The Assessment of the Atherogenic Lipoprotein Profile in Cardiovascular Diseases by Lipoprint System Analysis

Research focus: Identification of incidence of an atherogenic lipoprotein pheno‐ type B in four representative diagnoses of cardiovascular diseases: a) arterial hypertension, b) coronary heart disease, c) lower extremity arterial disease, d) ischemic stroke Research methods: A clinical study included 366 patients with a diagnosis of arterial hypertension (n=107), coronary heart disease (n= 104), lower extremity arterial disease (n= 100) and ischemic stroke (n= 55). The control group consisted of 150 healthy normotensive and normolipemic volunteers, all non-smokers, without signs of cardiovascular disease. In all tested individuals (or subjects) lipid parameters in serum: cholesterol and triglycerides were analyzed, using the enzymatic CHOD-PAP method, Roche Diagnostics Germany. Lipopro‐ teins in serum lipoprotein spectrum by Lipoprint LDL system were analyzed and an atherogenic and a non-atherogenic lipoprotein profile idetified. The Score of the Anti-Atherogenic Risk (SAAR) was calculated as the ratio between non-atherogen‐ ic and atherogenic lipoproteins. Results: More than 80 percent of tested patients with cardovascular diseases have an atherogenic lipoprotein profile, with a high level of strongly atherogenic small dense LDL. The aterogenic profile was found in arterial hypertension 78.5%, in coronary heart disease in 81.7%, in lower extremity arterial disease in 80 %, and in patients who survived an ischemic stroke in 85%. Main conclusion: The atherogenic lipoprotein profile was found to be the overwhelming lipoprotein profile in tested cardiovascular diseases A new phenomenon- atherogenic normolipidemia of cardiovascular disease, would be established as a new term used in the diagnostics of dyslipoproteinemias


Introduction
In the last few decades, lipoprotein research has focused on the phenomenon of atherogenic and non-atherogenic lipoproteins, specifically, atherogenic and non-atherogenic lipoprotein profiles phenotype A and phenotype B [7,18,60] after it was reported that more than 75% of patients with an acute coronary syndrome or myocardial infarction had normal plasma values of cholesterol, Low Density Lipoprotein cholesterol (LDL cholesterol) and High Density Lipoprotein cholesterol (HDL cholesterol) [15 -17].
Thus, it was necessary to look for other risks factors in plasma, the presence of which in relevant quantities could cause damage to endothelial cells and resultant endothelial dysfunction [59]. This called into question whether an increased total cholesterol level, or increased LDLcholesterol, as a criterion for the degree of atherogenic risk, provided a universal explanation for the origin of atherogenesis. A reasonable explanation was found in atherogenic lipoprotein subpopulations, the presence of which in plasma, even in very low concentrations, could impair the integrity of the vessel wall and lead to endothelial dysfunction with its fatal consequences [ Table 1]: formation of atherothrombotic plaques, acute myocardial infarction, ischemic stroke, or sudden death [39,59,64].
The predominance of atherogenic lipoproteins in plasma is characteristic for the atherogenic lipoprotein spectrum, phenotype B. When present in plasma in high concentrations, these lipoproteins contribute to ischemic vascular impairment [6,8,57,60]. The process of degenerative changes in vessels results in the formation of atheromatous vascular plaques. These later play an important role in the formation of stable or unstable angina (pectoris), and critical ischemia of peripheral and/or cerebral arteries as well [39,56]. When atherogenic lipoproteins in plasma are present in small quantities, we obtain a picture of a non-atherogenic lipoprotein profile, phenotype A.
Various methods have been developed (gradient gel electrophoresis, ultracentrifugation, magnetic resonance spectroscopy, endothelial models for testing lipoprotein cytotoxicity) to identify atherogenic lipoproteins [2,26,45,48], but because of technical and financial issues, long-term analyses and high operating costs, the previously mentioned methods were used primarily in basic research. Simple analytical procedures for routine distribution were lacking and the possibility of their implementation in every day laboratory practice remained limited.
An electrophoretic method by which to separate lipoproteins on polyacrylamide gel (PAG) with the use of Lipoprint LDL System [29,41] has become a milestone in routine laboratory analysis and in diagnosing metabolism disorders of lipoproteins. It enables the analysis of 12 lipoprotein subfractions: VLDL, IDL 1-3, LDL 1-7, and HDL. In our studies were analyzed serum lipoprotein spectrums in patients with newly recognized a) arterial hypertension, b) coronary heart disease, c) lower extremity arterial disease, and d) in patients who survived a stroke. As mentioned earlier, an analytical method for a quantitative evaluation of lipoprotein fractions was used, and the incidence of an atherogenic lipoprotein spectrum phenotype B (vs. phenotype A) in these four representatives of cardiovascular diseases was identified. At the same time, a lipoprotein spectrum of a control group of healthy individuals was examined and tested for the incidence of phenotype B.

