Tracking of Noninvasive Ultrasound Measurements of Subclinical Atherosclerosis in Adulthood: Findings from the Cardiovascular Risk in Young Finns Study

Ultrasound in Med. & Biol., Vol. 36, No. 8, pp. 1237–1244, 2010 Copyright Ó 2010 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter

doi:10.1016/j.ultrasmedbio.2010.05.003

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Original Contribution TRACKING OF NONINVASIVE ULTRASOUND MEASUREMENTS OF SUBCLINICAL ATHEROSCLEROSIS IN ADULTHOOD: FINDINGS FROM THE CARDIOVASCULAR RISK IN YOUNG FINNS STUDY JUHO R. H. RAIKO,* COSTAN G. MAGNUSSEN,*x MIKA KA¨HO¨NEN,k TOMI LAITINEN,{ LEENA TAITTONEN,#** JORMA S. A. VIIKARI,y OLLI T. RAITAKARI,z* and MARKUS JUONALAy * Research Center of Applied and Preventive Cardiovascular Medicine, University of Turku and Turku University Hospital, Turku, Finland; y Department of Medicine, University of Turku and Turku University Hospital, Turku, Finland; z Department of Clinical Physiology, University of Turku and Turku University Hospital, Turku, Finland; x Menzies Research Institute, University of Tasmania, Hobart, Australia; k Department of Clinical Physiology, University of Tampere and Tampere University Hospital, Tampere, Finland; { Department of Clinical Physiology, Kuopio University Hospital, Kuopio, Finland; # Department of Pediatrics, University of Oulu, Oulu, Finland; and ** Vaasa Central Hospital, Vaasa, Finland (Received 5 January 2010; revised 1 April 2010; in final form 3 May 2010)

Abstract—We examined tracking of ultrasound measurements of vascular structure and function in adulthood using data collected in the 2001 and 2007 follow-ups of Cardiovascular Risk in Young Finns Study. B-mode ultrasound measures of carotid artery intima-media thickness (IMT), carotid artery distensibility (CDist) and brachial artery flow-mediated dilatation (FMD) was obtained on 1809 apparently healthy Finnish adults aged 24 to 39 years in 2001 (1014 females; 795 males). Significant 6-year tracking was observed for IMT (males, r 5 0.56; females, r 5 0.46), CDist (males, r 5 0.35; females, r 5 0.36) and FMD (males, r 5 0.23; females, r 5 0.20). Subjects with 10-year risk of CVD (according to the SCORE risk score) above sex-specific median had improved IMT (r 5 0.44; r 5 0.57, p 5 0.0001) and CDist (r 5 0.31; r 5 0.40, p 5 0.03) tracking compared with those below median. Body mass index (BMI) $ 30 kg/m2 decreased tracking of CDist (r 5 0.36; r 5 0.19, p 5 0.01). In conclusion, ultrasound measurements tracked low to moderate over 6-years and was influenced by cardiovascular disease (CVD) risk factor status. (E-mail: [email protected]) Ó 2010 World Federation for Ultrasound in Medicine & Biology. Key Words: Cardiovascular, Atherosclerosis, Ultrasound, Intima-media thickness, Carotid artery distensibility, Flow-mediated dilatation, Tracking.

(CVD) in asymptomatic adults (Naghavi et al. 2006), early detection of atherosclerosis with imaging techniques may allow more effective therapeutic responses (Smith Jr. et al. 2000). Vascular ultrasound measurements seem suitable for clinical and research settings due to noninvasiveness and relation to clinical outcomes and CVD risk factors (Lane et al. 2006; Oliver and Webb 2003). Some of the most used methods are measurements of carotid intima-media thickness (IMT), carotid artery distensibility (CDist) and brachial artery flow-mediated dilatation (FMD). IMT is a well established structural marker of atherosclerosis that has been shown to predict coronary artery events (Lorenz et al. 2007) and extent of atherosclerosis (de Groot et al. 2008). CDist, the ratio between change in arterial diameter from diastolic to systolic and pulse pressure, is a marker of large artery elasticity (Oliver and Webb2003). Lower CDist signifies increased vascular

