Unit 5-Lipids PDF

LIPIDS & LIPOPROTEINS REAN STAR PAULINE C. CORTINA, RMT INSTRUCTOR, CSU-CAHS  LIPIDS Primary source of fuel, provide stability to cell membranes and allow transmembrane transport Insoluble in water, but soluble in organic solvents M ajor Lipids: Phospholip...

LIPIDS & LIPOPROTEINS REAN STAR PAULINE C. CORTINA, RMT INSTRUCTOR, CSU-CAHS  LIPIDS Primary source of fuel, provide stability to cell membranes and allow transmembrane transport Insoluble in water, but soluble in organic solvents M ajor Lipids: Phospholipids, cholesterol, triglycerides, fatty acid, fat soluble vitamins 1. PHOSPHOLIPID (Conjugated Lipid) MOST ABUNDANT LIPID from phosphatidic acid Two fatty acids and phosphorylated glycerol Sphingomyelin RV : 150-380 mg/dL (Serum) Forms of Phospholipids ü Lecithin/phospholipids chroline 70% ü Sphingomelyn 20% ü Cephalin 10% Phosphatidyl ethanomoline Phosphatidyl serine Lyscolecithin + Inositol phosphatide 2. CHOLESTEROL / 3-hydroxy-5,6-cholestene Unsaturated steroid alcohol containing four rings with a single C-H side chains tail similar to fatty acid Synthesized in the liver Not a source of energy Transport and excretion is promoted by estrogen Evaluates risk for atherosclerosis, myocardial infarction and coronary arterial occlusions  2 FORMS o Cholesterol eaters (CE) – 70% § Cholesterol bound to fatty acids and is found in plasma and serum § Neutral lipid- found in the center of lipid drops and lipoproteins § Esterified by LCAT § Excess cholesterol is re-esterified by the microsomal enzyme acyl cholesterol acyl transferase (ACAT) § Lecithin-Cholesterol Acyl Transferase (LCAT) – activated by Apo A-1 and enables HDL to accumulate cholesterol as cholesterol esters by promoting transfer of fatty acids from lecithin to cholesterol = lysolecithin and cholesterol ester o Free Cholesterol (FC) – 30% § Polar nonesterified alcohol found in plasma, serum and RBCs § Produced by lysosomal hydrolysis and becomes available for membrane, hormone and bile acid synthesis LABORATORY METHODS Patient should be seated at least 5 minutes prior collection Specimen: fasting (12-14 hrs) or non-fasting plasma or serum Use tube with gel separation 2 weeks prior to testing: patient should be in their usual diet and neither losing or gaining weight  CHEMICAL METHODS Principle: Dehydration and oxidation of cholesterol to form a colored compound (Colorimetry) 1. Saikowski Reaction End product: Cholestadienyl Disulfonic Acid (RED) 2. Libermann-Burchardt End product: Cholestadienyl Monosulfonic Acid (Green) Color developer: a) Glacial acetid acid b) Acetic anhydride c) Concentrated sulfuric acid  General Methods ONE STEP MTD Pearson, Stern, & Mac Gavack = Colometry TWO STEP MTD Bloors = Extraction + Colorimetry Cholesterol is extracted using an alcohol ether mixture Measured using L – B THREE STEP MTD Abell Kendall = Saponification +Extraction + Colorimetry Cholesterol saponified/hydrolyzed with alcoholic KOH Extracted with petroleum jelly Measured with L-B FOUR STEP MTD Schoenheimer Sperry, Parekh, & Jung =Precipitation + Saponification +Extraction + Colorimetry  ENZYMATIC METHOD Most common method of quantifying the cholesterol oxidase reaction is to measure the amount of hydrogen peroxide produced If the cholesteryl ester hydrolase step is omitted, it can be used to measure unesterified cholesterol Cholesterol ester + H20 – cholesterol esterase --> cholesterol + fatty acid Cholesterol + O2 –cholesterol-oxidase --> cholest – 4-en-3-one + H2O2 H2O2 + phenol + 4-aminoantipyrine –peroxidase ---> quinoneimine dye  REFERENCE METHOD CDC REFERENCE METHOD: Hydrolysis/saponification with alcoholic KOH + Abell, Levy, Brodie, Kendall Method Hexane extraction + Colorimetry (L-B reagent) CURRENT REFERENCE METHOD GCMS GOLD STANDARD IDMS NCEP Guideline Recommendation for Adults in Terms of Risk for CHD: DESIRABLE < 200 mg/dL (240 mg/dL (>6.72 mmol/L) AGE MODERATE RISK HIGH RISK 2-19 > 170 mg/dL >185 mg/dL 20-29 >200 mg/dL >220 mg/dL 30-39 >220 mg/dL >240 mg/dL 40-49 >240 mg/dL >260 mg/dL  TRIGLYCERIDE/TRIACYLGLYCEROL (Neutral Fat) Main storage lipid in man 3 molecules of fatty acids and one molecule of glycerol Laboratory measurement: based on hydrolysis of fatty acids to produce glycerol Specimen: fasting for 12-14 hours, plasma or serum Interferance: ascorbic aid, bilirubin and hemolysis < 200 mg/dL TG = Clear serum > 300 mg/dL TG = Turbid serum > 600 mg/dL TG = Opaque or milky serum  CLINICAL METHOD 1. COLOMETRIC Triglycerides –alc. –KOH > Glycerol fatty acids (Van Handel & Zilversmith) Glycerol oxidized by Periodic acid > Formaldehyde (HCHO) HCHO + Chromotropic Acid > (+) BLUE colored compound 2. FLUOROMETRIC Triglycerides –alc. –KOH > Glycerol + FA Glycerol oxidized by Periodic Acid > Formaldehyde (HCHO) (Hantzsch Condensation) HCHO + Diacetyl Acetone + NH3 > Diacetyl Lutidine Compound (Yellow) ENZYMATIC METHOD GLYCEROL KINASE METHOD Involves hydrolysis of TG to FA and glycerol followed by phosphorylation of glycerol to glycerophosphate Enzymes: Lipase, Glycerokinase, Glycerophosphate oxidase, Peroxidase  REFERENCE METHOD MODIFIED VAN HANDEL & ZILVERSMITH Involves alkaline hydrolysis with alc. KOH, solvent (COLORIMETRIC) extraction with choloroform and treatment with silicic acid to remove phospholipids and isolate TG Glycerol + sodium perodiate > Formaldehyde (HCHO) + formic acid HCHO + Chromotropic Acid > (+) PINK colored compound CURRENT REFERENCE METHOD GCMS CDC TRIGLYCERIDE LEVELS : NORMAL < 150 mg/dL BORDERLINE 150-199 mg/dL HIGH 200-499 mg/dL VERY HIGH ≥500 mg/dL LIPOPROTEINS Spherical lipid and protein complex, liquid core (TG and CE) and outer shell of phospholipids, proteins and free cholesterol Transport lipids throughout the body Attached with apolipoproteins 3 main functions of apolipoproteins: o activate enzymes to aid in lipid metabolism o Maintain structural integrity of lipoprotein molecule o enhance cellular uptake of lipoproteins Lipoprotein metabolism/lipolytic enzymes 1. Lipoprotein Lipase – hydrolyzes TG to glycerol, monoglycerol and FA which are rapidly removed by the liver 2. Hepatic lipase – hydrolyzes TAG and phospholipids from HDL; and lipids in VLDL and IDL 3. Lecithin cholesterol acyltranferase (LCAT) – converts free cholesterol to CE 4. Endothelial Lipase – Hydrolyzes HDL for the release of TG and phospholipids 5. ATP – binding cassette protein AI (ABCA1) – for efflux of cholesterol from peripheral tissues into HDL Lipid Metabolisms: o Dietary or exogenous pathway of lipid transport: Absorption of cholesterol and triglycerides Formation of chylomicrons (CM) into the lymph (chyle) and into the blood LPL liberates fatty acids from TAG Free Fatty acids are taken up by the muscles and adipose tissue o Endogenous pathway Production of TAG from free fatty acids by the liver with the synthesis of VLDL VLDL particles are converted to IDL LDL can be taken up by the liver or into other tissues or steroid or cell membrane synthesis o Reverse Cholesterol Transport pathway HDL