LIPIDS AND LIPOPROTEINS

Definition:

Lipids are heterogeneous group of compounds which are relatively insoluble in water but

soluble in nonpolar solvents such as ether, chloroform and benzene.

Lipids include fats oils, waxes, steroids and related compounds.

Biomedical importance/Functions:

Fats (Triglycerides) are the concentrated source of energy

They act as electrical insulators

They act as thermal insulators

Phospholipids and cholesterol are important membrane constituents

They protect internal organs from external shocks by acting as shock absorbing cushions

Lipoproteins form the important transport vehicles of lipids in blood.

Classification

Lipids are classified as

1. Simple lipids: Simple lipids are esters of fatty acids with various alcohols

a. Fats: Esters of Fatty acids with glycerol

b. Waxes: Esters of fatty acids with higher molecular weight monohydric

alcohols

2. Compound lipids: Esters of fatty acids containing group in addition to alcohol and

fatty acids

a. Phospholipids: Lipids containing in addition to fatty acids and an alcohol, a

phosphoric acid residue. They frequently have a nitrogen containing base

and other sustituens.

i. Glycerophospholipids: Glycerol is the alcohol group

ii. Sphingophospholipids: sphingosine is the alcohol

b. Glycolipids: Lipids containing a carbohydrate group in addition to fatty

acids and alcohol sphingosine.

c. Other compound lipids include sulfolipids and lipoproteins.

3. Derived Lipids: These are derived from simple and compound lipids by

hydrolysis. E.g. Cholesterol, fatty acids and glycerol

4. Miscellaneous lipids: They include lipid precursors and compounds having the

characteristic of lipids,. E.g. Fat soluble vitamins, carrotenoids

Neutral lipids: acyl glycerols, cholesterol and choleteryl ester are not charged, they are

also known as Neutral lipids

FATTY ACIDS

• Key building blocks for lipids

• Chains of carbon atoms with a carboxyl group at one end, and a methyl group at

the other

• May be “free” or attached to another compound

• Determine the characteristics of the fat

CLASSIFICATION OF FATTY ACIDS

Classification based on CHAIN LENGTH

• Short chain = less than 6 carbons

• Medium chain = 6-10 carbons

• Long chain = 12 or more carbons

• The shorter the carbon chain, the more liquid the fatty acid is

Based on degree of unsaturation

• SATURATED FATTY ACID - If all the carbon atoms in the chain are joined with

single bonds.

• UNSATURATED FATTY ACID = If adjoining carbons are joined by double

bonds

• Mono unsaturated : contain only one double bond

• Poly unsaturated: contain more than one double bond

Essential Fatty Acids (EFA)

The fatty acids that cannot be synthesized by the body and therefore, should be

supplied through diet are known as Essential Fatty Acids (EFA).

There are 3 EFA namely Linoleic, Linolenic and Arachidonic acid.

Functions of EFA

EFA are required for the membrane structure and function.

They are involved in the transport of cholesterol, formation of lipoproteins and also in the

prevention of fatty liver.

They are also needed for the synthesis of prostaglandins.

The deficiency of EFA leads to ‘Phrynoderma’ characterized by the presence of

dermatitis, loss of hair and poor wound healing.

TRIGLYCERIDES

Triglycerides are esters of fatty acids with Glycerol

Fats and oils are chemically triglycerides.

The difference between fats and oil is in consistency.

Oils are liquids where fats are solids.

STRUCTURE

• Triglyceride - three fatty acids attached to a glycerol backbone

• Diglyceride – two fatty acids +glycerol

• Monoglyceride – one fatty acid +glycerol

FUNCTIONS

• Major lipid in the body and diet

• Stored fat provides about 60% of the body’s resting energy needs.

• Insulation and protection

• Carrier of fat-soluble compounds

• Sensory qualities – flavor and texture

PHOSPHOLIPIDS

• Phospholipids belong to the class of compound lipids.

• They contain a phosphoric acid residue in addition to fatty acids , nitrogenous base

and alcohol.

• phospholipids are amphipathic in nature, each has a hydrophilic head and a long,

and a hydrophobic tail (containing fatty acids or fatty acid)

There are two classes of phospholipids:

A. Glycerophospholipids

Phospholipids that contain glycerol as the alcohol are called glycerophospholipids.

(or phosphoglycerides).

1. Phosphatidic acid :

This is the simplest phosphoglyceride.

