LECTURE FIVE

Practical Biochemistry
Lipid Profile Tests
Prepared by:
Dr.Dheaa Shamikh Zageer
Dr.Sundus Fadhil Hantoosh
1.Cholesterol:

Cholesterol is a 27 carbon compound with a unique structure with a hydrocarbon tail, a

central sterol nucleus made of four hydrocarbon rings, and a hydroxyl group. The center
sterol nucleus or ring is a feature of all steroid hormones.

Cholesterol is a lipophilic molecule that is essential for human life. Since cholesterol is
mostly lipophilic, it is transported through the blood, along with triglycerides, inside
lipoprotein particles (HDL, IDL, LDL, VLDL, and chylomicrons). These lipoproteins can be
detected in the clinical setting to estimate the amount of cholesterol in the blood.
Chylomicrons are not present in non-fasting plasma.

Figure (1): Cholesterol chemical structure (www.google.com)

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Cholesterol can be introduced into the blood through the digestion of dietary fat via
chylomicrons. However, since cholesterol has an important role in cellular function, it can
also be directly synthesized by each cell in the body. The synthesis of cholesterol begins from
Acetyl-CoA and follows a series of complex reactions. A primary location for this process is
the liver, which accounts for most de-novo cholesterol synthesis.

Since cholesterol is mostly a lipophilic molecule, it does not dissolve well in the blood. For
this reason, it is packaged in lipoproteins that have phospholipid and apolipoprotein.

Within the cell, cholesterol has several vital functions. Some of the primary uses for
cholesterol are related to the cell membrane. It is required for the normal structure of the
membrane; it contributes to its fluidity. This fluidity can influence the ability of some small
molecules to diffuse through the membrane, which ultimately changes the internal
environment of the cell. Also, within the membrane, cholesterol plays a role in intracellular
transportation. Beyond its place in the cell membrane, cholesterol has several other biological
functions. Of note, cholesterol is known to be an important precursor molecule for the
synthesis of vitamin D, cortisol, aldosterone, progesterone, estrogen, testosterone, bile salts,
among others.

While cholesterol is central to many healthy cell functions, it also can harm the body if it is
allowed to reach abnormal blood concentrations. Interestingly, when LDL-cholesterol levels
are too high, the condition referred to as hypercholesterolemia, the risk for premature
atherosclerotic cardiovascular diseases (ASCVD) increases.

Physicians can order a lipid panel (lipid profile) to determine the cholesterol concentrations
in a patient’s blood. A typical test result will include the concentrations of high-density
lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, and total cholesterol. These
values are used to screen patients for abnormalities in cholesterol and triglyceride blood
levels. With this information, physicians can estimate a patient's risk for certain health
problems such as coronary artery disease, peripheral arterial disease (PAD), and stroke. Most
laboratories report both LDL-cholesterol and non-HDL cholesterol, which is a secondary
target for treatment.

Hypercholesterolemia (high LDL-cholesterol) is one of the major risk factors contributing to

the formation of atherosclerotic plaques. These plaques lead to an increased possibility of
various negative clinical outcomes, including, but not limited to, coronary artery disease.

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peripheral arterial disease (PAD), aortic aneurysms, and stroke. A major contributor to the
increased risk of atherosclerotic lesion formation is high levels of low-density lipoprotein
(LDL) in the blood. Also, it has been shown that an elevated high-density lipoprotein (HDL)
blood concentration is correlated with a decreased risk in epidemiological studies.

The process through which atherosclerotic plaques develop begins with endothelial damage.
Endothelial damage leads to the dysfunction of endothelial cells, increasing the number of
LDL particles that can permeate through the vascular wall. Lipoproteins, especially LDL, can
then accumulate within the vessel wall trapped by the cellular matrix in the intima. LDL is
then modified and taken up via scavenger receptors on macrophages resulting in foam-cell
formation. As more lipid accumulates within the vessel wall, smooth muscle cells begin to
migrate into the lesion. Ultimately, these smooth muscle cells encapsulate the newly formed
plaque forming the fibrous plaque, the protector of the lesion, preventing the lipid core from
being exposed to the lumen of the vessel. Atherosclerotic plaques can lead to occlusion of the
vessel (decreasing blood flow distally and causing ischemia) or, more commonly because of
abundant lipid and macrophages (vulnerable plaque) rupture, inducing the formation of a
thrombus which can completely block the flow of blood (as occurs in acute myocardial
infarctions, unstable angina).

Hypercholesterolemia refers to the condition in which a patient has elevated blood

concentrations of LDL-cholesterol. High LDL is of particular clinical importance, but it
should be noted that hypercholesterolemia can also include very-low-density lipoprotein
(VLDL) and intermediate-density lipoprotein (IDL), i.e., non-HDL-cholesterol. High LDL
levels have been associated with an increased risk of atherosclerosis, potentially leading to
several other conditions such as coronary artery disease, stroke, and peripheral arterial
disease.

Several factors can lead to increased LDL levels. Some of these factors include genetics, diet,
stress, sedentary lifestyle, medications, and other disorders such as nephrotic syndrome and
hypothyroidism.

Genetic defects that lead to increased LDL levels in the blood include genes that regulate
LDL receptors in the liver. LDL receptors mediate the uptake of LDL into the liver.
Endocytosis of LDL is the primary way that the body decreases cholesterol levels, so it
follows that a decrease in LDL receptor function would also increase LDL concentrations in

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the blood. Patients with a genetic disposition for high cholesterol can be placed on
cholesterol-lowering medications like statins to decrease their risk.

Diet has a variable effect on cholesterol levels among individuals. However, it has been
shown that diets high in saturated fats and trans fats can increase cholesterol in the blood.
Lifestyle changes such as regular aerobic exercise can also help control cholesterol levels.
Saturated fats should not comprise any more than 7% to 10% of the diet.

Patient lifestyle changes like diet (reduction in saturated fat and trans fat with an increase in
fiber and total calories if obese and supplementation with plant stanols), smoking cessation,
and exercise are often a favorable approach to cholesterol reduction. However, in cases that
are refractory to these behavioral modifications, cholesterol-lowering drugs, such as statins,
should be used.

Hypercholesterolemia is often treated medically with lifestyle modification and medications.

The goal of lifestyle changes is typically for the patient to increase physical activity, lose
weight, and follow a heart-healthy diet. For patients with higher risk, a lipid-lowering drug
(often a statin) will be used in conjunction with these behavioral changes. Statins have been
shown to reduce atherosclerotic cardiovascular disease (ASCVD) in patients and are the
favored drugs because there are now generic formulations that make them cost-effective.

Table (1): Cholesterol levels in adults (www.google.com)

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2.Triglycerides:

Figure (2): Synthesis of triglycerides (www.google.com)

Structure of triglycerides: the triglyceride structure consists of 1 glycerol and 3 fatty

acids. Triglycerides are a special class of lipids which consist of one molecule
of glycerol which is covalently bonded to three molecules of fatty acids.

Glycerol: glycerol is an alcohol. Glycerol is a type of organic molecule referred to as an

alcohol because of the presence of hydrxyl (OH) groups in its structure.

Fatty Acids: fatty acids are carboxylic acids with long hydrocarbon chains. Fatty acids
contain a carboxyl group and a hydrocarbon tail. The fatty acid hydrocarbon tail can be of
variable length. Fatty acids are merely carboxylic acids with long hydrocarbon chains. The
hydrocarbon chain length may vary from 10-30 carbons (most usual is 12-18). Fatty acids are
highly hydrophobic and insoluble in water. The R-group of a fatty acid can be saturated or
unsaturated:

-Saturated fatty acids have no double bonds between carbons. They are hydrocarbons in
which all the carbons are bonded to each other via a single bond. There is the maximum
number of hydrogens for a given number of carbons.

