Lipids, lipoproteins, apolipoproteins and their disease association

Henry O. Ogedegbe, Ph.D., BB, C(ASCP)SC, CC(NRCC)

Department of Environmental Health, Molecular and Clinical Sciences,
Florida Gulf Coast University,
10501 FGCU Blvd. South,
Fort Myers, FL 33965-6565
Tel: 941-590-7486
Fax: 941-590-7474
E-mail: hogedegb@fgcu.edu
Abstract
Lipids are ubiquitous molecules, which are present in all tissues of the body and play major roles in a variety of
biological processes. They are primary source of fuel and are components of cell membranes and cell structures
where they provide stability and are involved in transmembrane transportation. Lipoproteins are the transport
vehicles for lipids. The apoliporoteins are the protein moieties of lipoproteins. Measurements of lipids,
lipoproteins and apolioproteins have been employed to determine susceptibility of individuals to coronary artery
diseases. Increased levels of total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B-100 are
directly related to risk for coronary heart disease while increased levels of high-density lipoprotein and
apolipoprotein A-I are negatively associated with coronary heart disease risk. Apo E 2 and 4 has been reported
to be independent risk factors for coronary artery disease and as predictors for the development of
atherosclerosis. Apo E 4 allele is associated with very high levels of low-density lipoprotein cholesterol and
total cholesterol while 2 allele is associated with decreased levels of low-density lipoprotein cholesterol and
higher triglyceride levels. An association between Alzheimers disease and the apo E 4 allele has been reported
in both familial and sporadic late-onset Alzheimers disease. The apo E 4 allele increases the risk and lowers
the age of onset distribution of Alzheimers disease in a close dependent fashion. The inheritance of each dose
of apo E 4 increases risk of disease and decreases age of onset; conversely, the apo E 2 allele appears to be
protective, by lowering the risk of disease and increasing age of onset. Genotyping of apo E may be helpful in
determining individuals who may be at risk for developing coronary artery disease and/or Alzheimers disease.
Introduction
Artherosclerotic vascular disease is a major cause of morbidity and mortality in the Western world.1-3 The
atherosclerotic lesion usually comprises of fatty streaks that eventually develop into fibrous plaques.1 Foam
cells consisting mainly of macrophages laden with lipids characterize the initial fatty streak which may regress
or progress via a transitional lesion to fibrous plaques. In the US, cardiovascular disease and osteoporosis
together account for most of the morbidity and mortality in the aging population in spite of improved treatment
modalities. Both diseases often appear together, especially in the elderly and are usually regarded as
independent entities.4 There is evidence however, which suggest that both diseases share etiologic factor
because hyperlipidemia contributes to atherosclerotic plaque formation and osteoporosis through similar
biologic mechanism involving lipid oxidation.5 Even though advances in lifestyle, risk modification,
pharmacotherapy, endovascular interventions and surgery have considerably improved clinical outcome, 4050% of adverse cardiovascular events continue to occur despite current strategies.6
Genetic and environmental factors contribute to the development and progression of atherosclerotic coronary
heart disease (CHD) processes. Major independent risk factors for CHD, including adverse levels of plasma
lipids and lipoproteins, are also influenced by genetic and potentially modifiable environmental factors.7 Not
much is currently known about lipoprotein metabolism in neurodegenerative diseases, but lipid alterations have
been repeatedly reported in Alzheimer brains in which neuronal loss and deafferentation are major features.
Although the mechanism underlying the link between the epsilon4 allele of the apolipoprotein E gene and
1

Alzheimer's disease (AD) is presently unclear, it may well be postulated that it is related to disturbances in brain
lipoprotein metabolism.8
In order to understand the significance of lipids, lipoproteins and apolipoproteins in relations to atherosclerosis,
arterial diseases and Alzheimers disease, their biochemistry must be understood. Lipids are a diverse group of
chemicals related primarily because they are insoluble in water, soluble in nonpolar solvents and found in
animal and plant tissue. They are ubiquitous molecules found in all tissues and cells of the body where they play
important functional roles in all aspects of biological life. They serve as hormones or hormone precursors, aid in
digestion, and provide fuel for metabolism and store energy. In addition they act as functional and structural
components in biomembranes, and form insulation for nerve cell membranes.9 There are five broad groups of
lipids, based on their chemical properties: fatty acids, sterol derivatives, glycerol esters, sphingosine and
terpenes Table 1. Dietary lipids are 98 to 99% triglycerides (TG), the remainder consists of cholesterol,
phospholipids, di and monoglycerides, fat-soluble vitamins, steroids, and terpenes.10 In order for the body to
utilize lipid molecules, they must first be processed through biochemical reactions that are catalyzed by specific
enzymes. This process takes place in three phases known as digestion, absorption, and transportation.
Table 1 Classification of Lipids
Fatty Acids
Short Chain (2 - 4
carbon)
Medium chain (6 10 carbon)
Long chain (12 - 20
carbon )
Prostaglandins

Sterols
Cholesterols and
Cholesteryl esters
Steroid hormones

Glycerols
Triglycerides

Sphingosines
Sphingomelins

Terpenes
Vitamin A

Diglycerides

Glycosphingolipids

Vitamin E

Bile acids

Monoglycerides

Cerebrosides

Vitamin K

Vitamin D

Phosphoglycerides
Plasmalogens

The digestive phase takes place in the lumen of the intestine and it involves the action of several enzymes such
as lipoprotein lipase (LPL), pancreatic lipase, lecithin cholesterol acyl transferase (LCAT), and acyl cholesterol
acyl transferase (ACAT). Emulsification of dietary fats with bile secretions to form small particles known as
micelles increases their surface area, rendering them more susceptible to digestion by pancreatic lipase.
Micelles are negatively charged polymolecules which have diameters of about 5 nm and which are able to
access the intestinal microvilli where they penetrate the mucosal wall.10 Triglycerides are hydrolyzed into diand monoglycerides, free fatty acids, and glycerol by the action of lipid digestive enzymes. Also during
digestion, cholesterol esters are hydrolyzed to free cholesterol and free fatty acids via a reaction that is catalyzed
by cholesterol esterase. Bile and lecithin are essential for solubilization of fat prior to digestion.10 Absorbed
micelles invariably reach the circulatory system via the lymphatic system and the thoracic duct.
Transportation of lipids throughout the bloodstream is accomplished through the use of specialized particles
known as lipoproteins Table 2. They are complex transport vehicles for fatty acids, cholesterol and their esters.
Lipoproteins include chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and
high-density lipoprotein (HDL). The different lipoproteins combined with protein molecules ferry the various
hydrophobic lipid molecules enclosed within the core of their biomembranes. The protein molecules are known
as apolipoproteins and are the purified protein components of lipoproteins.11 There are five main types of
apolipoproteins known as apolipoprotein A, B, C, D, and E and each of the main groups have sub groups. Minor
apolipoproteins include apo J, H, F and G. They function as cofactors such as C-II for LPL, A-I for LCAT, and
they act as lipid transfer proteins. In addition, they act as ligands for interaction with lipoprotein receptors in
tissues such as apo B-100 and apo E for the LDL receptor, apo E for the remnant receptor, and apo A-I for the
HDL receptor.12 The liver plays a vital role in lipid metabolism. The endogenous lipids produced by the liver
2

account for about 1.5 g of lipids daily and normal diet contributes about 150-300 mg of lipids per day. The
lipids produced in the liver are transported by lipoproteins to various sites in the body where they are needed.
The liver has both apo B and apo E receptors, which are involved in the catabolic clearance of the cholesterolladen LDL particles. The liver is also involved with the production of several of the enzymes that are used in the
biosynthesis and catabolism of the various lipid molecules. Bile salts, which are required for lipid
emulsification during the digestion of fats, are produced in the liver from cholesterol molecules.11
Table 2 Association of Human Plasma Lipids, Lipoproteins and Apolipoproteins
Lipoprotein
Chylomicrons
VLDL
IDL
LDL
HDL

Major Lipids
Exogenous triglycerides
Endogenous triglycerides
Endogenous triglycerides ,
cholesterol esters
Cholesterol esters, free
cholesterol
Phospholipids, cholesterol
esters

Major Apolipoproteins
A-I, B-18, C-I, C-II, C-III
B-100, C-I, C-II, C-III, E
B-100, E
B-100
A-, A-II

