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Lipoprotein (a), an Inflammatory Lipoprotein Recent Findings and a Better Approach for Measurement

Lipoprotein (a), an Inflammatory Lipoprotein Recent Findings and a Better Approach for Measurement. Joseph P. McConnell, Ph.D. DABCC Laboratory Director and Co-Founder Health Diagnostic Laboratory Inc. Best Practices in Primary Care Charlotte, North Carolina December 4, 2010. Disclosure.

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Lipoprotein (a), an Inflammatory Lipoprotein Recent Findings and a Better Approach for Measurement

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  1. Lipoprotein (a), an Inflammatory LipoproteinRecent Findings and a Better Approach for Measurement Joseph P. McConnell, Ph.D. DABCC Laboratory Director and Co-Founder Health Diagnostic Laboratory Inc. Best Practices in Primary Care Charlotte, North Carolina December 4, 2010

  2. Disclosure • Joe McConnell is the Laboratory Director and Co-Founder of Health Diagnostic Laboratory Inc. (HDL Inc.) • Focus of HDL Inc. • Disease state management with supporting clinical laboratory testing: tailoring of specific therapies to each individual guided by laboratory results. • Target • Patients with cardiovascular disease (CVD) • Metabolic syndrome (MS) • Diabetes mellitus (DM) • Nonalcoholic steatohepatitis (NASH) and fatty liver disease.

  3. Lipid Levels in Patients Hospitalized with Coronary Artery Disease 14 LDL-C 13 49.6% of patients hospitalized with CAD had admission LDL-C <100 mg/dL 17.6% had LDL-C <70 mg/dL. Less than one quarter of patients had an admission LDL-C >130 mg/dL. 12 11 10 9 8 7 Patients, % 6 5 46.4% of patients had HDL cholesterol > 40 mg/dL 4 3 2 1 0 220 200 100 20 30 210 40 160 50 60 70 80 90 150 110 170 190 140 180 130 120 136,905 hospitalizations LDL Cholesterol Level (mg/dL) Sachdeva A, et al. Am Heart J 2009;157:111-7.e2

  4. Cardiovascular Risk Factors • There is a need for additional risk factors. • Although traditional risk factors produce reasonably accurate estimates of risk in a population, they predict only 50-60% of events in individual patients. • Additional risk factors would improve accuracy of decisions regarding preventative therapies Rader DJ. NEJM. 2000;343:1179-82

  5. Inflamm. hsCRP Lp-PLA2 Cytokines MPO Lipids/Oxidation Platelet actv. Coagulation Fibrinolysis Endothelium Nutrition Fibrinogen PF 1+2 TAT TFPI TPP TXB2 12-HETE B-TBG P-selectin D-Dimer PAI-I TPA Plasminogen Endothelin Prostacyclin Nitric oxide Soluble ICAM VCAM LDL subclasses Lp (a) F2 isoprostanes MPO HCY B12,B6 Folate Vit. C,E, Homocysteine Thrombin monocyte chemotaxis ICAM VCAM E-selectin TXA2 Tissue factor VWF Native LDL Growth Factors slow O2 radicals smooth muscle cell mitogenesis Vasoconstriction rapid Oxidized LDL

  6. Elevation of Advanced Risk Markers in Patients with Normal Lipids Samples with optimal LDL-C(<100 mg/dL), HDL-C (>40 mg/dL), and Trig (<150 mg/dL) 50,000+ patient samples from HDL Inc. 15,000 with normal Lipids Although traditional lipid measures are optimal, advanced risk markers are elevated or in the intermediate range in 30% – 60% of our patients with optimal lipids. Optimal Int. risk At risk

  7. Case 45 yr Male: Family History • Maternal Grandfather died at age 57, two years after his first stroke (significant morbidity). • Maternal Grandmother died at age 61 of MI, apparently in her sleep. • Paternal Grandfather died at age 63, massive coronary. • Paternal grandmother died of a stroke at age 62. • Father survived first MI at age 61, died following 2nd MI at age 62.

  8. Case #2: A Concerned Young Woman A healthy 38 year old female is interested in a cardiovascular evaluation because her seemingly healthy father in his early 60s just had a myocardial infarction followed by triple vessel coronary bypass surgery. She appears quite healthy with a BMI of 24 (5’6 inches and 150 lbs) and is normotensive. There is no history of smoking. She has two children ages 3 and 5 and there was no gestational diabetes.

