There are six major classes of lipoproteins in blood that are classified based on their density. Different analytical techniques separate lipoproteins based on properties like density, size, and electric charge. The major lipoproteins are chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Apolipoproteins associated with each lipoprotein particle help maintain their structure and facilitate their metabolism and clearance from circulation.

2. Basic of LP classification
Different analytical separation procedures separate lipoproteins particles
according to different physical and chemical properties:
 Ultracentrifugation - density of particle;
 Gel electrophoresis - mobility in an electric field depending on electric charge;
 Gel filtration - the size of their molecule;
 Nuclear magnetic resonance spectroscoppy - different amplitude of the proton
NMR signal produced by each discrete LP particle.
 There are six major LPs in blood; the currently used nomenclature is based on
their different density

3. • Chylomicrons (CM) are formed in the small intestine and transport
exogenous triacylglycerols and cholesterol. After standing plasma or
serum for several hours at 4°C, CM settle on the surface in the form of a
white creamy layer.
• Very low-density lipoproteins (VLDL) are formed in the liver, they
transport the majority of endogenously produced TAGs.
• Intermediate-density lipoproteins (IDL) are formed from VLDL as
transient, short-living LP particles.
• Low-density lipoproteins (LDL) are products of VLDL degradation and
transport most of the endogenous and exogenous cholesterol from the
liver to peripheral tissues.
• High-density lipoproteins (HDL) are formed in the liver and small intestine
from their precursors - nascent discoid HDL particles, which acquire their
definitive spherical form in the bloodstream.
Classification of lipoprotein

4. Classification of lipoprotein
LP
Density
kg/L
% of all content
Apolipoproteins
CE Free C TAG PL Proteins
CM ≤0.940 3 1 90 4 2 B-48, C, E
VLDL 0.950 – 1.006 12 6 60 14 8 B-100, C, E
IDL 1.006 – 1.019 26 10 30 20 14 B-100, C, E
LDL 1.019 – 1.063 40 11 5 22 22 B-100
HDL 1.063 – 1.210 18 5 7 25 45 A, C, D, E
Lp(a) 33 9 3 22 33 B-100, (a)

5. Oil-drop of mixed micelle model of lipoprotein structure

6. APOLIPOPROTEINS
Apolipoproteins can be divided into integrals, which are a permanent part of
the lipoprotein particle (e.g. apoB) and freely associated, which LP particles
can exchange with each other (apoAI, AII, AIV, CI, CII, CIII, and E). Basic
functions of apolipoproteins are:
1. Structural:
– In CMs the structure is stabilized by the molecule apoB-48, which remains in
CM remnants until their uptake in the liver.
– ApoB-100 has a structural function in VLDL particles, which is also present in all
lipoprotein particles derived from VLDL (VLDL-remnants, IDL, LDL). In HDL
particles,
– apoprotein A (apo A) performs this function.
2. Specific receptor ligand: Part of the amino acid sequence of apoproteins is
the ligand for the receptor through which the LP particle enters the cell.
ApoB-100 and apoE have this function.
3. Activation or inhibition of the activity of enzymes involved in lipoprotein
metabolism: ApoA and apoC have this function concerning lecithin-
cholesterol acyl transferase (LCAT) and lipoprotein lipase (LPL).

7. Name Association
with LP
Function
Apo AI HDL
Structural protein for HDL; ligand for ABCA1 transporter,
activator of lecithin-cholesterol acyltransferase (LCAT).
Apo AII HDL Structural protein for HDL; inhibits cellular cholesterol efflux .
Apo IV HDL Activator of lipoprotein lipase (LPL) and LCAT .
Apo B48 CM Required for assembly and secretion of chylomicrons.
Apo
B100
VLDL, LDL
Structural protein for VLDL, IDL, LDL, and Lp(a); ligand for the
LDL receptor; required for assembly and secretion of VLDL.
Apo CI VLDL, HDL Activator of LCAT
Apo CII
CM, VLDL,
HDL
Essential cofactor for LPL.
Apo CIII
CM, VLDL,
HDL
Inhibition LPL and hepatic lipase.
Apo E
CM, VLDL,
HDL
Ligand for hepatic chylomicron and VLDL remnant receptor,
ligand for LDL receptor