INTRODUCTION Subclinical atherosclerotic changes are evident already in early childhood (Stary et al. 1994). Depending on risk factors, these changes might then progress into more developed atherosclerotic lesions during adolescence and young adulthood (McGill Jr. et al. 2000). Atherosclerotic progression can be highly unpredictable among individuals with comparable risk factors (Naghavi et al. 2006) and conventional risk factors explaining less than 50% of variability in quantitative measures of atherosclerosis (Peyser et al. 2002). Since subclinical atherosclerosis is a major determinant of risk for cardiovascular disease

Address correspondence to: Juho R.H. Raiko, Research Center of Applied and Preventive Cardiovascular Medicine, University of Turku and Turku University Hospital, P.O. Box 52, 20521 Turku, Finland. E-mail: [email protected] 1237

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stiffness and CVD risk (Oliver and Webb 2003). Dysfunction of arterial endothelium is an early and potentially reversible phase in development of atherosclerosis that can be assessed by examining FMD in the brachial artery (Lane et al. 2006). There is currently no ideal test for detection and measurement of preclinical atherosclerotic changes in vasculature (Raitakari 1999; Raitakari and Celermajer 2000). Furthermore, the need for screening and treatment of subclinical atherosclerosis remains controversial (Santos and Nasir 2008; Simon et al. 2006) and few guidelines exist (Urbina et al. 2009). Carotid plaque detection has been suggested to be incorporated in models estimating future CVD event risk (De Backer et al. 2004). Currently, according to the AHA Prevention Conference V, carotid IMT measurements can add incremental information to risk assessment in asymptomatic subjects .45 years (Greenland et al. 2000). Objectives of this study were to assess the tracking of noninvasive ultrasound methods (IMT, CDist and FMD) in examination of subclinical atherosclerosis over a 6year period in early adulthood. In addition, we examined possible factors (body mass index (BMI), hypertension and CVD risk according to Framingham and SCORE risk scores) causing variability in tracking. Analyses were performed using data collected in the Cardiovascular Risk in Young Finns Study. METHODS Subjects The Cardiovascular Risk in Young Finns Study is an ongoing observational multicenter follow-up into CVD risk factors and atherosclerosis precursors from childhood to adulthood. The study was started in 1980 and has been performed by five Finnish universities. In 1980, 3596 Finns aged 3, 6, 9, 12, 15 and 18 years participated in the first cross-sectional survey. Since then, follow-ups for the whole study group have been performed in 1983, 1986, 2001 and 2007. In 2001, we performed vascular ultrasound studies in 2265 study subjects (1247 females; 1018 males) aged 24 to 39 years, and in 2007, ultrasound studies were performed in 2197 subjects (1209 females, 988 males) aged 30 to 45 years. There were 1809 ultrasound studies performed at both time points. Fifty-seven subjects were re-examined 3 months after the original visit in 2001 to assess the intra-individual reproducibility of the ultrasound studies. In 2001, the use of lipid-lowering (N 5 7) and antihypertensive medication (N 5 43) was rare in subjects participating in the ultrasound examinations for both 2001 and 2007. The subjects gave written informed consent in 2001 and 2007 per the World Medical Association Declaration of Helsinki. The study was approved by local ethics committee.

