Radical-Trapping Activity, Blood Pressure, and Carotid Enlargement in Women
The aim of this study was to evaluate the influence of traditional and nontraditional (oxidation markers) cardiovascular risk factors on the degree of adaptive response of the carotid wall to atherosclerotic disease, a process known as arterial enlargement. Five thousand sixty-two clinically healthy, middle-aged women living in the area of Naples participated in the “Progetto Atena” study; 310 of these women (potentially at higher atherosclerotic risk) underwent a high-resolution ultrasound scan of the carotid arteries. In addition to routine biochemical tests, these women had the determination of serum IgG antibody titer against oxidized LDL and measurement of thiobarbituric acid reactive substances and total radical-trapping activity potential of plasma. Age, systolic blood pressure, body mass index, and radical-trapping activity were all positively correlated with external and internal common carotid diameters, whereas triglycerides (positively) and HDL cholesterol (inversely) were related only to external diameter. After controlling for traditional cardiovascular risk factors, associations still persisted for age, systolic blood pressure, and plasma radical-trapping activity with external carotid diameters. However, in the quartile of women with highest total cholesterol (>7.38 mmol/L), the slope of the regression line between systolic blood pressure and external diameter was significantly flatter than in the three other quartiles (test for difference, P=0.014). Outward carotid enlargement is related to traditional and nontraditional risk factors and comes even before plaque development. Women with poor resistance to oxidative stress potentially have a difficulty to remodel their arteries in response to atherosclerotic stimuli.
Quantitative determination of carotid intima-media thickness (IMT) by high-resolution B-mode ultrasound has been shown to be strongly correlated with traditional cardiovascular risk factors and is an independent predictor of clinical events, for example, myocardial infarction and stroke.1–4 B-mode ultrasound has also been used to prospectively evaluate the natural history of carotid atherosclerosis by monitoring lesion growth over time and plaque stabilization resulting from medical interventions in clinical trials.5–7 However, studies performed with carotid ultrasonography have preferentially involved men because of the lower prevalence of atherosclerotic lesions in premenopausal women.
B-mode ultrasound imaging allows noninvasive study not only of carotid intima-media thickening and plaques but also the degree of adaptive response of the arterial wall to atherosclerotic disease, a process known as carotid enlargement. Although arterial enlargement has been demonstrated to have an important role in atherosclerosis, for example, preserving lumen diameter despite plaque growth,8–10 few studies focused on variation in carotid diameters in response to arterial wall thickening.11–13
A recent study demonstrated a site-specific association of traditional risk factors and oxidation markers with early atherosclerotic lesions of the carotid arteries.14
In this study, we examined a cohort of middle-aged women by using high-resolution B-mode ultrasound to measure arterial thickness parameters and carotid diameters, to have an estimate of carotid artery enlargement in response to wall thickening, and to evaluate the impact of traditional (blood pressure, body mass index, hyperlipidemia) and nontraditional risk factors (serum oxidative markers) on this process.
Five thousand sixty-two clinically healthy, middle-aged women living in the area of Naples participated in the “Progetto Atena” study.15 The general objective of this population-based survey was to investigate the causes of chronic diseases that have a major impact in the female population. Participants with previous diagnosis of myocardial infarction, stroke, and cancer were not included. Subjects with acute diseases affecting blood and urine biochemistry were invited to delay their participation, after the recovery of the acute illness. During a 6-month period, the older 3 participants (potentially at higher risk of carotid atherosclerotic lesions), among those scheduled for the day, were asked to undergo a high-resolution ultrasound scan of the carotid arteries and additional biochemical tests. Of the 400 invited women, 310 (response rate, 77%) accepted the additional investigation and constituted the present study sample. The study was approved by the ethics committees of the institutions involved, and all the subjects recruited gave informed consent.
Anthropometry, Blood Pressure, Questionnaires
Body mass index (BMI) was calculated according to the formula BMI=weight (kg)/[height (m)]2.
Sitting brachial blood pressure was measured by trained research assistants with a standard mercury sphygmomanometer 2 times, 2 minutes apart, a procedure similar to other epidemiological studies. A standard questionnaire was used to collect information about smoking status and the presence of diabetes.
Blood was taken in the morning after a fasting period of 12 to 14 hours. Serum and plasma samples were prepared in part to be immediately analyzed and in part to be stored in liquid nitrogen. Serum samples were processed for determination of cholesterol and triglycerides by enzymatic methods.16,17 HDL was precipitated by phosfotungstate.18 LDL cholesterol was calculated according to the Friedewald formula: LDL cholesterol=Total cholesterol−(Triglycerides/5−HDL cholesterol).
