**2.3. Apolipoproteins in lipoproteins**

Apolipoproteins are the carrier proteins for lipoproteins and they consist of a single polypeptide chain often with relatively little tertiary structure. They are required to solubilise the non-polar lipids in the circulation and in some instances to recognise specific receptors. They are classified as Apo A1, A2, A4, A5, B48, B100, C1, C2, C3, D, E, H, J, L, M and Apo (a). Most apolipoproteins are synthesised by the liver and intestine.


**Table 2.** Classes of apolipoproteins, their molecular weight and functions

#### *2.3.1. Apolipoprotein A*

202 Lipoproteins – Role in Health and Diseases

**2.3. Apolipoproteins in lipoproteins** 

**Apo AIII** 46,000 CM

Apolipoproteins are the carrier proteins for lipoproteins and they consist of a single polypeptide chain often with relatively little tertiary structure. They are required to solubilise the non-polar lipids in the circulation and in some instances to recognise specific receptors. They are classified as Apo A1, A2, A4, A5, B48, B100, C1, C2, C3, D, E, H, J, L, M

> acyltransferase (LCAT) activation. Main structural protein. Binds ABCA1 on macrophages

RNA editing, lacks the LDL

receptor binding site

progesterone binding

B100 and similar to plasminogen, associated with premature

coronary artery disease and stroke

receptor

metabolism

phosphate

and Apo (a). Most apolipoproteins are synthesised by the liver and intestine.

**Apo AI** 29,100 HDL Lecithin: cholesterol

**Apo AII** 17,400 HDL Enhances hepatic lipase activity

**Apo AV** 39,000 HDL Enhances triacylglycerol uptake **Apo B48** 241,000 CM Derived from Apo B100 gene by

**Apo AIV** 46,000 HDL, CM Inhibits food intake in CNS

**Apo B100** 512,000 LDL, IDL, VLDL Binds to LDL receptor

**Apo CII** 8,900 VLDL, CM Activates lipoprotein lipase **Apo CIII** 8,750 VLDL, CM Inhibits lipoprotein lipase

**Apo D** 33,000 HDL Closely associated with LCAT,

**Apo E** 34,000 HDL At least 3 forms. Binds to LDL

**Apo(a)** 300,000 – 800,000 LDL, Lp(a) Linked by disulfide bonds to apo

**Apo H** 50,000 Chylomicrons Involved with triacylglycerol

**Apo M** HDL Transports sphingosine-1-

**Table 2.** Classes of apolipoproteins, their molecular weight and functions

**Apo CI** 7,600 VLDL, CM Activates LCAT

**Apolipoprotein Molecular weight Lipoprotein Function** 

Apo A are subdivided into apo AI, AII, AIII, AIV and AV. Apo AI and AII are the major apolipoproteins in the HDL particle. HDL is primarily saddled with the responsibility of removing excess cholesterol from peripheral tissues and delivering it to the liver for excretion in bile as bile acids in a process known as reverse cholesterol transport. Apo AI is synthesised mainly by the liver and also by the intestine. The protein consists of 243 amino acids arranged as eight α-helical segments of 22 amino acids which have 11 –mer repeats, no disulfide bonds or glycosylations. The helices are believed to be amphipathic in nature with both hydrophobic and hydrophilic faces. This nature enhances its interaction with the lipid and aqueous phases. Apo AII on the other hand is found as a twin chain of 77 amino acids each, linked by disulfide bonds. It enhances the activity of hepatic lipase, thus increasing lipoprotein metabolism. Apo III is found in chylomicrons.

#### *2.3.2. Apolipoprotein E*

Three isoforms of this apolipoprotein exist and they are all synthesised mainly by the liver and also by several tissues such as arterial wall, brain and adipose tissue. They are important for homeostasis of lipid and lipoproteins in blood circulation as well as their metabolism in these tissues. Apo E is required for the clearance of VLDL remnant (IDL) from circulation in the liver. Other suggestions on its involvement with immune response and inflammation have also been put forward.

#### *2.3.3. Apolipoprotein B*

Two types of apolipoprotein B are synthesised from the intestine and liver as apo B100 which has the full length of 4536 amino acid residues and a truncated form with 48% of the full length known as apo B48. These proteins which are synthesised on ribosomes, an organelle located on the surface of rough endoplasmic reticulum are translocated through the reticular membrane into the lumen of endoplasmic reticulum. Assembly of VLDL occurs here by accretion of lipids to the core of apo B particle. This occurs in three distinct stages as the apo B grows bigger, forming the pre-VLDL to VLDL 2 which eventually grows to become the triacylglycerol-rich VLDL 1 or chylomicrons, the most energy dense substances in the body. VLDL 2 is a triacylglycerol-poor version of VLDL and its assembly occurs in golgi bodies. This is transported to basolateral membrane of the intestinal cells where final assembly of VLDL 1 or chylomicrons occur and these are secreted into the lamina propria of the intestinal cells by reverse exocytosis.

Apo B100 and B48 are large and water-insoluble, and are the only non-exchangeable apolipoproteins. They are major components of chylomicrons and VLDL, and usually remain with their lipid aggregates throughout their passage in plasma and several metabolic changes occurring during their circulation in plasma. Chylomicrons are usually transported through the intestinal lymphatic system and flow into blood circulation via the left subclavian vein. As apo B carries the VLDL or chylomicrons through the blood stream, their triacylglycerol content are removed by peripheral tissues via enzymatic activity of lipoprotein lipase, located in the endothelial wall. This makes free fatty acids available for energy production in muscle and some are stored in adipose tissue. Apo B48 remains with the remnant of the lipoprotein particle, along with dietary cholesterol and apo E. the lipoprotein remnant is eventually cleared from circulation in the liver by an apo E dependent receptor-mediated reaction.