Triglyceride
The majority of fatty acids form esters known as glycerides with glycerol of which TG is the most abundant..
Plant triglycerides have linoleic acids, which are long chain fatty acids and are liquid at 4oC. Animal
triglycerides are solid at room temperature. For over three decades, epidemiological studies have investigated
the association of high TG with increased risk for CHD. Although the debate continues on the epidemiological
impact of TG on CHD, many surveys report that elevated serum triglyceride is an important risk factor. At the
same time, other studies have found inconclusive evidence of the association of TG with CHD. A potential bias
being that it cannot be determined if the increased TG preceded the onset of the disease.13 Consequently,
hypertriglyceridemia per se is probably not an appropriate therapeutic target for the prevention of
atherosclerotic cardiovascular disease because it is a poor marker of atherogenic risk and because there have
been no clinical trials that have directly addressed the question of whether lowering the triglyceride level
reduces the number of clinical events.14 In contrast to patients with hypercholesterolemia, no national guidelines
have been proposed for the treatment of patients with hypertriglyceridemia. However, high plasma triglyceride
concentrations in diabetic subjects have been found to increase their risk for developing CHD.15 More recent
case-control studies in patients with premature coronary artery disease have shown that total triglyceride and
VLDL levels discriminate better between subjects with and without coronary artery disease (CAD).
Angiographic studies demonstrate that elevated serum triglyceride is found in CAD patients and that elevated
VLDL or IDL is associated with severity.16
Cholesterol
Little was known about the structure of cholesterol until the pioneering research of A. Windaus and H. Wieland
in the first part of the 20th century. Its structure was completely elucidated in 1932.17 Cholesterol is implicated as
a risk factor for developing CHD and atherosclerosis. In spite of their atherogenic properties, sterols are
necessary for the synthesis of steroid hormones, manufacture of bile salts in the liver and maintenance of
membrane fluidity. Plant sterols are known as phytosterols and they may compete with cholesterol for uptake by
the mucosal cells of the gut. This competitive property of phytosterol may be used therapeutically to lower
cholesterol concentration in the blood. Lowering blood cholesterol levels decreases risk for CHD, especially in
the absence of other risk factors.18 According to the National Cholesterol Education Program (NCEP)
guidelines, total cholesterol of less than 200 mg/dl is desirable while levels between 200-239 mg/dl is
borderline risk for CHD. Levels greater than 240 mg/dl is considered high risk.19 Serum cholesterol traditionally
3

has been considered a poor predictor of total stroke risk; however, it is associated positively with ischemic
stroke risk and associated negatively with hemorrhagic stroke risk.20
Chylomicrons
Chylomicrons are produced exclusively in the intestine and are involved with the transportation of dietary fats,
especially TG. Thus the lipid content of chylomicrons is derived solely from dietary fat intake. The
chylomicrons usually traverse the lymphatic system to the thoracic duct and then move on to the systemic
circulation but do not enter the portal venous system. They pick up apo C-II from HDL, which then activates the
LPL hydrolysis of TG into monoglycerides and free fatty acids. The chylomicron particle contains exogenous
TG combined with cholesterol, small amounts of phospholipid and specific apolipoproteins A-I, A-II, B-48, C-I,
C-II, C-III, and E. An outer shell of phospholipids, free cholesterol and proteins surrounds the particle. 9,10 It has
been shown that n-3 Fatty acids treatment effectively lowers chylomicrons and VLDLs, but their effect on
chylomicron remnants was observed only in the late postprandial phase.21
Low-Density Lipoproteins
Low-density lipoprotein particles are the major cholesterol carriers in circulation and their physiological
function is to carry cholesterol to the cells. The LDL particle consists of a lipid core composed of 38%
cholesterol ester and 11% triglyceride. The outer coat of the molecule consists of 22% phospholipids, 8%
unesterified cholesterol and 21% apolipoprotein.10 It contains one molecule of apo B-100 and some variable
quantity of apo C-III and apo E. The receptor-mediated catabolism of the particle is an important determinant of
the concentration of LDL-C in plasma. Epidemiological, pathological and genetic studies show a strong positive
correlation between elevated plasma concentrations of LDL-cholesterol (LDL-C) and the risk of premature
CHD.22 Atherogenesis modifies the particles resulting in their accumulation in arterial walls.23 Further
delipidation of intermediate-density lipoprotein (IDL) by hepatic lipases results in the formation of LDL
particle. It is catabolized in the liver and the tissues and derived from VLDL. The particle is recognizable and its
breakdown is receptor-dependent. Extensive biochemical and genetic evidence indicate that the LDL receptor
(LDL-R) is a protein (most likely a glycoprotein) molecule.5 Endocytosis of the LDL particle is followed by its
degradation.
The major LDL subclasses include LDL1, LDL2, LDL3, LDL4 and LDL5. The particle may be separated from
VLDL and HDL by methods based on physical characteristics such as density, size, charge, or apolipoprotein
content. Estimation of LDL concentration may be obtained directly or with the Friedewalds formula.24
According to the NCEP guidelines; an LDL-C concentration of less than 130 mg/dl is desirable while
concentration between 130-159 mg/dl is considered borderline risk for CHD. Concentrations above 160 mg/dl
are considered high risk.19
Oxidative modification of LDL plays a key role in the pathophysiology of atherosclerosis. Isolevuglandins
(isoLGs) are extremely reactive gamma-ketoaldehydes that avidly bind covalently with proteins and cause
protein-protein as well as DNA-protein crosslinking. IsoLG-protein adducts are generated upon oxidation of
LDL, and may contribute to atherogenesis since such adducts cause recognition and endocytosis of the modified
LDL by macrophage cells.25 Myeloperoxidase (MPO), an abundant heme enzyme released by activated
phagocytes, catalyzes the formation of a number of reactive species that can also modify LDL to a form that
converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions.26
Familial hypercholesterolemia (FH), familial defective apoliporotein B-100 (FDB), familial combined
hyperlipidemia (FCHL) and cholesterol ester storage disease result in elevation of serum LDL-C. On the other
hand, serum LDL-C is decreased in abetalipoproteinemia and familial hypobetalipoproteinemia. Patients with
CHD may be managed by lowering their LDL-C concentration. Many patients with FH are unable to reach
targets because of drug intolerance or extremely high baseline LDL-C levels. Consequently, LDL apheresis has
become a useful modality for the treatment of patients with severe refractory hypercholesterolemia. Commonly
used LDL apheresis systems utilize immunoadsorption columns, dextran sulfate cellulose columns, or heparin
4

precipitation.27 In a study to determine the effects of estrogen use on lipoproteins by postmenopausal women,
Campos et al.28 showed that estrogen use in postmenopausal women is associated with significantly elevated
plasma apo A-l levels and decreased LDL-C concentrations. Thus estrogen use by postmenopausal women may
be a negative risk for CHD.
Lipoprotein(a)
Lipoprotein(a) is also known as the sinking pre- lipoprotein and was first described in 1963 by Kare Berg as
lipoprotein antigen or a genetic variant of LDL that was more prevalent in the plasma of myocardial infarction
survivors than in age-matched control group of Scandinavian men.29-31 It belongs to a class of apo B containing
lipoproteins that is highly associated with atherosclerotic diseases, stroke and myocardial infarction. 32 Lp(a) is a
macromolecular complex which is genetically determined, and has been identified as an independent risk factor
for premature CAD. It is elevated in diabetic and non-diabetic android obese subjects, and aggravates the
atherogenic effect of diabetes mellitus.33 It is an LDL molecule linked to apoprotein(a) and further linked by a
disulfide bond to apo B.34,35 It is a heterogeneous glycoprotein molecule, which shares at least 75% homology
with plasminogen including multiple tandem repeats of the kringle 4 of plasminogen and one repeat of kringle 5
and the protease domain which unlike plaminogen lacks enzyme activity.1,30 There are 10 subclasses of kringle 4
numbered 1 to 10 exhibiting differences in amino acid composition. All the kringles occurs as a single copy
except kringle 4-2 whose number varies among individuals and accounts for the size polymorphism of apo(a).36
The number of kringle 4 repeats in apo(a) is genetically determined and highly variable and apo(a) polymorphs
are inherited as an autosomal codominant genetic trait. The molecular mass of apo(a) which ranges from 420 to
840 kd according to the number of kringle 4 repeats is inversely related to Lp(a) concentration in serum.37 Both
genes are located on chromosome 6.1 It is highly glycosylated with numerous O-glycosidic linkages in the
regions between the kringle domains.38
Lipoprotein(a) is a Risk Factor fpr Coronary Heart Disease
The physiology and function Lp(a) are poorly understood but there is suggestion that it might contribute to the
thrombotic as well as to the atherogenic, aspects of CAD and mitogenesis.38,39 Both Lp(a) and apo(a) stimulate
smooth muscle cell proliferation in vitro. It appears to have a role in fibrinolytic system since myocardial
infarctions without obstructive CAD have a high frequency of Lp(a) concentration above 300 mg/dL, similar to
what is seen in myocardial infarction with angiographically documented CAD. High concentration of Lp(a) in
serum is associated with increased risk for CHD. Lp(a) is an independent risk factor for recurrent
atherosclerotic heart disease in men and women after menopause.40,41 Many studies have now demonstrated
increased Lp(a) concentrations in patients with CAD.31,42,43 High Lp(a) concentration may not be proportional to
the risk of CHD in African Americans.1 The distribution of Lp(a) concentrations varies in different population
groups.1,44 Lipoprotein(a) concentrations are lower in Caucasians than in people whose ancestry originated in
Africa or the Indian subcontinent.45 Gupta et al.44 investigated the relationship of plasma levels of Lp(a) and
other lipid values in patients undergoing coronary arteriography in India. The result showed that Lp(a)
concentration was higher in CAD group compared to normal coronary artery group. Plasma values of TC, TG,
apo A-1, apo-B, LDL-C, LDL/HDL-C ratio and apo A-1/B ratio were not significantly different in the groups
with normal coronary arteries and CAD. This may indicate that the measurement of Lp(a) provides a better
marker for predicting the presence of angiographically defined CAD as compared to traditional measures.44
Similar to increased Lp(a) concentrations seen in adults who are at risk for CAD, above-normal concentrations
of Lp(a) can be also be detected in five- to seven-day-old newborns.46 Vella et al.47 studied several risk factors in
relation to parental CAD in which TC, TG, HDL-C, LDL-C Apo A-I, Apo B and Lp(a) were determined in
subjects with and without parental CAD. The Lp(a) concentrations were similar to those seen in other white
populations with higher frequencies at low values. Subjects whose parents reported CAD had higher mean
Lp(a) values than than those who did not report CAD. The result indicated that Lp(a) is an important risk factor
for CHD and also that Lp(a) is more strongly related to the risk of CHD than HDL-C and LDL-C and apo A-I
and B.47
5

