Lipid Profile and Cardiovascular
Dr.
OMEED A. ALI
1
Determination of The Total
Cholesterol and TG in Serum
Dept.of Biochemistry
What is Cholesterol?
• It is
Amphipathic
• it has both a
water-soluble region
(a
polar -OH group) and a
fat-soluble
region
(a non-polar steroid ring
structure and hydrocarbon tail).
• Cholesterol
is a Fatty substance (lipid), which is
essential to healthy life.
Cholesterol
➢ Cholesterol can be (
exogenous
) derived from food.
➢ Cholesterol can be (
endogenous
) synthesized de
novo in liver, intestine, adrenal cortex and
reproduction tissues.
➢ 70 %
of cholesterol associated with
cellular
components
➢ 30 % is in the plasma ( ⅓ free form , ⅔ esterfied )
➢ Cholesterol is a very
hydrophobic
compound.
➢ It consists of four fused hydrocarbon rings called
steroid nucleus.
➢ Elevated levels of cholesterol are associated with
atherosclerosis
.
Synthesis of Cholesterol
➢ Synthesis of HMG-CoA
➢ Synthesis of mevalonic acid
➢ Synthesis of squalene
➢ Synthesis of cholesterol
HMG : Hydroxy Methyl Glutaryl
Synthesis of Cholesterol
Synthesis of Cholesterol
Cholesterol Functions
• Cholesterol
is in the
brain, nervous tissue,
skin
and
adrenal
glands.
– Membrane component
– Precurser to
• Bile acids
• Vitamin D
• Steroid hormones
Bile acids
Bile acids are polar derivatives of
cholesterol and aid in:
• lipid digestion
• lipid absorption
• cholesterol excretion
Steroids derived from
cholesterol
Cholesterol(Ch) and Cholesteryl
Ester(CE)
Cholester
ol
Cholesteryl
Ester
• Cholesterol is transported in the bloodstream of all animals.
• A large portion of the cholesterol in blood is in the form of
cholesteryl esters.
Transport of Cholesterol
➢ Cholesterol is relatively insoluble in aqueous media
and transported in body fluids as soluble protein
complexes called lipoproteins.
➢ Lipoproteins are composed of a lipid core
(containing
triglyceride
and
cholesterol
ester
)
surrounded by a hydrophilic shell (containing
Apo
lipoproteins
,
phospholipids
and
free cholesterol
).
What are lipoproteins?
• Lipoproteins are
Molecular complexes that consist of
lipids and proteins
.
• mainly
transport lipids in blood plasma
.
• Hydrophilic lipids (FC, PL) on surface.
• Hydrophobic lipids (TG, CE) in core.
•
Chylomicron (CM)
-
Transport of dietary triglycerides from the
Gastrointestinal tract to the liver.
•
Very low density lipoprotein
(VLDL)
- Transport of triglycerides from the liver to
tissues for storage and energy.
•
Intermediate density lipoprotein (IDL)
• Low density lipoprotein (LDL)
-
Transports cholesterol to peripheral tissues.
•
High density lipoprotein (HDL)
• Transports cholesterol away from the
peripheral tissues to the liver.
Classification of plasma
lipoproteins
according to their density
LDL and HDL
Normal
Artery
Clogged
Artery
Cholester
ol
• LDL:
carries cholesterol to the
peripheral tissues
•
it can be deposited and
increase
the
risk
of
arteriosclerotic
heart
and
peripheral vascular disease.
•
high levels of LDL are
atherogenic
.
“Bad cholesterol”.
•
HDL transports cholesterol
from the peripheral tissues to
the liver for excretion,
• hence HDL has a
protective
effect
.
“Good cholesterol”.
