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1. Laboratory procedures
1. Creatinine kinase
2. Blood Cholesterol
3. Triacylglycerol
4. LDL- cholesterol
5. HDL- cholesterol

2. Creatine kinase (CK)
• Three isoforms
– Ck-BB: dominant in the brain
– Ck- MB: dominant in the heart
– CK- MM: dominant in the skeletal muscle
• Healthy individuals typically contain the MM
isoform and a small amount of the MB isoform in
their serum
• In myocardial infarction, levels of both CK-total
and CK-MB increase significantly, but CK-MB is
considered a specific cardiac marker

3. Creatine kinase (CK)
Sample Requirement
• Serum or plasma (heparinized/EDTA) is used.
• Protect from light.
• Loss of activity occurs within 7 days at 4 ◦C

4. Creatine kinase (CK)…principle

5. Creatine kinase
Enzyme Reagents
• Imidazole buffer (10 mM, pH 7.7)
• Phosphocreatine (30 mM)
• N-acetylcysteine (20 mM)
• Magnesium acetate (10 mM)
• Glucose (20 mM)
• Glucose-6-phosphate dehydrogenase 1.5 KU/L
• Hexokinase 2.5 KU/L
• EDTA, ADP, and NADP (2 mM each)
• AMP (5 mM)

6. Creatine kinase…Procedure
• Keep enzyme reagent at 37 C before use.
• Take 1 ml of this enzyme reagent in a
thermostated cuvette; add 0.05 ml of diluted
serum/plasma, mix thoroughly, and incubate
exactly for 5 min at 37 C and then read
absorbance at 30 s intervals for further 3 min.

7. Creatine kinase…Calculation
• CK-MB activity/L of serum (IU/L)
= ΔOD/min * 6752 * dilution factor where, factor 6752
is obtained as:
• Note: If we use an antibody against CK-M, we get
value of only CK-B from this enzyme.

8. Creatine kinase… Clinical significance
• Normal range: 24–195 IU/L in males and 24–170 IU/L
in females.
• Increased CK-MB observed within 6–8 h after onset of
myocardial infarction
• The activity reaches to maximum levels after 12–24 h
• The increase is not observed in heart failure and
coronary insufficiency.
• The CK-MB % between 5.5 and 20% indicates
myocardial injury

9. Cholesterol estimation…Principle
• Cholesterol is present in blood both as free and
as esters
1. Cholesterol and cholesterol esters from the
serum are extracted into an alcohol-ether
mixture
2. The contents are centrifuged.
3. The protein free extract is evaporated to
dryness.
4. The cholesterol residue is dissolved in
chloroform and is measured colorimetrically by
Liebermann-Burchard reaction.

10. Cholesterol estimation…Reagents
1. Absolute alcohol
2. Diethyl ether
3. Chloroform
4. Acetic anhydride
5. Standard solution of cholesterol (100 mg%)
– Dissolve 100 mg of cholesterol in some amount of
alcohol. Make the volume to 100 ml with alcohol.
Alocohol-either mixture
3.1

11. Cholesterol estimation…Procedure
• Test
– In a centrifuge tube, take 8 ml of alcohol, 2 ml of
ether. Mix it.
– Add 0.2 ml of blood. Mix overall. Keep it in slanting
position for half an hour. Centrifuge it.
– The supernatant is collected (which contains
cholesterol) in another tube.
– This test tube is kept in a boiling water for the
evaporation of solvent and the residue, i.e. cholesterol
sticks to the bottom of the flask is left behind.
– Chloroform, acetic anhydride and sulphuric acid must
be of highest quality.

12. Cholesterol estimation…procedure
Standard
• In different tubes marked S1, S2, S3, and S4,
add 0.2, 0.4, 0.6 and 0.8 ml of standard
cholesterol solution.
• All should be kept in boiling water bath for
evaporation of solvent till the solvent
evaporates and residue is left behind.
• Blank: Clean dry test tube.

13. Cholesterol estimation…procedure
Black S1 S2 S3 S4 Test
CHCl3 5ml 5ml 5ml 5ml 5ml 5ml
Acetic anhydride 2ml 2ml 2ml 2ml 2ml 2ml
Concentrated
H2SO4
0.1m
l
0.1ml 0.1m
l
0.1ml 0.1m
l
0.1
ml
Now add the following reagents in test, different standard, and
blank
• Mix well, keep it in dark for 10 minutes.
• Read optical density at 610 nm.
The concentration
of cholesterol.

14. Cholesterol estimation…Interpretation
• Normal cholesterol: 150-
250 mg%.
• Hypercholesterolemia is
observed in
1. Diabetes mellitus.
2. Obstructive jaundice.
3. Nephrotic syndrome.
4. Cirrhosis of liver.
5. Hypoparathyroidism.
6. Xanthomatosis.
• Hypocholesterolemia is
observed in
1. Hyperthyroidism
2. Pernicious anaemia
3. Haemolytic anaemia
4. Malabsorption
syndrome.

15. Triglycerides Estimation…Principle
• What is TAG?
• Transporter?
1. Lipases split triglycerides into glycerol and fatty acids.
2. Glycerol kinase hydrolyzes glycerol in the presence of ATP to
form glycerol-3-phosphate and ADP.
3. Glycerol-3- phosphate oxidase enzyme splits glycerol-3-
phosphate in the presence of O2 to dihydroacetone
phosphate and H2O2.
4. Peroxide enzyme splits H2O2 to water and oxygen.
5. The oxygen oxidize 4-chlorophenol in the presence of 4-
aminoantipyrine to form red quinone imine, which is
measured at 520 nm and compared with that of triglyceride
standard.