ApoB-100 is one of the largest monomeric proteins known and it is the major apolipoprotein component common to the atherogenic lipoproteins [VLDL, LDL, IDL and Lp(a)] (Boerwinkle *et al.*, 1992; Carmena *et al.*, 2004). Apo B100 differs from apo B48 by the presence of LDL receptor site on the apo B100 molecule. Apo B-100 is encoded for by the Apo B gene and mutations in this gene cause familial hypercholesterolemia, an autosomal hereditary metabolic disorder. The level of apo B-100 is a stronger predictor of risk than LDL in humans. Apo B-100 is speculated to mediate delivery of the cholesterol content of lipoproteins to cells via an unknown mechanism. It is well established that Apo B-100 is associated with atherogenic lipoproteins, thus the number of Apo B-100 can be used to determine the risk of atherosclerosis and CHD in individuals. Apo B-100/apo A-I ratio are strongly and positively related to increased risk of fatal myocardial infarction. Apolipoprotein A-I (apo A-I) is the major apolipoprotein in the HDL structure. The Apo B-100/apo A-I ratio is especially valuable in patients with normal or low LDL concentrations, a frequent observation in type 2 diabetes mellitus which may present with hypertriglyceridemia and hyper-apo B concentrations (Carmena *et al.*, 2004).

#### *2.3.4. Apolipoprotein C*

Apolipoprotein C is subdivided into three and each has its own distinct function. Apo CI is involved with activation of Lecithin: cholesterol acyltransferase (LCAT) along with apo AI. This enzyme converts free cholesterol into cholesterol ester, which enhances the incorporation of cholesterol into the lipid core of a lipoprotein particle, particularly in assembly of HDL particle. The enzyme is mainly bound to HDL and LDL in plasma. Apo CII and CIII have antagonistic activity to each other which are required in regulation of lipoprotein lipase activity. Apo CII is required for activation of lipoprotein lipase, while CIII inhibits lipoprotein lipase activity. Apo CII is believed to open a lid-like region of the enzyme which allows the active site to hydrolyse the fatty acid ester bonds of triacylglycerols. In addition to inhibition of lipoprotein lipase, apo CIII also inhibits the binding of lipoproteins to receptors at the cell surface, thereby decreasing hydrolysis of triacylglycerols. High levels of apo CIII have been associated with elevated serum levels of triacylglycerols (hypertriglyceridemia).

#### *2.3.5. Transfer of apolipoproteins in lipoprotein homeostasis*

Lipids enter blood circulation bound to apolipoproteins as chylomicrons or VLDL which as secreted into the blood stream from the intestines. Chylomicrons or VLDL consist mainly of apo B100 and B48, but also consist of some apo AI, along with other apolipoprotein which will be discussed. These lipoproteins carry triacylglycerol-rich cholesterols to the peripheral tissues to provide sources of energy and for storage, while HDL carries excess cholesterol from peripheral tissue to the liver for excretion in bile acids. Immediately chylomicrons enter blood circulation, an exchange of apolipoproteins occurs between chylomicrons and HDL. The apo AI content of chylomicrons is exchanged for the apo C and E content of HDL. Apo C content is required for activation and inhibition of lipoprotein lipase which hydrolyses the triacylglycerol content of chylomicrons and VLDL, while apo E is needed for the receptor mediated clearance from circulation. Circulation of VLDL and chylomicrons in the blood stream exposes the particles to enzymatic release of triacylglycerols from the lipoprotein core and excess cholesterol is removed from cells. The triacylglycerol- poor and cholesterol rich LDL remnant produced is potentially toxic to the body and needed to be safely cleared from blood circulation. The main concern for this lipoprotein is its toxic effect on the cardiovascular system. The liver scavenges and disposes chylomicrons remnant more effectively than the LDL particles, a mechanism put in place by the body to get rid of the more atherogenic particle of the two; chylomicrons remnant. LDL particles are mostly removed by other mechanisms involving HDL.

#### *2.3.6. Apolipoprotein(a)*

204 Lipoproteins – Role in Health and Diseases

*2.3.4. Apolipoprotein C* 

triacylglycerols (hypertriglyceridemia).

*2.3.5. Transfer of apolipoproteins in lipoprotein homeostasis* 

dependent receptor-mediated reaction.

triacylglycerol content are removed by peripheral tissues via enzymatic activity of lipoprotein lipase, located in the endothelial wall. This makes free fatty acids available for energy production in muscle and some are stored in adipose tissue. Apo B48 remains with the remnant of the lipoprotein particle, along with dietary cholesterol and apo E. the lipoprotein remnant is eventually cleared from circulation in the liver by an apo E

ApoB-100 is one of the largest monomeric proteins known and it is the major apolipoprotein component common to the atherogenic lipoproteins [VLDL, LDL, IDL and Lp(a)] (Boerwinkle *et al.*, 1992; Carmena *et al.*, 2004). Apo B100 differs from apo B48 by the presence of LDL receptor site on the apo B100 molecule. Apo B-100 is encoded for by the Apo B gene and mutations in this gene cause familial hypercholesterolemia, an autosomal hereditary metabolic disorder. The level of apo B-100 is a stronger predictor of risk than LDL in humans. Apo B-100 is speculated to mediate delivery of the cholesterol content of lipoproteins to cells via an unknown mechanism. It is well established that Apo B-100 is associated with atherogenic lipoproteins, thus the number of Apo B-100 can be used to determine the risk of atherosclerosis and CHD in individuals. Apo B-100/apo A-I ratio are strongly and positively related to increased risk of fatal myocardial infarction. Apolipoprotein A-I (apo A-I) is the major apolipoprotein in the HDL structure. The Apo B-100/apo A-I ratio is especially valuable in patients with normal or low LDL concentrations, a frequent observation in type 2 diabetes mellitus which may present with

hypertriglyceridemia and hyper-apo B concentrations (Carmena *et al.*, 2004).