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Physical examination The physical examination consisted of the measurement of height, weight, systolic and diastolic blood pressure and waist and hip circumferences (Raiko et al. 2009). Height was measured with a Seca anthropometer to the nearest centimeter and weight was measured with Seca weighing scales to the nearest 0.1 kilogram. Body mass index (BMI) was calculated by dividing weight in kilograms by the square of height in meters. Waist circumference was taken at the point midway between the iliac crest and the lowest rib and hip circumference at the level of the greater trochanters as the average of two measurements with an accuracy of 0.1 cm. Blood pressure was measured at least three times with a random zero sphygmomanometer in sitting position after a 5 min rest. Blood pressure was estimated as the average of the three readings of systolic (SBP) and diastolic blood pressure (DBP). SBP $ 130 mmHg, DBP $ 85 mmHg or an existing diagnosis of hypertension were regarded as indications of hypertension. Lifestyle risk factors such as smoking were examined with questionnaires. Subjects that smoked on a daily basis were considered smokers. Blood tests In 2001 and 2007, venous blood samples were drawn primarily from the right antecubital vein after an overnight fast and serum was separated, aliquoted and stored at –70  C until analysis. If the sampling from the right arm failed the left antecubital vein was used. Serum total cholesterol levels were measured by the enzymatic cholesterol esterase, the cholesterol oxidase method (Cholesterol reagent, Olympus, Ireland) (Raiko et al. 2009). The same reagent was used for estimating high-density lipoprotein (HDL) cholesterol levels after precipitation of lowdensity lipoprotein (LDL) and very low-density lipoprotein (VLDL) cholesterol with dextran sulfate-Mg21 (Kostner 1976). LDL-cholesterol was estimated by the Friedewald formula (Friedewald et al. 1972) in subjects with triglycerides levels ,4.0 mmol. Serum triglyceride concentrations were assayed using the enzymatic glycerol kinase, the glycerol phosphate oxidase method (Triglyceride reagent, Olympus). The above mentioned analyses were all performed on an AU400-analyzer (Olympus, Tokyo, Japan). All the analyses were carried out in the Laboratory for Population Research of the National Institute for Health and Welfare (Turku, Finland). The following methods of the laboratory are accredited by the Finnish Accreditation Service according to standard ISO/IEC17025: total cholesterol, HDL-cholesterol and triglycerides. Ultrasound measurements In both follow-ups, ultrasound studies were performed on the carotid and brachial arteries with Acuson Sequoia 512 B-mode ultrasound mainframes (Acuson,

Cardiovascular Risk in Young Finns Study d J. R. H. RAIKO et al.

Mountain View, CA, USA) with 13.0 MHz linear array transducer. The left common carotid artery was scanned by ultrasound technicians following a standardized protocol. The image was focused on the posterior (far) wall, and gain settings were used to optimize image quality. A resolution box function (zoom) was used to record an image 25 mm wide and 15 mm high. A magnified image was recorded from an angle that showed the greatest distance between the lumen-intima interface and the media-adventitia interface. A moving scan with a duration of 5 s, which included the beginning of the carotid bifurcation and the common carotid artery, was recorded and stored in digital format on optical disks for subsequent off-line analysis. Digitally stored scans were manually analyzed by a single reader blinded to participants’ details, with analyses performed using ultrasonic callipers. From the 5-s clip image, the best-quality end-diastolic frame was selected (incident with the R wave on a continuously recorded electrocardiogram). Common carotid IMT was measured on the posterior wall of the left common carotid artery approximately 10 mm proximal to the carotid bifurcation. A minimum of four measurements were performed to calculate mean carotid IMT. CDist was assessed by measuring the common carotid artery diameter at end-diastole and end-systole. The proportional change between systolic and diastolic diameters was calculated and distensibility expressed as the ratio between change in diameter and pulse pressure derived from concomitant brachial blood pressure. CDist 5 [(systolic diameter – diastolic diameter)/ diastolic diameter]/pulse pressure. Brachial artery FMD was examined by measuring the left brachial artery diameter both at rest and during reactive hyperemia. Increased blood flow was induced by inflating a pneumatic tourniquet placed around the forearm to a pressure of 250 mm Hg for 4.5 min and then deflating the tourniquet. Measurement of arterial diameter was performed at end-diastole at a fixed distance from an anatomic marker at rest and 40, 60 and 80 s after cuff release. The vessel diameter during dilatation was expressed as the percentage relative to resting scan. The maximum diameter during dilatation was used in our analyses. In 2001, 57 participants were invited for reexaminations by ultrasound 3 months after the original measurements to assess reliability. The between-visit coefficients of variation (CV) for IMT, CDist and FMD were 6.4%, 16.3% and 26.0%, respectively (Juonala et al. 2008). Cardiovascular risk scores Framingham risk score depicts 10-year risk of CVD event (Anderson et al. 1991) and SCORE displays 10-year

