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A New Look at CHD Risk Factors
Total Cholesterol
LDL-Cholesterol
HDL-Cholesterol
Lipoprotein(a)
Homocysteine
Particle size/density
Lp subclasses
Triglycerides
Inflammatory factors
Oxidized-LDL (Ox-LDL) Metabolic syndrome Prothrombotic factors Apolipoproteins Glucose impairment Subclinical atherosclerotic
diseases Diabetes mellitus Life-habit risk factors
LDL = TC – (HDL + VLDL)LDL = TC – (HDL + VLDL) (TG/5 (TG/5 approximatesapproximates VLDL) VLDL)
Recommendation: Not valid with TG > Recommendation: Not valid with TG > 400 mg/dL400 mg/dL
LDL = Lp(a) + IDL + Remnant Lp + LDLLDL = Lp(a) + IDL + Remnant Lp + LDL
Total Error = Bias + (1.96 x CVTotal Error = Bias + (1.96 x CVImprecisionImprecision) ) < 12% < 12% (ideal: + 4% & CV < 4%)(ideal: + 4% & CV < 4%)
From: From: Clin ChemClin Chem 41:1414-1420(1995) 41:1414-1420(1995)
Measure LDL-C More AccuratelyMeasure LDL-C More Accurately
Friedewald Friedewald Calculation:Calculation:
Accuracy of LDL-c Calculation Method
Triglycerides Percent Correct PercentMg/dL (Within + 10%) Incorrect
< 200 93% 7%
201-300 75% 25%
301-400 61% 39%
401-500 41% 59%
> 500 < 20% > 80%
From: Warnick GR et. al. Clin Chem 36:15-19 (1990)
Triglycerides Correct LDL-CIncorrect LDL-Cmg/dL (Within + 10%)
< 200 85%
15%
201-300 77% 23%
301-400 59% 41%
401-600 41% 59%
Accuracy of LDL-c Calculation Method
From: Warnick GR et. al. Clin Chem 36:15-19 (1990)
10%
52%
46%
13%
0%
10%
20%
30%
40%
50%
60%
<130 130-160 160-190 > 190
% ClassifiedIncorrectly
Friedewald Misclassifies Risk Category
When Triglycerides > 177 mg/dL
Friedewald Misclassifies Risk Category
When Triglycerides > 177 mg/dL
Marniemi J et al. Clin Biochem 1995 June; 28:285-289.
LDL Risk Category
-4%-7%
-15%-19%
-20%
-15%
-10%
-5%
0%
% Error in LDL Chol.
Friedewald LDL Cholesterol
180 135 93 61
Directly Measured LDL Cholesterol 187 146 109 75
When LDL-C Target is < 100 Friedewald Should Not Be Utilized
When LDL-C Target is < 100 Friedewald Should Not Be Utilized
*Scharnagl H et al. Clin Chem Lab Med 2001 May;39(5):426-31.
Evaluation of Four Homogeneous Direct LDL-C Methods
Evaluation of Four Homogeneous Direct LDL-C Methods
Study: LDL-C methods from Genzyme, Reference Diagnostics (RD), Roche, and Sigma were evaluated for precision, accuracy, and specificity for LDL in the presence of abnormal lipoproteins.
Results: Precision was < 2% CV for all methods; and correlation to the CDC reference method was r = 0.955-1.067; total error was Genzyme = 12.6%, RD = 16.5%, Sigma = 38.3%, Roche = 41.6%.
Conclusion: The methods show nonspecificity toward abnormal lipoproteins, thus compromising accuracy. These direct methods are no better than the Friedewald LDL-C.
From: Miller, WG et.al. Clin Chem 48:489-498(2002)
1. Ultracentrifugation:Reference method, accuracy based
Time-consuming, tedious, technically difficult, costly
2. Electrophoresis:Simultaneous separation of major lipoprotein fractions, can quantitate, visualize some unusual bands
Technically difficult to do; unless automated, it can be tedious and time consuming, can be costly
Review of LDL-C MethodsReview of LDL-C Methods
+
+
-
-
Review of LDL-C Methods(Cont.)
Review of LDL-C Methods(Cont.)
