Directory UMM :Data Elmu:jurnal:A:Atherosclerosis:Vol153.Issue1.Nov2000:

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High plasma levels of

a

- and

b

-carotene are associated with a

lower risk of atherosclerosis

Results from the Bruneck study

Anna D’Odorico

_{, Diego Martines}

_{, Stefan Kiechl}

_{, Georg Egger}

_,

Friedrich Oberhollenzer

_{, Piero Bonvicini}

_{, Giacomo Carlo Sturniolo}

_,

Remo Naccarato

_{, Johann Willeit}

_*

a_{Department of Surgery and Gastroenterology}_,_Uni₆_{ersity of Padua}_,_Padua_,_Italy b_{Department of Neurology}_,_Uni₆_{ersity of Innsbruck}_,_Anichstr_35,_A_-6020_Innsbruck_,_Austria

c_Di₆_{ision of Internal Medicine}_,_Bruneck_,_Italy

d_{Department of Clinical Biochemistry}_,_Uni₆_{ersity of Padua}_,_Padua_,_Italy

Received 13 July 1999; received in revised form 29 November 1999; accepted 7 January 2000

Abstract

Background and purpose: A large number of studies have contributed to the hypothesis that carotenoids, vitamins A and E are protective against atherosclerosis by acting as antioxidants. The aim of this study was to assess the relationship between plasma levels of carotenoids (a- andb- carotene, lutein, lycopene, zeaxanthin,b-cryptoxanthin), vitamins A and E, and atherosclerosis in the carotid and femoral arteries. Methods: This prospective and cross sectional study involved a randomly selected population sample of 392 men and women aged 45 – 65 years. Carotid and femoral artery atherosclerosis was assessed by high-resolution duplex ultrasound.Results:a- andb- carotene plasma levels were inversely associated with the prevalence of atherosclerosis in the carotid and femoral arteries (P=0.004) and with the 5-year incidence of atherosclerotic lesions in the carotid arteries (P=0.04). These findings were obtained after adjustment for other cardiovascular risk factors (sex, age, LDL (low density lipoproteins), ferritin, systolic blood pressure, smoking, categories of alcohol consumption, social status, C-reactive protein). Atherosclerosis risk gradually decreased with increasing plasmaa- and b-carotene concentrations (P=0.004). No associations were found between vitamin A and E plasma levels and atherosclerosis. Conclusions: This study provides further epidemiological evidence of a protective role of higha- andb- carotene in early atherogenesis. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords:Antioxidants;a-Carotene;b-Carotene; Atherosclerosis; Carotid artery disease

www.elsevier.com/locate/atherosclerosis

1. Introduction

Cardiovascular disease is the leading cause of mor-bidity and mortality in industrialized countries and hyperlipidemia constitutes one of the main underlying risk conditions. A large number of epidemiological and experimental studies have contributed to the hypothesis that oxidative surface modifications of low-density lipo-protein (LDL) are crucial in the initiation of lipid-in-duced atherogenesis [1,2]. Modified LDL is more

readily taken up by macrophages through the scavenger receptor, giving rise to the formation of foam cells and fatty streaks. Antioxidant substances disolved in LDL may be expected to counteract lipid peroxidation and decelerate atherosclerosis progression [3]. Strong sup-port for this concept derives from an animal model in which large amounts of dietary carotenoids protected against the development of atherosclerosis [4].

Furthermore, a variety of epidemiological studies [5 – 13] suggest inhibitory effects of high vitamin E and carotenoids on myocardial infarction, ischemic stroke and progression of coronary heart disease. These stud-ies focused on clinical complications of advanced atherosclerosis, and thus cannot furnish the proof of an * Corresponding author. Tel.: +43-512-5044279; fax: +

43-512-5044260.

E-mail address:[email protected] (J. Willeit).

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A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239 232

antioxidant defense of carotenoids and vitamins [14]. Vitamin E, for example, could prevent progression of cardiovascular disease by potential blood clotting con-trol [15] and platelet adherence modulation properties [16,17]. Epidemiological surveys which directly address the effects of antioxidant levels on early atherosclerosis are needed to verify the key issue, but such evaluations are particularly scarce [18].

The current study was designed to investigate the association between carotenoids (a- and b- carotene, lutein, lycopene, zeaxanthin,b-cryptoxanthin), vitamins A and E, and carotid and femoral artery atherosclerosis in a large random sample of men and women aged 45 – 65 years.

2. Methods

2.1. Study population

Population recruitment was performed as a part of the Bruneck Study [19 – 23]. The survey area is located in northern Italy (province of Bolzano). Agriculture, tourism, commerce, and light industry are the main sources of income. Geographic remoteness determines low population mobility and maintenance of a tradi-tional lifestyle. At the 1990 baseline the study popula-tion was recruited as an age- and sex-stratified random sample of 1000 men and women aged 40 – 79 years (125 in each of the 5 – 8th decades). A total of 93.6% partic-ipated, with data assessment completed in 919 subjects. During the follow-up period between summer 1990 and 1995 a subgroup of 62 individuals died and one moved away and could not be traced. In the remaining popula-tion, follow-up was 96.5% complete (n=826). Plasma antioxidant levels were assessed as part of the 1995 evaluation in all subjects aged 45 – 65 years (n=452). After the exclusion of men and women with missing laboratory data (n=38) and those with temporary vitamin supplements (n=22), in whom a single antioxi-dant measurement may fail to adequately reflect long-term concentrations, 392 subjects were eligible for the current evaluation. All participants gave their informed consent before entering the study.