8. • There are three different apoE alleles in humans: E-2, the most frequent E-
3, and E-4.
• Compared to apoE3, apoE2 has reduced affinity and apoE4 has enhanced
affinity for the LDL (apoB/E) receptor.
• ApoE-2 phenotype is associated with familiar dysbetalipoproteinemia with
an accelerated atherosclerosis.
• Homozygotes for E-4 have higher risk of Alzheimer´s diseases.
ApoE phenotypes

9. Key enzymes of lipid transport
• Lipoprotein lipase (LPL): synthesized by adipose tissue and striated muscle.
Produced by parenchymal cells, secreted and transported to the endothelial
surface of blood capillaries where it is bound to heparan sulfate. Hydrolyzes
TGL rich lipoproteins.
• Hepatic lipase (HL): formed in hepatocytes, transported to hepatic
endothelial cells. Acts on TGL in IDL. Also on PL & TGL in HDL.
• Lecithin Cholesterol Acyl Transferase LCAT: synthesized by liver and
attached to HDL in blood. Concerned with removal of excess cholesterol
from peripheral tissues.

10. Lipid metabolism
• occurs in three major areas
– Intestine
– Liver
– Extrahepatic tissues (Muscle and adipose tissue)

12. • In a normal diet, 95% of lipids are triacylglycerols (TAG), the remaining 5%
are all other dietary fats, including cholesterol (CH).
• Gastric lipase partially hydrolyzes TAG and released fatty acids (FA) activate
contraction of the gallbladder with the subsequent expulsion of bile
and pancreatic juice into the duodenum.
Absorption of dietary lipid
• Bile emulsifies TAG to smaller droplets
which are more accessible to digestion
by pancreatic lipase.
• Glycerol, FAs, and partly also 2-monoacylglycerol enter the enterocyte,
together with free cholesterol. Short- and medium-chain FAs can be
absorbed from the enterocyte into the bloodstream directly and circulate
bound to albumin. The longer fatty acids in the enterocyte are used for TAG
re-synthesis and cholesterol esterification.

13. Chylomicron metabolism
• Dietary lipids, including fat-soluble
vitamins, are resorbed in the small
intestinal mucosa and incorporated into
lipoprotein particles - chylomicrons
(CM). The major apolipoprotein in CM is
apolipoprotein B-48 (apoB-48) ), which
is synthesized in enterocytes.
• Chylomicrons enter the bloodstream
through the lymph. The highest
concentration of CM in the blood
occurs 3–6 hours after eating and after
10 – 12 hours of fasting are no longer
present in the blood of a healthy
person.
TAG
FA+GLYCEROL
TG
CHOLESTEROL
NASCENT CHYLOMICRON
A1
B48
CE

14. Chylomicron metabolism
• In circulation, CMs receive additional apoE and apoC from HDL particles. ApoC-II
is an activator of lipoprotein lipase (LPL), which hydrolyzes TAG from the core of
CM to form free fatty acids (FFA) and glycerol. FFAs are used by muscle cells
mostly as a source of energy, while TAGs are resynthesized in adipocytes
• FFAs are used by muscle cells mostly as a source of energy, while TAGs are
resynthesized in adipocytes.
• After hydrolysis of TAG, the CM core shrinks and smaller chylomicron
remnants (CMR) are formed. When the amount of TAG in the nucleus drops to
∽20%, CM submits apoC-II to the HDL particle losing the ability to further
hydrolyse TAG.
• Excess envelope phospholipids together with apoA are incorporated into HDL
particles.
• CMRs containing the rest of TAG and all dietary cholesterol are taken up by the
liver via apoE binding receptors.

15. absorption of dietary lipids
formation of nascent
chylomicrons
plasma hydrolysis by LPL
loss of ApoA and ApoC
and formation of
chylomicron ramenant
hepatic sequestration by
interaction of Apo-E with LDL
receptor

16. VLDL metabolism
• VLDL particles synthesized in the liver transport TAG (and partially cholesterol) from the
liver to peripheral tissues.
• The rate of VLDL synthesis is limited by the rate of hepatic synthesis of apoB-100, which is
their essential structural apoprotein.
• Increased hepatic TAG synthesis from FA or carbohydrate precursors, accelerated in
metabolic syndrome, diabetes, obesity, produces larger VLDL1 particles, while
physiological metabolism produces smaller VLDL2 particles.
• Similarly to CM, VLDL particles receive apoC and apoE from HDL particles upon entry into
the circulation.
• Both apoC-II and insulin activate endothelial LPL, which hydrolyzes TAG in the particle core.
If the content of TAG in VLDL decreases to about 30%, a transient particle - intermediate-
density lipoprotein (IDL) is formed.
• IDLs bind with the receptor on hepatocytes via apoE and they are subsequently
degraded, or they lose additional TAG by activity HL and change to LDL particles.
• IDL particles have a very short biological half-life under physiological circumstance, but are
highly atherogenic