No pretreatment, full automation, improved analytical precision, fasting specimen not required, save on labor cost
Can have lack of LDL specificity; inaccuracy because of analytical interferences from TG, bilirubin, IDL-C, Lp-X, VLDL-C, Lp(a), apo E-rich HDL, and/or hemoglobin; can be costly
From: Nauck M et. al. Clin Chem 48:236-254 (2002)
3. Third Generation Direct Methods:+
-
HDL-c TestingHDL-c Testing
• NCEP Laboratory Goals
– Total Error = ≤ 13% (analytical bias +1.9% CV)
– TE = Ideal: ≤ 5% and ≤ 4% CV
• CDC Reference Method: 3 Stage Procedure
1. Ultracentrifuge = remove chylomicron + VLDL
2. Heparin MnCl2 ppt. = remove apoB lipoprotein
3. Abell-Kendall cholesterol assay on HDL
• CRMLN Designated Comparison Method
– Dextran Sulfate (50,000 DA) ppt.
– Abell Kendall cholesterol assay on HDL
From: Warnick et. al. Clin Chem 47:1579-1596 (2001)
There are 4 types of homogeneous HDL-C methods: immunologic, PEG, synthetic polymer, enzymatic.
All can be automated & directly measure HDL.
Most have excellent precision (< 3% CV).
Many have CRMLN certification, suggesting that accuracy is possible with proper instrument, reagents, and calibrator.
Specificity and interferences: most are robust and can tolerate TG < 900 mg/dL, but biases can occur with atypical lipoprotein patterns.
Homogenous HDL-c MethodsHomogenous HDL-c Methods
1. Ultracentrifugation:Comparison method for accuracy
Time-consuming, tedious, technically difficult, costly, can have interference from Lp(a) and others
2. Electrophoresis:Simultaneous separation of major lipoprotein fractions, can quantitate, visualize some atypical bands
Technically difficult to do; unless automated, it can be tedious and time consuming, can be costly
Review of HDL-c MethodsReview of HDL-c Methods
+
+
-
-
Review of HDL-C Methods(Cont.)
Review of HDL-C Methods(Cont.)
No pretreatment, full automation, improved analytical precision (<3% CV), fasting specimen not required, save on labor cost (20% savings)
Can have lack of HDL specificity; inaccuracy because of analytical interferences from bilirubin, hemoglobin, high TG (chylomicrons and VLDL-c), Lp(a), apo E-rich HDL, and/or HDL variants (Apo A-IMilano)
From: Nauck M et. al. Clin Chem 48:236-254 (2002)
3. Third Generation Direct Methods:+
-
• Completely automated; better precision
• Better gel/buffer system (high-resolution)
• Precision control of temperature during electrophoresis
• Densitometer designed for higher sensitivity
• Simultaneously measures VLDL-c, LDL-c, HDL-c & Lp(a)-c
• LDL-c and HDL-c compares well with CDC reference methods
• Lp(a) compares well with Lp(a) candidate reference method
New Approaches to Quantitative Lipoprotein Electrophoresis
Pool VLDL-C LDL-C HDL-Cmg/dL mg/dL mg/dL
B 8.1 ± 0.4 149.1 ± 2.0 41.4 ± 1.5(5.3% CV) (1.3% CV) (3.6% CV)
From: Naito HK, et al. Handbook of Lipoprotein Methods. AACC Press, Washington D.C., Pgs. 477-495(1997)
Within-Run with Helena Electrophoretic MethodWithin-Run with Helena Electrophoretic Method
Comparison of Methods: Helena Electrophoresis vs.
CDC Reference Method
Fractions Regression r
VLDL-C y=0734 + 0.389x 0.97682LDL-C y=1.783 + 1.07x 0.99809HDL-C y=2.449 + 1.1x 0.99241
From: Naito HK, et al. Handbook of Lipoprotein Methods. AACC Press, Washington D.C., Pgs. 477-495(1997)
Comparison of Methods: Helena vs. Ultracentrifugation
Fractions Regression r
VLDL-C y= 0.929x - 6.39 0.947LDL-C y= 1.006x + 5.27 0.962HDL-C y= 1.158x - 8.00 0.918Lp(a) y= 0.23x + 0.421 0.906
From: Nauck M, et al. Clin Chem 1995; 41:1761-1767.
LDL-C calculation method is not dependable when compared to some of the current direct LDL-C methods.
Ultracentrifugation methods for LDL and HDL still remains the gold standard for accuracy.
The 3rd-generation electrophoresis methods for lipoprotein-C quantitation have several attractive features.
