¹Division of Gerontology, Baltimore Veteran Affairs Medical Center, Baltimore, Maryland, USA

²Division of Cardiology, Department of Medicine, University of Maryland School of Medicine and Baltimore Veteran Affairs Medical Center, Baltimore, Maryland, USA

Correspondence to: L I Katzel, Baltimore Veterans Affairs Medical Center, Geriatric Research, Education, and Clinical Center BT/18/GR, 10 North Greene Street, Baltimore, MD, 21201, USA. E-mail: Les@grecc.umaryland.edu

Objective: Recent studies indicate that abdominal fat accumulation, in particular intra-abdominal fat, is related to impaired endothelial function in young healthy volunteers. The aim of this study was to examine whether the distribution of body fat depots is related to impaired endothelial function in older men.

Methods: Cross-sectional sample of 38 older (68±1 y) sedentary (VO_2max=2.4±0.1 l/min) men. Flow-mediated endothelial dependent vasodilation (EDD) was assessed in the brachial artery in response to reactive hyperemia using high-resolution ultrasound. Abdominal subcutaneous and visceral fat depots were assessed by computed tomography scan (CT-scan) at the L₄-L₅ region in the supine position. Percentage body fat was assessed via dual-energy X-ray absorptiometry (DEXA).

Results: Flow-mediated percentage change in brachial artery was 7.6±0.7%, suggesting an impaired flow-mediated EDD. Using simple linear regression analysis, there were no statistically significant relationship observed between flow-mediated EDD and the indices of total and abdominal adiposity (percentage body fat=29.3±0.9%, r=-0.11; total abdominal fat area=465±23 cm², r=-0.1; intra-abdominal fat area=200±14 cm², r=-0.14; subcutaneous fat area=265±13 cm², r=-0.05; BMI=29.3±0.9 kg/m², r=-0.07; and waist to hip ratio=0.98±0.01, r=-0.20).

Conclusion: These findings suggest that in older sedentary men there is no clear correlation between adiposity and body fat distribution and impairment of flow-mediated endothelium dependent vasodilation.

International Journal of Obesity (2002) 26, 663-669. DOI:10.1038/sj/ijo/0801972

endothelial function; vasodilation; obesity; abdominal fat

Introduction

Dysfunction of endothelium-dependent vasodilation is perceived as an early marker for developing atherosclerosis.¹ Nitric oxide (NO) is a major relaxing factor whose production and release have a net effect of inhibiting several components of the atherogenic process by affecting vascular smooth muscle migration and proliferation, platelet adhesion, and lipid peroxidation.¹ Consequently, factors that decrease NO production and bioavailability may lead to the development of atherosclerosis.

Impaired endothelial function is evident in young obese individuals and the distribution of body fat depots, in particular visceral type obesity, is strongly associated with the decrease in endothelial dependent vasodilation.^2,3,4,5,6 These results suggest that the increased risk of cardiovascular disease in obese subjects^7,8,9 could be linked to endothelial dysfunction through the mechanism of a decrease in NO production or bioavailability.¹

Because of the associations between endothelium function, obesity and fat distribution in younger individuals, it is conceivable that the increased total and abdominal body fat content with aging may account for some of the impairment in endothelium-dependent vasodilation.^{10,11,12,13,14,15} Therefore, this study examines whether total fat and the distribution of body fat depots may be related to impaired endothelial function in older men. We hypothesize that the decrease in endothelial function with age is related to total fat and the distribution of body fat depots.

Methods

Subjects

The University of Maryland Institutional Review Board approved this study and all patients signed informed consent. Healthy community dwelling older men were recruited via media advertisements for cross-sectional, weight loss and exercise intervention studies. At the initial visit, the subject underwent a screening evaluation that included a medical history questionnaire, physical examination, and fasting blood profile to identify diabetes (fasting blood glucose>6.4 mmol/l), cancer, liver, renal, hematologic or other metabolic disorders. An exercise treadmill test was also performed to exclude individuals with evidence of cardiac disease. Other exclusion criteria included: (1) a history of coronary artery disease, peripheral vascular disease or stroke; (2) poorly controlled hypertension (systolic blood pressure>180 and/or diastolic blood pressure>100 mmHg; (3) severe dyslipidemia (triglycerides>4.5 mmol/l, and/or low-density lipoprotein (LDL) cholesterol>4.9 mmol/l); (4) morbid obesity (body mass index, BMI>40 kg/m²); and (5) current smokers. Women were also excluded to avoid the confounding effects of gender on body fat distribution⁷ and endothelial function.^2,16 Data is reported in 38 elderly (68±1 y, mean±s.e.m.) sedentary (VO_2max=2.4±0.1 l/min) men.