2.2. Clinical history and examination

The study protocol included neurological and cardio-logical examinations as detailed previously [19,20]. A standardized questionnaire was completed by each par-ticipant on current and past exposure to candidate cardiovascular risk factors, sociodemographic variables, previous diseases, and use of medication. The systolic and diastolic blood pressure was taken in a sitting position after at least 10 min of rest (mean of three independent measurements). Hypertension was defined

by a systolic blood pressure ]160 mmHg or diastolic blood pressure ]95 mmHg. The average number of cigarettes smoked per day was noted for each smoker and ex-smoker. Diabetes mellitus was coded present for subjects on therapy with insulin or oral antidiabetic drugs and/or with fasting glucose plasma levels \7.8 mmol/l (140 mg/dl), or a 2-h value \11.1 mmol/l (200 mg/dl) after oral glucose loading. The body mass index was calculated as weight (kg) divided by height (m2_).

Assessment of regular alcohol consumption was per-formed with a standardized questionnaire [23]: Subjects were instructed to indicate their customary drinking frequency (days per week) and the average amount of alcoholic beverages ingested on a typical occasion or during a typical day. Beer (500-ml bottle, equivalent to about 25 g ethyl alcohol), white or red wine (250-ml glass, 25 g ethanol), and spirits and liqueurs (standard drink, 8 – 10 g alcohol each) were included as separate items. Average alcohol consumption was quantified in terms of grams per day (g/day) and classified in four categories: abstainers (0 g/day) and light (1 – 50 g/day), moderate (51 – 99 g/day), and heavy drinkers (]100 g/day). Socioeconomic status was defined with a two-category scale (1=low, 2=high) based on information about the occupational status of the person with the highest income in the household and the educational level of the proband. A high social status was assumed if the subject had ]12 years of education and/or the average monthly income of the subject or his/her spouse was $2000 or greater. Finally, subjects were asked to record frequency and duration of sports activ-ities over a 4 week period. Based on these data, three levels of leisure-time physical activity were differenti-ated: 1) no exercise at all, 2) regular physical activity of up to 2 h on average per week, and 3) regular physical activity of \2 h per week [20].

2.3. Laboratory measurements

Blood samples were taken from the antecubital vein after subjects had fasted and abstained from smoking for at least 12 h. For the measurement of carotenoid concentration, blood was collected in heparinized tubes covered with aluminium foil to avoid carotenoid oxida-tion and polymerizaoxida-tion by oxygen and light. Plasma was obtained by centrifugation of blood samples at 1500 g for 20 min and immediately stored at −70°C. After a storage time of 4 months, plasma samples were thawed and extracted by the addition of an equal volume of ethanol containing retinol acetate as an internal standard. After spinning for 45 s, the plasma was extracted with hexane and diethyl ether (1:1,v/v) and spun for 1 min and 15 s. The supernatant was removed and evaporated to dryness under nitrogen. The samples were reconstituted with 100 ml of acetone and injected into the HPLC system. The

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chromato-graphic system consisted of a Beckman 114 Mitzi au-tosampler with a Spherisorb S5 ODS 2 reversed phase column 259×4.6 mm (Phase Separation, Clwyd UK), and acetonitrile – water (9:1, v/v) and ethylacetate were used as the mobile phase at a flow rate of 1 ml/min. A diode array detector (Module 168, Beckman) was used. Carotenoids were detected at 450 nm, retinol and retinol acetate at 325 nm and a-tocopherol at 292 nm. Pure forms of each carotenoid were used as reference standards for quantification by ultraviolet or visible spectrophotometry (Uvicon Spectrophotometer 922, Kontron Instruments). Apolipoproteins were measured using a nephelometric fixed-time method (apolipo-protein B and AI, CV=5.7 and 2.4%). Total choles-terol, high-density lipoprotein cholesterol and triglycerides were determined enzymatically (CHOD-PAP and GPO-(CHOD-PAP methods, Merck, Germany; CV=

2.2 – 2.4%). Low-density lipoprotein cholesterol was calculated using the Friedewald formula, except for subjects with triglyceride concentrations \400 mg/dl. Fibrinogen and other parameters were measured by standard laboratory procedures [19].

2.4. Chemicals

Standards of a- and b-carotene, lutein, lycopene, retinol, retinol acetate anda-tocopherol were purchased from Sigma Chemical (St. Louis, MO). The zeaxanthin standard was kindly provided by Hoffman La Roche (Basel, Switzerland). HPLC-grade acetone, absolute ethanol, ethyl acetate, and hexane were purchased from Merck. Acetonitrile was obtained from Carlo Erba (Milan, Italy).

2.5. E6aluation of 6ascular status

At the 1990 baseline sonographic assessment was performed using a duplex ultrasound system (ATL8, Advanced Technology Laboratories) with a 10-MHz scanning frequency in B-mode and a 5-MHz scanning frequency in pulsed Doppler mode. All subjects were examined in a supine position. The scanning protocol included imaging of the right and left common (proxi-mal and distal segments) and internal carotid arteries (bulbous and distal segments) [19 – 22] and of the femoral arteries 40 mm proximal and 10 mm distal to the bifurcation into the superficial and deep branches. Pulsed Doppler was used to provide information on blood flow velocity and to identify the different arteries. Atherosclerotic lesions were defined by two ultrasound criteria: [1] wall surface (protrusion into the lumen or roughness of the arterial boundary) and (2) wall texture (echogenicity). The maximum axial diameter of the plaque was measured as the distance from the leading edge of the lumen-intima interface to the leading edge of the media-adventitia interface. As detailed previously

[20], an atherosclerosis score was calculated by adding the diameters of plaques (in millimeters) at each imag-ing site in the carotid arteries. Rescannimag-ing was per-formed in 1995 using the same ultrasound protocol. Incident carotid atherosclerosis was defined by the oc-curence of new plaque in previously normal segments. All the ultrasound methods applied were highly repro-ducible (for details see references [21,22]).