The 3rd- generation direct or homogenous LDL-C and HDL-C methods have better precision but may suffer from lack of LDL or HDL specificity; thus, accuracy may be compromised.
LDL-c and HDL-c SummaryLDL-c and HDL-c Summary
The selection of your instrument-reagent-calibrator system for LDL-C or HDL-C determinations should be based on the approved list of the CDC CRMLN certificate of traceability.
Periodic verification of accuracy of LDL-C and HDL-C should be done, particularly if the reagent and/or calibrator lot number changes.
More robust LDL-C and HDL-C methods should be employed that are not affected by interfering substances (i.e., abnormal lipoproteins, hypertrigly-ceridemia)
LDL-c and HDL-c Summary (cont)LDL-c and HDL-c Summary (cont)
Lipoprotein(a) ChemistryLipoprotein(a) Chemistry
Structurally resembles LDL
Has a second large polypeptide, Apo(a)
Is polymorphic in size; Molecular weight 420-840 kDa
Has 10 types of kringle 4, which is the basis of the different isoform size variability
Physiology of Lp(a) Physiology of Lp(a)
Is an acute phase protein. Apo(a) is made by the liver and is assembled with apo B-100 on the hepatocyte surface. Lp(a) catabolism is unclear.
Compete with plasma plasminogen for binding sites, resulting in decreased synthesis of plasmin and inhibition of fibrinolysis
Increases cholesterol deposition in the arterial wall
Enhances foam cell formation
Makes O2-free radicals in monocytes
Promotes SMC proliferation
Induces monocyte-chemotactic activity in subendothelial space
Is an acute phase protein. Apo(a) is made by the liver and is assembled with apo B-100 on the hepatocyte surface. Lp(a) catabolism is unclear.
Compete with plasma plasminogen for binding sites, resulting in decreased synthesis of plasmin and inhibition of fibrinolysis
Increases cholesterol deposition in the arterial wall
Enhances foam cell formation
Makes O2-free radicals in monocytes
Promotes SMC proliferation
Induces monocyte-chemotactic activity in subendothelial space
Physiology of Lp(a) (Cont.)Physiology of Lp(a) (Cont.) Mechanism of CAD: Atherogenesis and
Thrombogenesis
Emerging Risk Factor for Vascular Disease
Most prospective and retrospective studies suggest an independent association between Lp(a) and presence and extent of CAD, premature CAD, MI, restenosis after balloon angioplasty, and CVD.
There is evidence for a benefit of lowering Lp(a)
Response to Intervention Therapy
Diet and exercise have no effect: (maybe with monounsaturated fats or caloric restriction with weight loss)
Effect of statins are controversal
Niacin and aspirin will lower
Mechanism of CAD: Atherogenesis and Thrombogenesis
Emerging Risk Factor for Vascular Disease
Most prospective and retrospective studies suggest an independent association between Lp(a) and presence and extent of CAD, premature CAD, MI, restenosis after balloon angioplasty, and CVD.
There is evidence for a benefit of lowering Lp(a)
Response to Intervention Therapy
Diet and exercise have no effect: (maybe with monounsaturated fats or caloric restriction with weight loss)
Effect of statins are controversal
Niacin and aspirin will lower
Lp(a) and CHD: Meta-analysis of 27 Prospective Studies
Lp(a) and CHD: Meta-analysis of 27 Prospective Studies
The study (n = 5436) with a mean follow-up of 10 years showed that if an individual in the general population is in the upper third at baseline, you are at 70% increased risk for CHD compared to persons at the lower one-third.
The Lp(a) association to CHD risk is significant and is independent of the standard vascular risk factors.
From: Danesh J . Circulation 2000; 102: 1082-1085.
The study (n = 5436) with a mean follow-up of 10 years showed that if an individual in the general population is in the upper third at baseline, you are at 70% increased risk for CHD compared to persons at the lower one-third.
The Lp(a) association to CHD risk is significant and is independent of the standard vascular risk factors.
From: Danesh J . Circulation 2000; 102: 1082-1085.
