LIPIDS-AND-LIPOPROTEINS.pdf

CLINICAL CHEMISTRY LABORATORY BS MEDICAL TECHNOLOGY – MED229 1ST TERM – FINALS Prof. Debbie Ann C. Rivera-De Leon Transports exogenous (dietary TAG) to muscle...

CLINICAL CHEMISTRY LABORATORY BS MEDICAL TECHNOLOGY – MED229 1ST TERM – FINALS Prof. Debbie Ann C. Rivera-De Leon Transports exogenous (dietary TAG) to muscle and depot LIPIDS AND LIPOPROTEINS Apo B-48, Apo C & E Another primary source of fuel in body which provides ability to cell membrane and allows transmembrane transport LIPIDS Commonly referred to as fats, are ubiquitous constituents of all living cells and have a dual role. o First, because they are composed of mostly carbon-hydrogen (C-H) bonds, they are a rich source of energy and an Carry the exogenous triglycerides efficient way for the body to store Produced by the: INTESTINE excess calories. Post prandial specimen: turbidity and o They provide stability to cell white creamy float membrane. 2. Very Low Density LPP (Pre-β LPP) 65% TAG, 16-22% chole, 15-20% phospholipid Lipids – fats, oils, non polar 6-10% CHON Transports endogenous TAG TO muscles and Composed of carbon, hydrogen, and oxygen rich fat depot source of energy Apo B-100, Apo C& E Linked by ESTER BONDS Carry the endogenous triglycerides Produced in the: LIVER Classification of Lipids Classification of Lipoproteins Cause turbidity of fasting specimen From carbohydrate, saturated fatty acids and Chylomicrons (CM) trans fatty acids Triglyceride (TG) Very Low Density Apo E, and Apo C are also integral components Cholesterol Lipoproteins (VLDL) Phospholipid Low Density Adipocyte release of free fatty acids Free fatty Lipoproteins (LDL) acids/fatty acids High Density 3. High Density LPP (Alpha LPP) Lipoproteins (HDL) Smallest but the most dense LPP (1.063-1.21) 30% phospholipid, 15% CE, 45-50% CHON Transports chole from the tissues to the liver CLASSIFICATION OF LIPOPROTEINS Vehicle for reverse chole transport 1. Chylomicrons Apo A-I % Apo A-II; Apo C Largest but the least dense ( 240 mg/dL – high cholesterol Lecithin Cholesterol Acyl Transferase Catalyzes the esterification of cholesterol by promoting the Diagnostic significance: transfer of fatty acids from lecithin It evaluates the risk for: to cholesterol. Atherosclerosis Normally present in human plasma Myocardial and coronary arterial It is synthesized in the liver occlusions. Apo A-Q is the activator of LCAT It is also used as thyroid, liver and renal 2. Free Cholesterol – 30% function tests; and for DM Mellitus Plasma/serum/RBC Used to monitor effectiveness of lifestyle It is a polar non esterified form of alcohol changes and stress management. SOURCES OF CHOLESTEROL: 1. Exogenous (diet) – 15% 2. Endogenous (liver) – 85% MED229 I 3 I Alissa CLINICAL CHEMISTRY LABORATORY BS MEDICAL TECHNOLOGY – MED229 1ST TERM – FINALS Prof. Debbie Ann C. Rivera-De Leon Chemical Reactions: BENEFICIAL FUNCTIONS OF CHOLESTEROL: 1. Abell Kendal Bloor’s reagent extraction and KOH 1. It is used as a starting material for the saponification production of steroids Proceed to LB Method 2. It is converted to bile salts 3. It is an integral component of cell membrane 2. Liebermann Burchardt Reaction Extract with XEOLITE Reagent: Acetic anhydride and sulfuric acid Cholestadienyl monosulfonic acid End product = Cholestadienyl Monosulfonic Acid (green end color) Color developer Mixture: Glacial Acetic Acid Acetic anhydride Concentrated H2SO4 3. Salkowski Reaction Methodologies: End product = Cholestadienyl Either plasma or serum can be used for Disulfonic Acid ( red end color) measurements Reagent: choloroform and Sulfuric Acid 2 weeks prior to testing, the person should be Cholestadienyl disulfonic acid in its usual diet Total Cholesterol (TC) concentration is measured rather than its forms Methodologies: Precautions Cholesterol value is essential in diagnosis and ➔ Avoid hemolyzed blood management of lipoprotein disorders ➔ Avoid Icteric sample ➔ Avoid water contamination General Methods in Chemical Methods: ➔ Precise and Accurate timing for color development must be observed Two-Step Method – colorimetry + Extraction (Bloor’s method) Three-Step Method – C + E + E + Precipitation (Parekh and Jung, Schoenheimer Sperry) Colorimetric reaction is based on the presence of double bonds and hydroxyl group in the sterol’s structure MED229 I 4 I Alissa CLINICAL CHEMISTRY LABORATORY BS MEDICAL TECHNOLOGY – MED229 1ST TERM – FINALS Prof. Debbie Ann C. Rivera-De Leon Current CDC reference method: Hemolyzed Abell, Levy, and Brodie Method Hydrolysis with alc. KOH, hexane extraction and colorimetry with Liebermann Burchardt Reagent Recently, the reference method has changed to a gas chromatography – mass spectrometry (GC-MS) method that now specifically measures cholesterol and does not detect related sterols. This method shows good agreement with the gold standard method developed and applied in the U.S. National Institute of Standards and Technology, the so-called Definitive Method, using isotope