The New Gold Standard for Lipoprotein Analysis

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Cardiovascular Disease Mortality Cardiovascular Disease Mortality Trends for Males and FemalesTrends for Males and FemalesUnited States: 1979United States: 1979--20022002

Source: CDC/NCHS.

380400420440460480500520

79 81 83 85 87 89 91 93 95 97 99 01

Years

Dea

ths

in T

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ands

Males Females

“The Lipid Panel”

Evolution of Lipoprotein TestingEvolution of Lipoprotein Testing

Direct LDL is Better

Total Cholesterol = VLDL + LDL + HDL

Calculated LDL = TC – (HDL + TG/5)Friedewald Equation: VLDL = TG/5

Lipoprotein Particle Numbers by Subgroup is Best

Consensus Statements: American Diabetes Association & American College of Cardiology (2008)

and now the National Lipid Association (2011)

Lipoprotein Particles or Apo B-100 Predict Risk Better than Cholesterol

NCEP Guidelines for Cardiovascular Disease NCEP Guidelines for Cardiovascular Disease

NCEP Identified a Number of New LipoproteinRisk Factors – To Help Assess Those at Risk

NCEP - ATP III50% of at Risk Individuals are Not Identified

50% of Heart Attack Victims have Normal Cholesterol

ATP IV to come out soon

NCEP New Lipoprotein Risk FactorsNCEP New Lipoprotein Risk Factors

Small Dense LDL –- 3-fold Greater Risk of CVD than Buoyant LDL- Penetrates Arterial Endothelial Lining Easily- Less Recognized by LDL Receptors therefore Increases

HDL 2b & 3 –- HDL 3 Picks Up Excess Cholesterol and Becomes HDL 2b in Reverse Cholesterol Transport

Lp(a) -- Small Particles that are Easily Oxidized- Competes with Plasminogen, Prevents Fibrinolysis

RLP (Remnant Lipoprotein)-- One of the Most Atherogenic Lipoproteins- Skips Oxidation Step in Forming Plaque

High in 25% of population

High in 25% of population

High in 25% of population

Low in 25% of population

Lp(a) Competes with Plasminogen and Lp(a) Competes with Plasminogen and Prevents FibrinolysisPrevents Fibrinolysis

Fibrin

Fibrinogen

Blood Clot(MI, Stroke

or DVT)

Fibrinolysis

Plasmin

Plasminogen

Many of the over 40 genetic variations of Lp(a) mimic plasminogen

Lp(a)

Possible Antithrombotic Therapy Indicated

To Determine the NCEP New Risk FactorsLipoprotein Subgroup Information is Needed

Advanced Lipoprotein Testing Advanced Lipoprotein Testing

What are Lipoproteins and their Subgroups?

How do they Cause Cardiovascular Disease?

Lipoprotein ParticlesLipoprotein Particles

ApolipoproteinA-1 (HDL) or B-100

(LDL)

Unesterified Cholesterol

Cholesterol Ester

Phospholipid

Triglyceride

Atherogenic ParticlesAtherogenic Particles

Size (nm)Size (nm)

Density (g/ml)Density (g/ml)

VLDLVLDL

1.0041.0045050

TGTG--rich rich LipoproteinsLipoproteins

RLPRLP

2525

1.0131.013

BuoyantBuoyantLDLLDL

2222

1.0231.023

DenseDenseLDLLDL

1919

1.0441.044

Mean EndothelialMean EndothelialPore SizePore Size

CETP in Cholesterol MetabolismCETP in Cholesterol Metabolism

HL

LDL1-2

LPLIDL

HL

LDL3-4

HDL Enters Cells &

Arterial Intima NascentHDL

Apo A-1

LiverCETPTG’s

CE

VLDL

Apo B-100

Liver

LCAT

HDL3Reverse

Cholesterol Transport

LCAT

HDL2b

Brewer HB et al. Am J Cardiol 2003;92:10K-16K.

HDL REMOVESEXCESS LIPIDS

ARTERIAL INTIMA

MACROPHAGE CELL

FOAMCELLSBUILD

PLAQUE

RLP

Atherosclerotic Plaque FormationAtherosclerotic Plaque Formation

ARTERIAL LUMEN

DENSELDL

Lp(a)

INFLAMMATION RUPTURES PLAQUE

LDL

OXIDIZEDLDL

MONOCYTE CELL

FOAM CELL

Why Have the LPP Test ?Why Have the LPP Test ?Provides a More Complete Risk Answer

than the Standard Cholesterol Test

1. NCEP’s New Lipoprotein Risk FactorsLPP Has Three Risk Identifying Components

2. Lipoprotein Particle Numbers vs. Cholesterol Content

These Components IdentifyMany at Risk Individuals that are Missed

3. Non-Lipid Risk Factors: CRP, Insulin and Homocysteine

Lipoprotein Particle Profile (LPPLipoprotein Particle Profile (LPP™™) Process) Process