PROCAM Prospective Study: 788 males were followed for 10 yrs; [Lp(a) measured on fresh blood]
The overall risk of a coronary event was 2.7 times higher if Lp(a) was > 20 mg/dL. The risk increased further if there were other risk factors, I.e., LDL-C > 160 mg/dL, HDL-C < 35 mg/dL, BP > 140/90 mmHg
CONCLUSION: Lp(a) is an important independent CHD risk factor that aggravates the coronary risk exerted by elevated LDL-C, low HDL-C, hypertension or the combined effects of multiple risk factors (TG, smoking, diabetes, angina pectoris, and family history of MI)
PROCAM Prospective Study: 788 males were followed for 10 yrs; [Lp(a) measured on fresh blood]
The overall risk of a coronary event was 2.7 times higher if Lp(a) was > 20 mg/dL. The risk increased further if there were other risk factors, I.e., LDL-C > 160 mg/dL, HDL-C < 35 mg/dL, BP > 140/90 mmHg
CONCLUSION: Lp(a) is an important independent CHD risk factor that aggravates the coronary risk exerted by elevated LDL-C, low HDL-C, hypertension or the combined effects of multiple risk factors (TG, smoking, diabetes, angina pectoris, and family history of MI)
Lp(a) Increases CHD Risk In Men With Other Risk Factors
Lp(a) Increases CHD Risk In Men With Other Risk Factors
From: Am Coll Cardiol 2001:37:434-439
Who Should Have an Lp(a) Test Done?Who Should Have an Lp(a) Test Done?
Patients with a normal lipid profile, but have documentation of definite CHD (MI, angina, CABAG, angioplasty, stent implants)
Patients with parents or 1st-degree relatives who died of premature CHD
Patients with known elevation of Lp(a) or parents with elevation of Lp(a)
High-risk African American males
Postmenopausal women
Men with traditional and/or global CHD risk factors; diabetics and patients with renal disease
Patients with a normal lipid profile, but have documentation of definite CHD (MI, angina, CABAG, angioplasty, stent implants)
Patients with parents or 1st-degree relatives who died of premature CHD
Patients with known elevation of Lp(a) or parents with elevation of Lp(a)
High-risk African American males
Postmenopausal women
Men with traditional and/or global CHD risk factors; diabetics and patients with renal disease
It All Began with Some Observations by a Clinical Pathologist and a Cardiologist…
It All Began with Some Observations by a Clinical Pathologist and a Cardiologist…
Let’s prove it!
Clinical StudiesClinical Studies• Retrospective study of 1124 subjects with lipoprotein
electrophoresis (including Lp (a)) performed in a 26 month period. Lipoprotein electrophoresis performed only when abnormal lipids where found apriori and/or in cardiac patients with high hsCRP/histamine. These patients are followed to date.
• Prospective study of CHD compared with healthy subjects matched for age and sex: Distribution according to race and Lp(a).100 patients, (72 w, 28 aa), and 50 healthy subjects.
• Prospective study of 51 patients with poorly controlled T2DM before and after treatment with infusion with a external insulin pump.
Major DiagnosisMajor Diagnosis
T2DM CHD CVD Other
Caucasians 82 90 20 10
African Amer.
88 84 24 14
2 major groups: Caucasians + (Hispanics) and African Americans were considered for statistics.
CHD: coronary heart disease (MI, angina, coronary insufficiency, angioplasty, bypass surgery, restenosis)
CVD: cerebrovascular disease (ischemic stroke, TIA, brain infarction)
T2DM: Type 2 diabetes mellitus ( nephrotic syndrome)
Other: PAD, chronic renal failure
Distribution of Lp(a) Cholesterol According to Race
Distribution of Lp(a) Cholesterol According to Race
Lp(a) < 4.0 Lp(a) 4.1-10.0 Lp(a) > 10.1
