OUTLINE:

I.LIPOPROTEINS, APOLIPOPROTEINS & RELATED PROTEINSII.LIPID TRANSPORT AND LIPOPROTEIN METABOLISMIII. LIPID AND LIPOPROTEIN MEASUREMENTIV. THE NCEP GUIDELINESV. LIPIDS, LIPOPROTEINS AND DISEASE

Plasma lipoproteins-transport cholesterol and esterified lipids in the blood.4 major lipoproteinsChylomicrons, VLDL, LDL, HDL, IDLApolipoproteins-play impt.roles in lipid transport by activating or inhibiting enzymes involved in lipid metabolism, binding lipoproteins to cell surface lipoprotein receptors or both.

Large particles produced by intestine rich in triglycerides of exogenous origin and relatively poor in free cholesterol and phospholipidsContain 1-2% proteinLess dense than water/floats even w/o centrifugation due to its high lipid/protein High chylomicron content results in milky plasma in w/c chylomicrons accumulate as a floating creamy layer when left undisturbed for several hours. Apo-B48,apoA-I, IV;apoC-I,II,III,apoE

Smaller than chylomicrons; rich in triglyceridesLower lipid/protein ratio; float at higher densityLike chylomicrons, particles are large enough to scatter light; w/excessive VLDL, plasma is turbid.VLDL Triglycerides are endogenous, mainly hepatic origin and constitute half the particle mass.Cholesterol and phospholipids 40% of particle10% is protein apoB-100 and apo-C; apoESmaller particles poorer in 2 components

50% of total lipoprotein mass in plasma.Smaller than TG-rich lipoproteinsCholesterol-1/2 of LDL25% of LDL mass is protein, apoB-100 with traces of apoC

Small particles (50% protein) apoA-1, apoA-II but also apoC & apoE; 20% cholesterol (mostly esterified); 30% phospholipids & TG traces.HDL2 and HDL3 (differ in density, particle size and composition)

Density range 1.055-1.085 kg/L27% protein, 65% lipid, 8% CHO so like LDL but in lower concentrations.apoB, apo(a) bound thru disulfide bondsLpX Lipoprotein-abnormal lipoprotein found in patients with obstructive biliary disease.Lipids more than 90% of weight (phospholipids, unesterified chole, little esterified chole)B-VLDL (floating B lipoprotein) abN protein that accum. In Type III hyperlipopoteinemia

ApoA ApoA-I is 75% of apo of HDL; synthesized in the liver and intestine; activates lecithin cholesterol acyl transferase (LCAT) which esterifies cholesterol. ApoA-II is 20% of apo of HDL; 2 identical peptides linked by one disulfide bond. Unknown roleApoB-major protein 95% of LDL; 40% of protein of VLDL and chylomicrons. Major component is apoB-100; synthesized by liver; found in endogenous lipoproteins; one of d longest

ApoC-major protein component of VLDL; minor constituent of HDL & LDL. ApoC-I-IIIMinor lipoproteins- ApoD-minor constituent of HDL protein (5% or less). Unknown function but may be a cofactor for LCAT.ApoE-arginine-rich apolipoprotein; found in VLDL, IDL, remnant lipoproteins, chylomicrons and HDL.

Lipolytic enzymes: 2 triglyceride hydrolases (lipoprotein lipase LPL and hepatic triglyceride lipase) found in post-heparin plasma.LPL: derived from adipose tissue; hydrolyzes TG in chylomicrons and VLDL; located on the surface of capillary endothelial cells of adipose tissue and skeletal & heart muscles. Chylomicron TG is hydrolyzed ff attachment of these particles to capillary endothelial cells.PL & apoC-II are cofactors for TG hydrolysis by LPL.

Hepatic TG lipase (HTGL)-secreted by hepatocytes; assoc. with surface membrane of nonparenchymal liver cells. Limited capacity to hydrolyze TG in chylomicrons & VLDL; not require apoC-II as cofactor. May participate in conversion of VLDL remnants and IDL to LDL. Most active in hydrolysis of PL & TG of HDL and may play a role in HDL metabolism.LCAT-catalyzes the esterification of cholesterol by promoting transfer of FA from lecithin to cholesterol w/c results in the formation of lysolecithin and cholesterol ester; synthesized in the liver & circulates in plasma assoc. with HDL.

Lipid Transport in Lipoproteins: Transport of TG and chole from sites of origin in intestine (exogenous) and the liver (endogenous) to sites of energy storage and utilization.TG & chole enter plasma as TG-rich lipoprotein particles (chylomicrons & VLDL) that supply tissues with FA for energy reqts and storage.Chylomicrons & VLDL undergo intravascular change almost immed.after their entry into the circulation thru the action of lipoprotein lipase.

