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Chemistry

Principles

Lipid testing in the lab helps to assess cardiovascular risk, diagnose lipid disorders, and monitor treatment

  • Core Lipid Tests
    • Total Cholesterol: Measures all cholesterol in the blood
    • Triglycerides: Measures the amount of triglycerides in the blood
    • HDL Cholesterol: Measures high-density lipoprotein cholesterol
    • LDL Cholesterol: Measures low-density lipoprotein cholesterol
    • Apolipoproteins: Measures specific apolipoproteins associated with lipoproteins

Total Cholesterol

  • Principle: Enzymatic methods are commonly used. Cholesterol esterase hydrolyzes cholesterol esters to free cholesterol. Cholesterol oxidase then oxidizes free cholesterol to cholest-4-en-3-one and hydrogen peroxide (H2O2). H2O2 is then reacted with a chromogen in the presence of peroxidase (POD) to produce a colored product, which is measured spectrophotometrically
  • Reactions
    1. Cholesterol Esters –(Cholesterol Esterase)–> Cholesterol + Fatty Acids
    2. Cholesterol + O2 –(Cholesterol Oxidase)–> Cholest-4-en-3-one + H2O2
    3. 2 H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + 2 H2O
  • Detection: The intensity of the colored product is directly proportional to the total cholesterol concentration in the sample
  • Advantages: Accurate, precise, and widely available
  • Disadvantages: Can be affected by interfering substances such as bilirubin and hemoglobin

Triglycerides

  • Principle: Enzymatic methods are used. Lipoprotein lipase (LPL) hydrolyzes triglycerides to glycerol and free fatty acids. Glycerol is then phosphorylated by glycerol kinase (GK) to glycerol-3-phosphate (G-3-P). G-3-P is oxidized by glycerol-3-phosphate oxidase (GPO) to dihydroxyacetone phosphate (DAP) and H2O2. H2O2 is then reacted with a chromogen in the presence of peroxidase (POD) to produce a colored product, which is measured spectrophotometrically
  • Reactions
    1. Triglycerides –(Lipoprotein Lipase)–> Glycerol + 3 Fatty Acids
    2. Glycerol + ATP –(Glycerol Kinase)–> Glycerol-3-Phosphate + ADP
    3. Glycerol-3-Phosphate + O2 –(Glycerol-3-Phosphate Oxidase)–> Dihydroxyacetone Phosphate + H2O2
    4. 2 H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + 2 H2O
  • Detection: The intensity of the colored product is directly proportional to the triglycerides concentration in the sample
  • Advantages: Accurate, precise, and widely available
  • Disadvantages: Can be affected by interfering substances such as bilirubin and lipemia

HDL Cholesterol

  • Principle
    • Direct Methods: Use selective detergents or antibodies to block non-HDL lipoproteins (LDL, VLDL, chylomicrons), leaving only HDL to be measured. The HDL cholesterol is then measured using a cholesterol oxidase-peroxidase method similar to that used for total cholesterol
    • Indirect Methods: Use precipitation reagents to selectively precipitate non-HDL lipoproteins, followed by measurement of cholesterol in the supernatant containing only HDL
  • Reactions
    • Direct Methods
      1. Selective Detergents/Antibodies block non-HDL lipoproteins
      2. HDL Cholesterol + O2 –(Cholesterol Oxidase)–> Cholest-4-en-3-one + H2O2
      3. 2 H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + 2 H2O
    • Indirect Methods
      1. Non-HDL lipoproteins are precipitated
      2. HDL Cholesterol + O2 –(Cholesterol Oxidase)–> Cholest-4-en-3-one + H2O2
      3. 2 H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + 2 H2O
  • Detection: The intensity of the colored product is directly proportional to the HDL cholesterol concentration in the sample
  • Advantages
    • Direct methods are automated and less susceptible to interferences
    • Indirect methods are inexpensive but more labor-intensive
  • Disadvantages
    • Indirect methods can be affected by incomplete precipitation or carryover of non-HDL lipoproteins
    • Direct methods can be affected by certain interfering substances