Body composition

Anthropometrics: Weight (kg) and height (cm) were measured to calculate BMI (kg/m²). Waist circumference (measured at the narrowest point on the abdomen) was divided by the hip circumference (measured at the greatest gluteal protuberance of the buttock) to obtain the waist/hip ratio (WHR).

Dual-energy X-ray absorptiometry: Dual-energy X-ray absorptiometry (DEXA; model DPX-L; Lunar Radiation Corporation, Madison, WI, USA) was used to determine percentage body fat, fat mass, and fat-free mass (calculated as total bone mineral content plus lean tissue mass).

Computed tomography (CT): CT scan of the abdomen was performed to quantify visceral and subcutaneous fat areas using a PQ6000 scanner (Marconi Medical Systems, Cleveland, OH, USA). A single 5 mm scan was taken at the L₄-L₅ region with the subject in a supine position and arms stretched above the head. To quantify the relative portions of abdominal visceral and subcutaneous adipose tissue areas, a fat tissue highlighting technique was used. Hounsfield units (HU) of -190 to -30 were used to detect the cross-sectional area of adipose tissue in cm².¹⁷

Measurement of cardiac risk factors

Oral glucose tolerance test: Diabetic subjects did not undergo the glucose tolerance test (diabetes was defined as a fasting blood glucose>7.00 mmol/l, or use of oral hypoglycemic agents.¹⁸ The remaining subjects underwent a 75 g, 2 h oral glucose tolerance test after a 12 h overnight fast. Blood samples were drawn every 30 min during the 2 h test into chilled tubes containing EDTA for measurement of plasma glucose concentrations. Plasma glucose concentrations were analyzed using the glucose oxidase method on a Beckman analyzer (Beckman Glucose Analyzer II, Beckman Instruments, Fullerton, CA, USA). Immunoreactive insulin was determined by radio-immunoassay with an insulin-specific antibody whose cross-reactivity with proinsulin is <0.2% (Linco, St Louis, MO, USA). Total glucose and insulin area under the curve were calculated by the trapezoidal method. Diabetes following the glucose tolerance test was defined as a 2 h value >11.1 mmol/l.¹⁸

Lipoprotein lipids: Lipoprotein lipid concentrations were determined from the average of two or three blood draws taken on different days after a 12 h overnight fast. Blood samples were collected into chilled EDTA tubes. Total cholesterol, HDL-cholesterol and triglycerides concentrations were measured in our laboratory as previously described.^19,20 LDL-cholesterol was calculated by using the Friedwald equation.²¹ In our laboratory, the inter- and intra-assay variability for the measurements of total cholesterol were 6.2 and 1.5%, of triglycerides were 7.6 and 2.6%, and of HDL-cholesterol were 9.2 and 2.7%, respectively. Dyslipidemia was defined as a HDL-cholesterol concentration <0.90 mmol/l, or a LDL-cholesterol concentration >4.14 mmol/l, triglycerides concentration >4.5 mmol/l use of lipid lowering medications.

Blood pressure: Blood pressures were measured in both arms in the supine position after a 10 min rest period using a Dinamp. The reported values are the mean of two or three measurements made on separate days with patients taking their regular medications. Hypertension was defined as a systolic blood pressure 140 mmHg, or diastolic 90 mmHg on two or more occasions, or use of anti-hypertensive medications.

Treadmill testing

Maximal aerobic capacity (VO_2max) was measured using a modified Balke protocol exercise treadmill test as previously described.²² Peak volumes of oxygen, carbon dioxide, and ventilation were obtained every 20 s using a Sensormedics 2900 (Yorba Linda, CA, USA) metabolic cart. All VO_2max tests fulfilled at least two of the three the following criteria: (1) the heart rate at maximal exercise was >95% of the age-predicted maximal heart rate; (2) the respiratory exchange ratio was 1.10; and (3) the VO₂ reached a plateau during the final stage of exercise, ie the increase in VO₂ was <0.2 l/min during the final increase in work load.

