Dietary and circulating antioxidant vitamins in relation to carotid plaques in middle-aged women

¹ From the Dietology Unit, A Cardarelli Hospital, Naples (AI); the Epidemiology Unit, National Cancer Institute, Naples (EC); and the Departments of Clinical and Experimental Medicine (SP, RG, GC, MDM, and PR) and Biochemistry and Biotechnologies in Medicine (LS and FZ), Federico II University, Naples.

² Supported by funds from the Consiglio Nazionale delle Ricerche "Progetto finalizzato Biotecnologie," Rome; PRIN 1997, Ministero dell’Università e della Ricerca scientifica e tecnologica; and Regione Campania "Fondi ricerca sanitaria finalizzata." The Progetto Atena was supported by funds from the Consiglio Nazionale delle Ricerche "Progetto finalizzato FATMA."

³ Address reprint requests to P Rubba, Department of Clinical and Experimental Medicine, Federico II University, Via S Pansini, 5, 80131 Naples, Italy. E-mail: rubba{at}unina.it.

ABSTRACT  
Background: The results of the few studies conducted on the relation between antioxidant vitamins and carotid atherosclerosis have been inconclusive.

Objective: We evaluated the association between preclinical carotid atherosclerosis, as determined by high-resolution B-mode ultrasound, and both the intake amounts and plasma concentrations of antioxidant vitamins.

Design: Among 5062 participants in Progetto Atena, a population-based study on the etiology of cardiovascular disease and cancer in women, 310 women were examined by B-mode ultrasound to detect early signs of carotid atherosclerosis. The participants answered a food-frequency questionnaire, and their plasma concentrations of vitamin E, vitamin A, and carotenoids were measured. None of the women took vitamin supplements.

Results: The occurrence of atherosclerotic plaques at the carotid bifurcation was inversely associated with tertiles of vitamin E intake; the test for a linear trend across tertiles was significant (P < 0.05). Similarly, the ratio of plasma vitamin E to plasma cholesterol was inversely related to the presence of plaques at the carotid bifurcation; the test for a linear trend across tertiles was significant (P < 0.02). No association was found between the intake of other antioxidant vitamins (vitamins A and C and carotenoids) or their plasma concentrations and the presence of carotid plaques.

Conclusions: An inverse association was found between both the intake amount and plasma concentration of vitamin E and preclinical carotid atherosclerosis in middle-aged women. This association was independent of other cardiovascular risk factors, was not related to vitamin supplements, and supports the hypothesis that low vitamin E intake is a risk factor for early atherosclerosis.

Key Words: Vitamin E • antioxidants • carotid arteries • carotid bifurcation • atherosclerosis • ultrasound • Progetto Atena • women

INTRODUCTION  
In the oxidation hypothesis of atherosclerosis, antioxidant protection may play a role in preventing the formation of early lesions (1). However, the wealth of data regarding the preventive role of dietary or serum antioxidants has yielded conflicting results (2–7). A possible explanation for this inconsistency is that most authors considered overt ischemia (myocardial infarction, angina, stroke, transient ischemic attack) rather than preclinical atherosclerosis as an endpoint.

Carotid intima-media thickness (IMT) is a well-recognized marker of early, generalized atherosclerosis and is widely used in epidemiologic studies (8, 9). Increased IMT is correlated with coronary atherosclerosis (10). The relation between dietary antioxidants and the thickness of carotid artery walls has been evaluated in a few studies, some focusing on intakes and others on serum concentrations (11–14).

However, the relation between antioxidant vitamin intake, plasma vitamin concentrations, and carotid atherosclerosis has not yet been evaluated. The aim of this study was to investigate whether the dietary intake of antioxidant vitamins and the plasma concentrations of vitamin A, carotenoids, and vitamin E are associated with the presence of plaques in the common carotid arteries and carotid bifurcation in a sample of middle-aged women.

