High-resolution B-mode ultrasound has proven to be a valid and reliable method to detect and monitor changes in carotid intima-media thickness (IMT), a marker of subclinical atherosclerosis 1–3. An increased carotid IMT has been shown to correlate with the occurrence of atherosclerotic lesions in other vascular territories 4,5 and has been related to clinical events such as stroke and myocardial infarction in prospective studies 6,7. Furthermore, many population-based and case–control studies showed an increased carotid IMT in participants with ‘traditional’ and ‘nontraditional’ cardiovascular risk factors 8–13.
Controversy remains on the relationship between thyroid function and atherosclerotic risk. Both increased and decreased serum thyroid stimulating hormone (TSH) levels have been associated with an increased risk of carotid atherosclerosis 14–17. Furthermore, in normal, nonthyroxine-taking patients, the relationship between carotid IMT and TSH levels is doubtful 18,19.
In a currently ongoing female population-based study, we evaluated whether TSH levels were associated with subclinical carotid atherosclerosis.
Participants and methods
We studied a cohort of 310 middle-aged women living in the area of Naples (Southern Italy) and participating in a prospective population-based survey, the ‘Progetto ATENA’ study (n=5062) 20. Ten percent of the original cohort was randomly selected from the electoral roles, the rest were volunteers. The general objective of this study was to investigate the causes of those chronic diseases that have a major impact on the female population. Potential participants with previous diagnosis of myocardial infarction, stroke, and neoplastic disease were excluded. Myocardial infarctions and strokes were ascertained by questions and, additional information, including results of cardiac-enzyme measurements, serial ECG, cranial computed tomography, and cerebral MRI were obtained from medical records. Patients with viral or bacterial acute diseases affecting blood and urine biochemistry were asked to delay their participation. From the original cohort (n=5062), 85 participants with a history of previous thyroid disease or taking T4 were excluded.
During a 6-month period, the older three of 10 daily participants (those potentially at higher risk of atherosclerotic carotid lesions) were invited to undergo an ultrasound examination of the carotid arteries. Of the 400 women invited, 310 (response rate 77%) accepted the additional investigations and constituted the present study sample. After age adjustment, there were no statistically significant differences between the sample of women who underwent carotid ultrasound evaluation and the remaining cohort of Progetto ATENA with regard to BMI, systolic and diastolic blood pressure, the concentrations of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and the prevalence of current smokers (data not shown). The study was approved by the ethical committees of the institutions involved, and the participants recruited provided informed consent.
Clinical and biochemical assessment
Anthropometric measurements of the participants were taken with their indoor clothing and without shoes. BMI, used as a measure of general obesity, was calculated as weight (kg) divided by height (m2). Waist/hip ratio, an index of abdominal obesity, was calculated as the ratio of waist/hip circumferences. Brachial blood pressure was measured twice in a sitting position after a resting period of at least 5 min using a random zero sphygmomanometer.
Blood specimens were collected after a 12–14 h fast, between 8:00 and 9:30 a.m., to reduce the influence of circadian variation. Total cholesterol and triglyceride concentrations were measured using standard enzymatic methods 21,22. HDL cholesterol was measured after precipitation of VLDL and LDL cholesterol with phosphotungstic acid 23, and LDL cholesterol was calculated according to the Friedewald formula. Fasting glucose levels were enzymatically determined by the hexokinase method. Fasting insulin levels, used as a surrogate measure of insulin resistance, were determined by enzyme immunoassay (Boehringer Mannheim Immunodiagnostics on ES 300 instrument, Mannheim, Germany).
Serum TSH was measured in µU/ml using an ultrasensitive immunoradiometric assay (Immunotech) in an automatic analyzer (Beckman Coulter Inc., Brea, California, USA).