  9. Case #2: A Concerned Young Woman

  10. Lipoprotein (a) • Discovered by Kare Berg: 1963 • Particle structure containing a single copy of apolipoprotein B covalently linked to a protein of variable mass not found in other lipoproteins • He called the new particle lipoprotein (a) • Studies noted its association with vascular disease (coronary, cerebral and peripheral)

  11. Apo (a) 4 4 4 4 4 4 4 4 4 4 4 4 4 Lipoprotein (a) Lipoprotein (a) Structure Kringle 3 4 5 2 5 1 Active site (Protease) Apo-B Plasminogen Size heterogeneity (~250-1000 KDa) Why is Lp(a) atherogenic?

  12. Why is Lp(a) Atherogenic? • Has all the properties of LDL. • Has structural homology with plasminogen and inhibits fibrinolysis. • May serve as a sink for oxidized pholpholipids. • Inhibits tissue factor pathway inhibitor (TFPI) activity.

  13. Submitted to Journal of Thrombosis and Haemostasis • Elevated Lipoprotein(a) is associated with decreased plasma TFPI activity in human subjects. • Tissue Factor Pathway Inhibitor (TFPI), previously known as Lipoprotein Associated Coagulation Inhibitor (LACI)

  14. Submitted to Journal of Thrombosis and Haemostasis

  15. Why is Lp(a) Atherogenic?Quadruple Whammy • Has all the properties of LDL. • Has structural homology with plasminogen and inhibits fibrinolysis. • May serve as a sink for oxidized pholpholipids. • Inhibits tissue factor pathway inhibitor (TFPI) activity.

  16. Clinical Significance of Lp(a)Is Lp(a) Atherogenic? • Numerous retrospective case control studies • Virtually all show strong link between Lp(a) and vascular disease • Prospective studies with Discordant results • Several studies: • conclude Lp(a) is an independent risk factor • Some studies • reach the opposite conclusion

  17. Relative Risks of Future MI AmongApparently Healthy Middle-Aged Men Lipoprotein (a) Homocysteine Total Cholesterol Fibrinogen T-PA antigen TC/HDL CRP hsCRP + TC/HDL 0 1.0 2.0 3.0 4.0 5.0 6.0 Physicians Health Study Ridker: Ann Intern Med 130:933, 1999

  18. Danesh J, et al.,Circulation. 2000;102(10):1082-5

  19. Lp(a) and CHD Reykjavik Study (n=18 569) 2047 patients with first-ever MI or who died of CHD. vs. 3921 control participants in the Arch Int. Med. 2008;168(6):598-608

  20. Lp(a) Proven as Causal Factor for MI Copenhagen Heart Study

  21. Lp(a) Proven as Causal Factor for MI Copenhagen Heart Study

  22. European Atherosclerosis Society Recommends Screening for Lp(a) • Patients at moderate or high risk for CVD should be screened for Lp(a). • Bringing a Patients Lp(a) < 50 mg/dL should be a treatment priority. • 1-3 g niacin is best treatment for Lp(a) but further studies are needed to better define treatment and target level. June 2010 Press Release: European Atherosclerosis Society Consensus panel

  23. “Is there evidence that individuals identified by the biomarker of interest will benefit from an intervention or therapy they otherwise would not have received?” For hsCRP, the answer is a solid “yes,” and for Lp(a), a solid “no”.

  24. Clinical Significance of Lp(a) • Why the discordant results? • Analytical techniques not standardized • Size heterogeneity of apo (a) • Lp(a) is an acute phase reactant • should not be measured in the post-event period • Different populations have different distributions • skewed distribution in Whites and Asians • more normal distribution in blacks • Lp(a) in Blacks: nearly 2X values in Whites

  25. Lp(a) Analytical Standardization y = 0.2268x + 16.895 2 R = 0.3198 100 80 60 Lp(a) (mg/dL): Method 2 40 20 0 0 50 100 150 200 Lp (a) (mg/dL): Method 1

  26. Western Blotting Gel Electrophoresis Reduction and Denaturation Electrotransfer 4% Novex TG Gel T 2 Western Detection: Goat anti-Lp(a) 1o Ab Anti-goat IgG-AP 2o Ab Chromogenic Substrate BCIP/NBT for alkaline phospatase

