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

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, vv and ethylacetate were used as the mobile phase at a flow rate of 1 mlmin. 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-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 mgdl. 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 and a-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. Rescanning 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 andor 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 exclusion criteria P 1 B 0.05; P E \ 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- and b-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 of a + 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 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-tocopherolcholes- terol ratio.

4. Discussion