-Unsaturated fatty acids have at least one double bond between carbons. They are
hydrocarbons in which one or more carbons are bonded to each other through either double
or triple bonds, or a combination of both, in addition to single bonds.

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Figure (3): Saturated and unsaturated fatty acids (www.google.com)

Figure (4): Diseases associated with high triglycerides levels (www.google.com)

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Triglycerides normal range and high range:

A general blood test can show your triglyceride levels. Below are triglycerides normal range
by age:

-Triglycerides normal range for adults:

1-Healthy: less than 150 milligrams per deciliter (mg/dL) or less than 8.33 millimoles per
liter (mmol/L)

2-Borderline high: 150 to 199 mg/dL (8.32 to 11.05 mmol/L)

3-High: 200 to 499 mg/dL (11.11 to 27.72 mmol/L)

4-Very high: 500 mg/dL or above (27.77 mmol/L or above)

-Triglycerides normal range for children (10 to 19 years):

1-Healthy: less than 90 milligrams per deciliter (mg/dL) or less than 5 millimoles per liter
(mmol/L).

2-Borderline high: 90 to 129 mg/dL (5 to 7.17 mmol/L)

3-High: 130 or above mg/dL (7.2 mmol/L or above)

-Triglycerides normal range for children (below 10 years):

1-Healthy: less than 75 milligrams per deciliter (mg/dL) or less than 4.17 millimoles per liter
(mmol/L)

2-Borderline high: 75 to 99 mg/dL (4.17 to 5.5 mmol/L)

3-High: 100 or above mg/dL (5.56 mmol/L or above)

A healthcare provider might ask a person to avoid eating or fasting for about 12 hours before
the test. Every person must aim for a non-fasting level of TGs i.e., under 150 mg/dL. If a
physician has requested a person to fast for a test (10-14 hours), the levels must be under 30
mg/dL.

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Note: The fasting test results in a low number of triglycerides because it only looks at the
triglycerides made by the liver and sent to VLDL cholesterol, not the triglycerides from food.
Since the person hasn’t eaten anything, there are no chylomicrons in their blood.

Figure (5): Causes leading to high triglycerides (www.google.com)

Hypertriglyceridemia: high triglyceride levels, known as hypertriglyceridemia, can be

caused by various factors. Some of the common causes of high triglycerides include:

1-unhealthy diet: one of the primary causes of high triglycerides is an unhealthy diet.
Consuming a diet high in saturated fats, trans fats, sugar, and refined carbohydrates can lead
to elevated triglyceride levels. Excessive alcohol intake can also contribute to this.

2-Obesity and sedentary lifestyle: being overweight or obese and leading a sedentary lifestyle
can increase triglyceride levels.

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3-Genetics: causes of high triglycerides also extend to genetics. Some people may have a
genetic predisposition to higher triglyceride levels, which can be related to conditions like
familial hypertriglyceridemia.

4-Certain medical conditions: several medical conditions can be associated with causes of
high triglycerides, including diabetes (especially uncontrolled diabetes), thyroid problems,
kidney disease, liver disease, high blood pressure and certain rare genetic disorders.

5-Medications: certain medications, such as beta-blockers, diuretics, steroids, estrogen, and

immunosuppressants, may be the reason behind causes of high triglycerides.

6-Lifestyle factors: factors like smoking, alcohol consumption and stress may also contribute
to causes of high triglycerides.

7-Metabolic Syndrome: this condition is characterised by a combination of high blood

pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol levels,
including high triglycerides.

8-Pregnancy or menopause: triglyceride levels can rise during pregnancy, particularly in the
third trimester. During menopause, hormonal changes occur as a woman’s body produces
lower estrogen and progesterone levels, contributing to causes of high triglycerides.

9-Dietary choices: rapidly absorbing carbohydrates and sugary foods are also among causes
of high triglycerides. They can cause a surge in triglyceride levels after a meal.

3.Phospholipids:

Phospholipids are a class of lipids composed of a hydrophilic head group, a glycerol

molecule, and two hydrophobic fatty acid tails. The hydrophilic head group consists of
various combinations of functional groups, such as choline, ethanolamine, serine, or inositol,
linked to the phosphate moiety. The fatty acid tails can be saturated or unsaturated, and their
composition determines the physical properties of the phospholipid.

Structure of phospholipids:

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Phospholipids are amphipathic molecules, meaning they possess both hydrophilic (water-
loving) and hydrophobic (water-fearing) regions. This unique structure enables phospholipids
to form the foundation of biological membranes.

Let's explore the detailed structure of phospholipids:

1-Glycerol backbone: phospholipids consist of a glycerol molecule, which serves as the

central core of the molecule. The glycerol backbone contains three carbon atoms, labelled as
carbon 1, carbon 2, and carbon 3.
2-Fatty acid tails: two fatty acid tails are attached to the carbon atoms of the glycerol
backbone. The fatty acid tails are long hydrocarbon chains, typically consisting of 14 to 24
carbon atoms. These chains can be saturated, meaning they contain single bonds between
carbon atoms, or unsaturated, containing one or more double bonds.
3-Phosphate group: a phosphate group is attached to the third carbon atom (carbon 3) of the
glycerol backbone. The phosphate group consists of a phosphorus atom bonded to four
oxygen atoms. One of these oxygen atoms is also bonded to the glycerol backbone.
4-Hydrophilic head group: the phosphate group is further linked to a hydrophilic head group,
which determines the specific type of phospholipid. The head group can vary in composition
and includes various functional groups, such as choline, ethanolamine, serine, inositol, or
others. The type of head group present gives rise to different subclasses of phospholipids,
such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS),
and phosphatidylinositol (PI), among others.
Phospholipids are amphipathic molecules, meaning they possess both hydrophilic (water-
loving) and hydrophobic (water-fearing) regions. This unique structure enables phospholipids
to form the foundation of biological membranes.

Based upon the backbone, types of phospholipids are:

-Glycerophospholipids (or phosphoglycerides), or glycerol phospholipids: the backbone is

glycerol.

-Sphingophospholipids, the backbone of sphingomyelin is the amino alcohol sphingosine,

rather than glycerol.

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Figure (6): Phospholipids structure (www.google.com)

Figure (7): Glycerophospholipids (www.google.com)

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 Figure (8): Structure of sphingomyelin (www.google.com)

Functions of phospholipids:

1-Structural role in membranes: phospholipids are the major building blocks of biological
membranes. They form a lipid bilayer structure in which the hydrophilic head groups face the
aqueous environment, while the hydrophobic fatty acid tails are shielded from water. This
arrangement provides structural integrity to cell membranes, separating the internal contents
of cells from their surroundings. Phospholipids also contribute to the fluidity and flexibility
of membranes, allowing for membrane dynamics and cell movements.
2-Selective permeability: the phospholipid bilayer acts as a selective barrier that regulates the
entry and exit of substances into and out of cells. The hydrophobic core of the bilayer
restricts the passage of hydrophilic molecules and ions, while small hydrophobic molecules
can diffuse through the lipid tails. This selective permeability allows cells to maintain internal
conditions, control the transport of essential molecules, and prevent the entry of harmful
substances.
3-Cell signalling and communication: phospholipids play a key role in intracellular signalling
and communication processes in cells. Signals can be transmitted across cell membranes
more easily thanks to their use as platforms for the affixing and activation of signalling
proteins. Phospholipids like phosphatidylinositol 4,5-bisphosphate (PIP2) are used to activate
signalling pathways including the phosphoinositide signalling cascade, which controls cell
division, growth, and intracellular calcium signalling.
4-Energy storage: phospholipids can serve as a source of energy. When needed, the fatty acid
tails of phospholipids can be enzymatically cleaved from the glycerol backbone through a
process called lipolysis. This releases fatty acids that can be further metabolized to generate
energy through β-oxidation, providing fuel for cellular processes.
5-Precursors for bioactive lipids: for the creation of bioactive lipid mediators, certain
phospholipids function as precursors. For instance, the precursor for the production of
prostaglandins, leukotrienes, and thromboxanes is arachidonic acid, which is released from
phospholipids like phosphatidylcholine or phosphatidylinositol. Inflammation,
immunological responses, and blood coagulation are just a few of the physiological processes
in which these lipid mediators play a role.

