guidelines for Healthy American Adults. Compliance to the prescribed diet was monitored by periodic completion of a 3 days/week diet record during the study.
Office-BP measurements. Office-BP measurements were performed by trained laboratory personnel in a research setting with a conventional aneroid sphygmomanometer using the auscultatory technique in accordance with JNC-7 guidelines with the average of three sets of measurements in triplicate on three separate visits.17
24-h ABPM. Subjects underwent ABPM using a noninvasive monitor (Spacelabs 90219; Spacelabs Medical, Redmond, WA) beginning on the morning of their routine weekday, with the exclusion of weekend days. A detailed methodology for ABPM has been previously described by our group.12 Briefly, a standard adult BP cuff was fitted to the subject's nondominant arm with the cuff size determined by the subject's upper arm circumference. BP measurements were obtained at 30-min intervals during the day (6:00AM–10:00PM) and 60-min intervals at night (10:00PM–6:00AM). Only recordings of high technical quality (> 80% of valid BP measurements) were included in final analyses.
Hypertension phenotypes. Using office-BP and ABPM measurements, we defined masked-hypertension (office-BP: 120–139/80–89mmHg and ABPM: daytime ≥135/85mmHg or night-time ≥120/70mmHg); true-prehypertension (office-BP: 120–129/80–89mmHg and ABPM daytime <135/85mmHg or night-time <120/70mmHg) and sustained-hypertension (office-BP: ≥140/90mmHg and ABPM daytime ≥135/85mmHg or night-time ≥120/70mmHg).
Brachial artery FMD. Brachial artery diameter was measured at rest, and in response to reactive hyperemia and to nitroglycerin (NTG) stimulation. All measurements were performed in the morning following an overnight fast during which time subjects refrained from food, drink (with the exception of water), caffeine, alcohol, antihistamine, and anti-inflammatory medications. A 7.5-MHz linear phased array ultrasound transducer attached to a Sonos 5500 ultrasound machine (Philips Medical Systems, Bothell, WA) was used to image the brachial artery longitudinally. Electrocardiogram was continuously monitored during the FMD study. All images of brachial artery diameter were recorded after at least 10-min of lying in the supine position in a quiet and dim room at controlled ambient temperatures by a clinician, trained in ultrasound techniques at the Cardiovascular Research Center, Temple University School of Medicine. The clinician was blinded with the subjects' clinical data. The subject's right arm was comfortably immobilized in the extended position, for scanning the brachial artery 5–10cm above the antecubital fossa. Baseline images of the right brachial artery were first obtained. Then, reactive hyperemia was induced by forearm occlusion of the vessel using a pneumatic tourniquet inflated to supra-systolic pressure (200mmHg) for 5-min on the right forearm, followed by release.18 Three measurements of arterial diameter were performed at end-diastole at a fixed distance from an anatomic marker at rest and 40, 60, and 120s after cuff release. After at least 15-min of rest, new baseline images were obtained before a 0.4mg NTG tablet was administered sublingually to assess endothelium independent dilation. Images were then captured and recorded at rest, and at 40, 60, 120, 360, 480, and 600s.
Offline image analysis was performed by a single trained researcher blinded to image sequence and individual subject's details. Arterial diameter was measured from the anterior to the posterior “m” line (the interface between media and adventitia) at end-diastole, incident with the R-wave on the electrocardiogram. Brachial artery vasodilator response to reactive hyperemia and NTG was calculated as the maximal percent change in brachial artery diameter (FMD: at 1-min; NTG: at 4-min) from baseline. The coefficient of variation was 6.9% for FMD measurements.
Blood sampling and assays. Following a 12-h overnight fast, blood samples for hsCRP and TNF-α analysis were drawn into EDTA tubes. Samples were centrifuged at 3,000rpm for 20-min at 4°C. Plasma samples were then transferred to plastic micro-tubes and stored at −80°C until assay.
Plasma EDTA samples for hsCRP were sent to Quest Diagnostics as per the standard instructions. TNF-α levels were measured using a commercially available Kit (Pierce, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. The sensitivity of the assay kit was <2pg/ml. Inter and intra-assay coefficients of variation were 5.9% and 7.1%, respectively. Absorbance for the assay was read on an ELISA plate reader (Spectra Max Microplate Reader Molecular Devices, Sunnyvale, CA), set at 450nm and 550nm. The absorbance values obtained at 550nm values were subtracted from at 450nm values to correct for optical imperfections in the microplate.
Statistical analyses. Continuous variables are summarized as mean ± s.d. Categorical variables are described in terms of frequencies and percentages. The distribution of all variables was examined using the Shapiro–Wilke test of normality, and homogeneity of variances was determined using Levene's test. We used two sample t-tests to determine statistical difference and compared baseline subject characteristics, ABPM parameters, and FMD characteristics between true-prehypertension and masked-hypertension groups and sub-groups. Regression analysis was performed and linear regression models were applied to examine whether inammatory markers were related to FMD% after adjusting for age, BMI, and 24-h mean BP. The relationship between each clinical and BP variable was tested using univariate and multivariate regression analyses.