Apolipoprotein C is subdivided into three and each has its own distinct function. Apo CI is involved with activation of Lecithin: cholesterol acyltransferase (LCAT) along with apo AI. This enzyme converts free cholesterol into cholesterol ester, which enhances the incorporation of cholesterol into the lipid core of a lipoprotein particle, particularly in assembly of HDL particle. The enzyme is mainly bound to HDL and LDL in plasma. Apo CII and CIII have antagonistic activity to each other which are required in regulation of lipoprotein lipase activity. Apo CII is required for activation of lipoprotein lipase, while CIII inhibits lipoprotein lipase activity. Apo CII is believed to open a lid-like region of the enzyme which allows the active site to hydrolyse the fatty acid ester bonds of triacylglycerols. In addition to inhibition of lipoprotein lipase, apo CIII also inhibits the binding of lipoproteins to receptors at the cell surface, thereby decreasing hydrolysis of triacylglycerols. High levels of apo CIII have been associated with elevated serum levels of

Lipids enter blood circulation bound to apolipoproteins as chylomicrons or VLDL which as secreted into the blood stream from the intestines. Chylomicrons or VLDL consist mainly of apo B100 and B48, but also consist of some apo AI, along with other apolipoprotein which Apolipoprotein(a) itself is a large glycoprotein that exhibits size heterogeneity among individuals with isoforms that range between 180 – 700kDa in size. Apo(a) genotypes were determined using a newly developed pulsed-field gel electrophoresis method which distinguished 19 different genotypes at the apo(a) locus. The apo(a) gene itself was found to account for virtually all the genetic variability in plasma Lp(a) levels (Boerwinkle *et al.*, 1992). The apo(a) cDNA contains multiple tandem copies of a sequence that encodes a cysteine-rich protein motif called a kringle. The particular repeated kringle in apo(a) is designated kringle 4 because it closely resembles the fourth kringle in plasminogen, with the protease domain of apo(a) containing 88% amino-acid identity to plasminogen. McLean *et al.* (1992) proposed that the apo(a) isoforms are of different sizes because of variations in the numbers of kringle 4-encoding repeats in the apo(a) gene. The molecular mass of apo(a) protein varies from 187 kDa for an apo(a) that contains 12 kringle 4 domains, to 662 kDa for an apo(a) that contains 50 kringle 4 domains (Carmena *et al.*, 2004).

#### **2.4. Lipoprotein A [Lp(a)]**

Lipoprotein [Lp(a)] is a variant of LDL with an additional apolipoprotein in the structure. Lp(a) is essentially an LDL particle with a large glycoprotein, apolipoprotein (a) [apo(a)] attached to it (McLean *et al.*, 1987; Loscalzo *et al.*, 1990; Boerwinkle *et al.*, 1992; Palabrica *et al.*, 1995). Lp(a) resembles low density lipoprotein (LDL) in lipid composition, but it is distinguished by the presence of apo(a) which is bound by a disulfide linkage to apolipoprotein B-100, a ligand in the LDL molecule by which LDL binds to its receptor. Lp(a) levels has been demonstrated to have a clear association with development of atherosclerosis and other cardiovascular diseases (Zenker *et al.*, 1986; Danesh *et al.*, 2000; Berglund and Anuurad, 2008; Danik *et al.*, 2008). The postulated atherogenicity of Lp(a) is

probably due to the presence of apo(a) component of the Lp(a) molecule. A study showed that the removal of apo(a) from Lp(a) particles result in a lipoprotein with greatly enhanced affinity for the LDL receptor (Armstrong *et al.*, 1985).

#### *2.4.1. Synthesis of lipoprotein(a)*

Apo(a) is expressed by liver cells (hepatocytes), and the assembly of apo(a) and LDL particles seems to take place at the outer hepatocyte surface. The half-life of Lp(a) in the circulation is about 3 to 4 days (Rader *et al*., 1993) and this particle varies in blood concentration from one individual to another from <0.2 - >200 mg/dL. Ethnicity is a factor, with those of Asian and African origin averaging the highest concentrations. Within ethnic groups, individual elevation of Lp(a) is directly associated with increased risk of cardiovascular diseases (Sandholzer *et al.*, 1991; Chien *et al.*, 2008). Lp(a) is usually unaffected by factors like age, blood pressure, and total cholesterol.

#### *2.4.2. Similarity between lipoprotein(a) and plasminogen*

The structure of Lp(a) is similar to plasminogen, a naturally occurring glycoprotein that participates in dissolving of clots that form in the bloodstream, and tissue plasminogen activator (tPA). Lp(a) competes with plasminogen for its binding site, leading to reduced fibrinolysis (Loscalzo *et al.*, 1990; Palabrica *et al.*, 1995). Also because Lp(a) stimulates secretion of PAI-1 it leads to thrombogenesis. In addition, because of LDL cholesterol content, Lp(a) contributes to atherosclerosis (Schreiner *et al.*, 1993; Sotiriou *et al.*, 2006) and ultimately a cardiovascular risk factor (Berglund and Ramakrishnan, 2004).

#### *2.4.3. Correlation between apolipoprotein size and Lp(a) concentration*

There is a general inverse correlation between the size of the apo(a) isoform and the Lp(a) plasma concentration (Bowden *et al.*, 1994; Kraft *et al.*, 1996) which is caused by a variable rate of degradation before the apo(a) protein has matured for Lp(a) assembly (White *et al.*, 1994). The plasma concentration of Lp(a) is unaffected by many physiological, pharmacological, and environmental factors that affect the levels of other plasma lipoproteins (Albers *et al.*, 1977). A genetic determination of plasma Lp(a) levels was strongly suggested due to this lack of environmental and physiological influences. Consistent with this formulation, early genetic studies suggested that the presence of Lp(a) in plasma was inherited as a single autosomal dominant trait (Berg and Mohr, 1963; Iselius *et al.*, 1981), with an estimated heritability level ranging from 0.75 to 0.98 (Boerwinkle *et al.*, 1992).

Plasma Lp(a) concentrations vary 1000-fold between individuals and represent a continuous quantitative genetic trait with a skewed distribution in Caucasian populations (Utermann, 1989). A study was conducted by Lackner *et al.* (1991) in which the apo(a) gene of members of 12 Caucasian families were segregated. It was found that within a given family, sibling pairs with identical apo(a) genotypes tended to have very similar plasma Lp(a) levels (Lackner *et al.,* 1991). However, individuals with the same apo(a) genotypes who were members of different families often had significantly different plasma concentrations of Lp(a). Taken together, these observations suggest that the apo(a) gene is the major determinant of plasma Lp(a) levels and that cis-acting DNA sequences at or near the apo(a) locus, other than the number of kringle 4 repeats, contribute importantly to plasma Lp(a) concentrations (Boerwinkle *et al.*, 1992). Variation in the hypervariable apo(a) gene on chromosome 6q2.6-q2.7 and interaction of apo(a) alleles with defective LDL-receptor genes explain a large fraction of the variability of plasma Lp(a) concentrations (Utermann, 1989).