Screening for Lp(a)

Screening for Lp(a) should be considered under the following circumstances: (a) patient or family history of
premature atherosclerotic heart disease, (b) familial history of hyperlipidemia, (c) established atherosclerotic
heart disease with a normal routine lipid profile, (d) hyperlipidemia refractory to therapy, and (e) history of
recurrent arterial stenosis.40 Oxidized - Lp(a) can induce P-selectin expression in cultured human umbilical vein
endothelial cells (HUVECs), which may thereby influence the pathogenesis of athersclerosis.48 In a study to
investigate the effect of hormone replacement therapy (HRT) on Lp(a) levels in postmenopausal women with
CAD, Falco et al.49 showed that HRT induced a significant decrease in Lp(a) level. In a study to examine the
correlation of Lp(a) to the extent and severity of CAD and its relation to unstable clinical events, Zampoulakis
et al.50 found that elevated Lp(a) predisposes to the extent of CAD and total occlusion but not to the severity of
lesion. Thus patients with increased Lp(a) levels and unstable angina are at increased risk of suffering
myocardial infarction.
In a prospective study to establish whether elevated Lp(a), detected as a sinking pre-beta-lipoprotein band on
electrophoresis of fresh plasma, is an independent risk factor for the development of premature CHD in men,
Bostom et al.51 discovered that elevated plasma Lp(a) is an independent risk factor for the development of
premature CHD in men, comparable in magnitude and prevalence to a TC level of 240 mg/dL or more, or an
HDL-C level less than 35 mg/dL. LDL receptor does not appear to have a role in the clearance of Lp(a) thus
HMG-CoA which upregulates LDL-R does not have appreciable effect on Lp(a) concentration.
Lipoprotein(a) Isoforms
There is an inverse correlation between the size of apo(a) isoform and Lp(a) concentration: the largest isoforms
are associated with the lowest concentrations. As a result of the observed differences in the distribution of Lp(a)
levels across apo(a) sizes in different racial and ethnic groups, Paultre et al.52 tested the hypothesis that apo(a)
isoform size determines the association between Lp(a) and CAD.They related Lp(a) levels, apo(a) isoforms, and
the levels of Lp(a) associated with different apo(a) isoforms to the presence of CAD. They concluded from their
study that elevated levels of Lp(a) with small apo(a) isoforms independently predict risk for CAD in African
American and white men. The results further suggested that small apo(a) size confers atherogenicity to Lp(a).104
In a study to test the hypothesis that elevated Lp(a) concentrations may be associated with recurrence of
symptoms and restenosis after balloon angioplasty, Desmarais et al.39 evaluated 240 consecutive patients
undergoing coronary balloon angioplasty with measurements of Lp(a), TC, HDL-C, LDL-C, TG, Apo A-I, Apo
B-100 concentrations from fresh specimens. The result showed that patients with recurrence had significantly
greater Lp(a) concentrations compared with those without. Thus they concluded that an elevated Lp(a)
concentration was a risk factor for clinical recurrence after percutaneous transluminal balloon coronary
angioplasty. Other lipid levels were not significantly associated with recurrence.39
High-Density Lipoproteins
High-density lipoprotein is involved with its major protein constituent apo A-I in the reverse cholesterol
transport (RCT). The efficiency of RCT depends on the specific ability of apo A-I to promote cellular
cholesterol efflux, bind lipids, activate LCAT.53 Thus the levels of HDL-C and composition of HDL subclasses
in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins,
receptors, and cellular transporters.54 The HDL molecule usually contains 50% protein and 50% lipids. About
30% of the lipids of the HDL particles are phospholipids while the remaining 70% is made up of cholesterol.10 It
is the smallest of the lipoproteins (9 to 12 nm) but has the highest density (1.063 to 1.21 g/ml) of any of the
lipoproteins, due to its high protein content. It may be fractionated further into subfractions known as HDL2
and HDL3.55 HDL2 is present in premenopausal women at about three times the concentration found in men.
Low HDL2 has been implicated in the predisposition to development of CHD. The HDL2 are larger in size and
richer in lipids than HDL3 and are more efficient vehicles for transfer of cholesterol from peripheral tissue to
the liver.56 The major apolipoproteins found in HDL are A-I and A-II, some apo C and a small amount of apo E.
A major function of HDL is to act as a repository for apo C and E that are needed in the metabolism of
chylomicrons and VLDL.12 Both the liver and the intestine are responsible for HDL production. Only HDL-C,
6

particularly HDL2-C, show an independent inverse relationship to the incidence of atherosclerotic CAD.57
Studies employing laboratory animals have suggested that HDL administration not only inhibits progression of,
but even reduces, established atherosclerotic lesions.58
It has been shown that HDL isolated from subjects with non-insulin-dependent diabetes mellitus (NIDDM)
exhibits a decreased capacity to induce cholesterol efflux. In a study Gowri et al.15, examined the effect of
HDL2 and HDL3 subfractions from poorly controlled NIDDM and control subjects on THP-1 macrophagemediated LDL-oxidation and found that the composition and protective effects of HDL2 but not HDL3, differed
significantly between control and diabetic subjects. They concluded that compositional alterations in HDL2
from poorly controlled NIDDM subjects might reduce its antiatherogenic properties. High-density lipoproteincholesterol concentration greater than 60 mg/dL is a negative risk factor associated with CHD. Women
generally have higher levels of HDL-C and lower TC than men due to higher estrogen levels. However, after
menopause, this difference disappears due to decreased estrogen.59
Both HDL and LDL transport cholesterol in either the free form or as a fatty acid ester in the blood. Genetic
abnormalities in either the apo B, E, LDL receptor may lead to an increased plasma LDL-C concentration.60
High-density lipoproteins are involved with the transport of excess cholesterol from the peripheral tissues back
to the liver, either directly via the HDL receptor, which recognizes and binds to apo A-I or via remnant
receptors, which recognize apo E. Other lipoprotein receptors, termed 'scavenger receptors', are found on the
surface of macrophages. Expression of these receptors is not regulated by intracellular cholesterol
concentration, as are LDL receptors, thus allowing the buildup of large amounts of cholesterol in the
macrophages. This can lead to the formation of foam cells and a build up of plaque in blood vessels, which
might set in motion an almost irreversible process of lumen closure, or atherosclerosis.1
Apolipoprotein B
Apo B-100 is associated with IDL, VLDL, and LDL. The LDL and IDL particle are directly related to the
development of atherosclerosis. It is the sole protein component of LDL and most circulating apo B is
associated with LDL. It is the ligand responsible for the receptor-mediated uptake and clearance of LDL from
the circulation.22 Apo B-100 is possibly the longest single polypeptide chain known having 4536 amino acids.12
Apo B is recognized by the LDL-R which functions in the delivery of cholesterol to peripheral tissues for
membrane and or steroid hormone synthesis and to the liver for removal or reuse.61 Measurement of apo B-100
and LDL-concentrations in serum or plasma may be employed to assess the presence of CHD risk in
individuals. Both apo B and LDL are increased in patients with CHD. Apolipoprotein B is the least well
characterized of the apolipoproteins and the presence of at least four major molecular forms in circulating
plasma has been reported. The LDL contains three of these forms: apo B-100, apo B-74 and apo B-26.62 A
fourth form, apo B-48, is produced by the intestine and is the major apolipoprotein found in chylomicrons. Apo
B-100, which is the protein component of LDL and apo E, which is present in intermediate lipoproteins and
some forms of HDL, are the two proteins through which particular lipoproteins bind to the LDL-R.1 The domain
of apo B-100 that interacts with the LDL-R comprise two clusters [A(3147-3157) and B (3359-3367)] of basic
amino acids linked through a disulfide bond between residues 3167 and 3297.63 Several point mutations of the
putative receptor binding domain of apo B-100 have been identified. The first and most frequent substitution to
be identified is apo B-100 (Arg3500Gln). The other two substitutions; apo B-100 (Arg3500Trp) and apo B100 (Arg3531Cys) occur less frequently.63
Effect of Apolipoprotein B on Diabetes Mellitus and Coronary Heart Disease
In a study by Mero et al.64, to examine if there is a relationship between the severity of CAD and postprandial
lipemia in patients with NIDDM they found that postprandial apo B-48 and apo B-100 metabolism in
triglyceride rich lipoproteins is distorted in NIDDM patients, even in those with only mild CAD. Igua et al. 65
compared the structural and biological characteristics of apo B-100 containing particle subfractions isolated
from poorly controlled diabetic patients with insulin dependent diabetes mellitus (IDDM) and healthy controls
and found that IDDM is not associated with any significant abnormalities in the apo B containing lipoprotein
7