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risk of fatal CVD event (Conroy et al. 2003). Framingham equation was developed using data on US cardiovascular research cohort (Anderson et al. 1991) and SCORE is based on European cohort (Conroy et al. 2003). Estimation of risk in Framingham risk score is based on age, smoking status, systolic blood pressure, total cholesterol, HDL-cholesterol and diabetes status and different equations exist for males and females (Anderson et al. 1991). SCORE algorithm was similar but excluded diabetes status and HDL-cholesterol (Conroy et al. 2003). Statistical methods Ultrasound measurements and risk factor levels are expressed as mean 6 SD unless stated otherwise. Comparisons of mean ultrasound measurement levels were performed with Mann Whitney test due to skewed distributions of IMT, CDist and FMD. Trends by age were examined with linear regression analyses. Tracking of ultrasound measurements Two approaches were used to examine tracking of vascular ultrasound measurements. First, IMT, CDist and FMD were divided into age- and sex-specific quintiles at both time-points and the probability of remaining in quintiles expressed. Second, the correlation between measurements in 2001 and 2007 was examined with Spearman’s partial correlation analysis. The partial correlation analyses were standardised for sex and age unless analyses were stratified by these variables. Tracking of conventional risk factors was examined with Spearman’s correlation. All studies were standardized by age except analyses on Framingham risk score that includes age as a CVD risk factor. The degree of tracking was estimated for correlation coefficients as follows: ,0.30 for low, 0.30–0.60 for moderate, 0.60–0.90 for moderately high and .0.90 for high (Ulmer et al. 2003). Factors affecting tracking of ultrasound measurements Spearman’s partial correlation models between ultrasound measurements in 2001 and 2007 were standardised by age and stratified by BMI, blood pressure and Framingham risk score groups in analyses concerning the effect of categorical risk factors on tracking. Comparisons between tracking correlation coefficients were examined using Fisher r-to-z transformation (Altman and Gardner 1988). Effect of cardiovascular risk was studied by measuring tracking in groups below and above sexspecific median of 10-year CVD risk according to Framingham (Anderson et al. 1991) and SCORE (Conroy et al. 2003) risk scores based on risk factors in 2001. Median for Framingham risk score was 0.66% in females and 1.70% in males and median for SCORE was 0.010% in females and 0.14% in males.

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Comparison of 3-month and 6-year tracking of ultrasound measurements Short-term reproducibility of ultrasound measurements was examined using Spearman’s partial correlations between ultrasound measurements in 2001 and the re-examination 3 months later and the measurements in 2007. Correlation analyses were also performed between mean values of the measurements in 2001 and the reexaminations and measurements 6 years later. Analyses were standardised by sex and age. All statistical analyses were performed using the SAS (version 9.1.3) software and statistical significance was inferred at a two-tailed p value ,0.05. All the correlation analyses were re-performed using Pearson’s correlation analysis with essentially similar results. RESULTS Table 1 contains baseline descriptive data for study subjects who had ultrasound measurements in 2001 and 2007. Table 2 shows parameter estimates and 95% confidence intervals from regression analyses for trend in ultrasound measurement by age. There was an increasing trend by age in IMT in both sexes and a decreasing trend in CDist in 2001 and 2007 (always p , 0.0001). No statistically significant age trends were observed for FMD in 2001 (p 5 0.83 in males, p 5 0.18 in females) or in 2007 (p 5 0.07 in males; p 5 0.15 in females). Correlation between ultrasound measurements and age did not differ between sexes in either follow-up (p for interaction always .0.05). Table 1. Descriptive data on study subjects in follow-up in 2001

N Age (years) BMI (kg/m2) SBP (mm Hg) DBP (mm Hg) Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triglycerides (mmol/l) Smokers Diabetics IMT (mm) CDist (%/ 10 mm Hg) FMD (%) Framingham score* (%) SCOREy (%)