Separates by Density using Analytical Ultracentrifugation

The New Gold Standard - Enhancements

Clinically Relevant Lipoprotein Particlesare Detected Not the Surrogate Marker Cholesterol

the CDCCDC’’s Gold Standards Gold Standard

Continuous Gradient

Lipoprotein Particle MeasurementLipoprotein Particle MeasurementFluorescent Dye – a Phospholipid Analog

Hydrophobic EndFluorescent - Hydrophilic End

When Hydrophobic EndImbeds into the Phospholipid Shell of the Lipoprotein the Hydrophilic End Fluoresces

The Fluorescence is a Direct Measurement of Particle Number

Separation by DensitySeparation by DensityCentrifuge Tube with Mixture of Serum, Gradient and Dye

Proteins

LDL

VLDL

HDL

Intense Gravitational Force

Separated Lipoproteins

600,000 G’s

Density (g/ml)

1.030

1.006

1.300

1.000

1.100

1.063

RLP LDLLDLVLDLVLDL HDLHDL

LPP Test LPP Test -- Lipoprotein Groups and SubgroupsLipoprotein Groups and Subgroups

Accurate Densities

Particle Measurement

Dense -Small

HDL3HDL2bBuoyant - Large

Atherogenic ProfileHealthy ProfileLPP showing NCEPLPP showing NCEP’’s New Lipoprotein Risk Factorss New Lipoprotein Risk Factors

Low RLPHigh HDL 2b High RLP Dense LDL Low HDL 2bBuoyant LDL

30

50

70

90

110

130

150

170

190

300 500 700 900 1100 1300 1500 1700 1900

LPP (VLDL + LDL) Particle Numbers (nmol/L)

Apo

B-1

00 (m

g/dL

)LPP Non-HDL Particle Numbers vs Apo B-100

Correlation of Apo B and LPPnon-HDL Particle Numbers is Excellent

One Apo BMolecule per non-HDL Particle

ADA and ACC Consensus: Apo B More Predictive of CVD Risk than Cholesterol*

* Diabetes Care, Volume 31, Number 4, April 2008

r = 0.93n = 2990

Lipoprotein Particle NumbersLipoprotein Particle Numbers

125 mg/dL

Risk

All of These Patients have the Same LDL “Cholesterol”

125 mg/dL125 mg/dL125 mg/dLDCBA

IncreasedLowParticle Numbers or Apo B-100

Increased High

Large - BuoyantDepleted

TG’s &CETP

Large - Buoyant

LDL Particles with Varying Cholesterol Content

Small - DenseEnriched

-TG’s &CETP

Small - DenseDepleted

TG’s &CETP

Relevance of Lipoprotein Particle NumbersRelevance of Lipoprotein Particle Numbers

The LPP Test is Like Having Apo B-100 Broken Down into Subgroups for a Better Therapeutic Approach

30% of the Population is Cholesterol Depleted20% of the Population is Cholesterol Enriched

A Disconnect Exists between Lipoprotein Particle Number and Cholesterol Number

Reference Value

Green is NormalYellow is BorderlineRed is High

The Magnitude of the Result is Easy to Judge with Respect to the Reference Value

Color Highlighted Numerical Results

Test Report Page 1Test Report Page 1

Test Report Page 2Test Report Page 2

Primary Risk Assessment& Therapeutic Approach

Includes Apo B-100, Lp(a), Metabolic Syndrome LPP+ C-Reactive Protein, Insulin and Homocysteine

Lipid Panel

Risk Modification

Direct Measurement of Lipoprotein Particle Numbers

Graph – Quick Assessment

Interrelation Between Atherosclerosis and Interrelation Between Atherosclerosis and Metabolic Syndrome Metabolic Syndrome -- Insulin ResistanceInsulin Resistance

HypertensionHypertension

ObesityObesity

High GlucoseHigh GlucoseMetabolic Syndrome

InsulinResistance

Metabolic Syndrome Metabolic Syndrome InsulinInsulin

ResistanceResistanceAtherosclerosisRisk Increases

AtherosclerosisAtherosclerosisRisk IncreasesRisk Increases

Six Possible Traits

High TriglyceridesHigh Triglycerides

Small, Dense LDLSmall, Dense LDL

Low HDLLow HDL

The Role of CETP and TGThe Role of CETP and TG’’s s in Metabolic Syndromein Metabolic Syndrome

Bays H. Expert Rev Cardiovasc Ther 2004;2:89-105.

