Lipids and Lipoproteins (CC lab)
Lipids and Lipoproteins
Classification of lipids
Fatty acids (palmitic, linoleic, etc.)
Glycerol esters (triglycerides)
Sterols (cholesterol, hormones, vitamin D)
Terpenes (vitamins A, E, K)
Sphingosine derivatives (sphingomyelin)
Fatty acids
Even-numbered fatty acids predominate
Most common saturated fatty acids are palmitic (16:0) and stearic (18:0)
Unsaturated fatty acids are more common in nature
Lauric acid (C12, 12:0, n-dodecanoic acid) is an example
Unsaturated fatty acids
Double bonds in fatty acids are nearly always cis
Palmitoleic acid (16:1^9, 9-hexadecanoic acid) is an example
Essential fatty acids
Mammals can synthesize saturated and mono-unsaturated fatty acids
Linoleic (18:2) and linolenic (18:3) fatty acids cannot be synthesized and must be obtained from the diet
Both are required for the biosynthesis of prostaglandins
Clinical importance of fatty acids
Fecal fatty acids are sometimes measured to detect malabsorptive and pancreatic disorders
Serum free fatty acids help distinguish between hyperinsulinemic hypoglycemia and disorders of fatty acid oxidation
Glycerol esters (Triacylglycerols)
Triglycerides are the most abundant family of lipids in plant and animal cells
Major components of the human diet
Measuring triglycerides (reference method)
Triglycerides are extracted into chloroform prior to analysis
Measuring triglycerides (enzymatic method)
Triglycerides are converted to glycerol and free fatty acids by lipase
Glycerol is further converted to glycerophosphate and ADP by glycerokinase
Dihydroxyacetone and H2O2 are produced by glycerophosphate oxidase
Quinoneimine dye is used to measure the reaction at lmax ~500 nm
Sterols (cholesterol)
Sterols are steroid backbones with a hydroxyl group at position 3 and a branched aliphatic chain at position 17
Cholesterol biosynthesis
Approximately 2% of total body cholesterol is replenished each day
Dietary sources account for less than half
Cholesterol is synthesized from Acetyl CoA
90% of in vivo synthesis occurs in the intestine and liver
Absorption of dietary cholesterol appears to have a maximum of approximately 1 g/day
Measuring cholesterol by L-B
The Liebermann-Burchard method is used by the CDC to establish reference materials
Cholesterol esters are hydrolyzed and extracted into hexane prior to the L-B reaction
Enzymatic cholesterol methods
Enzymatic methods are most commonly adapted to automated chemistry analyzers
The reaction is not entirely specific for cholesterol, but interferences in serum are minimal
Lipoproteins
Lipids must combine with water-soluble compounds, such as phospholipids and proteins, to be transported in blood
Lipoprotein classes
Chylomicrons: 86% TG, 3% Chol
VLDL: 55% TG, 12% Chol
IDL: 23% TG, 29% Chol
LDL: 6% TG, 42% Chol
HDL: 3% TG, 15% Chol
Lp(a) (LDL): Pre-b
Appearance of hyperlipidemia
Standing Plasma Test for chylomicrons
Chylomicrons accumulate as a floating "cream" layer in refrigerated plasma
Chylomicrons in fasting plasma are abnormal
Lipoprotein electrophoresis
LEP is no longer a common laboratory test
Total cholesterol, TG, HDL, and LDL can be measured directly
Chylomicrons, LDL, VLDL, and Lp(a) can be identified by migration
Fredrickson classification
Type I: Pos, clear (Normal)
Type IIa: Neg, clear (b band, LDL)
Type IIb: Neg, cloudy (b, pre-b, LDL, VLDL)
Type III: Occ., cloudy (pre-b, Chol, TG, VLDL)
Type IV: Neg, cloudy (a-2, VLDL)
Type V: Pos, cloudy (a-2, VLDL)
Measuring HDL cholesterol
Ultracentrifugation is the most accurate method
Routine methods precipitate apolipoprotein B with a polyanion/divalent cation
Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol
Indirect LDL cholesterol
Friedewald formula assumes that all cholesterol is VLDL, LDL, and HDL lipoproteins
Chylomicrons are usually low in normal, fasting subjects
IDL and Lp(a) are usually insignificant contributors to total cholesterol
[LDL Chol] = [Tot Chol] – [HDL Chol] – [TG]/5
Direct LDL cholesterol
Older direct methods involve precipitation with heparin or polyvinyl sulfate
Newer methods involve precipitation of VLDL, IDL, and HDL with polyvalent antibodies to Apo A and Apo E
LDL is almost exclusively Apo B-100
Direct vs. Indirect LDL
The Friedewald equation assumes that chylomicrons, IDL, and Lp(a) are not significant
Non-fasting specimens can have chylomicrons
TG > 400 mg/dL indicates the presence of chylomicrons (or remnants)
Type III hyperlipidemia is characterized by high b-VLDL, which has a 3:1 TG:C ratio
Apolipoproteins
