LIPID PROFILES (block 12)

Clinical Pathology Department 2008

Measurement of lipids and lipoproteins

Many analytical techniques have been developed : chemical, enzymatic and immunochemical methods as well as physical methods, such as ultracentrifugation, electrophoresis, column chromatography, precipitation method. Enzymatic method: accurate, precise, simple to use

TOTAL CHOLESTEROL (CHOD-PAP method)

Principle: hydrolisis & oxydation Serum + reagents

Incubation

measure the absorbance

The reaction is as follow:

Chol ester hydrolase

Cholesterol ester + H2O Cholesterol + O2

Chol oxydase

cholesterol + fatty acid

Cholest-4-en-3one + H2O2

H2O2 + phenol + 4 aminoantipyrine

peroxidase

Quinoneimine dye + 2H2O

Reagents:
Goods buffer pH 6.7 Phenol 4-Aminoantipyrine Cholesterol esterase (CHE) Cholesterol oxidase (CHO) Peroxidase (POD)
Standard :

50 mmol/l 5 mmol/l 0.3 mmol/l 200 U/l 50 U/l 3 kU/l

200 mg/dl (5.2 mmol/l)

Specimen
Serum, heparin plasma or EDTA plasma. Stability : 7 days at 20-250C 3 months at - 200C

Assay Procedure
Sample or standard Dist. Water Reagent Blank 10 l 1000 l Sample or standard 10 l 1000 l

Mix, incubate for 20 min. at 20 250C or for 10 min. at 370C . Read absorbance within 60 min against reagent blank.

Calculation With standard or calibrator

Cholesterol [mg/dl] =
A Sample A Std / Cal

x Conc.Std / Cal [mg/dl]

Measuring range
3 750 mg/dl (0.08 19.4 mmol/l).

Specificity / Interferences
No interference was observed by: - ascorbic acid up to 5 mg/dl, - bilirubin up to 20 mg/dl, - hemoglobin up to 20 g/dl and - lipemia up to 2,000 mg/dl triglycerides.

Sensitivity / Limit of Detection The lower limit of detection is 3 mg/dl (0.08 mmol/l). Reference Range Desirable Borderline high risk
High risk

200 mg/dl (5.2 mmol/l) 200 240 mg/dl (5.2 6.2 mmol/l) > 240 mg/dl (> 6.2 mmol/l)

HDL-CHOLESTEROL (CHOD-PAP method)

principle
measure clear supernatant
centrifuge

Sample + precipitation reagen

Precipitate VLDL, IDL,Lp(a), LDL

Reagents Precipitating reagent (1.4 mmol/l phosphotungstic acid, 8.6 mmol/l magnesium chloride), 250 ml. Storage 150 C - 250 C until expiry date.

Sample Material Serum , heparin-or EDTA plasma The HDL-Cholesterol in serum is stable - 7 days (150C - 250 C) -14 days ( 20 C - 80 C)

Procedure
Precipitation : Pipette into centrifuge tubes: Sample Precipitating reagent 1

200 l 500 l

Mix well and incubate for 10 min at RT, then centrifuge for 5 min at 5000g. 0.1 ml clear supernatant cholesterol determination (CHOD-PAP method)

Photometric measurement
Wavelength:500nm,546nm

Sample Pippete : Supernatant Water Reaction solution 0.1 ml 1.0 ml

reagent blank 0.1 ml 1.0 ml

Mix well and incubate for 10 min (RT) or 5 min ( 370 C) Then measure the absorbance (A) against the reagent blank value.

Calculation:
HDL-Cholesterol concentration =AxF

F = Factor (obtained from reagent kit) e.g. F (mg/dl) = 222 [CHO-PAP Prod.No.1.14366.1.14165-67]

Notes The supernatant after the centrifugation should be a clear solution. Sera with a triglyceride content over 1000 mg/dl tend to turbid supernatants or flotating precipitates. In this case a predilution of sample with the same volume of physiologic sodium chloride solution ( 9 g/l =^ 154 mmol/l) followed by the precipitation procedure is recommended. Multiply the result by 2.

TRIGLYCERIDES

Principle: determination of triglycerides after enzymatic splitting with lipoprotein lipase Serum + reagents

Incubation

measure the absorbance

Method

Colorimetric enzymatic test using glycerol-3phosphate-oxidase (GPO)

Reagents :
Goods buffer pH 7.2 4-Chlorophenol ATP Mg2+ Glycerokinase (GK) Peroxidase (POD) Lipoprotein lipase (LPL) 4-Aminoantipyrine Glycerol-3-phosphate-oxidase (GPO) Standard : 50 mmol/l 4 mmol/l 2 mmol/l 15 mmol/l 0.4 kU/l 2 kU/l 2 kU/l 0.5 mmol/l 2 kU/l

200 mg/dl (2.3 mmol/l)

Specimen Serum, heparin plasma or EDTA plasma Stability : 2 days at 20-250C 7 days at 4-80C at least one year at -200C Discard contaminated specimens.

Assay Procedure
Sample or standard Dist. Water Reagent Blank 10 l 1000 l Sample or standard 10 l 1000 l

Mix, incubate 20 min (20 250C) or 10 min (370 c). Read absorbance against reagent blank within 60 min.

Calculation With standard or calibrator

A Sample

Triglycerides [mg/dl] = A Std /

Cal

x Conc.Std / Cal [mg/dl]

Measuring range
1 1000 mg/dl (0.01 11.3 mmol/l).

Specificity / Interferences
No interference was observed by -bilirubin up to 40 mg/dl. -Ascorbic < 6 mg/dl, -Haemoglobin < 25 g/dl.

Sensitivity / Limit of detection

The lower limit of detection is 1 mg/dl

Reference Range
Desirable : < 200 mg/dl (fasting) (2.3 mmol/l) 200-400 mg/dl (2.3 4.5mmol/l) > 400 mg/dl (4.5 mmol/l)

Borderline high : Elevated :

LDL-Cholesterols

measurement : - assume that total cholesterols is composed primarily of cholesterols in VLDL, LDL and HDL - method : indirect or direct

Indirect method

Friedewald equation LDL = Total chol. HDL ( TG/5)

Direct Method LDL

Methods: 1. Immunoseparation (IS) - Anti-human lipoprotein antibody binds & inactivates chylomicrons, HDL, and VLDL - LDL then measured using enzymatic method 2. Zwitterionic detergents (ZI) - Zwitterionic detergents mask the VLDL - After addition of reagen, a LDL polyanion complex is formed which can be measured turbidimetrically

Direct Method LDL

Methods: 3. Clearance Method - selective detergents release cholesterol from chylomicrons, HDL, and VLDL - this cholesterol is then removed by the action of chol-esterase and oxidase - LDL- chol is then released in the 2nd step, and measured

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