Prevalent hypertension was defined as systolic blood pressure of more than 140 mmHg or diastolic blood pressure of more than 90 mmHg or use of antihypertensive drugs. Hypercholesterolemia was defined by a total cholesterol level of more than 200 mg/dl or the use of lipid lowering drugs and hypertriglyceridemia by a triglyceride level of more than 160 mg/dl. Diabetes mellitus was defined if the participant reported a medical history of the disease or use of antidiabetic drugs or with fasting glucose levels of more than 126 mg/dl. The National Cholesterol Education Program Adult Treatment Panel III criteria 24 were used to classify women as having the metabolic syndrome on the basis of the presence of at least three of the following risk factors: (a) waist circumference of more than 88 cm, (b) fasting triglycerides of at least 150 mg/dl, (c) HDL cholesterol less than 50 mg/dl, (d) hypertension (systolic blood pressure≥130 mmHg, diastolic blood pressure≥85 mmHg), and (e) fasting glucose of at least 110 mg/dl.
High-resolution carotid ultrasound
Carotid B-mode ultrasound examinations were performed using the Biosound 2000 II SA equipped with an 8 MHz annular array mechanical transducer (Esaote, Indianapolis, Indiana, USA). This system provides high-resolution ultrasonic images with 0.3 mm axial resolution. Scans were performed according to a standardized protocol developed by the Division of Vascular Ultrasound Research at the Wake Forest University School of Medicine 25. The key features of the protocol were the identification of two anatomical landmarks, that is the dilation of the bulb and flow divider, and the use of different scanning angles, that is anterior, lateral, and posterior, to measure IMT in three carotid segments: the distal 1 cm of the common carotid artery, the carotid bifurcation, and the proximal 1.0 cm of the internal carotid artery. Statistical analyses were based on mean IMT of the near and far wall for three carotid segments for both sides (means of 12 sites).
The occurrence and the number of atherosclerotic plaques, defined as localized echo structures encroaching into the vessel lumen with an IMT of more than 1.2 mm, were used as other ultrasound end-points of the study. A cut-off point of 1.2 mm for IMT was chosen because it corresponded to the 90th percentile of the mean IMT in a random sample of 170 Neapolitan women 11.
In our vascular laboratory, which takes part in interventional clinical trials using ultrasound, the within-subject coefficient of variation previously reported for IMT is less than 6%.
Statistical analyses were performed using the statistical package for social sciences (SPSS version 17.0; SPSS Inc., Chicago, Illinois, USA).
Continuous variables were described as mean and SD and discrete variables were reported as percentage. Logarithmic transformation of triglycerides and insulin was performed to normalize the distribution before analyses. Univariate comparisons between women in the upper decile of TSH and others were carried out using Student’s t-test for continuous variables and the distribution of categorical variables was analyzed using the χ 2-test. Pearson’s and Spearman’s correlation coefficients were used to describe the association of cardiovascular risk factors with carotid mean IMT. Analysis of covariance was performed to examine the association of carotid IMT and plaque number with TSH levels, using age, triglycerides, LDL cholesterol, HDL cholesterol, BMI, and systolic blood pressure entered as covariates. A logistic regression analysis was also performed to test the relationship between TSH levels and increased IMT, after adjustment for diabetes mellitus, hypertriglyceridemia, low HDL cholesterol, waist/hip ratio, and hypertension.
In our sample, the TSH range was 0.15–11.10 μU/ml. The clinical and biochemical characteristics of the study participants stratified according to TSH deciles (upper decile vs. 1–9 deciles) are presented in Table 1. Women in the upper TSH decile (n=30; TSH>3.13 mU/l) had a significantly higher prevalence of diabetes mellitus and hypertriglyceridemia compared with women in the other TSH deciles (Table 2).
Carotid mean IMT was significantly correlated with BMI (r=0.21), LDL cholesterol (r=0.17), HDL cholesterol (r=0.16), triglycerides (r=0.22), systolic blood pressure (r=0.37), and age (r=0.50).