Results
In this cohort of relatively healthy African Americans (N = 50), we identified prehypertension in 41 of 50 subjects. Amongst the 41 subjects with prehypertension, we identified 24 subjects with masked-hypertension, and 17 subjects with true-prehypertension through ABPM. Parameters for ABPM for the two groups are reported in Table 1. Student t-tests revealed no significant differences between the true-prehypertension and masked-hypertension groups for any of the ABPM parameters except for the daytime SBP which was higher in subjects with masked-hypertension (P = 0.04).
Baseline BP measurements among true-prehypertensive and masked-hypertensive groups
Baseline BP measurements among true-prehypertensive and masked-hypertensive groups
Next, we assessed brachial artery endothelial function in a subset of 28 subjects with prehypertension using brachial artery FMD. Within this subset, 11 subjects had true-prehypertension and 17 subjects had masked-hypertension. Demographic characteristics of the subjects who underwent brachial artery FMD study are reported in Table 2. Between the two sub-groups, there was no difference in age, BMI, office-BP measurements, serum creatinine, lipid profile, or blood glucose. Parameters for ABPM for the two sub-groups are reported in Table 3. There was no statistical difference between the sub-groups; true-prehypertension and masked-hypertension for any of the ABPM parameters except for daytime SBP, which remained higher in subjects with masked-hypertension (P = 0.04). FMD characteristics for the two sub-groups (true-prehypertension and masked-hypertension) are reported in Table 4. The two sub-groups differed in FMD%, with the masked-hypertension sub-group having significantly lower FMD% than the true-prehypertension sub-group (6.5 ± 4.0% vs. 8.4 ± 3.8%; P = 0.03) indicating diminished endothelial-dependent dilation in the masked-hypertensives. Between the two sub-groups, there was no statistical difference for the baseline brachial artery diameter, pre-NTG baseline brachial artery diameter or percent change in brachial artery after the administration of NTG.
ABPM parameters among true-prehypertensive and masked-hypertensive subjects with FMD
ABPM parameters among true-prehypertensive and masked-hypertensive subjects with FMD
Descriptive statistics among true-prehypertensive and masked-hypertensive sub-groups on whom FMD study was performed
Descriptive statistics among true-prehypertensive and masked-hypertensive sub-groups on whom FMD study was performed
For the inflammatory markers, WBC count, TNF-α, and hsCRP, no significant differences were found between the true-prehypertension sub-group and masked-hypertension sub-groups except that, the masked-hypertension sub-group had a significantly higher hsCRP (3.9 ± 3.1 vs. 2.4 ± 2.1mg/l; P = 0.04) compared to the true-prehypertension sub-group as shown in Table 3.
Thus, the masked-hypertension sub-group had significantly lower FMD% (P = 0.03) and significantly higher hsCRP (P = 0.04) compared to true-prehypertensives. The mean hsCRP for the masked-hypertension group was 3.90mg/l which falls within the high CV risk category.19 On further analysis, the hsCRP inversely correlated with FMD% amongst the masked-hypertensive sub-group (R2 = 0.160; P = 0.04). The relationship remained statistically significant after adjusting for confounding factors such as age BMI and 24-h SBP. TNF-α and WBC count did not significantly correlate with FMD%.
Discussion
Diagnosis of masked-hypertension would be achievable only if ABPM is performed routinely, which explains why a large portion of the population with masked-hypertension is still under-diagnosed. At present, most studies were performed on subjects who were on medications and very few studies have reported data in essentially untreated subjects with masked-hypertension.5,6,12,20,,,,–25 In the African-American Study of Kidney Disease and Hypertension (AASK) Cohort study, a prospective observational study of African Americans with chronic kidney disease, 70% of subjects with controlled clinic BP were identified as having masked-hypertension. The prevalence of masked-hypertension in the general population has been reported to be ~10%, thus the 70% prevalence of masked-hypertension in African Americans reported in the AASK Cohort study is strikingly high, and warrants further investigation as it's likely that the underlying chronic kidney disease in the AASK Cohort study could have influenced their study findings.13 To the best of our knowledge, the present study is the first to evaluate the prevalence of masked-hypertension in a cohort of African Americans without overt clinical disease.
Our study showed that masked-hypertension was prevalent in 58% (24 of 41) of prehypertensive African-American subjects. To confirm that the identification of masked-hypertension in our cohort represents a true pathological state, and is not an artifact of ABPM, we assessed endothelial function and inflammatory markers in a subset of our cohort. The masked-hypertension sub-group had a significantly lower FMD response indicating that endothelial-dependent dilation was diminished in the masked-hypertensive sub-group. There was no difference in endothelial-independent dilation (NTG-mediated) between the two sub-groups indicating that the vascular smooth muscle responsiveness and function were similar.