Furthermore, the size of the apo(a) glycoprotein varies in individuals and this size is inversely correlated with the plasma level of Lp(a). The reason for the inverse correlation between the size of the apo(a) gene and level of plasma Lp(a) is not known, but a variation of length within the kringle 4-encoding region of the apo(a) gene may account for a greater proportion of the inter-individual variation in plasma Lp(a) concentrations. Also, the number of kringle 4 repeats in the gene may not have a direct effect on plasma Lp(a) concentration (Boerwinkle *et al.*, 1992; Brunner *et al.*, 1996). A study conducted on apo(a) gene of mamorset monkeys showed a plasma Lp(a) concentration of a very wide range of over a 100-fold, but only one apo(a) isoform (Guo *et al.*, 1991). This may be explained by the differences in the composition of kringle 4 sequence of apo(a) genes in which individuals may have same sizes of apo(a) alleles but different plasma Lp(a) concentrations. The frequency of recombination activity in this locus may be responsible for the variation in their kringle 4 composition and number which may have marked effect on synthesis and /or degradation of Lp(a) (Boerwinkle *et al.*, 1992).

#### **2.5. Role of oxidation in atherogenesis**

206 Lipoproteins – Role in Health and Diseases

*2.4.1. Synthesis of lipoprotein(a)* 

affinity for the LDL receptor (Armstrong *et al.*, 1985).

unaffected by factors like age, blood pressure, and total cholesterol.

ultimately a cardiovascular risk factor (Berglund and Ramakrishnan, 2004).

*2.4.3. Correlation between apolipoprotein size and Lp(a) concentration* 

*2.4.2. Similarity between lipoprotein(a) and plasminogen* 

ranging from 0.75 to 0.98 (Boerwinkle *et al.*, 1992).

probably due to the presence of apo(a) component of the Lp(a) molecule. A study showed that the removal of apo(a) from Lp(a) particles result in a lipoprotein with greatly enhanced

Apo(a) is expressed by liver cells (hepatocytes), and the assembly of apo(a) and LDL particles seems to take place at the outer hepatocyte surface. The half-life of Lp(a) in the circulation is about 3 to 4 days (Rader *et al*., 1993) and this particle varies in blood concentration from one individual to another from <0.2 - >200 mg/dL. Ethnicity is a factor, with those of Asian and African origin averaging the highest concentrations. Within ethnic groups, individual elevation of Lp(a) is directly associated with increased risk of cardiovascular diseases (Sandholzer *et al.*, 1991; Chien *et al.*, 2008). Lp(a) is usually

The structure of Lp(a) is similar to plasminogen, a naturally occurring glycoprotein that participates in dissolving of clots that form in the bloodstream, and tissue plasminogen activator (tPA). Lp(a) competes with plasminogen for its binding site, leading to reduced fibrinolysis (Loscalzo *et al.*, 1990; Palabrica *et al.*, 1995). Also because Lp(a) stimulates secretion of PAI-1 it leads to thrombogenesis. In addition, because of LDL cholesterol content, Lp(a) contributes to atherosclerosis (Schreiner *et al.*, 1993; Sotiriou *et al.*, 2006) and

There is a general inverse correlation between the size of the apo(a) isoform and the Lp(a) plasma concentration (Bowden *et al.*, 1994; Kraft *et al.*, 1996) which is caused by a variable rate of degradation before the apo(a) protein has matured for Lp(a) assembly (White *et al.*, 1994). The plasma concentration of Lp(a) is unaffected by many physiological, pharmacological, and environmental factors that affect the levels of other plasma lipoproteins (Albers *et al.*, 1977). A genetic determination of plasma Lp(a) levels was strongly suggested due to this lack of environmental and physiological influences. Consistent with this formulation, early genetic studies suggested that the presence of Lp(a) in plasma was inherited as a single autosomal dominant trait (Berg and Mohr, 1963; Iselius *et al.*, 1981), with an estimated heritability level

Plasma Lp(a) concentrations vary 1000-fold between individuals and represent a continuous quantitative genetic trait with a skewed distribution in Caucasian populations (Utermann, 1989). A study was conducted by Lackner *et al.* (1991) in which the apo(a) gene of members of 12 Caucasian families were segregated. It was found that within a given family, sibling pairs with identical apo(a) genotypes tended to have very similar plasma Lp(a) levels (Lackner *et al.,* 1991). However, individuals with the same apo(a) genotypes who were members of different families often had significantly different plasma concentrations of Oxidative stress, especially LDL oxidation has been suggested for almost three decades as the most probable aetiology of atherosclerosis (Steinbrecher *et al.*, 1984). Markers of LDL oxidation in plasma, particularly circulating oxidized LDL and auto-antibodies against oxidized LDL, could be used to assess the development of atherosclerosis in patients (Carmena *et al.*, 2004). Circulating oxidized LDL is additive to the global risk score based on age, sex, total and HDL cholesterol, diabetes mellitus, hypertension, and smoking as a useful marker for identifying persons at risk for CAD (Holvoet *et al.*, 2001; Toshima *et al.*, 2000).

A study has associated circulating oxidized LDL with both subclinical atherosclerosis (clinically silent ultrasound assessed atherosclerotic changes in the carotid and femoral arteries) and inflammatory variables (C-reactive protein and the inflammatory cytokines interleukin-6 and tumor necrosis factor-α). This conclusion supports the concept that oxidatively modified LDL may play a major role in development of atherosclerosis (Hulthe and Fagerberg, 2002). It has been proposed that, because of the antigenic properties of oxidized LDL, the anti-oxidized LDL antibody titer could represent a useful index of in vivo LDL oxidation. Autoantibodies against oxidized LDL have been reported to be associated with atherosclerosis, but existing reports are still conflicting. Some studies have reported a positive relationship between autoantibodies against oxidized LDL and CHD (Sherer *et al.*, 2001) whereas another did not (Leinonen *et al.*, 1998). There is a strong cross-reactivity between autoantibodies against oxidized LDL and anticardiolipin antibodies, which have been positively associated with CHD (Erkkila *et al.*, 2000).

#### **2.6. Relationship between insulin resistance, diabetes, and small, dense LDL**

Cardiovascular heart disease risk is usually significantly increased when elevated levels of small, dense LDL accompanied by hypertriglyceridemia, reduced HDL-cholesterol levels, abdominal obesity, and insulin resistance. Results from the Que´bec Cardiovascular Study have indicated that persons displaying elevated plasma concentrations of insulin and apo B together with small, dense LDL particles showed a remarkable increase in CHD risk (Lamarche *et al.*, 1999).