17. FFA
TAG
VLDL
Nascent VLDL
APO B100
APO E,C (HDL)
PLASMA
In the liver: synthesis of VLDL by
incorporation of triglycerides with Apo B100,
and small amount of Apo C and Apo E.
After their secretion into plasma VLDL will
be enriched with Apo-E and Apo-C
transferred from HDL

18. VLDL
IDL
TG FFA
LPL
LPL TG
APO C,E(HDL)
LDL
CELL MEMBRANE
VIT D
MYELIN SHEATH
STEROID HORMONE
CHOLESTEROL
APO-B100
1
2
3
Transformation of
VLDL to IDL by loosing
Triglycerides and FFA IDL are normally transient in plasma and will be
rapidly eliminated
- by fixation on hepatic receptors and degradation
- or by plasma hydrolysis with loss of the
triglycerides, Apo-E and Apo-C and formation of
LDL rich in cholesterol
Interaction with LDL cell
receptor and Apo-B100
than release of cholesterol
into the cell

19. Metabolism of LDL
• LDL particles originate from the last stage of VLDL metabolism. They contain
only 10% TAG from the original VLDL particles, but all cholesterol and
structural apoB-100, the ligand for LDL receptors (LDL-R) both on the surface
of liver cells and cells of various peripheral tissues.
• The size of the LDL particles allows them to pass through the endothelium
into the interstitial space and can theoretically come into contact with any
cell.
• Approximately 30 – 40% of LDL are catabolized within 24 hours,
predominantly by LDL-R. The LDL particle bound to LDL-R is internalized into
the cell and, after cleavage of LDL-R, is hydrolyzed to cholesterol and amino
acids.
• The released LDL-R returns to the cell membrane surface.
• Intracellular cholesterol is a signal for the subsequent regulation of the
amount of LDL- R on the cell membrane so that the cell is not overfilled with
cholesterol. The entry of cholesterol into cells via LDL-R does not depend on
the level of cholesterol in the blood but the number of LDL-R on the cell
surface

21. • LDL particles are currently considered to be the most
pathogenic population of lipoproteins.
• LDL particles in the bloodstream are heterogeneous in
composition and size.
– Large, lighter LDL particles containing more TAGs are less atherogenic.
They are eliminated from the circulation by LDL-R; the amount
entering the cell is strictly regulated.
– The smaller and heavier LDL particle are more atherogenic. Slowed
catabolism, prolonged circulation of particles, and various
pathological conditions lead to the modification of LDL particles (e.g.
glycation, oxidation).
Metabolism of LDL

22. Formation of sdLDL particles

23. sdLDL and process of atherogenesis
• Small dense LDLs (sdLDL)
are not recognized by LDL-
R, due to their size they
enter the subendothelial
spaces by transcytosis,
where they aggregate and
easily oxidize.
Oxidized LDL particles are internalized into macrophages (as well as
other cells) via scavenger receptors (SR). A macrophage in the
subendothelial space crowded with LDL particles is known as a foam cell,
which is the first step in the process of atherogenesis.

24. Lp(a)
• Lp(a) binding to apo B100 has a structural similarity to plasminogen,
• Lp(a) interferes with fibrinolysis by competing with plasminogen
binding to plasminogen receptors on fibrinogen, and fibrin, leading
to decreased thrombolysis.
• Lp(a) has often been considered as an independent non modifiable
cardiovascular risk factor with atherogenic and also
thrombogenic potential

25. • The most quantity of cholesterol not used by cells is returned to the
liver via a process called reverse transport of cholesterol mediated by
HDL particles.
• The first particle of HDL is formed in an immature form (nascent
HDL) from phospholipids and apo A-I, and minimal cholesterol content.
• In the bloodstream they are enriched for the enzyme LCAT. These
small, discoid particles enter the subendothelial spaces of cells with
excess cholesterol by transcytosis, remove cholesterol from the cells,
and after its esterification with the LCAT enzyme, they move it to the
core of LP particle, thus acquiring a spherical shape (HDL2 and HDL3
particles)
• Excess cholesterol is transported by HDL particles back to the liver
• Due to the involvement of HDL particles in reverse cholesterol
transport, it has long been assumed that the higher the HDL-C level, the
lower the cardiovascular risk.
HDL metabolism