It is the precursor of the other members of this group.

2. Lecithine (Phosphatidyl choline):

These are the most abundant group of phospholipids in cell membrane.

It contain choline as the base.

3. Cephaline (phosphatidyl ethanolamine):

Ethanolamine is the nitrogenous base present in cephaline.

4. Phosphatidyl inositiol.

Inositol is attached to phosphatidic acid.

Action of certain hormones are mediated through phosphatidyl inositol

5. Cardiolipin:

Two molecules of PA esterified through their phosphate groups to an

additional molecule of glycerol is called cardio lipin (diphosphatidylglycerol).

6. Plasmalogens:

When the fatty acid at carbon 1 of a glycero -phospholipid is replaced by an

unsaturated alkyl group attached by an ether linkage

B. Sphingo phospholipids (phosphor shingosides).

Phospholipids that contain sphingosine as the alcohol.

Sphingomyelin is an important constituent of the myelin of nerve fibers.

Functions of phospholipids.

Phospholipids forms the structural components of cell membrane.

Phospholipids are the predominant lipids of cell membranes.

Phospholipids Lecithin and Cephalin are responsible for maintaining the electron

transport chain in mitochondria.

They are essential for the synthesis of lipoproteins.

Phospholipids prevent fatty liver. So they are known as lipotropic factors.

Phosphatidyl inositol is involved in the signal transmission across cell membrane

GLYCOLIPIDS

Glycolipids are important constituents of cell membrane and nervous tissue.

They contain sphingosine as the alcohol.

They contain a carbohydrate residue.

Two types:

1. Cerebrosides -

Simplest form of Glycolipids

They contain Sphingosine , Fatty acid and one or more sugar residue.

2. Gangliosides. –

Complex form glycolipids.

They contain Sphingosine, Fatty acids and one or molre molecules of N – acetyl

neuraminic acid(NANA) a sialic acid.

STEROIDS

Steroids are compounds containing a cyclic steroid nucleus (or ring) namely

cyclopentanoperhydrophenanthrene.

Cholesterol is the best-known sterol, found only in animal products .

Other steroids include

Bile acids

Steroid hormones

Ergosterol. Etc.

CHOLESTEROL: Functions

• Major component of cell membranes (especially abundant in nerve and brain

tissue)

• Precursor molecule: Example - Vitamin D and estrogen are synthesized from

cholesterol

• Important in the synthesis of bile acids

LIPOPROTEINS

Lipoproteins are lipid - protein complexes found in blood.

They are the principal means for the transport of water insoluble (non polar) lipids in the polar medium of Blood.

They are classified according to their densities and chemical qualities

The four Major Classes of Lipoproteins are

Chylomicrons

HDL

VLDL

IDL

LDL

Structure of Lipoproteins

Lipoproteins are composed of lipids and proteins.

The protein part of a lipoprotein is known as apolipoprotein.

Amphipathic lipids like phospholipids and cholesterol along with apolipoproteins form the surface monolayer of lipoproteins.

The central or core region of lipoprotein consists of hydrophobic (non polar) lipids like Triglycerides and cholesteryl ester.

Apolipoproteins

Apolipoproteins are found on the surface monolayer of lipoprotein molecules

They are of two types

- Peripheral apolipoproteins – located on the periphery of monolayer and loosely attached

- Integral apolipoproteins – buried in to the depth of surface monolayer and they are tightly bound to the lipoprotein molecules

Apolipoproteins are classified by alphabetical designation (A thru E)

The use of Roman numeral suffix describes the order in which the apolipoprotein emerge from a chromatographic column

Apolipoproteins are responsible for recognition of lipoprotein particle by cell surface receptors.

Major Apolipoprotein classes

Apolipoprotein

Origin Mol. Wt.

kD

Plasma conc. mg/dl

Major lipoprotein location

Function

Apo AI Liver, Small Intestine

28000

100- 200

HDL Structural; activator of lecithin:cholesterol acyltransferase (LCAT)

Apo A II liver 17400

20-50 HDL Structural; inhibitor of hepatic lipase; component of ligand for HDL binding

Apo A-IV small 4400 10-20 Chylomicrons, Activator of LCAT;

intestine 0 VLDL, HDL modulator of lipoprotein lipase (LPL)