Sensitivity to insulin in diabetic and non-diabetic individuals was assessed using nuclear magnetic resonance (NMR) spectroscopy. Insulin resistance had profound effects on lipoprotein size and an increase in serum triglycerides. The lipid profile revealed a 2- to 3 fold increase in concentrations of large VLDL particles with no change in medium or small VLDL, increase in overall LDL particle concentration with more small LDL particle size and reduced large LDL. In type 2 diabetes, these alterations could be attributed primarily to the underlying insulin resistance. These changes in the NMR lipoprotein subclass profile predictably increased the risk of cardiovascular disease but were not fully apparent in the conventional lipid profile (Garvey *et al.*, 2003). The Diabetes Atherosclerosis Intervention Study (DAIS) (Vakkilainen *et al.*, 2003) showed that lipid-modifying treatment decreased the angiographic progression of coronary atherosclerosis in subjects with type 2 diabetes. This effect was related in part to the correction of lipoprotein abnormalities. Compared with placebo, fenofibrate treatment significantly increased LDL particle size and HDL cholesterol and decreased plasma total cholesterol, LDL cholesterol, and triglyceride concentrations. The final LDL particle size was inversely correlated with the increase in percentage diameter stenosis (Vakkilainen *et al.*, 2003).

The Pittsburgh Epidemiology of Diabetes Complications Study on whether NMR lipoprotein spectroscopy improves the prediction of coronary artery disease (CAD) in patients with childhood-onset type 1 diabetes, independently of conventional lipid and other risk factors showed that both lipid mass and particle concentrations (NMR spectroscopy) of all VLDL subclasses, small LDL, medium LDL, and medium HDL were increased in CAD cases compared with controls, whereas large HDL was decreased. Mean LDL and HDL particle sizes were also less in CAD cases (Soedamah-Muthu *et al.*, 2003; Carmena *et al.*, 2004).

#### **2.7. Dyslipoproteinaemia**

Dyslipoproteinaemia is a term broadly used for derangement in lipid and lipoprotein metabolism, which may either be hyperlipoproteinaemia or hypolipoproteinaemia. Dyslipoproteinaemias are generally classified as familial (primary) or acquired (secondary). This chapter discusses hyperlipoproteinaemia with its close relevance to development of cardiovascular diseases. Primary hyperlipoproteinaemias are of genetic origin and may be due to a mutation in a receptor protein which presents as inborn errors of lipid metabolism, and includes common hypercholesterolemia, combined familiar hyperlipidemia, familiar hypercholesterolemia, familiar hypertriglyceridemia, VLDL remnants hyperlipidemia and primary chylomicronaemia (Garmendia, 2003). Primary hyperlipoproteinaemia was first classified by Fredrickson and Lees (1965) and this classification was adopted by World Health Organization (WHO). They divided primary hyperlipoproteinaemias into four types and details are show in Table 3 below.

The secondary hyperlipoproteinemias also mimic primary types and may present with similar symptoms. Secondary dyslipoproteinaemias are usually due to other underlying causes that lead to alterations in plasma lipid and lipoprotein metabolism, including hypothyroidism, diabetes mellitus, nephrotic syndrome, chronic biliary obstruction, renal insufficiency. Some drugs modify lipid metabolism and these include alcohol, betaadrenergic blockers, diuretics, progestagens, corticosteroids (Garmendia, 2003). Treatment of the underlying cause or discontinuation of offending drug may resolve the dyslipoproteinaemia. Lipid and lipoprotein abnormalities are common observations and are regarded as modifiable risk factors for development of cardiovascular diseases.


Adapted from Fredrickson classification of hyperdyslipoproteinaemia (Fredrickson and Lees, 1965).

**Table 3.**

208 Lipoproteins – Role in Health and Diseases

(Lamarche *et al.*, 1999).

stenosis (Vakkilainen *et al.*, 2003).

Carmena *et al.*, 2004).

**2.7. Dyslipoproteinaemia** 

**2.6. Relationship between insulin resistance, diabetes, and small, dense LDL** 

Cardiovascular heart disease risk is usually significantly increased when elevated levels of small, dense LDL accompanied by hypertriglyceridemia, reduced HDL-cholesterol levels, abdominal obesity, and insulin resistance. Results from the Que´bec Cardiovascular Study have indicated that persons displaying elevated plasma concentrations of insulin and apo B together with small, dense LDL particles showed a remarkable increase in CHD risk

Sensitivity to insulin in diabetic and non-diabetic individuals was assessed using nuclear magnetic resonance (NMR) spectroscopy. Insulin resistance had profound effects on lipoprotein size and an increase in serum triglycerides. The lipid profile revealed a 2- to 3 fold increase in concentrations of large VLDL particles with no change in medium or small VLDL, increase in overall LDL particle concentration with more small LDL particle size and reduced large LDL. In type 2 diabetes, these alterations could be attributed primarily to the underlying insulin resistance. These changes in the NMR lipoprotein subclass profile predictably increased the risk of cardiovascular disease but were not fully apparent in the conventional lipid profile (Garvey *et al.*, 2003). The Diabetes Atherosclerosis Intervention Study (DAIS) (Vakkilainen *et al.*, 2003) showed that lipid-modifying treatment decreased the angiographic progression of coronary atherosclerosis in subjects with type 2 diabetes. This effect was related in part to the correction of lipoprotein abnormalities. Compared with placebo, fenofibrate treatment significantly increased LDL particle size and HDL cholesterol and decreased plasma total cholesterol, LDL cholesterol, and triglyceride concentrations. The final LDL particle size was inversely correlated with the increase in percentage diameter

The Pittsburgh Epidemiology of Diabetes Complications Study on whether NMR lipoprotein spectroscopy improves the prediction of coronary artery disease (CAD) in patients with childhood-onset type 1 diabetes, independently of conventional lipid and other risk factors showed that both lipid mass and particle concentrations (NMR spectroscopy) of all VLDL subclasses, small LDL, medium LDL, and medium HDL were increased in CAD cases compared with controls, whereas large HDL was decreased. Mean LDL and HDL particle sizes were also less in CAD cases (Soedamah-Muthu *et al.*, 2003;

