CLINICAL CHEMISTRY 1: LIPIDS AND LIPOPROTEINS LABORATORY DETERMINATION

PROFESSOR: Kristina Kaira B. Lacia, RMT – FACULTY OF MEDICAL LABORATORY SCIENCE

2nd Semester I A.Y. 2023-2024 I PRE-FINAL

LIPIDS AND LIPOPROTEINS LABORATORY DETERMINATION

LABORATORY DETERMINATION - END COLOR: GREEN
1. FATTY ACID o GAS CHROMATOGRAPHY – MASS SPECTROMETRY (MOST RECENT).
2. PHOSPHOLIPIDS
3. CHOLESTEROL TRIGLYCERIDE
4. TRIGLYCERIDE o Fasting specimen is required! (12-14hrs).
5. LIPOPROTEINS o SPECIMEN: Serum/Plasma
o Measurement is dependent on glycerol.
CHOLESTEROL Saponification in triglyceride would get glycerol and fatty acid.
o Does not require a fasting specimen if tested alone. o METHODS:
o For lipid profile, a 12hrs fasting specimen is a must! 1. Chemical Methods
o SPECIMEN: Serum/Plasma 2. Enzymatic Methods
o If analysis is delayed, refrigerate at 4 degree Celsius.
o METHOD: CHEMICAL METHODS
1. Chemical Methods 1. Van Handel & Zilversmith
2. Enzymatic Methods • PINCIPLE: Colorimetry
• END COLOR: BLUE
CHEMICAL METHODS • REAGENT:
1. Liebermann Burchardt 1. Alcoholic KOH
• PRINCIPLE: Dehydration and oxidation of cholesterol to form a 2. Periodic acid (+ glycerol = formaldehyde)
colored compound. 3. Chromotropic acid (+ formaldehyde = blue colored
• END PRODUCT: Cholestadienyl Monosulfonic Acid (green end compound)
color).
• Lieberman Burchardt Reagent: ENZYMATIC METHOD
1. Glacial acetic acid • Commonly used in the laboratory.
2. Acetic anhydride • Glycerol present in the serum/plasma can become an interference.
3. Concentrated H2SO4 - “Double-cuvette blank”
- “Single-cuvette blank”
CHEMICAL METHOD (we uses blanking technique, zero the instrument to make sure no
2. Salkowski interference by using water).
• PRINCIPLE: Dehydration and oxidation of cholesterol to form a • Measures the amount of hydrogen peroxide produced in the
colored compound. reaction.
• END PRODUCT: Cholestadienyl Disulfonic Acid (red end color). • Measured at 500nm.
• Salkowski Reagent: - ATP: 3 PHOSPHATE (- PHOSPHATE = ADP)
1. Glacial acetic acid
2. Ferric chloride
3. Concentrated H2SO4

CHEMICAL METHOD
1. One-step method (colorimetry) (add reagent to have colored
compound)
2. Two-step method (extraction + colorimetry).
3. Three-step method (saponification2 + extraction1 + colorimetry3)
(saponification – conversion of cholesterol to cholesterol ester by
the use alcoholic potassium hydroxide). o CDC REFERENCE METHOD – Modified Van Handel, Zilversmith.
4. Four-step method (saponification2 + extraction1 + colorimetry3 + - Uses chloroform for extraction.
precipitation4) (the most complex but the most reliable). - Alcoholic KOH for saponification.
- Uses silicic acid – to remove phospholipids.
ENZYMATIC METHODS - END COLOR: PINK
• Most common method for measuring cholesterol. o GAS CHROMATOGRAPHY – MASS SPECTROMETRY (MOST RECENT).
• Measures the amount of hydrogen peroxide produced in the Lipids is soluble with water and blood however insoluble with inorganic
reaction. solvent such as chloroform.
• Measured at 500nm.
• Intensity of color produced is directly proportional to cholesterol LIPOPROTEINS
concentration. o Fasting specimen is required! (12-14hrs).
• ADVANTAGE: NO extraction step needed. o SPECIMEN: Serum/Plasma
• INTERFERENCES: Vitamin C, Bilirubin, Hemoglobin. o METHODS:
- Water (H2O) is for hydrolysis. 1. Ultracentrifugation (based on density)
- Oxygen (O2) is for oxidation. 2. Electrophoresis
- Hydrogen peroxide (H2O2). 3. Chemical Precipitation
*centrifuge lipoproteins for 24hrs.