Apo B100 Liver 5.4 * 10 ^5

70- 125 LDL, VLDL Structural; ligand for LDL-receptor

Apo B48 Small Intestine

2.6 * 10 ^ 5

< 5 Chylomicrones Structural, Ligand for receptor

ApoC I Liver 6630 5-8 Chylomicrones, VLDL,HDL

Activator of LCAT, inhibitor of hepatic TGRL uptake

Apo C II Liver 8900 3-7 Chylomicrones, VLDL,HDL

Activator of LPL, inhibitor of hepatic TGRL uptake

Apo C III Liver 9400 10-12 Chylomicrones, VLDL,HDL

Inhibitor of LPL, inhibitor of hepatic TGRL uptake

Apo E Liver Macrophage Brain

34400

3-15 VLDL, HDL Ligand for apoE receptor; mobilization of cellular cholesterol

Apo (a) (3-7)*

10^5

<30 Lp (a) Structural, Plasminogen inhibitor

In addition to being a constituent of various lipoproteins, e.g. VLDL & HDL, a variant of apolipoprotein E, designated apoE4, is implicated in Alzheimer's disease and other neurological conditions

Lipoprotein Classes

Lipoproteins are classified based on their density and Electrophoretic mobility

Based on density and size they are classified as

• Chylomicrons

• very low density lipoproteins (VLDL)

• intermediate density lipoproteins (IDL)

• low density lipoproteins (LDL)

• high density lipoproteins (HDL)

Chylomicrons (derived from diet)

Chylomicrones are the largest and least dense of the lipoprotein particles (density <<1.006).

- Account for the turbidity of post prandial plasma.

- Readily float to the top of stored plasma

- Produced by the intestine.

- diameter 80 - 1200 nm

- Mainly transports dietary triglycerides

- Major apolipoproteins present : apoB-48, apoA-I, apoA-II, apoA-IV, apoC-II/C-III, apoE

- remains at origin in electrophoretic field

VLDL

– density >1.006

– diameter 30 - 80nm

– endogenous triglycerides

– Account for the turbidity of fasting hyperlipidemic serum.

– apoB-100, apoE, apoC-II/C-III

– prebeta in electrophoresis

– formed in the liver as nascent VLDL (contains only triglycerides, apoE and apoB)

IDL (intermediate density lipoproteins)

– density: 1.006 - 1.019

– diameter: 25 - 35nm

– cholesteryl esters and triglycerides

– apoB-100, apoE, apoC-II/C-III

– slow pre-beta

LDL (low density lipoproteins)

– density: 1.019 - 1.063

– diameter: 18-25nm

– cholesteryl esters

– apoB-100

– beta (electrophoresis)

HDL (high density lipoproteins)

– density: 1.063-1.210

– diameter: 5-12nm

– cholesteryl esters and phospholipids

– apoA-I, apoA-II, apoC-II/C-III and apoE

– alpha (electrophoresis)

Lipoprotein class

Source Density (g/mL)

Diameter (nm)

Protein % of dry wt

Phospholipid %

Triacylglycerol % of dry wt

HDL Liver and Intestine, VLDL, Chylomicrones

1.063-1.21

5 – 15 33 29 8

LDL VLDL 1.019 – 1.063

18 – 28

25 21 4

IDL VLDL 1.006-1.019

25 - 50 18 22 31

VLDL Liver 0.95 – 1.006

30 - 80 10 18 50

chylomicrons

Intestine < 0.95 100 - 1200

1 - 2 7 84

Chylomicron Metabolism

Chylomicrons are found in the chyle formed by the lymphatic system draining the

intestine.

They are responsible for the transport of exogenous triglycerides.

They transport triglycerides from the intestine to liver.

Apolipoprotein B48 is only synthesized from intestinal cells and which combine with

absorbed dietary triglycerides (exogenous) along with other lipids (mainly cholesterol

and phosphor lipids) from the intestine and released into circulation as nascent

chylomicrons.

In circulation they receive Apo C II and Apo E from HDL to become mature

chylomicrons.

Apolipoprotein CII is an activator of the enzyme Lipoprotein lipase which is located on

the walls of the capillaries.

They are found in tissues like heart, adipose tissue, spleen, lung, renal medulla, lactating

mammary gland etc.

Lipoprotein lipase once activated by apo CII act on chylomicrons to hydrolyze the

triglyceride content of the lipoproteins.

Triglycerol is hydrolyzed progressively through decay glycerol and monoacyl glycerol

and finally into glycerol and free fatty acids.