particles. Therefore they concluded that the excessive occurrence of CHD and other atheroslerotic vascular
diseases in patients with IDDM must have other causes. In adults, a low level of LDL-C/apo B-100 ratio is an
indicator of apo B-enriched small dense LDL, which is associated with premature CAD.66 Islam et al.66
investigated whether a low LDL-C/apo B-100 ratio was associated with a positive family history of premature
CAD in young children. The result showed that in young children, a low LDL-C/apo B-100 ratio and high apo
B-100 levels were associated with a positive family history of CAD in white girls suggesting that they were at
increased risk of genetically mediated CAD. Apo B concentration less than 1.04 g/L indicates low risk for CHD,
from 1.04 g/L to less than 1.22 g/L is moderate risk, from 1.22 g/L to less than 1.40 g/L is high risk and 1.40 g/L
or greater is very high risk.67
Apolipoprotein A
Apolipoprotein A-I is the major apolipoprotein of HDL. It sets the plasma level of HDL and appears to confer
protection against the development of atherosclerosis. Measurement of apo A-I may be a better marker than
HDL-C in CHD risk assessment.68 A small amount of apo A-I can be found unassociated with HDL, some of
which may be associated with nascent HDL that are newly secreted from the liver and intestine. It plays a role
in the mobilization of excess cholesterol from cells that cannot metabolize or otherwise dispose of it. The
transportation process of HDL and apo A-I may involve both passive and second messenger pathways.61 The
products of apo A-I, A-II, C-III and A-IV genes are the major proteins components of HDL. Each of them
modifies the activities of LCAT in vitro. The genes coding for apo A-I, C-III, and A-IV occur in a tight cluster
spanning ~15 kb on the long arm of human chromosome 11 where the apo C-III is transcribed in the opposite
direction to the apo A-I and A-IV genes.69 More than 10 common polymorphisms within the apo A-I, C-III, and
A-IV have been detected.
Tangier Disease
Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway
controlled by an ATP binding cassette transporter called ABCA1. Mutations in ABCA1 cause Tangier disease, a
severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation
of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I
by the ABCA1 pathway is required for generating HDL particles and clearing of sterol from macrophages.
Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from
tissue macrophages and protecting against atherosclerosis.70
To test this hypothesis, that apo A-I in combination with apo A-II and C-I remove free cholesterol from the
extrahepatic tissues to the liver for subsequent elimination, Eriksson et al.71 measured fecal steroid excretion
before and after intravenous infusion of human proapo A-I liposome complexes in subjects. The result showed
that infusion of proapo A-I liposomes in humans promotes net cholesterol excretion from the body, implying a
stimulation of reverse cholesterol transport, a mechanism that may prove useful in treatment of artherosclerosis.
Apo A is produced in the intestine and liver and appears to play structural roles in the HDL. The action on apo
A-I is inhibited by apo A-II and 50% of HDL mass is protein with apo A-I and apo A-II constituting almost
90%. The ratio of apo A-I and apo A-II is approximately 3:1 and apo A-I contains 243 amino acids while apo AII contains 154 amino acids.72
Apolipoprotein A is a Negative Risk for Coronary Heart Disease
Apo A-I is inversely related to a predisposition to CHD.73 A study by Schaefer and associates75 showed that
treatment of patients with 3-hydroxyl-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors such as
pravastatin increases fractional synthetic rate of apo A-I as a result of increased production rate. Their result
demonstrated that increased production of HDL apo A-I is the metabolic cause of the increase in HDL and apo
A-I levels under inhibition of HMG-CoA reductase in man.74 Therefore treatment with HMG-CoA reductase
inhibitors are effective in lowering LDL-C levels and increasing the level of HDL-C and A-I in patients with
hypercholesterolemia. In a study to compare pre- and post-Ramadan lipid and lipoprotein profiles in stable
Kuwaiti hyperlipidemic subjects attending a Lipid Clinic, Akanji et al.75 found that the most consistent changes
8

post-Ramadan were increased levels of apo A-1, apo A-1/apo B and apo A-1/HDL ratios and reduced uric acid
levels which suggest that Ramadan fasting in hyperlipidemic subjects might favorably influence CHD risk.
Apolipoprotein A Mutants
A novel mutant of apo A-I associated with low HDL has been identified in a Japanese family. Apo A-I
(Glu235-->0) Nichinan is caused by a 3-bp deletion of nucleotides 1998 through 2000 in exon 4 of the apo A-I
gene. This induces a critical structural change in the carboxyl-terminal domain of apo A-I for cellular
cholesterol efflux and increases the catabolism of apo A-I resulting in low HDL-C levels.76 A Finnish family
with premature CHD and decreased HDL-C levels was identified as having an apo A-I variant, apo A-I
(Lys107-->0), caused by a 3-bp deletion of nucleotides 1396 through 1398 in exon 4 of the apo A-I gene. These
subjects were heterozygous for this mutation. The heterozygotes had reduced apo A-I and apo A-II
concentrations compared with normal family members. However LDL-C, TG, VLDL, IDL, LDL subclasses
were similar in both groups. The low HDL-C in the heterozygotes results from the decreased concentration of
apo A-I and A-II containing particles and the smaller size and reduced cholesterol content.77 This puts the
heterozygotes at greater risk for CHD.
ApoA-I (R151) Paris is a natural apo A-I variant that is associated with low levels of HDL-C and the partial
deficiency of LCAT in the plasma of heterozygous carriers. Defective LCAT cofactor activity but normal lipid
binding and cholesterol-efflux-promoting abilities characterize it.78 Carriers of the apo A-IMilano (A-IM)
mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature CHD. The high
efficiency of apo A-IM containing HDL for cell cholesterol uptake would result in an improved reverse
cholesterol transport in the apo A-IM carriers which may explain the low susceptibility to atherosclerosis
development.79 Apo A-I of less than 1.20 g /L is a risk factor and 1.65 g/L or greater is an antirisk factor for
CHD.67 Various hormones influence apo A-I expression such as those belonging to the thyroid and steroid
family. Thyroid hormone, glucocorticoids, and estradiol enhance activity of the gene while retinoic acid and
androgens decrease it.80
Apolipoprotein E
Apolipoprotein E was first isolated from plasma in 1973 and originally was known as arginine-rich
apolipoprotein. It plays a role in the transport and redistribution of lipids, including cholesterol in the liver and is
implicated in growth and repair of injured neurons in the nervous system. The apo E gene is located on
chromosome 19q13.2 in a cluster of related apolipoprotein genes that span approximately 48 kb, and which
includes the apo C-I and C-II loci which code for activating cofactors of LCAT and LPL. Apo E is most likely
synthesized in the liver and initially enters the plasma as part of nascent HDL. It is a 299-amino acid
glycoprotein with a molecular weight of 34,200 and has a central role in lipid metabolism in both physiological
and pathophysiological processes. Levels of apo E in plasma range from 2-6 mg/dl.9
Apo E promotes the binding of lipoproteins (LDL, VLDL, and apo E-HDL) to the LDL-R and a specific
chylomicron remnant receptor, and is also associated both with transport of cholesterol ester in plasma and with
the redistribution of cholesterol in the tissues. It is a major protein constituent of TG rich lipoproteins, including
chylomicrons, VLDL and their remnants. A major function of apo E is to serve as a high affinity ligand for
several hepatic lipoproteins receptors, including LDL-R and LDL receptor-related protein and for cell surface
HSPG. By interacting with these receptors, or with HSPG, apo E mediates the clearance of chylomicrons,
VLDL and their remnants from circulation.81,82
Apolipoprotein E Isoforms
Apo E phenotype is an important variable that has been used in large-scale population studies.68 There are three
major isoforms of apo E, namely apo E2, E3, and E4. Individuals may have any one or a combination of any
two of these isoforms.57 Apo E3 and E4 both bind well to the LDL-R whereas E2 is defective in binding. Apo E
is genetically polymorphic and has 3 codominant alleles (apo 2, 3, and 4) at the apo E gene locus. The three
9

