Relationship between plasma antioxidant concentrations and carotid intima-media thickness: the Asymptomatic Carotid Atherosclerotic Disease In Manfredonia Study

Abstract

Background

Few studies have examined the relationship among carotid atherosclerosis, vascular risk factors, and antioxidant plasma concentrations, and those that have reported conflicting results. The aim of this study was to assess the relationship between asymptomatic carotid atherosclerosis, as defined by carotid intima-media thickness (CIMT), and inflammatory markers, plasma lipids and serum antioxidant vitamins.

Methods and results

We examined baseline characteristics of the 640 participants in the Asymptomatic Carotid Atherosclerotic Disease In Manfredonia Study. All participants were asymptomatic with respect to carotid artery disease in 2006-2007 and underwent physical examination with carotid ultrasound investigation, the collection of medical history and laboratory data. Analysis of variance methods were used to examine differences between participants by category of CIMT. Of the 640 participants, 291 did not have evidence of carotid atherosclerosis (CIMT < 0.8mm), 232 were found to have some atherosclerosis (0.8 mm ≤ CIMT < 1.2 mm), and 117 were found to have extensive atherosclerosis (CIMT > 1.2 mm). Among participants with CIMT ≥ 0.8mm, body mass index, blood pressures, total cholesterol, LDL cholesterol, triglycerides, uric acid, C-reactive protein, and fibrinogen were significantly higher, whereas concentrations of vitamin A, vitamin E, lycopene, and β-carotene were all significantly lower when compared with participants who did not show evidence of carotid atherosclerosis (P < 0.001).

Conclusion

The optimal control of hypertension, diabetes, and dyslipidemia, in addition to smoking cessation and an adequate intake of antioxidant micronutrients from foods represent a key for the prevention of atherosclerotic disease.

Keywords

antioxidants atherosclerosis beta-carotene carotid arteries intima-media thickness lycopene vitamin A vitamin E

Introduction

Atherogenesis represents a complex process initiated by the oxidative damage of oxidized low-density lipoprotein cholesterol (LDL-C_ox) in the artery wall [1–3], which then stimulates monocyte recruitment and their differentiation to macrophages, resulting in the formation of foam cells and increased thickness of the arterial walls [4, 5]. The consequences of this atherosclerotic process are ischemic heart and cerebrovascular diseases. In particular, increased carotid intima-media thickness (CIMT) represents an early phase of the atherosclerotic process [6–8] and is a widely accepted marker of atherosclerosis that correlates with established coronary artery disease [9, 10].

Antioxidants have been hypothesized to inhibit lipid peroxidation and therefore may play a protective role in the development of cardiovascular diseases [11, 12] by preventing the formation of early atherosclerotic lesions [13]. Data from some population studies, including descriptive, case-control, and cohort studies, have shown that dietary [14, 15] and serum concentrations of antioxidants such as vitamin E [16], vitamin C [17, 18], and carotenoids [19–21] are inversely associated with cardiovascular mortality. However, other epidemiological studies have reported no association between cardiovascular events and serum antioxidant concentrations [22, 23]; in particular, negative results have been reported in intervention trials of β-carotene [24, 25] and vitamin E [26, 27] to prevent cardiovascular events.

Few studies have examined the relationship among CIMT, vascular risk factors, and antioxidant plasma concentrations. The aim of this study was to examine the relationship between asymptomatic carotid atherosclerosis (CIMT and carotid plaque) and serum plasma levels of inflammatory markers, plasma lipids and serum antioxidant vitamins.

Patients and methods

Patients

The Asymptomatic Carotid Atherosclerosis Disease In Manfredonia Study (ACADIM Study) is a prospective, cross-sectional study conducted between August 2006 and May 2007 in a randomly selected population of 640 participants who were asymptomatic with respect to carotid artery disease and seen at the Cardiology Unit of ‘San Camillo de Lellis' Hospital (Manfredonia, Foggia, Italy). The ACADIM Study was conducted in collaboration with the Department of Biomedical Sciences (Section of Human Nutrition and Clinical Biochemistry) of University ‘G. D'Annunzio’ (Chieti, Italy), and Laboratory Analyses of ‘San Camillo de Lellis' Hospital (Manfredonia, Foggia, Italy).

