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Atherogenic Versus Non-atherogenic Lipoprotein Profiles in Healthy Indi-viduals. Is There a Need to Change Our Approach to Diagnosing Dyslipi-demia?

S. Oravec1, A. Dukat1, P. Gavornik1, M. Kucera1, K. Gruber2, L. Gaspar1, M. Rizzo3, P.P. Toth4, D.P. Mikhailidis5 and M. Banach*

,6

12

nd Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic;

2Department of Internal Medicine, Landesklinikum, Thermenregion Baden, Austria;

3Biomedical Department of Internal

Medicine and Medical Specialties, University of Palermo, Italy; 4University of Illinois College of Medicine, Peoria, IL,

USA; 5

Department of Clinical Biochemistry, Royal Free Campus, University College London Medical School, Univer-

sity College London (UCL), London; 6Department of Hypertension, Chair of Nephrology and Hypertension, Medical

University of Lodz, Poland

Abstract: The electrophoretic separation of lipoproteins on polyacrylamide gels enables the quantification of non-atherogenic and atherogenic plasma lipoproteins including small dense low density lipoprotein (sdLDL) particles, which represent the atherogenic lipoprotein subpopulations in plasma. This methodology could help distinguish between non-atherogenic hyperlipidemia, normolipidemia with an atherogenic lipoprotein profile, non-atherogenic normolipidemia, and atherogenic hyperlipidemia. According to our pilot research of a normolipidemic population, the atherogenic lipopro-tein profile might be present in about 6% of normolipidemic young healthy individuals. Therefore, if confirmed by other studies, it will be necessary to consider a different diagnostic approach and risk stratification for patients with atherogenic normolipidemia (as well as non-atherogenic hypercholesterolemia).

Keywords: Atherogenic normolipidemia, lipoprotein profile, low density lipoprotein, small dense LDL, very low-density lipo-protein.

INTRODUCTION

Low density lipoprotein cholesterol (LDL-C) is an im-portant risk factor for the development of atherosclerotic disease [1, 2]. The goal of hypercholesterolemia therapy in primary and secondary prevention is the reduction of athero-genic lipoprotein burden in serum [3, 4]. According to the European Society of Atherosclerosis (EAS)/European Soci-ety of Cardiology (ESC) guidelines (2011) LDL-C and non-high density lipoprotein cholesterol (non-HDL-C) are the primary and secondary targets of therapy in patients at risk for cardiovascular disease (CVD). However, the most recent recommendations of the American College of Cardiology (ACC)/American Heart Association (AHA) Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults do not recommend any spe-cific target of hypolipidemic therapy. Rather, they recom-mend the intensity of statin therapy to be initiated based on the risk for atherosclerotic cardiovascular disease (ASCVD) [5, 6].

In the last three decades, lipoprotein research has focused on the differentiation of atherogenic and non-atherogenic lipoproteins and characterizing atherogenic lipoprotein

*Address correspondence to this author at the Department of Hypertension, Chair of Nephrology and Hypertension, Medical University of Lodz, Ze-romskiego 113; 90-549 Lodz, Poland; Tel: +48 42 639 37 71; Fax: +48 42 639 37 71; E-mail: maciejbanach@aol.co.uk

profiles (i.e. phenotype A vs. phenotype B) [7-9]. Many pa-tients with coronary artery disease (CAD) do not meet crite-ria for the diagnosis of dyslipidemia according to current guidelines.. Castelli published evidence that more than 75% of patients with an acute coronary syndrome (ACS) or a myocardial infarction (MI) had normal plasma values of LDL-C and/or HDL-C [10-12]. It was apparent that a more sophisticated approach to lipoprotein characterization was needed.