Men

Women

794 31.9 25.7 6 4.0 121 6 12 73 6 11 5.19 6 0.95 3.43 6 0.92 1.15 6 0.27 1.50 6 0.95 209 (26.3%) 6 (0.8%) 0.59 6 0.09 2.00 6 0.77 6.84 6 4.50 2.53 6 2.65 0.22 6 0.26

1015 31.9 24.4 6 4.5 112 6 12 69 6 10 5.08 6 0.93 3.16 6 0.76 1.41 6 0.31 1.17 6 0.69 177 (17.4%) 6 (0.6%) 0.57 6 0.10 2.33 6 0.66 8.78 6 4.04 1.00 6 1.02 0.020 6 0.025

Statistics are mean 6 SD for continuous variables or N (percentage) for dichotomous variables. BMI 5 body mass index; SBP 5 systolic blood pressure; DBP 5 diastolic blood pressure; IMT 5 intima-media thickness; CDist 5 carotid artery distensibility; FMD 5 flow-mediated dilatation. *10-year risk for CVD event (Anderson et al.1991). y10-year risk for fatal CVD event (Conroy et al.2003).

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Tracking of ultrasound measurements Table 3 displays tracking of ultrasound measurements between 2001 and 2007 stratified by age and sex. Correlations in IMT (all p , 0.0001) and CDist (all p , 0.006) were significant in all subgroups. With the exception of nonsignificant correlations in 24-yearold females and 33-year-old males, FMD in 2001 was associated with FMD 6 years later (p always ,0.03). In analyses combining data on all age groups, Spearman’s nonpartial correlations were statistically significant in males and females for IMT (r 5 0.61, p , 0.0001 vs. r 5 0.52, p , 0.0001), CDist (r 5 0.41, p , 0.0001 vs. r 5 0.41, p , 0.0001) and FMD (r 5 0.23, p , 0.0001 vs. r 5 0.20, p , 0.0001). Similarly, correlations standardised by age were significant in both males and females for IMT (r 5 0.56, p , 0.0001 vs. r 5 0.46, p , 0.0001), CDist (r 5 0.35, p , 0.0001 vs. r 5 0.36, p , 0.0001) and FMD (r 5 0.23, p , 0.0001 vs. r 5 0.20, p , 0.0001). Figure 1 displays the probability of subjects to remain in their original fractile between follow-ups. Probabilities tended to be the highest in the lower and upper quintiles in both sexes. Males with IMT, CDist and FMD in the highest quintile in 2001 were most likely to maintain the same quintile in 2007 (57.8%, 38.5% and 31.9% respectively). Females in the fifth quintile for IMT and CDist (45.6% and 38.2%, respectively) and the first quintile for FMD (33.7%) were most likely to maintain their status in 2007. Factors affecting tracking of ultrasound measurements In males, 6-year tracking of IMT was better in those aged 33 to 39 years at baseline compared with those aged 24 to 30 years (r 5 0.60 vs. r 5 0.50, p 5 0.03). An apparent sex difference in tracking of IMT was observed, with males tracking more strongly compared with females (r 5 0.56 vs. 0.46, p 5 0.0059). Figures 2a and b display tracking of ultrasound measurements between 2001 and 2007 in BMI and SCORE risk score groups using risk factor levels in 2001. Tracking of CDist tended to be decreased in subjects with baseline BMI $ 30k g/m2 (r 5 0.36 vs. r 5 0.19, p 5 0.01). Tracking of IMT (r 5 0.44 vs. R 5 0.57, p 5 0.0001) and CDist (r 5 0.32 vs. r 5 0.40, P 5 0.03) were significantly higher in subjects with 10-year CVD risk above median according to SCORE risk score at baseline. Tracking of IMT displayed tendency toward increasing in subjects with 10-year CVD risk above median according to Framingham risk score (r 5 0.47 vs. r 5 0.53, p 5 0.06), whereas no difference was observed in tracking of CDist (r 5 0.32 vs. r 5 0.38, p 5 0.14) and FMD (r 5 0.21 vs. r 5 0.22, p 5 0.83). Tracking in normotensive and hypertensive subjects was equally strong (IMT: r 5 0.49 vs. 0.55 , P 5 0.14; CDist: r 5 0.33 vs.