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6-Membrane protein function: the insertion and stability of integral membrane proteins take
place in a phospholipid environment. Some phospholipids, such as phosphatidylinositol
phosphates, have the ability to interact directly with particular membrane proteins,
controlling their activity, location, and function.
7-Cellular trafficking and membrane remodelling: phospholipids take involved in cellular
activities involving membrane trafficking, including vesicle production, exocytosis, and
endocytosis. In addition, they participate in membrane remodelling processes such membrane
fusion and fission, which are essential for cell division, organelle dynamics, and intracellular
transport.
8-Cellular signatures and recognition: in order to differentiate one cell type from another,
phospholipids can serve as biological signatures by supplying distinctive lipid profiles. These
lipid profiles may have an impact on processes including immunological responses, tissue
formation, and cell migration by affecting cell recognition and cell-cell interactions.

4.LIPOPROTEINS:

Figure (9): Lipoprotein structure (www.google.com)

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 Figure (10): A phospholipid particle (www.google.com)

Lipoproteins are made up of a lipid core (which can contain cholesterol esters and
triglycerides) and a hydrophilic outer membrane comprising phospholipid, apolipoprotein,
and free cholesterol. In a lipoprotein, the polar ends of all the phospholipid molecules face
outwards, so as to interact with water, itself a polar molecule. This enables the lipoprotein to
be carried in the blood rather than rising to the top, like cream on milk. The non-polar fat
balled up inside the phospholipid layer, at the center of the lipoprotein, is thus transported to
the place where it must be stored or metabolized, through the bloodstream, despite being
insoluble in blood. Thus, lipoproteins are molecular level trucks to carry fats wherever they
are required or stored.

Notably, LDL particles are thought to act as a major transporter of cholesterol; at least two-
thirds of circulating cholesterol resides in LDL to the peripheral tissues. Conversely, HDL
molecules are thought to do the opposite. They take excess cholesterol and return it to the
liver for excretion. Clinically, these two lipoproteins are significant since high LDL and low
HDL increase patients' risk of atherosclerotic vascular diseases.

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Different lipoproteins are differentiated based on specific proteins attached to the
phospholipid outer layer, called the apolipoprotein. This also helps to make the fatty
molecule more stable, and also binds to cell surface receptors in some cases, to enable the cell
to take up the lipoprotein by receptor-mediated endocytosis.

The types of lipoproteins:

1-Chylomicrons

2-Chylomicron Remnants

3-Very-low-density lipoprotein (VLDL)

4-Intermediate-density lipoprotein (IDL)

5-Low-density lipoprotein (LDL)

6-High-density lipoprotein (HDL)

7-Lipoprotein (a)

Plasma lipoproteins are divided into seven classes based on size, lipid composition, and
apolipoproteins. Plasma lipoprotein particles contain variable proportions of four major
elements: cholesterol, triglycerides, phospholipids and specific proteins called apoproteins.
An alphabetical nomenclature (A, B, C, D, E.) is generally used to designate the apoproteins.
The varying composition of these elements determines the density, size, and electrophoretic
mobility of each particle. These factors in turn have been used for the clinical and
biochemical classification of lipoprotein disorders. Schematically, lipoproteins have been
described as globular or spherical units in which a nonpolar core lipid (consisting mainly of
cholesterol esters and triglycerides) is surrounded by a layer containing phospholipids,
apoproteins, and small amounts of unesterified cholesterol. Apoproteins, in addition to
serving as carrier proteins, have other important functions such as being co-factors for
enzymes involved in lipoprotein metabolism, acting as specific ligands for binding of the
particles to cellular receptor sites, and intervening in the exchange of lipid constituents
between lipoprotein particles.

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1-Chylomicrons:
Chylomicrons are large triglyceride rich particles made by the intestine, which are
involved in the transport of dietary triglycerides and cholesterol to peripheral tissues and
liver. Chylomicrons contain apolipoproteins A-I, A-II, A-IV, A-V, B-48, C-II, C-III, and
E. Apo B-48 is the core structural protein and each chylomicron particle contains one
Apo B-48 molecule. The size of chylomicrons varies depending on the amount of fat
ingested. A high fat meal leads to the formation of large chylomicron particles due to the
increased amount of triglyceride being transported whereas in the fasting state the
chylomicron particles are small carrying decreased quantities of triglyceride.
2-Chylomicron remnants:
The removal of triglyceride from chylomicrons by peripheral tissues results in smaller
particles called chylomicron remnants. Compared to chylomicrons these particles are
enriched in cholesterol and are pro-atherogenic.
3-Very-low-density lipoprotein (VLDL):
Very-low-density lipoproteins (VLDL) particles are produced by the liver and are
triglyceride rich. Very-low-density lipoproteins (VLDL) contain apolipoprotein B-100,
C-I, C-II, C-III, and E. Apo B-100 is the core structural protein and each very-low-
density lipoprotein (VLDL) particle contains one Apo B-100 molecule. Similar to
chylomicrons the size of the very-low density-lipoprotein particles can vary depending
on the quantity of triglyceride carried in the particle. When triglyceride production in the
liver is increased, the secreted very-low-density lipoprotein particles are large. However,
very-low-density lipoprotein particles are smaller than chylomicrons.

4-Intermediate-density lipoprotein (IDL):

The removal of triglycerides from very-low-density lipoprotein (VLDL) by muscle and
adipose tissue results in the formation of intermediate-density lipoprotein (IDL) particles
which are enriched in cholesterol. These particles contain apolipoprotein B-100 and E.
These intermediate-density lipoprotein particles are pro-atherogenic.

5-Low-density lipoprotein (LDL):

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Low-density lipoprotein (LDL) particles are derived from very-low-density lipoprotein
(VLDL) and intermediate-density lipoprotein (IDL) particles and they are even further
enriched in cholesterol. Low-density lipoprotein (LDL) carries the majority of the
cholesterol that is in the circulation. The predominant apolipoprotein is B-100 and each
low-density lipoprotein (LDL) particle contains one Apo B-100 molecule. Low-density
lipoprotein (LDL) consists of a spectrum of particles varying in size and density. An
abundance of small dense low-density lipoprotein (LDL) particles are seen in association
with hypertriglyceridemia, low high-density lipoprotein (HDL) levels, obesity, type 2
diabetes (i.e. patients with the metabolic syndrome), and infectious and inflammatory
states. These small dense low-density lipoprotein (LDL) particles are considered to be
more pro-atherogenic than large low-density lipoprotein (LDL) particles for a number of
reasons. Small dense low-density lipoprotein (LDL) particles have a decreased affinity
for the low-density lipoprotein (LDL) receptor resulting in a prolonged retention time in
the circulation. Additionally, they more easily enter the arterial wall and bind more
avidly to intra-arterial proteoglycans, which traps them in the arterial wall. Finally, small
dense low-density lipoprotein (LDL) particles are more susceptible to oxidation, which
could result in an enhanced uptake by macrophages.