More advanced laboratory separation methods have shown that some LDL fractions are more atherogenic than others [13-17]. There are many different methodological approaches for measuring the size, distribution and number of LDL particles/subpopulations: gradient gel electrophoretic separation of LDL subclasses, density gradient ultracentrifu-gation, proton nuclear magnetic resonance spectroscopy, and others [18-22]. However, these methods are technically diffi-cult and time-consuming, which restricts their application in clinical settings. Another drawback of these methods is that the predictive values of the results were not sufficient, com-pared with polyacrylamide gel electrophoresis (PAGE). (Ta-ble 1) presents in detail the advantages and disadvantages of all available analytical methods [19-23]. It is also worth mentioning that all these methods are relatively expensive to perform.

The Lipoprint LDL and HDL Systems use PAGE to iden-tify and quantify lipoprotein subfractions, i.e. atherogenic

2 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Oravec et al.

and non-atherogenic lipoproteins, and distinguish an athero-genic from a non-atherogenic lipoprotein profile [24, 25]. Lipoprint LDL enables the analysis of 12 lipoprotein sub-fractions – VLDL, IDL 1-3, LDL 1-7, and HDL – and dis-tinguishes an atherogenic lipoprotein profile phenotype B from a less atherogenic lipoprotein profile phenotype A. Phenotype A is characterized by a major peak of large, buoyant LDL particles and a minor peak of smaller, denser LDL subspecies [7]. Atherogenic lipoprotein profile (phe-notype B) is characterized by a predominance of atherogenic lipoproteins: very low density (VLDL), intermediate density (IDL1 and IDL2), and particularly by the presence of sdLDL (LDL 3-7 subfractions) [17, 27-33] (Table 1). As their name suggests, they are small, with a diameter <26.5 nm (265 Å), and the density range of 1.048–1.065 g/ml, i.e. higher den-sity than LDL1 and LDL2. On the PAGE they are detected as subtle bands on the anodic end of the gel immediately behind HDL that migrate at the head of separated lipopro-teins. Their atherogenic potential is a consequence of their chemical composition and biological characteristics (Table 2) [14, 30-32]. On the basis of lipoprotein separation by the LDL System, non-atherogenic normolipidemia (Figs. 1, 2), non-atherogenic hypercholesterolemia (Figs. 3, 4), athero-genic normolipidemia (Figs. 5, 6), and atherogenic hyper-lipoproteinemia (Figs. 7, 8) can be characterized [12, 33-36].

NON-ATHEROGENIC HYPERCHOLESTEROLEMIA

Dyslipidemia is one of the primary causes of atheroscle-rosis with its complications: myocardial infarction, stroke, peripheral arterial disease and cardiac mortality. Early diag-nosis and effective therapy for dyslipidemia with lipid modi-

fying medications and lifestyle modification can reduce CVD risk [15-18, 37-39].

Table 2. The Atherogenic Properties of Small Dense LDL Cho-

lesterol [29, 31, 63, 64]

Reasons for Small Dense LDL Higher Atherogenicity:

* Low recognition by LDL receptors (alteration in three-dimensional conformation of apoB results in reduced affinity for the LDL receptor)

* Enhanced predisposition for oxidation and acetylation

* Oxid-LDL release of pro-inflammatory cytokines and interleukins

from macrophages and T helper cells smooth muscle cell apoptosis

* Oxid-LDL release of metalloproteinases from macrophages collagen and elastin degradation

* Oxid-LDL enhanced aptitude trapping in subendothelial space by

intercellular matrix proteins and activates lipid

scavenging by macrophages (scavenger-receptors)

stimulation of foam cell formation

* Easier penetration (smaller diameter) into the subendothelial space

Table 1. Comparison of Different Methods Used for Analysis of Lipoprotein Subfractions [22, 23]

Component Lipoprint Berkely VAP NMR

TEST METHOD Polyacrylamide Gel Elec-

trophoresis Gradient Gel Electrophore-

sis Density Gradient

Ultracentrifugation Nuclear Magnetic Reso-

nance

RESOLUTION

Physical and measurable separation of lipoprotein

subfractions

Physical, non-measurable separation of LDL proteins

Mathematical deconvolu-tion of aggregate bands

Mathematical deconvolu-tion of aggregate NMR

signal

FDA CLEARED YES NO YES YES

NORMAL REFERENCE

RANGE FOR SUB-

CLASSES

YES (quantitative based on ATP III guidelines)

