1. Lipids and Lipoproteins
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Director of Clinical Chemistry & Toxicology

2. Classification of lipids
Fatty acids (palmitic, linoleic, etc.)
Glycerol esters (triglycerides)
Sterols (cholesterol, hormones, vitamin D)
Terpenes (vitamins A, E, K)
Sphingosine derivatives (sphingomyelin)

3. Fatty acids Even-numbered fatty acids predominate  
Lauric acid (C12, 12:0, n-dodecanoic acid)
Even-numbered fatty acids predominate
The most common saturated fatty acids are palmitic (16:0) and stearic (18:0), but unsaturated fatty acids are more common in nature

4. Unsaturated fatty acids
Palmitoleic acid (16:19, 9-hexadecanoic acid)
Double bonds in fatty acids are nearly always cis

5. 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 therefore must be obtained from the diet (plants).
Both are required for the biosynthesis of prostaglandins

6. Clinical importance of fatty acids
Fecal fatty acids are sometimes measured to detect malabsorptive and pancreatic disorders—the test is mostly considered obsolete
Serum free fatty acids help distinguish between hyperinsulinemic hypoglycemia (FFA normal) and disorders of fatty acid oxidation (FFA elevated and negative ketones)

7. Glycerol esters (acylglycerols)
Triglyceride
Triglycerides are the most abundant family of lipids in plant and animal cells, and are major components of the the human diet

8. Measuring triglycerides (reference method)
fatty acids + glycerol
KOH
formic acid + formaldehyde
Periodate
chromogen
=570 nm
chromotropic acid
Triglycerides are extracted into chloroform prior to analysis

9. Measuring triglycerides (enzymatic method)
Glycerol + FFAs
Lipase
Glycerophosphate + ADP
Glycerokinase
ATP
Glycerophasphate
oxidase
Dihydroxyacetone + H2O2
Quinoneimine dye
max 500 nm
Peroxidase

10. Sterols (cholesterol)
Sterols are steroid backbones that have a hydroxyl group at position 3 and a branched aliphatic chain of 8 or more carbons at position 17

11. Cholesterol biosynthesis
About 2% (approximately 1 g) 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 (although all cells have the capability)
Absorption of dietary cholesterol appears to have a maximum of approximately 1 g/day

12. Cholesterol biosynthesis
Acetyl-CoA
3-Hydroxy-3-methylglutaryl-CoA
“Statin” drugs inhibit this enzyme
HMG-CoA reductase
Squalene
Mevalonate
Cholesterol + Lecithin
Cholesterol ester
LCAT

13. 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

14. Enzymatic cholesterol methods
Cholesterol esters
Cholesterol
Cholesteryl
ester
hydroxylase
Choles-4-en-3-one + H2O2
Cholesterol
oxidase
Quinoneimine dye (max500 nm)
Phenol
4-aminoantipyrine
Peroxidase
Enzymatic methods are most commonly adapted to automated chemistry analyzers
The reaction is not entirely specific for cholesterol, but interferences in serum are minimal

15. Lipoproteins
In order to be transported in blood, lipids must combine with water-soluble compounds, such as phospholipids and proteins.

16. Lipoprotein classes %TG %Chol LPE Chylomicrons 86 3 Origin VLDL 55 12
Pre-
IDL 23 29
Pre-/
LDL 6 42 
HDL 15 
Lp(a)
(LDL)

17. Appearance of hyperlipidemia
Standing Plasma Test for chylomicrons
Plasma is placed in refrigerator (4°C) overnight
Chylomicrons accumulate as floating “cream” layer
Chylomicrons in fasting plasma are abnormal

18. Lipoprotein electrophoresis 
Pre- - +
Migration
Chylomicrons
LDL
IDL
VLDL
Lp(a)
HDL
LEP is no longer a common laboratory test
Standing plasma test for chylomicrons
Total cholesterol, TG, HDL, and LDL can be measured directly

19. Fredrickson classification
Type
Refrig.
LPE
LPs I
Pos, clear
Normal
TG (chylos)
IIa
Neg, clear
  band
LDL
IIb
Neg, cloudy
 , pre- 
LDL, VLDL
III
Occ., cloudy
 pre- 
Chol, TG, VLDL IV
-2
VLDL V
Pos, cloudy
VLDL Chylos