SUBJECTS AND METHODS  
Subjects
Progetto Atena is a prospective study carried out in the area of Naples. The general objective of the project is to investigate the causes of chronic diseases that have a major impact on women. The total study cohort, who were enrolled over a 4-y period, comprised 5062 healthy women aged 30–69 y (15). Potential participants with a previous diagnosis of myocardial infarction, stroke, or major cancers were excluded. During a 6-mo period, the oldest 3 participants (potentially at higher risk for carotid lesions) among those scheduled for the day were asked to undergo a free vascular examination (high-resolution ultrasound of the carotid arteries); 310 women underwent this additional investigation. In 3 women, vitamin intakes and concentrations were not determined; thus, the remaining 307 women were included in the present analysis. The study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983, and all of the women gave their informed, written consent.

Questionnaires
The choice of information collected through questionnaires (15) about demographic background, occupation, medical history, drug use, and personal habits such as smoking and alcohol consumption was largely based on the type of information that was collected in the major Italian population studies on cardiovascular disease. In particular, usual dietary intakes, defined as the average intake over the past year, were estimated with the use of a 138-item semiquantitative food-frequency questionnaire that was designed on the basis of several validity and reliability studies (16, 17). A random sample (10%) of the cohort was interviewed with the use of a 24-h recall method to improve the final estimation of frequency and quantity (18, 19).

The questionnaires were administered by trained interviewers. Participants were asked how often, on average, they consumed a specified portion size of each food during the previous year.

Daily nutrient intake was calculated by multiplying the nutrient content of the specified portion of each food item by the frequency of its daily consumption and then summing the results of all the items. Food values for energy and vitamins were taken from the food composition tables and databases for epidemiologic studies in Italy (20).

Clinical and biochemical assessment
Standard procedures were used for blood pressure measurements, and the training and supervision of observers were identical with those used in similar epidemiologic studies (21). Body mass index was calculated as weight (in kg) divided by height squared (in m²).

To reduce the influence of circadian variation, all blood specimens were collected between 0800 and 0930 after the subjects had fasted overnight. Samples were processed for determination of serum cholesterol and triacylglycerol by enzymatic methods (22, 23). HDL cholesterol was precipitated by phosphotungstate (24). LDL cholesterol was calculated according to the Friedewald formula.

RESULTS  
Of the 307 women who participated in the present study, 12% had atherosclerotic plaques localized only at the common carotid artery, 25% had plaques only at the carotid bifurcation, 29% had plaques on both sites, and 34% had no plaques. The cardiovascular risk profile of the Progetto Atena study population, divided into the subsample who underwent a high-resolution ultrasound examination of the carotid arteries (age range: 35–69 y) and the remaining cohort, is shown in Table 1. After adjustment for age, there were no significant differences in cardiovascular risk profile between the subsample and the remaining cohort.

View this table:
TABLE 1 . Cardiovascular risk profile of the Progetto Atena study population (n = 5062 women) divided into the subsample who underwent a high-resolution ultrasound examination of the carotid artery and the remaining cohort¹  
The daily dietary intakes of antioxidant vitamins, the plasma antioxidant vitamin concentrations, and the daily total energy values of the study sample are shown in Table 2. None of the women took dietary supplements containing antioxidants. Only the women who were in the highest tertile of vitamin E intake had a dietary intake in the range suggested by the 10th edition of Recommended Dietary Allowances (32).

View this table:
TABLE 2 . Daily dietary intakes of antioxidant vitamins, plasma antioxidant vitamin concentrations, and daily total energy values of the study sample (n = 307 women)¹  
Pearson’s correlation coefficient between plasma vitamin E and plasma total cholesterol was 0.59 (P < 0.001). Spearman’s correlation coefficient between the dietary intake of vitamin E and the ratio of the plasma concentrations of vitamin E and cholesterol was 0.15 (P < 0.01).

The percentages of women with plaques at the carotid bifurcation or at the common carotid artery are shown in Table 3 by tertile of daily intake of vitamin C, vitamin A, and vitamin E; by tertile of plasma concentrations of vitamin A and carotenoids; and by tertile of the ratio of plasma vitamin E to plasma cholesterol. Also shown in this table are the associations between plaques at these 2 locations and the various tertiles after adjustment for age, smoking status, body mass index, systolic blood pressure, alcohol intake, and glucose concentration.