26. • FUNCTIONS
– Serves as a circulating reservoir for apo-C and apo-E.
– Delivering-chol. esters to liver for uptake by SR-B1
(reverse cholesterol transport).
– Protective effect - anti-inflammatory, anti-oxidative,
platelet anti-aggregatory, anticoagulant, and
profibrinolytic activities
HDL metabolism

27. • HDL and revers cholesterol transport

28. Lipid profile
Systematic lipid profile
• Total cholesterol
• HDL cholesterol
• LDL cholesterol
• Triglycerides
Extended lipid profile
• Apo A and Apo B
• Lipoprotein
electrophoresis
• Lp(a)

29. Preparation:
 Blood should be collected after a 12-hour fast (no food or drink,
except water).
 For the most accurate results, wait at least 2 months after a heart
attack, surgery, infection, injury or pregnancy to check cholesterol
levels.
 Cholesterol level is elevated during pregnancy (till 6 weeks after
delivery)
 Some drugs are known to increase cholesterol levels as anabolic
steroids, beta blockers, epinephrine, oral contraceptives and vit, D.
Specimen
• Serum
• Heparinized plasma

30. Normal rare common common rare common rare
Normal CM LDL LDL+VLDL IDL VLDL CM+VLDL
Aspect of fasting serum
Normal I IIa IIb III
IV V
1 2 3 4 4 5 6

31. Triglycerides determination
• GOP method
Enzymatic hydrolysis of triglycerides with subsequent
determination of glycerol by colorimetric method.
Triglycerides + H2O glycerol + fatty acids
Lipase
glycerol + ATP glycerol-3 P + ADP
Glycerol Kinase
Glycerol-3 P + O2 dihydroxyacetone phosphate + H2O2
Glycerol P-Oxydase
H2O2 + 4-AAP + 4-chlorophenol quinoneimine dye + 4 H2O
Peroxydase (POD)
The intensity of the red color produced is directly proportional to
the concentration of triglycerides in the sample when measured
at 510nm. Normal value < 1.5 g/l

32. Cholesterol determination
• GOP method
Enzymatic hydrolysis and oxydation of cholesterol with
subsequent determination by colorimetric method.
Esterified Cholesterol NE cholesterol + fatty acids
Cholesterol Esterase
NE chloesterol Cholestenon + H2O2
Cholesterol oxydase
H2O2 + 4-AAP + 4-chlorophenol quinoneimine dye + 4 H2O
Peroxydase (POD)
The intensity of the red color produced is directly proportional to
the concentration of cholesterol in the sample when measured at
510nm.
Normal value < 2 g/l

33. • Precipitation method
– after selective precipitation of LDL and VLDL with a polyanion cation
complex or phosphotungstic acid in the presence of divalent cations. HDL
cholesterol is assayed on the supernatant after centrifugation by
enzymatic technique.
• Directly determination
– The method depends on the properties of a detergent which solubilizes
only the HDL so that the HDL-c is released to react with the cholesterol
esterase, cholesterol oxidase and Chromogens to give color .The non HDL
lipoproteins LDL, VLDL and chylomicrons are inhibited from reacting with
the enzymes due to absorption of the detergents on their surfaces . The
intensity of the color formed is proportional to the HDL-c concentration in
the sample .
HDL-c determination
Referance range:
Men > 0.45 g/l
Women > 0.55g/l
TC/HDL-C= (atherogenicity index) is
used as a cardiovascular risk factor:
< 4.9 for Men
< 4.2 for Women.

34. • calculation method
LDL chol. = Total Chol. – (Trig /5 + HDL chol)
Not valid if triglycerides level is > 400 mg/dl
• Direct determination
after selective destruction of non LDL-cholesterol, LDL Cholesterol is
evaluated by the enzymatic couple Oxydase/peroxidase.
LDL-c determination
LDL Cholesterol reference value < 140 mg /l.
the pathological threshold is different depending on the number of risk
factors presented by the patient: HDL cholesterol< 0.35 g/l, high blood
pressure, ongoing smoking, diabetes, family coronary history or arterial
diseases, age (≥ 45 years in men, ≥ 55 years in women), early menopause .

