**2. Patients and methods**

The hypercholesterolemic individuals with untreated hypercholesterolemia were tested by Lipoprint LDL analysis. In this group of hypercholesterolemic subjects, 145 individuals with a non-atherogenic lipoprotein profile were identified.

Of the total number, 15 individuals were under 40 years of age without clinically apparent impairment and no laboratory signs of cardiovascular disease. These subjects formed one subgroup of younger people (34 years +- 5 years). The subgroup of younger subjects was separated from the older individuals with hypercholesterolemia because a separate analysis of the older subjects with hypercholesterolemia was performed to confirm that undamaged vessels in older individuals persist even into old age, and that diagnosed hypercholesterolemia does not cause an atherogenic impairment in the vessels. The subgroup of older subjects consisted of 130 individuals (32 males, 57 +-11 years of age; and 98 females, 62 +- 9 years of age).

The medical examination, which included a physical examination, blood pressure, and ECG examination, bicycle stress test, echocardiography, and duplex ultrasound examination of the carotid arteries, confirmed that there was no impairment of the cardiovascular system. Only mild signs of clinically irrelevant aortic valve sclerosis were found in the subgroup of older subjects.

Individuals with hyperglycemia, diabetics, and those individuals who were being treated with lipid-lowering drugs were excluded from the study.

The control group consisted of 165 normolipidemic volunteers, all nonsmokers, who had no clinically apparent impairment, or laboratory signs of cardiovascular disease. Volunteers were recruited from medical students at the medical facility. The average age of the subjects was 21.5 ± 2.5 years, and the group involved 65 males and 100 females. All subjects gave written, informed consent, and the study was approved by the local ethics committee.

A blood sample from the antecubital vein was collected in the morning after a 12-hour fasting period. EDTA-K2 plasma was obtained and the concentration of total cholesterol and triglycerides in plasma was analyzed, using an enzymatic CHOD PAP method (Roche Diagnostics, Germany).

The quantitative analysis of lipoprotein families and lipoprotein subfractions included: VLDL; IDL1; IDL2; IDL3; LDL1; LDL2; LDL3-7; and HDL. A non-atherogenic lipoprotein profile, phenotype A, versus an atherogenic lipoprotein profile, phenotype B, was determined using the Lipoprint LDL System (Quantimetrix Corp., USA; (Hoefner *et al.*  2001). The analysis of HDL subclasses, with their subpopulations, including large HDL-, intermediate HDL-, and small HDL- subclasses in plasma, was also performed using the Lipoprint HDL System (Morais *et al.* 2003).

The Score of the Anti-Atherogenic Risk (SAAR) was calculated as the ratio between nonatherogenic and atherogenic lipoproteins in plasma (Oravec 2007a). SAAR values over 10.8 represented a non-atherogenic lipoprotein profile, whereas values under 9.8 represented an atherogenic lipoprotein profile. The cut-off values for a non-atherogenic lipoprotein profile and an atherogenic lipoprotein profile were calculated from the results of 940 Lipoprint LDL analyses. Using the Quantimetrix Lipoprint LDL system interpretation, all 940 individuals were examined (general group of subjects) and tested for the occurrence of atherogenic and non-atherogenic lipoprotein profiles, and were then divided into the two subgroups of subjects with an LDL profile:


For practical interpretation of the analysed lipoprotein profiles using the Lipoprint LDL System, for the non-atherogenic lipoprotein phenotype A, the subtypes 1a, 1b, 2a, 2b, 3, and 4 were introduced, because of the large profile heterogeneity in the non-atherogenic lipoprotein profile phenotype A. With regard to the atherogenic lipoprotein phenotype B, only subtype 5 and subtype 6 were introduced. (Oravec 2007b). (Tab.2)

Statistical evaluation of obtained values was performed by an unpaired student´s t-test. The level of significance was set at p < 0.05.