  27. Apolipoprotein (a) Isoform Identification by Western Blotting 23 and 31 S 12 20 and 27 S 21 16 S >S4 S4 S3 S1 B 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 Samples M: Protein Size Marker, Myosin band (207 kDa)

  28. Effect of Kringle Number on Lp(a) Analysis

  29. Marcovina SM et al, Clinical Chemistry: 2000 • “To various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a).” • “The major problem in the lack of accuracy is the over- or underestimation of Lp(a) values as a result of apo(a) size heterogeniety.“

  30. Apo(a) Isoform size and Coronary Artery Disease

  31. Apo (a) IsoformSize Effect • Large isoforms: • Likely not as atherogenic as small isoforms • Produce increased signals on immunoassay • False positive • Small isoforms • Likely the more atherogenic form • Produce decreased signals on immunoassay • False negative

  32. Is There An Isoform Independent Method? • Immunologic methods • All commercially available immunologic methods exhibit some isoform bias • In general, immunoturbidimetric are less affected than ELISA • Lp(a) cholesterol determination • Measure the cholesterol content of Lp(a) just like HDL or LDL cholesterol are measured.

  33. LDL VLDL Lp (a) HDL Lp (a) Cholesterol Measurement Electrophoresis and enzymatic cholesterol staining Lp (a) cholesterol measurement is not influenced by apo (a) size

  34. 3V None 2V Mild 1V Novel Risk Markers Relation to Angiographic CAD and Events 504 consecutive patients undergoing coronary angiography Vessel Disease Mean Age 60 ± 11 years, 62% Male 46% patients have none or mild coronary occlusion 54% patients have significant occlusion (>50% stenosis)

  35. Lp(a) and Oxidized phospholipids Inflammatory Lp(a) N ENGL J MED 353;1 July 7, 2005

  36. Oxidized Phospholipids, Lp(a) and Coronary Artery Disease Conclusions • Circulating levels of oxidized LDL and Lp(a) are associated with angiographically documented CAD, particularly in patients < 60years of age. • Oxidized LDL and Lp(a) levels are highly correlated. • Lp(a) may serve as a “sink” for oxidized phospholipids • The atherogenicity of Lp(a) may be mediated in part by associated pro-inflammatory oxidized phospholipids.

  37. LIPOPROTEIN PARTICLE and Inflammation *Spherical macromolecular complex Apolipoprotein Phospholipid *Core: nonpolar lipids *Surface: polar lipids *Specific protein:  1 * Function: lipid transportation from liver, intestine to target tissues Free cholesterol Cholesterol ester Triglycerides

  38. Oxidized Phospholipids, Lp(a) Lipoprotein, and Coronary Artery Disease Phosphatidylcholine (PC) Oxidation Oxidatively-Modified PC E06 Antibody recognizes an epitope of Phosphatidylcholine (PC) exposed on the surface of Lipoprotein particles when PC is oxidized.

  39. Lp-PLA2 and LDL Oxidation Phosphatidylcholine (PC) Oxidation Oxidatively-Modified PC Lp-PLA2 Putative Inflammatory Mediators + Oxidized Fatty Acid Lyso-PC

  40. Pro-Atherogenic Activities of Lysophosphatidylcholine (Lyso-PC) Lyso-PC Expression of adhesion molecules Upregulation of cytokines and CD40 ligand Stimulation of macrophage proliferation Induction of MCP-1 Inhibition of endothelial derived nitric oxide Migration of vascular smooth muscle cells Chemoattractant for macrophage and T-cells 1. Dada N, et al. Expert Rev Mol Diagn. 2002;2(1):89-94. 2. Quinn MT, et al. Proc NatlAcadSci USA. 1988;85:2805-2809.. 3. MacPhee CH, et al. Biochem J. 1999;338:479-487. 4. Carpenter KL, et al. FEBS Lett. 2001;505:357-363

  41. Lp(a), Oxidized phospholipids (OxPLs), and Lp-PLA2 • Lp(a) has been reported to be enriched in Lp-PLA2 (2X increased mass and several fold inceased activity). • OxPLs in plasma appear to be preferentially sequestered on Lp(a) • Association between Lp(a), Lp-PLA2 and OxPLs deserves further study

  42. Angiography Study Measurements Lp(a) mass by two methods -Diasorin -Polymedco (Denka Seiken) Lp(a) Cholesterol -Electrophoresis and enzymatic stain -Helena Laboratories

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