particles mobilize Ch from tissues and reintroduces it into the circulation for exchange VLDL for further metabolism or back to the liver for excretion into the bile  Largest and least dense, produced in the intestine Delivers dietary lipids to hepatic and peripheral calls Triglycerides are the predominant lipid component CHYLOMICRONS (CM) Major apolipoprotein: Apo B- 48, Apo A1, Apo C and Apo E Cleared 6-9 hours post prandial; NON-ATHEROGENIC Density: < 0.95 kg/L AKA: Pre-beta-lipoprotein Secreted in the liver Major apolipoprotein: Apo B- 100, Apo C and Apo E VLDL Transports endogenous TAG from the liver to peripheral tissues: ATHEROGENIC Density: 0.95 – 1.006 kg/L AKA: Beta-lipoprotein Major catabolic end-product of VLDL Constitutes about 50% of the total lipoproteins in plasma LDL Primary target of cholesterol lowering therapy and primary marker for CHD risk Major apolipoprotein: Apo 100, Apo E Transports cholesterol to peripheral tissues: MOST ATHEROGENIC Density: 1.019 – 1.06.3 kg/L Research method: Beta Quantification  AKA: Alpha-lipoprotein Smallest (5-12nm) and most dense Produced in the liver and the intestine Major apolipoprotein: Apo AI, Apo A-II, Apo C Transports excess cholesterol from tissues back to the liver HDL HDL2 transports more affectively and is more CARDIOPROTECTIVE Density: 1.063 – 1.21 kg/L CDC Reference method: Ultracentrifugation precipitated with heparin-MnCI2 and Abel-Kendall assay MINOR LIPOPROTEINS Product of VLDL catabolism / “VLDL remnant” Converted to LDL: “Subclass of LDL Migrates either in the pre-B or Beta Region IDL Defective clearance of IDL: Type 3 hyperlipoproteinemia due to deficiency of Apo E – III Major apolipoprotein: Apo B- 100 Density: 1.006 – 1.019 kg/L  AKA: “sinking pre-B Lipoprotein” LDL variant that has a molecule of Apo (a) linked to Apo B-0100 by a disulfide bond Lp(a)/Lipoprotein (a) Independent risk factor for atherosclerosis Increased levels: premature CHD and stroke Electrophoretic mobility: VLDL (sometimes between LDL and albumin) Density: like LDL (1.045 – 1.080 kg/L) Isolation in the LDL – HDL density range by ultracentrifugation and measure by immunoassay ABNORMAL LIPOPROTEINS Found in obstructive jaundice and LCAT deficiency Specific and sensitive indicator of cholestasis Lipoprotein X (LpX) Lipid content is mostly phospholipid and free cholesterol (90%) Contains albumin and Apo C AKA: “Floating B-Lipoprotein” Abnormally migrating B- VLDL, cholesterol – rich VLDL B-VLDL Electrophoretic mobility: w/ LDL Density: Like VLDL (400 mg/dL or for patients with B- VLDL 2. DeLong Equation More accurate than Friedewald when TG > 400 mg/dL LDLc = TC – (HDLc + VLDL) VLDL = TC + 6.5 (mg/dL) VLDL = TG + 2.825 (mmol/L) 3. Beta Quantification Uses ultracentrifugation (at least 18 hours at 105 K x g ) to separate VLDL and CM Remaining solution is measured for cholesterol LDL is precipitated out and the solution is again measured for cholesterol The difference between the 2 measurements is the concentration of LDL  LDL MEASUREMENT 4. HOMOGENOUS DIRECT LDL-C METHOD TG elevated 2 reagents  FREDICKSON CLASSIFICATION OF HYPERLIPIDEMIAS TYPE SYNONYM DEFACT Serum Clinical Features Treatment Serum Abnormality Appreance TYPE 1 Famillial Low LDL Altered ApoC2 Chylomicron Pancreatitis, Lipemia retinalis, Diet Creamy top Hyperchylomicronemia ↑ skin eruptions, Xanthoma, layer Hepatospienomegaly TYPE IIa Famillial Hypercholestrolmeia ↓LDL receptor LDL ↑ Xanthelasma, Arcus senilis, Cholestryramine or Clear Tendon