Taken together, our finding of similar endothelial-independent dilation between the two sub-groups in conjunction with diminished endothelial-dependent dilation in the masked-hypertensive sub-group are suggestive of reduced production and/or bioavailability of endothelial derived nitric oxide (NO) in the masked-hypertensive subgroup. To explain this, various mechanisms such as a generalized defect in vasodilator function, impaired NO-dependent relaxation which could be related to a reduced bioavailability of NO/NTG to the smooth muscle cells, a selective defect of vascular smooth muscle cell function, in particular of the soluble guanylate cyclase/cyclic guanosine monophosphate signaling system have been postulated.15,16 Moreover, for any given BP value, endothelial function is diminished, tends to occur early, is more severe and is associated with higher CV risk including premature onset of CV complications in African Americans when compared to their white counterparts.15,16 It is also possible that impairment of endothelial-dependent dilation which seems to occur early in the evolution of high BP, namely, in the prehypertension BP range, may in fact be representative of a more advanced pathological process within the vasculature of African-American subjects with masked-hypertension.
Mean hsCRP level in the masked-hypertension sub-group was >3.0mg/l, which falls within the high relative CV risk category. Using regression analysis, we also showed that there was a modest but significant negative relationship between hsCRP and FMD%. This suggests that the vascular endothelium contributes to, and is affected by the systemic inflammatory process.21 Previously, we have shown that African Americans tend to have a subclinical heightened state of systemic inflammation in the vasculature as assessed hsCRP12 which can activate endothelial cells and reduce the bioavailability of endothelium-derived NO.21 Thus, we speculate that low-grade systemic inflammation may likely influence the diminished endothelial function in African Americans with masked-hypertension; however the present study cannot demonstrate a cause–effect relationship.
There are probable reasons that might be responsible for the occurrence of masked-hypertension in our relatively small cohort of African-American subjects. We considered both daytime and night-time ABPM values to define masked-hypertension in our cohort.13 Our cohort consisted of predominantly women (~80%), who have been shown to have a higher prevalence of masked-hypertension.3,22 In addition, masked-hypertension may also be more common in subjects with prehypertension.4,9 Obesity also likely plays an important role in the phenomenon of masked-hypertension.23,–25 In our study, the BMI for the masked-hypertensive sub-group was ~31kg/m2.
It must be noted that there are a number of limitations to our study. First, our sample size is relatively small and this is due to our strict and extensive exclusion criteria. Many possible confounding variables that influence vascular function were well controlled by the experimental design. This is in sharp distinction to prior studies performed on subjects with masked-hypertension in African Americans and in the general population; wherein the disease process was fairly advanced, and in some studies subjects were on treatment and in others, the subjects had many complications.5,6,12,20,,,,–25 However, even with a relatively small sample size we still observed a statistically significant negative relationship between hsCRP and FMD%, due to a rather heightened inherent low-grade systemic inammatory status in this cohort of seemingly healthy African Americans. Second, our subjects consisted of African Americans who are at-risk for CV disease as indicated by hsCRP levels, and hence the results may not be completely representative of the general population. Third, only prehypertensives were considered for analysis. The rationale for selecting prehypertensives was based on the premise that, screening for masked-hypertension amongst all individuals presenting with normal clinic BP is impractical and cost ineffective. Also, detection of masked-hypertensives who are at-risk for CV disease within the prehypertensive population, may point towards a need for vigilant BP screening. Furthermore, we could promote vigorous lifestyle adjustments that are effective in favorably altering the cardio-metabolic profile, and therefore attempting to modify the CV risk. Fourth, it may seem that the high levels of hsCRP in this cohort may be consequential to the high BMI; however, in our analyses, we accounted for BMI along with the other potential confounding factors such as age and 24-h SBP. Fifth, only statistical differences and associations are reported. These associations potentially point to areas for investigating the underlying mechanisms. Of course, the results of our cross-sectional study need to be confirmed through longitudinal studies. Despite these limitations, we believe that our preliminary data are indicative of greater morbidity amongst a sub-group of prehypertensive African Americans.
In conclusion, in our preliminary study we identified masked-hypertension in 58% of our study cohort of prehypertensive African Americans. The masked-hypertensives exhibited higher levels of hsCRP suggesting an elevated systemic inflammation. Also, masked-hypertensives had a decreased FMD response indicating diminished endothelial-dependent dilation. These results suggest that amongst African Americans with prehypertension there exists a subset of masked-hypertensives in whom the CV risk is augmented. Importantly, the prevalence of masked hypertension in our study was strikingly high in comparison to previously reported prevalence data. Though this finding will need to be confirmed by larger studies, it may point towards a need for vigilant BP screening and out-of-office BP monitoring in prehypertensive African Americans to accurately estimate their CV risk.
This research was supported by NIH/NHLBI Grant RO1 HL085497 (PI, Michael Brown) and by NIH/NIA Grant KO1 AG019640 (PI, Michael Brown). The abstract was presented at the American Heart Association, Scientific Sessions 2010.
Disclosure:
The authors declared no conflict of interest.
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