Walter B. Bayubay CLS (ASCP), AMT, MA Ed, CPI

Biochemical Analysis (Lipid Panel) Analyte Total Cholesterol Reference Range Patient A < 200 241 LDL-C <130 180 HDL-C >/= 40 38 Triglycerides <150 267 Patient B 180 100 42 140

LIPOPROTEINS are macromolecular complexes that serve as transport vehicle of insoluble lipids in the plasma.

Structure of Lipoprotein

Five Main Classes of Lipoproteins: Chylomicrons Very Low Density Lipoprotein (VLDL) Intermediate Density Lipoprotein (IDL) Low Density Lipoprotein (LDL) High Density Lipoprotein (HDL)

RELATIVE SIZES OF LIPOPROTEINS

RELATIVE COMPOSITION OF LIPOPROTEINS

HDL vs. LDL

Methods of Lipoprotein Estimation Gradient Gel Electrophoresis Ultracentrifugation - Vertical Auto Profile (VAP) Nuclear Magnetic Resonance (NMR) Ion Mobility - Gas-phase Electrophoresis

Gradient Gel Electrophoresis HDL and LDL run in separate polyacrylamide gels Gradients are segmented into different regions LDL 3 segments (S 3 GGE) HDL - 10 segments (S 10 GGE) Segmenting gradient permits better separation of subfractions HDL analysis requires purification of HDL before GGE Lipoproteins identified by lipid (LDL) or protein (HDL) stain Concentrations or relative concentrations determined based on scan of separated lipoproteins

Ultracentrifugation Vertical Auto Profile (VAP) Vertical auto profile (VAP) method is a direct single test for measuring comprehensive lipoprotein cholesterol profile. It is based on a well-established method of ultracentrifugation that uses vertical rotor and single density gradient spin.

Ultracentrifugation Vertical Auto Profile (VAP) VAP provides cholesterol concentrations of total lipoprotein, high-density lipoprotein (HDL), lowdensity lipoprotein (LDL), very-low-density lipoprotein (VLDL), lipoprotein(a) (Lp(a)), intermediate-density lipoprotein (IDL), HDL subclasses (HDL2 and HDL3), LDL subclasses (LDL1, LDL2, LDL3, and LDL4), VLDL subclasses (VLDL1, VLDL2, and VLDL3), and LDL maximum time, which is directly proportional to LDL size.

Ultracentrifugation Vertical Auto Profile (VAP) Because VAP measures additional lipoprotein classes, such as Lp(a), IDL, and subclasses of HDL, LDL, and VLDL, it can identify patients at high risk for coronary heart disease who cannot be identified using the standard lipid panel. In addition, the VAP method is compliant with the National Cholesterol Education Program's Adult Treatment Panel III guidelines.

Nuclear Magnetic Resonance (NMR) Nuclear magnetic resonance (NMR) spectroscopy measures the number and size of lipoprotein particles instead of their cholesterol or triglyceride content, but its clinical utility is uncertain. Lipoprotein concentrations and size determined from a H-NMR scan of serum No sample preparation required

Nuclear Magnetic Resonance (NMR) In one study, cardiovascular disease risk prediction associated with lipoprotein profiles evaluated by NMR was comparable but not superior to that of standard lipids or apolipoproteins. In another study, the magnitude of predictive value of LDL particle concentration (NMR) was not substantively different from that of the total cholesterol to HDL cholesterol ratio and was less than that of C-reactive protein.

LIPOPROTEIN FRACTIONATION

Ion Mobility Ion mobility lipoprotein fractionation is a technology that uses gas-phase (laminar flow) electrophoresis to separate unmodified lipoproteins on the basis of size. Involves electrophoretic separation of particles in laminar air-flow (Gas-Phase Electrophoretic Macromolecular Mobility Analyzer GEMMA) Technique used routinely to determine particle distributions in aerosol analysis

Ion Mobility The size of the lipoprotein particles detected and counted are not affected by particle modifications. (unique property of this method) Ionized lipoprotein particles are electrophoretically separated in a gas phase, and lipoprotein particles are distinguished on the basis of size.

Ion Mobility Following the separation, each lipoprotein particle is directly detected and counted as it exits the separation chamber. Size-separated particles are detected and counted by light scattering.