Endothelial reactivity

Flow-mediated endothelium-dependent vasodilation (EDD) of the brachial artery (endothelial function) and Doppler blood flow velocity were obtained in the left arm, approximately 5 cm above the antecubital fossa using a linear array broad-band frequency (7.5-11 MHz) transducer attached to an ATL Apogee 800 ultrasound system (Advanced Technology Laboratories Inc., Bothell, WA, USA) using the method of Corretti.²³ Vasoactive drugs were withheld the morning of the test. Following baseline measurements a reactive hyperemic stimulus was performed by inflating an upper arm blood pressure cuff (12.5 cm width) to an occlusive pressure between 180 and 200 mmHg for a duration of 5 min. The blood pressure cuff was then deflated to 0 mmHg and brachial arterial diameter and flow velocity were recorded 1 min after cuff deflation. Hyperemic velocity was recorded upon immediate blood pressure cuff release. Ultrasound images of end-diastolic frames were obtained for off-line analysis on the same ultrasound system, and arterial diameter and blood flow velocity were subsequently measured. Arterial blood flow was measured as mean Doppler flow velocity integrated over five consecutive cardiac cycles for each time point multiplied by the cross-sectional area of the artery (Diameter²/4). The intra-observer coefficients of variation for brachial artery diameter and blood flow velocity as measured in our laboratory are 1.9 and 9.9%, respectively.²³ The correlation coefficient between paired measurements were 0.912 for brachial artery diameter and 0.984 for flow velocity measurements.²³

Statistics

Data was entered in a customized relational database and transferred to Statview (Abacus Concepts, Berkeley, CA, USA) for analysis. The distribution of all variables was tested for normality. Relations between variables were determined by linear regression analyses and calculations of Pearson correlation coefficients. Statistical significance was set as P<0.05. Data are presented as mean±standard error of the mean.

Results

Subject characteristics are presented in Tables 1 and 2. Overall, the men were obese (BMI=29.3±0.9 kg/m²; WHR=0.98±0.01) and had a low aerobic capacity (VO_2max=2.4±0.1 l/min). Metabolic risk factors for cardiovascular disease were also prevalent in this study group despite the fact that the average values for total cholesterol (4.71 mmol/l), HDL (0.92 mmol/l), LDL (2.99 mmol/l), triglycerides (1.74 mmol/l), fasting glucose (5.5 mmol/l) and blood pressure (134/79 mmHg) were within the normal range. Therefore, 24 were hypertensive (12 of the men were on hypertensive medication), 22 had dyslipidemia (16 with low HDL, five on lipid lowering medications), six had diabetes (three with diabetic OGTT and three on oral agents), and nine had impaired glucose tolerance by OGTT.

The flow-mediated 1 min post-occlusion percentage change in brachial artery diameter (7.6±0.7%) and the absolute change in 1 min diameter (0.28±0.03 mm) were low in this sample of older men (Table 3), suggesting an impaired flow mediated EDD. By comparison, young controls studied in our laboratory have a mean percentage change in 1 min diameter of 11.5% and a mean absolute change of 0.45 mm.¹⁵

Pearson correlation demonstrated that there was no relationship between flow-mediated EDD and age (68±1 y; r=0.03), VO_2max (r=0.07), BMI (r=-0.07), WHR (r=-0.20) and percentage body fat (r=-0.11) in this group of older men (Figures 1,2 and 3). Additionally, despite a six-fold range in intra-abdominal fat area (64-386 cm²) flow-mediated EDD did not correlate with any of the measures of abdominal fat area (total abdominal fat area, r=-0.1; intra-abdominal fat area, r=-0.14; subcutaneous fat area, r=-0.05; Figures 4,5 and 6). There was a significant relationship between glucose area under the curve obtained from OGTT and flow-mediated EDD (r=-0.39; P=0.02) in this group of older men. However, the other cardiovascular risk factors such as fasting plasma glucose and insulin, systolic blood pressure, diastolic blood pressure, total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides did not correlate with flow-mediated EDD (r=-0.16-0.32; Table 4).

Discussion

A decline in endothelial function is evident with advancing age.^11,13,14,15 The decline may be due to age-associated increases in adiposity, decline in physical activity and/or the presence of cardiac risk factors rather than age per se. This study investigates whether the distribution of body fat depots is related to the impairment in endothelium function in older men. These data demonstrate that in older, sedentary men with cardiac risk factors, visceral and subcutaneous abdominal fat areas are not related to flow mediated endothelium-dependent vasodilation. In addition, cardiovascular risk factors such as systolic and diastolic blood pressure, and fasting concentrations of cholesterol, HDL-cholesterol, LDL-cholesterol, insulin and glucose did not correlate with endothelial dysfunction.