Arterial hypertension
Arterial hypertension (AH) (Fig. 3) is one of the most serious cardiovascular diseases. More than 20% of the adult population suffers from this disease. AH is one of the risk factors for atherosclerosis development of coronary, brain, and peripheral arteries, together with the main cardinal risk factors, that is, dyslipoproteinemia and tobacco smoking [14,20,38]. Atherogenic LDL subfractions also play a role in the development of the arterial hypertension [32,43].
AH is a permanent, long-lasting increase in blood pressure of more than 140/90 mmHg in people of middle age. In people older than 70 years of age, values higher than 160/95 mmHg are considered increased. For more extensive guidelines see the Statement of WHO/ISH (International Society of Hypertension) on the management of hypertension [61].
Dyslipoproteinemia, which frequently accompanies AH and multiplies the risk of atherosclerosis development, can also be considered one of the multiple sources that give rise to AH [35,64].

Patients
In our study 107 patients with newly diagnosed arterial hypertension were examined. Repeated blood pressure (BP) examination confirmed an increased blood pressure more than 150 mmHg for systolic and more than 90 mmHg for diastolic blood pressure in all hypertensive patients. Average systolic blood pressure was 172 ±19 mmHg and average diastolic blood pressure was 102 ±10 mmHg. The group of hypertensive patients comprised 66 men and 41 women. The average age of the men was 50 ± 17.6 years and the average age of the women was 51.0 ± 13.4 years.
The control group consisted of 150 healthy normotensive and normolipemic volunteers, all non-smokers, without signs of cardiovascular disease and without biochemical signs of lipid metabolism disorders. The average age of the subjects was 21 years, and the control group involved 50 males and 100 females. Volunteers were recruited from medical students at the Medical Faculty, who gave written, informed consent, and the study was approved by the local ethics committee.

Methods
A blood sample from an antecubital vein was obtained in the morning after a 12-hour fasting period. Total cholesterol and triglycerides in serum were analyzed from lipid parameters, using the enzymatic CHOD-PAP method, Roche Diagnostics Germany. To determine the nonatherogenic lipoprotein phenotype A and the atherogenic lipoprotein phenotype B, the Lipoprint LDL System Quantimetrix CA, USA, was used.
The Score of the Anti-Atherogenic Risk (SAAR) was calculated as the ratio between nonatherogenic and atherogenic lipoproteins in serum [42]. 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 vs. nonatherogenic lipoprotein profile and were divided into the two subgroups of subjects with an LDL profile: • Indicative of Type A, that is, a non-atherogenic lipoprotein profile phenotype A • Not indicative of Type A, that is, an atherogenic lipoprotein profile, phenotype B [29] Statistical evaluation of obtained values was performed with an unpaired student's t-test. The level of significance was accepted at p < 0.05.