The released fatty acids and glycerol are taken up by the tissues.

Chylomicrones becomes smaller in size with reduced lipid content and the apo C return

to HDL forming chylomicron remnant.

Chylomicron remnants are taken up by liver through apoE receptor mediated endocytosis.

The cholesteryl ester and triglycerols are metabolized and hydrolyzed.

VLDL, IDL and LDL metabolism

VLDL mainly transports the endogenous triglycerides (Synthesized by the body).

They are the vehicles for the transport of triglycerides from liver to extra hepatic tissues.

Liver synthesize apolipoprotein B100 which incorporates triglycerides, phospholipids

and cholesterol and released in circulation as nascent VLDL.

In circulation they acquire apo C and apo E from HDL to form mature VLDL.

Activation of lipoprotein lipase by apoCII results in the hydrolysis of the triglyceride

content to glycerol and free fatty acid which is taken up by the tissues.

Upon action by Lipoprotein lipase VLDL loses is Triglyceride content and becomes

smaller in size to form IDL with the transfer of Apo C back to HDL.

A portion of IDL is taken by the Liver via apoB100, E receptor.

Further loss of triglycerides and ApoE from IDL leads to the formation of cholesterol rich

LDL.

LDL has only a single apolipoprotein i.e. apoB100 which is the ligand for apo b100 , E

receptors on liver and extra hepatic tissues like arterial smooth muscles, lymphocytes and

fibroblasts.

Metabolism of HDL

Major function of HDL is the transport of cholesterol from extra hepatic tissues to Liver

(Reverse cholesterol pathway).

HDL also acts as a repository for the apo C and apo E that are required in the metabolism

of chylomicrons and VLDL. HDL is synthesized from both Liver and Intestine.

Nascent HDL from Intestine does not contain ApoC and Apo E but only Apo A.

In addition to ApoA Liver HDL contain Apo E and ApoC which is transferred to HDL

from Intestine.

Nascent HDL is discoid in shape and consists of phospholipids and free cholesterol along

with apolipoproteins.

The enzyme LCAT (Lecithin Cholesterol Acyl Transferase) binds to the disk and

catalyses the transfer of Acyl groups from surface phospholipids to the cholesterol

forming cholesterol ester.

The nonpolar cholesteryl ester moves to the hydrophobic interior (core ) region of

lipoprotein.

As the reaction continues more and more cholesterol moves to the interior it acquires a

spherical shape.

The surface layer also acquires cholesterol from the tissues to which it comes in contact.

Now the HDL is known as HDL3. Further uptake of cholesterol by HDL 3 and

esterification transforms HDL3 to a less dense HDL2.

Action of hepatic lipase hydrolyzes phospholipids and triglycerides of HDL2 and

releasing its cholesteryl ester to the liver reform HDL3. This is known as HDL cycle.

Dyslipoproteinaemias (Dyslipidemia)

Dyslipidemia or dyslipoproteinaemia are charecterised by either an increase or decrease in the blood levels of various lipids.

Classification

Based on the cause of the disease it is classified as.

Primary

• Inherited disorders of lipoproteins

Secondary

• Associated with other disorders

• Diabetes mellitus, nephrotic syndrome, hypothyroidism etc

Dyslipoproteinaemias can be.

• Hypolipoproteinaemias

• Hyperlipoproteinaemias

Hypolipoproteinemias

Low lipoprotein levels in the plasma are seen

1. Abetalipoproteinemia

Beta lipoprotein (LDL) is absent

Fat soluble vitamins are not absorbed

Mental and physical retardation

Blindness- due to degenerative changes in retina

2. Familial α – lipoprotein deficiency

Accelarated catabolism of Apo A-I

Apo A-I absent

Plasma HDL low

Increased risk for cardiovascular disease

3. Tangier’s disease

Absence of ATP binding cassette transporter-1

Plasma HDL low

Cholesterol ester accumulates in tissues

Large orange yellow tonsils, muscle atrophy, recurrent peripheral neuropathies, atherosclerosis

Hyperlipoproteinaemias

In the hyperlipidemias, the blood levels of cholesterol or triacylglycerols, or both, are elevated resulting from overproduction of lipoproteins or defects in various stages of their degradation.