alleles give rise to six genotypes, of which three are homozygous (2/2, 3/3, and 4/4) and three are
heterozygous (2/3, 2/4, and 3/4). The 2 allele is associated with lower levels of serum TC, LDL-C, and apo
B compared to the 3 allele. The 4 allele is associated with higher levels of TC, LDL-C and apo B and higher
risk for cardiovascular disease and AD.83,84 The 2 allele is present in type III hyperlipoproteinemia.
Epidemiological evidence suggest that the apo E4 phenotype conveys a higher cardiovascular risk than does apo
E2 or apo E3.85 In addition, an 2/3 genotype appears to confer relative protection compared with an 3/3
genotype.86
Effect of Apolipoprotein E Genotype on Drug Treatment
There is evidence, which suggest that individual with different genotypes have inter-individual response to diets
and lipid lowering drugs. For example, there is indication that apo E2 individuals are more likely to respond
favorably to gemfibrozil and cholestyramine therapy.87 With probucol, apo E4 genotype individuals may show
improved plasma lipoprotein-lipid profiles more than apo E3 individuals. Apo E2 and E3 genotype
perimenopausal women appear to show improved plasma lipoprotein-lipid profiles more with HRT than apo E4
women.119 Low-fat diet interventions tend to reduce plasma LDL cholesterol and, perhaps, plasma total
cholesterol levels more in apo E4 than in apo E2 or E3 individuals.87 Most studies generally indicate that apo E2
and E3 individuals improve plasma lipoprotein-lipid profiles more with exercise training than apo E4
individuals. Thus the future utility of apo E genotype to determine optimal therapy to improve plasma
lipoprotein-lipid profiles and stratify CAD risk is very possible.87
In a study, Ballantyne et al.88 examined the association of apo E genotypes with baseline plasma lipid levels and
severity of CAD, and response to treatment with fluvastatin in the Lipoprotein and Coronary Atherosclerosis
Study (LCAS). The result indicated that subjects with the 3/4 or 4/4 genotype had an increased frequency of
previous angioplasty, but other measures of baseline CAD severity and baseline lipids did not differ
significantly among the genotypes, nor did CAD progression or clinical events. They concluded that although
subjects with the epsilon4 allele had less reduction in LDL-C with fluvastatin, they had similar benefit in terms
of CAD progression.
Apolipoprotein E Genotype and Atherosclerosis
Atherosclerosis involves the hardening of arteries due to the deposit of cholesterol in the cells of the smooth
muscle layers of the vascular tissue and may be age and/or environment related. Apo E 2 and 4 has been
reported to be independent risk factors for CAD and as predictor for the development of atherosclerosis.89 Apo
E 4 allele is associated with very high levels of LDL-C and TC while 2 allele is associated with decreased
levels LDL-C and higher TG levels. The common apo E isoforms are the result of amino acid substitutions in
codons 112 and 158 (cysteine to arginine). The presence of these isoforms leads to differences in the affinity of
the resulting individual phenotypes for LDL and apo E receptors, LDL-R activity, apo E distribution among
lipoproteins, LDL formation rate and cholesterol absorption, all of which may promote atherosclerosis and
increase the risk of developing CAD. Thus, given the similarity between risk factor for heart disease and risk
factor for stroke, a positive association between apo E and stroke would be expected. Patients with apo E 4
allele who have hemorrhagic strokes have poorer prognosis, and after head injury 4 patients seem to be
associated with poorer recovery than non-4 patients.90
Apolipoprotein E Genotype and Coronary Heart Disease Risk
In a study to determine whether Apo E 4 allele is an independent predictor of coronary event, Scuteri et al.121
measured apoE genotypes in 730 participants in the Baltimore Longitudinal Study of Aging who were free of
preexisting CHD. They concluded that the apo E 4 genotype is a strong independent risk factor for coronary
events in men, but not women. The association does not appear to be mediated by differences in total
cholesterol levels. In a meta-analysis to assess the impact of apo E alleles in coronary disease in 14 published
10

observational studies, Wilson et al.91 observed that 4 allele was associated with greater odds for CHD in both
men and women compared with 3 allele or 2 allele.
Apolipoprotein E and Alzheimers Disease
The ability of Apo E to modulates the risk for AD, CHD and cerebral atherosclerosis is well established.92,93
Alzheimers disease is an age-associated neurodegenerative disorder, which is characterized by a progressive
decline of the cognitive functions. It is the most common cause of dementia in elderly people. Apo E 4 allele
has been implicated in AD. Most cases of AD are sporadic and the factors involved are unknown. However, a
small percentage of cases are hereditary and are typically due to mutations in one or more of the three separate
genes. Mutation in the amyloid precursor protein (APP) gene on chromosome 21, and the presenilin-1 (PS1)
gene on chromosome 14 are fully penetrant and cause early-onset AD. Mutations in the presenilin-2 (PS2) gene
a PS1 homologue on chromosome 1 cause partially penetrant autosomal dominant AD with onset age beginning
at 40 years and extending past 75 years. A fourth gene which encodes the protein apo E is implicated in lateonset AD.94,95 An association between AD and the apo E 4 allele has been reported in both familial and
sporadic late-onset AD. The apo E 4 allele increases the risk and lowers the age of onset distribution of AD in
a close dependent fashion. The inheritance of each dose of apo E increases risk of disease and decreases age of
onset; conversely, the apo E 2 allele appears to be protective, by lowering the risk of disease and increasing age
of onset.96
To evaluate the genetic factors for AD among a Chinese population in Taiwan, Hu et al.97 studied the
polymorphisms of six candidate genes of AD, including the regulatory region of apo E (Apo-E, G-186T), the
promoter of apo E (Apo-E, A-491T), the bleomycin hydrolase gene (BH, A1450G), a mutation of alpha(2)macroglobulin gene (A2M G2998A), LDL-R-related protein gene (LRP, C766T), and alpha(1)antichymotrypsin gene (ACT, -15Ala/Thr) in AD patients and non-affected elder individuals among Taiwanese
Chinese. Eighty-two AD patients and 110 non-affected individuals were recruited for the study. Their result
showed that among the nine candidate genes of AD that they studied the apo E 4 allele was the only
independent genetic risk factor for AD. The other candidate genes in the study were not associated with the
occurrence of AD. In addition, there was no gene-gene interactions.97 The apo E is a multifunctional molecule
with potential roles in amyloid deposition and clearance, microtubule stability, intracellular signaling, immune
modulation, glucose metabolism oxidative stress and other cellular process.98 However, the association with AD
is not absolute since only about 50% of all patients with AD have the apo E 4 allele and a substantial number
of persons with the apo E 4 allele do not develop AD.98 Consequently, another gene in combination with the
apo E 4 allele may be involved in sporadic AD. Furthermore, the link of apo E 4 allele to both sporadic and
familial late-onset AD raises the possibility that a dysfunction of the lipid transport system could seriously
affect lipid homeostasis in the brain.
The abnormally low concentration of apo E observed in the brains of some AD subjects with the apo E 4 allele
could compromise cholesterol, fatty acid, and phospholipid transport in the central nervous system. This in turn
could impair the cholinergic system, which, in contrast to other neurotransmitters in the central nervous system,
relies heavily on lipids to synthesize acetylcholine. An inverse relationship has been observed between apo E 4
allele copy number and residual brain choline acetyltransferase activity and nicotinic-receptor binding sites in
the brains of subjects with AD.99 Apo E in the presence of the protein amyloid-beta may also promote the
development of hair-shaped fibrils and neuritic plaques containing arms of damaged nerve cells in the brain.
These plaques, which contain damaged nerve cells, are the hallmark of AD. Thus, when combined with clinical
criteria, apo E genotyping improves specificity of the diagnosis of AD.100
Apolipoprotein D
Apolipoprotein D is a 29-kDa glycoprotein that is primarily associated with HDL in human plasma. It is an
atypical apolipoprotein and, based on its primary structure, it is predicted to be a member of the lipocalin
family. Lipocalins adopt a beta-barrel tertiary structure and transport small hydrophobic ligands. Although apo
11