NO Quantitative Classification (based on population dis-

tribution)

Quantitative Classification (based on population dis-

tribution)

NUMBER OF HDL

FRACTIONS

3 (Large, intermediate, small HDL = 10 subfrac-

tions)

5 (HDL2b, HDL2a HDL3a,3b,3c)

3 (HDL-C, HDL2-C, and HDL3-C)

3 (large, medium, small HDL-P = 26 subpopula-

tions)

NUMBER OF LDL

FRACTIONS 7 7 4 3 (47 subpopulations)

NUMBER OF IDL

FRACTIONS 3 Does not report IDL 1 (IDL) 1 (total IDL)

NUMBER OF VLDL

FRACTIONS 1 (total VLDL) Does not report VLDL

3 (VLDL-C, VLDL1+2-C, VLDL3)

4

QUANTITATIVE

ANALYSIS Cholesterol mg/dL LDL-peak particle size Cholesterol mg/dL Particle Number

Atherogenic Versus Non-atherogenic Lipoprotein Profiles in Healthy Individuals Current Medicinal Chemistry, 2014, Vol. 21, No. 1 3

Fig. (1). Distribution of lipoproteins using polyacrylamide gel method. An example of patients with non-atherogenic normolipidemia. Opti-mal distribution of lipoproteins under a normolipemia conditions, atherogenic lipoproteins are not present.

Fig. (2). Distribution of lipoproteins using polyacrylamide gel method. An example of patients with non-atherogenic normolipidemia (HDL subfractions). Optimal concentration of non-atherogenic large HDL (HDL1-3), small HDL (HDL8-10) are present in low concentration.

4 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Oravec et al.

Fig. (3). Distribution of lipoproteins using polyacrylamide gel method. An example of patients with non-atherogenic hypercholesterolemia. Cholesterol is transported by LDL1 and LDL2 subfractions, which are hypothesized to be non-atherogenic. Atherogenic lipoproteins (sdLDL) are not present in this lipoprotein spectrum.

Fig. (4). Distribution of lipoproteins using polyacrylamide gel method. An example of patients with non-atherogenic hypercholesterolemia (HDL subfractions). A high concentration of non-atherogenic large HDL (HDL1-3) is present, small HDL (HDL8-10) are presented in traces concentration.

Atherogenic Versus Non-atherogenic Lipoprotein Profiles in Healthy Individuals Current Medicinal Chemistry, 2014, Vol. 21, No. 1 5

Fig. (5). Distribution of lipoproteins using polyacrylamide gel method. An example of a patient with atherogenic normolipidemia. Athero-genic sdLDL are present in high concentration (LDL3, 4 subfractions).

Fig. (6). Distribution of lipoproteins using polyacrylamide gel method. An example of a patient with atherogenic normolipidemia (HDL sub-fractions). Small HDL (HDL8-10) – a hypothesized atherogenic part of HDL family is present in higher concentration.

6 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Oravec et al.

Fig. (7). Distribution of lipoproteins using polyacrylamide gel method. An example of a patient with atherogenic hypercholesterolemia. High concentration of atherogenic sdLDL (LDL3, 4) is present in lipoprotein spectrum.

Fig. (8). Distribution of lipoproteins using polyacrylamide gel method. An example of a patient with atherogenic hypercholesterolemia (HDL subfractions). High concentration of small HDL (HDL8-10) subfractions is present in lipoprotein spectrum.