In a multivariate analysis, carotid IMT values were significantly greater in the higher TSH decile women group (0.92±0.02 vs. 0.86±0.01 mm; P=0.037) compared with women in the other deciles. Women in the upper TSH decile had an increased risk of carotid IMT (odds ratio 16.0, 95% confidence interval 1.08–237.9, P=0.04), after adjustment for comorbidities.
The prevalence of carotid plaques was not significantly different in the two groups (75 vs. 68%, P=0.29), whereas the total number of plaques was higher in the group of women in the upper TSH decile (2.3±0.3 vs. 1.7±0.1; P=0.025) (Fig. 1).
In a Mediterranean female population, we found a positive, independent association of TSH levels with carotid artery IMT and the number of atherosclerotic plaques, indicators of subclinical atherosclerosis.
Previous studies have examined a possible association between thyroid function and atherosclerotic risk, mainly in patients with overt or subclinical hyperthyroidism or hypothyroidism 14–17. A few data have been obtained in a population-based sample. Our results are in agreement with and complement the findings of a recent analysis carried out in 643 Japanese adults with euthyroid status, showing that both free thyroxine and TSH levels were associated with carotid IMT and suggesting an increased cardiovascular risk in participants with low normal thyroid function 19. In contrast to these results, in the Tromso study, no significant relationship was found between carotid IMT and serum TSH levels in normal, nonthyroxine-taking individuals, whereas carotid IMT was increased in participants taking thyroxine 18.
Noninvasive, ultrasound-based assessment of carotid wall structure is increasingly performed to identify individuals with asymptomatic subclinical atherosclerosis 3. However, as there remains no clear consensus on which carotid parameter constitutes the best indicator of vascular risk and studies emphasize the predictive value of plaque thickness and number 26,27, in the present analysis we used different ultrasound end-points.
In our cohort of women, the prevalence of carotid plaques was higher than in other female population-based surveys 28,29. The diverse definition of atherosclerotic plaque adopted in our study and a worse cardiovascular risk profile may account for the disparity in values.
Our finding of a different association of THS levels with carotid IMT and plaque has no clear explanation, but could be related to the evidence that processes leading to IMT and plaque formation may not be similar 30, and patterns of risk factors may be different. In a population of hyperlipidemic patients, average IMT was significantly greater in patients having overt hypothyroidism than in patients with subclinical hypothyroidism, whereas the prevalence of carotid plaques was higher in patients with subclinical hypothyroidism than in patients having overt hypothyroidism 31.
The mechanism that could link TSH levels with carotid IMT and plaque number within our euthyroid population remains to be elucidated. The positive relationship remained statistically significant after adjustment for lipid parameters, smoking, and other traditional atherosclerotic risk factors. In patients with elevated TSH levels (subclinical hypothyroidism), recent evidences indicate a high mean platelet volume and platelet distribution width values 32, an increased oxidative stress 33, and a reduced endothelium-dependent vasodilation 34, all factors potentially involved in the pathogenesis of atherosclerosis.
Some major limitations of the present study should be taken into account. Small transient increases in TSH are common within the population and often revert to normal; thus, whether these individuals have a sustained increase in TSH is not known. As an association with TSH would likely be mediated by changes in circulating thyroid hormone concentrations, the lack of measurement of total or free T4 on the same specimens is a limitation of our study, as are lack of thyroid antibodies and evaluation of the iodine status of the population. In a cross-sectional and relatively small epidemiological study, an association may be found, and it is only longitudinal data with hard outcomes that will determine whether this is a true association. We are carrying out a follow-up study in these women and, in the near future, this information could be available.
Conflicts of interest
The ‘Progetto Atena’ was supported by funds from the Consiglio Nazionale delle Ricerche (Rome, Italy), ‘Progetto finalizzato Biotecnologie’, and ‘Progetto finalizzato FATMA’; the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) 1998, no. 9806174392-008; Fondazione Banco di Napoli.