LDL Cholesterol

  • Principle
    • Friedewald Equation (Calculation): LDL cholesterol is estimated using the Friedewald equation:
    • LDL Cholesterol = Total Cholesterol - HDL Cholesterol - (Triglycerides / 5) (all in mg/dL)
    • LDL Cholesterol = Total Cholesterol - HDL Cholesterol - (Triglycerides / 2.2) (all in mmol/L)
    • This equation is valid only when triglycerides are < 400 mg/dL (4.5 mmol/L)
    • Direct Methods: Use selective detergents or antibodies to block non-LDL lipoproteins (HDL, VLDL, chylomicrons), leaving only LDL to be measured. The LDL cholesterol is then measured using a cholesterol oxidase-peroxidase method similar to that used for total cholesterol
  • Reactions
    • Direct Methods
      1. Selective Detergents/Antibodies block non-LDL lipoproteins
      2. LDL Cholesterol + O2 –(Cholesterol Oxidase)–> Cholest-4-en-3-one + H2O2
      3. 2 H2O2 + Chromogen –(Peroxidase)–> Oxidized Chromogen (Colored) + 2 H2O
  • Detection: The intensity of the colored product (for direct methods) is directly proportional to the LDL cholesterol concentration in the sample
  • Advantages
    • Friedewald equation is inexpensive and widely used
    • Direct methods are more accurate and can be used when triglycerides are elevated
  • Disadvantages
    • Friedewald equation is not accurate when triglycerides are > 400 mg/dL (4.5 mmol/L) or in certain dyslipidemia
    • Direct methods are more expensive

Apolipoproteins

  • Principle: Immunochemical methods are commonly used to measure specific apolipoproteins. Antibodies specific to the apolipoprotein are used to capture and quantify the apolipoprotein in a blood sample
  • Methods
    • Turbidimetric Immunoassay (TIA): Antibody-apolipoprotein complex formation is measured by the change in turbidity (light scattering)
    • Nephelometry: Antibody-apolipoprotein complex formation is measured by the amount of light scattered at an angle
    • Enzyme-Linked Immunosorbent Assay (ELISA): Enzyme-labeled antibodies are used, and the enzyme activity is measured to quantify the antibody-apolipoprotein complex
  • Detection: The amount of antibody-apolipoprotein complex formed is measured, and is proportional to the apolipoprotein concentration
  • Advantages: Specific and automated
  • Disadvantages: Can be affected by interfering substances and requires specialized equipment

Lipoprotein (a)

  • Principle: Immunochemical methods are commonly used to measure Lp(a). Antibodies specific to Lp(a) are used to capture and quantify the Lp(a) in a blood sample
  • Methods
    • Turbidimetric Immunoassay (TIA): Antibody-Lp(a) complex formation is measured by the change in turbidity (light scattering)
    • Nephelometry: Antibody-Lp(a) complex formation is measured by the amount of light scattered at an angle
    • Enzyme-Linked Immunosorbent Assay (ELISA): Enzyme-labeled antibodies are used, and the enzyme activity is measured to quantify the antibody-Lp(a) complex
  • Detection: The amount of antibody-Lp(a) complex formed is measured, and is proportional to the Lp(a) concentration
  • Advantages: Specific and automated
  • Disadvantages: Can be affected by interfering substances and requires specialized equipment

Lipid Electrophoresis

  • Principle: Separates lipoproteins based on their electrical charge and size
  • Procedure
    • Serum is applied to a support medium (e.g., agarose gel or paper)
    • An electric field is applied, causing lipoproteins to migrate at different rates
    • Lipoproteins are stained with a lipid-specific dye
  • Detection: The separated lipoprotein bands are visualized and quantified
  • Clinical Significance
    • Helps identify specific lipoprotein abnormalities, such as:
      • Elevated chylomicrons
      • Increased VLDL
      • Increased LDL
      • Decreased HDL
    • Used to diagnose and classify hyperlipidemias

Key Terms

  • Cholesterol Esterase: An enzyme that hydrolyzes cholesterol esters
  • Cholesterol Oxidase: An enzyme that oxidizes cholesterol
  • Peroxidase: An enzyme that catalyzes the reaction between hydrogen peroxide and a chromogen
  • Lipoprotein Lipase (LPL): An enzyme that hydrolyzes triglycerides in lipoproteins
  • Glycerol Kinase: An enzyme that phosphorylates glycerol
  • Glycerol-3-Phosphate Oxidase: An enzyme that oxidizes glycerol-3-phosphate
  • Chromogen: A substance that produces a colored product
  • Turbidimetric Immunoassay (TIA): An immunoassay that measures the change in turbidity
  • Nephelometry: An immunoassay that measures the amount of light scattered
  • Enzyme-Linked Immunosorbent Assay (ELISA): An immunoassay that uses enzyme-labeled antibodies
  • Friedewald Equation: An equation used to estimate LDL cholesterol
  • Apolipoproteins: Proteins that bind to lipoproteins