Inclusion and exclusion criteria, and definitions

Potential participants were considered to be asymptomatic for carotid artery disease if they had never experienced a transient ischemic attack, amaurosis fugax, or stroke. Potential participants were excluded if they had symptomatic carotid artery disease, current infectious or inflammatory disease, recent operations or endovascular interventions, bilateral carotid occlusion, monolateral/bilateral stent implantation, or monolateral/bilateral endoarterectomy.

During the study period, 800 potential participants of general population asymptomatic for carotid artery disease underwent a carotid ultrasound investigation (CUI) of the extracranial carotid arteries (common, internal, and external). Of these persons, 160 were not eligible for inclusion. The most common reasons for ineligibility were bilateral carotid artery occlusions (n = 47), recent endovascular interventions (n = 58), and monolateral endoarterectomy (n = 55). After CUI, demographic characteristics, lifestyle characteristics, medical history, data from physical examination, and laboratory data, were collected. A participant was defined a smoker if he had smoked cigarettes, cigars, or a pipe within the past 30 days.

Body weight was measured by using a balance scale. During the height and weight measurements, the participants wore light clothing and no shoes. Body mass index (BMI) was computed as the ratio of weight to the square of height (kg/m²). Arterial hypertension was defined as having blood pressure values ≥ 140/90 mmHg measured at least twice and was assumed to be present in patients taking antihypertensive drugs [28]. Diabetes mellitus was defined according to the 2002 clinical practice recommendations of the Expert Committee of the American Diabetes Association [29]. Hyperlipidemia was defined as an elevation of LDL-C values above 130 mg/dl and was assumed to be present in all patients taking lipid-lowering therapies [30]. The diagnosis of peripheral artery disease was classified according to the American College of Cardiology/the American Heart Association Task Force on Practice Guidelines [31]. Stroke was defined as a neurological deficit after 24h, evaluated by neurologist or internist, according to the American Heart Association/the American Stroke Association Stroke Council Guidelines [32]. History of myocardial infarction (MI) was defined according to the consensus document of the Joint European Society of Cardiology/the American College of Cardiology Committee for the redefinition of MI [33].

After receiving information about the purpose of the study, participants signed an informed consent before participating in the ACADIM Study. The study was carried out in accordance with the Helsinki Declaration of 1975 as revised in 1983, and approved by the Ethics Review Committee and by the Medical Direction of ‘San Camillo de Lellis' Hospital, Manfredonia (Foggia, Italy).

Ultrasound examination

CUIs were performed by means of a color-coded ACUSON Sequioia^C512 carotid duplex ultrasound machine with a 7.5-MHz linear transducer (SIEMENS, New York, USA). The investigation included longitudinal and transverse examinations of the carotid arteries. Both diameter reductions were measured and calculated at the site of maximal stenosis in the extra cranial common carotid arteries (CCA) according to the European Carotid Surgery Trial method [34]. The measurements of IMT were made at 10 mm proximal to the carotid bulb or 20 mm proximal to the flow divider. CIMTwas measured between the leading edge of the first echogenic line (lumen-intima interface) and the second echogenic line (upper layer of the adventitia) in the far (deeper) artery wall. All measurements were made on frozen, enlarged images (2 ×) at the end of a heart cycle (end diastole), with the transducer in the mediolateral position [35]. Measurements were made in both CCA, and the larger of the two values was used in data analysis. Off-line analysis of CCA-IMT was made on video images based on the Atherosclerosis Risk in Communities Study protocol [36]. Carotid atherosclerosis was defined as a CIMT between 0.8 and 1.2 mm, whereas carotid plaque was defined as focal echogenic structures encroaching into the vessel lumen where the CIMTwas greater than 1.2 mm. These cutoffs were chosen because they were used in previous randomized clinical trials [37].