Dyslipoproteinaemia is a term broadly used for derangement in lipid and lipoprotein metabolism, which may either be hyperlipoproteinaemia or hypolipoproteinaemia. Dyslipoproteinaemias are generally classified as familial (primary) or acquired (secondary). This chapter discusses hyperlipoproteinaemia with its close relevance to development of cardiovascular diseases. Primary hyperlipoproteinaemias are of genetic origin and may be due to a mutation in a receptor protein which presents as inborn errors of lipid metabolism, and includes common hypercholesterolemia, combined familiar hyperlipidemia, familiar

#### **2.8. Theories of atherogenesis**

Arteries are blood vessels that carry oxygenated blood from the heart to all tissues of the body. The arterial wall is composed of three layers, namely the intima (inner lining), media and adventitia. A single layer of endothelial cells line the inner surface of the intima, forming a barrier to blood cells and plasma flowing within the blood vessel. Atherosclerosis is characterized by lesions in the intima of arteries, seen as raised fibrous plagues ranging in colour from pearly gray to yellowish gray. The cellular components of the plaque include a cell similar to the adjacent endothelial cell, macrophages, fibrinogen from which fibrin is formed and white blood cells intersparsed between dense connective tissue which consist majorly of collagen fibers. The cells within and around the plaque are usually lipid ladened. Atherosclerosis poses a high risk not just because it can close up an artery, slowing down or entirely restricting blood flow, but may also lead to thrombus formation. A thrombus is a complex aggregation of platelets, red and white blood cells in a fibrin network. Several theories have emanated, suggesting the actual pathogenesis of atherosclerosis.

Schoenhagen (2006) documented the different theories that have been postulated in the course of history. In 1851, a scientist, Rokitansky suggested the encrustation theory or thrombogenesis in which it is said that atherosclerosis began in the intima of arteries with the deposition of thrombus. This is followed by the organisation of the thrombus through infiltration of fibroblast, secondarily followed by deposition of lipid. The German pathologist, Rudolf Virchow postulated the insudation or inflammation theory in 1856, a different initiation of atherosclerosis. It was suggested that infiltration of fatty substances from the blood stream into the arterial wall leads to deposition of cholesterol which acts as an irritant, causing inflammation and the proliferation of cells. The cholesterol deposits act as irritant in the arterial intima, initiating inflammatory process as macrophages are incriminated as key role players in the phases of the disease. This theory was further supported by the work of N.N. Anitschkow in 1933 where he discovered that a disease resembling human atherosclerosis could be reproduced in rabbits with high serum cholesterol or LDL levels. He thus stated this occurrence may be as a result of defects in metabolism of lipids and lipids Schoenhagen, 2006).

The flow theory relates the circulation of blood in vessels to its effect on arterial wall. It stated that lesions occurred more often at curved, branching, or bifurcated sites, generally at regions of perturbed blood flow. Other hypotheses that arose from the flow theory include the stagnation point hypothesis by Fox and Hugh (1966), high wall shear stress hypothesis proposed by Fry (1968), low wall shear stress hypothesis by Caro *et al.* (1969), diminished lateral pressure hypothesis by Texon (1980) and the convection-diffusion hypothesis.

All the theories above were established based on three methods. Atherosclerotic plaques from autopsy findings from individuals of both sexes, various ages and race with different diseases which included hyperlipidaemia, diabetes and hypertension were considered. Epidemiological studies of factors which promote or prevent development of atherosclerosis, and finally experimental pathology which established the sequence of lesion development or regression were considered. None of these theories entirely explains the pathogenesis of atherosclerosis, but each has explained an aspect of this process.

#### **2.9. Prevention and treatment of cardiovascular diseases**

210 Lipoproteins – Role in Health and Diseases

**2.8. Theories of atherogenesis** 

Arteries are blood vessels that carry oxygenated blood from the heart to all tissues of the body. The arterial wall is composed of three layers, namely the intima (inner lining), media and adventitia. A single layer of endothelial cells line the inner surface of the intima, forming a barrier to blood cells and plasma flowing within the blood vessel. Atherosclerosis is characterized by lesions in the intima of arteries, seen as raised fibrous plagues ranging in colour from pearly gray to yellowish gray. The cellular components of the plaque include a cell similar to the adjacent endothelial cell, macrophages, fibrinogen from which fibrin is formed and white blood cells intersparsed between dense connective tissue which consist majorly of collagen fibers. The cells within and around the plaque are usually lipid ladened. Atherosclerosis poses a high risk not just because it can close up an artery, slowing down or entirely restricting blood flow, but may also lead to thrombus formation. A thrombus is a complex aggregation of platelets, red and white blood cells in a fibrin network. Several

theories have emanated, suggesting the actual pathogenesis of atherosclerosis.

metabolism of lipids and lipids Schoenhagen, 2006).

Schoenhagen (2006) documented the different theories that have been postulated in the course of history. In 1851, a scientist, Rokitansky suggested the encrustation theory or thrombogenesis in which it is said that atherosclerosis began in the intima of arteries with the deposition of thrombus. This is followed by the organisation of the thrombus through infiltration of fibroblast, secondarily followed by deposition of lipid. The German pathologist, Rudolf Virchow postulated the insudation or inflammation theory in 1856, a different initiation of atherosclerosis. It was suggested that infiltration of fatty substances from the blood stream into the arterial wall leads to deposition of cholesterol which acts as an irritant, causing inflammation and the proliferation of cells. The cholesterol deposits act as irritant in the arterial intima, initiating inflammatory process as macrophages are incriminated as key role players in the phases of the disease. This theory was further supported by the work of N.N. Anitschkow in 1933 where he discovered that a disease resembling human atherosclerosis could be reproduced in rabbits with high serum cholesterol or LDL levels. He thus stated this occurrence may be as a result of defects in

The flow theory relates the circulation of blood in vessels to its effect on arterial wall. It stated that lesions occurred more often at curved, branching, or bifurcated sites, generally at regions of perturbed blood flow. Other hypotheses that arose from the flow theory include the stagnation point hypothesis by Fox and Hugh (1966), high wall shear stress hypothesis proposed by Fry (1968), low wall shear stress hypothesis by Caro *et al.* (1969), diminished

All the theories above were established based on three methods. Atherosclerotic plaques from autopsy findings from individuals of both sexes, various ages and race with different diseases which included hyperlipidaemia, diabetes and hypertension were considered. Epidemiological studies of factors which promote or prevent development of atherosclerosis, and finally experimental pathology which established the sequence of lesion development or regression were considered. None of these theories entirely explains the

lateral pressure hypothesis by Texon (1980) and the convection-diffusion hypothesis.

pathogenesis of atherosclerosis, but each has explained an aspect of this process.