20. Measuring HDL cholesterol
Ultracentrifugation is the most accurate method
HDL has density – 1.21 g/mL
Routine methods precipitate apolipoprotein B with a polyanion/divalent cation
Includes VLDL, IDL, Lp(a), LDL, and chylomicrons
HDL, IDL, LDL, VLDL
HDL + (IDL, LDL, VLDL)
Dextran sulfate
Mg++
Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

21. Indirect LDL cholesterol
Friedewald formula assumes that all cholesterol is VLDL, LDL, and HDL lipoproteins
Chylomicrons are usually low in normal, fasting subjects, and IDL and Lp(a) are usually insignificant contributors to total cholesterol
Since VLDL is 55% TG and 12% Chol:
[LDL Chol] = [Tot Chol] – [HDL Chol] – [TG]/5

22. Direct LDL cholesterol
Older direct methods for LDL involved 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

23. 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 -VLDL, which has a 3:1 TG:C ratio

24. Apolipoproteins
The protein composition differs from one lipoprotein class to another, and the protein constituents are called Apolipoproteins

25. 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

26. Apolipoprotein content of LPs
Apolipoprotein(s)
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

27. Cholesterol metabolism (exogeneous)
Dietary cholesterol,
triglycerides
Apo-C, E from HDL
C,TG B A
Chylomicron
C,TG B A C E
Endothelium
LPL
C,TG B E
Hepatocyte B/E receptors
Chylomicron
remnant

28. Cholesterol metabolism (endogeneous)
Endothelium
C,TG
LPL C E B
VLDL
Hepatocyte B-100
receptors C
C,TG B E B
LDL
IDL

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

30. Dyslipoproteinemias Hyperchylomicronemia LPL deficiency
Apo C-II deficiency

31. Hyperchylomicronemia
Dietary cholesterol,
triglycerides
Apo-C, E from HDL
C,TG B A
Chylomicron
C,TG B A C E
Endothelium
LPL
Chylomicrons
Triglycerides
HDL
LDL
C,TG B E
Hepatocyte B/E receptors
Chylomicron
remnant

32. Dyslipoproteinemias Hyperchylomicronemia Hyperbetalipoproteinemia
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

33. Hyperbetalipoproteinemia
Endothelium
C,TG
LPL C E B
VLDL
Hepatocyte B-100
receptors C
C,TG B E
LDL
Normal TG B
LDL
IDL

34. Dyslipoproteinemias Combined hyperlipoproteinemia Normal LDL receptors
Overproduction of VLDL and Apo B-100

35. Combined hyperlipoproteinemia
Endothelium
C,TG
LPL C E B
VLDL
Hepatocyte B-100
receptors C
C,TG B E
LDL
Normal TG B
LDL
IDL

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

37. Dysbetalipoproteinemia
Dietary cholesterol,
triglycerides
Apo-C, E from HDL
C,TG B A
Chylomicron A
Endothelium
C,TG
LPL C E B
Cholesterol TG
C,TG E B
Hepatocyte B/E receptors
Chylomicron
remnant

38. Dyslipoproteinemias Familial hypercholesterolemia
Defect in LDL receptor
Absent
Defective
Incidence = 1:500

39. Familial hypercholesterolemia
Endothelium
C,TG
LPL C E B
VLDL
Hepatocyte B-100
receptors C
C,TG B E
LDL
or n TG
HDL B
LDL
IDL

40. Dyslipoproteinemias Familial hypercholesterolemia
Defect in LDL receptor
Absent
Defective
Incidence = 1:500
Familial defective Apolipoprotein B-100

41. Familial hypercholesterolemia
Endothelium
C,TG
LPL C E B
VLDL
Hepatocyte B-100
receptors C
C,TG
or n LDL E B B
LDL
IDL

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

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

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)

45. Low cholesterol, low/normal HDL
Abetalipoproteinemia (ApoB degraded after synthesis causes fat malabsorption)
Hypobetalipoproteinemia (genetically defective ApoB)
Chylomicron retention disease (unknown cause)

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 degredation)
LCAT deficiency

47. High HDL Lifestyle (ethanol)
2° to phenytoin, phenobarbitol, rifampicin (p-450 inducers) and estrogens
Cholesteryl Ester Transfer Protein defects