ULTRACENTRIFUGATION
• REFERENCE METHOD for quantitation of lipoproteins.
• Based on the protein and triglyceride contents of lipoproteins.
• EXPRESSED in Svedberg (S) units.
• REAGENT: Potassium bromide solution with 1.063 density.

o CDC REFERENCE METHOD – Abell Levy and Brodie Method (cdc)

- Uses hexane for extraction.
- Alcoholic KOH for saponification.
- Reaction with Liebermann Burchardt Reagent.

:) DGS
 CLINICAL CHEMISTRY 1: LIPIDS AND LIPOPROTEINS LABORATORY DETERMINATION
PROFESSOR: Kristina Kaira B. Lacia, RMT – FACULTY OF MEDICAL LABORATORY SCIENCE
2nd Semester I A.Y. 2023-2024 I PRE-FINAL

LIPIDS AND LIPOPROTEINS LABORATORY DETERMINATION

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ELECTROPHORESIS REFERENCE VALUES:

• Allow separation of major lipoproteins.
• Preferred supporting medium: Agarose gel.
• Based on size and chart
• HDL migrate to ALPHA REGION during electrophoresis so the other
name is (HLD) ALPHA LIPOPROTEIN.
• VLDL migrate to PRE-BETA REGION during electrophoresis so the
other name is (HLD) PRE-BETA LIPOPROTEIN.
• LDL migrate to BETA REGION during electrophoresis so the other
name is (HLD) BETA LIPOPROTEIN.
• CM migrate to GAMMA REGION during electrophoresis.
• IDL (INTERMIDIATE DENSITY LIPOPROTEIN) migrates between the
LDL and VLDL.

Conversion factor of glucose: mmol-mg/Dl

ADDITIONAL NOTES:
CHEMICAL PRECIPITATION • Initial screening must be done at the age of 20 or older.
• Uses polyanions (heparin, dextran sulfate) and divalent cations • Total cholesterol, HLD-C, LDL-C, TRIGLYCERIDES.
(manganese, magnesium). • Testing should be repeated at least once every 5 years.
• Most commonly used for HDL.
• Aggregation of lipoproteins relies on charges.

HDL-C Method
HDL-C (carries cholesterol).
• Homogenous Assay
• Allows automation: Highly precise and accurate.
1. First Reagent – “block” non-HDLs.
2. Second Reagent – quantifies HDL.
• CDC REFERENCE MTHOD:
• 3-STEP PROCEDURE:
1. Removal of VLDL and chylomicrons via
ultracentrifugation.
2. Heparin in manganese precipitation to remove LDL.
3. Analysis via Abell – Kendall Assay (Cholesterol
Measurement).

LDL-C Method
LDL-C (carries exogenous cholesterol)
• Beta-quantification (reference method)
✓ Combination of ultracentrifugation and chemical
precipitation.
1. Ultracentrifugation – remove VLDL and CM (PRODUCT
1).
2. Chemical precipitation – separate HDL from LDL
(PRODUCT 2),
3. Cholesterol is measured in PRODUCT 1 and 2.
4. LDL is calculated by subtracting cholesterol
measurements in PRODUCT 2 from PRODUCT 1.
• Friedewald Equation
Example: lingling test lipids panel, TC = 230mg/Dl HDL = 25mg/Dl
triglyceride = 120mg/Dl (use the 3rd formula) (230-25)+(120/5)
=229md/Dl

:) DGS