Results
In the control group shown in Table 2, along with the individuals with non-atherogenic normolipidemia, that is, an ideal lipoprotein profile (Fig. 1), a subgroup of normolipidemic individuals with an atherogenic lipoprotein profile was also identified. This group represented people with an atherogenic normolipidemia (Fig. 2). These people are clinically healthy, without clinical or laboratory signs of cardiovascular diseases, but with a positive familial history for cardiovascular diseases (myocardial infarction) in the parents' or grandparents' generation. The triglycerides and LDL3-7 concentrations in the control group with the atherogenic profile, compared to the individuals with a non-atherogenic lipoprotein profile, were increased (p < 0.05, respectively, p < 0.0001). The Score of the Anti-Atherogenic Risk (SAAR) for a non-atherogenic lipoprotein profile is a sensitive indicator by which to differentiate between an atherogenic and non-atherogenic plasma lipoprotein constellation (nonatherogenic vs. atherogenic: p < 0.0001). Non-atherogenic profile, 93.4 % vs. atherogenic profile, 6.6 %, in control group Table 2. Serum concentration of lipids, lipoproteins, and SAAR-score in the control group *Reference ranges derived from 125 serum samples that met the NCEP ATPIII guidelines for desirable lipid status **LDL-C comprised of the sum of cholesterol in Md bands C through A as well as all the subfractions A non-atherogenic lipoprotein profile in the control group was confirmed in 93.4% healthy normolipidemic individuals, and an atherogenic lipoprotein profile was found in 6.6%. Table 3 shows high statistical significance for the analyzed lipid and lipoprotein parameters between the control group and the group of subjects with arterial hypertension (p < 0.0001, and for HDL, p <0.03).
In Table 4, 78.5% of patients with arterial hypertension have an atherogenic lipoprotein profile. There is a highly significantly increased concentration of small dense LDL (subfractions LDL3-7) in a subgroup of AH-patients, who have an atherogenic profile, compared to the concentration of small dense LDL in the subgroup of AH-patients with a non-atherogenic profile, which confirms the predominance of atherogenic lipoproteins in AH-patients and the creation of atherogenic lipoprotein profile, phenotype B, as well. SAAR in patients with AH is low, that is, 9.2 (cut off is 10.8), and confirms also the predominance of atherogenic lipoproteins in serum.
*Reference ranges derived from 125 serum samples that met the NCEP ATPIII guidelines for desirable lipid status **LDL-C comprised of the sum of cholesterol in Md bands C through A as well as all the subfractions

Coronary heart disease
Coronary heart disease (CHD) (Fig. 4) is a common manifestation of cardiovascular diseases and is frequently associated with lipid and lipoprotein metabolism disorders. Hypercholesterolemia and hypertriglyceridemia, as well as combined hyperlipoproteinemia are regular features that accompany CHD [22,50,51]. Pathophysiologically, the cause of myocardial ischemia is a disproportion, or imbalance, between myocardial oxygen supply and oxygen demand. Ischemia in stable angina is generally due to fixed atheromatous stenosis of one or more coronary arteries as a consequence of impaired lipoprotein metabolism and the formation of lipid atheromas in the coronary arteries [5,33,34,49].
However, clinically, stable angina is not the only form of manifestation of coronary heart disease. Stable angina, as an ischemia due to fixed atheromatous stenosis, can turn into a myocardial ischemia due to plaque rupture with thrombosis and spasm of the artery (instable angina). In addition, myocardial necrosis (myocardial infarction), caused by acute occlusion of a coronary artery (due to plaque rupture and thrombosis), can have fatal consequences for disabled persons. It can be supposed that the modified forms of lipoproteins can play an important role in any form of clinical manifestation of coronary heart disease. Recently, clinical studies reported that the atherogenic lipoprotein populations (lipoprotein subfractions), presented in the plasma lipoprotein spectrum in high concentrations, play an important role in the development of atherosclerotic changes in the arterial wall [14,38,39]. We distinguish facultative atherogenic very low density lipoproteins, VLDL, and their remnants, intermediate density lipoproteins, IDL, low density lipoproteins, LDL (considered a lipoprotein family with high atherogenicity), and high density lipoproteins, HDL. Modified lipoprotein entities in all these lipoprotein families can play a role in the formation of atherogenic lipoproteins, which accelerate the atherogenesis in the artery walls, including in the coronary arteries.
In our study, we focused on the determination of the incidence of an atherogenic lipoprotein phenotype in patients with coronary heart disease -in stable angina patients.