2.6. Statistical analysis

The association of carotenoid plasma concentrations with demographic characteristics and potential vascular risk factors was analyzed by means of Pearson’s corre-lation coefficients and analysis of variance. The associa-tion between carotenoid levels and ultrasonographic outcome measures (prevalent carotid and/or femoral artery atherosclerosis, incident carotid atherosclerosis) was examined by logistic regression analysis, with the hypothesis test based on likelihood ratio statistics. a+ b-carotene (sum of alphacarotene and betacarotene) concentrations were modeled either as a continuous variable or as a set of indicator variables. Multivariate logistic regression models were constructed with a for-ward stepwise selection procedure using standard inclu-sion and excluinclu-sion criteria (P1 B0.05;PE\0.10).

3. Results

Proportions and mean values of selected demo-graphic parameters, vascular risk factors and carotenoid plasma concentrations are given in Tables 1 and 2; Fig. 1. Levels of a-carotene, b-carotene and cryptoxanthin were significantly higher in women. An-tioxidant concentrations did not show clear age trends (P\0.05, each). Notably,a- andb-carotene levels were reduced by half in heavy smokers and drinkers (Table 3). Two-factorial analysis of variance demonstrated that effects of smoking and alcohol consumption were at least in part independent of each other (P=0.005 and 0.034). Relationships with other vascular risk fac-tors are summarized in Table 4.

Logistic regression analysis revealed a strong and independent association ofa+b-carotene plasma levels with prevalent carotid and femoral artery atherosclero-sis and with 5-year incidence of atherosclerotic lesions in the carotid arteries (Table 5). In the latter analysis, dietary habits in the Bruneck Study population were assumed to be consistent during the follow-up period. All analyses were adjusted for age, sex, ferritin, systolic blood pressure, diabetes, cigarette smoking, alcohol consumption, social status and levels of C-reactive protein. In addition, the prospective model considered pre-existing disease status (atherosclerosis score). The figure illustrates the risk of carotid and femoral

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A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239 234

atherosclerosis according to quintiles ofa+b-carotene. This graph shows a gradual decrease in atherosclerosis risk with increasing carotene levels (P value for linear trend, 0.0049). Split analyses in genders, in smokers and non-smokers, and in abstainers and regular alcohol consumers all yielded consistent relations between atherosclerosis anda+b-carotene levels (data not pre-sented). Thus, alcohol consumption and cigarette smok-ing neither confounded nor modified the association obtained. Other carotenoids did not significantly pre-dict the risk of atherosclerosis. This finding applied to all subfractions (cryptoxanthin, etc.) and to their sum (‘other carotenoids’). Likewise, retinol and a -tocopherol were unrelated to atherosclerosis in our population (Table 5) as was the a-tocopherol/ choles-terol ratio.

4. Discussion

Oxidative modifications of LDL are widely accepted to represent a key event in lipid-induced atherogenesis. The efficacy of lipid peroxidation depends on LDL composition, residence time of LDL in the sub-en-dothelial space and, most important, the balance of pro-oxidants involving ferrous iron [19] and antioxi-dants. Carotenoids, a group of lipid-soluble pro-vita-mins contained in LDL particles, are experimentally well-founded to protect against lipid peroxidation [24 – 26]. b-carotene represents 25 – 50% of total carotenoids and may be the fraction with the most pronounced antioxidant effects [27].

The current population study is the first to demon-strate a strong inverse association between a+b -carotene plasma levels and the prevalence of atherosclerosis in carotid and femoral arteries. This association was internally consistent in genders and across the whole age range (45 – 65 years) and emerged as independent of other vascular risk factors. It was particularly relevant to cigarette smokers and heavy drinkers, whose a+b-carotene levels were markedly reduced (Table 3). As for other carotenoids, we ob-served a tendency towards lower atherosclerosis risk in subjects with high plasma levels, though statistical sig-nificance was not achieved.

To the best of our knowledge, there is only one previous epidemiological study addressing the potential protective effects of carotenoids on atherogenesis. This study attempted to quantify nutritional carotene intake and revealed an inverse association of this variable with early stages of carotid atherosclerosis in men [28]. A protective role of carotene in atherogenesis is further substantiated by an animal model [4] and various stud-ies on symptomatic coronary artery disease: With a few exceptions [10,13] low plasma b or total carotene pre-dicted an increased cardiovascular risk [9,11,12,29]. In-tervention trials, however, exhibited effects of a regular intake of 20 – 50 mg of synthetic b-carotene on cardio-vascular risk and mortality that were at times beneficial [30,31], at others non-existent [32] or even unfavorable [33,34]. Several clues to explain this discrepancy may be inferred from the inconsistencies observed between and within observational and interventional studies. i) Plasma antioxidant concentrations are the result of long-term dietary habits. In clinical intervention trials the period of antioxidant supplementation may be too short for lipid-soluble antioxidants to accumulate in the lipid core of plaques [14]. ii) Commercial supplements of b-carotene were higher in the trans- than in the cis-isomer, whereas natural b-carotene showed an op-posite ratio. Recently, the nine-cis form of b-carotene was shown to have a higher antioxidant capacity in vitro [35] and in vivo [36]. Furthermore, the ability of these isomers to accumulate in the lipid core of Table 1

Characteristics of the study population (n=392)a

Women (n=187) Men (n=205)

53.995.6

Age (years) 54.195.9

0.69890.47 1.27190.975

Carotenoidsa+b,mmol/l

Other Carotenoids,mmol/l 1.50490.681 1.29990.492 Retinol,mmol/l 2.28990.911 2.64291.039