View this table:
TABLE 3 . Percentages of women (n = 307) with plaques at the carotid bifurcation or at the common carotid artery and odds ratios (ORs) by tertile of daily intake of vitamin C, vitamin A, and vitamin E; by tertile of plasma concentrations of vitamin A and carotenoids; and by tertile of the ratio of plasma vitamin E to plasma cholesterol¹  
In logistic regression analyses, there was a significant inverse association between tertiles of vitamin E intake and the presence of plaques at the carotid bifurcation; the linear trend across tertiles was significant (P < 0.05). In a separate analysis on postmenopausal women (226 of 307), after adjustment for age, smoking status, body mass index, systolic blood pressure, alcohol intake, and glucose concentration, there was a significant association between the presence of plaques at the carotid bifurcation and the lowest tertile of vitamin E intake (OR: 2.79; 97.5% CI: 1.10, 7.07). The linear trend across tertiles was significant (P < 0.02). In the postmenopausal group only, 7 women were undergoing hormone replacement therapy. No consistent association was found between the intake of vitamins A (expressed as mg retinol equivalents) or C and the presence of carotid plaques.

A significant association was found between low ratios of plasma vitamin E to plasma cholesterol and ultrasound evidence of plaques at the carotid bifurcation. The lowest tertile had an OR of 2.16 (97.5% CI: 1.06, 4.39). There was a significant linear trend across tertiles (P < 0.02). In the subgroup analysis of postmenopausal women, the OR of the lowest tertile was 2.01 (97.5% CI: 0.90, 4.51) and the P value for the linear trend across tertiles was 0.06.

There was no significant association between different tertiles of plasma vitamin A concentrations and carotid plaques, even when considering the ratio of plasma vitamin A to plasma cholesterol: the ORs and 97.5% CIs for the middle and lowest tertiles were 1.72 (0.85, 3.48) and 1.32 (0.67, 2.61), respectively. No association was found between either the intakes or the plasma concentrations of antioxidant vitamins and the presence of plaques in the common carotid artery.

DISCUSSION  
To our knowledge, this is the first study that combines information on the dietary intakes of antioxidant vitamins and determination of their plasma concentrations with high-resolution ultrasound imaging of carotid atherosclerosis. It shows an inverse association between both the intake and the plasma concentration of vitamin E and the presence of carotid plaques, independently of other conventional risk factors.

Only a few studies have addressed the relation between antioxidant vitamins and carotid atherosclerosis. However, the methods used in those studies were quite different from ours: some studies focused on antioxidant vitamin intake as assessed by food-frequency questionnaires, some analyzed serum concentrations, and some verified the effect of vitamin E supplements on carotid atherosclerosis. In a cross-sectional study of the Atherosclerosis Risk in Communities (ARIC) population, dietary consumption of vitamins C and E was inversely associated with the thickness of the carotid artery wall in older women, although the use of dietary supplements partly accounted for this association (11). In a case-control study of subjects selected from the ARIC study cohort, serum -tocopherol concentrations were unrelated to carotid IMT (14). The effect of supplementary antioxidant vitamin intake on carotid atherosclerosis was analyzed in primates with experimentally induced atherosclerosis (33) and in humans and yielded conflicting results: reduced progression of carotid IMT (34), no effect on carotid IMT changes in women but a beneficial effect of vitamin E + vitamin C in men (35), and a neutral effect on the progression of atherosclerosis (36). Vitamin E supplementation had various effects with regard to the modification of cardiovascular risk in patients with clinically overt heart diseases (37–41).

In the present study, a low dietary intake of vitamin E was associated with an increased risk of plaques at the carotid bifurcation, particularly in postmenopausal women, whereas no relation was found for plaques at the common carotid artery. From the analysis of the Progetto Atena food-frequency questionnaires and on the basis of dietary survey data (42), women in southern Italy obtain their vitamin E mainly from fresh vegetables, legumes, and olive oils. In addition, there is a high intake of monounsaturated fatty acids from olive oil, whereas the consumption of polyunsaturated fatty acids is relatively low (43).