1. Pignoli P, Tremoli E, Poli A, Oreste P, Paoletti R. Intimal plus medial thickness of the arterial wall: a direct measurement with ultrasound imaging. Circulation. 1986;74:1399–1406
2. Kanters SD, Algra A, van Leeuwen MS, Banga J. Reproducibility of in vivo
carotid intima-media thickness measurements. Stroke. 1997;28:665–671
3. Greenland P, Abrams J, Aurigemma GP, Bond MG, Clark LT, Crique MH, et al. Prevention conference V: beyond secondary prevention: identifying the high risk patient for primary prevention. Circulation. 2000;101:111–116
4. Bots MI, Hofman A, Grobbee DE. Carotid intima-media thickness and lower extremity arterial atherosclerosis. The Rotterdam study. Arterioscler Thromb. 1994;14:1885–1891
5. Crouse JR, Craven TE, Hagaman AP, Bond MG. Association of coronary disease with segment-specific intimal-medial thickening of the extracranial carotid artery. Circulation. 1995;92:1141–1147
6. Bots ML, Hoes AW, Koudstaal PJ, Hofman A, Grobbee DE. Common carotid intima-media thickness and risk of stroke and myocardial infarction: the Rotterdam study. Circulation. 1997;96:1432–1437
7. O’ Leary DH, Polak JF, Kronmal R, Manolio TA, Burke GL, Wolfson SK. Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. N Engl J Med. 1999;340:14–22
8. Heiss G, Sharett 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;134:250–256
9. Folsom AR, Wu KK, Shahar E, Davis CE. Association of hemostatic variables with prevalent cardiovascular disease and asymptomatic carotid artery atherosclerosis. Arterioscler Thromb. 1993;13:1829–1836
10. Gaeta G, De Michele M, Cuomo S, Guarini P, Foglia MC, Bond MG, Trevisan M. Arterial abnormalities in the offspring of patients with premature myocardial infarction. N Engl J Med. 2000;343:840–846
11. Rubba P, Panico S, Bond MG, Covetti G, Celentano E, Iannuzzi A, et al. Site-specific atherosclerotic plaques in the carotid arteries of middle-aged women from Southern Italy. Associations with traditional risk factors and oxidation markers. Stroke. 2001;32:1953–1959
12. Pauciullo P, Giannino A, De Michele M, Gentile M, Liguori R, Argiriou A, et al. Increased carotid artery intima-media thickness is associated with a novel mutation of low-density lipoprotein receptor independently of major cardiovascular risk factors. Metabolism. 2003;52:1433–1438
13. De Michele M, Iannuzzi A, Panico S, Celentano E, Sacchetti L, Mazzaccara C, et al. Effect of high-density lipoprotein cholesterol levels on carotid artery geometry in a Mediterranean female population. Eur J Cardiovasc Prev Rehabil. 2004;11:403–407
14. Hak AE, Pols HA, Visser TJ, Drexhage HA, Hofman A, Witteman JC. Subclinical hypothyroidism is an independent risk factor for atherosclerosis and myocardial infarction in elderly women: the Rotterdam study. Ann Intern Med. 2000;132:270–278
15. Monzani F, Caraccio N, Kozakowa M, Dardano A, Vittone F, Virdis A, et al. Effect of levothyroxine replacement on lipid profile and intima-media thickness in subclinical hypothyroidism: a double-blind, placebo-controlled study. J Clin Endocrinol Metab. 2004;89:2099–2106
16. Volzke H, Robinson DM, Schminke U, Ludemann J, Rettig R, Felix SB, et al. Thyroid function and carotid wall thickness. J Clin Endocrinol Metab. 2004;89:2145–2149
17. Dorr M, Empen K, Robinson DM, Wallaschofski H, Felix SB, Volzke H. The association of thyroid function with carotid artery plaque burden and strokes in a population-based sample from a previously iodine-deficient area. Eur J Endocrinol. 2008;159:145–152