Serum IgG antibody titer against oxidized LDL was determined with the use of an enzyme-linked immunosorbent assay on microwells coated with Cu2+ oxidized LDL (ELISA, Biomedica o-LAB, Biomedica Gruppe). The error of the method was evaluated on both sera at low and high content of oxidized LDL and was <10%.
The measurement of thiobarbituric acid reactive substances (TBARS) was used as a screening method for lipid peroxidation. A modified thiobarbituric acid (TBA) test19 was used to minimize artifactual oxidative degradation of lipids during the assay. A description of the method has been published elsewhere.14
The total radical-trapping activity potential of plasma (TRAP), which is indicative of plasma antioxidant potential, was measured with the use of a spectrophotometric end-point method20,21 on a Cobas Fara centrifugal analyzer (Roche). The error of the method was evaluated by daily analyzing a plasma pool and was <5%. The method has been described elsewhere.14
High-Resolution Carotid Ultrasound
A Biosound 2000 II s.a. (Biosound Inc) was used to examine the extracranial carotid arteries. Examinations were performed by an experienced and certified sonographer following a standardized protocol developed by the Division of Vascular Ultrasound Research at the Wake Forest University School of Medicine. The segment under study was the distal common carotid artery defined as the 1-cm segment proximal to the beginning of the dilation associated with the carotid bifurcation. The protocol required the near and far walls of this segment to be visualized on both the right and left sides in a sequential manner, using anterior, lateral, and posterior scanning angles. Three arterial interfaces were associated with the near wall. In a longitudinal B-mode arterial image, the first arterial interface corresponds anatomically to the periadventitial-adventitial boundary (I), the second boundary is the adventitial-medial interface (II), and the third boundary is the intima-lumen interface (III). Similarly, three arterial interfaces were also demonstrated on the far wall. The first is the intimal-lumen interface (IV), the second is the medial-adventitial interface (V), and the third is the adventitial-periadventitial interface (VI).
The scanning protocol was specifically designed to measure internal and external arterial diameters. A 2-minute additional recording of the common carotid artery at the angle that allowed the best definition of all the interfaces was included. The distance between interfaces III and IV was used for internal diameter measurement, whereas the distance between interfaces II and V was used for measuring external diameter (Figure 1).
Scans were recorded on super half-inch VHS videotape and sent to the ultrasound reading center. At the Division of Vascular Ultrasound Research, all reading stations used the same computer hardware and software. The hardware allowed for frames to be chosen and digitized and the software allowed ultrasound images to be measured and stored together in a mainframe. The software used to process the ultrasound images was a morphometry program that enabled the reader to easily make many pairs of measurements from digitized ultrasound images of arteries.22 Certified ultrasound readers reviewed the examinations and made quantitative IMT and diameter measurements. Atherosclerotic plaques were considered as localized echogenic structures encroaching into the vessel lumen with an IMT >1.2 mm. Diameter measurements were made during the systole, that is, at their maximum over the cardiac cycle.
Continuously monitored quality control data derived from a large international multicenter trial being conducted during this time at the Division of Vascular Ultrasound Research show a coefficient of reliability for the common IMT of 0.85. This figure includes instrument, subject, sonographer, and reader variabilities.23 The coefficients of variation in replicate blind determination were 4.5% for the internal diameter and 2.2% for the external diameter.
Procedures of descriptive statistics were used to evaluate variable distribution. The Pearson coefficient and linear regression analyses were used to investigate the univariate and multivariate associations between carotid diameters and cardiovascular risk factors. Linear regression analysis was used to test the association between carotid diameters considered as dependent variables and IMT.
After the observation of the association between carotid wall thickness and diameters, ANCOVA was performed with the use of a general linear model approach to determine the association between traditional cardiovascular risk factors, oxidation markers, and carotid diameters. In these analyses, carotid diameters were considered the dependent variables, quartiles of traditional risk factors and of oxidation markers were considered the fixed factors, and carotid wall thickness was added as covariate.
To test the possible influence of high cholesterolemia on the carotid enlargement related to the blood pressure levels, we developed a statistical model that compared the different coefficients of a regression analysis of SBP on external diameters in 2 groups of women. The first group comprised the women in the lowest 3 quartiles of cholesterolemia and the second group comprised women in the highest quartile of cholesterolemia.24
We chose a probability value <0.05 for single comparison and applied the Bonferroni formula for multiple comparisons.25
All statistical analyses were performed using SPSS for Windows (release 10.0) statistical package for personal computers.