6-High-density lipoprotein (HDL):

High-density lipoproteins are a heterogeneous group of macromolecules with different
physical properties and chemical components; two subclasses of HDL have been
identified (HDL2 and HDL3) within which several subspecies have also been
demonstrated. The predomination function of HDL seems to be the reverse transport of
cholesterol from different tissues into the liver, where it is eventually removed, which is
one potential mechanism by which high-density lipoprotein (HDL) may be anti-
atherogenic. Subclass HDL2 has been reported to have a better correlation with coronary
artery disease protection than total high-density lipoprotein (HDL) cholesterol. In
addition, high-density lipoprotein (HDL) particles have anti-oxidant, anti-inflammatory,
anti-thrombotic, and anti-apoptotic properties, which may also contribute to their ability
to inhibit atherosclerosis. High-density lipoprotein (HDL) particles are enriched in
cholesterol and phospholipids. Apolipoproteins A-I, A-II, A-IV, C-I, C-II, C-III, and E
are associated with these particles. Apo A-I is the core structural protein and each high-
density lipoprotein (HDL) particle may contain multiple Apo A-I molecules. High-

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density lipoprotein (HDL) particles are very heterogeneous and can be classified based
on density, size, charge, or apolipoprotein composition.
The serum concentration of high-density lipoprotein (HDL) and its components derives
from various complex intravascular and cellular metabolic events. These events include
secretion of precursor high-density lipoprotein (HDL) particles from the liver and small
intestine, interaction of these particles with lipids and proteins released during the
catabolism of triglyceride-rich lipoproteins, and production of cholesteryl esters (the core
substance in HDL) from the action of lecithin–cholesterol acyltransferase (LCAT), an
enzyme that originates in the liver. This enzyme acts on unesterified cholesterol released
into plasma from cellular turnover. The cholesterol esters formed in this reaction are in
turn transferred to very-low-density lipoprotein (VLDL) and subsequently appear in low-
density lipoprotein (LDL). The end result is a system that allows the transfer of
cholesterol through low-density lipoprotein (LDL) to peripheral cells and its return to the
liver through high-density lipoprotein (HDL), and that prevents excessive accumulation
of cholesterol in the body.

7-Lipoprotein (a):
Lipoprotein (a) is a low-density lipoprotein (LDL) particle that has apolipoprotein (a)
attached to Apo B-100 via a disulfide bond. Lipoprotein (a) particle is pro-atherogenic.
The physiologic function of this lipoprotein (a) is unknown.
Lipoprotein (a) contain Apo (a) and Apo B-100 in a 1:1 molar ratio. Like Apo B-100,
apo (a) is also made by hepatocytes. The levels of lipoprotein (a) in plasma can vary
more than a 1000-fold ranging from undetectable to greater than 100mg/dl. Lipoprotein
(a) levels largely reflect lipoprotein (a) production rates, which are primarily genetically
regulated. Individuals with high molecular weight Apo (a) proteins tend to have lower
levels of lipoprotein (a) while individuals with low molecular weight Apo (a) tend to
have higher levels. It is hypothesized that the liver is less efficient in secreting high
molecular weight Apo (a). The mechanism of lipoprotein (a) clearance is uncertain but
does not appear to involve LDL receptors. Elevated plasma lipoprotein (a) levels are
associated with an increased risk of atherosclerosis. The kidney appears to play an
important role in lipoprotein (a) clearance as kidney disease is associated with delayed
clearance and elevations in lipoprotein (a) levels.

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Apolipoproteins:
Apolipoproteins have four major functions including 1) serving a structural role, 2) acting as
ligands for lipoprotein receptors, 3) guiding the formation of lipoproteins, and 4) serving as
activators or inhibitors of enzymes involved in the metabolism of lipoproteins.
Apolipoproteins thus play a crucial role in lipoprotein metabolism.

The importance of lipoproteins:

Lipoproteins show varying patterns that correlate with the risk of having a fatal
cardiovascular event. High LDL, VLDL and triglyceride levels are associated with a high risk
of atherosclerosis and heart disease. High HDL is correlated with reduced cholesterol levels,
and a lower cardiovascular risk. Thus, a high measurement of apo-A-1 correlates with a low
atherosclerosis risk. HDL levels drop with cigarette smoking, and rise with regular exercise,
alcohol use, estrogen levels, and weight loss.

- Lipoprotein Lipid Transport System:

The lipoprotein lipid transport system in plasma has been described as involving two
pathways: an exogenous route for the transport of cholesterol and triglycerides absorbed from
dietary fat in the intestine, and an endogenous system through which cholesterol and
triglycerides reach the plasma from the liver and other nonintestinal tissues.
1-Exogenous lipoprotein pathway:
The exogenous pathway starts with the intestinal absorption of triglycerides and cholesterol
from dietary sources. Its end result is the transfer of triglycerides to adipose and muscle tissue
and of cholesterol to the liver. After absorption, triglycerides and cholesterol are re-esterified
in the intestinal mucosal cells and then coupled with various apoproteins, phospholipids, and
unesterified cholesterol into lipoprotein particles called chylomicrons. The chylomicrons in
turn are secreted into intestinal lymph, enter the bloodstream through the thoracic: duct, and
bind to the wall of capillaries in adipose and skeletal muscle tissue. At these binding sites the
chylomicrons interact with the enzyme lipoprotein lipase, which causes hydrolysis of the
triglyceride core and liberation of free fatty acids. These fatty acids then pass through the
capillary endothelial cells and reach the adipocytes and skeletal muscle cells for storage or
oxidation, respectively.

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After removal of the triglyceride core, remnant chylomicron particles are formed. These are
high in cholesterol esters and characterized by the presence of apoproteins B, CIII, and E.
These remnants are cleared from the circulation by binding of their E apoprotein to a receptor
present only on the surface of hepatic cells. Subsequently, the bound remnants are taken to
the inside of hepatic cells by endocytosis and then catabolized by lysosomes. This process
liberates cholesterol, which is then either converted into bile acids, excreted in bile, or
incorporated into lipoproteins originated in the liver (VLDL).

Under normal physiologic conditions, chylomicrons are present in plasma for 1 to 5 hours
after a meal and may give it a milky appearance. They are usually cleared from the
circulation after a 12-hour fast.

Figure (11): Exogenous lipoprotein pathway (www.google.com)

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2-Endogenous lipoprotein pathway:
The liver constantly synthesizes triglycerides by utilizing as substrates free fatty acids and
carbohydrates; these endogenous triglycerides are secreted into the circulation in the core of
very-low-density lipoprotein particles (VLDL). The synthesis and secretion of very-low-
density lipoprotein particles (VLDL) at cellular level occur in a process similar to that of
chylomicrons, except that a different B apoprotein (B-100 instead of B-48) together with
apoproteins C and E intervene in their secretion. Subsequent interaction of the VLDL
particles with lipoprotein lipase in tissue capillaries leads to hydrolysis of the core
triglycerides and production of smaller remnant VLDL particles rich in cholesterol esters
(intermediate-density lipoproteins, IDL) and liberation of free fatty acids. Around half of
these remnant particles are removed from the circulation in 2 to 6 hours as they bind tightly
to hepatic cells. The rest undergo modifications with detachment of the remaining
triglycerides and its substitution by cholesterol esters and removal of all the apoproteins
except apoprotein B. This process results in transformation of the remnant VLDL particles
into low-density lipoprotein particles (LDL) rich in cholesterol. In fact, these last particles
contain around three-fourths of the total cholesterol in human plasma, although they
constitute only some 7% of the total cholesterol pool. Their predominant function is to supply
cholesterol to cells with LDL receptors, like those in the adrenal glands, skeletal muscle,
lymphocytes, gonads, and kidneys.

