individuals of Italian heritage within that age bracket living in the municipality of Veranópolis in 1998.

biochemical examinations were as follow: blood samples of a peripheral vein were withdrawn according to the venocly- sis system with a disposable vacuum device (Vacutainer) and stored in tubes with 0.1% EDTA (final volume at the concentration of 1mg/dL). After that, the material collected was maintained at 4ºC until DNA extraction for up to 24 hours. DNA extraction was performed according to the tech- nique reported by Debomoy 18. The general protocol of ex- traction comprised the following steps: 1) the blood sam- ples collected were centrifuged at 5,000 rpm for 5 minutes; 2) after centrifugation, the region containing the leukocytes was transferred to tubes of 1.5 mL and washed with PBS (phosphate buffered saline, pH 7.5); 3) for extracting DNA from the leukocytes (in 200 µL PBS), 100 µL of the lysis buf- fer (10 mM Tris, 100 mM EDTA, 0.5% SDS, 200 µg/mL pro- teinase K), 2.5% SDS, and 25 mM EDTA pH 8.0 were added, and that was followed by incubation at 55ºC for 3 hours; 4) after incubation, the DNA of the sample was extracted by 2 washes with a solution of phenol, chloroform, and isoamyl alcohol (25:24:1); 5) the DNA present in the aqueous portion was then precipitated by adding 0.1 volume of sodium ace- tate and 0.7 volume of isopropanol and incubated at -20ºC; 6) the DNA, observed as a clear and gelatinous sediment, was dissolved in 50 µL of Milli-Q water and maintained at - 20ºC for later use in the reaction of gene amplification called polymerase chain reaction (PCR).

The relevance of this type of study is based on the fact that the phenomenon of population aging will markedly increase the number of elderly individuals older than 80 years, who constitute a group especially susceptible to car- diovascular events. However, recent studies, including the Framingham Study, have shown that the traditional risk fac- tors of cardiovascular diseases do not apply with the same intensity observed in younger age groups 14,15. Therefore, we need to determine which risk factors really influence the susceptibility to cardiovascular diseases in this population group.

Polymerase chain reaction was performed according to the method reported by James et al 19 using the primers apo EF 5'- TAA GCT TGG CAC GGC TGT CCA AGG A-3' (25-mer) and apo ER 5'- ACA GAA TTC GCC CCG GCC TGG TAC AC- 3' (26-mer). The mixture of the reaction (total of 50 µL) was prepared with 10 pmol of each primer, 100 µM dNTPs, 1U of Taq Polymerase (CenBiot-RS), buffer containing 1.5 mM MgCl , 1% DMSO, and 2 mmL of the DNA of the sample. 2

Based on these considerations, we analyzed the as- sociation between different ApoE genotypes, cardiovascu- lar risk factors, and morbidity in the elderly individuals aged 80 years or older living in the municipality of Veranópolis, in the State of Rio Grande do Sul, and who took part in the Projeto Veranópolis. That project analyzed the interaction of genetic and environmental variables related to successful aging and to cardiovascular diseases. The basic demogra- phic and epidemiologic profile of the municipality of Veranó- polis is shown in table I 16.

The amplification process consisted of an initial dena- turation at 95ºC for 5 minutes, followed by 30 cycles of denaturation at 95ºC for 1 minute, coiling at 61ºC for 1 minu- te, extension at 70ºC for 1 minute, and a final extension at 72ºC for 10 minutes.

Methods

The efficacy of PCR was visualized through electro- phoresis in agarose gel (2%) with ethidium bromide, when a band of 240 base pairs (bp) was observed under ultraviolet light.

We carried out a cross-sectional, individual, observa- tional, analytical study 17 on 64 elderly individuals aged 80 years or more, who were chosen according to criteria of re- presentativity and randomization in a total population of 220

Genotyping was performed by digesting the PCR pro- duct (20 µL) with 5 U of the restriction enzyme Hha I (Gibco)

in a specific buffer. The solution undergoing digestion was incubated at 370C for 3 hours, and then analyzed in polya- crylamide gel (15%) using a molecular weight marker of 10- bp ladder. The characteristic bands of each allele were then analyzed and identified.

c, and triglycerides (TG) for adults (> 20 years)19 Lipids Values (md/dL) Desirable Borderline Alterated TC < 200 200-239 ≥ 240 LDL-c < 130 130-159 ≥ 160 HDL-c ≥ 35 _ _ TG < 200 _ ≥ 200

For biochemical assessment, samples of venous blood were collected after study participants had fasted for 12 hours. The venoclysis system with the disposable vacuum device (Vacutainer) was used. Blood was collected in tubes with no anticoagulant to quantify the lipid profile, Lp(a), glucose, and uric acid. For fibrinogen quantification, blood was collected in tubes with sodium citrate. The blood samples were centrifuged for 15 minutes in a Kubota KC-70 centrifuge (Japan) at the velocity of 3,000 rpm to separate plasma from the remaining cells. Plasma was then divided into five 1.0-mL aliquots in Eppendorf tubes with micropipe- ttes. The plasma aliquots were then refrigerated between -8 and 4°C for a maximum of 24 hours 20.

Source: II Consenso Brasileiro sobre Dislipidemias.

commercial kit (Paraná, Brazil), and the mean of 2 measure- ments performed for each plasma sample was considered the final value 20. Glycemic levels above 126 mg/dL were considered increased. Plasma fibrinogen was quantified according to the turbidimetric method using the Tecnow 7000 spectrophotometer (Tecnow Instrumentos Científicos, São Paulo). Fibrinogen levels above 300 mg/dL were consi- dered increased. Measurement of plasma uric acid was per- formed using the enzymatic method (UV, Method GIDH) with the Merck commercial kit (Germany). Levels above 7.0 mg/dL were considered increased 22.