A New Look at CHD Risk Factors Total Cholesterol LDL-Cholesterol HDL-Cholesterol Lipoprotein(a) ...

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