The contributing factors to development of cardiovascular diseases are numerous as mentioned in the risk factors above. Lowering of plasma cholesterol levels is usually the first line of intervention for prevention and treatment of cardiovascular diseases. Dramatic successes have been recorded with cholesterol-lowering therapy which may suggest that maintenance of low cholesterol levels is sufficient to prevent development of atherosclerosis or reversing an established disease condition (Brunzell *et al.*, 2008). Different approaches have been used for prevention and treatment of this condition, some are enumerated below.

#### *2.9.1. Role of High density lipoprotein-cholesterol (HDL-C)*

High density lipoprotein-cholesterol (HDL-C) is the smallest of the lipoprotein subfractions. It is however, the most complicated and diverse of the lipoproteins. It is the major lipoprotein which transports excess cholesterol from the plasma to the liver for excretion or utilization in the liver and other hormone producing regions of the body. Excess cholesterol is eliminated from the body via the liver, which secretes cholesterol in bile or converts it to bile salts (Toth, 2005; Tall, 2008). Also, its anti-inflammatory property protects LDL from oxidation and limits the concentrations of oxidized components, which may pose as atherogenic treats. HDL-C have been associated with reduced risk of cardiovascular events (Duffy and Rader, 2009; Khera *et al.*, 2011). HDL-C plays a key role in the reverse transportation of cholesterol by accepting cholesterol from lipid-laden macrophages (Lehrke *et al.*, 2007). In the study conducted by Khera *et al.* (2011), the ability of HDL to promote cholesterol efflux from macrophages was strongly and inversely associated with both subclinical atherosclerosis and obstructive coronary artery disease. It was also discovered that the associations persisted after adjustment for traditional cardiovascular risk factors, including the levels of HDL cholesterol and apolipoprotein A-I. HDL has several protein constituents which are exchangeable with other lipoproteins, and it acquires different apolipoproteins in the process of maturation such as apo AII, AIV, AV, CI, CII, CIII and E which results in generation of diverse HDL particles with various metabolic functions. In addition to being carrier proteins for the HDL particle, these proteins have protective roles which they play against cardiovascular diseases, such as by acting as anti-inflammatory regulators to limit the activity of pro-inflammatory cytokines.

Nascent HDL is synthesised and secreted by the liver and small intestine. It travels in the circulation where it gathers cholesterol to form mature HDL, which then returns the cholesterol to the liver via various pathways. Apolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) in plasma. The protein is encoded for by APOAI gene (Breslow *et al.*, 1982, Arinami *et al.*, 1990). Defects in this gene have been associated with HDL deficiencies (HUGO Gene Nomenclature Committee, 2011). This protein increases the efflux of cholesterol from tissue to liver where it is excreted. A few individuals were reported to produce a HDL ApoA-I protein variant called ApoA-I Milano, an abnormal and apparently more efficient apolipoprotein. It has low measured HDL-C levels yet very low rates of cardiovascular events even with high blood cholesterol values

(Franceschini *et al.*, 1981). Apo CI, CII and apo E also accumulate in the nascent HDL particle, which serves as a store for these apolipoproteins in circulation.

Phospholipids are transferred from macrophages by a specific transporter molecule known as ATP-binding cassette transport protein A1 (ABCA-1) into the core of the lipoprotein, and cholesterols are extracted from the cells by a transporter protein derived from macrophages in the sub-endothelial spaces of tissues; ABCG-1 transporter. The eventual maturation of the HDL particle is dependent on the lecithin: cholesterol acyltransferase (LCAT), an enzyme activated by apoAI, which catalyses the formation of cholesterol esters from cholesterol. Mobilization of free cholesterol and phospholipids from IDL and LDL continues until a matured, spherical HDL particle is formed. Endocytosis of the matured HDL into hepatocytes occurs and the cholesterol and cholesterol esters are transported via a facilitated transfer to distinct pools within the cell. The modified HDL particles are secreted back into circulation where they can further acquire cholesterol before they re-circulate to the liver. The complete reverse cholesterol transport occurs with the addition of apo E to the HDL particle which facilitates their uptake and catabolism.

#### *2.9.2. Lipoprotein Lipase as an anti-atherogenic agent*

Activation of lipoprotein lipase (LpL) activity has been reported to have anti-atherogenic activity. Lipoprotein lipase (LpL) is a rate-limiting enzyme found on the surface of endothelial cells. It is polypeptide with 839 amino acids and an extracellular domain which binds to apo B100 and apo E. LpL catalyses the hydrolysis of the triacylglycerol (TAG) component of circulating chylomicrons and very low density lipoproteins (VLDL). The enzyme digests the TAG to fatty acids and monoglycerides. This provides non-esterified fatty acids and 2-monoacylglycerol which can be utilised immediately by cells for energy production or synthesis of other lipids. Unutilized fatty acids may be bound to circulating albumin and released slowly to meet future cellular requirements. Glycerol produced from LpL activity is transported back to the liver and kidneys, where it is converted to dihydroxyacetone phosphate in the alternative glycolytic pathway. The fatty acids from LpL activity in the muscle may diffuse into cells to be oxidized to two-carbon units or used to resynthesis TAG which are stored in adipose cells (Clee *et al.*, 2000; Tsutsumi, 2003). Significant LpL activity occurs in muscle, adipose tissue and lactating mammary glands. Accumulation of VLDL remnants (IDL with apo B100 and apo E are converted to LDL with further loss of triacylglycerols. Both carrier proteins are necessary for recognition of IDL and LDL by the LDL receptors in the liver, after which they are taken up into hepatocytes by endocytosis and catabolized.