The protein composition of lipoproteins differs from one class to another
The protein constituents are called Apolipoproteins
Functions of apolipoproteins
Activate enzymes involved in lipid metabolism (LCAT, LPL)
Maintain structural integrity of lipid/protein complex
Delivery of lipids to cells via recognition of cell surface receptors
Apolipoprotein content of LPs
Chylomicron: AI, B-48, CI, CII, CIII
VLDL: B-100, CI, CII, CIII, E
IDL: B-100, E
LDL: B-100
HDL: AI, AII
Lp(a): (a), B-100
Cholesterol metabolism (exogenous)
Dietary cholesterol and triglycerides are transported as chylomicrons
Chylomicrons acquire Apo-C and Apo-E from HDL
Lipoprotein lipase (LPL) hydrolyzes TG in chylomicrons
Chylomicron remnants are taken up by hepatocytes via B/E receptors
Cholesterol metabolism (endogenous)
VLDL is synthesized in the liver and secreted into the bloodstream
VLDL acquires Apo-C and Apo-E from HDL
LPL hydrolyzes TG in VLDL, forming IDL
IDL can be taken up by hepatocytes or further metabolized to LDL
LDL is taken up by cells via LDL receptors
Lipoprotein classes
Chylomicrons: 86% TG, 3% Chol
VLDL: 55% TG, 12% Chol
IDL: 23% TG, 29% Chol
LDL: 6% TG, 42% Chol
HDL: 3% TG, 15% Chol
Lp(a) (LDL): Pre-b
Page 29: Dyslipoproteinemias
Causes can be primary or secondary
Secondary causes include starvation, liver disease, renal failure, diabetes, hypothyroidism, lipodystrophies, drugs
Primary causes of hyperlipidemia:
Increased production
Defective processing
Defective cellular uptake
Inadequate removal
Page 30: Dyslipoproteinemias Hyperchylomicronemia
LPL deficiency
Apo C-II deficiency
Page 31: Hyperchylomicronemia
Dietary cholesterol, triglycerides
Chylomicron
Apo-C, E from HDL
LPL
Endothelium
Chylomicron remnant
Hepatocyte B/E receptors
Chylomicrons
Triglycerides
HDL
LDL
Page 32: Dyslipoproteinemias Hyperchylomicronemia
LPL deficiency
Apo C-II deficiency
Hyperbetalipoproteinemia
Overproduction of VLDL
Enhanced conversion of VLDL to LDL
LDL enriched with cholesteryl esters
Defective LDL structure
Decreased LDL receptors
Page 33: Hyperbetalipoproteinemia
VLDL
LPL
Endothelium
LDL
IDL
Hepatocyte B-100 receptors
LDL
Normal TG
Page 34: Dyslipoproteinemias Combined hyperlipoproteinemia
Normal LDL receptors
Overproduction of VLDL and Apo B-100
Page 35: Combined hyperlipoproteinemia
VLDL
LPL
Endothelium
LDL
IDL
Hepatocyte B-100 receptors
LDL
Normal TG
Page 36: Dyslipoproteinemias
Combined hyperlipoproteinemia
Normal LDL receptors
Overproduction of VLDL and Apo B-100
Dysbetalipoproteinemia
Both cholesterol and triglyceride elevated
Mutant form of Apo E
Page 37: Dysbetalipoproteinemia
Dietary cholesterol, triglycerides
Chylomicron
Apo-C, E from HDL
LPL
Endothelium
Chylomicron remnant
Hepatocyte B/E receptors
Cholesterol
TG
Page 38: Dyslipoproteinemias Familial hypercholesterolemia
Defect in LDL receptor
Absent
Defective
Incidence = 1:500
Page 39: Familial hypercholesterolemia
VLDL
LPL
Endothelium
LDL
IDL
Hepatocyte B-100 receptors
LDL
or n TG
HDL
Page 40: Dyslipoproteinemias
Familial hypercholesterolemia
Defect in LDL receptor
Absent
Defective
Incidence = 1:500
Familial defective Apolipoprotein B-100
Page 41: Familial hypercholesterolemia
VLDL
LPL
Endothelium
LDL
IDL
Hepatocyte B-100 receptors
or n LDL
Page 42: High cholesterol, high LDL
Diet/Lifestyle
2° to hypothyroidism or nephrotic syndrome (disruption of Apo-B metabolism)
Polygenic: (means we don’t know)
Familial hypercholesterolemia
Familial defective Apo-B
Rare disorders
Page 43: High TG, normal cholesterol
Diet/Lifestyle
2° to diabetes, thiazide diuretics, Cs, beta-blockers, CRF/Nephrotic syndrome
Familial hypertriglyceridemia (etiology unknown)
ApoC-III excess (interferes with LPL)
LPL deficiency
ApoC-II deficiency
Page 44: High cholesterol, TG
Obesity
2° to steroids, Cs, hypothyroidism, CRF
Familial combined hyperlipidemia (multifactorial)
Peroxisome proliferator-activator receptor
Dysbetalipoproteinemia (Type III)
Hepatic lipase deficiency (rare)
Page 45: Low cholesterol, low/normal HDL
Abetalipoproteinemia (ApoB degraded after synthesis causes fat malabsorption)
Hypobetalipoproteinemia (genetically defective ApoB)
Chylomicron retention disease (unknown cause)
Page 46: Low HDL
Lifestyle
2° to steroids, beta-blockers, progestogens
Familial hypoalphalipoproteinemia (ApoA- I, C-III, or A-IV defects)
ApoA-I variants
Tangier disease (enhanced HDL degradation)
LCAT deficiency
Page 47: High HDL
Lifestyle (ethanol)
2° to phenytoin, phenobarbitol, rifampicin (p-450 inducers) and estrogens
Cholesteryl Ester Transfer Protein defects