Cholesterol Measurement Methods

Lipids

• Lipids are constituents of all living cells, composed mostly of C-H bonds
• They serve as energy storage for excess calories, and are precursors for steroid hormones, prostaglandins, leukotrienes, and lipoxins

Classification of Fatty Acids

• Based on length:
  • Short: 4-6 carbon atoms
  • Medium: 8-12 carbon atoms
  • Long: more than 12 carbon atoms
• Based on the number of C=C double bonds:
  • Saturated: no double bonds
  • Monounsaturated: one double bond
  • Polyunsaturated: contain 2 or more double bonds

Triglycerides

• Contain 3 fatty acids attached to 1 molecule of glycerol by ester bonds
• No charged groups, water insoluble, neutral lipid

Phospholipids

• Contain 2 fatty acids attached to 1 molecule of glycerol
• 3rd position contains phospholipid head groups, which are amphipathic (hydrophobic head and hydrophilic tail)

Cholesterol

• Unsaturated steroid alcohol containing 4 rings and a single C-H side chain tail
• Synthesized from acetyl-CoA
• Also an amphipathic lipid

Classification of Lipoproteins

• Chylomicrons:
  • Largest lipoproteins, lowest density
  • Contain 86% triglyceride, 5% cholesterol, 7% phospholipid, and 2% apolipoprotein
  • Apoproteins B-48, A-I, C-I, C-II, and C-III on their surface
• VLDL (Very-Low-Density Lipoprotein):
  • Carries endogenous triglycerides synthesized in the liver
  • Composed of 55% triglycerides, 19% cholesterol, 18% phospholipid, and 8% apolipoprotein
  • Apoproteins B-100, C-I, C-II, C-III, and E on their surface
• IDL (Intermediate-Density Lipoprotein):
  • Transitional form, formed from VLDL and modified in the liver to LDL
  • Carries endogenous triglycerides and cholesterol esters
  • Composed of 23% triglycerides, 38% cholesterol, 19% phospholipid, and 19% apolipoprotein
  • Apoproteins B-100, and some E on their surface
• LDL (Low-Density Lipoprotein):
  • Also known as "bad cholesterol"
  • Transports a large amount of endogenous cholesterol
  • Composed of 50% cholesterol, 22% phospholipids, 6% triglycerides, and 22% protein
  • Apoprotein B-100 on their surface
• HDL (High-Density Lipoprotein):
  • Also known as "good cholesterol"
  • Synthesized in the intestine and liver cells
  • Recycled chylomicron and VLDL molecules
  • Composed of 50% protein, 28% phospholipids, 19% cholesterol, and 3% triglycerides
  • Apoproteins A-I, mainly, and A-II on their surface
• Lp(a) (Lipoprotein(a)):
  • Composed primarily of cholesterol esters, phospholipids, and apolipoprotein (a) and B-100
  • Elevated levels are associated with increased risk for coronary heart disease, myocardial infarction, and cerebrovascular disease

Lipid Disorders

• Arteriosclerosis: deposition of esterified cholesterol in artery walls
• CAD, PVD, CVD: associated with abnormal lipid metabolism
• Dyslipidemia: abnormal levels of lipids in the blood
• Hypercholesterolemia: high levels of cholesterol in the blood
• Hypertriglyceridemia: high levels of triglycerides in the blood
• Combined hyperlipidemia: high levels of both cholesterol and triglycerides in the blood

Lipid and Lipoprotein Analyses

• Decision cutpoints used to characterize CHD risk developed by NCEP
• Standardization programs facilitate reliable classification of patients
• Pre-analytical considerations:
  • Age: cholesterol levels increase with age
  • Sex: women have lower levels than men except in childhood and early 50's
  • Season: cholesterol levels are slightly higher in cold periods
  • Food intake: daily intake of fat increases cholesterol levels
  • Acute illness: recommended to wait 8 weeks after any form of trauma or acute bacterial/viral infection and 3-4 months after child birth
  • Lifestyle: higher in sedentary and poor diet habits
  • Prolonged venous occlusion: leads to an increase in cholesterol by 10-15%