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Table 2. The effect of age on ultrasound measures in 2001 and 2007

Carotid IMT (mm) Men Women Carotid distensibility (%/10 mm Hg) Men Women Brachial FMD (%) Men Women

2001

2007

b (95% CI) 0.0063 (0.0051 to 0.0074)* 0.0052 (0.0043 to 0.0061)*

b (95% CI) 0.0073 (0.0061 to 0.0085)* 0.0058 (0.0048 to 0.0067)*

20.041 (20.049 to 20.034)* 20.042 (20.050 to 20.033)*

20.031 (20.039 to 20.024)* 20.043 (20.051 to 20.035)* 20.04 (20.09 to 0.003) 20.04 (20.09 to 0.01)

0.01 (20.04 to 0.06) 0.04 (20.01 to 0.09)

The b-values are parameter estimates from regression analyses for 1-year increase in age. IMT 5 intima-media thickness; FMD 5 flow-mediated dilatation. *P , 0.0001

r 5 0.27, p 5 0.25; FMD: r 5 0.21 vs. r 5 0.23, p 5 0.65; p for all correlation coefficients ,0.0001).

difference between 3-month and 6-year tracking was nonsignificant in all methods.

Comparison of 3-month and 6-year tracking of ultrasound measurements Figure 3 demonstrates Spearman’s correlations between the original measurements in 2001 and the reexaminations conducted in 57 subjects 3 months later and the measurements among the same subjects in the follow-up in 2007. In IMT, there was little short-term variability but long-term tracking between 2001 and 2007 was only moderate (r 5 0.64 vs. r 5 0.49, p , 0.0003 in both). CDist demonstrated poorer reproducibility and tracking (r 5 0.51 vs. r 5 0.48, p , 0.0005 in both). We noticed significant short-term variability and moderate tracking in FMD (r 5 0.39 vs. r 5 0.36, p , 0.02 in both). The

DISCUSSION In this study, we observed that of the noninvasive ultrasound measurements, the consecutive IMT measurements had the strongest 6-year tracking in both sexes. IMT tracked better in males and its tracking was stronger in 33 to 39 year-old males than in 24 to 30-year-olds. IMT displayed the highest and FMD the lowest 3-month tracking. For IMT and CDist, tracking was higher in subjects with baseline 10-year CVD risk above median level according to SCORE risk score. Baseline BMI was associated with the tracking of CDist.

Table 3. Spearman’s correlation coefficients displaying correlation between ultrasound measurements in 2001 and 2007 stratified by sex and age IMT

CDist

FMD

Men

Men

Men

Age

n

r

p value

Age

n

r

p value

Age

n

r

p value

24 27 30 33 36 39

115 111 142 147 148 131

0.45 (0.29–0.58) 0.39 (0.22–0.54) 0.61 (0.50–0.70) 0.49 (0.36–0.60) 0.62 (0.51–0.71) 0.69 (0.59–0.77)

,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001

24 27 30 33 36 39

113 110 142 146 143 130

0.31 (0.13–0.47) 0.46 (0.30–0.60) 0.30 (0.14–0.44) 0.23 (0.07–0.38) 0.43 (0.29–0.55) 0.40 (0.25–0.54)

0.0007 ,0.0001 0.0003 0.0056 ,0.0001 ,0.0001

24 27 30 33 36 39

107 105 132 135 124 120

0.24 (0.05–0.41) 0.19 (20.01–0.37) 0.33 (0.17–0.47) 0.10 (20.07–0.26) 0.25 (0.08–0.41) 0.21 (0.03–0.38)

0.011 0.057 0.0001 0.24 0.0055 0.022

IMT

CDist

FMD

Women

Women

Women

Age

n

r

p value

Age

n

r

p value

Age

n

r

p value

24 27 30 33 36 39

132 170 159 199 190 165

0.52 (0.38–0.63) 0.44 (0.31–0.55) 0.33 (0.18–0.46) 0.45 (0.33–0.55) 0.48 (0.36–0.58) 0.59 (0.48–0.68)