24.999.4

Tocopherol,mmol/l 24.8910.9 25.293.7

Body mass index, kg/m2 _25.8₉_3.4

142.4917.7 Systolic blood pressure, 145.2921.0

mmHg

87.299.7

Diastolic blood pressure, 86.498.3 mmHg

5.8491.02 Serum total cholesterol, 6.1191.02

mmol/l

Serum HDL cholesterol, 1.6290.41 1.4190.40 mmol/l

3.5991.07 Serum LDL cholesterol, 3.8790.91

mmol/l

1.6290.27 1.290.7

Apolipoprotein A-I, g/l

1.1790.32 Apolipoprotein B, g/l 1.1390.30

1.3490.73

Serum Triglycerides, mmol/l 1.5191.04 2.6790.59 Fibrinogen, g/l 2.7890.56

Serum Ferritin,mg/l 58.7973.8 191.09176.1 0.05390.006 0.005490.007 HbA1c%, proportion of Hb

5.3%

Diabetes mellitus 4.9%

20.3%

Current smokers 32.2%

Hypertension 29.9% 23.9%

High social status 10.2% 28.8%

Physical acti6ity

0 h/week 21.4% 29.3%

40.1%

B2 h/week 43.4%

\2 h/week 38.5% 27.3%

Alcohol categories

15.6%

0 g/day 56.7%

42.8% 51.2%

1–50 g/day

51–100 g/day 0.5% 17.1%

\100 g/day 0.0% 16.1%

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Odorico

Atherosclerosis

153

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231

–

239

235

Mean values9SD of serum Retinol,a-tocopherol and carotenoids in men and women aged 45–64 years Lutein (mmol/l) Zeaxanthin

Retinol a-Tocopherol Cryptoxanthin

Age (years) Lycopene a-carotene b-carotene

(mmol/l) (mmol/l)

(mmol/l)

(mmol/l) (mmol/l) (mmol/l) (mmol/l)

Men

0.3190.13 0.0990.03 0.2190.14 0.6690.35 0.1290.13 0.5990.47

2.8790.92 24.0910.4 45–49

0.2990.22 0.5890.28 0.1390.13 0.6490.36

0.1090.06

50–54 2.5090.93 20.698.80 0.3390.15

0.1090.05

55–59 2.5890.93 22.099.57 0.3690.20 0.2290.15 0.6790.38 0.1190.11 0.5890.40

0.2390.17

60–64 2.5191.22 19.8914.6 0.3590.19 0.1090.05 0.5390.34 0.0990.06 0.5490.30

Women

0.3190.21 0.7690.49 0.2490.36

2.1490.81 1.1590.91

45–49 20.698.70 0.3790.21 0.0990.06

0.3690.15 0.1090.05 0.3690.19 0.7190.29 0.1990.17 1.1190.67

2.3690.95 21.899.08 50–54

0.3690.21 0.6690.33 0.1990.32 1.1190.81

55–59 2.3590.91 23.498.98 0.3890.20 0.1090.08

0.3790.27 0.6290.42 0.1390.09 0.9590.68

0.1090.05 0.3990.17

60–64 2.3291.00 24.1911.4

0.772

Pvalue for sex B0.001 0.704 0.070 B0.001 0.038 B0.001 B0.001

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A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239 236

Fig. 1. Risk of carotid and femoral artery atherosclerosis according to quintiles ofa- andb-carotene (n=392) Odds ratios of carotid and femoral atherosclerosis decreased with increasing plasmaa+b-carotene concentrations. The horizontal bars represent 95% confidence intervals.

Table 3

Mean plasma values of antioxidant according to various categories of cigarette smoking and alcohol consumption (n=392)a

Cigarette smoking (number of cigarettes/day)

Antioxidant 0 1–10 11–20 \20 Pvalueb

2.3990.96

Retinol (mmol/l) 2.8790.83 2.4290.91 2.8791.24 0.230

30.1910.0 23.798.05

24.290.37 27.0910.7

a-Tocopherol (mmol/l) 0.015

1.4691.70 0.7090.43 0.6890.86

a+bCarotene (mmol/l) 1.0190.70 0.022

1.5490.60 1.2990.54 1.2290.56

1.4290.67 0.287

Other carotenoids (mmol/l)

Alcohol consumption (g/day)

1–10

Antioxidant 0 11–20 \20 Pvaluea

2.4890.95

Retinol (mmol/l) 2.2490.90 2.9391.12 2.9190.17 0.011

24.799.73 27.3913.2

23.998.73 26.9914.5

a-Tocopherol (mmol/l) 0.086

1.0690.81 0.6090.42 0.4090.03

a+bCarotene (mmol/l)1.0890.88 1.0890.88 0.013

1.4590.70 1.2590.55 1.2090.61

1.4190.59 0.341

Other carotenoids (mmol/l)

a_{Values are mean}₉_SD.

b_P_{values for differences of antioxidant levels across smoking or alcohol categories after adjustment for age and sex.}

Table 4

Correlation of plasma antioxidants levels with vascular risk factors

Variables Retinol a-Tocopherol Carotenoidsa+b Other Carotenoids

Women Men Women Men Women Men

Men Women

0.08 −0.02 0.13

Age −0.11 −0.09 −0.13 −0.04 −0.03

Total cholesterol 0.15 0.10 0.32* 0.24* 0.01 −0.07 0.19 0.01

−0.26* 0.33* 0.23*

Triglycerides 0.17 −0.24* −0.20 −0.09 −0.07

0.17 0.14 0.08 −0.28*

0.16 −0.21

Serum ferritin −0.12 −0.13

−0.2

Systolic BP −0.03 0.09 −0.01 −0.09 −0.10 −0.03 −0.13

−0.02 0.02 0.03 −0.19 −0.11 0.01 −0.08

C-reactive protein −0.02

0.00 −0.03 0.06 −0.16

−0.07 −0.17

Fibrinogen −0.10 −0.13

−0.11 0.13 −0.06 −0.17

Body mass index 0.03 −0.23* −0.02 −0.24*

0.17 0.08 0.11 −0.32*

0.17 0.04

Alcohol g/day −0.14 0.15

Physical activity −0.03 −0.04 −0.05 0.01 0.11 0.09 0.02 0.09

0.04 −0.05 0.09 −0.10

−0.12 −0.21

HbA1c −0.15 −0.07

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atherosclerotic plaque may differ and awaits future clarification.