LDL-2/3 removed via hepatic LDL-receptorsHDL-secreted from liver and intestine as disk-shaped particles that contain cholesterol and phospholipid; thought to be vehicle for reverse chole transport where excess chole is removed from tissues peripheral to the liver & transported back to the liver for reuse or disposal in the bile.

ComponentCVp (%)CVp (%)Total Cholesterol5.06.4 Triglycerides17.823.7 LDL-cholesterol 7.88.2 HDL-cholesterol 7.1 7.5 ApoA-1 7.1 -- ApoB 6.4 --

Cholesterol-all of sterol in plasma; mixture of unesterified (30-40%) & esterified (60-70%)Enzymatic Methods-less subject to interference by nonsterol substances; not absolutely specific for cholesterol coz chole oxidase can react w/other sterol. Reducing subs. Like ascorbic acid and bilirubin can interfere w/measurements by consuming H2O2. Bili may react w/an intermed. in the peroxidase reaction. Turbidity (inc.TG) can interfere. Uric acid, Hgb and other subs. not affect chole.

Formaldehyde: reaction w/sulf.acid soln of chromotropic acid to produce pink chromophore.Peroxidate oxidation not specific for glycerol.a-glycerophosphate oxidized by periodate to form formaldehyde.Enzymatic methods: specific, rapid and easy to use. Directly in plasma or serum; not subj to interference by PL or glucose. TG are hydrolyzed and glycerol formed is converted to glycerophosphate and measured. Rgts are commercially available as lyophilized prepns still for reconstitution.TG blanks: Free glycerol interfere w/enzymatic methodOmits hydrolysis step. Use for standardization &QC of TG

Phosphatidyl choline and sphingomyelin 90% of PL & 80% of this is phosphatidyl choline.Others: phosphatidyl serine & ethanolamineDses: obstructive jaundice, a/hypo betalipoproteinemia, Tangier dse or LCAT def.Where conc., composition or LP distr. of PL Released phosphate converted to phosphomolybdate by rxn w/ammonium molybdate & mixture is treated w/mild reducing agent.

Ultracentrifugal methods: Lipid content w/ lower densities than other plasma macromolecules. Different densities so chylomicrons and VLDL float at plasma densityLDL&HDL sedimented.Analytical ultracentrifugation: reference method, rates of flotation under specific condPrep.ultracentrif: separated at diff.densitiesElectrophoretic methods: Support med. Is agarose gel due to speed, sensi & resolutionMigration rates HDL, VLDL, LDL Lipid-staining dye Oil Red O, fat red 7B or Sudan BlackBReact w/ester bonds in TG & cholesteryl esters.

Polyanion precipitation methods: Heparin sulfate, dextran sulfate, phosphotungstate in(+)of Ca,Mg, Mn.Influenced by rgt conc, pH, ionic strength, (+)serum proteins and anticoag, relative amts of lipid&protein in LP particles & duration and conds of sample storage.Not gained wide acceptanceMC used to remove apoB-cont. LP prior to analysis of HDL-chole.

ApoB-cont. LP (chylomicrons, VLDL, IDL, LDL, Lp(a)) are removed by polyanion-divalent cation precipitation &HDL-chole is analyzed directly in the supernatant.Combined methods: Hyperlipidemic to measure plasma chole, VLDL, LDL, HDL, TGAssessment if w/chylomicrons in fasting state, (+) or (-)B-VLDL seen in Type III hyperlipoprot.Lp(a) indep. Risk factor for coronary dse.Combi of preparative ultracentrif, polyanion precip, & electrophoresis.Plasma Total Chole, HDL-chole, TG

HDL-chole: direct measurementLDL-chole = infranatant chole HDL-choleVLDL-chole = TC infranatant chole4. LDL-chole = TC HDL-chole Plasma TG 2.175VLDL-chole = plasma TG2.175Plasma TG 6.5

Standing plasma test: for chylomicronsFloating ice cream layer and detected visuallyPlasma sample turbid after standing overnight has VLDL so if (+) so chylomicrons are presentDetection of B-VLDL and Lp(a): electrophoretically examined for B-VLDL (floating b-lipo)VLDL-chole/plasma ratio

Consistent withAnalyteTotal ErrorBiasCVCholesterolLess than 9%Less than 3%Less than 3%TGLess than 15%Less than 5%Less than 5%HDL-choleLess than 22%Less than 10%Less than 6%

1. Unlike chole, none of these analytes has unique chem. structure.2. In the case of LDL, clinical & epid. Database in w/c estimates of coronary risk are based are estab. using either combined ultracentrifug.-polyanion precipn of Friedewald methods, HDL method is heparin Mn procedure,3. Either combi method or Friedewald equation, some non-LDL LP primarily IDL &Lp(a) are atherogenic.