With respect to overall obesity, Steinberg et al⁶ reports a 40-50% reduction in endothelium dependent vasodilation in young obese individuals with and without type 2 diabetes compared to lean control subjects. However, various studies indicate that the distribution of body stores rather than obesity per se results in impaired endothelial function.^2,3,4,5 To this effect, Hashimoto et al⁵ report a significant relationship between indices of body fat distribution and impaired endothelium function in young and middle-aged men and women. In that study, flow-mediated vasodilation was significantly impaired in subjects characterized with visceral obesity compared with non-obese subjects and obese subjects with subcutaneous obesity. Arcaro et al² also concur that in obese pre-menopausal women free of diabetes, hypertension and dyslipidemia, flow-mediated endothelial-dependent vasodilation was inversely correlated with the visceral to subcutaneous adipose tissue ratio, and not with body weight, height or blood pressure.

However, our data surprisingly failed to show any significant correlation between flow-mediated endothelium-dependent vasodilation and any measure of body composition such as subcutaneous and visceral abdominal fat area, waist-to-hip ratio, percentage body fat, and body mass index. This may be due in part to the range of adiposity in this group of older men. Some of the other studies that demonstrated a relationship between adiposity and endothelial function recruited younger subjects with morbid obesity to extend the range of adiposity.^2,4,5,6 Despite the fact that the BMI range (24-34 kg/m²) in the present study encompasses typical values observed in sedentary, older men, it is important to note that within our group of older men, in spite of an six-fold range of intra-abdominal fat area as assessed by CT scan, there was no relationship between abdominal adiposity and endothelial function.

At the present moment, we have no definitive explanation for the discrepancies in the results from the present data and the other studies that report a significant relationship between body composition and endothelial function.^2,3,4,5,6 It is possible, however, that primary aging may in part account for our findings. Various studies document that chronological aging is associated with progressive loss of flow-mediated dilation in systemic arteries of healthy subjects with no risk factors for cardiovascular disease.^11,13,14,15 Healthy adult subjects who did not possess any confounding risk factors for atherosclerosis (such as diabetes, hypertension and hyperlipidemia), showed that endothelium-dependent vasodilation in forearm resistance vessels declined progressively with increasing age with the infusion of methacholine chloride.¹³ In addition, vasodilation to acetylcholine, an endothelium-dependent relaxant agent, decreased with advancing age in the forearm of both normotensive control subjects and patients with essential hypertension.¹¹ Similar impairment of endothelium function with advancing age was demonstrated with reactive hyperemia. One study reports that flow-mediated dilation was preserved in men less than 40 y but declined thereafter at a rate of 0.21% per y.¹⁴ Comparing a group of men older than 40 y with younger subjects. Corretti et al¹⁵ demonstrated that among normal subjects with no cardiac risk factors, percentage diameter change in response to a flow-mediated stimulus was less in older men than in young men (6.8 vs 11.5%). It is important to note that, although our subjects had cardiovascular risk factors, the abnormality with the endothelium in previous studies was present in healthy older adults with no risk factors. These results suggest that advancing age appears to be an independent factor that causes progressive alterations of endothelium-dependent vascular activity.

Various factors may explain the mechanisms responsible for the age-associated impairment of endothelial function. Possibilities include an age-related decrease in the release and production of endothelium derived relaxing factors such as NO, increased accumulation of glycosylated end products that reduce the activity of NO, or increased production of endothelium derived constricting factors.^1,24 During reactive hyperemia, vasodilation of the brachial artery is partly dependent on the release of endothelium-derived nitric oxide.¹ Therefore, impairment in the vasodilatory effect may be due to either diminished released NO from the endothelium or decreased action of NO at the vascular smooth muscle site. Tschudi et al²⁵ showed that the initial rate of release of NO and peak NO concentration was reduced in rat aorta of middle-aged and old rats compared to young rats. The authors concluded that, since NO has a very short half-life, a smaller amount of NO produced coupled with a slower rate of release will reduce the biological effect of NO on the vascularture with aging.²⁵ Therefore, it is conceivable that the aging human vascularture is similar to animals and that subjects with impaired endothelium function may also have a decrease in NO production. That abnormal endothelium-dependent vasodilation to agents that stimulate the release of endothelium-derived NO is observed in older individuals supports the theory that aging per se does affect endothelium function.^11,13 Oxidative stress may also be considered a mechanism by which endothelial function is impaired in aging humans. Intrabrachial artery infusion of vitamin C significantly increased the vasodilatory response to acetylcholine in obese subjects.²⁶ Flow-mediated vasodilation was also improved in smokers and non-smokers with impaired glucose tolerance with vitamin C infusion.²⁷ Although we did not measure humoral marker of oxidative stress, we speculate that increased reactive oxygen species may be one of the mechanism affecting endothelium function in the present study group.