Our study failed to obtain a significant relation be-tween plasma concentrations of vitamin E and atherosclerosis. In the EVA study, in contrast, erythro-cyte vitamin E was negatively associated with common carotid artery intima-media thickness in elderly volun-teers (59 – 71 years) [18]. In addition, there is limited epidemiological evidence from the ARIC study to show that high nutritional intake of a-tocopherol protects against carotid atherosclerosis [28]. Studies of the asso-ciation between vitamin E intake/levels and onset of cardiovascular disease yielded contradictory results [5,6,8,10,12,13,37 – 39].

There are several potential reasons for the inconsisten-cies in the results of the above studies: 1) The method of vitamin E determination was highly variable. The EVA study measured erythrocyte vitamin E, which reflects long-term dietary intake better than do plasma concentrations. Other surveys attempted to quantify vitamin E content in nutrition as a surrogate of the actually important plasma levels. 2) Levels of vitamin E in various populations are highly variable based upon differences in diet. Likewise, distinct proportions of subjects have plasma levels low or high enough to be protective or deleterious. In the urban population of India [10], in whom low vitamin E significantly predicted an increased risk of cardiovascular disease, meana

-toco-pherol was 40% lower than in Bruneck. The Basel study in turn accorded with our survey in that the plasma concentration of vitamin E were usually high and a protective role of a-tocopherol could not be demon-strated [9]. Lack of significant associations in this and in our survey may be due to the low variation in vitamin E concentration and does not rule out the possibility of consistently high vitamin E levels contributing to a generally low rate of atherosclerosis in these populations. 3) Recently, vitamin E has been reported to exert antioxidant, neutral or even prooxidant (‘tocopherol-me-diated peroxidation’) activities, depending on the pres-ence of compounds which have not yet been fully characterized [40]. This finding may contribute to the inconsistencies in the results of previous surveys.

The merits of the current study include its randomized recruitment strategy encompassing men and women and the measurement of atherosclerosis in various vascular territories. All plasma sampling was performed within a limited time, between July and early October. Concentrations of antioxidants obtained are thus not subject to seasonal variations. Because samples were immediately processed and stored at −70°C for 3 months, variable storage conditions are unlikely to confound the results. The main weakness of the current analysis is the simultaneous assessment of antioxidant levels and atherosclerosis in the carotid and femoral arteries. However, plasma levels of carotenoids reflect Table 5

Association of plasma antioxidant levels with prevalent/incident atherosclerosis in the carotid and femoral arteries

Pvalue Odd ratios (95% CI) per 1 SD-unit changeb

Mean9SD

AS−a

Atherosclerosis AS+a

Pre6alent carotid atherosclerosis

2.4391.0

Retinol (mmol/l) 2.5951.0 1.06 (0.82–1.37) 0.63

a-Tocopherol (mmol/l) 24.199.4 26.3911.6 1.02 (0.99–1.04) 0.19 0.79 (0.65–0.96)

0.7790.5 0.02

1.0890.9

a+bCarotene (mmol/l)

0.48 0.92 (0.73–1.16)

1.3490.6 1.4390.7

Other carotenoids (mmol/l)

Pre6alent femoral atheroscleosis

2.4090.9 2.6191.2 0.81

Retinol (mmol/l) 1.03 (0.80–1.32)

23.898.5 26.9912.8 0.26

a-Tocopherol (mmol/l) 1.01 (0.99–1.04)

0.02 0.80 (0.66–0.95)

0.7390.6

a+bCarotene (mmol/l) 1.1090.8

0.98 (0.78–1.23) 0.83

1.4290.6

Other carotenoids (mmol/l) 1.3490.7 Carotid and femoral atherosclerosis

0.94 (0.70–1.27) 2.5791.2

2.4590.9 0.71

Retinol (mmol/l)

a-Tocopherol (mmol/l) 24.599.3 26.4913.7 1.01 (0.98–1.04) 0.65 0.004 1.0690.9 0.5090.4 0.59 (0.41–0.85)

a+bCarotene (mmol/l)

1.4290.6 1.2990.6

Other carotenoids (mmol/l) 0.92 (0.71–1.18) 0.48

Incident carotid atherosclerosis

2.4490.97 2.5791.06

Retinol (mmol/l) 1.05 (0.81–1.35) 0.69

0.99 1.00 (0.98–1.02)

25.9911.8

a-Tocopherol (mmol/l) 24.599.65

1.0490.86 0.7090.51

a+bCarotene (mmol/l) 0.58 (0.35–0.97) 0.04

1.4390.66 1.3190.58 0.80 (0.52–1.22)

Other carotenoids (mmol/l) 0.30

a_AS₋_{, no prevalent or incident atherosclerosis; AS}₊_{prevalent or incident atherosclerosis.}

b_{Odds ratio were derived from logistic regression analysis of prevalent}_/_{incident atherosclerosis on antioxidants and covariates (age, sex, LDL}

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A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239 238

long-term dietary habits that proved consistent in the Bruneck population over a longer period. When 1995 plasma levels were used as a surrogate for the 1990 levels and the analysis was repeated with a prospective design, the results were virtually unchanged (Table 5). In this context it is important to note that the inherent assessment variability of this ‘prospective’ approach may tend to bias results towards a null association but does not create spurious association. Theoretically, an artificial relation could emerge when atherosclerosis per se affects carotene levels. At this time, however, there are no data to support such a conjecture. As a further limit of our study (and of all previous epidemiological surveys in this field) it can not be ruled out that high carotene concentration acts as a marker of other ingre-dients of fruits and vegetables or of a healthy life style, rather than being protective in itself.