35. Apoproteins AI and B determination
– Different immunological techniques are used for determination
of these two main apoproteins with specific antiserums and
purified standards (immunonephelometricy,
immunoturbidimetry, electro-immuno-diffusion….).
– Reference value: apoB = 0.6 -1.40 g
apoA1 > 1.10 g / l
By immunological techniques (immunonephelometry,
immunoturbidimetry).
Reference value: < 0.30 g / l.
Lp(a) derermination

36. LIPOPROTEIN ELECTROPHORESIS
this test is practical on agarose or
polyacrylamide gel.
After electrophoretic migration of LP and
staining, it allows to visualize qualitatively:
- an overload of chylomicron
- an increase in VLDL and LDL
- the presence of IDL or Lpa
- a relative decrease in HDL

38. Dyslipoproteinemias
• Dyslipoproteinemias (DLP) is a group of metabolic diseases that are
manifested by changes in the quality or quantity of serum lipoprotein
particles. They have a significant impact on cardiovascular and
cerebrovascular mortality, especially in developed countries.
• Classification of dyslipidemia
– The first classification was the Fredrickson classification based on
phenotypes observed in electrophoresis.
– Etiological classification : congenital / acquired
– European Atherosclerosis Society (EAS) classification, based on levels
of total cholesterol and triacylglycerols.

39. FREDRICKSON CLASSIFICATION OF HYPERLIPIDEMIA
Type I IIa IIb III IV V
Lipoprotein
pattern
↑CM ↑LDL
↑LDL
↑VLDL
↑IDL or
remnants ↑VLDL ↑VLDL
Cholesterol N/↑ ↑↑↑ ↑↑ ↑ ↑ ↑↑
TAG ↑↑↑ N/↑ ↑ ↑ ↑↑
↑↑
↑
Risk of AS - increased
Sings of MS - - central obesity, DM, hypertension, hyperuricemia
Pancreatitis,
abdominal
symptoms
+ - - - + +

40. Etiological classification
• The development of genetics and molecular biology makes it
possible to divide DLP into two basic groups:
– Primary : congenital, genetically determined
– Secondary: arising from other acute and chronic diseases,
drugs or nutritional factors.
• Most cases of lipoprotein disorders are due to an interaction
between genetic and environmental factors. The same disease
may not cause DLP in all patients and may lead to different DLP
phenotypes in different individuals.

41. Primitive hyperlipidemia
• Fredrickson's classification defined 6 classes of hyperlipidemia. It is
a phenotypic classification, based on abnormalities of lipid
metabolism objectified by lipoprotein electrophoresis.
 Type I : hyperchylomicronemia
 Type IIa : essential high cholesterol.
 Type IIb : Mixed hyperlipidemia
 Type III: dysbetalipidemia
 Type IV: endogenous hypertriglyceridemia
 Type V: mixed hypertriglyceridemia.

45. endothelial cell
transporter for
lipoprotein
lipase

46. Pathologie
Métabolique
Type selon
Fredrickson
Caractéristiques
Diabetes (type I ou II ) IV ou IIb ↓ Activité de la LPL (↓ insuline )
↑Synthèse des VLDL (↓ insuline )
Obesity IV ↑ VLDL
L’association : Obésité, hypertension,
diabète type caractérisé par :↓
cholestérol HDL
↑ risque cardiovasculaire
Hyperuricemia, gouts IV ou IIb
Cholestasis
intra- or extrahepatic
IIa ou IIb Lipoprotéines anormales : lipoprotéine X
↓ activité LCAT (présence des sels
biliaires ?
SECONDARY HYPERLIPIDEMIA

47. Pathologie
Métabolique
Type selon
Fredrickson
Caractéristiques
Insuffisance
rénale chronique
IV ↑ Synthèse VLDL
↓ Catabolisme des VLDL (↑ apoCIII ,
déficit de la lipase hépatique )
Syndrome
néphrotique
IV ou IIb ↑ Synthèse VLDL et ↓ Catabolisme des
VLDL (↓activité de la lipase hépatique)
Hypothyroïdie IIa ou IIb ↓ catabolisme des LDL et du cholestérol
Traitement par b-
bloquants
IV ↓ activité LPL
Traitement par des
corticoïdes
IV ou IIb ↓ activité LPL
SECONDARY HYPERLIPIDEMIA