D can bind cholesterol, progesterone, pregnenolone, bilirubin and arachidonic acid, it is unclear if any, or all of
these, represent its physiological ligands.101 The apo D gene is expressed in many tissues, with high levels of
expression in spleen, testes and brain. It is present at high concentrations in the cyst fluid of women with gross
cystic disease of the breast, a condition associated with increased risk of breast cancer.101,102,103 It also
accumulates at sites of regenerating peripheral nerves and in the cerebrospinal fluid of patients with
neurodegenerative conditions, such as AD. It may, therefore, participate in maintenance and repair within the
central and peripheral nervous systems. While its role in metabolism has yet to be defined, it is likely to be a
multi-ligand, multi-functional transporter. It could transport a ligand from one cell to another within an organ,
scavenge a ligand within an organ for transport to the blood or could transport a ligand from the circulation to
specific cells within a tissue.101 Apo D is present in the human brain, especially in glial cells, and has increased
abundance in the elderly and AD subjects.104
It is a protein component of the human plasma lipid transport system but is also associated with a more
favorable prognosis in women with breast cancer.102 Vazquez et al.102 examined the tumoral expression of apo D
in epithelial ovarian cancer so as to analyze the possible correlation with tumor and patient characteristics as
well as to androgen receptors and their prognostic significance. They found the existence of apo D expression to
be significant in ovarian carcinomas, and and that the protein expression might be of clinical usefulness for
identifying lesions with different evolution.102 Apo D is expressed in normal human prostate and elevated Apo D
staining is associated with advanced prostate cancer.105 Apo D has been implicated in the transport of several
small hydrophobic molecules including sterols and steroid hormones. Defects in apo D metabolism in NiemannPick disease type C (NPC) appears to be linked to the known defects in cholesterol homeostasis in the
disorder.106
Apolipoprotein C
The primary activity of apolipoprotein C is to activate LPL leading to lipolysis of TG. There are three major
forms namely apo C-I, C-II, C-III and they vary in amino acid content having 57, 78, and 79 amino acids
respectively. They are synthesized in the liver and associate with VLDL and HDL particles.55 The metabolic
function of apo C-I is its inhibitory action on the uptake of VLDL via hepatic receptors, particularly the LDL
receptor-related protein. Consequently, the presence of apo C-I on the lipoprotein particle may prolong its
residence time in the circulation and subsequently facilitate its conversion to LDL. Apo C-II, on the other hand,
is a major activator of LPL, which is required for an efficient processing of triglyceride-rich lipoproteins (TRL)
in the circulation. However, an excess of apo C-II on the lipoprotein particle has been sugg suggested to inhibit
the LPL-mediated hydrolysis of triglycerides. Apo C-III inhibits the lipolysis of TRL by hampering the
interaction of these lipoproteins with the heparan sulfate proteoglycan-lipoprotein lipase complex. 107 Poorly
lipolyzed apo C-III-containing lipoprotein particles may accumulate in plasma because of their lower binding
affinity towards hepatic receptors due to a change in lipid composition, particle size or the presence of apo C-III
on the particle itself.
All apo C specifically modulate the metabolism of TRL, which may contribute to the development of
hyperlipidemia and other lipoprotein abnormalities in humans.107 Apo C-III plays an important role in the
metabolism of plasma triglyceride, which can delay the catabolism of TRL by interfering with apo E-mediated
receptor clearance of remnant particles from plasma. Apo C-III and apo E play a central role in controlling the
plasma metabolism of TRL and apo C-III has been implicated as a potential determinant of the TG lowering
effect of fibrates, which down-regulate its expression.108
Li et al.109 investigated the protective effect of apoA-I, A-II, C-I and C-II, the main proteins in HDL on the
morphology and function of human umbilical vein endothelial cells (HUVEC) injured with LDL in vitro. They
cultured human endothelial cells derived from umbilical veins and exposed them to LDL, HDL, and apo A-I, AII, C-I and C-II. They then examine the morphology of the endothelial cells and measured the amount of lactate
dehydrogenase (LDH) and 6-keto-prostaglandin F1 alpha (PGF1 alpha) released. The result showed contration
of the endothelial cells, increased release of LDH and decreased secretion of prostacyclin (PGI2).109 However,
12

on addition of HDL, and apo A-I, A-II, C-I and C-II before incubation with LDL, cellular injury induced by
LDL was inhibited. They concluded that apo A-I, A-II, C-I and C-II, as well as HDL, may play an important
role in combating atherogenesis by protecting endothelial cells from damages induced by LDL.109
Hyperlipoproteinemia
Hyperlipoproteinemia is the result of malfunction in the synthesis and/or catabolism of lipoproteins. The
Fredrickson classification of hyperlipoproteinemia is based on the appearance of plasma as well as the total
cholesterol and triglyceride values in the plasma (Table 3). The first comprehensive classification of the
dyslipoproteinemias,which was formulated in 1965 and 1966 and adopted by the World Health Organization
(WHO) described hyperlipoproteinemia as plasma phenotypes. The Fredrickson type are important because they
allow focus on metabolic diseases related to hyperlipoproteinemia, identify hyperlipoproteinemia as disorders
that affect particular lipoproteins, associate lipoprotein types with distinctive clinical features and provide the
basis for successful diet and drug therapy. Often the only way to tell a type I from type V was the plasma
appearance or a type III from type IV was to use a lipoprotein electrophoresis pattern. It is now more convenient
to talk about where the metabolic defect is in lipoprotein metabolism. Lipoprotein disorders may be classified as
primary or secondary. The primary disorders are either genetic or nongenetic. The secondary disorders may
have their origin in diet, use of alcohol or drugs or disease of metabolic hormonal, infectious or malignant
etiology.9,10 The origin of CAD which causes a high morbidity and mortality in most western countries
involves a significant genetic component. The identity of some of these genetic mutations include those of
LDL-R, apo B-100 and Lp(a). Other genetic loci that have been implicated include LPL, apo CII, cholesteryl
ester transfer protein (CETP), apo A-I and LCAT.110
Hypertriglyceridemia
Hypertriglyceridemia can have a primary or secondary etiology. In primary forms the origin is essentially
genetic, while the secondary ones are consequent upon metabolism of various pathologies including renal,
thyroid, diabetes mellitus etc.111 Diabetic dyslipoproteinemia characterized by hypertriglyceridemia, low
HDL-C, and often elevated LDL-C with predominance of small, dense LDL is a strong risk factor for
atherosclerosis.112 Hypertriglyceridemia is not a common finding in well controlled patients with IDDM;
however, in NIDDM, hypertriglyceridemia and coronary heart disease are a well recognized clinical
triad.113 In the latter setting, hypertriglyceridemia is usually the result of an associated inherited
hyperlipidemia, most commonly familial hypertriglyceridemia but also familial combined hyperlipidemia
(FCHL). In the former, one sees elevated TG and a low HDL-C, in the latter the same phenotype may be
present but often there is a high LDL-C.113
Irrespective of the pathogenesis of the primary hypertriglyceridemic disorder, the occurrence of poorly
controlled diabetes will enhance the hypertriglyceridemia and even in the NIDDM subject, with
triglycerides in the thousands, dietary and glycemic control, alone, will strikingly ameliorate the
hypertriglyceridemia.111,113 Familial hypertriglyceridemia (FHT) has been suggested to be an autosomal
dominant condition with age-dependent penetrance, but so far the underlying defective gene has not been
elucidated. Patients with type IV hyperlipoproteinemia, particularly those with FHT, have impaired
absorption of bile acid, which may contribute to the hypertriglyceridemia. Increased production of
cholesterol has been associated with type IV hyperlipidemia, but the influence of the confounding variable
of obesity has been difficult to ascertain. Moreover, cholesterol metabolism has not been systematically
evaluated in patients with FHT, one of the two major subsets of type IV patients.114
Familial Hyperchylomicronemia
The familial hyperchylomicronemia may be divided into type I and V hyperlipoproteinemia. 10 The familial
hyperchylomironemia syndrome is a hereditary disorder of lipoprotein metabolism caused by LPL deficiency,
apo-CII deficiency or LPL inhibition.115 The syndrome is characterized by hyperchylomicronemia,
hypertriglyceridemia, pancreatitis and attacks of epigastric pain. The presence of eruptive xanthomas, may
eventually lead to necrotizing pancreatitis or pancreatic insufficiency.116 Treatment consists of lifelong
13