In conclusion, our study is among the first to docu-ment an inverse association between plasma levels ofa -and b-carotene and the risk of atherosclerosis in vari-ous vascular territories. It provides further support for the hypothesis that antioxidants protect against atherosclerosis and arterial disease and intends to stim-ulate further research efforts in this field.

Acknowledgements

We are grateful to Milena Minotto, Daniela Pizzuti and Carla Pasini Venturi for technical assistance and support. The study was supported by a grant from the Italian National Research Council (CNR), Progetto finalizzato FATMA.

References

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[38] Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett MC. Vitamin E consumption and the risk of coro-nary heart disease in men. N Engl J Med 1993;328:1450 – 6. [39] Spencer AP, Carson DS, Crouch MA. Vitamin E and coronary

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A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239

234

atherosclerosis according to quintiles of

a

+

b

-carotene.

This graph shows a gradual decrease in atherosclerosis

risk with increasing carotene levels (P

value for linear

trend, 0.0049). Split analyses in genders, in smokers and

non-smokers, and in abstainers and regular alcohol

consumers all yielded consistent relations between

atherosclerosis and

a

+

b

-carotene levels (data not

pre-sented). Thus, alcohol consumption and cigarette

smok-ing neither confounded nor modified the association

obtained. Other carotenoids did not significantly

pre-dict the risk of atherosclerosis. This finding applied to

all subfractions (cryptoxanthin, etc.) and to their sum

(‘other

carotenoids’).

Likewise,

retinol

and

a

-tocopherol were unrelated to atherosclerosis in our

population (Table 5) as was the

a

-tocopherol/choles-terol ratio.

4. Discussion

Oxidative modifications of LDL are widely accepted

to represent a key event in lipid-induced atherogenesis.

The efficacy of lipid peroxidation depends on LDL

composition, residence time of LDL in the

sub-en-dothelial space and, most important, the balance of

pro-oxidants involving ferrous iron [19] and

antioxi-dants. Carotenoids, a group of lipid-soluble

pro-vita-mins contained in LDL particles, are experimentally

well-founded to protect against lipid peroxidation [24 –

26].

b

-carotene represents 25 – 50% of total carotenoids

and may be the fraction with the most pronounced

antioxidant effects [27].

The current population study is the first to

demon-strate a strong inverse association between

a

+

b

-carotene

plasma

levels

and

the

prevalence

of

atherosclerosis in carotid and femoral arteries. This

association was internally consistent in genders and

across the whole age range (45 – 65 years) and emerged

as independent of other vascular risk factors. It was

particularly relevant to cigarette smokers and heavy

drinkers, whose

a

+

b

-carotene levels were markedly

reduced (Table 3). As for other carotenoids, we

ob-served a tendency towards lower atherosclerosis risk in

subjects with high plasma levels, though statistical

sig-nificance was not achieved.

To the best of our knowledge, there is only one

previous epidemiological study addressing the potential

protective effects of carotenoids on atherogenesis. This

study attempted to quantify nutritional carotene intake

and revealed an inverse association of this variable with

early stages of carotid atherosclerosis in men [28]. A

protective role of carotene in atherogenesis is further

substantiated by an animal model [4] and various

stud-ies on symptomatic coronary artery disease: With a few

exceptions [10,13] low plasma

b

or total carotene

pre-dicted an increased cardiovascular risk [9,11,12,29].

In-tervention trials, however, exhibited effects of a regular

intake of 20 – 50 mg of synthetic

b

-carotene on

cardio-vascular risk and mortality that were at times beneficial

[30,31], at others non-existent [32] or even unfavorable

[33,34]. Several clues to explain this discrepancy may be

inferred from the inconsistencies observed between and

within observational and interventional studies. i)

Plasma antioxidant concentrations are the result of

long-term dietary habits. In clinical intervention trials

the period of antioxidant supplementation may be too

short for lipid-soluble antioxidants to accumulate in the

lipid core of plaques [14]. ii) Commercial supplements

of

b

-carotene were higher in the trans- than in the

cis-isomer, whereas natural

b

-carotene showed an

op-posite ratio. Recently, the nine-cis form of

b

-carotene

was shown to have a higher antioxidant capacity in

vitro [35] and in vivo [36]. Furthermore, the ability of

these isomers to accumulate in the lipid core of

Table 1

Characteristics of the study population (n=392)a

Women (n=187) Men (n=205) 53.995.6

Age (years) 54.195.9

0.69890.47 1.27190.975

Carotenoidsa+b,mmol/l

Other Carotenoids,mmol/l 1.50490.681 1.29990.492 Retinol,mmol/l 2.28990.911 2.64291.039

24.999.4

Tocopherol,mmol/l 24.8910.9

25.293.7

Body mass index, kg/m2 _25.8₉_3.4 142.4917.7 Systolic blood pressure, 145.2921.0

mmHg

87.299.7

Diastolic blood pressure, 86.498.3 mmHg

5.8491.02 Serum total cholesterol, 6.1191.02

mmol/l

Serum HDL cholesterol, 1.6290.41 1.4190.40 mmol/l

3.5991.07 Serum LDL cholesterol, 3.8790.91

mmol/l

1.6290.27 1.290.7

Apolipoprotein A-I, g/l

1.1790.32 Apolipoprotein B, g/l 1.1390.30

1.3490.73

Serum Triglycerides, mmol/l 1.5191.04 2.6790.59 Fibrinogen, g/l 2.7890.56

Serum Ferritin,mg/l 58.7973.8 191.09176.1 0.05390.006 0.005490.007 HbA1c%, proportion of Hb

