A total of 275 overweight (BMI ≥ 27 kg/m²) Caucasian Italian subjects (211 males and 64 females; mean age 61.36 ± 10.39 years; BMI 29.67 ± 2.56 kg/m²) were recruited among the patients attending the Cardiology Division, IRCCS San Matteo Hospital, Pavia. For each enrolled subject, body weight was measured to the nearest kg and height to the nearest centimeter. BMI was calculated as body weight divided by height² (kg/m²). According to previous methodology 222324, we used a BMI ≥ 27 kg/m² as the criterion for inclusion in the overweight group. Furthermore, this cut-off was chosen since it has been previously adopted as the criterion to indicate overweight in a Western Europe population 25.

We then assembled an age- and gender-matched normal weight control group, consisting of 103 subjects (74 males and 29 females; mean age 61.95 ± 14.79 years; BMI 23.92 ± 1.88 kg/m²). Controls were, mainly, individuals who visited the outpatients' internal medicine department of the same hospital and at the same period with the coronary patients, for routine clinical or laboratory examinations. Controls were subjects without any clinical symptoms, signs or any suspicion of cardiovascular disease in their medical history, as determined by a physician 26.

Among the overweight subjects, 107 (38.9%) subjects suffered from diabetes mellitus, which was diagnosed according to the ADA criteria 27, or the use of insulin or oral hypoglycemic drugs. 173 (62.9%) overweight subjects were hypertensive. Diagnosis of hypertension was performed as either the blood pressure measured was higher than 140/90 mmHg or by the need of antihypertensive medications. Furthermore, 121 (44%) subjects showed hypercholesterolemia, defined as a serum total cholesterol major than 200 mg/dl, or the use of lipid lowering drugs 28. Smoking was considered to be present if the subject had smoked more than three cigarettes a day for at least one year.

The overweight subjects were further divided into two subgroups according to whether they had CHD or not. The retrospective diagnosis of CHD was performed on the basis of a documented history of myocardial infarction (clinical history of retrosternal pain that lasted for at least 30 min without response to nitroglycerine, ST segment elevation on a standard twelve-lead electrocardiogram and an increase in serum creatine kinase to at least twice the normal upper limit); coronary artery disease documented by angiography (stenosis > 75% in at least one coronary artery); coronary artery bypass grafting or a positive history of anginous chest pain together with a positive exercise test. Among the overweight subjects, 155 (56.3%) had CHD; for the other patients the possibility of CHD was excluded on the grounds of their medical history and an exercise stress test, as described previously 29.

In order to assess possible differences in the Lp(a)-associated risk between overweight and normal weight subjects, 337 normal weight subjects with CHD (251 males and 86 females; mean age 62.01 ± 11.18 years; BMI 23.85 ± 2.01 kg/m²), were recruited from the cardiology section.

The study protocol followed the guidelines of our local ethics committee and all subjects gave full informed consent to participate in the study.

Venous blood was collected from subjects after an overnight fast of about 12 hours. Quantification of Lp(a) and phenotyping of apo(a) protein were done using plasma obtained from blood collected in EDTA tubes, which were centrifuged at 4°C, at low-speed, for 12 minutes. After centrifugation, the plasma aliquots were frozen and stored at -80°C.

Lp(a) plasma concentrations were determined by a sandwich-ELISA technique, using the commercially available kit Macra-Lp(a) (SDI, Delaware, USA). The intra-assay and inter-assay coefficients of variation of this method is 5 and 9%, without cross-reaction with plasminogen.

Apo(a) isoforms phenotyping was performed by an immunoblotting method, as previously described in detail 30, with slight modifications.

Briefly, 15 μL of EDTA-plasma samples were pretreated with 30 μL of a reducing solution. The submarine electrophoretic run was performed on 1% sodium dodecyl sulfate-agarose gel and electrophoresis was carried out in tank buffer for 14 h at 80 V and 0.04 A. Reduced samples (20 μL) were applied in wells, at 3 cm from the cathode of the gel. The separated proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Segrate, Italy) by a capillary blotting technique and tested with a polyclonal antihuman Lp(a) antiserum from rabbit (DAKO, Glostrup, Denmark). A peroxidase-conjugated goat antirabbit immunoglobulin (DAKO, Glostrup, Denmark) was used as the second antibody. Relative band mobility was determined as referred to apo(a) standard isoform mobility included in each blot (values: 35, 27, 23, 19, and 14 K-IV repeats; Immuno AG, Wien, Austria). Thus the estimated number of K-IV repeats in each sample was calculated.