adherence to a low-fat diet to prevent hyperchylomicronemia and its sequel. Apolipoprotein C-II plays a major
role as a cofactor for lipoprotein lipase, the enzyme involved in the hydrolysis of triglyceride-rich particles.
Human apo C-II consists of 79 amino acid residues and the amino-terminal two thirds of the molecule binds to
lipid through the formation of amphipathic helixes, while the carboxy-terminal third is engaged in activation of
LPL.117
Most cases of type I hyperlipoproteinemia are due to genetic defects in the LPL gene or in its activator, the apo
C-II gene. Several cases of acquired type I hyperlipoproteinemia have also been described in the course of
autoimmune diseases due to the presence of circulating inhibitors of LPL.118 Primary hyperchylomicronemia is
known as a syndrome in which the accumulation of chylomicron occurs in the circulation. The main clinical
symptoms of this disorder are the large increase in plasma triglyceride and cholesterol, and the presence of
xanthomatous eruption, lipemia retinalis, hepatosplenomegaly, and the complication of acute pancreatitis. With
gene analysis, a deficiency of LPL or apolipoprotein C-II is revealed as a main cause of primary
chylomicronemia. In addition, in some cases, abnormalities of remnant receptors, the presence of antibody
against LDL, apolipoprotein C-II, and LDL-R are seen as causes of chylomicronemia syndrome.119 Most
individuals presenting with chylomicronemia have the familial forms of hypertriglyceridemia in combination
with secondary acquired disorders.115
Type V hyperchylomicronemia is characterized by the presence of increased VLDL and chylomicrons in the
plasma of fasting individuals on normal diet. Triglyceride levels similar to type I hyperlipoproteinemia may be
present and the patients may experience abdominal syndromes including pancreatitis, eruptive xanthomas,
lipemia retinalis and hepatosplenomegaly due to the increased TG level.10 Most cases occur in adulthood with
females presenting later than men. Plasma cholesterol levels are usually slightly increased while LDL-C and
HDL-C are usually normal to low. The defect appears to be associated with inadequate clearance of
chylomicrons.
Familial Hyperlipoproteinemia
Familial type III hyperlipoproteinemia is a rare disorder, which affects 1 to 10 in 10,000 people in the general
population.120 People with this disorder have premature atherosclerosis in peripheral vessels and coronary
arteries. In addition, they have accumulations of chylomicron remnant and VLDL in the fasting state.
Homozygosity for apolipoprotein 2 and accumulation of TRL in plasma are characteristic of this disease.
Compared with other hyperlipoproteinemias, type III hyperlipoproteinemia usually responds to therapy. As a
result of its clinical significance, the apo E polymorphism has been studied in several different human
populations where, despite the wide variation observed in allele frequency distribution, apo E 3 allele was
found to be the most common apo E allele.121 In addition to the common apo E isoforms, several rare apo E
variants have been identified in hyperlipidemic patients and their kindred. Most of these variants are
characterized by replacements of one or more charged amino acids by uncharged amino acids or vice versa.92
Some replacements in the LDL receptor-binding region (positions 136 to 150) cause defective binding to the
LDL receptor and are associated with the recessive form of familial type III hyperlipoproteinemia. In addition,
some replacements of basic amino acids with neutral or acidic amino acids lead to heparan sulfate proteoglycan
(HSPG) binding which is associated with the dominant form of familial type III hyperlipoproteinemia
Familial Combined Hyperlipidemia
Familial combined hyperlipidemia (FCHL) is a complex disorder with several environmental factors, which
interact with multiple genes. Elevated levels of total serum cholesterol and/or TG characterize it. The disorder is
estimated to be common in Western populations with a prevalence of 1% to 2%. In addition, 14% of patients
with premature CHD have FCHL which makes the disorder one of the most common genetic dyslipidemias
underlying premature CHD.122 Familial combined hypertriglyceridemia has been suggested to be an autosomal
dominant condition with age-dependent penetrance, but so far the underlying defective gene has not been
elucidated. Lipoprotein lipase gene and apo A-I/C-III/A-IV gene cluster might be involved in familial clustering
14

of hypertriglyceridemia. Heterozygous LPL deficiencies caused by several types of gene mutation are known to
result in a partial defect in catabolism of VLDL, causing mild to moderate hypertriglyceridemia. However,
although the mutation of LPL gene results in reduced lipolytic activity, this type of dyslipidemia appears to
manifest only if VLDL-TG production is also increased. These suggest that overproduction of VLDL-TG is a
more important cause of hypertriglyceridemia than is the LPL deficiency.123
Familial Hypercholesterolemia
Familial defective apolipoprotein B-100 (FDB) and familial hypercholesterolemia (FH) are the common causes
of monogenic primary hypercholesterolemia. They are both associated with severe hypercholesterolemia and
cannot always be distinguished from one another phenotypically. Familial defective apolipoprotein B-100 is the
most common known mutation causing primary hypercholesterolemia.124 More than 150 mutations exist in the
LDL-R gene, associated with FH an autosomal dominant inherited disorder characterized by severe
hypercholesterolemia, frequent presence of tendon xanthomas and an elevated risk of premature CAD.125
Familial hypercholesterolemia is caused by different mutations in the LDL-R gene or by a guanine to adenine
mutation in exon 26 of the apolipoprotein B gene which causes FDB.126 Familial defective apo B-100 is an
autosomal codominant disorder leading to plasma LDL-C elevation and CAD. It is caused by substitution of
glutamine for arginine at amino acid residue 3500 of apo B-100, in the putative LDL-R binding domain of the
mature protein. This results in reduced affinity of LDL for the LDL-R. The amino acid substitution decreases
the binding capacity of the LDL for the LDL-R, which then leads to an increase in levels of plasma TC and
LDL-C. The frequency of the mutation may be as high as 1 in 600 in the normal population and 1 in 500 to 1 in
700 in Europe and in North America.127,128
Table 3 Fredricksons Classification of Hyperlipoproteinemia
Fredricksons Cholesterol
Classification
I
Normal to
increased

Triglycerides

Iia

Increased to very
increased

Normal

LDL very
increased

Iib

Increased

Increased

VLDL and LDL

increased

III

Normal to
increased

Increased

VLDL and LDL

very increased

IV

Normal to
increased

Increased

VLDL very
increased, LDL
increased

Normal to
increased

Very
increased

Chylomicrons,
VLDL and LDL
very increased

Very
increased

Lipoprotein
Pattern Changes
Chylomicrons
very increased

Appearance
of Specimen
Milky top
layer

Some Causes of
hyperlipoproteinemia
Insuliopenic diabetes
mellitus, lupus
erythematosus,
Pancreatitis
Clear
Nephrotic syndrome,
hypothyroidism,
porphyria
stress
Slightly turbid Nephrotic syndrome,
hypothyroidism,
porphyria
stress
Turbid
hypothyroidism,
myxedema, diabetic
acidosis, primary
biliary cirrhosis
Turbid
Diabetes mellitus,
nephrotic syndrome,
pregnancy,
alcoholism
Milky
Insuliopenic diabetes
mellitus, nephrotic
syndrome,
alcoholism,
15

myeloma,
pancreatitis
The vast majority of affected heterozygotes have TC and LDL-C levels well above the 95th percentile for age
and gender; in contrast, HDL-C, VLDL-C and plasma TG are not affected by the mutation.128 In FDB
heterozygotes, about 70% of the LDL particles are mutant, which may alter their atherogenicity relative to LDL
containing normal apo B.129 Maher et al.129 compared CAD in heterozygous FDB with CAD in heterozygous FH
and found that there was no significant difference between the FDB and FH patients in the type of cardiac
symptoms or their age of onset and coronary angiographic appearance was similar in both groups. They then
surmised that the LDL particles with the R3500Q mutation in apo B have the same atherogenicity as the LDL
particles with normal apo B. To compare the phenotypic expression of either defects, Brugger et al.126 studied
patients with FH and FDB from Germany and found that the average TC level in plasma was 413.7 mg/dL in
FH and 321.8 mg/dL in FDB patients. Patients with FH had a significantly higher risk of myocardial infarction,
coronary artery bypass graft, positive coronary angioplasty, atherosclerotic plaques in the carotid arteries and
CAD than patients with FDB. This finding is contrary to those of Maher et al.129 who found no significant
difference between similar groups of patients. Because FDB is one of the independent causes of early onset
CHD, the R3500Q mutation should be considered in families with a high frequency of cardiovascular diseases.
Abetalipoproteinemia
Abetalipoproteinemia is a rare disease in which apo B is not synthesized and as a result, lipoproteins containing
this apolipoprotein are not formed resulting in the accumulation of lipid droplets in the intestine and the liver,
due to an inability to produce chylomicrons and VLDL in the intestine and liver, respectively. The disease
results from mutations in the gene encoding the 97-kd subunit of the microsomal triglyceride transfer protein,
which plays a central role on secretion of lipoprotein from the liver and the intestine. It catalyzes the transfer of
TG, cholesteryl ester and phosphatidylcholine between membranes and lipoproteins. Downstream effects
resulting from this defect include malnutrition, very low plasma cholesterol and TG levels, altered lipid and
protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated,
abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological
abnormalities.130,131
Hypobetalipoproteinemia
Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of
LDL-C. It can be caused by mutations in the gene encoding apo B-100, leading to the formation of truncated
apo Bs which have a reduced capacity to export lipids from the hepatocytes as lipoprotein constituents.132 It is
characterized by less than fifth percentile age- and sex-specific levels of apo B and LDL-C. In a minority of
cases, FHBL is due to truncation-producing mutations in the apo B gene on chromosome 2p23-24.133 Low LDLC and apo B levels in plasma cosegregate with mutations of apo B in some kindreds with FHBL. Approximately
35 apo B mutations, many specifying apo B truncations, have been described. Based on the percentile
nomenclature where the full-length nature apo B consisting of 4536 amino acids is designated as apoB-100,
only those truncations of apo B >25% of normal length are detectable in plasma.133
Familial hypoalphalipoproteinemia
Isolated deficiency of HDL-C is genetic in presentation and is known as familial hypoalphalipoproteinemia
(FHA). Hypoalphalipoproteinemia (HA) is a common finding in patients with premature CAD. It is present in
about 5% of patients with premature CHD. Some patients with the disease have decreased production of HDL.
Plasma half-life of HDL in normal individuals ranges from 3.3 to 5.8 days. Catabolism of HDL is enhanced in
nephrotic patients but decreased in hypertriglyceridemic subjects and greatly enhanced in familial HDL
deficiency (Tangier disease).10 Familial hypoalphalipoproteinemia syndromes are phenotypically heterogeneous.
One form is associated with abnormal cellular cholesterol efflux caused by heterozygous mutations at the
ABCA1 gene, that defines familial HDL deficiency while homozygous mutations or compound heterozygosity
causes Tangier disease.134 Tangier disease is characterized by deficiency of HDL and their major protein
16