Patients
In our study, 104 patients with newly diagnosed coronary heart disease were examined. The diagnosis of CHD (stable angina pectoris grade I or II) was confirmed by medical examination, laboratory results, resting ECG, results of echocardiography, and duplex ultrasound of the carotid arteries.

Methods
See methods published in the section "Arterial hypertension (AH)."

Results
The results of lipid parameters presented in Table 5  In Table 6, an atherogenic lipoprotein phenotype B is present in 81.7% of patients with CHD. An increased concentration of small dense LDL (LDL3-7) in the CHD-patient subgroup with an atherogenic lipoprotein profile, compared to the results of the CHD-patient subgroup with a non-atherogenic lipoprotein profile (p<0.0001), confirms a predominance of atherogenic lipoproteins in the serum of patients with CHD.
*Reference ranges derived from 125 serum samples that met the NCEP ATPIII guidelines for desirable lipid status **LDL-C comprised of the sum of cholesterol in Md bands C through A as well as all the subfractions Almost all lower extremity arterial disease is due to atherosclerotic changes in artery vessels, and the pathology of LEAD is also similar to coronary heart disease. The most important risk factor for the development and progression of atherosclerotic LEAD are tobacco smoking, arterial hypertension, and hyperlipidemia. Other risk factors include diabetes mellitus, low physical activity, and a diet rich in lipids and carbohydrates. However, dyslipidemia plays an important role. Increased lipid levels of cholesterol and triglycerides are generally accepted as important risk factors for the development of atherosclerosis [14,25,47].
In the last few decades, there has been much discussion about which atherogenic lipoproteins participate in the formation of the atherogenic lipoprotein profile, phenotype B. Atherogenic lipoproteins in relevant concentration in the blood serum are responsible for the acceleration of the development of atherogenic cardiovascular diseases, including the development of LEAD. The LDL subpopulations of small dense LDL are considered to be strongly atherogenic lipoprotein entities in the plasma/serum lipoprotein spectrum [38,59] with consequent acceleration of endothelial dysfunction and formation of the atheromatous subendothelial plaques in the arteries [21]. In the present study, we have focused on determining the incidence of an atherogenic lipoprotein phenotype, along with determining the role of atherogenic serum lipoproteins, in patients with lower extremity arterial disease.

Patients
In the clinical study, 100 patients with newly diagnosed lower extremity arterial disease were examined. The study included 55 males and 45 females: the average age of males was 56.0 years ±11 years and the average age of females 52.5 years ± 14 years. The patients had C2a degree, according to the Claudication classification: [proximal type (AP), the first degree (P1) with dyslipidemia]. Patients were ex-smokers.
LEAD was diagnosed according to the history of disease, intermittent claudication, the medical examination, including physical examination (Ratschow's test in the modification according to Linhart, see the Angiological Section of Slovak Medical Chamber) [23,24,27,28] and examination of the ankle-brachial (pressure) index (ABPI) [40,55,57].

Results of lipid parameters presented in
An increased concentration of small dense LDL (LDL3-7 subgroups) in the LEAD-patients with an atherogenic lipoprotein profile, compared to the results from the LEAD-patients with a nonatherogenic lipoprotein profile (p<0.0001), confirms the predominance of atherogenic lipoproteins in serum in the subgroup of patients with an atherogenic lipoprotein profile.