Clinical and laboratory data

After CUI, demographic characteristics (age, sex), life-style characteristics (smoking habits), medical history (presence of hypertension, diabetes, family history of atherosclerosis, MI, hyperlipidemia, angina pectoris, peripheral artery disease, history of prior cerebral accident, current medications), data from physical examination (BMI, blood pressure), and laboratory data were collected.

Venous blood samples from participants on fast were obtained for the measurement of laboratory values. Measurements included: total cholesterol, high-density lipoprotein cholesterol (HDL-C), LDL-C, triglycerides, C-reactive protein (CRP), fibrinogen, and uric acid (measured at Analysis Laboratory of the ‘San Camillo de Lellis' Hospital, Manfredonia, Foggia, Italy) and plasma concentrations of vitamin A, vitamin E, lycopene, and β-carotene (measured at the Department of Clinical Biochemistry, University ‘G. D'Annunzio’, Chieti, Italy). All investigators and laboratory personnel were blinded to the participant's CIMT. Antecubital venous blood samples from all participants were handled identically and blindly through all stages of blood collection, storage, retrieval, and analytic processes.

Antioxidants measurements

Blood samples were collected between 08:00 and 10:00 h in polypropylene tubes containing EDTA 1 mmol/l. Samples were stored in an icebox before centrifugation at 3000g for 10 min at 4°C. Two hundred microleter aliquots of plasma were transferred into foil-wrapped polypropylene tubes. Plasma samples were either used for extraction immediately or stored in the dark at −80°C until analysis was performed. Sample preparation is as described in the study by Lee et al. [38].

Vitamin A, vitamin E, lycopene, and β-carotene were determined by HPLC (Whatman, Clifton, New Jersey, USA). Two Water Pumps 515 HPLC equipped with Waters auto injector (model 717 plus auto sampler) and Waters 996 photodiode array detector were used as HPLC system. Data acquisition and processing were performed using chromatography software, Empower-Pro, by Waters (Whatman, Waters Corporation, Milford, Massachusetts, USA). Analysis was performed by isocratic elution. The flow rate was 1.5 ml/min. The mobile phases used were: A — methanol/n-butanol/water (89.5/5/5.5 v/v/v) premixed and vacuum filtered through a 0.45 μm polypropylene membrane filter (Whatman) before use, and B — methanol/n-butanol/water (76/19.5/4.5 v/v/v) premixed and vacuum filtered before use as for A. The gradient elution profile was as follows: 0-7 min 100% A; 7-15 min 100% B; 15-20 min 100% A for column reequilibration. Auto injections of 20 μl were performed at 5°C. The analytical column used was a replaceable Partisphere 5 C-18 cartridge (110 × 4.6 mm inner diameter, 5 μm particle size; Whatman, Waters Corporation) protected by a guard cartridge (C-18, 5 μm) system and maintained at 45°C. Photodiode array wavelength range was from 270 to 460 nm and the chromatograms were extracted at 340 nm for vitamin A, 288 nm for vitamin E and vitamin E-acetate (used as internal standard), and 441 nm for lycopene and β -carotene. The run time was 20 min.

Statistical analysis

Study participants were grouped into three categories by CIMT: less than 0.8 mm, 0.8-1.2 mm, and greater than 1.2 mm. For each baseline characteristic, the mean value or corresponding percentage of study participants was calculated by category of CIMT. The statistical significance of differences was examined by analysis of variance (continuous variables) and by the χ² test (categorical variables).

Associations among demographic, lifestyle, medical history, and laboratory characteristics and having CIMT in the elevated range (≥ 0.8 mm) were examined using logistic regression models. A P value of less than 0.05 was considered statistical significant. Data were analyzed by using SPSS statistical software (version 10.0 for Windows; SPSS Inc., Chicago, Illinois, USA).

