Lipids and Lipoproteins

• Lipids are more commonly referred to as fats
• Insoluble in water but soluble in organic solvents
• Major forms of lipids:
  • FATTY ACIDS
    • Simplest
    • Building blocks of lipids
    • Saturated (no double bonds) or unsaturated (with double bonds)
  • TRIGLYCERIDES
    • Tri – three molecules of fatty acids + one molecule of glycerol
    • Breakdown is facilitated by lipoprotein lipase
    • Primary cause of turbid serum
    • Main storage form of lipid
    • Requires a fasting specimen (12-14 hours)
    • > 500mg/dL highg risk for CAD
    • RV: <500 mg/dL                 –           normal
                  150-199 mg/dL           –           borderline high
                  200-499 mg/dL           –           high TAG
                  >500 mg/Dl                 –           very high TAG (acute / recurrent pancreatitis)
  • CHOLESTEROL
    • Not readily catabolized = not a source of fuel
    • No fasting is required
    • Four ringed structure made by hepatocytes
    • Constituent of cell membranes and precursor of some hormones (steroids: progestin, glucocorticoids, mineralocorticoids, androgen and estrogen).
    • Estrogen promotes transport and excretion of CHOLE
    • Should be measured in adults 20 y/o at least once every 5years.
    • RV:
      • <200 mg/dL                 =          desirable
      • 200 – 239 mg/dL         =          borderline high         
      • 240                           =          high cholesterol
    • Two forms: esterified (60-70%) and free cholesterol (30-40%)
    • TAG and Chole most important lipids in management of CAD
  • PHOSPHOLIPIDS
    • Structure: 2 fatty acids + phospholipid attached to glycerol
    • Most abundant lipid
    • Can also be found as surfactants in lungs. Def in neonates: RDS
    • Forms: Lecithin/phosphatidylcholine (major, 70-75%), sphingomyelin (18-20%), phosphatidylserine and phosphatidylethanolamine (3-6%) and lysophosphatidylcholine (4-9%)
    • RV: 150 – 380 mg/dL (serum)
    •  Sphingomyelin
      • Component of cell membranes (RBC and nerve sheath)
      • Niemann-pick dxs: accumulation in the liver and spleen. (lipid storage disorder)

• LIPOPROTEINS
  • Carrier proteins for lipids
  • Major lipoproteins
    1. Chylomicrons: largest and least dense.
       • Contains mostly TAG.
       • Produced in the intestines.
    2. VLDL/Pre-beta lipoprotein. Made in the liver.
    3. HDL/ Alpha Lipoprotein: smallest but most dense lipoprotein.
       • Removes excess cholesterol from cells.
       • Produced by liver and intestine.
       • Maintains balance of cholesterol.
       • CDC Reference method for determination: ultracentrifugation, precipitation with heparin-MnCl₂ and Abell-Kendal assay.
    4. LDL/Beta Lipoprotein: Marker of CHD risk.
       • most cholesterol-rich and most atherogenic.
       • major end-product of VLDL catabolism.

• Minor lipoproteins:
  1. IDL – Subclass
     • Migrates either in the pre-beta or beta region
     • Major apolipoprotein: Apo B-100
  2. Lp(a) aka sinking pre-beta, linked to atherosclerosis
• Abnormal lipoproteins: LpX – linked to obstructive jaundice, β-VLDL aka floating β lipoprotein
  • Indicator of cholestasis.
• Beta-VLDL: floating beta lipoprotein
  • Migrates with LDL in beta region
    found in type 3 hyperlipoproteinemia or dysbetalipoproteinemia.
  • VLDL rich in cholesterol

APOLIPOPROTEINS

• Apo A – major protein component of HDL
  • Apo A-I: LCAT activator
  • Apo A-II: may inhibit hepatic & lipoprotein lipases; increases plasma TAG
• Apo B – major protein component of LDL
  • Apo B-48: found in chylomicron
  • Apo B-100: synthesized in liver; found in VLDL & LDL
• Apo C – major protein component of VLDL; minor in HDL and LDL
  • Apo C-I: may inhibit the hepatic uptake of VLDL and cholesterol ester transfer protein
  • Apo C-II: if deficient – there would be reduced clearance of TAG-rich lipoproteins
  • Apo C-III: main form found in HDL. Lipolysis of TAG-rich lipoproteins is inhibited by this form
• Minor apolipoproteins
  • Apo D: aids in the activation of LCAT
  • Apo E: Arginine rich
    • Apo E-I
    • Apo E-II: associated with type III hyperlipoproteinemia
    • Apo E-III: most common isoform
    • Apo E-IV: associated with high levels of LDL, increased risk for Alzheimer’s and CHD
    • Apo F, Apo H and Apo J

LIPID QUANTITATION

1. TRIGLYCERIDES
   1. CHEMICAL METHOD (Van Handel and Zilversmit method and Modified Van Handel Zilversmit method)

STEP 1: EXTRACTION BY ORGANIC SOLVENT

• This is for the removal of lipids from proteins
• There is an additional adsorption step to remove non-triglycerides

STEP 2: SAPONIFICATION OR HYDROLYSIS BY KOH IN ETOH

• TAG à fatty acids + glycerol

STEP 3: OXIDATION

• Oxidizes glycerol to measurable compounds

STEP 4:  COLORIMETRY

• 500-600nm

1. 1. ENZYMATIC METHOD – lipase and glycerokinase serve in the initial enzymatic reaction
2. TOTAL CHOLESTEROL
   1. COLOR REACTION
      • Liebermann Burchardt Reaction
        • Principle: Dehydration and Oxidation of cholesterol to form a colored compound
        • Reagents: Acetic anhydride-sulfuric acid
        • End product: Cholestadienyl monosulfonic acid – GREEN
      • Salkowski Reaction
        • Methods:
          • Bloor’s method – extraction of cholesterol by Bloor’s, L-B reaction
      • Abell-Kendall method – extraction of cholesterol by Zeolite, L-B reaction
   2. Enzymatic Method
      • Cholesterol oxidase reaction measures amount of hydrogen peroxide produced.
      • Interference: (+) hemoglobin, (-) Bilirubin and ascorbic acid.

CDC reference method: Abell, Levy and Brodie method (3 step method: Saponification, extraction, and colorimetry)

3. HDL
   • Methods: Electrophoresis & Modified Bloor’s
4. Ultracentrifugation: density gradient
   • Reference method for quantitation of lipoprotein.
   • Svedverg (s) units
   • Reagent: potassioum bromide solution with 1.063 density.
5. Electrophoresis
6. Chemical precipitation (HDL and LDL)

*plasma is placed in a test tube and stored at 4°C overnight. Presence of “cream” floating and turbidity of plasma is observed for presence of chylomicron and VLDL respectively