Normally distributed data are presented as means ± SD. For Lp(a) levels, which showed a skewed distribution, we used medians and interquartile ranges. Student's t test was exploited for comparison in normally distributed data between 2 groups and the Mann-Whitney U test was performed for comparison of Lp(a) concentrations between 2 groups. Categorical variables were compared by means of the chi-square test. The significance of clinical and biochemical parameters as independent predictors of CHD in overweight subjects was tested in stepwise regression analysis. Predictors or independent variables were: gender, age, BMI, smoking, diabetes, hypertension, hypercholesterolemia, Lp(a) levels and apo(a) phenotypes. The criterion for inclusion into the model was a P value less than 0.05, as determined with the F test. A probability value below 0.05 was considered to indicate statistical significance. All statistical analyses were performed using the SPSS statistical package, version 11.0 for Windows (SPSS Inc., Chicago, IL, USA).

Clinical characteristics of overweight subjects, normal weight subjects with CHD and controls are summarized in Table 1. Among the overweight subjects, the prevalence of diabetes mellitus, hypertension and hypercholesterolemia was significantly higher than in controls. Among the normal weight CHD subjects, the prevalence of smoking and diabetes mellitus was significantly higher than in controls, whereas no significant differences in the prevalence of hypertension and hypercholesterolemia were found. Furthermore, no difference in median Lp(a) level was found between the three study groups.

The features of overweight subjects with and without CHD are shown in Table 2. The two groups show no statistically significant differences in age, gender, hypertension, smoking status and BMI. The CHD group included significantly more subjects with diabetes mellitus and hypercholesterolemia. Overweight subjects with CHD had Lp(a) plasma significantly higher than those without [median (interquartile range): 20 (5–50.3) versus 12.6 (2.6–38.6) mg/dl, P < 0.05].

Out of the 715 subjects recruited, we identified twenty-three different apo(a) isoforms with molecular weight varying from 400 and 835 kDa. The detection method showed good sensitivity, specificity and reproducibility, as described 30.

Because of the high degree of apo(a) polymorphism, we divided apo(a) isoforms in two subgroups according to a previously identified cut-off between 640 and 655 kDa. This cut-off seems to discriminate well apo(a) isoforms associated with higher atherothrombotic predisposition 31. According to previous methodology 193233, we decided to use only the smaller apo(a) isoform detected for categorization.

Table 3 shows the apo(a) phenotype distribution in the study groups. In the group of normal weight subjects with CHD, low molecular weight apo(a) isoforms prevailed (57%) and the difference in comparison with controls was significant (P < 0.01).

In the subgroup of overweight patients with CHD low molecular weight apo(a) isoforms were more prevalent (55.5%), whereas among subjects without CHD apo(a) isoforms of high molecular weight prevailed (59.2%). The difference between the two groups of overweight subjects was statistically significant (P < 0.05).

The frequencies of apo(a) isoforms for the overweight subjects with CHD and the CHD normal weight subjects were similar. Furthermore, there was no significant difference in the distribution of apo(a) isoforms between overweight subjects without CHD and controls.

The common cardiovascular risk factors, Lp(a) concentrations and apo(a) isoforms of low molecular weight were tested as predictors of CHD in overweight and normal weight subjects in a stepwise regression analysis (Table 4). The presence of diabetes mellitus, small-sized apo(a) isoforms, hypercholesterolemia, and BMI were found to be significant predictors of CHD in overweight subjects. In normal weight subjects the presence of diabetes mellitus, smoking, hypercholesterolemia, hypertension and at least one small-sized apo(a) isoform were found, in the order they entered into the model, to be independent predictors of CHD.