Caucasians 344 85 184
African Amer. 40 89 210
Hispanics 49 12 20
Asians 42 6 4
Pakistani+
Indians
34 2 3
Total (%) 509 (45.28) 194 (17.26) 421 (37.45)
Distribution of Lipids in the Cohort with Lp(a) > 4.1
Distribution of Lipids in the Cohort with Lp(a) > 4.1
Chol > 200 TG > 150 HDL < 40
# (%) # (%) # (%)
Caucasians 130 (37.8) 110 (32.0) 86 (25)
African Amer. 29 (72.5) 14 (35) 16 (40)
Hispanics 15 (30.6) 10 (20.4) 14 (8.6)
Asians 12 (28.6) 8 (19.1) 6 (14.3)
Pakistani+
Indians30 (88.2) 14 (41.2) 2 (5.9)
Distribution of Increased Lipoproteins in the Cohort with Lp (a) > 4.1
Distribution of Increased Lipoproteins in the Cohort with Lp (a) > 4.1
VLDL > 35 LDL > 120 HDL < 40 BROAD B
Caucasians #(%)
48 (23.5) 62 (30.4) 40 (19.6) 34 (16.6)
African Amer. #(%)
50 (23.8) 71 (33.8) 38 (18.1) 10 (4.8)
Distribution According to Race & Lp(a) (36w, 21aa)Distribution According to Race & Lp(a) (36w, 21aa)
Lp(a) Caucasians African American
# (%) # (%)
0 – 4 25(69.4) 12(57.1)
4.1 – 10 4(11.1) 4(19.1)
> 10.1 7(19.5) 5(23.8)
Normal Lp(a) ValuesNormal Lp(a) Values
Caucasian African AmericanAll Males Females All Males Females
Mean 2.6 2.4 2.9 3.4 3.2 3.8
SD 2.8 2.8 2.7 3.4 3.4 3.6
Median 1.9 1.6 2.3 2.2 2.0 2.4
95th Percentile 6.2 5.2 6.3 8.6 8.3 8.3
% ≥10 mg/dL 98.3 98.8 97.7 97.9 98.2 97.0
Min 0 0 0.2 0.4 9.2 0.4
Max 11.4 10.9 11.8 11.3 10.9 11.5
N 254 146 108 102 56 46
Lp(a) Non-Diseased Population (Caucasian & African American)
Lp(a) Stratified by Risk ScoreLp(a) Stratified by Risk Score
Framingham Risk AssessmentAll ≤4 5 and 6 7 ≥8
Mean 11.1 3.2 6.6 10.9 14.9
SD 8.7 3.4 5.9 8.8 8.0
Median 7.2 1.9 4.4 9.0 10.6
5th Percentile 2.4 1.5 2.3 4.2 5.4
95th Percentile 22.4 9.8 14.8 18.0 26.3
% ≥10 mg/dL 51.6 7.2 22.5 59.4 65.2
Min 0.2 0 0.6 1.6 3.3
Max 24.4 14.6 22.4 23.2 28.8
N 530 103 105 195 127
Diseased Population
ROC of Lp(a) Cut-offs vs Clinical Sensitivity & Specificity at Framingham Risk Assessment Cut-off of < 4
ROC of Lp(a) Cut-offs vs Clinical Sensitivity & Specificity at Framingham Risk Assessment Cut-off of < 4
0.22.03.95.98.19.912.51518.220
25
28
0
20
40
60
80
100
0 20 40 60 80 100
Area under the curve = 0.944 ± 0.007
Se
ns
itiv
ity
(%
)
1-Specificity (%)
CP1009715-2
Cardiovascular Disease is the Leading Cause of Death in the US
Cardiovascular Disease is the Leading Cause of Death in the US
CV Cancer Accident Pneumonia AIDSdisease Influenza
0.0
0.2
0.4
0.6
0.8
1.0
Mill
ion
sM
illio
ns
Ischemic Events in the U.S.Ischemic Events in the U.S.
1,500,000 heart attacks: 500,000 deaths
500,000 strokes: 150,000 deaths
One third of individuals who experience an ischemic event will die as a result of that event.
Many who have an event have no prior symptoms.
Prevention of Ischemic Cardiovascular Events is Key!!
Prevention of Ischemic Cardiovascular Events is Key!!
• How do we determine who will have an ischemic event?
Risk Factors
• Age, smoking status, hypertension, diabetes, Cholesterol, HDL.
• The ATP-III: NCEP–Provide risk factor screening
guidelines and treatment guidelines based on risk factor analysis.
Cholesterol Screening: NCEPCholesterol Screening: NCEP
• Everyone over 20 years of age
– Every 5 years
• Total Cholesterol• HDL cholesterol• Triglyceride• LDL cholesterol
• Treatment guidelines based on LDL cholesterol concentration
Cholesterol and Cardiovascular DiseasesCholesterol and Cardiovascular Diseases
Prevention of Ischemic Cardiovascular Events is Key!!
Prevention of Ischemic Cardiovascular Events is Key!!
One-third to one-half of ischemic events occur in individuals with LDL < 130 mg/dL and in current guidelines for primary prevention the target is < 130 mg/dL.
