**1. Introduction**

72 Lipoproteins – Role in Health and Diseases

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> The 1985 Nobel Prize in Medicine was awarded to American lipidologists Goldstein and Brown for their work in identifying the role of the LDL receptor pathway in lipoprotein metabolism and in maintaining the homeostasis of blood cholesterol (Goldstein & Brown 1985).

> The discovery of the LDL receptor and an understanding of its role in lipid metabolism in health and illness were a milestone in research into metabolic disorders in lipids. At the same time, some other successes in lipoprotein research were also reported: a new understanding of the role of oxidized LDL in atherosclerosis pathogenesis (Steinberg 1987, Witztum & Steinberg 1991); an update of the Ross theory on atherosclerosis genesis (Ross 1986); studies with hypolipidemics; a cholestyramine study, the Coronary Drug Project with niacin, and the Helsinki Heart Study with gemfibrosil. The next two decades was devoted to the effort to create sophisticated criteria for determining risk groups in populations, developing a consensus about cholesterol, and adopting pharmacological uniformity to achieve so-called target lipid values in at-risk individuals with dyslipidemia. The welldefined criteria as a result of these efforts gave hope to at-risk individuals for longer-term survival without ischemic vascular accidents (Canner *et al.* 1986, Frick *et al.* 1987, Expert panel 2001).

> Generally, it was confirmed that hypercholesterolemia represents a risk factor for the development of cardiovascular diseases. In addition to arterial hypertension and nicotine abuse, hypercholesterolemia is considered one of three cardinal risk factors.

> Cholesterol in plasma is transported by a sophisticated lipoprotein complex system and is also an active part of this lipoprotein system. Different parts of the lipoprotein system are called lipoprotein families. Every lipoprotein family transports different concentrations of

cholesterol in blood plasma, but the major conveyor of cholesterol in plasma is the family of Low Density Lipoproteins, i.e., the LDL family. LDL is considered an atherogenic part of the lipoprotein system (Kwiterovich 2002a, 2002b).

LDL transports a major cholesterol load from the liver to the peripheral cells of the body. Under conditions of impaired LDL catabolism in the periphery, LDL particles persist in the circulation, their physical-chemical characteristics are modified, and the physiological pathway of LDL degradation - via LDL receptors - fails. The consequence of this sequence of events is the formation of an alternative metabolic pathway of LDL degradation through scavenger receptors and the formation of cholesterol deposits in the subendothelial space of the arterial wall. In this way, the process of atherogenesis and atherothrombosis begins; and LDL particles play a crucial role at the beginning and in the development of this injury process in the vessel walls (Berneis & Krauss 2002, Haffner 2006, Fruchart *et al.* 2008).

LDL-cholesterol became a criterion for the degree of atherogenic risk for the development of atherothrombosis. A high LDL-cholesterol concentration in plasma correlates positively with the premature onset of cardiovascular diseases, and is considered a strong cardiovascular risk factor. From this point of view, the aim of treatment of hypercholesterolemia, in secondary as well as in primary prevention, is the reduction of LDL concentration in plasma and a lowering of the cholesterol level to the ´´target reference values" (Expert panel 2001, Backers 2005).

However, in the last few decades, lipoprotein research has focused on the phenomenon of atherogenic and non-atherogenic lipoproteins, atherogenic and non-atherogenic lipoprotein profiles, and on the phenotype A vs. phenotype B characterization (Austin *et al.* 1990, Chait *et al.* 1993, Van *et al.* 2007). The traditional approach to hypercholesterolemia as an atherogenic risk factor for the development of degenerative diseases of the cardiovascular system became a target of criticism. Castelli published evidence that more than 75 percent of patients with an acute coronary syndrome or a myocardial infarction had normal plasma values of cholesterol, LDL cholesterol and/or HDL cholesterol (Castelli 1988, 1992, 1998). Thus, it was necessary to look for other risk factors in plasma, at levels that could cause an acute coronary event. An increased cholesterol level, as an universal explanation for the origin of atherogenesis, was no longer valid.

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: formation of atherothrombotic plaques, acute myocardial infarction, stroke, and sudden death (Nichols & Lundmann 2004, Rizzo & Berneis 2006, Shoji *et al.* 2009, Zhao *et al.* 2009).

Those laboratory analysis methods became an essential contribution to the identification of atherogenic lipoprotein entities, which simplified the analysis and quantification of the atherogenic lipoprotein subfractions. Gradient gel elecrophoretic separation of LDL and HDL subclasses or proton nuclear magnetic resonance spectroscopy were the methods of choice for the analysis of these entities (Rainwater *et al.* 1997, Alabakovska *et al.* 2002, Otvos *et al.* 2003).

Recently, electrophoresis of plasma lipoproteins on the polyacrylamide gel (PAG) Lipoprint LDL System is one of several diagnostic analytical methods for the identification and quantitative evaluation of lipoprotein subfractions, i.e., the atherogenic and non-atherogenic lipoproteins (Hoefner *et al.* 2001).

The LDL System has become a staple in routine laboratory analysis and in the diagnosis of lipoprotein metabolism disorders, and has also been recommended by the FDA for human medicine. Lipoprint LDL enables the analysis of 12 lipoprotein subfractions: VLDL; IDL1; IDL2; IDL3; LDL1; LDL2; LDL 3-7; HDL; and determines an atherogenic lipoprotein profile phenotype B versus a non-atherogenic lipoprotein profile phenotype A.

