Abnormalities in circadian blood pressure variability and endothelial function: pragmatic markers for adverse cardiometabolic profiles in asymptomatic obese adults

Disease-free (or otherwise healthy) obese men and women with no co-morbidities, screening for a weight loss study at the Outpatient Clinic, Pennington Biomedical Research Center (PBRC), were offered an opportunity to have 7-day automatic ambulatory blood pressure (BP) monitoring and endothelial function assessment. The Pennington Biomedical Research Center is a campus of the Louisiana State University System and conducts basic, clinical and population research. The research enterprise at the Center includes 80 faculty and more than 40 post-doctoral fellows who comprise a network of 57 laboratories. These are supported by lab technicians, nurses, dieticians, and support personnel, and 19 highly specialized core service facilities. The Center's nearly 600 employees occupy several buildings on a 234-acre campus.

Each candidate was provided with the PBRC Institutional Review Board approved informed consent and followed standard consenting procedures. Fifteen obese adults agreed to participate in the automatic ambulatory BP monitoring and endothelial function assessment. Demographic and anthropometric information along with a resting EKG were obtained, followed by a comprehensive medical history and a complete physical examination. The initial visit ended with teaching and demonstration of the use, of an automatic blood pressure monitor for ambulatory use. Upon completion of 7-day ambulatory BP monitoring, a fasting blood draw, and a resting endothelial function assessment were done. Comparison groups of 10 disease-free normal-weight, and 10 disease-free overweight adults were obtained from other clinical trials. They also consented for automatic ambulatory BP monitoring and endothelial function assessment and were processed in a similar fashion.

Healthy (disease-free) normal-weight, overweight and obese (n = 35) non-smoking men and women between the ages of 30-75 years, with no personal history of or ongoing treatment for any chronic medical conditions.

Apparently healthy men and women between the ages of 30-75 years, with no history of DM, HTN, DysL, MetS, and/or CVD were included. Adults with a personal history of or ongoing treatment for diabetes, hypertension or any other chronic cardiac, renal, gastro-intestinal, pulmonary or any other systemic disease process requiring chronic intake of prescription medications, were excluded.

An automatic BP monitoring device (Spacelabs^® Medical) for ambulatory use was attached to a BP cuff to obtain BP and heart rate (HR) readings at 30-min intervals during the day (6:30 AM to 9:30 PM) and 60-min intervals at night (10 PM to 6 AM) while the participants went about their activities. Data were downloaded into the database about mid-way and at the end of the 7-day recording span.

Assessment of resting endothelial function was done in a fasting state, after having avoided stimulants for 12 hours, at the same fixed clock hour (range 8-10 AM), using a Toshiba^® brachial ultrasound device. This device uses 7.5 MHz multi-frequency linear array transducer and a MIA Vascular Tools Brachial Analyzer Version 5.8.1 to determine brachial artery diameter. This technique has been previously validated and is in use with our clinical core. After obtaining reference (resting) brachial artery diameter measures, a forearm BP cuff was used to occlude the brachial artery for 5 minutes. The increase in brachial artery diameter (over the reference measure) due to the flow-mediated dilatation of the brachial artery after the release of the occlusion, served as a measure of endothelial function. One subject from each group (normal-weight, over-weight and obese without circadian blood pressure variability abnormality) had a measure of endothelial function. One obese subject with each of the circadian variability abnormality (M-hypotension, CHAT and EPP) also had endothelial function assessment.

These non-invasive devices used a BP cuff that is inflated and released to obtain BP readings and data on endothelial function. The automatic ambulatory BP device attached to a BP cuff under the clothes was placed on the belt or carried in a pouch. Repeated measurements of BP and HR at timed intervals over 7 days, while the participant was at work or at home, allowed for acclimatization to the minimal discomfort, which is similar to having BP measured. Endothelial function was also obtained by using a BP cuff, coupled with an ultrasound probe that measures brachial artery diameter at rest (reference), during occlusion and the post-occlusion increase after release.

Standard demographic and anthropometric measures were obtained for all the participants. Waist circumference (a surrogate marker for central adiposity), serum hs-CRP and fibrinogen (for the assessment of systemic inflammation), fasting serum glucose and HbA1C (for the assessment of glycemic status) and fasting complete lipid profile (for the assessment of serum lipid sub-fractions, and obtaining cardiac risk ratios) were obtained.