Age-adjusted cardiovascular risk profile in the population under study compared with the entire cohort of the ATENA project is described in Table 1. No statistically significant differences were detected between the sample of the present study and the entire cohort.
B-mode ultrasound variables in the sample are presented in Table 2. In univariate analyses, age, systolic and diastolic blood pressures (SBP and DBP), BMI, and TRAP were all positively correlated with external and internal common carotid diameters, whereas triglycerides (positively) and HDL cholesterol (inversely) were related only to external diameters. No correlation was found between glucose, total cholesterol, LDL cholesterol, antibody against oxidized LDL, lipid peroxidation, and carotid diameters (Table 3). In multivariate analyses, after controlling for traditional cardiovascular risk factors (age, blood glucose, blood pressure, total cholesterol, triglycerides, HDL cholesterol, BMI, cigarette smoking), a significant correlation was still found between external diameters and age (P<0.001), SBP (P=0.002), and TRAP (P= 0.02) and between internal diameters and SBP (P=0.004) and TRAP (P=0.004).
Carotid wall thickness showed a strong positive association with the external diameter (R=0.59, Figure 2), whereas a weaker relation was found with the internal diameter (R=0.37).
We decided to test the influence of the above-mentioned cardiovascular risk factors on carotid diameters, taking into account the influence of IMT. After adjustment for carotid wall thickness, associations with carotid diameters still persisted for SBP and TRAP (Table 4).
In particular, a statistically significant difference was observed between the means of the highest quartile of SBP versus the third, the second, and the lowest quartiles of both external (4th versus 3rd, P<0.01; 4th versus 2nd, P<0.001; 4th versus 1st, P<0.001) and internal diameters (4th versus 3rd, P<0.01; 4th versus 2nd, P<0.01; 4th versus 1st, P<0.001). All these differences were calculated after Bonferroni correction for multiple comparisons.
Since previous studies demonstrated an inverse relation between total cholesterol and carotid lumen diameters12,13 and we did not find a significant association between cholesterol levels and carotid diameters, we also tested for a possible influence of high cholesterolemia on the carotid enlargement related to the blood pressure levels.
In the quartile of women with the highest total cholesterol (>7.38 mmol/L; mean age, 57±0.7 years), the slope of the regression line between SBP and external diameter was significantly flatter than in the other three quartiles (total cholesterol, <7.38 mmol/L; mean age, 57±0.5 years), and the coefficients of regression were significantly different in the two groups (Figure 3).
In a subset analysis, women were divided into two groups: women with and women without plaques at the level of the common carotid arteries. In the first group, TRAP and SBP were associated with external and internal diameters, whereas BMI was associated only with external diameters (Table 5). In the group without common carotid plaques, only TRAP, SBP, and age reached statistical significance in the association with both external and internal diameters (Table 6).
The main findings of this study are that SBP (among the traditional risk factors) and the antioxidant capacity of the plasma (among the nontraditional risk factors) were associated with outward carotid enlargement. This association was independent of IMT and was present both in women with and women without plaques, suggesting that the adaptive response of the arterial wall to atherosclerotic stimuli comes even before plaque development.
It has been thought for several years that arterial stenoses were the end result of the progressive growth of atherosclerotic plaques leading to lumen obstruction. This model has been seriously questioned after the pathoanatomic demonstration in the coronary arteries of a process known as compensatory enlargement.9
It is possible to evaluate carotid artery enlargement in human beings by ultrasound B-mode imaging. The procedure involves quantitative measurement of the internal diameter of the artery, using intima-lumen interfaces and the external (interadventitial) diameter, making reference to media-adventitia interfaces. This methodology was adopted for evaluating young male subjects with homozygous familial hypercholesterolemia compared with healthy control subjects of comparable age and with normal serum cholesterol concentration.26 In patients with familial hypercholesterolemia, a severe and generalized intima-media thickening was demonstrated, with premature atherosclerotic plaques involving the common carotid arteries on both sides. However, the presence of atherosclerotic plaques did not lead to a significant reduction of the internal diameter but rather to an increase of the external diameter.