18. Jorde R, Joakimsen O, Stensland E, Mathiesen EB. Lack of significant association between intima-media thickness in the carotid artery and serum TSH level. The Tromso Study. Thyroid. 2008;18:21–25
19. Takamura N, Akilzhanova A, Hayashida N, Kadota K, Yamasaki H, Usa T, et al. Thyroid function is associated with carotid intima-media thickness in euthyroid subjects. Atherosclerosis. 2009;204:e77–e81
20. Panico S, Dello Iacovo R, Celentano E, Galasso R, Muti P, Salvatore M, Mancini M. Progetto ATENA, a study on the etiology of major chronic diseases in women: design, rationale and objectives. Eur J Epidemiol. 1992;8:601–608
21. Siedel J, Schlumberger H, Klose S, Ziegenhorn J, Wahlefeld AW. Improved reagent for enzymatic determination of serum cholesterol. J Clin Chem Biochem. 1981;19:838–839
22. Wahlefeld AWBergmeyer HU. Triglyceride determination after enzymatic hydrolysis. Methods of enzymatic analysis. 1974.2nd ed. Weinheim Verlag Chemie and New York Academic Press:1831
23. Lopes-Virella MF, Stone P, Ellis S, Colwell JA. Cholesterol determination in high-density lipoproteins separated by three different methods. Clin Chem. 1977;23:882–884
24. . Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel III): final report. Circulation. 2002;106:3143–3421
25. 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. J Cardiovasc Pharmacol. 1990;13:S30–S33
26. Rundek T, Arif H, Boden-Albala B, Elkind MS, Paik MC, Sacco RL. Carotid plaque, a subclinical precursor of vascular events: the Northern Manhattan Study. Neurology. 2008;70:1200–1207
27. Stork S, van den Beld AW, von Schacky C, Angermann CE, Lamberts SW, Grobbee DE, Bots ML. Carotid artery plaque burden, stiffness, and mortality risk in elderly men: a prospective, population-based cohort study. Circulation. 2004;110:344–348
28. Bonithon-Koop C, Scarabin PY, Taquet A, Touboul PJ, Almejac A, Guize L. Risk factors for early carotid atherosclerosis in middle-aged French women. Arterioscler Thromb. 1991;11:966–972
29. Lassila HC, Sutton Tyrrel K, Matthews KA, Wolfson SK, Kuller LH. Prevalence and determinants of carotid atherosclerosis in postmenopausal women. Stroke. 1997;28:513–517
30. Finn A, Kolodgie F, Virmani R. Correlation between carotid intimal/medial thickness and atherosclerosis. A point of view from pathology. Arterioscler Thromb Vasc Biol. 2010;30:177–181
31. Chiche F, Jublanc C, Coudert M, Carreau V, Kahn JF, Bruckert E. Hypothyroidism is not associated with increased carotid atherosclerosis when cardiovascular risk factors are accounted for in hyperlipidemic patients. Atherosclerosis. 2009;203:269–276
32. Erikci A, Karagoz B, Ozturk A, Caglayan S, Ozisik G, Kaygusuz I, Ozata M. The effect of subclinical hypothyroidism on platelet parameters. Hematology. 2009;14:115–117
33. Torun AN, Kulaksizoglu S, Kulaksizoglu M, Pamuk BO, Isbilen E, Tutuncu NB. Serum total antioxidant status and lipid peroxidation marker malondialdehyde levels in overt and subclinical hypothyroidism. Clin Endocrinol (Oxf). 2009;70:469–474
34. Biondi B, Galderisi M, Pagano L, Sidiropulos M, Pulcrano M, D’ Errico A, et al. Endothelial-mediated coronary flow reserve in patients with mild thyroid hormone deficiency. Eur J Endocrinol. 2009;161:323–329
Keywords:© 2013Wolters Kluwer Health Lippincott Williams Wilkins
carotid atherosclerosis; thyroid stimulating hormone; ultrasound; women