A possible explanation for the association between vitamin E and plaques at the carotid bifurcation and for the lack of association for plaques at the common carotid artery is that early atherosclerosis usually begins at the bifurcations. The common carotid artery and the carotid bifurcation have different geometries, shear stresses, extracellular matrices, and cell compositions, and the mechanisms that regulate lesion development are also different. In addition, atherosclerotic plaques are known to develop mostly at the carotid bifurcation, where there is, on average, a greater wall thickness and therefore a higher likelihood of atherosclerotic lesion development (44).

Interesting data also come from the analysis of plasma antioxidant vitamin concentrations. It should be kept in mind that in epidemiologic studies it is important to adjust the plasma concentrations of vitamins A and E in relation to the plasma concentrations of lipoproteins. In particular, plasma concentrations of vitamin E are markedly influenced by both LDL and VLDL concentrations not only in hyperlipidemic patients but also in the general population. A simple way of dealing with this problem is to use the ratio of vitamin E to total cholesterol (27).

In the present study, lower plasma concentrations of vitamin E (adjusted for cholesterol) were found to be associated with an increased risk of plaques at the carotid bifurcation. Other plasma antioxidant vitamin concentrations (and the plasma ratio of vitamin A to cholesterol) showed no association with carotid plaques.

We could not rule out the possibility that the intake and the plasma concentration of vitamin E could reflect a healthier diet and that something other than vitamin E may be responsible for the apparent benefit on the development of carotid plaques. However, the lack of association between higher intakes of vitamin E or plasma concentrations of other antioxidant vitamins (ie, vitamins A and C, which are also considered markers of a healthier diet) and carotid plaques argue against this hypothesis. It should be remembered that none of the women in our study population took vitamin supplements.

Although recent studies on the association between vitamin E and atherosclerosis have been inconclusive, we have provided consistent evidence that both low intakes and low plasma concentrations of vitamin E are associated with early atherosclerotic carotid lesions. Therefore, a possible explanation for the benefit of vitamin E intake found in epidemiologic studies on healthy people without established atherosclerotic lesions and for the lack of benefit in clinical trials studying the effect of vitamin E supplementation in patients with clinically overt cardiovascular disease may be that the vascular protection occurs at early stages of atherosclerosis. The evidence of detectable arterial damage in the subjects in the lowest tertile of vitamin E intake and plasma concentration indicates that only individuals with an inadequate intake or a low plasma concentration of vitamin E are expected to benefit from an increase of vitamin E intake, either through dietary changes or supplementation. Thus, before advising subjects to change their diet or take antioxidant vitamin supplements, it would be helpful to evaluate their intakes and plasma concentrations, because knowing which individuals have the lowest intakes or plasma concentrations of vitamin E would help us identify those who could benefit the most from this type of intervention (45).