Results

Means (± SD) or percentages of various demographic characteristics and established cardiovascular disease risk factors are summarized in Table 1 by category of CIMT. Of the 640 participants, 291 had CIMT less than 0.8 mm, 232 had CIMT between 0.8 and 1.2 mm, and 117 had CIMT greater than 1.2 mm. As compared to participants with CIMT less than 0.8 mm, those in higher categories of CIMT were older, more often male, had a BMI in the overweight range, and were more often smokers. The proportion of participants with prevalent cardiovascular diseases (hypertension, angina pectoris, and MI) and diabetes by category of CIMT are also shown in Table 1. As compared to participants with CIMT less than 0.8 mm, the proportion with arterial hypertension and diabetes was significantly greater among participants with CIMT between 0.8 and 1.2 mm, but not among those with CIMT greater than 1.2 mm. In contrast, the proportion of patients with hyperlipidemia, family history of coronary heart disease, or family history of stroke was significantly greater among both groups with elevated CIMT as compared to those with CIMT less than 0.8mm.

Table 1

Demographic characteristics and comorbid conditions in the study population


	Carotid intima-media thickness

	< 0.8 mm (n = 291)	0.8-1.2 mm (n = 232)	> 1.2 mm (n = 117)

Demographic characteristics
Age (years)	47.48 ± 12.31	60.98 ± 8.84	65.33 ± 13.31
Males/females	110/181	118/114∗∗	69/48∗∗
Body mass index (kg/m²)	25.98 ± 3.18	27.97 ± 2.11∗∗	28.61 ± 2.13∗∗
Smoking status (yes/no) Medical history	20/271 (6.78%)	26/206 (11.20%)∗∗	32/85 (27.35%)∗∗∗, †
Hypertension (yes/no)	32/259 (11%)	63/169 (27%) ‡	11/106 (9.5%)
Diabetes (yes/no)	1/290 (0.34%)	6/226 (2.58°/o) §	1/116 (0.85%)
Angina or myocardial infarction (yes/no)	1/290 (0.34%)	2/230 (0.86%)	1/116 (0.85%)
Hyperlipidemia (yes/no)	14/227 (4.81%)	66/166 (28.44%) ‖	25/92 (21.36%)∗∗∗
Family history of atherosclerosis (yes/no)	3/288 (1.03%)	20/212 (8.62%)∗∗	35/82 (29.91°/o)∗∗∗, †
Family history for stroke (yes/no)	2/289 (0.68%)	15/217 (6.46%)∗∗	18/99 (15.38%)∗∗, †

∗P < 0.001 for 0.8-1.2 mm group and > 1.2 mm group vs. < 0.8 mm group.

∗∗P < 0.01 for 0.8-1.2 mm group vs. < 0.8 mm group.

∗∗∗P < 0.001 for > 1.2 mm group vs. < 0.8 mm group.

^† P < 0.01 for > 1.2 mm group vs. 0.8-1.2 mm group.

^‡ P < 0.001 for 0.8-1.2 mm group vs. > 1.2 mm group and < 0.8 mm group.

^§ P <0.01 for 0.8-1.2 mm group vs. > 1.2 mm group and < 0.8 mm group.

^‖ P < 0.001 for 0.8-1.2 mm group vs. < 0.8 mm group.

Means (± SD) of inflammatory markers, plasma lipids, and serum antioxidant vitamins/carotenoids are summarized in Table 2 by category of CIMT. Levels of inflammatory markers tested in this study (uric acid, CPR, and fibrinogen) were all significantly higher among participants with elevated CIMT as compared to those with CIMT less than 0.8 mm. In addition, plasma lipids (total cholesterol, LDL-C, and triglycerides) were often significantly higher among participants with elevated CIMT as compared to those with CIMT less than 0.8 mm. All antioxidant vitamins/carotenoids evaluated in the study (vitamins A and E, lycopene, and β-carotene) had significantly lower concentrations among participants with elevated CIMT as compared to those with CIMT less than 0.8 mm.

Odds ratios and 95% confidence intervals for having CIMT ≥ 0.8 mm are presented for selected risk factors including inflammatory markers and plasma lipid levels. In multivariate-adjusted models, participants with elevated levels of inflammatory factors and plasma lipids were significantly more likely to have CIMT ≥ 0.8 mm (P < 0.01 for CRP, uric acid, fibrinogen, plasma LDL-C, and triglycerides) (Table 3).