Atherogenic lipoprotein profiles are characterized by a predominance of atherogenic lipoproteins, namely very low density (VLDL), intermediate density IDL1, and IDL2, and particularly by the presence of small dense lipoproteins with low density (LDL). Profiles identify highly atherogenic LDL subfractions that form the LDL 3-7 fractions (Tab.1). These subfractions are smaller, with a diameter < 26.5 nm (265 Angström) and they float within a density range of 1.048 – 1.065 g/ml, i.e., a higher density than LDL1 and LDL2. On the PAG they are detected as subtle bands on the anodic end of the gel right behind HDL that migrate to the head of separated lipoproteins.


#### **Table 1.**

74 Lipoproteins – Role in Health and Diseases

lipoprotein system (Kwiterovich 2002a, 2002b).

values" (Expert panel 2001, Backers 2005).

origin of atherogenesis, was no longer valid.

cholesterol in blood plasma, but the major conveyor of cholesterol in plasma is the family of Low Density Lipoproteins, i.e., the LDL family. LDL is considered an atherogenic part of the

LDL transports a major cholesterol load from the liver to the peripheral cells of the body. Under conditions of impaired LDL catabolism in the periphery, LDL particles persist in the circulation, their physical-chemical characteristics are modified, and the physiological pathway of LDL degradation - via LDL receptors - fails. The consequence of this sequence of events is the formation of an alternative metabolic pathway of LDL degradation through scavenger receptors and the formation of cholesterol deposits in the subendothelial space of the arterial wall. In this way, the process of atherogenesis and atherothrombosis begins; and LDL particles play a crucial role at the beginning and in the development of this injury

process in the vessel walls (Berneis & Krauss 2002, Haffner 2006, Fruchart *et al.* 2008).

LDL-cholesterol became a criterion for the degree of atherogenic risk for the development of atherothrombosis. A high LDL-cholesterol concentration in plasma correlates positively with the premature onset of cardiovascular diseases, and is considered a strong cardiovascular risk factor. From this point of view, the aim of treatment of hypercholesterolemia, in secondary as well as in primary prevention, is the reduction of LDL concentration in plasma and a lowering of the cholesterol level to the ´´target reference

However, in the last few decades, lipoprotein research has focused on the phenomenon of atherogenic and non-atherogenic lipoproteins, atherogenic and non-atherogenic lipoprotein profiles, and on the phenotype A vs. phenotype B characterization (Austin *et al.* 1990, Chait *et al.* 1993, Van *et al.* 2007). The traditional approach to hypercholesterolemia as an atherogenic risk factor for the development of degenerative diseases of the cardiovascular system became a target of criticism. Castelli published evidence that more than 75 percent of patients with an acute coronary syndrome or a myocardial infarction had normal plasma values of cholesterol, LDL cholesterol and/or HDL cholesterol (Castelli 1988, 1992, 1998). Thus, it was necessary to look for other risk factors in plasma, at levels that could cause an acute coronary event. An increased cholesterol level, as an universal explanation for the

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: formation of atherothrombotic plaques, acute myocardial infarction, stroke, and sudden death (Nichols &

Those laboratory analysis methods became an essential contribution to the identification of atherogenic lipoprotein entities, which simplified the analysis and quantification of the atherogenic lipoprotein subfractions. Gradient gel elecrophoretic separation of LDL and HDL subclasses or proton nuclear magnetic resonance spectroscopy were the methods of choice for the analysis of these entities (Rainwater *et al.* 1997, Alabakovska *et al.* 2002, Otvos *et al.* 2003).

Lundmann 2004, Rizzo & Berneis 2006, Shoji *et al.* 2009, Zhao *et al.* 2009).

On the basis of lipoprotein separation by the Lipoprint LDL System, a non-atherogenic normolipidemia, an atherogenic normolipidemia, a non-atherogenic hyperbetalipoproteinemia and an atherogenic hyperlipoproteinemia can be characterized (Oravec 2006a, 2006b, 2007a, 2007b).

#### Two of these are identified as new lipoprotein profiles with high clinical significance: an **atherogenic normolipidemia** and a **non-atherogenic hyper-betalipoproteinemia LDL1,2.**

A non-atherogenic hyperbetalipoproteinemia LDL1,2 involves individuals with a high concentration of plasma cholesterol, predominantly transported by LDL1 and LDL2 subfractions. However, these individuals are at low risk for a cardiovascular event based on

cardiologic and angiologic examimation results, and have familial history negative for cardiovascular diseases.

Conversely, an atherogenic normolipidemia was identified in a group of individuals with normal cholesterol and triglyceride concentrations in plasma, who had a high concentration of strongly atherogenic small dense LDL in the lipoprotein profile. These individuals could be at higher risk for a cardiovascular event despite normolipidemia.

In our clinical study, we characterized hypercholesterolemic individuals with untreated hypercholesterolemia, who had a non-atherogenic hyperbetalipoproteinemia, as well as normolipemic individuals who were currently without clinical or laboratory signs of damage to the cardiovascular system, but who, nevertheless, had an atherogenic lipoprotein profile. All these subjects underwent a medical examination to identify the extent of the arterial vessel damages caused by hypercholesterolemia, or dyslipidemia.