constituent apo A-I as well as presence of low molecular mass lipoproteins and a high concentration of apo CIII in the lipoprotein fraction. ABCA1 heterozygotes have decreased HDL-C and increased TG. Age is an
important modifier of the phenotype in heterozygotes with higher proportion of them aged 30-70 years having
HDL-C greater than the 5th percentile for age and sex compared with carriers less than 30 years if age.135
Hyperalphaliporoteinemia
Cholesteryl ester transfer protein is a plasma glycoprotein that mediates the transfer of cholesteryl ester from
HDL to triglyceride-rich lipoproteins in exchange for triglycerides.136 Since CETP regulates the plasma levels of
HDL cholesterol and the size of HDL particles, it is considered to be a key protein in RCT, which is a protective
system against atherosclerosis. Cholesteryl ester transfer protein and plasma phospholipid transfer protein
belong to members of the lipid transfer and lipopolysaccharide-binding protein gene family, which include the
lipopolysaccharide-binding protein (LBP) and bactericidal and permeability-increasing protein. The proteins
possess different physiological functions, but share marked biochemical and structural similarities. The
importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient
subjects with a marked hyperalphalipoproteinemia.137
There is no agreement about whether plasma cholesteryl ester transfer protein (CETP) deficiency is associated
with an antiatherogenic state or not, although this disorder was reported to be one of the major causes of marked
hyperalphalipoproteinemia.138 However, hyperalphalipoproteinemia is regarded by some as a beneficial state
accompanied by a longevity syndrome.139 Hirano et al.138 performed a large population based study concerning
the atherogenicity of markedly elevated HDL-C levels in a genetically homogeneous population to determine
whether CETP deficiency is associated with decreased atherogenicity. The study revealed that the frequency of
the CETP gene mutation was higher in patients with CHD than in control patients. This indicated that marked
hyperalphalipoproteinemia caused by CETP gene mutation may not represent a longevity syndrome. There
might therefore be a need to revisit and reevaluate the clinical significance and pathophysiology of a marked
hyperalphalipoproteinemia.138
Familial hyperalphalipoproteinemia
Familial hyperalphalipoproteinemia (FHA) is a heritable trait associated with elevated plasma concentrations of
HDL-C and possibly with longevity and protection against CHD. Individual with this condition are known to
have elevated plasma levels of HDL-C and apo A-I, which may be due to a selective upregulation of apo A-I
production, which might have antiantherogenic properties. Rader et al.140 studied the production rate of apo A-I
and apo A-II in FHA individuals and control subjects and found that the production rate of apo A-I was
markedly increased in FHA subjects than in control subjects while the production rate of apo A-II was not
substantially increased. Hyperalphalipoproteinemia due to complete deficiency of cholesteryl ester transfer
activity is characterized by the presence of both small polydisperse LDL and markedly large HDL enriched with
cholesteryl ester and apo E.
Laboratory Measurements of Lipids
Phospholipids
Measurement phospholipids have in the past been done by thin-layer chromatography. Theseparation of the
phospholipids is based on the side chains groups and involves extraction of the of the phospholipids into
solvent, drying for concentration, spotting onto silica gel plates, separation with a chloroform-methanolwater solvent and visualizing through charring and iodination.141 Spectrophotometric methods can also be
employed to quantitate the phospholipid but inorganic phosphorus are used as indicators.141
Cholesterol
Measurement of cholesterol includes both the ester and the free forms of the steroid. In serum or plasma, two
thirds of the total cholesterol exist in the esterified form with the rest in the free form.10 This can result in greater
intensity in the color produced with the cholesterol ester than with the free cholesterol. The implication of
which is a large positive bias. In enzymatic reactions, the hydrolysis of the longer-chain cholesterol esters, such
17

as cholesterol arachidonate is not complete which may lead to a negative bias.10 The methods of cholesterols are
usually modifications of (1) Liebermann-Burchard, (2) iron-salt-acid, (3) p-toluene-sulfonic acid, or (4)
enzymatic end point. The Lierbermann-Burchard procedure is the most widely used method and early analytical
methods used strong acids such as sulfuric and acetic acids and chemicals such as acetic anhydride or ferric
chloride, which produced a measurable color with cholesterol. The current reference method uses hexane
extraction after hydrolysis with alcoholic potassium hydroxide followed by reaction with Liebermann-Burchard
color reagent, which comprises sulfuric and acetic acid and acetic anhydride.56
An enzymatic method for cholesterol employs the enzyme cholesteryl ester hydrolase, which cleaves the fatty
acid residue and thus convert cholesteryl ester to unesterified or free cholesterol. The free cholesterol is reacted
by cholesterol oxidase, which produces cholest-4en-3-on and hydrogen peroxide. The hydrogen peroxide
produced is a substrate for an enzymatic color reaction, which employs horseradish peroxidase to couple two
colorless chemicals into a colored compound.56 The enzymatic method, has been applied to automated
procedures including dry-chemistry approach. Use of electrode systems is another approach for quantifying
cholesterol. The oxygen selective membrane is used to measure the rate of oxygen consumption when the serum
is reacted with a reagent containing cholesterol oxidase.141
Triglycerides
The measurement of triglycerides and cholesterol is usually used to detect genetic and other metabolic problem
such as hyperlipoproteinemias. The measurement of triglycerides is also required for the estimation of LDL
cholesterol concentration by the Friedewald equation.68 Most triglyceride methods employ lipases and proteases
to cleave fatty acids from glycerol and the glycerol is converted to glycerol-3-phosphate and adenosine
diphosphate (ADP) in the presence of adenosine triphosphate (ATP) and glycerol kinase. The ADP is reacted
with phosphoenolpyruvate in the presence of pyruvate kinase to ATP and pyruvate. The pyruvate is reacted with
nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase and converted to lactate
and reduced NADH and the absorbance is measured at 340 nm. Other methods using fluorometric measurement
in which the disappearance of NADH fluorescence is read at 460 nm after excitation at 355 nm. The method is
usually direct, rapid, and specific one of several enzymatic sequences.
Lipoproteins
The lipoproteins HDL and LDL are usually quantified based on their cholesterol content. The lipoproteins may
be separated and quantified based on their densities, sizes, charge, and apolipoprotein contents. The range of
observed densities among lipoprotein classes are a function of their lipid and protein content and they allows for
fractionation by ultracentrifugation. Separation by electrophoresis is made possible by the differences in charge
and size. Antibodies that are specific for particular apolipoproteins can be used to bind and separate lipoprotein
classes.56
Apolipoproteins
The apolipoproteins may be measured by immunoassay methods using turbidimetric or nephelometric assays.
Other methods that have been used to quantify apolipoproteins include radioimmunoassay (RIA)
radioimmunodetection (RID) and Enzyme labeled immunosorbent immunoassay (ELISA).56 Either polyclonal
antibodies or monoclonal ones may be employed in these assays. Various molecular methods may be employed
to genotype apolipoprotein E including genotyping of the apo E locus by isoelectric focusing, by restriction
digestion of a PCR fragment of the gene, and by allele-specific oligonucleotide hybridization. The isoelectric
focusing method is long and cumbersome, while earlier restriction digestion methods using the enzyme HhaI
produce too many small fragments to permit easy interpretation.
Conclusion
Epidemiological, clinical, genetic, experimental and pathological studies have established primary roles for
lipids, lipoprotein and apolipoproteins in atherogenesis.142,143 It is well established that lipids, lipoproteins and
apoliporoteins are useful in diagnosing and prognosticating therapeutic intervention in the management of
hyperlipidemic conditions in patients.144,145 There is increased interest in the measurement of apolipoproteins
18

because of the suggestion that they might be better predictors of coronary heart disease than lipid parameters
currently used.68,146 According to Jungner et al.145 apo B appears to give more accurate predictive estimate of
atherogenic risk than TC or LDL-C. Apo B and A-I can be determined with high precision and accuracy and
with automation can reduce cost. Low levels of apo A-I/B ratios in individuals has been found to be indicative
of susceptibility to CHD.68 Thus measurement of apo A-I/B ratio may be useful in the assessment of CHD risk
in individuals. Numerous clinical studies have indicated the usefulness of Lp(a) as a risk marker for
atherosclerotic diseases.39,48,50,51 Therefore patients with increased levels of Lp(a) and unstable angina may be at
greater risk of suffering a myocardial infarction. Studies have shown that apo E genotyping might be helpful in
predicting susceptibility to AD.98-100 Apo E has also been implicated in CHD. 83,90,91 Low-density lipoproteincholesterol and apo B-100 concentrations in serum have direct relationship with risk for CAD.146-148 At the same
time, elevated levels of HDL-C and apo A-I are inversely related to CHD risk.74,75,76 It is therefore obvious that
information about lipid, lipoprotein and apolipoprotein and their biochemistry and metabolism are very relevant
to the health of the population at large. This information may be applied to successfully diagnosis and
management of CAD cases. The desire to reduce lipid levels in individuals should be embraced by all as part of
the overall program designed to achieve the healthy people 2010 initiative.149 People should be aware of the
relationships between the various lipid components and the disease processes.
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