Table 2

Blood serum and plasma levels of inflammatory markers, lipids, and antioxidant vitamins/carotenoids


	Carotid intima-media thickness

	< 0.8mm (n = 291)	0.8-1.2 mm (n = 232)	>1.2mm (n = 117)

Inflammatory markers
Uric acid (μmol/l)	249.81 ± 86.84	307.51 ± 91.59∗	334.87 ± 108.25∗∗,∗∗∗
CRP (g/dl)	2.90 ± 1.30	3.84 ± 1.75∗	4.86 ± 2.20∗∗,∗∗∗
Fibrinogen (mg/dl)	297.38 ± 93.66	349.46 ± 80.62∗	369.60 ± 76.53∗∗,∗∗∗
Plasma lipids
Total cholesterol (mg/dl)	174.46 ± 14.90	231 ± 35.25 †	179 ± 13.21
HDL-C (mg/dl)	48.18 ± 4.64	59.62 ± 4.5 †	46.15 ± 3.59
LDL-C (mg/dl)	119.72 ± 10.39	164.25 ± 36.20	155.72 ± 23.39 ‡
Triglycerides (mg/dl)	127.20 ± 24.22	160.62 ± 35.27∗	182 ± 22.51 ∗∗,∗∗∗
Antioxidants
Vitamin A (μmol/l)	3.82 ± 0.65	2.03 ± 0.85	2.09 ± 0.96 §
Vitamin E (μmol/l)	62.64 ± 11.75	30.37 ± 5.48	32.93 ± 9.27 §
Lycopene (μmol/l)	0.82 ± 0.33	0.33 ± 0.63	0.34 ± 0.21 §
Beta-carotene (μmol/l)	2.21 ± 0.42	0.91 ± 0.35	0.80 ± 0.39 §

CRP, C-reactive protein; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

∗P < 0.01 for 0.8-1.2 mm group vs. < 0.8 mm group.

∗∗P < 0.001 for >1.2 mm group vs. < 0.8 mm group.

∗∗∗P < 0.01 for > 1.2 mm group vs. 0.8-1.2 mm group.

^† P < 0.01 for 0.8-1.2 mm group vs. > 1.2mm group and < 0.8mm group.

^‡ P < 0.01 for 0.8-1.2mm group and > 1.2 mm group vs. < 0.8 mm group.

^§ P < 0.001 for 0.8-1.2 mm group and > 1.2 mm group vs. < 0.8 mm group.

Table 3

Odds ratios and 95% confidence intervals of selected risk factors for elevated carotid intima-media thickness (≥ 0.8 mm)


	Odds ratio	95% Confidence interval	P value

Risk factor a
Age (years)	1.13	1.09-1.18	< 0.001
Body mass index (kg/m²)	1.10	1.01-1.20	0.023
Inflammatory markers
Uric acid (μmol/l)	2.00	1.47-2.71	< 0.001
CRP (g/dl)	3.64	2.35-5.62	< 0.001
Fibrinogen (mg/dl)	1.010	1.006-1.014	< 0.001
Plasma lipids
Total cholesterol (mmol/l)	1.68	1.09-2.59	0.018
LDL-C (mmol/l)	3.37	1.87-6.07	< 0.001
Triglycerides (mmol/l)	3.60	1.40-9.27	0.008

CRP, C-reactive protein; LDL-C, low-density lipoprotein cholesterol.

All risk factors were simultaneously adjusted for each other in a single regression model.

Discussion

The primary findings of our study indicate that low plasma levels of antioxidant vitamins (A, E) and carotenoids (lycopene and β-carotene) are associated with carotid atherosclerosis. In our study, elevated CIMT was significantly associated with having a low concentration of all antioxidants evaluated (vitamin A, vitamin E, lycopene, and β-carotene) and a higher concentration of inflammatory factors including serum uric acid, CRP, and fibrinogen.