dilution mass spectrometry. Methodologies: Enzymatic Methods 3) Triglyceride (Neutral Fat) Comprises one molecule of glycerol and 3 FA Do not contain charged or hydrophilic groups- very hydrophobic and water insoluble. Main storage lipid in man (adipose tissues) Hydrolysis of TAG will yield FA and converted to energy – excellent insulator Fasting requirement: 12-14 hours Reference vaues: 10-190 mg/dL 500 mg/dL – very high residue from cholesterol esters, (recurrent pancreatitis) Comprise about two thirds of circulating cholesterol Diagnostic Significance: Converting them to unesterified or free It evaluates suspected atherosclerosis and measures cholesterol the body’s ability to metabolize fats The free cholesterol is reacted by cholesterol oxidase, producing hydrogen peroxide, A common enzymatic color reaction using Methodologies: horseradish peroxidase to couple two colorless chemicals into a colored compound Either plasma or serum can be used for The intensity of the resulting color, measurements proportional to the amount of cholesterol MED229 I 5 I Alissa CLINICAL CHEMISTRY LABORATORY BS MEDICAL TECHNOLOGY – MED229 1ST TERM – FINALS Prof. Debbie Ann C. Rivera-De Leon 4) Fatty Acids Are linear chain of carbon – hydrogen bonds Prior to venipuncture, ideally patient should that terminate with a carboxyl group. undergo fasting for 12-14hours/ It is mostly found as a constituents of Postural changes dec. TAG levels by almost phospholipids or triglycerides 50% (UPRIGHT TO SUPINE POSITION) Mainly derived from hydrolysis of triglycerides in adipose tissue. Lipemic Serum They are very important source of energy Methodologies: Provide the substance for conversion of glucose (gluconeogenesis) Lipemic serum will be Only small form found in plasma, most is frozen – 20 ° C for assay at a bound to albumin. later date, the specimen should be warmed thoroughly to 37 ° C and mixed before the test. Reference value: 9-15 mg/dL Methods: LIPOPROTEINS 1. Van Handel and Zilversmith – colorimetric Large molecular complexes of lipids with Formaldehyde + Chromotropic acid = Red specialized proteins known as chromophore apolipoproteins. 2. Hantzsch Condensation – Fluorometric Transport lipids (chole and TAG) to sites of Formaldehyde + Diacetyl acetone = Diacetyl energy storage and utilization. Iutidine 3. Glycerol Kinase – Enzymatic Apolipoproteins helps to keep the lipids in solution interact with specific cell-surface receptors Current CDC reference method Enzymes that participates in Lipoprotein metabolism Modified Van Handel and Zilversmith (Lipolytic enzymes): - It involves alkaline hydrolysis by alc. KOH, 1. Lipoprotein Lipase (LPL) chloroform extraction and the extract is treated Hydrolyzes TAG in lipoproteins and released of with silicic acid, colorimetry with fatty acid and glycerol. chromotropic acid. 2. Hepatic Lipase - (+) result: pink colored product Hydrolyze TAG and phospholipid from HDL; Hydrolyzes lipids on VLDL and IDL 3. Lecithin Cholesterol Acyl Transferase (LCAT) Methods of Analysis (Triglycerides) Enzymatic Catalyzes the esterification of cholesterol from HDL; enables HDL to accumulate cholesterol as cholesterol ester. 4. Endothelial Lipase Hydrolyzes phospholipids and TAG in HDL. MED229 I 6 I Alissa CLINICAL CHEMISTRY LABORATORY BS MEDICAL TECHNOLOGY – MED229 1ST TERM – FINALS Prof. Debbie Ann C. Rivera-De Leon Methodologies: Sample collected using serum separator tubes is the preferred sample EDTA plasma is the choice for research studies for lipoprotein fractions, If testing is done in non-fasting samples, only Summary of Specimen Considerations TC and HDL-C can be measured. In fasting samples Most plasma TAG is present Prolonged tourniquet application: false in VLDL; in non-fasting state in chylomicrons. elevated (10-15% elevation) Lipemic samples occurs in non-fasting blood, Fasting requirement: least 12 hours in patients with hyperlipidemia or receiving Required: TG and LDL total parenteral nutrition therapy (TPN) Not required: TC and HDL Lipemic samples may be pretreated by Serum has been the fluid of choice for ultracentrifugation or enzymatic cleavage to lipoprotein measurement in the routine remove lipids because it can interfere with clinical laboratory spectrophotometric, turbidimetric and Pregnancy: elevated immunoassays. Age: proportional with age Lipoproteins are separated based on Sex: female have lower normal range electrophoresis and density than male (Ultracentrifugation) 1. Ultracentrifugation (density gradient) Reference method for quantitation of lipoproteins 2. Electrophoresis HDL, VLDL, LDL, Chylomicrons (electrophoretic pattern) Supporting medium: agarose gel- sensitive; reo;ve lipoprotein classes 3. Chemical precipitation It uses polyanions (heparin and divalent cations), polyethylene glycol LDL and HDL Measurement “LDL – Cholesterol and VLDL – Cholesterol can be precipitated using polyanions like dextran sulfate magnesium, heparin with manganese or phosphotungstate with magnesium” MED229 I 7 I Alissa