Tapeshwar Yadav

(Lecturer & Head, MBAHS)

BMLT, DNHE,
M.Sc. Medical Biochemistry
&
Clinical Diagnostic Laboratory Head
(Hetauda Hospital
 Indications

Arterial diseases
Family history of arterial disease
Diabetes mellitus
Hypertensions
Obesity etc.

Preanalytical considerations
The blood sample should be collected after 12-14 hours of fasting
The subject should be in normal diet (two weeks before the test )
The subject shouldn’t take hypolipidemic drugs before the test
The test shouldn’t be done within 3 months of MI , major surgery
No heparin therapy is given before the test
 Important lipid profile tests

 Measurement of Serum Total cholesterol

 Measurement of serum HDL cholesterol

 Measurement of serum LDL cholesterol

 Measurement of serum total triglycerides

 Measurement of serum Apo B level

 Measurement of serum Apo A-I level

 Measurement of Lp(a) concentration

 Determination of total cholesterol/HDL ratio

 Clinical significances of serum cholesterol
MEASUREMENT OF SERUM
TOTAL CHOLESTEROL Increased serum cholesterol
 Type II familial hypercholesterolemia
NCEP classification of serum Cholesterol  Obesity

In adults  Poorly controlled diabetes mellitus

 Desirable : <200 mg/dl  Alcoholism

 Borderline : 200-239 mg/dl  Nephrotic syndrome

 Von Gierke’s disease
 High : ≥240 mg/dl
 Hypothyroidism
In children :
 Cholestasis
 Desirable : <170 mg/dl
Decreased serum cholesterol
 Borderline : 170-199 mg/dl  Abetalipoproteinemia
 High : ≥200 mg/dl  Familial hypobetalipoproteinemia
 Malabsorption of lipids
 Hyperthyroidism
Methods for serum cholesterol estimation
 Lieberman-Burchard reaction

 Iron-salt-acid reaction

 Para-toluene sulfonic acid reaction

 Enzymatic end point reaction

 Enzymatic method

Principle

The intensity of colored complex is measured using green filter. The color intensity is
directly proportional to serum cholesterol concentration.
 Reagent Test (T) Standard (S) Blank (B)

Procedure Reagent R1 1000μl 1000μl 1000μl

Serum 10μl ------ ------
Cholesterol standard ------- 10μl ------
Distilled water ------- ------- 10μl

 Keep at 37 0C for 5 minutes. Measure the optical density values of test, standard
& blank in colorimeter using green filter.

Calculation
O.D. of Test – O.D. of Blank
Serum Cholesterol(mg/dl) = X 200
O.D. of Standard – O.D. of blank

Interferring Substances
 Hemolysis

 Hyperlipemic samples

 No interferrence by : bilirubin upto 10 mg/dl, ascorbic acid upto 7.5 mg/dl, uric
acid upto 20 mg/dl, hemoglobin upto 200 mg/dl
Classification of serum TG
Enzymatic method
 Normal : <200 mg/dl
 NADH consumption (decreased
 Borderline high : 200-400
absorbance at 340nm)
 High : 400-1000 mg/dl
 End point colorimetric method
 Very high : >1000 mg/dl
 Formazan colorimetric method
Nonenzymatic method
 Fluorescent method
 Eegriwe’s reaction

 Schryver’s reaction

 Pay’s reaction

 Hantzsch’s reaction
Enzymatic method
 Specimen : 10-12 hrs fasting serum sample

Principle
Lipase
Triglycerides 3-Fatty acids + Glycerol

Glycerol
Glycerol + ATP Glycerol-3-phosphate + ADP
Kinase

Pyruvate
ADP + Phosphoenol Pyruvate ATP + Pyruvate
Kinase

Lactate
NADH + H+ + Pyruvate NAD + Lactate
Dehydrogenase

 The decrease in absorbance is determined colorimetrically at 340nm

Principle of endpoint colorimetry for serum TG

Lipoprotein
Triglyceride Glycerol + Fatty Acid
Lipase

Glycerol
Glycerol + ATP Glycerol-3-phosphate + ADP
Kinase

Oxidase
Glycerol-3-Phosphate + O2 Dihydroxyacetone Phosphate + H2O2

Peroxidase
H2O2 + 4-Aminoantipyrine Quinineimine dye + 4H2O

 The intensity of colored complex is measured colorimetrically using green filter

Procedure for endpoint colorimetric method

Reagents Test (T) Standard (s) Blank (B)

Triglyceride reagent 1000μl 1000μl 1000μl
Serum 10μl ----- ------
TG standard ----- 10μl ------
Distilled water ------ ------- 10μl

 Incubate at 37oC for10 minutes. Take reading in colorimeter using green filter.

Calulation :

O.D. of Test – O.D. of Blank

Serum Triglyceride(mg/dl) = X 200
O.D. of Standard – O.D. of blank
Clinical significance Determination of serum LDL cholesterol
Hypertriglyceridemia NCEP classification of serum LDL
 Obesity In adults :
 Smoking  Desirable : <130 mg/dl
 Physical inactivity  Borderline : 130-159 mg/dl
 Alcohol consumption  High : ≥160 mg/dl
 Heavy meal In children
Hypotriglyceridemia  Desirable : <110mg/dl
 Strenous exercise  Borderline: 110-120 mg/dl
 Weight loss  High : ≥130 mg/dl
Method

Calculation
Clinical significances : Similar to serum total cholesterol
 Determination of serum HDL cholesterol

NCEP classification of serum HDL cholesterol

 Desirable : >60 mg/dl

 Borderline : 35-60 mg/dl

 High : <35 mg/dl Method

Clinical significance
Increased HDL Decreased HDL

 Estrogen therapy  Obesity

 Insulin therapy  Smoking

 Moderate alcohol intake  Lack of exercise

 Hypertriglyceridemia

 Tangiers disease

 Diabetes mellitus

 Malnutrition
 DETERMINATION OF APOPROIEINS

 A lipid-binding protein that is a constituent of the plasma lipoprotein.

Total cholesterol/HDL Ratio
 An optimal : less than 3.5

 Low risk : 3.3-4.4

 Average risk : 4.4-7.1

 Moderate risk : 7.1-11.0

 High risk : >11.0

Calculation

 Ratio of total cholesterol-to-HDL is important; the smaller the number the better.

 For example, someone with a total cholesterol of 200 and an HDL of 60 would have a
ratio of 3.3 (200 ÷ 60 = 3.3).

Significance

 HDL wins its laurels as the "good" cholesterol because it sponges up cholesterol from
blood vessel walls and ferries it to the liver for disposal.

 In contrast, LDL deposits the harmful fat in vessel walls.

Non-HDL Cholesterol (Non-HDL-C)

 It is the cholesterol content in LDL+VLDL+IDL+Lp(a)

NCEP value for non-HDL cholesterol

 <130 mg/dl : Very little risk

 <160 mg/dl : Borderline high

 <190 mg/dl : High risk

 >190 mg/dl : Very high risk

Significance

Recently, it has become a commonly used marker for a blood lipid pattern associated
with increased risk of heart disease.