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 andor with fasting glucose plasma levels \ 7.8
mmoll 140 mgdl, or a 2-h value \ 11.1 mmoll 200 mgdl after oral glucose loading. The body mass index
was calculated as weight kg divided by height m
2
. 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 gday and classified in four
categories: abstainers 0 gday and light 1 – 50 gday, moderate 51 – 99 gday, and heavy drinkers ] 100
gday. 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 andor the
average monthly income of the subject or hisher 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 polymerization 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,vv
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 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, 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