The quantity of cholesterol freed from LDL is said to control cholesterol metabolism in the
cell through the following mechanisms:

(1) increased LDL cholesterol in the cell decreases synthesis of the enzyme 3-hydroxy-3
methylglutaryl coenzyme A (HMG-CoA) reductase, which modulates the intracellular
synthesis of cholesterol;

(2) increased LDL cholesterol may enhance the storage of cholesterol within the cell by
activation of another enzyme; and

(3) increased cholesterol within the cell diminishes the synthesis of LDL receptors through a
negative feedback process.

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Besides the above described route for low-density lipoprotein (LDL) degradation in
extrahepatic sites, a so-called scavenger cell pathway has been described. This consists of
cells in the reticuloendothelial system which, by phagocytosis, dispose of the excess
concentrations of this lipoprotein in plasma.

Figure (12): Endogenous lipoprotein pathway (www.google.com)

Clinical significance of lipoprotein:

Cholesterol and triglycerides, like many other essential components of the body, attract
clinical attention when present in abnormal concentrations. Increased or decreased levels
usually occur because of abnormalities in the synthesis, degradation, and transport of their
associated lipoprotein particles. When hyperlipidemia or hypolipidemia are defined in terms
of the class or classes of increased or decreased plasma lipoproteins, the names
hyperlipoproteinemia or hypolipoproteinemia are preferentially employed.

1-High lipoprotein:
22
Hyperlipoproteinemia is the lipid disturbance of major relevance clinically because of its
association with an increased risk of atherosclerotic cardiovascular disease. Multiple
epidemiologic studies have demonstrated that increased levels of plasma total cholesterol and
low-density lipoproteins are strongly and directly related to a greater incidence of coronary
heart disease. Elevated plasma triglycerides and very-low-density lipoproteins are directly
associated with the risk of atherosclerotic heart disease, although not as independent risk
factors. In contrast, high levels of high-density lipoprotein cholesterol have been found to be
a protective factor for the development of that disease, so that decreased levels constitute a
risk factor.

Clinical manifestations of hyperlipoproteinemia include a greater incidence of ischemic

vascular disease, acute pancreatitis, and visible accumulations of lipid deposits (xanthomas
and xanthelasmas). The localization of these lesions is of great help in many instances to
categorize the lipoprotein dysfunction present.

Increased concentration of plasma lipids is etiologically related mainly to genetic disorders,

dietary factors (such as ingestion of excessive calories, saturated fatty acids and cholesterol),
or ingestion of drugs, or it may occur as a secondary phenomenon in a large variety of
diseases. In any of these instances the elevation of the different plasma lipoproteins usually
occurs in a number of combinations that have led to their classification into six different
patterns or phenotypes. Genetic or acquired disorders may be related to one or more of these
lipoprotein patterns, so the identification of a particular pattern gives no specific information
regarding the cause of the hyperlipidemic disorder in question. A practical approach is to
classify the different hyperlipidemic: states into those that mainly cause hypercholesterolemia
or those that predominantly cause hypertriglyceridemia. In each of these categories, primary
(genetic) as well as secondary disorders are included. The genetic disorders in turn could
result from a single gene disturbance either of dominant or recessive inheritance, or a
polygenic derangement in which multiple genes interact with environmental factors.

High alpha lipoprotein (Hyperalphalipoproteinemia):

23
Another clinical condition associated with elevation in plasma lipoproteins is
hyperalphalipoproteinemia, characterized by elevated plasma levels of high-density
lipoproteins. The elevation in high-density lipoprotein (HDL) leads to slight increase in total
plasma cholesterol values. Other plasma lipid components (LDL, VLDL, and triglycerides)
are normal.

The majority of cases of hyperalphalipoproteinemia are genetic with either a dominant or

polygenic inheritance. Secondary elevations of high-density lipoprotein (HDL) have been
related to various factors such as weight reduction, regular exercise, moderate alcohol intake,
estrogen administration, exposure to chlorinated hydrocarbon pesticides, and biliary cirrhosis.
Patients with this condition usually do not present any distinguishing clinical features.
Hyperalphalipoproteinemia is associated with a decreased risk of coronary atherosclerosis
and with increased longevity.

Low lipoprotein:
Hypoalphalipoproteinemia probably is the most clinically significant hypolipoproteinemia in
view of the fact that considerable evidence suggests that low levels of plasma HDL
cholesterol are related to an increased incidence of coronary heart disease in high-risk
populations.

According to the NIH Consensus Conference, the finding of high-density lipoprotein (HDL)
cholesterol values below 35 mg/dl constitutes an independent risk factor for coronary artery
disease. Several factors have been identified as causing a decrease in high-density lipoprotein
(HDL) cholesterol. These include ill-defined genetic factors, obesity, cigarette smoking,
physical inactivity, hypertriglyceridemia, oral contraceptives, beta-adrenergic blocking drugs,
thiazide diuretics, and cholesterol-reducing diets.

Other hypolipoproteinemias comprise two rare disorders characterized by a decrease in the

concentration of lipids in plasma and an autosomal recessive inheritance.

Abetalipoproteinernia usually appears early in childhood, and because of a defective

production of apoprotein B, there is absence of chylomicrons, VLDL, and LDL in the plasma.
The plasma cholesterol level is usually less than 75 mg/dl and that of triglycerides less than

24
15 mg/dl. The main clinical features are malabsorption of fats, peripheral neuropathy, ataxia,
retinitis pigmentosa, and acanthocytosis.

Tangier disease is a condition that also manifests in childhood and is characterized by the
absence of HDL from the plasma. This defect leads to the production of abnormal
chylomicron remnants, which are stored as cholesterol esters in cells of the phagocytic
system. Levels of plasma cholesterol are usually less than 100 mg/dl and that of triglycerides
range from 100 to 250 mg/dl. The main clinical features are enlarged orange tonsils, corneal
opacities, and infiltration of the bone marrow and the intestinal mucosa. Patients with this
illness are at increased risk for premature atherosclerosis.

Dyslipoproteinemia, is the term utilized for conditions in which structurally abnormal

lipoproteins circulate in plasma. Such a defect is seen in lecithin cholesterol acyltransferase
(LCAT) deficiency. This is a rare disorder in which decreased activity of this enzyme leads to
a large accumulation of unesterified cholesterol in plasma and body tissues. Laboratory
findings include a variable level of total plasma cholesterol with decreased esterified
cholesterol, an increase in unesterified cholesterol and increased VLDL. The structure of all
the lipoproteins is abnormal. The condition usually presents in young adulthood with corneal
opacities, renal insufficiency, hemolytic anemia, and premature atherosclerosis.

Lipoprotein levels:
Plasma cholesterol and triglyceride values provide information concerning the lipoprotein
particles that are increased. Plasma cholesterol values represent total cholesterol, which
includes both unesterified cholesterol and cholesteryl esters. Isolated elevation of plasma
cholesterol usually indicates that low-density lipoprotein (LDL) is increased. Isolated
elevation of plasma triglyceride points to an elevation in chylomicrons, very-low-density
lipoprotein (VLDL), and/or remnants.