Cardiovascular Risk FactorsCardiovascular Risk Factors
• There is a need for additional risk factors.
• Additional risk factors would improve accuracy of decisions regarding preventative therapies
Rader DJ. NEJM. 2000;343:1179-82
monocytechemotaxis
Oxidized LDL
Native LDL
slow
rapid
ICAMVCAME-selectin
O2 radicals
GrowthFactors
Thrombin
smooth musclecell mitogenesis
Homocysteine
Tissue factorVWF
Vasoconstriction
TXA2
Platelet actv.TXA212-HETEB-TBGP-selectin
EndotheliumEndothelinProstacyclinNitric oxideICAM,VCAMADMA
FibrinolysisD-DimerPAI-IPlasminogen
CoagulationFibrinogenPF 1+2TATTFPITPP
Lipids/Oxidation
LDL subclassesLp (a)F2 isoprostanesOxLDLParaoxonase
NutritionHCYB12,B6FolateVit. C,E,Carotene
Inflamm.hsCRPcytokineschlamydiaCMVLp-PLA2
Lp-PLA2Lp-PLA2
LPC, OxFALPC, OxFA
Emerging Risk FactorsProposed Panel
Emerging Risk FactorsProposed Panel
• Homocysteine
• Lipoprotein(a) [Lp(a)]
• High-sensitivity CRP
• Fibrinogen
• Small dense LDL
Currently no guidelines for measurement; may guide intensity of risk reduction therapy in selected patients.
The NCEP ATP-III panel identified these novel markers and indicated that clinicians may utilize them in selected persons to guide intensity of risk reduction therapy and modulate clinical judgment when making therapeutic decisions.
They do not however, identify in which group of patients these markers are best used or how to respond to elevated values.
Emerging Risk FactorsEmerging Risk Factors
JAMA 2001:285;2486-2497.
Use of Novel Risk Markers
Mayo Test Volumes: 2003
– Homocysteine 58,000– hsCRP 26,000– Lp(a) 22,000– LDL subfractions 3,600– Fibrinogen ?????
Mayo Recommendations for use of Extended Risk Marker Panel
Mayo Recommendations for use of Extended Risk Marker Panel
Increasing risk Acute coronary syndrome
CAD and CAD risk equivalents
Low risk population
Extended Marker Panel
6%-9% 10 year risk
>20% 10 year risk
10% - 20% 10 year risk
Mayo Recommendations for Use of Extended Risk Marker Panel
Mayo Recommendations for Use of Extended Risk Marker Panel
• When to Measure– Use to enhance clinical decision making in
persons at intermediate risk for developing an ischemic event as assessed by the Framingham 10 year risk score: 10-20% risk.
• Provide with Request– Age, gender, smoking status, blood pressure
(treated or untreated)
Mayo Recommendations Clinical Response to Elevated Markers
Mayo Recommendations Clinical Response to Elevated Markers
• Interpretations are provided with every report of the novel risk marker panel.
• Interpretations are made by doctoral level staff in Laboratory Medicine or in the Mayo Cardiovascular Health Clinic.
• Interpretations include a description of abnormal values, as well as suggestions for appropriate treatment, given the noted abnormalities.
• A separate document describing up to date information on the background, interpretation and recommended therapy for abnormalities of each of the risk markers is included with the report.
Novel Risk Markers Relation to Angiographic CAD and Events
Novel Risk Markers Relation to Angiographic CAD and Events
Mean Age 60 ± 11 years, 62% Male46% patients have none or mild coronary occlusion
54% patients have significant occlusion (>50% stenosis)Median Follow-up: 4 years
None
Mild1V
2V
3V
Vessel Disease
504 consecutive patients undergoing coronary angiography
Patient DemographicsPatient Demographics
Age (yr) 60±11
Male (%) 62
Hypertension (%) 46
BMI (kg/m2) 29±6
Prior infarction (%) 15
Heart failure (%) 12
Significant CAD (%) 54
ACS (%) 34
Study PopulationStudy Population
• Indication for coronary angiography
–Acute coronary syndrome (34%)
–Positive stress test (25%)
–Exertional dyspnea (27%)
–Other (14%)
AimsAims
• Other CAD risk Factors
– (both traditional and emerging)
• Acute coronary syndromes
• Angiographic CAD
• Clinical outcomes
To analyze for associations between novel risk factors including Lp-PLA2 and:
Lipoprotein Associated Phospholipase A2 (Lp-PLA2)
Lipoprotein Associated Phospholipase A2 (Lp-PLA2)
• AKA: Platelet-activating factor acetylhydrolase
• 50kDa, Ca-insensitive lipase
• Produced predominantly by macrophages and contributes to foam cell formation
• > 80% bound to LDL
• Not responsive to IL-1, IL-6, TNF-alpha.