,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001 ,0.0001

24 27 30 33 36 39

131 170 158 196 190 163

0.31 (0.15–0.46) 0.28 (0.14–0.41) 0.35 (0.21–0.48) 0.34 (0.21–0.46) 0.41 (0.29–0.52) 0.48 (0.35–0.59)

0.0003 0.0002 ,0.0001 ,0.0001 ,0.0001 ,0.0001

24 27 30 33 36 39

125 160 143 187 179 155

0.03 (20.15–0.20) 0.20 (0.05–0.34) 0.32 (0.17–0.46) 0.18 (0.04–0.32) 0.22 (0.08–0.36) 0.24 (0.09–0.38)

0.73 0.013 ,0.0001 0.012 0.0027 0.0029

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a

Females

IMT CDist FMD

65

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55

Correlation coefficient

Probability (%)

50 45 40 35 30 25 20 15 10 5 0 II

I

III

IV

1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

BMI<30 kg/m2 BMI ≥30 kg/m2

P=0.10

P=0.01 P=0.49

IMT

V

CDist

FMD

Quintile

b 70

Males

60

Correlation coefficient

IMT CDist FMD

65 55

Propability (%)

50 45 40 35 30 25 20 15

1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

CVD risk below median CVD risk above median

P=0.0001

P=0.03

P=0.97

IMT

10 5 0 I

II

III

IV

V

Quintile

Fig. 1. Probability of ultrasound variables to remain in the same fractile from 2001 to 2007.

IMT and CDist had moderate or moderately high 6year tracking. For FMD, tracking was low or moderate and poor tracking in FMD has been shown in other studies as well (Roman et al. 2006). Thus, predictive capabilities of FMD measurements are highly limited whereas IMT and CDist measurements at one time point reflect reasonably well the situation 6 years later. Furthermore, maximum FMD percentage change seems to be the most reproducible method for assessing FMD (Donald et al. 2008). According to our results on FMD, examination of endothelial dysfunction as a sole indicator of subclinical atherosclerosis would thus be unreliable. Nevertheless, FMD has shown similar hourly variation as blood pressure (Ja¨rvisalo et al. 2006). Tracking of IMT was stronger and more independent on interfering factors than tracking of CDist and FMD. Measurement of IMT is simply based on assessment of arterial structure whereas CDist and FMD are more functional and complex indices. Both blood pressure and arterial diameter are used to derive CDist (Juonala et al. 2008)

CDist

FMD

Fig. 2. (a) Correlation between ultrasound measurements in 2001 and 2007 in body mass index (BMI) groups (BMI , 30 kg/m2 and BMI $ 30 kg/m2) based on BMI values in 2001. Significant p values indicate a difference in correlation between BMI groups. (b) Correlation between ultrasound measurements in 2001 and 2007 in groups below and above median of estimated 10-year risk of cardiovascular disease (CVD) event according to the SCORE risk score based on data in 2001. Significant p values indicate a difference in correlation between CVD risk score groups.

and variation in FMD is affected by several factors (Corretti et al. 2002; Ja¨rvisalo et al. 2006). Thus, poor tracking in the latter ultrasound methods may be partly caused by physiologic fluctuation in the constitutive factors instead of measurement error (Juonala et al. 2008). Analyses on the interference of categorical risk factors on ultrasound studies revealed decreased tracking of CDist in subjects with BMI $ 30 kg/m2. Increased 10year CVD risk (.0.010% in females and .0.14% in males) at baseline according to SCORE risk score seemed to increase tracking in both IMT and CDist. Moreover, tracking of IMT displayed tendency toward decreasing (p 5 0.06) in subjects with Framingham risk score above median. The SCORE risk score (Conroy et al. 2003) might have better predictive capabilities on tracking of ultrasound measurements in our Finnish cohort due to its European origin. The Framingham risk score is based on US

Correlation coefficient

Cardiovascular Risk in Young Finns Study d J. R. H. RAIKO et al. 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

IMT re-examination IMT 2007 CDist re-examination CDist 2007 FMD re-examination FMD 2007