REFERENCES  

1. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witzum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915–24.
2. Gey KF, Puska P, Jordan P, Moser UK. Inverse correlation between plasma vitamin E and mortality from ischemic heart disease in cross-cultural epidemiology. Am J Clin Nutr 1991;53(suppl):326S–34S.
3. Riemersma RA, Wood DA, Macintyre CCH, Elton RA, Gey KF, Oliver MF. Risk of angina pectoris and plasma concentrations of vitamin A, C and E and carotene. Lancet 1991;337:1–5.
4. Diaz MN, Frei B, Vita JA, Keaney JF Jr. Antioxidants and atherosclerotic heart disease. N Engl J Med 1997;337;408–16.
5. Gaziano JM, Manson JE, Buring JE, Hennekens CH. Dietary antioxidants and cardiovascular disease. Ann N Y Acad Sci 1992;669:249–59.
6. Jha P, Flather M, Lonn E, Farkouh M, Yusuf S. The antioxidant vitamins and cardiovascular disease. A critical review of epidemiologic and clinical trial data. Ann Intern Med 1995;123:860–72.
7. Steinberg D. Clinical trials of antioxidants in atherosclerosis: are we doing the right thing? Lancet 1995;346:36–8.
8. Greenland P, Abrams J, Aurigemma GP, et al. Prevention Conference V: beyond secondary prevention: identifying the high-risk patient for primary prevention: non-invasive tests of atherosclerotic burden: Writing Group III. Circulation 2000;101:E16–22.
9. Heiss G, Sharret AR, Barnes R, Chambless LE, Szklo M, Alzola C. Carotid atherosclerosis measured by B-mode ultrasound in populations: associations with cardiovascular risk factors in the ARIC study. Am J Epidemiol 1991;134:250–6.
10. O’Leary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Wolfson SK Jr. Carotid artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group. N Engl J Med 1999;340:14–22.
11. Kritchevsky SB, Shimakawa T, Tell GS, et al. Dietary antioxidants and carotid artery wall thickness. The ARIC study. Circulation 1995;92:2142–50.
12. Raal FJ, Pilcher GJ, Veller MG, Kotze MJ, Joffe BI. Efficacy of vitamin E compared with either simvastatin or atorvastatin in preventing the progression of atherosclerosis in homozygous familial hypercholesterolemia. Am J Cardiol 1999;84:1344–6.
13. Bonithon-Kopp C, Coudray C, Berr C, et al. Combined effects of lipid peroxidation and antioxidant status on carotid atherosclerosis in a population aged 59–71 y: the EVA Study. Etude sur le Veillisement Arteriel. Am J Clin Nutr 1997;65:121–7.
14. Iribarren C, Folsom AR, Jacobs DR, Gross MD, Belcher JD, Eckfeldt JH. Association of serum vitamin levels, LDL susceptibility to oxidation, and autoantobodies against MDA-LDL with carotid athersoclerosis. A case-control study. The ARIC Study Investigators. Atherosclerosis Risk in Communities. Arterioscler Thromb Vasc Biol 1997;17:1171–7.
15. Panico S, Dello Iacovo R, Celentano E, et al. Progetto Atena, a study on the etiology of major chronic diseases in women: design, rationale and objectives. Eur J Epidemiol 1992;8:601–8.
16. Trevisan M, Krogh V, Ferro-Luzzi A, et al. Questionario alimentare per gli studi epidemiologici con coorti di grande numerosità da condurre in Italia. (Food questionnaire for epidemiological studies on large cohorts for use in Italy.) Ann Ist Super Sanita 1992;28:397–401 (in Italian).
17. Celentano E, Galasso R, Panico S. L’indagine nutrizionale in studi su grandi numeri di persone: necessità di integrazione tra accuratezza e semplicità. (Nutritional research in studies of large numbers of people: the need for integrating accuracy and simplicity.) Ann Ist Super Sanita 1992;28:403–8 (in Italian).
18. Kaaks R, Riboli E, van Staveren W. Calibration of dietary intake measurements in prospective cohort studies. Am J Epidemiol 1995;142:548–56.
19. Wahrendorf J, Boeing H, Heinemann L, et al. Results from a comparative dietary assessment in Europe: II. feasibility of pooling individual-based dietary data between countries. Eur J Clin Nutr 1989;43:379–90.
20. Salvini S, Parpinel M, Gnagnarella P, Maissonneuve P, Turrini A. Banca dati di composizione degli alimenti per studi epidemiologici in Italia. (Food composition tables and databases for epidemiological studies in Italy.) Milan, Italy: Istituto Europeo di Oncologia, 1997 (in Italian).
21. The ARIC Investigators. The Atherosclerosis Risk In Communities (ARIC) Study: design and objectives. Am J Epidemiol 1989;129:687–702.
