TriglycerideTriglyceride-Rich Lipoproteins
and Cardiovascular Disease
Tao Wang, Ph.D.
Otsuka Maryland Research Institute
Rockville, Maryland, USA
Plaque of Coronary Atherosclerosis
Endothelial Cells
Basement Membrane
Lipids
Smooth Muscle Cells
Adventitia
Media
Intima
Lipid Accumulation
Fatty Core of Plaque
Thrombosis
Fat Swollen Muscle Cells
Fibrous Cap of Plaque
1
Triglycerides and CHD Risk
A metameta-analysis of prospective population studies (multivariate model)
Men
PROCAM
CSCHDS
N=
2,536
2,966
3,395
4,129
4,407
4,860
Summary
22,293
FHS
WCGS
ROG
LRC
Women
FHS
LRC
2,969
3,376
Summary
6,345
0.5
1.0
2.0
3.0
5.0
Relative Risk
From Hokanson JE, Austin MS. J Cardiovascular Risk 1996;3:213-9.
Combined Effects of Cholesterol and Triglycerides on
Risk of Fatal Myocardial Infarction in the AMORIS Study
From Walldius G, et al. Blood Pressure 1992; 1 (Suppl. 1): 35-42.
2
Association between Hypertriglyceridemia and
Cardiovascular Disease via Other Mechanisms
Small, Dense LDL
Oxidized Lipoproteins
TG
Remnant Lipoproteins
CVD
Reduced HDL
Thrombogenic Factors
Triglyceride Concentration as an Estimate of
Total TriglycerideTriglyceride-rich Lipoproteins
Triglycerides
Chylomicrons
VLDL
VLDL remnants
Chylomicron
remnants
Remnant
Lipoproteins
IDL
LDL
3
Questions on the Use of Triglyceride
To Assess CHD Risk
• Should it be measured?
• What does it mean?
• Should it be treated if elevated?
• What are the treatments for elevated TG?
Limitations of Using Triglyceride
To Assess CHD Risk
• Lack of independent effects
• Patients with grossly elevated plasma triglyceride
concentration (e.g., type I or V hyperlipoproteinemia)
are not typically associated with higher CHD risk
• Measurement is fraught with problems
• Reflection of total triglyceride-rich lipoproteins which
are very heterogeneous
4
TriglycerideTriglyceride-rich Lipoproteins and Their Components
Neither cholesterol nor triglyceride is freely circulated in blood.
They are carried by lipoproteins. Different lipoproteins have
different atherogenic potentials.
LDL-C
Atherosclerosis Progression
HDL-C
Atherosclerosis Regression
Cholesterol
Lipoprotein
Remnants
Atherosclerosis
Progression
VLDL-C &
Chylomicrons
?
Triglycerides
Metabolism of TriglycerideTriglyceride-rich Lipoproteins
Peripheral
circulation
CM
remnants
CM
B48
B E
B
A
A
EC
E
A C
HDL A
E
C
B100
E C
Lipoprotein
lipase
C EC
B
C
E
B
B E
C
E C
ApoB/E
ApoB/E
receptors
E
C
VLDL
Liver
VLDL
remnants
E
B
E
C
E
B
B
C
IDL
LDL
5
Key Steps in Metabolism of
TriglycerideTriglyceride-Rich Lipoproteins
Peripheral
circulation
CM
remnants
CM
B
A
B E
E CC
EC
A E
A C
HDL
Lipoprotein
lipase
E
C
C
B
C
VLDL
E
E
B
C
1.
Exchange of apo C with HDL
Apo C-II is an essential cofactor for
lipoprotein lipase
2.
Hydrolysis by lipoprotein lipase
Chylomicrons and VLDL are depleted
with TG and enriched in cholesterol.
They are transformed to chylomicron
remnants and VLDL remnants,
respectively.
3.
Removal of apo C from remnants
and enrichment with remnants with
apo E
Apo E is a ligand for apo B/E
receptors which remove lipoprotein
remnants from circulation, whereas
Apo C-III impairs this process
Liver
B E
EC
ApoB/E
ApoB/E
receptors
E
B
C
VLDL
remnants
E
Physical and Chemical Properties
of Lipoprotein Remnants
• Smaller and denser
• Have altered eletrophoretic mobility
• Depleted with triglycerides and enriched in cholesterol
• Enriched in apo E
Words of caution:
All these properties are relative to their lipoprotein precursors.
Remnants are a group of very heterogeneous lipoproteins.
They are probably best described by their physiological
properties than their physical or chemical properties.
6
Biological Effects of Lipoprotein Remnants
• Impair endothelium-dependent vasorelaxation
• More likely to be retained by heparan sulfate proteoglycans
within the arterial intima
• Bind to chylomicron receptors on macrophages and being
taken up without modification
• Enhance in vitro aggregation of platelets
Measurement of Lipoprotein Remnants
• Ultracentrifugation: mostly for IDL
• Agarose gel electrophoresis: mostly for double pre-beta, or
beta VLDL
• Calculation: VLDL-C / TG or (VLDL-C - 0.17 x TG) / 0.521
• Polyacrylamide gel electrophoresis: mostly for “mid-band”,
which is similar to IDL
• Chemical precipitation followed by apo C measurement
• Immunoseparation of remnant-like particles (RLP): mostly
for apo-E enriched chylomicron remnants and VLDL remnants.