Research carried out over the past two decades have not only established a central role for LpL in the overall lipid metabolism and transport but have also identified additional, noncatalytic functions of the enzyme. Furthermore, abnormalities in LpL function have been found to be associated with a number of pathophysiological conditions, including atherosclerosis, chylomicronaemia, obesity, Alzheimer's disease, and dyslipidaemia associated with diabetes, insulin resistance, and infection (Mead *et al.*, 2002).

**Figure 3.** Summary of the fate of Lipoprotein sub-fractions (Adapted from http://courses.washington.edu/conj/bess/cholesterol/liver.html)

endocytosis and catabolized.

(Franceschini *et al.*, 1981). Apo CI, CII and apo E also accumulate in the nascent HDL

Phospholipids are transferred from macrophages by a specific transporter molecule known as ATP-binding cassette transport protein A1 (ABCA-1) into the core of the lipoprotein, and cholesterols are extracted from the cells by a transporter protein derived from macrophages in the sub-endothelial spaces of tissues; ABCG-1 transporter. The eventual maturation of the HDL particle is dependent on the lecithin: cholesterol acyltransferase (LCAT), an enzyme activated by apoAI, which catalyses the formation of cholesterol esters from cholesterol. Mobilization of free cholesterol and phospholipids from IDL and LDL continues until a matured, spherical HDL particle is formed. Endocytosis of the matured HDL into hepatocytes occurs and the cholesterol and cholesterol esters are transported via a facilitated transfer to distinct pools within the cell. The modified HDL particles are secreted back into circulation where they can further acquire cholesterol before they re-circulate to the liver. The complete reverse cholesterol transport occurs with the addition of apo E to the HDL

Activation of lipoprotein lipase (LpL) activity has been reported to have anti-atherogenic activity. Lipoprotein lipase (LpL) is a rate-limiting enzyme found on the surface of endothelial cells. It is polypeptide with 839 amino acids and an extracellular domain which binds to apo B100 and apo E. LpL catalyses the hydrolysis of the triacylglycerol (TAG) component of circulating chylomicrons and very low density lipoproteins (VLDL). The enzyme digests the TAG to fatty acids and monoglycerides. This provides non-esterified fatty acids and 2-monoacylglycerol which can be utilised immediately by cells for energy production or synthesis of other lipids. Unutilized fatty acids may be bound to circulating albumin and released slowly to meet future cellular requirements. Glycerol produced from LpL activity is transported back to the liver and kidneys, where it is converted to dihydroxyacetone phosphate in the alternative glycolytic pathway. The fatty acids from LpL activity in the muscle may diffuse into cells to be oxidized to two-carbon units or used to resynthesis TAG which are stored in adipose cells (Clee *et al.*, 2000; Tsutsumi, 2003). Significant LpL activity occurs in muscle, adipose tissue and lactating mammary glands. Accumulation of VLDL remnants (IDL with apo B100 and apo E are converted to LDL with further loss of triacylglycerols. Both carrier proteins are necessary for recognition of IDL and LDL by the LDL receptors in the liver, after which they are taken up into hepatocytes by

Research carried out over the past two decades have not only established a central role for LpL in the overall lipid metabolism and transport but have also identified additional, noncatalytic functions of the enzyme. Furthermore, abnormalities in LpL function have been found to be associated with a number of pathophysiological conditions, including atherosclerosis, chylomicronaemia, obesity, Alzheimer's disease, and dyslipidaemia

associated with diabetes, insulin resistance, and infection (Mead *et al.*, 2002).

particle, which serves as a store for these apolipoproteins in circulation.

particle which facilitates their uptake and catabolism.

*2.9.2. Lipoprotein Lipase as an anti-atherogenic agent* 

LpL encodes lipoprotein lipase, which is expressed in heart, muscle, and adipose tissue. LpL functions as a homodimer, and has the dual functions of triglyceride hydrolysis and ligand/bridging factor for receptor-mediated lipoprotein uptake. Through catalysis, VLDL is converted to IDL and then to LDL. Severe mutations that cause LpL deficiency result in type I hyperlipoproteinemia, while less extreme mutations in LpL are linked to many disorders of lipoprotein metabolism.

LpL isozymes are regulated differently depending on the tissue. For example, insulin is known to activate LpL in adipocytes and its placement in the capillary endothelium. By contrast, insulin has been shown to decrease expression of muscle LpL (Kiens *et al.*, 1989). The form that is in adipocytes is activated by insulin, whereas that in muscle and myocardium is activated by glucagon and epinepherine. This helps to explain why during fasting, LpL activity increases in muscle tissue and decreases in adipose tissue. After feasting, the opposite occurs (Braun and Severson, 1992; Mead *et al.*, 2002).

The concentration of LpL displayed on endothelial cell surface cannot be regulated by endothelial cells, as they neither synthesize nor degrade LpL. Instead, this regulation occurs by managing the flux of LpL arriving at the lipolytic site and being released into circulation attached to lipoproteins (Braun and Severson, 1992; Goldberg, 1996). The typical concentration of LpL in plasma is in the nanomolar range. Lipoprotein lipase deficiency leads to hypertriglyceridemia (elevated levels of triglycerides in the bloodstream) and decreased high density lipoprotein activity (Clee *et al.*, 2000; Tsutsumi, 2003; Okubo *et al.*, 2007). Diets high in refined carbohydrates have been shown to cause tissue-specific overexpression of LpL. This has been implicated in tissue-specific insulin resistance and consequent development of type 2 diabetes mellitus & obesity.

### *2.9.3. Influence of Hormones on plasma LDL-C and Lp(a) levels*

Several studies have reported conflicting reports on the effect of hormonal replacement therapy on plasma LDL-C and Lp(a) levels (Taskinen *et al.*, 1996; Shlipak *et al.*, 2000; Vigna *et al.*, 2002). In a cohort study conducted by Danik *et al*. (2008), the effect of hormone replacement therapy (HT) on Lp(a) and cardiovascular risk was investigated. It was reported that the relationship of high Lp(a) levels with increased cardiovascular disease is modified by hormonal therapy. These data suggest that the predictive utility of Lp(a) is markedly attenuated among women taking HT and may inform clinicians' interpretation of Lp(a) values in such patients. It was noteworthy that the effect of hormonal therapy was observed only in women with high LDL cholesterol levels, in agreement with previous studies suggesting an interaction between Lp(a) and LDL cholesterol (Berglung and Anuurad, 2008).