-Lipoprotein (a) test:

Lipoprotein (a) test is used to identify an elevated level of lipoprotein (a) help evaluate a
person’s risk of developing cardiovascular disease (CVD). Lipoprotein (a) test may be used
in conjunction with a routine lipid profile to provide additional information about a person’s
risk for cardiovascular disease.

25
The lipoprotein (a) level is genetically determined and remains relatively constant over an
individual’s lifetime. Since it is usually not affected by lifestyle changes or by most drugs, it
is not the target of therapy. Instead, when lipoprotein (a) is high, the presence of this added
risk factor may suggest the need for more aggressive treatment of other, more treatable risk
factors such as an elevated low-density lipoprotein (LDL).

Like low-density lipoprotein (LDL), lipoprotein (a) is considered a risk factor for
cardiovascular disease. The amount of lipoprotein (a) that a person has is genetically
determined and remains relatively constant over an individual’s lifetime. A high level of
lipoprotein (a) is thought to contribute to a person’s overall risk of cardiovascular disease,
making this test potentially useful as a cardiovascular risk marker.
Since about 50% of the people who have heart attacks have a normal cholesterol level,
researchers have sought other factors that may have an influence on heart disease. It is
thought that lipoprotein (a) may be one such factor. Lipoprotein (a) has two potential ways to
contribute. First, since lipoprotein (a) can promote the uptake of low-density lipoprotein
(LDL) into blood vessel walls, it may promote the development of atherosclerotic plaque on
the walls of blood vessels. Secondly, since apo(a) has a structure that can inhibit enzymes
that dissolve clots, lipoprotein (a) may promote accumulation of clots in the arteries. For
these reasons, lipoprotein (a) may be more atherogenic than low-density lipoprotein (LDL).

-When Is Lipoprotein-A Test Ordered?

Lipoprotein (a) test is not routinely ordered as part of a lipid profile. However, lipoprotein (a)
test may be ordered, along with other lipid tests, when an individual has a strong family
history of cardiovascular disease at a young age that is not explained by high low-density
lipoprotein (LDL) or low high-density lipoprotein (HDL).

Some health practitioners may also order these tests when:

-A person has existing heart or vascular disease, especially those individuals who have
healthy lipid levels or ones that are only mildly elevated

-Someone may have an inherited predisposition for high cholesterol level

-A person has had a stroke or heart attack but has normal or only mildly elevated lipids

26
 In rare cases, a lipoprotein (a) level may be ordered when a woman is postmenopausal to
see if elevations in lipoprotein (a), tied to decreasing estrogen levels, have significantly
increased her risk of developing cardiovascular disease.

In general, lipids should not be measured during a fever or major infection, within four
weeks of an acute myocardial infarction (heart attack), a stroke, or major surgery, right
after excessive alcohol intake, with severely uncontrolled diabetes, when a woman is
pregnant, or during rapid weight loss.

Lipoprotein (a) Levels

Normal lipoprotein (a) less than or equal to 30 mg/dL

Lipoprotein (a) values >30 mg/dL may suggest increased risk of coronary heart disease. High
lipoprotein (a) can occur in people with a normal lipid profile. An elevated level of
lipoprotein (a) is thought to contribute to risk of heart disease independently of other lipids.

Lipoprotein (a) concentrations >30 mg/dL are associated with 2- to 3-fold increased risk of
cardiovascular events independent of conventional risk markers.

The level of lipoprotein (a) is genetically determined and is not easily modified by lifestyle
changes or drugs. However, some non-genetic conditions may also lead to elevated
lipoprotein (a). These include estrogen depletion, familial hypercholesterolemia, severe
hypothyroidism, uncontrolled diabetes, chronic renal failure, and nephrotic syndrome.

There are no known problems associated with low lipoprotein (a). Many individuals have no
detectable lipoprotein (a) in their blood.

27
 Figure (13): Definitions of abnormal lipid levels (www.google.com)

Hyperlipidemia:

The hyperlipidemia is traditionally defined as conditions in which the concentration

of cholesterol or triglyceride-carrying lipoproteins in plasma exceeds an arbitrary normal
limit. These lipoproteins deposit in the interstitial space of arteries arising from aorta,
restricting the blood supply to the heart. This phenomenon is known as atherosclerosis.
Higher deposition of lipoproteins completely blocked the blood supply to the heart, and thus
myocardial infarction (MI) occurs, which is commonly known as heart attack.

28
Classification of hyperlipidemia:

1-On the basis of lipid type:

-Hypercholesterolemia-In this the level of cholesterol is elevated.

-Hypertriglyceridemia-It is defined as an elevated level of triglycerides.

Figure (14): Classification of hyperlipidemia (www.google.com)

2-On the basis of causing factor:

1-Familial (Primary) hyperlipidemia-On the basis of causing factors hyperlipidemia can be

designated as either primary or secondary. According to Fredrickson familial hyperlipidemia
is classified into five types on the basis of electrophoresis or ultracentrifugation pattern of
lipoproteins.

 Type I–Raised cholesterol with high triglyceride levels.

 Type II–High cholesterol with normal triglyceride levels.
 Type III–Raised cholesterol and triglycerides.
 Type IV–Raised triglycerides, atheroma and uric acid.

29
  Type V–Raised triglycerides.

This classification was later adopted by WHO. This method does not directly account for
HDL and also does not distinguish among the different genes that may be partially
responsible for some of these conditions. It remains a popular system of classification but is
considered dated by many.

2-Acquired (Secondary) hyperlipidemia-Acquired hyperlipidemia (secondary

dyslipoproteinemias) results from underlying disorders and lead to alterations in plasma lipid
and lipoprotein metabolism. This type of hyperlipidemia may mimic primary forms of
hyperlipidemia and can have similar consequences. They may result in increased risk of
premature atherosclerosis, pancreatitis and other complications of the chylomicronemia
syndrome. The most common causes of acquired hyperlipidemia are given below.

 Diabetes Mellitus
 Use of drugs such as diuretics, β-blockers and estrogens.
 Alcohol consumption.
 Some rare endocrine disorders and metabolic disorders.
 Hypothyroidism
 Renal failure
 Nephrotic syndrome

-Complications of hyperlipidaemia

1-Atherosclerosis: It is a common disorder and occurs when fat, cholesterol and calcium
deposits in the arterial linings. This deposition results in the formation of fibrous plaques. A
plaque normally consists of three components:

1) atheroma which is a fatty, soft, yellowish nodular mass located in the centre of a larger
plaque that consists of macrophages, which are cells that play a role in immunity;

2) a layer of cholesterol crystals; and,

3) calcified outer layer. Atherosclerosis is the leading cause of cardiovascular disease.

30
2-Coronary Artery Disease (CAD): Atherosclerosis is the major cause of CAD. It is
characterised by the narrowing of the arteries that supply blood to the myocardium and
results in limiting blood flow and insufficient amounts of oxygen to meet the needs of the
heart. The narrowing may progress to the extent that the heart muscle would sustain damage
due to lack of blood supply. Elevated lipid profile is correlated to the development of
coronary atherosclerosis.

3-Myocardial Infarction (MI): MI is a condition which occurs when blood and oxygen
supplies to the cardiac arteries are partially or completely blocked, resulting in damage or
death of heart cells. The blockage is usually due to the formation of a clot in an artery. This
condition is commonly known as heart attack. The studies show that one-fourth of survivors
of myocardial infarction were hyperlipidemic.