Fasting, posture, venous occlusion, anticoagulants, recent MI, stroke, cardiac catheterization, trauma, acute infection, pregnancy.

Total Chole Desirable less than 200 mg/dL Borderline High 200-239 High more than 240

HDL-chole Low HDL-choleless than 35

NCEP Lab Standardization Panel recommendations for chole used separate specifications for bias and precision (CV) & consistent w/total error of 8.9% or less. Set in single value, total error considers both bias & imprecision at the same time.% total error = % bias + 1.96 (% CV)2. Recom. HDL-chole apply to levels of 42 mg/dL3. More rigorous guidelines for HDL-chole for adoption provided that HDL-chole measurement method is available.

Diet TherapyInit. LevelLDL GoalW/o CHD &*fewer than 2 risk factors more 160less 160W/o CHD w/2 or fewmore 130less 130 W/ CHDmore 100less 100Drug TxW/o CHD fewer than 2 risk factorsmore 190less 160W/o CHD & 2 or more more 160less 130W/ CHDmore 130less 100

Apolipoprotein immunoassaysRadioimmunoassay (RIA)Radial immunodiffusion (RID)ElectroimmunoassayImmunonephelometryEnzyme-linked (ELISA) & Fluorescence Immunoassay Probs with (1)

Qualitative apolipoprotein analysis Simple immunodiffusion technique can determine (+) or (-) of apolipoprotein Evaluation of LP disorders where particular apoLP may not be present (Tangier, abetalipo, apoC-II deficiency Gel electrophoresis techniques; isoelectric focusing (separates by charge).

Total chole Desirable blood choleless than 200 mg/dL Borderline-high blood chole 200-239 High-blood cholemore than 240

HDL-choleLow HDL-choleless than 35

Positive risk factors Age Male more than 45 Female more 55yo or premature menopause w/o ERT Family Hx of prem. CHD (defin MI or sudden death before 55yo in father or other male first-degree relative Current cigarette smoking HPN (more than 140/90 mmHg on antiHPNsive meds Low HDL-chole (more than 35mg/dL) DMNegative risk factor High HDL-chole (more than 60)

CategoryTC (mg/dL)LDL-chole (mg/dL)AcceptableLess than 170Less than 110Borderline170-199110-129HighMore than 200More than 130

LP PhenotypePrem.CADXanthomasPancreatitisI. Disorders in exog. LP pathwayA.Defective or absent LPLINoEruptiveYesB.def.of apoLP C-III or VNoEruptiveYesII.Disorders of endog,LPA.Familial combined hyperlipidemiaIIa, IIIB, IV rarely VYesIsolated xanthelasmaRarely 1. Hyperapobeta-LpnemiB.Familial hyperTGN, IVYessameNo

IV, occ IIbCan occurNo (occ.eruptive)Occ.

LP PhenotypePrem.CADXanthomasPancreatitisC.Familial hypercholeIia (occ. Iib)YesTendon, tuberousNoD. Familial defective apoB-100N, IIaYesOcc. tendonNoIII. Disorders of both exo & endo LP pathwayA. Type V hyperLPnemiaVCan occurEruptiveYesB.DysLPtenemia (Type III)IIIYesYesNoC.Def,pf apoB-cont LP

1.AbetaLP/HypoHypo or abetaNoNo

LP PhenotypePrem.CADXanthomasPancreatitisIV. Disorders of reverse chole transport pathway A. Dec.HDL synthesisHypo aYesPlanarNoB. Inc.HDL catabolism 1.ApoLP A-1 variantsHypo aNoNoNo 2. Tangier diseaseHypo aCan occurNoNo C.LCAT deficiencyHypo aCan occurNoNo

D.CETP defHyperDecreasedNo No

Although ultracentrifugation and electrophoretic techniques are of historical significance, most useful lipid and lipoprotein testing methods are now enzymatic.Low-density lipoprotein cholesterol can be measured directly but is usually calculated using the Friedewald formula. Calculated values require evaluation of fasting samples.Low-density lipoprotein cholesterol is currently considered the most important value in assessing cardiac risk and directing therapy.

The profile currently recommended for initial screening in adults, age 20 or older, includes total cholesterol, LDL, HDL cholesterol, and triglycerides. Testing should be repeatedat least once every 5 years.Other tests, including apolipoprotein levels and lipoprotein subclasses,may prove valuable in fine-tuning risk assessment and evaluating response to therapy.New guidelines elevate the perceived atherosclerotic risk of diabetes and support aggressive intervention in diabetic patients and patients with metabolic syndrome.