A commercial kit (Paran, Brazil) was used for lipid quantification, and the mean of two measurements performed for each plasma sample was considered the final value. 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 considered increased. Measurement of plasma uric acid was performed using the enzymatic method (UV, Method GIDH) with the Merck commercial kit (Germany). Levels above 7.0 mg/dL were considered increased. 

To analyze total cholesterol, the manual technique of the enzymatic colorimetric reaction was used with the commercial enzymatic reagent Cholesterin/Cholesterol Chod-Pap (MPR2, Boehringer-Mannheim, Germany). Readings of standard cholesterol levels were performed with the Preciset Cholesterol Calibrator 125512 (Boehringer-Mannheim, Germany), and the final value was the mean of two measurements performed for each plasma sample. To analyze HDL-cholesterol (HDL-c), the heparin-Mn2+ precipitation method by Gildez was used with some adaptations. The lipoproteins containing apolipoproteins B-100 (VLDL and LDL) were precipitated according to the heparin-Mn2+ precipitation method (Sigma Chemical Co, USA) and then incubated and centrifuged (refrigerated centrifuge, RB-1811 Model, Tomy Seiko Co. Ltd, Japan). The floating material was then collected for quantification of HDL-c particles through the colorimetric enzymatic reaction for cholesterol; the mean of two measurements for each plasma sample was considered the final value. The LDL-c value was obtained with the Friedewald formula for triglyceride levels below 400 mg/dL. Samples with triglyceride levels above 400 mg/dL were excluded. Lipoprotein(a) levels were obtained with the Boehringer nephelometric system (Boehringer Nephelometer 100); Lipoprotein(a) levels above 30 mg/dL were considered increased. For glucose level determination, the colorimetric enzymatic method was performed using the Bio Diagnóstica ICQ Ltda commercial kit.

Complementary clinical examinations were performed, and structured interviews were applied to detect cardiovascular risk and morbidity. Three clinical parameters were used for obesity: body mass index, waist/hip ratio, and percent body fat (% fat). Individuals who were classified as obese in the three parameters or those classified as obese in at least two parameters and overweight according to the body mass index were considered obese. Body mass index was calculated by dividing the weight in kilograms by the square of the height in meters squared (kg/m²). The waist/hip ratio was calculated by dividing the minimum abdominal circumference by the maximum abdominal circumference. Percent fat was obtained with fat quantification by electrical bioimpedanciometry. 

The manual technique of colorimetric enzymatic reaction was used for triglyceride quantification with the commercial enzymatic kit Labtest TG Gpo-Ana (Argentina). Readings of standard triglyceride levels were performed with the Preciset Triglyceride Calibrator 125512 (Boehringer-Mannheim, Germany), and the final value was the mean of two measurements performed for each plasma sample. Levels above 200 mg/dL were considered increased.genotype individuals had a significantly higher mean LDL-c concentration than E3E3 individuals, while the mean uric acid concentration was significantly higher in E3E3 individuals than in E3E4 individuals. Fibrinogen levels were statistically higher in E3E4 individuals, but all means observed in the remaining genotypes were not considered indicative of cardiovascular risk. The frequency of the E4 allele in the population studied was above 10%, even though the frequencies were within the Hardy-Weinberg equilibrium. Individuals with the E3E4 genotype were twice more likely to have LDL-c and fibrinogen levels above that expected than individuals with the E3E3 genotype were. On the other hand, E3E4 individuals were less likely to have more elevated uric acid levels than E3E3 individuals were. The ApoE frequencies in Veranpolis were compared with those of different populations or different age groups of these populations, or both. Further genetic studies may help to elucidate this phenomenon.Elderly individuals tend to have higher rates of familial morbidity and mortality due to cardiovascular diseases, diabetes mellitus, and dyslipidemia. However, these differences were not statistically significant (Fig. 1). Despite the high frequency of the E4 allele in the elderly, no E4E4 genotype was observed. Several studies carried out in other countries suggest a frequency-dependent effect of the E4 allele, i.e., the presence of two copies of the E4 allele is associated with early mortality. As no elderly individual with this genotype was observed, the data obtained confirm this hypothesis.

Discussion:

The analysis of the results suggests a more complex interaction between ApoE polymorphism and the variables related to the cardiovascular events studied. Genotype-dependent relations, which have not yet been explored, may exist.

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Regarding gene and genotypic frequencies related to ApoE polymorphism, it is important to emphasize that these frequencies vary according to the population studied. Review studies have shown that gene frequencies related to this polymorphism are extremely variable, mainly concerning the E4 allele frequency 1,2. In Europe, this gene has a gradient distribution, with the E4 allele being more frequent in the northern region and less frequent in the southern region. In Asian populations, this allele has a low frequency unlike that of African and New Guinea populations 31-33. However, specific studies on possible causes of this variation are still in their infancy (Fig. 1).