Stroke
Stroke (Fig. 6) is the leading cause of mortality and of long-term morbidity in the populations of developed industrialized countries in the world. The atherogenic serum lipoproteins in high concentrations create an atherogenic lipoprotein profile, which plays a key role in the acute onset of cardiovascular and cerebrovascular events, that is, stroke [54,55]. Cerebral stroke attack remains a frequent medical problem and is the third most frequent cause of mortality all over the world. It represents a heterogeneous group of diseases with more than 150 known causes. In 25-39% of strokes, the cause leading to the acute cerebrovascular event cannot usually be definitively explained [4]. hyperlipoproteinemia, that is, of dyslipidemia, is to reduce the lipid concentration in serum to established target values of lipids (cholesterol and triglycerides), but the primary goal is to reduce the atherogenic potential of serum lipids [9,21,53]. Dyslipoproteinemia is also the key phenomenon in the pathogenesis of the onset of atherosclerotic alterations in brain vessels [64]. Accompanied by high cholesterol levels -a classic risk factor for the development of cardiovascular diseases -an increased concentration of triglycerides in the blood serum can also play an important role in atherogenesis [3,58].
There are several studies that have provided evidence for the relation between carotid artery stenosis and an ischemic cerebral event [55]. However, the causal inter-relation between dyslipidemia and stroke has not been explained sufficiently [3,4,63]. Relapsing ischemic strokes account for one-fourth of all strokes in a year and are a strong evidence for a failure of secondary prevention [10]. This hard reality leads rightly so to the idea of optimal stroke prevention through the selection of individuals, who are at risk of stroke [13]. The aim of this pilot study was to identify the atherogenic lipoproteins and determine the lipoprotein profile in subjects who had suffered an ischemic cerebrovascular event, that is, stroke.

Patients
The study included 55 patients, 23 men, with an average age of 64 years ± 13 years, and 32 women, average age 74 years ± 13 years, who survived an ischemic cerebrovascular event, that is, a large-artery atherosclerosis subtype of stroke. To determine the subtype of ischemic stroke, the original TOAST (Trial of ORG 10172 in Acute Stroke Treatment) [1] criteria were used. The diagnosis of subtype was based on the risk factor profiles, clinical features, and results of diagnostic tests, including CT scan/MRI, vascular imaging (carotid duplex, transcranial Doppler), EEG -electroencephalography, echocardiography (transesophageal/ transthoracic), assessment of prothrombotic syndromes [1,30], activated partial thromboplastin time (aPTT), and international normalized ratio (INR).

Methods
See methods published in the section "Arterial hypertension (AH)." A blood sample from the antecubital vein was obtained throughout the 24 hours after the onset of cerebrovascular event.

Results
The results of lipid parameters presented in Table 9 confirm a highly significantly increased concentration of analyzed lipid and lipoprotein parameters (p<0.0001) in people who survive a stroke, compared to control values, and also a low value on the SAAR, which is generally low (< 10.8) in an atherogenic lipoprotein phenotype.
In Table 10, an atherogenic lipoprotein phenotype was identified in 85.5 % of the patients who survive a cerebral ischemic stroke. The increased concentration of small dense LDL (LDL3-7 subgroups) in the atherogenic lipoprotein profile of patients with stroke, compared to the results in a non-atherogenic lipoprotein profile, is mild, but significant (p<0.05). The difference in the SAAR between the two subgroups was highly significant (p<0.0001), which also confirmed the overwhelming atherogenic lipoprotein constellation in patients who survived a stroke. The concentration of LDL1 was significantly higher in the subgroup of stroke-patients with a non-atherogenic lipoprotein profile (p<0.0001), however, the difference in the LDL2 lipoprotein subfraction was not significant.