❧
15
❧
Curr Cardiol Rev. 2010 May; 6(2): 82–90
Clinical significance
• Risk evaluation
– Normal: 3.0
～5.20mmol/L
– Borderline: 5.20
～6.20mmol/L
– High blood cholesterol: ≥6.20mmol/L
• Hypercholesterolemia
• Hypocholesterolemia
Hypercholesterolemia
• High blood cholesterol (≥6.20mmol/L)
• Usually a result of
high LDL/low HDL
cholesterol levels
Leads to
• atherosclerosis: narrowing of artery walls
• decreased blood and oxygen supply to heart
• coronary heart disease(CHD)
•
Observed in many disorders
•
nephrotic syndrome, hypothyroidism, Diabetes
mellitus, extrahepatic biliary obstruction
Primary
Hypercholesterolaemia
➢ Familial hypercholesterolemia
➢ Familial defective apoB
➢ Familial combined hyper
lipidaemia
➢ Polygenic hyper cholesterolaemia
➢ Hyper alpha lipoproteinaemia
Secondary
Hypercholesterolaemia
➢ Hypothyroidism
➢ Diabetes mellitus
➢ Nephrotic syndrome
➢ Cholestasis
➢ Acute intermittent porphyria
➢ Anorexia nervosa
➢ Certain drugs e.g. Anticonvulsants, oral
contraceptives
➢ Alcohol
Hypocholesterolemia
• A decrease in the plasma cholesterol: less
common.
• Observed in: severe liver injury, hyperthyroidism,
pernicious anemia, malabsorption syndrome.
Lipoprotein Profile
Includes:
✓ Total Cholesterol
✓ LDL Cholesterol
✓ HDL Cholesterol
✓ Triglycerides
• Lipid profile is a measure of the lipid contents of the
blood.
Who needs to have their
Cholesterol checked?
Cholesterol screening is used as part of assessing
someone's risk of heart disease. It is necessary to check
your cholesterol if you fit any of these groups:
•
If you have evidence of heart disease
• If you have a family history of high cholesterol
• If you are a diabetic
• If you have high blood pressure
• If you have a family history of heart disease
What Are Some Ways To Control
Blood Cholesterol and Triglycerides?
• Healthy Eating
• Physical Activity
• Weight Loss
• Medication
Principle (CHOD-POD method)
Cholesterol
+O2 Cholestenone+
H
2
O
2
Cholesterol oxidase(CHOD)
Cholesterolester
+ H
2
O
Cholesterol
+Fatty acid
Cholesterolesterase(CHE)
H
2
O
2
+ 4-Aminoantipyrine + Phenol
Quinoneimine
+ 4 H
2
O
Peorxidase(POD)
This kit uses a
coupled enzymatic reaction
scheme:
• By the catalysis of CHE and CHOD, Cholesterol ester is catalyzed to
yield H
2
O
2
, which oxidates 4-Aminoantipyrine (4-AAP) with phenol to
form
a colored dye of quinoneimine
.
•
The absorbency increase is directly proportional to the concentration
of cholesterol.
Specimens & Materials
• Specimen:
serum
• Reagent
:
– R
1
: Phosphate buffer (pH 7.4)
，Phenol(酚)
– R
2
: 4-AAP, Cholesterolesterase(CHE),
Cholesterol oxidase(CHOD), Peroxidase (POD)
• Working reagent
:
– R
1
(10ml) + R
2
(1ml)
• Standard: cholesterol
；
C
S
=5.2mmol/L
• Water bath, Test tubes, Pipettes
• Spectrophotometer
Method
B
S
T
Working reagent
0.6
ml
0.6
ml
0.6
ml
dH
2
O
10
μl
-
-
Standard
-
10
μl
-
Serum
-
-
10
μl
Mix well
, incubate for
15
mins at 37°C
dH
2
O
1.0
ml
1.0
ml
1.0
ml
• Mix
well, measure the absorbance of T and S
setting zero with B,
λ=510nm
.
1. Switch on , for 20 min before using.
2. Select
Wave length
of Maximal Absorption
3. Prepare test sample, blank sample, standard sample .
put them into Spectrophotometry.