Atherogenic Versus Non-atherogenic Lipoprotein Profiles in Healthy Individuals Current Medicinal Chemistry, 2014, Vol. 21, No. 1 7

The findings of hypercholesterolemia in clinically healthy subjects, without clinically apparent signs or labora-tory confirmation of CVD, and with the lack of history of CV events, stimulated an active search for hypercholes-terolemic individuals and undertaking a detailed medical examination of these subjects [42]. The identification of a non-atherogenic hypercholesterolemia offers new informa-tion, which suggests a reevaluation of the present statement, considering the whole LDL family as an atherogenic lipopro-tein part of the plasma lipoprotein spectrum.

‘Buoyant’ LDL1 and LDL2, even in higher concentra-tions in plasma, seems to be less atherogenic than sdLDL – LDL3-7 [7]. The evidence of cardiologic examination and confirmation of common normal results with only mild signs of clinically irrelevant aortic valve sclerosis supports and confirms the reduced atherogenicity of large ‘buoyant’ LDL subfractions in individuals with hyperbetalipoproteinemia LDL1,2. Small dense LDL are specifically associated with the premature development of CVD [16, 26-31, 43-46].

LDL1 and LDL2 subfractions represent an important physiological conveyor of cholesterol in plasma [47, 48]. They are an important source for the biosynthesis of steroid hormones, bile acids, vitamin D3, membranes of cells and of subcellular structures [48-50]. The lowering of LDL1 and LDL2 subfractions by hypolipemic drugs might cause a non-specific lowering of cholesterol, which might have a nega-tive effect on several physiological processes [33-35, 51]. The non-specific lowering of total cholesterol (TC) does not form a non-atherogenic lipoprotein profile [40-54] (Figs. 3

and 4).

The Lipoprint HDL System analysis may also reveal (within 10 analysed subfractions) a reduced small density HDL subpopulation, which is confirmed to be more athero-genic. In the non-atherogenic hypercholesterolemia, small values of small HDLs support even more the non-atherogenic lipoprotein profile of plasma lipids [26, 55-57] (Figs. 3 and 4).

ATHEROGENIC NORMOLIPIDEMIA

Generally, normolipidemia is characterized by TC and TG values within reference ranges [3, 33-36]. We know that patients with normolipidemia are better protected from de-velopment of CVD. As dyslipidemia represents a risk factor of development of CVD, it is a driver of atherogenesis. Therefore, treatment of dyslipidemia has the goal of correct-ing lipid concentrations in serum, reaching target values of lipids, but first of all reducing the atherogenic potential of serum lipids [14, 35, 49-59]. Normolipidemia, it might be assumed, leads to the creation of a non-atherogenic lipopro-tein profile and to lowering or removal of the risk of athero-sclerosis development and prevention of CV events. How-ever, the existence of atherogenic normolipidemia casts doubt on the theory of normolipidemia protection against the development of atherosclerosis.

Atherogenic normolipidemia was identified as a new lipoprotein profile with high clinical significance (in addition to the generally well-known non-atherogenic normolipide-mia) in a group of young individuals with normal cholesterol and triglyceride concentrations in the plasma, which had a

high concentration of highly atherogenic sdLDL. These indi-viduals may be at a higher risk of CV events in spite of nor-molipidemia [33-36].

Our previous studies included individuals which under-went detailed medical examination to identify the extent of the arterial vessel damage, which could be caused by athero-genic lipoprotein subfractions. The medical investigation, which included a physical examination, blood pressure and ECG examination and duplex ultrasound examination of the carotid arteries, confirmed that there was no impairment of the CV system. These young normolipemic individuals were without clinical or laboratory signs of CVD albeit with an atherogenic lipoprotein profile (phenotype B with a pre-dominance of VLDL, IDL1 and IDL2, and particularly by the presence of sdLDL - LDL 3-7 subfractions) and a posi-tive family history of CVD. Additionally, using the Lipoprint HDL System we confirmed high concentration of small HDL (mostly atherogenic and with impaired functionality), which multiply the atherogenicity of the lipoprotein spectrum of atherogenic normolipidemia [25, 36, 55-61] (Figs. 5 and 6). This study was limited due to the fact that no investigation on subclinical atherosclerosis (ankle-brachial pressure index, exercise ECG, arterial stiffness or flow mediated dilation) was performed.