• Hyperlipidemia related to

– Increased lipoprotein production

– Abnormal intravascular processing

– Defective cellular uptake of lipoprotein

General characteristics

Elevation of plasma lipids

Deposition of cholesterol on arterial walls- atherosclerosis

Ischemic heart disease and cerebrovascular accidents

Deposition of lipids in subcutaneous tissues-Xanthomas

Lipid deposits under periorbital areas- Xanthalesma

Deposition of lipids around cornea- Corneal arcus.

Hyperlipoproteinaemias classification

Fredrickson classified hyper lipoproteinaemias based on electrophoretic pattern of plasma lipoproteins

Type I (Familial LPL deficiency)

It is rare .

Due to the deficiency of lipoprotein lipase.

A chylomicron band in fasting is the characteristic finding.

Increased triglyceride levels in plasma.

Type II A (Primary familial Hypercholesterolemia)

The cause is LDL receptor defect

There is elevation of LDL.

Patients seldom survive the second decade of life.

Serum contain increased cholesterol level.

Type II B hyperlipoproteinemia

There is elevation of both cholesterol and triglycerides with excessive production of ApoB.

Both LDL and VLDL are elevated.

The abnormalities are manifested only by the third decade of life.

Type III

It is very rare.

It is due to increased levels of LDL and IDL.

Broad beta band is observed on electrophoresis.

Palmar Xanthomas and vascular disease are noticed.

Typy IV (Familial hypertriglycerolaenaemia)

This is due to over production of triglycerides by Liver.

The VLD level in plasma is elevated.

It may be associated with diabetes mellitus, obesity and impared glucose tolerance.

Type V

Chylomicrones and VLDL are increased.

Hypertriglyceridemia usually secondary to other disorders like alcohol intake, renal failure, pancreatitis etc.

Fredrickson’s Classification

Type LP elevated Metabolic defect TAG Cholesterol

Type I

Familial LPL def

CM LPL deficiency N

Type II A

Familial hypercholesterolaemia

LDL LDL receptor defect N

Type II B LDL,VLDL Excess of apo B

Type III VLDL &CM Abnormal apo E

Type IV

Familial hypertriglycerolaenaemia

VLDL Over production of VLDL. Associated with glucose intolerance and hyperinsulinaemia

¯LDL, ¯ HDL

Type V CM & VLDL Secondary to other disease like alcoholism, renal failure, pancreatitis

N

Plasma Lipid Profile

1. Total Cholesterol

2. HDL Cholesterol

3. LDL Cholesterol

4. Triglycerides

5. Phospholipids

6. LDL:HDL ratio

7. ApoB: Apo A ratio

Total & LDL Cholesterol and the risk of coronary heart disease. ( American Heart Foundation)

Total Cholesterol LDL cholesterol

Desirable < 200 <130

Borderline 200 -239 130 - 160

High >240 >160

LDL:HDL ratio >2.5 High risk

Total Cholesterol/HDL >3.5 High risk

Apo B: Apo A ratio >3 high risk

Lp(a) levels >30mgms increase the risk 3 times

Management of lipopoprotein disorders

1. Control of the cause

2. Dietary Restriction

• About 20% of total calories from fat

– 1/3 from UFA

– 1/3 from MUFA and

– 1/3 from PUFA

• Lower levels of animal fat

• Increase consumption of vegetable oils and PUFA

• Diet with high fiber content

• Exercise

• Weight reduction

• Avoid smoking

• Abstain from alcohol

• Drug therapy- Cholesterol lowering drugs

ATHEROSCLEROSIS

Arterioscelrosis

This is the general term given to various conditions leading to the hardening of arteries

due to thickening and loss of elasticity of arterial walls.

Atherosclerosis

This is the most common form of arteriosclerosis and occurs over a period of years.

• Atherosclerosis derived from the Greek words

• athere – meaning “gruel” or paste)

• skleros, meaning “hardness”

• Atherosclerosis refers to the process in which deposits of fatty substances, cellular

waste, calcium, cholesterol and other substances (atheroma) build up and harden

in the endothelium (inner lining) of an artery.

• Leading cause of morbidity and mortality

• Atherosclerosis is a slow, complex disease that starts early in childhood and

progresses throughout life.

Structure of normal Arterial wall

Three Layers

1) Intima

single layer of endothelial cells

acts as a metabolically active layer between blood and the vessel wall

2) Media

thickest layer

contractile and elastic function of the vessel

3) Adventitia

contains the nerves, lymphatics and blood vessels (vasa vasorum) that

nourish the cells of the arterial wall

Stages in atherogenesis.