Participants were placed into normal weight (BMI < 25 kg/m²) overweight (BMI between 25-29.9 kg/m²) and obese (BMI > 30 kg/m²) categories. Normoglycemia and prediabetes, for the purpose of CVD risk assessment, were defined as a fasting serum glucose less than 100 mg/dL and a fasting serum glucose of more then 100 mg/dL but less then 126 mg/dL, respectively (impaired fasting glucose or IFG: ADA diagnostic criteria [26]). The diagnosis of prehypertension was based on resting (after a 5-minute rest) mean of (two successive assessments 1 minute apart) clinic BP measures of systolic (S) BP > 120 but < 139 and/or diastolic (D) BP > 80 but < 89 mm Hg (JNC 7 criteria [27]). Premetabolic syndrome was defined using the diagnostic criteria for the metabolic syndrome (NCEP ATP III criteria), with the substitution of prediabetes and prehypertension criteria for glucose and BP measures.

ABPM data gathered at the PBRC were electronically sent to the Halberg Chronobiology Center, University of Minnesota for statistical analysis. Endothelial function data were analyzed at the PBRC and then merged with the ABPM data sent back from Dr. Halberg's laboratory. A summary in time (sphygmochron) was prepared that reported, among other measures, the midline-estimating-statistic of rhythm (MESOR or M), the timing of high values (acrophase), and the extent of predictable change within a day (double circadian amplitude) for SBP, DBP and HR. Normal circadian BP variability for SBP and DBP over a 24-hour span is shown in Figure 1. Seven-day monitoring ensures the consistency of either normality or abnormality over each one of the seven days and reports the average over the seven-day span.

MESOR Hypotension (M-Hypotension) or MESOR Hypertension (M-Hypertension) in this system is defined as a BP MESOR below the lower 5% or above the upper 95% prediction limit of peers matched by age and gender. M-Hypertension for SBP and DBP is shown in Figure 2.

Excessive circadian BP excursion (circadian hyper amplitude tension or CHAT) is a circadian double amplitude for SBP and/or DBP above the upper 95% prediction limit for peers. BP ecphasia is defined as an acrophase (timing) for BP (but not HR) occurring outside of the anticipated 90% prediction limits. HR variability is deficient (DHRV) when the standard deviation (SD) of HR is < 7.5 bpm. Pulse pressure is elevated (EPP) when it exceeds 60 mmHg. DHRV and EPP are shown in Figure 3 and Figure 4, respectively.

Day-night ratios were also computed for the 7-day record as a whole and for each day separately, for a classification in terms of "dipping". Due to the normal daytime BP being more than the nighttime BP, a negative day-night ratio was classified as "reverse dipping". A positive day-night ratio between 10% and 20% was classified as "dipping", whereas a ratio of less than 10% or greater than 20% was classified as "non-dipping" or "excessive dipping", respectively.

The occurrence of any of the above abnormal circadian patterns of BP and/or HR, overall and for each day separately, in terms of circadian characteristics and in terms of the day-night ratio, was determined for each individual in the three groups (normal-weight, overweight and obese). Serial measurement over 7 days ensured the consistency of either normality or abnormality. The groups were compared using an Exact Test or Poisson regression for frequency of occurrence data and Analysis of Variance of quantitative measurements.

The effect of abnormal circadian BP variability upon untoward cardiovascular events is both separate and additive. Having more than one abnormality increases this risk: In a reference population of 214 patients (some with M-Hypertension) presenting with none of the 3 abnormal variability measures (decreased heart rate variability (DHRV), EPP, or CHAT), morbidity within a 6-year follow-up was found in 8 cases (3.7%). The presence of one abnormality (DRHV or EPP) alone raised the incidence of morbidity to 30.8%. When these two risk factors (DRHV and EPP) were both present, morbidity was doubled (66.7%). The presence of CHAT increased morbidity from 3.7% to 23.5% in the absence of the other two risk factors, from 30.8% to 50% when either DHRV or EPP was also present, or from 66.7% to 100% when all 3 risk factors are present [28]. Halberg et al. [29] have shown that even in the absence of conventional CVD risk factors (like DM, HTN, and DysL); abnormalities in circadian BP variability are risk factors for CVD and early death. Others have shown the superior predictive ability of ambulatory BP monitoring data vs. conventional office BP measures in adults with hypertension.

The endothelium is a highly active organ which regulates intravascular homeostasis by integrating numerous functions such as glycemia, blood pressure, pro-inflammatory/anti-inflammatory processes and coagulation. Endothelial dysfunction, the initial perturbation in the process of atherosclerosis, in asymptomatic individuals portends increased vascular disease risk [30]. Obese adults with abnormal circadian BP variability (M-Hypotension, CHAT, and EPP) also had endothelial dysfunction (flow mediated brachial diameter increase of 0.18(0.07), 0.12(0.08), and 0.13(0.05) mm over resting diameter or flow mediated dilation at only 78, 52, and 56% of the resting diameter, respectively, p <0.05). Abnormalities of both these measures: abnormal circadian BP variability (reflective of the functional aspects of the cardiovascular system), and endothelial dysfunction (reflective of a sum total of various effectors including glycemia, blood pressure, coagulation, pro-inflammatory and anti-inflammatory factors) in asymptomatic (disease free) obese adults are novel non-invasive methods for assessing the dynamic aspects of cardiovascular disease risk.