4. To “
Blank
”, mode “
T
or
A
” , Set
T =100
or
A=0
5. Pull the pole once time
6. Change mode to “
T
”, Set
T =0
7. Change mode to “
A
”.
8. pull the pole second time, record A1;third time ,record
A2, forth time ,record A3.
Operating steps of Spectrophotometry
Blan
k
Standar
d
Test
1
Test
2
Blan
k
Standar
d
Test
1
Test
2
Blan
k
Standar
d
Test
1
Test
2
Blan
k
Standar
d
Test
1
Test
2
Blan
k
Standar
d
Test
1
Test
2
T
handle 0
set
T100%
：
Blank
T
handle 1
set
T 0%
：
A=1.-----
A
handle2
、
3
、
4
assay A
：
Standard
、
test1
、
2
Rest state
：
handle 1
A=1.-----
（
3
）
Determine
Photometric Mode:
Transmittance,
T
0% ~
100%
Absorbance,
A
0 ~ 1
Concentration,
C
Calculation
• C
T
(mmol/L)=A
T
/A
S
x C
S
• Normal value of
T-cho
:
3.1~5.7
mmol/L
HDL-Cholesterol determination
- In the plasma, cholesterol is transported by three lipoproteins
:
high
density lipoprotein (HDL-Cholesterol), low density lipoprotein (LDL-
Cholesterol), and very low density lipoprotein (VLDL- Cholesterol).
- The concentration of total cholesterol in serum has been associated
with metabolic, infectious and coronary heart diseases.
- Introduction:
LPL : Lipoprotein Lipase , CETP: cholesterol Ester Transfer protein,
HDL (high density lipoprotein) :
• HDL
: good cholesterol, carry cholesterol from
organs and blood to liver to get rid of it
• It removes excess cholesterol from tissues (it cleans
blood).
• High levels linked to a reduced risk of heart and
blood vessel disease. The higher your HDL level,
the better.
- The concentration of HDL-cholesterol in serum has important in diagnosis of
the how the level of
risk to get coronary heart diseases.
- The measurement of HDL Cholesterol and triglyceride provides valuable
information for the prediction of coronary heart disease and for lipoprotein
phenotyping.
- Specimen collection:
1. Specimen should be
serum
and free from
hemolysis
.
2. Patient should be
fasting for 12-14 hours
.
- Principle:
- HDL cholesterol determination
- Enzymatic methods, involving cholesterol esterase and oxidase and Trinders color system.
- The enzymatic reaction sequence employed in the assay of cholesterol is as follows:
. ESTERASE
Cholesterol Esters
Cholesterol + Fatty Acids
.OXIDASE
Cholesterol + O
2
Cholesten-3-one + H
2
O
2
PEROXIDASE
2 H
2
O
2
+ 4-Aminoantipyrine + Phenol
Quinoneimine + 4 H
2
O
(red dye)
- Cholesterol Esters are hydrolyzed to produce cholesterol, Hydrogen peroxide is then produced
from the oxidation of cholesterol by cholesterol oxidase. In a coupled reaction catalyzed by peroxidase,
quinoneimine red colored dye is formed from 4-aminoantipyrine, phenol and hydrogen peroxide. The
absorption of light at 505 + 5 nm of the solution of this dye is proportional to the concentration of
cholesterol in the sample.
- Preparing HDL-Cholesterol sample:
- When serum is reacted with the polyethylene glycol reagent, all
the low and very low-density lipoproteins (LDL and VLDL) are
precipitated.
- The HDL fraction remains in the supernatant.
- The supernatant is then used as a sample for cholesterol assay.
Method :
- HDL Cholesterol:
- Follow the Table:
Blank
Standard
Test
Cholesterol liquid
enzymatic reagent
1 ml
1 ml
1 ml
Pre-worm at 37
◦C for 2 min and add:
Distelled water
100 µl
---
---
Standard (50 mg/dl)
---
100 µl
---
Supernatant (serum)
---
---
100 µl
Mix and incubate at 37ºC for 10 min.