Ion Mobility Separation of Lipoprotein

Ion Mobility Components Sample Introduction Separation Detection Spray & Dry Aerosol analyzer Detector analyzer voltage light beam Air + CO 2 + HV - a source +1 ions Condenser Liquid Sample filter blower Saturated vapor in detector

Unit of Measure The ångström (Å) (0.1 nm) is a unit of measure denoting the diameter (size) of a particle. This diameter is used to determine the pattern A vs pattern B LDL phenotype.

Pattern A is classified as optimal, and pattern B is classified as high risk. This is based on large population studies showing that people without coronary heart disease tend to have an abundance of large, buoyant LDL particles (pattern A), and people with coronary heart disease tend to have an abundance of smaller, dense LDL particles (pattern B). 7

Ideal profile

Fractionation by Ion Mobility HDL (alpha) - small - large IDL - small - large LDL (beta) - very small - small plus medium - large VLDL (pre-beta) - small - medium - large

Fractionation by Ion Mobility HDL LDL IDL VLDL - small - large - very small - small plus medium - large - small - large - small - medium - large

CVD Risk Associations The following CVD risk associations were observed in the Malmo Diet and Cancer Study cardiovascular cohort 2 : High number of small LDL particles: 1.3 times increased risk High number of medium LDL particles: 1.4 times increased risk Low number of large HDL particles: 1.8 times increased risk

Ion Mobility vs. Electrophoresis

Ideal profile

HDL Large equivalent to Apo A1 Apolipoprotein A1 A major protein component of HDL in plasma Promotes fat efflux (including cholesterol), from tissues to the liver for excretion Also isolated as a prostacyclin (PGI2) stabilizing factor, and thus may have an anticlotting effect A stronger prognostic marker for cardiovascular disease

Ideal profile

LDL Medium + Small concentration - gives better information than LDL-C - principally made of Apo B Apolipoprotein B (Apo B) Major apolipoprotein component of LDL and chylomicrons Responsible for carrying cholesterol to tissues Can lead to plaques that cause vascular disease (atherosclerosis) leading to heart disease Better indicator of heart disease risk than LDL or Total Cholesterol

Pattern A vs Pattern B

Type B Profile indicates high risk for coronary heart disease

Type A Profile indicative of low risk for coronary heart disease

Before After

Analyte Reference Range Patient B Total Cholesterol <200 180 LDL-C <130 100 HDL-C >/= 40 42 Triglycerides <150 140

Lipoprotein Sub-fraction Reference Ranges Lipoprotein Subtype Females LDL, Peak Diameter 216.0-234.3 Angstrom Males 216.0-234.3 Angstrom LDL, Total 396-1461 nmol/l 440-1600 nmol/l LDL, Medium + Small 105-521 nmol/l 144-787 nmol/l LDL, Very Small 77-293 nmol/l 75-419 nmol/l Non-HDL 571-2003 nmol/l 582-2142 nmol/l HDL, Large 1153-7796 nmol/l 469-5258 nmol/l

Lipoprotein Sub-fraction Reference Ranges GOALS Lipoprotein Subtype LDL, Peak Diameter LDL, Total LDL, Medium + Small LDL, Very Small HDL, Large Females and Males > 216.0 Angstrom < 915 nmol/l < 304 nmol/l < 218 nmol/l < 3017 nmol/l

Conclusions: Eighty percent of individuals suffering from coronary heart disease (CAD) have normal cholesterol levels. Half of the 1.5 million heart attacks in the U.S. each year strike people without symptoms also have normal cholesterol levels. Lipid analysis is the only screening test for determining at risk individuals BUT it misses 50% of at risk individuals

Lipoprotein fractionation by Ion Mobility offers ability to interrogate lipoproteins within specific groups, HDL, LDL LDL medium + small concentration gives better information than LDL-C (Apo B) HDL large equivalent to HDL-C (Apo A1) LDL particle diameter and concentration used in evaluating therapeutic options

Ion mobility provides accurate, reproducible, direct determination of size and concentration for a broad range of lipoprotein particles.

Balance Diet

Foods that elevate levels of HDL Cholesterol Raw onions ½ of a medium size raw onion can raise HDL as much as 25% Omega-3 fatty acids Include fish, green vegetables and canola oil Soluble fiber Beans, oats, whole grains, fruits like apples, pears and raspberries Monounsaturated fats Olive oil and canola oil

Exercise

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