Stage I: Formation of Foam Cells

Increased level of cholesterol for prolonged periods will favor deposits in the subintimal

regions of arteries.

LDL Cholesterol, especially oxidized LDL particles are deposited in the walls of the

arteries.

The macrophages takeup the cholesterol and overloaded with cholesterol to form foam

cells.

Stage II Progression of atherosclerosis

Smooth muscle cells containing lipid droplets are seen.

During early stages of athero sclerosis, the condition is reversible if plasma lipid levels

are lowered

But as the lipid accumulates, the lesion progresses unchecked and the arterial changes

become irreversible.

Stage III Fibrous Proloferation

Liberation of various growth factors by macrophages and platelets leads to accumulation

of lipoproteins, glycosaminoglycans and collagen.

There is a definte component of inflammation in atherogenesis.

This chronic inflammation leads to increased plasma high sensitive CRP.

Stage IV Advancing fibrous plaque

This leads to narrowing of vessel wall when proliferative changes occur.

The blood flow through the narrow lumen become more turbulent and there is a tendency

for clot formation.

Atherosclerotic plaque

CLINICAL MANIFESTATIONS OF ATHEROSCLEROTIC DISEASE

Atherosclerotic disease has a variety of clinical manifestations, including

carotid artery disease, coronary artery disease, renovascular disease, and

peripheral arterial disease (PAD).

Symptoms of carotid artery disease include transient ischemic attack and

stroke.

Symptoms of coronary artery disease include stable and unstable angina

pectoris.

Symptoms of PAD include intermittent claudication (IC), critical limb

ischemia (CLI), rest pain, and gangrene.

Risk Factors

Advanced age

Male sex

Having diabetes

Dyslipidemia (elevated serum cholesterol or triglyceride levels)

Tobacco smoking

Having high blood pressure

Being obese

A sedentary life-style

Having close relatives who have had some complication of

atherosclerosis

Elevated serum levels of homocysteine

Stress or symptoms of clinical depression

Chronic sub-clinical scurvy (vitamin C deficiency)

Treatment

Atherosclerosis is best treated by prevention through simple “lifestyle changes”.

However, there are medications and surgeries that can also slow down or reverse the

effects of atherosclerosis.

• Cholesterol-lowering drugs that lower the amount of low-density lipoprotein

(LDL), such as statins and fibrates, which can slow, stop, or even reverse the build

up of plaques.

• Anti-platelet medications, such as aspirin, which reduce the probability of

platelets clotting in an artery causing further blockage.

• Anticoagulants such as heparin may be prescribed to thin the blood, which may

help prevent blood clots.

• Blood vessel dilators, such as prostaglandins, may be prescribed to prevent the

muscles within an artery from tightening and narrowing the lumen.

• If a patient presents with severe symptoms of a blockage that may threaten the

survival of the muscle or tissue, then surgery might be an option.

• First is an angioplasty, which is when a doctor inserts a catheter into the blocked

or narrowed artery and a wire with a deflated balloon on it is passed though the

catheter to the part of the vessel that is narrowed or blocked. Then the balloon is

inflated and the plaque gets squished against the vessel’s walls. The doctor may

then choose to leave a stent (mesh tube) in the artery to keep it open.

• Endarterectomy is another surgery in which the doctor will make an incision into

the diseased artery and remove plaques off the walls, then close the artery.

• Vascular surgery is when the doctor creates a bypass around the blockage using a

synthetic vessel or a vessel from another part of the body.

• The primary way to treat atherosclerosis is by reducing the risk factors though

simple lifestyle changes.

• Doctors often recommend proper daily exercise to use oxygen efficiently, and

improve circulation.

• Smoking contributes to damaged arteries, so quitting smoking will reduce the

progression of plaque build up.

• Keeping cholesterol, blood pressure, diabetes, and stress under control by eating a

healthy will greatly reduce the likeliness of atherosclerosis.

References

Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles,

Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

Lawrence A. Kaplan and Amadeo J. Pesce, St. Louis, Clinical chemistry: theory, analysis,

correlation, 3rd Edition. MO. Mosby, 1996.

Burtis CA, Ashwood ER and Bruns DE, Tietz Textbook of Clinical Chemistry and Molecular

Diagnostics. 4th ed. St. Louis, Missouri: Elsevier Saunders; 2006