Transcript Lipids - University of Florida

Lipids and Lipoproteins
Roger L. Bertholf, Ph.D.
Associate Professor of Pathology
Director of Clinical Chemistry & Toxicology
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


OH

O
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
Unsaturated fatty acids
OH
O
Palmitoleic acid (16:19, 9-hexadecanoic acid)
• Double bonds in fatty acids are nearly
always cis
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
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)
Glycerol esters (acylglycerols)
H
H
H
C
OH
H
C
O
CO
(CH2)n
CH3
H
C
OH
H
C
O
CO
(CH2)n
CH3
H
C
OH
H
C
O
CO
(CH2)n
CH3
H
Glycerol
H
Triglyceride
• Triglycerides are the most abundant family
of lipids in plant and animal cells, and are
major components of the the human diet
Measuring triglycerides
(reference method)
Triglycerides
KOH
fatty acids + glycerol
Periodate
formic acid + formaldehyde
chromogen
=570 nm chromotropic acid
• Triglycerides are extracted into chloroform
prior to analysis
Measuring triglycerides
(enzymatic method)
Triglycerides
Lipase
Glycerol + FFAs
Glycerokinase
ATP
Glycerophosphate + ADP
Glycerophasphate
oxidase
Dihydroxyacetone + H2O2
Quinoneimine dye
max 500 nm
Peroxidase
Sterols (cholesterol)
12
11
HO
13
17
C 14 D 16
15
1
9
2
10
8
A
B
3
5
7
6
4
• 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
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
Cholesterol biosynthesis
Acetyl-CoA
3-Hydroxy-3-methylglutaryl-CoA
HMG-CoA reductase
Squalene
Cholesterol + Lecithin
LCAT
Mevalonate
Cholesterol ester
“Statin” drugs inhibit this enzyme
Measuring cholesterol by L-B
L-B reagent
H2SO4/HOAc
HO
HOO2S
Cholesterol
Cholestahexaene sulfonic acid
max = 620 nm
• 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
Cholesterol esters
Cholesteryl
ester
hydroxylase
Cholesterol
Cholesterol
oxidase
Choles-4-en-3-one + H2O2
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
Lipoproteins
• In order to be transported in blood, lipids must
combine with water-soluble compounds, such as
phospholipids and proteins.
Lipoprotein classes
%TG
%Chol
LPE
Chylomicrons
86
3
Origin
VLDL
55
12
Pre-
IDL
23
29
Pre-/
LDL
6
42

HDL
3
15

Lp(a)
(LDL)
(LDL)
Pre-
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
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
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
Neg, cloudy -2
VLDL
V
Pos, cloudy
-2
VLDL Chylos
Measuring HDL cholesterol
• Ultracentrifugation is the most accurate method
– HDL has density 1.063 – 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
Dextran sulfate
Mg++
HDL + (IDL, LDL, VLDL)
• 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, 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
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
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
Apolipoproteins
• The protein composition differs from one
lipoprotein class to another, and 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
Lipoprotein
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
Cholesterol metabolism (exogeneous)
Dietary cholesterol,
triglycerides
C,TG
B
A
C,TG C
B
Chylomicron
C,TG
Hepatocyte B/E receptors
B
Chylomicron
remnant
LPL
A
Endothelium
Apo-C, E from HDL
B
Hepatocyte B-100
receptors
VLDL
C
C,TG
B
B
LDL
IDL
Endothelium
C,TG C
LPL
Cholesterol metabolism (endogeneous)
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
Dyslipoproteinemias
• Hyperchylomicronemia
– LPL deficiency
– Apo C-II deficiency
Hyperchylomicronemia
Dietary cholesterol,
triglycerides
C,TG
B
A
C,TG C
B
Chylomicron
Chylomicrons
Triglycerides
HDL
LDL
Hepatocyte B/E receptors
C,TG
B
Chylomicron
remnant
LPL
A
Endothelium
Apo-C, E from HDL
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
B
Hepatocyte B-100
receptors
LDL
Normal TG
VLDL
C
C,TG
B
B
LDL
IDL
Endothelium
C,TG C
LPL
Hyperbetalipoproteinemia
Dyslipoproteinemias
• Combined hyperlipoproteinemia
– Normal LDL receptors
– Overproduction of VLDL and Apo B-100
B
Hepatocyte B-100
receptors
LDL
Normal TG
VLDL
C
C,TG
B
B
LDL
IDL
Endothelium
C,TG C
LPL
Combined hyperlipoproteinemia
Dyslipoproteinemias
• Combined hyperlipoproteinemia
– Normal LDL receptors
– Overproduction of VLDL and Apo B-100
• Dysbetalipoproteinemia
– Both cholesterol and triglyceride elevated
– Mutant form of Apo E
Dysbetalipoproteinemia
Dietary cholesterol,
triglycerides
C,TG
B
A
C,TG C
B
Chylomicron
Cholesterol
TG
Hepatocyte B/E receptors
C,TG
B
Chylomicron
remnant
LPL
A
Endothelium
Apo-C, E from HDL
Dyslipoproteinemias
• Familial hypercholesterolemia
– Defect in LDL receptor
• Absent
• Defective
– Incidence = 1:500
B
Hepatocyte B-100
receptors
LDL
or n TG
HDL
VLDL
C
C,TG
B
B
LDL
IDL
Endothelium
C,TG C
LPL
Familial hypercholesterolemia
Dyslipoproteinemias
• Familial hypercholesterolemia
– Defect in LDL receptor
• Absent
• Defective
– Incidence = 1:500
• Familial defective Apolipoprotein B-100
B
Hepatocyte B-100
receptors
or n LDL
VLDL
C
C,TG
B
B
LDL
IDL
Endothelium
C,TG C
LPL
Familial hypercholesterolemia
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
High TG, normal cholesterol
• Diet/Lifestyle
• 2° to diabetes, thiazide diuretics, Cs, betablockers, CRF/Nephrotic syndrome
• Familial hypertriglyceridemia (etiology
unknown)
• ApoC-III excess (interferes with LPL)
• LPL deficiency
• ApoC-II deficiency
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)
Low cholesterol, low/normal HDL
• Abetalipoproteinemia (ApoB degraded after
synthesis causes fat malabsorption)
• Hypobetalipoproteinemia (genetically
defective ApoB)
• Chylomicron retention disease (unknown
cause)
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
High HDL
• Lifestyle (ethanol)
• 2° to phenytoin, phenobarbitol, rifampicin
(p-450 inducers) and estrogens
• Cholesteryl Ester Transfer Protein defects