• Hydrolyzes oxidized phospholipids
Multivariate Logistic Regression Analysis Multivariate Logistic Regression Analysis
• After adjusting for age, gender, smoking history, hypertension, cholesterol, HDL cholesterol, and triglyceride, Lp-PLA2 was no longer was no longer independently predictive of angiographic CAD.
• CRP was not predictive of CAD in either the univariate or multivariate model.
• Data Presented at American College of Cardiology Annual Meeting, March 2003.
HR For Angiographic CAD: MultivariateHR For Angiographic CAD: MultivariateVariable HR 95% CI P-Value
Age 1.05 1.03 - 1.07 <0.0001
Male Gender 4.03 2.46 – 6.60 <0.0001
Hypertension 1.60 1.06 - 2.42 0.026
Smoking 1.45 0.95 - 2.22 0.087Total cholesterol 1.01 1.004 - 1.016 0.001
HDL cholesterol 0.96 0.94 - 0.98 0.0002
Log triglyceride 0.84 0.52 - 1.37 0.481
Log CRP 1.06 0.87 - 1.30 0.532
Fibrinogen 1.16 0.95 - 1.42 0.154
Lp-PLA2 0.91 0.71 - 1.17 0.466
Lp(a) protein (per 20 mg/dL) 1.14 0.91 - 1.43 0.25Lp(a) cholesterol (per 5 mg/dL) 1.56 1.15 - 2.11 0.004
Lp(a) Cholesterol Electrophoresis Helena Laboratories
Lp(a) Cholesterol Electrophoresis Helena Laboratories
LDLVLDLHDL Lp(a)
UC
Non-UC
UC
Non-UC
UC
Non-UC
UC
Non-UC
UC
Non-UC
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
UC Lp(a) = 6Non-UC Lp(a) = 8Lp(a) mass = 125
UC Lp(a) = 8Non-UC Lp(a) = 19Lp(a) mass = 131
UC Lp(a) = < 2Non-UC Lp(a) = < 2Lp(a) mass = <7
UC Lp(a) = 19Non-UC Lp(a) = 21Lp(a) mass = 72
UC Lp(a) = <2Non-UC Lp(a) = <2Lp(a) mass = 11
Baudhuin, et.al., Clinical Biochemistry: 2004
Lp(a) cholesterol can be measured in whole serum
Verified in samples from 470 subjects
Immunologic Lp(a) vs. Lp(a) Cholesterol
Immunologic Lp(a) vs. Lp(a) Cholesterol
y = 8.0083x + 16.934
R2 = 0.558
0
50
100
150
200
250
0 5 10 15 20 25 30
UC Lp(a) cholesterol (mg/dL)
Lp
(a)
mas
s (m
g/d
L)
HR for Events in 425 Non-AMI: UnivariateHR for Events in 425 Non-AMI: UnivariateVariable HR 95% CI
Age 1.6 1.19 – 2.13
Male Gender 1.2 0.70 - 2.01
Hypertension 1.5 0.90 - 2.48
Smoking 1.2 0.73 - 2.03
LDL cholesterol 0.95 0.74 - 1.23
HDL cholesterol 0.82 0.62 - 1.07
Log triglyceride 1.2 0.91 - 1.51
Log CRP 1.5 1.11 - 1.97
Log HCY 1.4 1.10 - 1.74
LDL size 1.0 0.76 - 1.28
Fibrinogen 1.7 1.39 - 2.19
Lp-PLA2 1.3 1.07 - 1.60
Lp(a) protein: > 30 mg/dL 1.3 0.76 - 2.27
Lp(a) cholesterol: detectable 2.3 1.33 – 4.03
HR For CVD in 425 non-AMI: MultivariateHR For CVD in 425 non-AMI: MultivariateVariable HR 95% CI
Age 1.3 0.96 - 1.88
Male Gender 1.2 0.62 - 2.49
Hypertension 1.3 0.74 - 2.30
Smoking 1.2 0.68 - 2.23
LDL cholesterol 1.0 0.69 - 1.45
HDL cholesterol 0.83 0.61 - 1.13
Log triglyceride 0.97 0.70 - 1.34
Log CRP 1.1 0.73 - 1.55
Log HCY 1.2 0.92 - 1.59
LDL size 1.9 0.72 - 1.37
Fibrinogen 1.6 1.16 - 2.29
Lp-PLA2 1.3 1.05 - 1.57
Lp(a) protein: > 30 mg/dL 0.60 0.27 - 1.33
Lp(a) cholesterol: detectable 3.2 1.45 - 7.09