4-Angina Pectoris: Angina is not a disease but a symptom of an underlying heart condition.
It is characterised by chest pain, discomfort or a squeezing pressure. Angina occurs as a result
of a reduction or a lack of blood supply to a part or the entire heart muscle. Poor blood
circulation is usually due to CHD when partial or complete obstruction of the coronary
arteries is present.

5-Ischemic stroke or Cerebrovascular Accident (CVA): It occurs when blood circulation

in part of the brain is blocked or diminished. When blood supply, which carries oxygen,
glucose, and other nutrients, is disrupted, brain cells die and become dysfunctional. Usually,
strokes occur due to blockage of an artery by a blood clot or a piece of atherosclerotic plaque
that breaks loose in a small vessel within the brain. Clinical trials revealed that lowering of
LDL and total cholesterol by 15% significantly reduced the risk of first stroke [39].

Causes of hyperlipidemia:

1-A diet rich in saturated fat and cholesterol increases blood cholesterol and triglyceride
levels.

2-Other disorders as obesity, diabetes mellitus and hypothyroidism increase the risk of
hyperlipidemia.

3-Smoking and not exercising may lead to hyperlipidemia.

31
4-Excessive use of alcohol also increases the risk of hyperlipidemia.

5-Certain drugs as steroids and β–blockers may cause hyperlipidemia.

6-Hereditary factor is also one of the common causes for hyper-lipidemia.

7-In some cases hyperlipidemia occurs during pregnancy.

8-Lipoprotein lipase mutations.

Lipids Desirable value Borderline High risk

Cholesterol Less than 200 mg/dl 200-239 mg/dl 240 mg/dl
Triglycerides Less than 140 mg/dl 150-199 mg/dl 200-499 mg/dl
HDL cholesterol 60 mg/dl 40-50 mg/dl Less than 40 mg/dl
LDL cholesterol 60-130 mg/dl 130-159 mg/dl 160-189 mg/dl
Cholesterol/HDL ratio 4.0 5.0 6.0

Table (2): Normal levels for a lipid profile (www.google.com)

Hypolipidemia:

Hypolipidemia is a decrease in plasma lipoprotein caused by primary (genetic) or secondary

factors. It is usually asymptomatic and diagnosed incidentally on routine lipid screening.

Hypolipidemia is defined as a total cholesterol (TC) < 120 mg/dL (< 3.1 mmol/L) or low-
density lipoprotein cholesterol (LDL-C) < 50 mg/dL (< 1.3 mmol/L).
Causes may be primary (genetic) or secondary. Secondary causes are far more common
than primary causes and include all of the following:

1-Chronic infections (including hepatitis C infection) and other inflammatory states

2-Hematologic and other cancers

3-Hyperthyroidism
4-Malabsorption
5-Undernutrition (including that accompanying chronic alcohol use)
The unexpected finding of low cholesterol or low LDL-C in a patient not taking a lipid-
lowering drug should prompt a diagnostic evaluation, including measurements of AST

32
(aspartate aminotransferase), ALT (alanine aminotransferase), and thyroid-stimulating
hormone; a negative evaluation suggests a possible primary cause.

There are 3 primary disorders in which single or multiple genetic mutations result in
underproduction or increased clearance of LDL:

1-Abetalipoproteinemia:
This autosomal recessive condition is caused by mutations in the gene for microsomal
triglyceride (TG) transfer protein, a protein critical to chylomicron and very-low-density
lipoprotein (VLDL) formation. Dietary fat cannot be absorbed, and lipoproteins in both
metabolic pathways are virtually absent from serum; total cholesterol is typically < 45
mg/dL (< 1.16 mmol/L), TGs are < 20 mg/dL (< 0.23 mmol/L), and LDL is undetectable.
2-Chylomicron retention disease:

Chylomicron retention disease is a very rare autosomal recessive condition caused by

deficient apo B secretion from enterocytes. Mutations in a gene encoding a protein
important in transport of chylomicrons through enterocytes have been linked to this
disorder.

3-Hypobetalipoproteinemia:

Hypobetalipoproteinemia is an autosomal dominant or codominant condition caused by

mutations in the gene coding for apo B.

Heterozygous patients have truncated apo B, leading to rapid LDL-C clearance.

Heterozygous patients manifest no symptoms or signs except for TC < 120 mg/dL (< 3.1
mmol/L) and LDL-C < 80 mg/dL (< 2.1 mmol/L). Triglycerides are normal. Some patients
may have hepatic steatosis.
Homozygous patients have either shorter truncations, leading to lower lipid levels (TC < 80
mg/dL [< 2.1 mmol/L], LDL-C < 20 mg/dL [< 0.52 mmol/L]), or absent apo B synthesis,
leading to symptoms and signs of abetalipoproteinemia.

Serum lipid profile: fasting or non-fasting?

33
Serum lipid profile is measured for cardiovascular risk prediction and has now become
almost a routine test. The test includes four basic parameters: total cholesterol, HDL
cholesterol, LDL cholesterol and triglycerides. It is usually done in fasting blood specimen.
Fasting refers to 12–14 h overnight complete dietary restriction with the exception of water
and medication.

This may hold true due to two main reasons:

(1) post prandial triglycerides remain elevated for several hours,

(2) most reference values for serum lipids are established on fasting blood specimen. NCEP
and European guidelines also recommend doing lipid profile in fasting blood specimen for
assessment of cardiovascular risk. However, these guidelines allow total and HDL cholesterol
in the non-fasting specimen as these lipids are not much different in fasting and non-fasting
specimens. In addition, non-HDL cholesterol (total cholesterol − HDL cholesterol), a
secondary target of therapy in adult treatment panel III, may also be used in the non-fasting
state.

Basically, fasting state is essential for triglycerides estimation because as mentioned above it
remains high for several hours after meal and the Friedewald equation, used for calculation of
LDL cholesterol (LDL cholesterol = total cholesterol − HDL cholesterol − [triglycerides/5]),
uses fasting triglycerides value. If non-fasting triglycerides value is used in this equation the
LDL cholesterol, the primary target of lipid lowering therapy, will be underestimated.

In addition to fasting/non-fasting state there are other factors (pre-analytical) which may
affect lipid components:

1. A change from an upright to a supine position due to dilutional effect can reduce the
cholesterol levels by 10% and triglycerides by 12%.
2. Prolonged tourniquet application (2–5 min) can increase cholesterol from 5 to 15%.
3. Cholesterol is slightly higher in winter than in summer and the opposite is true for
triglycerides.
4. The disease conditions like nephrotic syndrome increase total cholesterol, LDL
cholesterol and VLDL cholesterol and hypothyroidism increases LDL cholesterol and

34
 total cholesterol. Infection and inflammation may decrease total cholesterol and HDL
cholesterol and increase triglycerides. Lipids alter following myocardial infarction
and these changes may persist for several weeks. That is why it is better to do lipid
profile in such patients within 24 h of myocardial infarction. The study showed that
all individual values of the lipid profile in patients admitted with acute illness vary
significantly during and after hospital stay, whereas the ratio of total cholesterol to
HDL remains relatively stable.

It is therefore, important that all these factors should be kept in mind while interpreting the
lipid profile.