P=0.25 P=0.84

P=0.87

N=54

N=53

N=46

Examination

Fig. 3. Correlation between original ultrasound measurements in 2001 and re-examinations in 2001 (3-month tracking) and measurements in 2007 (6-year tracking). The p values indicate whether there is significant difference between 3-month tracking and 6-year tracking.

cohort (Anderson et al. 1991) and might, thus, be less suitable for assessment of CVD risk in our cohort. One explanation for the effect of CVD risk on tracking might be that subjects with high CVD risk have higher IMT and lower Cdist. Thus, the effect of measurement error might become less important in these subjects. Effect of measurement error was examined by comparing the re-examination results with the original measurements. We discovered that even though shortterm variability was lower in IMT than in the other methods there was no difference between 3-month and 6-year tracking. This might indicate that tracking of ultrasound variables depends mostly on measurement errors and physiologic fluctuation while long-term vascular changes have less effect on tracking. FMD has been shown to have 25% day-to-day variation (Ja¨rvisalo et al. 2006). Suggesting the validity of our study protocol, the intra-individual variability/reproducibility values reported previously in this cohort (Juonala et al. 2008) are in line with the findings from other cohorts (De Roos et al. 2003;Jensen-Urstad and Rosfors 1997; Magnussen et al. 2009). Carotid IMT measurement is the most documented noninvasive vascular ultrasonographic method in the assessment of subclinical atherosclerosis (de Groot et al. 2008; Lane et al. 2006) whereas fewer publications exist on the clinical validation of CDist (Oliver and Webb2003) and FMD (Lane et al. 2006) examinations. Currently, assessment of cardiovascular risk is based on measurement of conventional risk factors such as blood pressure, serum lipids and BMI. Although vascular ultrasound measurements seem to lack high tracking and longterm risk assessment capabilities, long-term tracking of conventional risk factors in adult population is somewhat similar (Ulmer et al. 2003;Wilsgaard et al. 2001). IMT has

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been recommended as a screening method for subclinical atherosclerosis in the Screening for Heart Attack Prevention and Education (SHAPE) guidelines (Naghavi et al.2006). No guidelines currently exist for clinical utility of CDist and FMD in young adults. Low reproducibility of ultrasound measurements might render them less suitable for follow-up of single patients but they might still be proper parameters for evaluations of effects on populations. Moreover, effects of interfering factors on tracking of conventional risk factors were weak and no major sex differences were found in Tromsø study (Wilsgaard et al. 2001) whereas in our study age, sex and BMI and 10-year CVD risk at baseline had varying effects on tracking of ultrasound variables. Limitations Re-examinations were performed in 57 subjects and limited size of this cohort diminishes reliability of the studies on reproducibility of ultrasound measurements. However, there was no difference in long-term tracking between re-examined subjects and the rest of the cohort indicating that tracking in re-examination seems to represent tracking in the total cohort. Although there are specific techniques to manage analysis based on grouped data, correlation analyses comparing tracking coefficients between different groups were performed between mean values in the present study. We acknowledge that there are multiple methods for measuring IMT, Cdist and FMD and our results might be generalised only to our methods. CONCLUSIONS According to our study, IMT measurements seemed to track better than CDist and FMD examinations. Baseline BMI decreased tracking of CDist. Tracking of IMT and CDist displayed significant increase in subjects with elevated baseline SCORE risk score. Similar tracking analyses on ultrasound measurements have not been published yet indicating that further studies on the topic are required for proper validation of ultrasound measurements in clinical and scientific use. Acknowledgments—This study was financially supported by the Academy of Finland (grants no. 117832, 121584 and 126925), the Social Insurance Institution of Finland, Tampere and Turku University Hospital Medical Funds, Research Foundation of Orion Corporation, the Margaretha Foundation, the Lydia Maria Julin Foundation, the Research Foundation of Clinical Chemistry, the Valto Takala Foundation, the Juho Vainio Foundation, the Finnish Foundation for Cardiovascular Research, the Ida Montini Foundation, the Turku University Foundation and the Foundation of Outpatient Care Research.

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