HL

TG’s

Small DenseHDL3

TG’s

HL

SmallDenseLDL 3-4

CETPCECECE

TGTGTG’’’sss

HDL

Depleted

CECECE

CETP

TGTGTG’’’sss

LDL1-2

Depleted

Low HDL fromRenal Clearance

of Apo A1

TG’sRLP

LPL

VLDLApo B-100

LiverHigh TG’s

Atherogenic Profile with High TG’s, Low HDL and Dense LDL. Patient has Metabolic Syndrome

High Triglycerides

Dense LDL

Low HDL

Three Lipid Traits is a Probable Diagnosis of Metabolic Syndrome

Most Likely the Patient has One or More non-Lipid Traits:1. Hypertension2. Obesity3. High glucose

Risk Level Increases

Check for Insulin Resistance

Primary Risk Assessment using Non-HDL

Particle Numbers

1

Modify Risk Level Using Metabolic Syndrome Traits

Lp(a), CRP and Insulin

2

Direct Therapeutic Approach Based on LDL and HDL

Subgroup Distributions and Lp(a)

3

LPP Treatment StrategyLPP Treatment Strategy

Important Benefits of the LPPImportant Benefits of the LPP™™ TestTest

Identification of the NCEP New Risk Factors

Measurement of Lipoprotein Particles Numbers with Lipid Panel in Cholesterol Equivalents

Identification of Lipid Metabolic Syndrome Traits

LPP+ Adds Additional Risk Factors hs-CRP plus Insulin and Homocysteine

Male Patient on 4 gm EPA & DHA OmegaMale Patient on 4 gm EPA & DHA Omega--33’’s, s, adding 10 mg adding 10 mg RosuvastatinRosuvastatin and then 145 mg and then 145 mg TricorTricor

10 mg Statin

10 mg RosuvastatinLDL decreased

(48%)145 mg Tricor

145mg TricorDense LDL

decreased 29%

TG’s decreased57%

Metabolic Syndrome Traits

from 2 to 0

62 year old female MD with a healthy profile like shown found that her calcium score was in the 90th

percentile

Examples of the Atherogenic Apo CExamples of the Atherogenic Apo C--1 Enriched HDL1 Enriched HDLand Low Birth Weight Babies (~6 lb and Low Birth Weight Babies (~6 lb vsvs ~7 lb)* ~7 lb)*

Apo C-1 HDL at d = 1.06

Low TG’s, VLDL and RLP

Apo C-1 HDL Characteristics Observed in 1-2% of Specimens

High and Very Buoyant HDL

ProinflammatoryHDL Associated with Endothelial Dysfunction

Kwiterovich P. et al. JAMA 2005;293(15) 1891.

High Very Buoyant HDL High Very Buoyant HDL –– Apo CApo C--1 or Lp(a) ?1 or Lp(a) ?Profile Most Likely has Apo C-1 Enriched HDL Since Lp(a) is Low

Lp(a) = <5 mg/dL

If Patient History Indicates a Low Birth Weight the Probability Would Increase

Confirmation with an Apo C-1 assay is Currently Only Done as Research

Mass Spectrometry shows two isoforms of Apo C-1, one appears to always be associated with CVD*.

*Moore D, McNeal C, Macfarlane R, Biochem Biophys Res Commun 2011;404(4),1034-8.

Lipoprotein Therapeutic Statins Niacin Fibrates Estrogens Resins Absorption Omega-3’s Alcohol Life Style Inhibitors EPA & DHA (moderate) Changes (diet & exercise)

VLDL (Triglycerides) ♥ ♥ ♥♥ ♥♥ X X ♥ ♥♥ ■

RLP (IDL) ♥ ♥ ♥ ♥ X X ♥ ♥♥** ■

LDL I & II - buoyant ♥ ♥♥ ♥ ♥ ♥ ♥ ♥ ♥ ** ■

LDL III - dense ♥ ♥** ♥♥ ♥ ♥♥ ♥ ♥ ■ ■

LDL IV or Lp(a) ■ ■ ♥♥ ■ ♥ ■ ■ ■ ■

HDL 2b - buoyant ♥♥ ♥ ♥ ♥ ♥ ■ ■ ■ ♥

HDL 2a & 3 ♥ ♥ ♥♥ ♥ ♥ ■ ■ ■ ♥ __________________________________________________________________________________________________________________

♥♥ Therapeutic ♥ Beneficial ■ Little or No Effect X Negative Result *These guidelines provide some of the treatment options available to modify lipoprotein particle numbers determined by the LPPTM test. **Spectracell Laboratories observed response to treatment. The National Cholesterol Education Program (NCEP) guidelines provide dosage information on the treatment options.

Lipoprotein Particle Numbers Lipoprotein Particle Numbers Therapeutic GuidelinesTherapeutic Guidelines