The association between common carotid arterial diameters and some traditional cardiovascular risk factors (ie, blood pressure, BMI, smoking) has been previously evaluated in few population-based studies. In the Atherosclerosis Risk In Communities (ARIC) study, hypertension, smoking, and diabetes were related to both larger external and internal common carotid diameters, whereas LDL cholesterol was associated with smaller carotid diameters.12 In the Etude sur le Vieillissement Arteriel (EVA) study, a positive association was found between both common carotid diameters and age, SBP and DBP, body weight, alcohol consumption, triglycerides, and blood glucose, whereas a weak negative association was observed between HDL cholesterol and external diameter and between LDL cholesterol and internal diameter.13 In a community-based study of adults ≥65 years of age, common carotid diameters were directly related to SBP and to echocardiographically determined left ventricular mass.14
In this study, the impact of traditional and nontraditional cardiovascular risk factors on carotid enlargement was evaluated by taking into account the influence of arterial wall thickness. Our results are partially consistent with those of previous studies,12,13 since blood pressure was associated with both external and internal diameter increase, whereas total cholesterol was not related to diameter increase. Interestingly, in the upper quartile of total cholesterol, there was a significantly reduced influence of SBP on carotid diameters as compared with the significant influence documented by the regression line in the other quartiles. It seems that higher cholesterol values (>7.38 mmol/L) inhibit the possibility of carotid arteries to further expand their diameters in response to a potent stimulator such as the systolic blood pressure.
Interesting data came from the evaluation of the relation between oxidation parameters and carotid diameters. Antioxidant capacity of the plasma (TRAP) was associated with an increase in arterial external diameter. This association was independent of traditional risk factors and persisted even after controlling for wall thickness. In particular, the lowest values of TRAP were associated with lack of carotid outward expansion. Since the total antioxidative potential of plasma reflects the ability of an individual to resist oxidative stress, it is conceivable that women with poor resistance to oxidative stress also have a resistance to remodeling the arteries in response to atherosclerotic stimuli.
The present finding complements the recent observation of an inverse association between both the intake amount and plasma concentration of vitamin E and preclinical carotid atherosclerosis in this sample of middle-aged women.27 A possible explanation for the lack of benefit in clinical trials studying the effect of antioxidant supplementation in patients with clinically overt cardiovascular disease may be that the vascular protection occurs at earlier stages of atherosclerosis. It could be that only individuals with inadequate intake or low plasma concentration of antioxidants are expected to benefit from an increase in antioxidant vitamin intake, either through dietary changes or supplementation.
In a previous study, we showed a site-specific association of atherosclerotic plaques in the carotid arteries of middle-aged women with traditional risk factors and oxidation markers.14 Common carotid plaques were associated with higher SBP, higher BMI, and higher titers against oxidized LDL. The present study demonstrated that the same traditional cardiovascular risk factors acted on common carotid diameters, whereas the main influence of oxidation parameters on common carotid enlargement was exerted by TRAP.
This study confirms a strong association of blood pressure with carotid diameters, recognizes an oxidation marker (TRAP) as a powerful determinant of carotid enlargement, and suggests that carotid enlargement accompanying high blood pressure may be partially counteracted by high cholesterol levels. Future interventional studies are needed to test whether the use of antioxidant vitamins and lipid-lowering agents may be beneficial to promote outward remodeling.
The “Progetto Atena” was supported by funds from the Consiglio Nazionale delle Ricerche (Rome, Italy), “Progetto finalizzato Biotecnologie,” and “Progetto finalizzato FATMA”; the Ministero dell’Università e della Ricerca Scientifica e Tecnologica, “Progetto Ricerche Interesse Nazionale,” 1997; and Fondazione Banco di Napoli.
- Received June 26, 2002.
- Revision received July 18, 2002.
- Accepted November 15, 2002.
Heiss G, Sharett AR, Barnes R, Chambless LE, Szklo M, Alzola C, ARIC Investigators. Carotid atherosclerosis measured by B-mode ultrasound in populations: associations with cardiovascular risk factors in the ARIC study. Am J Epidemiol. 1991; 134: 250–256.
Bots ML, Hoes AW, Koudstaal PJ, Hofman A, Grobbee DE. Common carotid intima-media thickness and risk of stroke and myocardial infarction. The Rotterdam study. Circulation. 1997; 96: 1432–1437.
Chambless LE, Heiss G, Folsom A, Rosamond W, Szklo M, Sharrett AR, Clegg LX. Association of coronary heart disease incidence with carotid arterial wall thickness and major risk factors the Atherosclerosis Risk In Communities (ARIC) Study, 1987–1993. Am J Epidemiol. 1997; 146: 483–494.