5.3%

Diabetes mellitus 4.9%

20.3%

Current smokers 32.2%

Hypertension 29.9% 23.9%

High social status 10.2% 28.8%

Physical acti6ity

0 h/week 21.4% 29.3%

40.1%

B2 h/week 43.4%

\2 h/week 38.5% 27.3%

Alcohol categories

15.6%

0 g/day 56.7%

42.8% 51.2%

1–50 g/day

51–100 g/day 0.5% 17.1%

\100 g/day 0.0% 16.1%

(2)

Atherosclerosis

153

(2000)

231

–

239

235

(mmol/l) (mmol/l)

(mmol/l)

(mmol/l) (mmol/l) (mmol/l) (mmol/l)

Men

0.3190.13 0.0990.03 0.2190.14 0.6690.35 0.1290.13 0.5990.47

2.8790.92 24.0910.4

45–49

0.2990.22 0.5890.28 0.1390.13 0.6490.36

0.1090.06

50–54 2.5090.93 20.698.80 0.3390.15

0.1090.05

55–59 2.5890.93 22.099.57 0.3690.20 0.2290.15 0.6790.38 0.1190.11 0.5890.40

0.2390.17

60–64 2.5191.22 19.8914.6 0.3590.19 0.1090.05 0.5390.34 0.0990.06 0.5490.30

Women

0.3190.21 0.7690.49 0.2490.36

2.1490.81 1.1590.91

45–49 20.698.70 0.3790.21 0.0990.06

0.3690.15 0.1090.05 0.3690.19 0.7190.29 0.1990.17 1.1190.67

2.3690.95 21.899.08

50–54

0.3690.21 0.6690.33 0.1990.32 1.1190.81

55–59 2.3590.91 23.498.98 0.3890.20 0.1090.08

0.3790.27 0.6290.42 0.1390.09 0.9590.68

0.1090.05 0.3990.17

60–64 2.3291.00 24.1911.4

0.772

Pvalue for sex B0.001 0.704 0.070 B0.001 0.038 B0.001 B0.001

(3)

A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239

236

Table 3

Mean plasma values of antioxidant according to various categories of cigarette smoking and alcohol consumption (n=392)a Cigarette smoking (number of cigarettes/day)

Antioxidant 0 1–10 11–20 \20 Pvalueb

2.3990.96

Retinol (mmol/l) 2.8790.83 2.4290.91 2.8791.24 0.230

30.1910.0 23.798.05

24.290.37 27.0910.7

a-Tocopherol (mmol/l) 0.015

1.4691.70 0.7090.43 0.6890.86

a+bCarotene (mmol/l) 1.0190.70 0.022

1.5490.60 1.2990.54 1.2290.56

1.4290.67 0.287

Other carotenoids (mmol/l)

Alcohol consumption (g/day)

1–10

Antioxidant 0 11–20 \20 Pvaluea

2.4890.95

Retinol (mmol/l) 2.2490.90 2.9391.12 2.9190.17 0.011

24.799.73 27.3913.2

23.998.73 26.9914.5

a-Tocopherol (mmol/l) 0.086

1.0690.81 0.6090.42 0.4090.03

a+bCarotene (mmol/l)1.0890.88 1.0890.88 0.013

1.4590.70 1.2590.55 1.2090.61

1.4190.59 0.341

Other carotenoids (mmol/l)

a_{Values are mean}₉_SD.

b_P_{values for differences of antioxidant levels across smoking or alcohol categories after adjustment for age and sex.} Table 4

Correlation of plasma antioxidants levels with vascular risk factors

Variables Retinol a-Tocopherol Carotenoidsa+b Other Carotenoids

Women Men Women Men Women Men

Men Women

0.08 −0.02 0.13

Age −0.11 −0.09 −0.13 −0.04 −0.03

Total cholesterol 0.15 0.10 0.32* 0.24* 0.01 −0.07 0.19 0.01

−0.26* 0.33* 0.23*

Triglycerides 0.17 −0.24* −0.20 −0.09 −0.07

0.17 0.14 0.08 −0.28*

0.16 −0.21

Serum ferritin −0.12 −0.13

−0.2

Systolic BP −0.03 0.09 −0.01 −0.09 −0.10 −0.03 −0.13

−0.02 0.02 0.03 −0.19 −0.11 0.01 −0.08

C-reactive protein −0.02

0.00 −0.03 0.06 −0.16

−0.07 −0.17

Fibrinogen −0.10 −0.13

−0.11 0.13 −0.06 −0.17

Body mass index 0.03 −0.23* −0.02 −0.24*

0.17 0.08 0.11 −0.32*

0.17 0.04

Alcohol g/day −0.14 0.15

Physical activity −0.03 −0.04 −0.05 0.01 0.11 0.09 0.02 0.09

0.04 −0.05 0.09 −0.10

−0.12 −0.21

HbA1c −0.15 −0.07

(4)

Our study failed to obtain a significant relation

be-tween

plasma

concentrations

of

vitamin

E

and

atherosclerosis. In the EVA study, in contrast,

erythro-cyte vitamin E was negatively associated with common

carotid artery intima-media thickness in elderly

volun-teers (59 – 71 years) [18]. In addition, there is limited

epidemiological evidence from the ARIC study to show

that high nutritional intake of

a

-tocopherol protects

against carotid atherosclerosis [28]. Studies of the

asso-ciation between vitamin E intake/levels and onset of

cardiovascular disease yielded contradictory results

[5,6,8,10,12,13,37 – 39].