16. Triglycerides Estimation…Reagents
1. Buffer solution
• PIPES buffer (50 mM, pH
7.5)
• 4-chlorophenol (5 mM)
• Magnesium ions (4.7 mM)
• ATP (1 mM)
• Lipases (1 U/ml)
• Peroxidase (0.5 U/ml)
• Glycerol kinase (0.4 U/ml)
• Sodium azide (0.05%)
2. Enzyme reagent
• 4-amineantipyrine (0.4 mM)
• Glycerol-3-phosphate
oxidase (1.5 U/ml)
• Sodium azide (0.095%)

17. Triglycerides Estimation…Reagents
3. Working triglyceride
reagent
• Add 250 μl of enzyme
reagent to 30 ml of
buffer solution.
• Mix gently, and keep
the reagent for at least
10 min at room
temperature before
use.
4. Triglyceride standard:
200 mg/dl

18. Triglycerides Estimation…procedure
1. Label three tubes as test (T), standard (S), and blank
(B).
2. Then add 10 μl of sample to the tube labelled as “T.”
3. Add 10 μl of 200 mg/dl triglyceride standard to the
tube labelled as “S.”
4. Add 1 ml of working triglyceride reagent to all the
tubes,
5. mix the contents, and incubate for 5 min at 37C.
6. Then measure the absorbance of the samples against
the reagent blank within 60 min.

19. Triglycerides Estimation…interpretation
• A normal level of triglycerides is less than 150
mg/dl in fasting condition
• High levels of triglycerides increase the risk of
developing pancreatitis.

20. HDL Estimation
• Composition?
• Apoproteins?
• Types?

21. HDL Estimation…principle
• Chylomicrons, LDL, and VLDL are precipitated from the
sample by adding phosphotungstic acid and
magnesium ions to the sample
• Centrifugation leaves only the HDL in the supernatant.
• The cholesterol content is determined by enzymatic
method (cholesterol oxidase/peroxidase method).

22. HDL Estimation…Reagents
1. Precipitating reagent: 0.55 mM phosphotungstic
acid and 25 mM magnesium chloride.
2. Cholesterol reagent: Same as that used in
cholesterol estimation.
3. Working precipitating reagent prepared by
mixing precipitating reagent four parts with one
part distilled water.

23. HDL Estimation…Procedure
1. Mix 500 μl of working precipitating reagent with 200 μl
serum, and allow it to stand at room temperature for 10
min.
2. Centrifuge this reagent at 4000 rpm for 10 min or at
12000 rpm for 3 min.
3. After centrifugation, separate the clear supernatant
within 2 h
4. Label two tubes as test and blank.

24. HDL Estimation…Procedure
5. Pipette 100 μl of supernatant into the tube labelled as
“test.” Add 100 μl of distilled water into blank tube.
6. Add 1 ml cholesterol reagent to both tubes and mix.
Now, incubate the tubes at 37C for 5 min
7. Measure absorbance at 500 nm against blank in a
spectrophotometer, and calculate the HDL
cholesterol.

25. HDL Estimation…Precautions
1. The sample should be collected from fasting patient
and free from hemolysis.
2. After precipitation the supernatant should be clear
and estimate the HDL cholesterol within 2 h after
precipitation with enzymatic method only.
3. Serum from the blood clot should be separated as
rapidly as possible. HDL
4. cholesterol remains stable in serum for 6 days at 2–
25C and 4 months at 20C.

26. HDL Estimation…interpretation
• Normal range of HDL cholesterol is 30–60
mg/dl
• HDL less than 40 mg/dl indicates high risk of
heart disease

27. LDL Estimation
• Metabolic function?
• Composition?
• Apoproteins?
• Conditions related with increased blood LDL?

28. LDL Estimation…principle
• Firstly, polyvinyl sulfate is added to sample to precipitate
the LDL,
• The cholesterol content of HDL, VLDL, and chylomicron is
measured.
• LDL cholesterol is calculated on the basis of difference
between total cholesterol levels of serum and the
cholesterol levels in the supernatant after centrifugation.

29. LDL Estimation…reagents
1. LDL-cholesterol precipitant: Stable for 4
weeks at 20–25C
2. Cholesterol reagent: Same as that used in
cholesterol estimation

30. LDL Estimation…precidure
1. Mix 200 μl of serum with 100 μl of precipitating
reagent and allow standing for 15 min at room
temperature and then centrifuge at 10000 rpm
for 2 min
2. Take two test tubes, and mark them as test and
blank.
3. Add 25 μl of supernatant in tube labelled as test
and 25 μl of distilled water in blank tube.
4. Add 1 ml of cholesterol reagent to these tubes,
mix, and incubate at 37C for 5 min.

31. LDL Estimation…procedure
1. Measure absorbance at 500 nm against blank
in a spectrophotometer,
2. calculate the cholesterol value in the
supernatant. From total cholesterol value,
the value obtained in the supernatant is
subtracted to get the LDL cholesterol value

32. LDL Estimation…interpretation
• LDL cholesterol below 100 mg/dl is desirable.
• LDL greater than 150 mg/dl indicates
increased risk to cardiovascular diseases