Furberg CD, Adams HP Jr, Applegate WB, Byington RP, Espeland MA, Hartwell T, Hunninghake DB, Lefkowitz DS, Probstfield J, Riley WA, Young B. Effect of lovastatin on early carotid atherosclerosis and cardiovascular events. Circulation. 1994; 90: 1679–1687.
Mercuri M, Bond MG, Sirtori CR, Veglia F, Crepaldi G, Feruglio FS, Descovich GC, Ricci G, Rubba P, Mancini M, Gallus G, Bianchi G, D’Alo’ G, Ventura A. Pravastatin reduces intima-media thickness progression in an asymptomatic hypercholesterolemic Mediterranean population: the Carotid Italian Ultrasound Study. Am J Med. 1996; 101: 627–634.
Losordo DW, Rosenfield K, Kaufman J, Pieczek A, Isner JM. Focal compensatory enlargement of human arteries in response to progressive atherosclerosis: in vivo documentation using intravascular ultrasound. Circulation. 1994; 89: 2570–2577.
Crouse JR, Goldbourt U, Evans G, Pinsky J, Sharrett AR, Sorlie P, Riley W, Heiss G. Risk factors and segment-specific carotid arterial enlargement in the Atherosclerosis Risk in Communities (ARIC) cohort. Stroke. 1996; 27: 69–75.
Bonithon-Kopp C, Touboul PJ, Berr C, Magne C, Ducimetiere P. Factors of carotid arterial enlargement in a population aged 59 to 71 years: the EVA study. Stroke. 1996; 27: 654–660.
Polak JF, Kronmal RA, Tell GS, O’ Leary DH, Savage PJ, Gardin JM, Rutan GH, Borhani NO, on behalf of the Cardiovascular Health Study. Compensatory increase in common carotid diameter: relation to blood pressure and artery intima-media thickness in older adults. Stroke. 1996; 27: 2012–2015.
Rubba P, Panico S, Bond MG, Covetti G, Celentano E, Iannuzzi A, Galasso R, Belisario MA, Pastinese A, Sacchetti L, Mancini M, Salvatore F. Site-specific atherosclerotic plaques in the carotid arteries of middle-aged women from Southern Italy: associations with traditional risk factors and oxidation markers. Stroke. 2001; 32: 1953–1959.
Siedel J, Schlumberger H, Klose S, Ziegenhorn J, Wahlefeld AW. Improved reagent for enzymatic determination of serum cholesterol. J Clin Chem Biochem. 1981; 19: 838–839.
Wahlefeld AW. Triglyceride determination after enzymatic hydrolysis. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis.Vol 4. 2nd ed. Weinheim: Verlag Chemie/New York, Academic Press; 1974: 1831.
Lopes-Virella MF, Stone P, Ellis S, Colwell JA. Cholesterol determination in high-density lipoproteins separated by three different methods. Clin Chem. 1977; 23: 882–884.
Miller NJ, Rice-Evans C, Gopinathan V, Davies MJ, Milner A. A new method for estimating plasma antioxidant activity and its application to the investigation of antioxidant status in premature neonate. In: Free Radicals and Antioxidants in Nutrition. London: Richelieu Press; 1993.
Tang R, Hennig M, Thomasson B, Sherz R, Ravinetto R, Catalini R, Rubba P, Zanchetti A, Bond MG. Baseline reproducibility of B-mode ultrasonic measurement of carotid artery intima-media thickness: the European Lacidipine Study on Atherosclerosis (ELSA). J Hypertens. 2000; 18: 197–201.
Glantz SA, Slinker BK. Primer of Applied Regression Analysis of Variance. New York: McGraw-Hill Inc; 1990.
Armitage P, Berry G. Statistical Methods in Medical Research. Oxford, UK: Blackwell Scientific Publications Limited; 1994.
Rubba P, Mercuri M, Faccenda F, Iannuzzi A, Irace C, Strisciuglio P, Gnasso A, Tang R, Andria G, Bond MG, Mancini M. Premature carotid atherosclerosis: does it occur in both familial hypercholesterolemia and homocystinuria? Ultrasound assessment of arterial intima-media thickness and blood velocity. Stroke. 1994; 25: 943–950.
Iannuzzi A, Celentano E, Panico S, Galasso R, Covetti G, Sacchetti L, Zarrilli F, De Michele M, Rubba P. Dietary and circulating antioxidant vitamins in relation to carotid plaques in middle-aged women. Am J Clin Nutr. 2002; 76: 582–587.