Cholesterol test results

Ideal resultsTrusted Source for most adults are as follows:

 LDL: less than 100 mg/dL

 HDL: 40 to 60 mg/dL (a higher number is better)
 Total cholesterol: less than 200 mg/dL
 Triglycerides: less than 150 mg/dL
 VLDL levels: under 30 mg/dL

Figure (15): Ideal levels for lipid profile (www.google.com)

Lipid Profile Tests:

Lipid profile is usually done in fasting blood specimen. Fasting refers to 12-14 hrs overnight
complete dietary restriction with the exception of water and medication. This may hold true
due to two main reasons:

1-postprandial triglycerides remain elevated for several hours.

2-most reference values for serum lipids are established on fasting blood specimen.

35
Basically, fasting state is essential for triglycerides estimation because as mentioned above it
remains high for several hours after meal, and the equation used for calculation of low-
density lipoprotein cholesterol (LDL)

LDL=total cholesterol-(HDL cholesterol+VLDL)

This equation uses fasting triglycerides value.

Low-density lipoprotein is bad cholesterol.

So, if non-fasting triglycerides value is used in this equation, the LDL cholesterol, the
primary target of lipid lowering therapy, will be underestimated.

To calculate very low-density lipoprotein is

VLDL=triglycerides/5

To calculate LDL

LDL=Total cholesterol-(HDL cholesterol+VLDL)

First:

Total Cholesterol (TC) Test:

Total cholesterol is directly linked to risk of heart and blood vessel disease.

Enzymatic Colorimetric Method:

This method is for the measurement of total cholesterol in serum. It involves the use of three
enzymes: cholesterol esterase (CE), cholesterol oxidase (CO), and peroxidase (POD).

CE
Cholesterol esters→ Cholesterol + fatty acids
CO
4-AA +phenol→ Quinoneimine+ 4H2O
POD

4-AA is 4-aminoantipyrine
The intensity of the color formed is directly proportional to the concentration of total
cholesterol in the sample.

36
-Reagent Composition:

-R1 (Monoreagent): sodium cholate, cholesterol esterase, cholesterol oxidase, peroxidase, 4-

aminoantipyrine, phenol.

-Standard: cholesterol standard 200mg/dl

Sample:

Serum, EDTA or heparinized plasma free of hemolysis.

Wavelength:

500nm

Procedure:
Tubes Blank Sample Standard
-R1 1ml 1ml 1ml
-Sample - 10µL -
-Standard - - 10µL
Mix and incubate the tube 10 minutes at room temperature or 5 minutes at 37Cº

Read the absorbance of the samples and the standard at 500nm against the reagent blank.

Calculation:
Concentration (sample) = Absorbance(sample)-Absorbance(blank) × Concentration(standard)
Absorbance (standard)-Absorbance(blank)

=( )mg/dL total cholesterol

This test may be measured any time of the day without fasting. However, if the test is drawn

as part of total lipid profile, it requires a 12hrs fast for most accurate results.

Too much cholesterol leads to coronary artery disease.

Reference Values:
<200mg/dL: desirable (<5.18mmol/L)
(200-239)mg/dL: borderline high (5.18-6.2)mmol/L

37
>240mg/dL: high (>6.2mmol/L)
Second
Triglycerides Test:
-Enzymatic colorimetric method.

Principle of the test:

LPL
Triglycerides + H2O→ Glycerol + free fatty acids
GK
Glycerol + ATP → G3P + ADP
GPO
G3P + O2 → DAP+H2O2
POD
H2O2 + 4-AP + p-chlorophenol →Quinone + H2O

The intensity of the color formed is proportional to the triglycerides concentration in the
sample.
GK: Glycerol kinase
LPL: Lipoprotein lipase
ATP: Adenosine triphosphate
G3P: Glycerol-3-phosphate
ADP: Adenosine diphosphate
GPO: Glycerol-3-oxidase
DAP: Dihydroxyacetone phosphate
POD: Peroxidase
Reagents:
-R1: buffer, GOOD, p-chlorophenol
-R2: Enzymes: lipoprotein lipase (LPL), glycerol kinase (GK), glycerol-3-oxidase (GPO),
peroxidase (POD)
-Standard: triglycerides 200mg/dL
Sample:
Serum or heparinized or EDTA plasma

38
Procedure:
-Wavelength: 505nm (490-550)
-Adjust the instrument to zero with distilled water.
Tubes Blank Standard Sample
-Working reagent 1ml 1ml 1ml
-Standard - 10µL -
-Sample - - 10µL
Mix and incubate for 5minutes at 37 Cº or 10minutes at room temperature. Read the
absorbance of the sample and standard against the blank.
Calculation:
Concentration(sample)= Ab (sample)-Ab (blank)/Ab (standard)-Abs(blank) ×Conc (standard)
Concentration(sample)= ( )mg/dL concentrations of triglycerides in sample
Conversion factor:
( )mg/dL ×0.0113=( )mmol/L
Reference values:
Men: (40-160)mg/dL
Women: (35-135)mg/dL
High-Density Lipoprotein (HDL) Good Cholesterol:
HDL Test:

This test may be measured any time of the day without fasting. However, if the test is drawn
as part of a total lipid profile, it requires 12 hours fast for most accurate results.

Principle:

The technique uses a separation method based on the selective precipitation of apolipoprotein
B containing lipoproteins [VLDL, LDL, and LP(a)] by phosphotungstic acid MgCl2,
sedimentation of the precipitant by centrifugation, and subsequent enzymatic analysis of high
density lipoproteins (HDL) as residual cholesterol remaining in the clear supernatant.

39
The Reagents:
-R1: Precipitating reagent: phosphotungstic acid, magnesium chloride.
-CAL: Cholesterol standard: cholesterol 50mg/dL
-R2: Cholesterol monoreagent

Sample:
Serum or plasma, free of hemolysis, obtained from the patient after an overnight fast.
Procedure:
-Precipitation:
1-Bring reagents and samples to room temperature.
2-Pipette into labelled centrifuge tubes:
Sample or standard (0.2ml)
Precipitating reagent (0.4ml)
Ratio:
Sample=1
Reagent=2
Dilution Factor=3
Mix well; allow to stand for 10minutes at room temperature. Centrifuge at 4000rpm for 20
minutes or 2minutes at 12000rpm. Collect the supernatant.
Test:
Colorimetry.
Bring the cholesterol (MR) monoreagent and the cholesterol standard (50mg/dL) of the kit to
room temperature.
Tubes Blank Sample (supernatant) Standard (supernatant)
Monoreagent 1ml 1ml 1ml
Sample (supernatant) - 50µL -
Standard (supernatant) - - 50µL
Mix and let the tubes stand for 10minutes at room temperature or 5minutes at 37Cº.
Read the absorbance of the supernatant and the standard at 500nm against the reagent blank.
Calculations:
Conc(sample) =Ab(sample)-Ab(blank)/Ab(standard)-Ab(blank) × Conc(standard)
Conc(sample)= ( )mg/dL concentration HDL-cholesterol

40
If results are to be expressed as SI unit apply:
( )mg/dL × 0.0259 =( ) mmol/L

Reference Values:
-Men
>55mg/dL: low risk
(35-55) mg/dL: moderate risk
<40mg/dL: high risk
-Women:
>65mg/dL: low risk
(45-65)mg/dL: moderate risk
<45mg/dL: high risk
Clinical significance:
Low HDL-cholesterol is a strong predictor of coronary heart disease.
SVLDL=Triglycerides/5
Normal values: <40mg/dL
SLDL=Total cholesterol-(HDL+VLDL)
Values:
Suspected if above 150mg/dL
Increased if above 190mg/dL
Blood should be collected after 12 hours fast

41