xanthomas Cholestipol, Statins, Niacin TYPE IIb Famillial Combined ↓LDL receptor & LDL & VLDL ↑ Stains, Niacin, Fibrate Clear Hypercholestrolemia ↑Apo B TYPE III Famillial Apo E2 synthesis defect IDL ↑ Turbo-eruptive xanthomas, Fibrate, Statins Turbid dybetalipoproteinemia palmar xanthoma TYPE IV Famillial Hyperlipemia ↑VLDL production VLDL ↑ Statins, Niacin, ↓elimination Fibrate TYPE V Endogenous ↑VDL production VLDL & Niacin, Fibrate Creamy top hypertriglyceridemia ↓LPL Chylomicron Turbid ↑ bottom Type 1 hyperlipoproteinemia: Elevated chylomicrons 1) Serum appearance: Creamy layer of chylomicrons over clear serum 2) Total cholesterol: Normal to Moderately elevated 3) Triglyceride: Extremely elevated 4) ApoB- 48 increased, ApoA-IV increased Type IIa hyperlipoproteinemia: Increased LDL 1) Serum appearance: Clear 2) Total Cholesterol: Generally elevated 3) Triglyceride: Normal 4) Apo-B 100: increased Type IIb hyperlipoproteinemia: Increased LDL and VLDL 1) Serum appearance: Clear or slightly turbid 2) Total Cholestrol: Elevated 3) Triglyceride: Elevated 4) Apo B-100 increased Type III hyperlipproteinemia: Increased LDL 1) Serum appearance: Creamy layer sometimes present over a turbid layer 2) Total cholesterol: Elevated 3) Triglyceride: Elevated 4) Apo E- II increased, Apo E-III decreased, and Apo E-IV decreased Type IV hyperlipoproteinemia: Increased VLDL 1) Serum appearance: Turbid 2) Total cholesterol: Normal to slightly elevated 3) Triglyceride: Moderately to severely elevated 4) ApoC-II either increased or decreased, and ApoB-100 increased Type V hyperlipoproteinemia: Increased VLDL with increased chylomicrons 1) Serum appearance: Turbid with creamy layer 2) Total cholesterol: Slightly to moderately elevated 3) Triglyceride: Severely elevated 4) Apo C-II increased or decreased, Apo B-48 increased, and Apo B-100 increased  HYPOLIPOPROTEINEMIAS a. Abetalipoproteinemia: (also known as Bassen-Kornzweig syndrome) Total cholesterol level very low, trglyceride level nearly undetectable, LDL and Apo B-100 absent b. Hypobetalipoproteinemia: Unable to synthesize apo B- 100 and apo B- 48, low total cholesterol level and normal to low triglyceride level c. Hypoalphalipoproteinemia: Severely elevated triglyceride level and low HDL level d. Tangier disease: HDL absent, apo A-I and Apo A-II very low levels, LDL low, total cholesterol level low, triglyceride level normal to slightly increased. Due to a mutation in the ABCA1 gene on chromosome 9. Arteriosclerosis – hardening and narrowing of arteries Atherosclerosis – narrowing of arterial due to plaque build up on arterial walls due to deposition of cholesterol and TG Coronary Artery Disease Peripheral vascular disease Cerebrovascular disease Sitostrerolemia - is an extremely rare auto somal recessive disorder wherein phytosterois (plant sterois) are absorbed and accumulate in plasma and peripheral tissues. CETP deficiency – is an autosomal recessive disorder in which the transfer of cholesterol esters in inhibited. As a result, HDL particles are large and laden with cholesterol ester, and apoA-I is increased, as is HDL-C (typically >100mg/dL). Chylomicron retention disease (Anderson’s disease) – presents in childhood with fat malabsorption and low levels of plasma lipids  METABOLIC SYNDROME: - Group of risk factors that seem to promote development of atherosclerotis cardio vascular disease & type of 2 diabetes mellitus - Risk Factors: ↓ HDL –C ↑ LDL – C ↑ triglycerides ↑ blood pressure ↑ blood glucose