Several epidemiological and case-control studies have failed to identify an association between plasma vitamin A concentration and CIMT [39–43]. We found only one case-control study that documented a significantly lower vitamin A (retinol) plasma concentration in the case group with carotid atherosclerosis as compared with healthy control participants, independent of fruit and vegetable intake [44]. With regard to vitamin E, the results of studies to date are inconclusive. A significant association between having lower vitamin E plasma concentrations and carotid atherosclerosis has been identified by several epidemiological studies [42, 44, 45]. In contrast, many studies have not identified this association [35, 41, 43, 45]. Even for the well-studied relationship between β-carotene and carotid atherosclerosis, the results of published studies are unclear and conflicting. Many studies have found that the consumption of β-carotene was inversely related with coronary heart disease risk [45, 46] and that the risk of carotid and femoral atherosclerosis decreased with increasing plasma β-carotene concentration, suggesting aprotectiveroleforβ-carotene in early atherogenesis [39]. However, several well-conducted studies have found no association between β-carotene plasma concentration and CIMTor peripheral vascular disease [42–44].

In contrast, the results of published studies of lycopene and carotid atherosclerosis are generally concordant and shown a significant inverse relationship between serum concentration of lycopene and CIMT, supporting the hypothesis that plasma lycopene may decrease the risk of atherosclerosis and play an important role in early stage of atherogenesis [42, 44, 47].

Our study confirms the role of plasma lipid profiles in the pathogenesis of the atherosclerotic process, with a significantly higher serum concentration of total cholesterol, LDL-C, and triglycerides among participants with CIMT ≥ 0.8 mm. These findings suggest that the first and earliest manifestation of the atherosclerotic process characterized by elevated CIMT depends partly on the presence of an abnormal plasma lipid profile, with a low concentration of protective lipoproteins (HDL-C) and higher concentrations of LDL-C and triglycerides. The plasma lipid patterns of participants with the highest levels of CIMT (> 1.2 mm) was associated with an abnormal lipid profile that had significantly higher concentrations of LDL-C, and triglycerides, and lower levels of HDL-C than participants with a lesser degree of carotid atherosclerosis.

We also identified a strong positive association between values of the inflammatory markers tested in our study and CIMT. There was a progressive increase related to the degree of carotid atherosclerosis present in the participant. This increase was independent of age, BMI, and plasma lipids in logistic regression analyses. In particular, this strong, positive relationship may represent an important indicator of the oxidative stress and proinflammatory milieu involved in the atherosclerotic process.

As expected, we identified arterial hypertension and diabetes as two of the strongest and most significant risk factors for the development of early atherosclerosis as characterized by CIMT between 0.8 and 1.2 mm. Both hypertension and diabetes have been shown to contribute to remodeling of the arterial wall. These findings are in accordance with the findings of a community-based study in Taiwan, including 3602 participants, which showed that hypertension was the major determinant of carotid atherosclerosis, particularly among those older than 35 years [48].

A family history of atherosclerosis and stroke represent a significant risk factor carotid atherosclerosis. One important limitation of this study is the cross-sectional nature of our findings. As antioxidant values, lipid profiles, anthropometrics, and questionnaires were collected concurrently with the ultrasound assessment of carotid arteries, temporality cannot be inferred from these findings. Further studies that examine this important relationship are warranted.

In summary, we found a strong inverse relationship between antioxidant plasma levels and the presence of carotid atherosclerosis. In addition, we identified risk factors for carotid atherosclerosis, such as hypertension, diabetes, smoking, overweight status, a positive family history of atherosclerosis or stroke, and elevated inflammatory markers such as uric acid, CRP and fibrinogen, which may represent a risk factor profile for individuals at the highest risk of developing carotid atherosclerosis as evaluated by CUI. This noninvasive investigation is inexpensive compared with other modalities of imaging, and may be a reasonable routine procedure among patients with hypertension, diabetes, or other important risk factors identified in our study. The optimal control of hypertension, diabetes, dyslipidemia, and the cessation of smoking represent key elements in a strategy for the prevention of atherosclerotic disease, particularly in countries with a high prevalence of cardiovascular and cerebrovascular diseases.

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