7
Measurement of IDL by Ultracentrifugation
d = 1.019 kg/L
d = 1.006 kg/L
T6
Chylomicron
+ VLDL
T9
IDL
B6
LDL
B9
HDL
IDL = B6 - B9
Measurement of betabeta-VLDL by Ultracentrifugation
followed by Agarose Gel Electrophoresis
Normal
Normal
Type III
HDL
VLDL
LDL
6
m
6
6
m
m 06
6
6
ru
0
00 eru .00 .00 eru
00 .00
1
1.
S <1 >1
S
Se < 1. > 1.
<
>
d
d
d
d
d
d
β-VLDL
Measurement of β -VLDL requires the use of both ultracentrifugation
(separation based on hydrated density) and agarose gel electrophoresis
(separation based on charges)
8
Lipoprotein Measurement by LipoPhor
HDL
LDL
VLDL
VLDL
LDL
HDL
IDL
VLDL
LDL
IDL (“mid-band”)
HDL
LipoPhor is a commercial product based on polyacrylamide gel
electrophoresis which separates lipoproteins by charge and molecule size
Principle of the RLPRLP-Cholesterol Assay
Immunoseparation Gel
+
Test Sample
RLP
LDL + nascent VLDL
HDL
Sepharose 4B +
anti-apo B-100
Sepharose 4B +
anti-apo A-1
Incubation at RT for 2 hours
Cholesterol
Quantification
9
RLPRLP-C as an Independent Risk Factor for CHD
Variable
p
OR
Hypertension
< 0.0001
3.72
Age > 65 yr
< 0.0001
2.66
0.0001
2.14
< 0.0005
2.17
< 0.01
1.59
0.71
1.07
HDL-C < 35 mg/dL
Sex
th
RLP-C > 75 %ile
LDL-C > 130 mg/dL
# of CHD Patients = 213, # of Control = 2761
from cycle 4 of the Framingham Offspring Study
RLPRLP-C Values in “Normolipidemic” CAD Patients
6.6 mg/dL
All CAD cases and controls had total cholesterol < 240 mg/dL and TG < 200 mg/dL
From Devaraj, et al. Am J Med 1998;104:445-50.
10
RLPRLP-C Values in Type II Diabetic Patients
with or without Macrovascular Diseases
Control
(N = 24)
Type II DM without
MVD (N =24)
Type II DM with
MVD (N = 24)
Age (yr)
55.4 ± 7.2
57.3 ± 7.4
60.0 ± 6.5
BMI (kg/m²)
27.5 ± 6.8
31.6 ± 6.0
31.7 ± 7.8
Cholesterol (mmol/L)
5.1 ± 0.8
5.0 ± 1.2
4.9 ± 0.9
TG (mmol/L)
1.3 ± 0.5
1.9 ± 1.2
1.6 ± 1.0
LDL-C (mmol/L)
3.4 ± 0.8
3.2 ± 1.0
3.2 ± 0.9
HDL-C (mmol/L)
1.3 ± 0.4
1.1 ± 0.3
1.2 ± 0.4
RLP-C (mmol/L)
0.15 ± 0.06
0.20 ± 0.10
0.23 ± 0.08 a,b
RLP-C/TG
0.06 ± 0.03
0.06 ± 0.02
0.08 ± 0.03 b,c
a,b: Compared with controls: a, p < 0.05 ; b, p < 0.005
c:
Compared with type II DM without MVD: p < 0.05
From Hirany, et al. Clin Chem 2000;46:667-72.
Predictability of Future Coronary Events By RLPRLP-C
135 patients with pre-existing CAD were followed-up for up to 3 years. Elevated RLP-C
concentrations were the only independent predictor for future coronary events in a
multivariate Cox proportional hazard analysis model including stenosis of the left main
artery, number of diseased arteries, age, gender, history of smoking, diabetes mellitus,
hypertension, hypercholesterolemia, low HDL-C, high LDL-C and hypertriglyceridemia.
From Kugiyama, et al. Circulation 1999;99:2858-60.
11
Conclusions
• Hypertriglyceridemia has been found to be associated with increased
CHD risk. But the independent effect was not proven.
• Triglyceride concentrations only reflect total triglyceride-rich lipoproteins.
Measurement of more atherogenic lipoprotein remnants offers better
performance in assessing CHD risk.
• Lipoprotein remnants can be measured by ultracentrifugation, agarose
gel electrophoresis, polyacrylamide gel electrophoresis and
immunoseparation. Each method measures a unique fraction of
lipoprotein remnants.
• Lipoprotein remnants isolated by the RLP-Cholesterol Assay have been
found to have atherogenic properties in in vitro experiments and proven
to be an independent risk factor for CHD in clinical studies.
• Measurement of lipoprotein remnants provides additional information on
individuals’ risk for CHD. It should not be used to replace other other
lipid and lipoprotein tests.
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