Cholesterol Measurement

• Non-enzymatic (chemical) method: Abell-Kendall or Bloors method
• Enzymatic method: Glycerol Kinase method
• Reference method: GC-MS

Triglyceride Measurement

• Non-enzymatic (chemical) method: chloroform and phospholipids removal by zeolite absorption
• Enzymatic method: Glycerol Kinase method
• Reference method: GC-MS

Lipoprotein Measurement

• Polyanion precipitation: lipoproteins are precipitated with polyanions (heparin sulfate, dextran sulfate, and phosphotungstate)

• Electrophoresis: used to identify rare familial disorders

• Ultracentrifugation: preparative ultracentrifugation for quantitative purposes and preparative isolations

• Immunochemical methods: use antibody-coated plates for specific epitopes on apolipoproteins### Lipoprotein Measurement

• Immunoturbidimetry is the most common method used to measure apolipoproteins, which can be easily adapted to spectrophotometric analyzers.

• Immunonephelometry measures light scattering, but may be affected by larger triglyceride-rich lipoproteins and VLDL.

• Other immunochemical methods include ELISA, Radial Immunodiffusion, and RIA.

Hyperlipoproteinemias

• Type I: Elevated chylomicrons, characterized by a creamy layer of chylomicrons over clear serum, extremely elevated triglycerides, and normal to moderately elevated total cholesterol.
• Type IIa: Increased LDL, characterized by clear serum, generally elevated total cholesterol, and normal triglycerides.
• Type IIb: Increased LDL and VLDL, characterized by clear or slightly turbid serum, elevated total cholesterol, and elevated triglycerides.
• Type III: Increased IDL, characterized by a creamy layer sometimes present over a turbid layer, elevated total cholesterol, and Apo E-II increased.
• Type IV: Increased VLDL, characterized by turbid serum, moderately to severely elevated triglycerides, and normal to slightly elevated total cholesterol.
• Type V: Increased VLDL with increased chylomicrons, characterized by turbid serum with a creamy layer, severely elevated triglycerides, and slightly to moderately elevated total cholesterol.

Familial Hyperlipoproteinemias

• Familial combined hyperlipidemia (FCHL): Characterized by increased plasma levels of total cholesterol, triglyceride, or both, and increased Apo B-100.
• Hyperapobetalipoproteinemia: Characterized by normal or moderate elevation of LDL cholesterol, elevated apo B-100, and VLDL and apo B-100 overproduction in the liver.
• Familial hypertriglyceridemia: Characterized by moderate elevation of triglyceride with excess production of VLDL.
• Familial hypercholesterolemia: Characterized by increased LDL cholesterol, normal or slightly increased triglyceride, and slightly decreased HDL cholesterol.

Secondary Lipoproteinemia

• Many conditions can cause abnormal lipoprotein metabolism, including diabetes mellitus, hyperthyroidism, obesity, pregnancy, nephrotic syndrome, pancreatitis, alcoholism, and myxedema.

Hypolipoproteinemias

• Abetalipoproteinemia: Characterized by very low total cholesterol, undetectable triglyceride, and absent LDL and Apo B-100.
• Hypobetalipoproteinemia: Characterized by inability to synthesize apo B-100 and apo B-48, low total cholesterol, and normal to low triglyceride.
• Hypoalphalipoproteinemia: Characterized by severely elevated triglyceride and low HDL.
• Tangier disease: Characterized by absent HDL, very low apo A-I and apo A-II, low LDL, low total cholesterol, and normal to slightly increased triglyceride.

Apo A-1, Apo B, and Lp(a) Clinical Significance

• Apo A-1 is antiatherogenic, activates lecithin-cholesterol acyltransferase (LCAT), and removes free cholesterol from extrahepatic tissues.
• Apo B-100 is associated with increased risk of coronary artery disease.
• Lp(a) is an independent risk factor associated with impaired plasminogen activation and decreased fibrinolysis.