There are several potential reasons for the

inconsisten-cies in the results of the above studies: 1) The method

of vitamin E determination was highly variable. The

EVA study measured erythrocyte vitamin E, which

reflects long-term dietary intake better than do plasma

concentrations. Other surveys attempted to quantify

vitamin E content in nutrition as a surrogate of the

actually important plasma levels. 2) Levels of vitamin E

in various populations are highly variable based upon

differences in diet. Likewise, distinct proportions of

subjects have plasma levels low or high enough to be

protective or deleterious. In the urban population of

India [10], in whom low vitamin E significantly predicted

an increased risk of cardiovascular disease, mean

a

-toco-concentration of vitamin E were usually high and a

protective role of

a

-tocopherol could not be

demon-strated [9]. Lack of significant associations in this and in

our survey may be due to the low variation in vitamin

E concentration and does not rule out the possibility of

consistently high vitamin E levels contributing to a

generally low rate of atherosclerosis in these populations.

3) Recently, vitamin E has been reported to exert

antioxidant, neutral or even prooxidant

(‘tocopherol-me-diated peroxidation’) activities, depending on the

pres-ence of compounds which have not yet been fully

characterized [40]. This finding may contribute to the

inconsistencies in the results of previous surveys.

The merits of the current study include its randomized

recruitment strategy encompassing men and women

and the measurement of atherosclerosis in various

vascular territories. All plasma sampling was performed

within a limited time, between July and early October.

Concentrations of antioxidants obtained are thus not

subject to seasonal variations. Because samples were

immediately processed and stored at

−

70°C for 3

months, variable storage conditions are unlikely to

confound the results. The main weakness of the current

analysis is the simultaneous assessment of antioxidant

levels and atherosclerosis in the carotid and femoral

arteries. However, plasma levels of carotenoids reflect

Table 5

Association of plasma antioxidant levels with prevalent/incident atherosclerosis in the carotid and femoral arteries

Pvalue Odd ratios (95% CI) per 1 SD-unit changeb

Mean9SD

AS−a

Atherosclerosis AS+a

Pre6alent carotid atherosclerosis

2.4391.0

Retinol (mmol/l) 2.5951.0 1.06 (0.82–1.37) 0.63

a-Tocopherol (mmol/l) 24.199.4 26.3911.6 1.02 (0.99–1.04) 0.19

0.79 (0.65–0.96)

0.7790.5 0.02

1.0890.9

a+bCarotene (mmol/l)

0.48 0.92 (0.73–1.16)

1.3490.6 1.4390.7

Other carotenoids (mmol/l)

Pre6alent femoral atheroscleosis

2.4090.9 2.6191.2 0.81

Retinol (mmol/l) 1.03 (0.80–1.32)

23.898.5 26.9912.8 0.26

a-Tocopherol (mmol/l) 1.01 (0.99–1.04)

0.02 0.80 (0.66–0.95)

0.7390.6

a+bCarotene (mmol/l) 1.1090.8

0.98 (0.78–1.23) 0.83

1.4290.6

Other carotenoids (mmol/l) 1.3490.7

Carotid and femoral atherosclerosis

0.94 (0.70–1.27) 2.5791.2

2.4590.9 0.71

Retinol (mmol/l)

a-Tocopherol (mmol/l) 24.599.3 26.4913.7 1.01 (0.98–1.04) 0.65

0.004 1.0690.9 0.5090.4 0.59 (0.41–0.85)

a+bCarotene (mmol/l)

1.4290.6 1.2990.6

Other carotenoids (mmol/l) 0.92 (0.71–1.18) 0.48

Incident carotid atherosclerosis

2.4490.97 2.5791.06

Retinol (mmol/l) 1.05 (0.81–1.35) 0.69

0.99 1.00 (0.98–1.02)

25.9911.8

a-Tocopherol (mmol/l) 24.599.65

1.0490.86 0.7090.51

a+bCarotene (mmol/l) 0.58 (0.35–0.97) 0.04

1.4390.66 1.3190.58 0.80 (0.52–1.22)

Other carotenoids (mmol/l) 0.30

a_{AS−, no prevalent or incident atherosclerosis; AS+}_{prevalent or incident atherosclerosis.}

b_{Odds ratio were derived from logistic regression analysis of prevalent/incident atherosclerosis on antioxidants and covariates (age, sex, LDL} cholesterol, ferritin, systolic blood pressure, categories of alcohol consumption, social status, C-reactive protein, cigarette smoking).

(5)

A.D’Odorico et al./Atherosclerosis153 (2000) 231 – 239

238

long-term dietary habits that proved consistent in the

Bruneck population over a longer period. When 1995

plasma levels were used as a surrogate for the 1990

levels and the analysis was repeated with a prospective

design, the results were virtually unchanged (Table 5).

In this context it is important to note that the inherent

assessment variability of this ‘prospective’ approach

may tend to bias results towards a null association but

does not create spurious association. Theoretically, an

artificial relation could emerge when atherosclerosis per

se affects carotene levels. At this time, however, there

are no data to support such a conjecture. As a further

limit of our study (and of all previous epidemiological

surveys in this field) it can not be ruled out that high

carotene concentration acts as a marker of other

ingre-dients of fruits and vegetables or of a healthy life style,

rather than being protective in itself.

In conclusion, our study is among the first to

docu-ment an inverse association between plasma levels of

a

-and

b

-carotene and the risk of atherosclerosis in

vari-ous vascular territories. It provides further support for

the

hypothesis

that

antioxidants

protect

against

atherosclerosis and arterial disease and intends to

stim-ulate further research efforts in this field.

Acknowledgements

We are grateful to Milena Minotto, Daniela Pizzuti

and Carla Pasini Venturi for technical assistance and

support. The study was supported by a grant from the

Italian National Research Council (CNR), Progetto

finalizzato FATMA.

References