Read Ab. at 505nm against blank.
- Calculation :
*
Determine the HDL Cholesterol conc.
Ab Test
Ab Std.
Conc. =
X conc. of Std (50mg/dl)
- Normal value of :
- HDL-Cholesterol :
Triglyceride determination
Introduction:
Triglycerides are esters of fatty acids and are hydrolyzed to glycerol and free fatty
acids (by lipase)
Triglyceride is body storage form of fat and energy
- Most TG found in adipose tissue
Give energy in case of absence of carbohydrates
- Triglyceride determinations when performed in conjunction with other lipid
assays are useful in the diagnosis of primary and secondary
hyperlipoproteinemia.
prepare for the test?
- Hyperlipoproteinemia: abnormally elevated of fat in blood ( disorder
in lipid metabolism).
- Standard methods for the measurement of triglyceride concentrations
involved either enzymatic or alkaline hydrolysis to liberate glycerol.
- TG test needs 9-12 hrs fasting because its level is effected by meal (fatty
meal, high carbohydrates meal)
Indication of the test
▶
Family history of
heart disease
▶
IHD
▶
Smoking
▶
Being overweight, sedentary life style
▶
Diabetes
▶
High blood pressure
▶
Age. Men 45 years or older
and women 50 years or older.
NORMAL RANGE
▶
normal fasting: 150 mg/dL
▶
borderline high: 150 to 199 mg/dL
▶
high: 200 to 499 mg/dL
▶
very high: >500 mg/dL
medical conditions that can cause high
triglyceride levels, including:
▶
cirrhosis in the liver
▶
diabetes, especially if it is not well controlled
▶
genetic factors
▶
hyperlipidemia
▶
hypothyroidism
▶
nephrotic syndrome or kidney disease
▶
pancreatitis
Low triglyceride level may be due to:
▶
low-fat diet
▶
hyperthyroidism
▶
malabsorption syndrome
▶
malnutrition
- Specimen collection and storage:
1.
Fresh
,
non-hemolyzed
serum from fasting patients is recommended.
2. Triglycerides in serum appears stable for
three days
when stored at 2-8
◦C.
3. Prolonged storage of the samples at room temperature is not recommended
since other glycerol containing compounds may hydrolyze, releasing free glycerol
with an apparent increase in total triglycerides content.
- Principle:
The enzymatic reaction sequence employed in the assay of Triglycerides is as follows:
H
2
O
Lipase
>
Triglycerides +
Glycerol + Fatty Acids
Glycerol Kinase
>
Glycerol + ATP
Glycerol-3-Phosphate + ADP
O
2
G-1-P
>
Glycerol-3-Phosphate +
DAP + H
2
O
2
oxidase
H
2
O
2
+ 4AAP + 4 chlorophenol
Peroxidase
>Quinoneimine Dye + 2H
2
O
- The present procedure involves hydrolysis of triglycerides by lipase.
- The glycerol concentration is then determined by enzymatic assay coupled with Trinder reaction that
terminates the formation of a quinoneimine dye.
- The amount of the dye formed, determined by its absorption at 505 nm, is directly proportional to
the concentration of triglycerides in the samples.
- Method :
- By Triglyceride reagent kit.
-Follow the table:
Blank
Standard
Test
Reconstituted Reagent
1 ml
1 ml
1 ml
Pre-worm at 37
◦C for 2 min and add:
Standard
Sample
---
---
0.01 ml (10 µl)
---
---
0.01 ml (10
µl)
Mix and incubate at 37ºC for 10 min
Read the absorbance of standard and sample at 505 nm against blank
-
Calculation
:
Ab Test
Ab Std.
Conc. of TG =
X
conc.
of Std. ( 200mg/
dl)
- Normal range:
10 -190 mg/dl