Discussion
In the last few decades, lipoprotein research has focused on the phenomenon of atherogenic and non-atherogenic lipoproteins, and on the phenotype A vs. phenotype B characterization, as a consequence of the published evidence that the majority of the patients with an acute coronary syndrome or patients who survive a myocardial infarction had normal plasma values of cholesterol, LDL-cholesterol, and HDL-cholesterol [15][16][17]. A reasonable explanation for this fact was to posit a new, active atherogenic substance in plasma, an atherogenic lipoprotein subfraction, the presence of which in plasma, even in very low concentrations, could impair the integrity of the vessel wall and lead to endothelial dysfunction with its fatal consequences. Several clinical studies reported observations that in the plasma of patients with coronary heart disease there are subfractions of lipoproteins, which could play a crucial role in atherodegenerative processes and form the atherothrombotic plaques [5,33,34,37,39,49]. The Quebec Cardiovascular Study, a prospective study of 2,103 men [33,34] concluded that " a significant proportion of the risk for heart disease associated with small, dense LDL particles may be independent of variations in plasma lipid concentrations. Small LDL particles and elevated apo B levels were found to be the most predictive indications for ischemic heart disease ".
For this reason, patients who were suffering from cardiovascular diseases were examined in order to quantify the atherogenic lipoproteins in serum and to determine the incidence of an atherogenic lipoprotein profile in patients who had a diagnosis of cardiovascular diseases.
The clinical studies included 366 patients with a diagnosis of arterial hypertension (n=107), coronary heart disease (n= 104), lower extremity arterial disease (n= 100), and ischemic stroke (n= 55). Patients were tested with the diagnostic method Lipoprint LDL System, which quantifies atherogenic lipoproteins and identifies an atherogenic and a non-atherogenic lipoprotein profile [29]. This was a fundamental methodological contribution of this new analytical and diagnostic method.
Our study confirmed that more than 80% of tested patents with cardiovascular diseases have an atherogenic lipoprotein profile, with a high level of strongly atherogenic small dense LDL. The atherogenic lipoprotein profile was found to be the overwhelming lipoprotein profile in tested cardiovascular diseases. Such a profile was found in arterial hypertension in 78.5%, in coronary heart disease in 81.7%, in lower extremity arterial disease in 80%, and in patients who survived an ischemic stroke in 85%. The average atherogenic lipoprotein profile in all these tested diagnoses in the study was found to be of 81.3%.
This study also highlights the observation that, in the atherogenic lipoprotein profiles, in all diagnoses, compared to the non-atherogenic profiles, the concentration of total cholesterol is lower (n.s.) and the concentration of triglycerides is higher (even statistically significant; in AH, CHD, LEAD, as well as in the control group, up to p<0.002). Hypertriglyceridemia accompanied the hypercholesterolemia in all tested diagnoses, that is, in AH, CHD, LEAD, and stroke). The concentration of triglycerides, compared to the control group, was significantly increased (p<0.0001) and proportionally even higher than cholesterol. From this result, it can be assumed that triglycerides/hypertriglyceridemia can play a much more important role than was generally accepted, as until now the most important role in the pathogenesis of vascular degenerative atherosclerotic injury was attributed to cholesterol and hypercholesterolemia. Our present results are in agreement with other authors, who have called attention to hypertriglyceridemia as a risk factor for cardiovascular diseases [8,12,19,36,58], as triglyceriderich lipoproteins can generate small dense LDL in high quantities [46].
The strong atherogenic lipoproteins -small dense LDL -have been found in the lipoprotein profile of all diagnostic groups [25,52,55,56]. Their presence is decisive for an atherogenic profile declaration. This is a rule that is valid not only for a hyperlipidemia, but also for a normolipidemia.
In the case of normolipidemia (see the atherogenic lipoprotein profile in the control group), a new phenomenon could be established -atherogenic normolipidemia [44] -as a risk factor for the development of cardiovascular disease. A special form of normolipidemia can also be atherogenic. This is new knowledge, and this new knowledge could help in the prevention and treatment of cardiovascular disease.