22. Siedel J, Schlumberger H, Klose S, Ziegenhorn J, Wahlefeld AW. Improved reagent for enzymatic determination of serum cholesterol. J Clin Chem Clin Biochem 1981;19:838–9.
23. Wahlefeld AW. Triglyceride determination after enzymatic hydrolysis. In: Bergmeyer HU, Gawehn K, eds. Methods of enzymatic analysis. 2nd ed. Weinheim: Verlag Chemie, 1974:1831–974.
24. Lopes-Virella MF, Stone P, Ellis S, Colwell JA. Cholesterol determination in high-density lipoproteins separated by three different methods. Clin Chem 1977;23:882–4.
25. Vuilleumier JP, Keller HE, Gysel D, Hunziker F. Clinical chemical methods for the routine assessment of the vitamin status in human populations. Part I: the fat-soluble vitamins A and E, and beta-carotene. Int J Vitam Nutr Res 1983;53:265–72.
26. Belisario MA, Azar G, Oriani G, Pizzuti GP, Sacchetti L. Simultaneous evaluation of vitamins A and E in human plasma by normal phase HPLC. Boll Soc Ital Biol Sper 1993;69:641–7.
27. Rubba P, Mancini M, Fidanza F, Leccia G, Riemersma RA, Gey KF. Plasma vitamin E, apolipoprotein B and HDL-cholesterol in middle-aged men from southern Italy. Atherosclerosis 1989;77:25–9.
28. Bond MG, Strickland HL, Wilmoth SK, Safrit A, Phillips R, Szostack L. Interventional clinical trials using noninvasive ultrasound end points: the Multicenter Isradipine/Diuretics Atherosclerosis Study. The MIDAS Research Group. J Cardiovasc Pharmacol 1990;15(suppl): S30–3.
29. Crouse JR III, Byington RP, Bond MG, et al. Pravastatin, lipids and atherosclerosis in the carotid arteries (PLAC II). Am J Cardiol 1995;75:455–9.
30. Mercuri M, Bond MG, Sirtori CR, et al. Pravastatin reduces intima-media thickness progression in an asymptomatic hypercholesterolemic Mediterranean population: the Carotid Atherosclerosis Italian Ultrasound Study. Am J Med 1996;101:627–34.
31. Pauciullo P, Iannuzzi A, Sartorio R, et al. Increased intima-media thickness of the common carotid artery in hypercholesterolemic children. Arterioscler Thromb 1994;14:1075–9.
32. National Research Council. Recommended dietary allowances. 10th ed. Washington, DC: National Academy Press, 1989.
33. Verlangieri AJ, Bush MJ. Effects of d-alpha-tocopherol supplementation on experimentally induced primate atherosclerosis. J Am Coll Nutr 1992;11:131–8.
34. Azen SP, Qian D, Mack WJ, et al. Effect of supplementary antioxidant vitamin intake on carotid arterial wall intima-media thickness in a controlled clinical trial of cholesterol lowering. Circulation 1996;94:2369–72.
35. Salonen JT, Nyyssonen K, Salonen R, et al. Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) study: a randomized trial of the effects of vitamin E and C on 3-year progression of carotid atherosclerosis. J Intern Med 2000;248:377–86.
36. Lonn EM, Yusuf S, Dzavik V, et al. Effects of ramipril and vitamin E on atherosclerosis: the study to evaluate carotid ultrasound changes in patients treated with ramipril and vitamin E (SECURE). Circulation 2001;103:919–25.
37. Stephens NG, Parsons A, Schofield PM, Kelly F, Cheeseman K, Mitchison MJ. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study. Lancet 1996;347:781–6.
38. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto miocardico. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSI-Prevenzione trial. Lancet 1999;354:447–55.
39. Yusuf S, Dagenais G, Pogue J, Bosch J, Sleight P. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med 2000;342:154–60.
40. Ascherio A, Rimm EB, Hernan MA, et al. Relation of consumption of vitamin E, vitamin C, and carotenoids to risk for stroke among men in the United States. Ann Intern Med 1999;130:963–70.
41. Rimm EB, Stampfer MJ, Ascherio A, Giovannucci E, Colditz GA, Willett WC. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 1993;328:1450–6.
42. Fidanza F, Rubba P, Cozzolino G. Food habits of a traditional Mediterranean population in southern Italy—socioeconomic influences. Nutr Metab Cardiovasc Dis 1991;1:71–6.
43. Mancini M, Rubba P. The Mediterranean diet in Italy. World Rev Nutr Diet 2000;87:114–26.
44. Stary HC, Blankenhorn DH, Chandler AB, et al. A definition of the intima of human arteries and of its atherosclerotic-prone regions. Arterioscler Thromb 1992;12:120–34.
45. Krauss RM, Eckel RH, Howard B, et al. AHA dietary guidelines: revision 2000: a statement for healthcare professionals from the Nutrition Committee of the American Heart Association. Circulation 2000;102:2284–99.