Lipoprotein (a) StructureLipoprotein (a) Structure
Apo (a): -- Sequence homology to plasminogen (genetic variant)-- Size heterogeneity (~300-700 KDa; 3-40 K4-T2 repeats)
Kringle (~13 KDa, ~80 AA)
43
52
1
Cleavage siteby pg activator
Plasminogen (pg)Inactivecleavagesite
H2N
HOOC
H2N
HOOC
SS
K5
K4
T10
T 9T 8 T 7
T 6
T 5
T 4
T 3
T 2
ApoB-100
T1
M 1 S 2 3 S 4 5 S
>S4
S4
S3
S1
B
Apolipoprotein (a) Isoform Identification by Western Blotting
Apolipoprotein (a) Isoform Identification by Western Blotting
Apo (a) Size Standards: >S4: 35 KIV repeats, >700 kDa S4: 27 KIV, ~700 kDa S3: 23 KIV, ~650 kDa S1: 19 KIV, >Apo B-100 (512kDa) B: 14 KIV, <Apo B-100 1- 5: Patient Serum SamplesM: Protein Size Marker, Myosin band (207 kDa)
Apolipoprotein (a) IsoformsApolipoprotein (a) Isoforms
23 and 31
20 and 27
16 12 21 S SS
Overview and ConclusionsOverview and Conclusions
• Prevention of Cardiovascular events is Key!!!
• Know Your Cholesterol, Know Your Risk– Lipid profile in everyone > 20 years of age– Use ATP III guidelines– Determine Framingham Risk Score– Educate Others
• Novel/Emerging Risk Markers– Primarily in patients at intermediate risk– Guide intensity and type of therapy– Much more work needs to be done
ConclusionsConclusions
• Lp-PLA2 is emerging as an independent risk marker for cardiovascular events: orally active specific inhibitors make it a potential therapeutic target.
• Lp(a) cholesterol is a strong marker for angiographic coronary disease as well as cardiovascular events.
• Differences observed between Lp(a) cholesterol and Immunologic Lp(a) mass assays need to be further investigated, but may be due to dependance of the mass assays on apo(a) isoform size
• Efforts to standardize Lp(a) methods need to continue.
Lipid ProfilesLipid Profiles
• Medicare-approved panel– Total Cholesterol, Triglycerides, LDL-c, HDL-c
• Candidate additional analytes– ApoA, ApoB, IDL, Lp(a), LDL Sub, HDL Sub, RLP
Panel ComparisonPanel Comparison
• 266 consecutive subjects• 45% had desirable TC, TG by NCEP
– Of those, 44% had Lp(a) above 55th percentile– 28% had Lp(a) above 75th percentile
• 28% had desirable TC, TG, LDL-c, HDL-c– Of those, 49% had Lp(a) above 55th percentile
Lp(a) CorrelatesLp(a) Correlates
• None of the following are correlates or predictors of Lp(a) values:– Total cholesterol– LDL-c– HDL-c– TG– Apo A1– Apo B100
Lp(a)/Lp(a)-c and Platelet FunctionLp(a)/Lp(a)-c and Platelet Function
• Study of 100 subjects with existing Lp(a) and Lp(a)-c measurements– Lp(a) ranged from <5-231 mg/dL– Lp(a)-c ranged from 0.1-33 mg/dL
• Measured platelet aggregation to collagen/ADP and collagen/EPI by PFA
• Medication history: 34% on ASA or NSAIDS
ResultsResults
• Closure times with either EPI/collagen or ADP/collagen were not decreased as Lp(a) concentrations increased
• No difference between Lp(a) and Lp(a)-c