Gradual weight gain in clinically healthy overweight and obese adults is preferentially manifest as an enlarging waist circumference [31]. Abdominal obesity, clinically measured as an increased waist circumference and suggestive of an expanding visceral adipose tissue compartment, has an independent association with coronary heart disease [32]. Visceral adipose tissue, which strongly correlates with most metabolic risk factors [33], upon expansion, alters its usual and customary adipokine secretion menu [34]. There is an increased flux in the factors influencing the pro-inflammatory and renin-angiotensin-aldosterone system, in parallel with attenuation in the anti-inflammatory factors. The altered pro-inflammatory (increased) and anti-inflammatory (decreased) balance, among other reasons promotes insulin resistance [35]. Elevated HbA1c is related to new onset CVD over a relatively short follow-up period in both men and women without diabetes, who do not develop diabetes, even after adjustment for other major risk factors [36]. Dysregulated pro-inflammatory: anti-inflammatory balance, increased serum hs-CRP [37], total leukocyte count [38], serum uric acid [39], and decreased adiponectin [34] are associated with increased cardiovascular disease. Disease-free obese with an exacerbated proinflammatory milieu exhibit prediabetes and prehypertension [16]. Prediabetes is associated with abnormal circadian BP variability [15], prehypertension clusters with other CVD risk factors [40] and metabolic syndrome more strongly predicts CVD than its individual components [41]. An imbalance between the central and the peripheral clock mechanisms has recently been suggested as the cause for the endothelial function [42]. 24-hour ambulatory blood pressure measures have been used to predict target-organ disease and clinical outcome in patients with hypertension [43] and more recently, elevations in nocturnal BP have been shown to precede diabetic nephropathy in hypertensive patients with T2DM [44].

Overweight or obese adults with larger than normal waistline, along with subtle metabolic alterations, either with elevated FSG (prediabetes [26], elevated SBP and/or DBP (prehypertension [27]), and/or a combination of risk factors (premetabolic syndrome) are commonplace in routine clinical practice. These adults with unrecognized elevated CVD risk, more often than not, are lost to regular follow-up. This results in a lost opportunity for primary prevention of CVD. We believe that increased CVD in asymptomatic normal-weight, overweight and/or obese adults can readily recognized by the abnormal circadian BP variability, and endothelial dysfunction. Non-pharmacologic, as well as pharmacologic measures can be utilized to reverse these early abnormalities. Non-pharmacologic measures: a 7% weight loss from reference (improving glycemia), and increasing physical activity up to 150 minutes per week, (improving both glycemia and blood pressure) could be advocated. Pharmacologic measures: treatment with thiazolidinediones (reducing insulin resistance and pro-inflammatory milieu, along with remodeling of the adipose tissue), biguanides (reducing insulin resistance), angiotensin converting enzyme inhibitors or angiotensin receptor blockers (reducing blood pressure, improving glycemia and systemic inflammation) and HMG-CoA reductase inhibitors or statins (systemic inflammation, improving cardiac risk ratios and endothelial function) could be utilized.

The results from this study show that latent CVD risk in disease-free (healthy) obese adults assessed with no or low risk by conventional risk assessment methods, can be unmasked by simple non-invasive measures. The obese participants exhibiting normal circadian BP variability had normal endothelial function, normotension, normoglycemia and were within the desirable limits for systemic inflammation, triglycerides, HDL-C, and cardiac risk ratios. Asymptomatic obese participants with abnormal circadian BP variability and endothelial dysfunction also had: an increased visceral adipose tissue, a heightened pro-inflammatory profile, prediabetes, prehypertension and abnormal cardiac risk ratios. Abnormal circadian BP variability and endothelial dysfunction, taken together with the altered adverse cardiometabolic profile, are indicative of an unrecognized CVD risk in disease free obese men and women.

The study has several limitations that warrant discussion. The study participants were adult asymptomatic volunteers who were screening for various clinical studies at the Pennington Center, and may not be representative of the general population. Further, it is a cross-sectional study in which the temporal sequence of emergence of dysregulated assessments is unknown. Finally, the small sample size may have compromised the power to detect population differences among the normal-weight, overweight, and obese groups. Despite these shortcomings, this investigation documents statistically significant novel findings of a clinical correlation between circadian BP variability and endothelial function abnormalities and systemic proinflammation, prediabetes, prehypertension, elevated cardiac risk ratios, and establishes a foundation for further investigation of the underlying mechanisms.