There are some methodological limitations to this study that need to be addressed. First, as noted above, despite the wide range of BMI (10 units), we did not have substantial numbers of lean subjects (percentage body fat<20%). In our experience, very few non-smoking older individuals who are not trained athletes or exercises regularly, have a percentage body fat<20%. We previously reported enhanced endothelial function in older, lean master athletes compared to sedentary older men (8.9 vs 5.7%; P=0.02).²⁸ It is conceivable that, because the lean athletes were highly trained, the increased fitness and decreased body fat accounted for the difference in endothelial function compared to their sedentary, more obese peers. Therefore, inclusion of lean athletes and morbidly obese men could have affected the relationship between adiposity and endothelial function in the present population. Secondly, unlike other studies^2,5 that specifically recruited healthy subjects with uncomplicated obesity, this present group of individuals possessed a variety of well-controlled cardiovascular risk factors (such as hypertension, hyperlipidemia, metabolic insulin resistance) that are associated with endothelial dysfunction that could have masked the effect of obesity per se in this particular group of older men. However, there was no relationship between flow mediated endothelium function and the major cardiovascular risk factors in this sample of older men, probably because the cardiac risk factors were not as severe as those documented in previous studies. As reported in the present study, the overall values for cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, blood pressure, glucose and insulin were within the normal range. These results may suggest that aging itself played a more significant role in endothelium function in the present study. Due to the sample size and age range of the population, we were unable to address the interaction between aging, body composition and flow-mediated endothelial-dependent vasodilation. Therefore, the question of whether obesity in and of itself affects endothelial function may only be addressed in large cross-sectional studies that encompass subjects across a wide age span with and without cardiac risk factors or longitudinally following interventions that reduces body weight and fat mass.

In conclusion, the results of this study demonstrate that in older sedentary men with cardiovascular risk factors, total and abdominal adiposity did not account for the impairment in endothelium-dependent vasodilation. The contribution of primary aging, the presence of hypertension, dyslipidemia, insulin resistance, impaired NO release and activity, and free radical production to impaired endothelium-dependent vasodilation in the elderly remains to be determined.

Acknowledgements

We would like to thank the patients who participated in this study. We would also like to thank the staff at the Department of Veterans Affairs Baltimore Geriatric Research, Education and Clinical Center for screening and recruiting. This work was supported by the Department of Veteran Affairs Baltimore Geriatric Research, Education and Clinical Center, Baltimore, Maryland; the University of Maryland Claude D Pepper Older Americans Independence Center (NIA P60 AG12583), a K24 mid-career development grant (K24 AG00930), an NIH training grant (T32 AG00219), and by a Special Emphasis Research Career Award (NIA K01 AG00657).

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Figure 1 Linear regression analysis of the relationship between body mass index (kg/m²) and endothelial function. Endothelial function is reported as the percentage change in the brachial artery diameter after reactive hyperemia (r=-0.17; P=NS).

Figure 2 Linear regression analysis of the relationship between waist-to-hip ratio and endothelial function. Endothelial function is reported as the percentge change in the brachial artery diameter after reactive hyperemia (r=-0.20; P=NS).

Figure 3 Linear regression analysis of the relationship between percentage body fat (%) and endothelial function. Endothelial function is reported as the percentage change in the brachial artery diameter after reactive hyperemia (r=0.12; P=NS).

Figure 4 Linear regression analysis of the relationship between total abdominal fat area (cm²) and endothelial function. Endothelial function is reported as the percentage change in the brachial artery diameter after reactive hyperemia (r=-0.11; P=NS).

Figure 5 Linear regression analysis of the relationship between subcutaneous abdominal fat area (cm²) and endothelial function. Endothelial function is reported as the percentage change in the branchial artery diameter after reactive hyperemia (r=-0.05; P=NS).

Figure 6 Linear regression analysis of the relationship between intra-abdominal fat area (cm²) and endothelial function. Endothelial function is reported as the percentage change in the brachial artery diameter after reactive hyperemia (r=-0.14; P=NS).

Table 1 Physical characteristics and body composition measures of the 38 older men

Table 2 Cardiac risk factors of the 38 older men

Table 3 Flow-mediated endothelial function measurements of the 38 older men

Table 4 Correlation analysis between flow-mediated endothelial-dependent vasodilation and various cardiovascular risk factors in 38 older men