These findings have been recently supported by the pilot results of the ongoing study conducted in a group of healthy young sportsmen with endurance training (rowing; mean age: 14.2±1.2 years, mean body mass index: 21.5±3.6 kg/m2, mean fat-free body mass: 82.2±4.3%) vs standard regular trening (lawn tennis; mean age: 14.2±0.8 years, mean body mass index: 19.0±2.3 kg/m2, mean fat-free body mass: 84.6±2.7%) [62]. We noticed that sdLDL (LDL3,4 subfrac-tions) were present in 31.5% of all participants at baseline (in 23% in the reasearch group and 41% in the control group, respectively). Interesingly, in both groups the changes in resting ECG and echocardiography (in 27% in the research group and 44% in control group, respectively) were ob-served. Interesingly, it has been also observed that in the group with the endurance traning the ratio of sdLDL has still increased, and significantly decreased in the group with the standard trening. This increase in the group with endurance traning has correlated with the increase of N-terminal pro-brain natriuretic peptide (NT-proBNP) and C-reactive pro-tein levels [62].

DISCUSSION

The identification of atherogenic and non-atherogenic lipoproteins in the plasma lipoprotein spectrum represents new diagnostic approach with deeper analysis of lipoprotein parameters than current existing routine analysis of plasma TC, TGs or lipoproteins such as LDL, HDL and VLDL. These lipid parameters bring limited information about the percentage of subjects who are at risk of atherosclerosis pro-gression and endothelial dysfunction due to atherogenic dyslipidemia with their consequences - cardiovascular or cerebrovascular events [63, 64]. The individuals with athero-genic normolipidemia are at risk of CV events. Therefore, it is desirable recognize these individuals, perform detailed examination and recommend optimal therapy (non-pharmacological, and potentially pharmacotherapy). The

8 Current Medicinal Chemistry, 2014, Vol. 21, No. 1 Oravec et al.

existence of even 6% of normolipidemic young healthy indi-viduals with an atherogenic lipoprotein profile among clini-cally healthy volunteers puts in doubt our former belief and generally accepted statement that normolipidemia per se represents an optimal healthy lipid profile. When a strong atherogenic sdLDL persist, the atherogenicity of the plasma significantly increases [44-50].

Atherogenic normolipidemia in the lipoprotein profile of clinically examined healthy subjects represents a completely new phenomenon [36, 55, 56]. Moreover, it has important medical consequences, as these individuals may be at risk for premature CVD and therefore should undergo regular medi-cal screening examination with targeted preventive manage-ment.

Identification of the type of lipoprotein profile (athero-genic vs. non-atherogenic) represents a beneficial contribu-tion to lipid diagnostics and an advance in quality of inter-pretation in lipoprotein analysis. Obviously many questions still need to be answered, especially why in some healthy young individuals we can observe an atherogenic lipid pro-file, what factors influence it (genetically based?), and fi-nally what would be an optimal therapeutic strategy, espe-cially in connection to current discussion regarding the man-agement of lipid disorders in primary prevention.

DECLARATION OF INTEREST

This review was written independently; no company or institution supported it financially. Some of the authors have given talks, attended conferences and participated in trials and advisory boards sponsored by various pharmaceutical companies. No professional writer was involved in the preparation of this review.

ACKNOWLEDGEMENTS

This study was support by the EU structural research fund Interreg III AT-SR, project code 1414-02-000-28, dur-ing 2006-2008 (Dr Oravec et al.) and Polish Ministry of Sport and Tourism Research Grant No. 2013.055/40/BP/ DWM (Dr. Banach et al.). We acknowledge the excellent technical assistance of MTA Olga Reinoldova, 2nd Depart-ment of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.

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Received: January 26, 2014 Revised: February 04, 2014 Accepted: February 25, 2014