Clinical Chemistry Lab - 05 Lipid and Lipoprotein Methodologies

CLINICAL CHEMISTRY LABORATORY • LIPID AND LIPOPROTEIN METHODOLOGIES OUTLINE • Cholesterol Methodologies • Lipoprotein o Notes to Remember Methodologies o Chemical Methods o Notes to Remember o General Methods o Patient Preparation o Colorimetric Non-Enzymatic Method o Ultracentrifugation o Enzymatic Methods o Electrophoresis o CDC Reference Method o Polyanion Precipitation o Clinical Significance Method o HDL • Triglyceride Methodologies o LDL o Notes to Remember o Other Methods o Chemical Methods o Enzymatic Method o CDC Reference Method o Clinical Significance GENERAL METHODS • • • • LIPID & LIPOPROTEIN METHODOLOGIES • • Important in assessing risk for atherosclerosis which may lead to myocardial infarction Lipid profile o Measured in laboratory for lipid & lipoproteins determination o Includes: ▪ Cholesterol ▪ TAG ▪ Lipoprotein (VLDL, LDL, HDL) • Cholesterol o Most atherogenic ▪ Not metabolized as energy ▪ Majority of cholesterol in synthesized by liver • Diet is not a significant factor in cholesterol management ▪ Can be transported to lived by LDL • Can be deposited to blood vessels Only method in CC involving multiple steps* One-step Method (Pearson, Stern and Mac Gavack) o Colorimetry Two-step Method (Bloors) o Extraction + Colorimetry Three-step Method (Abell-Kendal Assay) o Saponification + Extraction + Colorimetry o Most commonly used method Four-step Method (Schoenheimer, Sperry, Parekh, Jung) o Precipitation (acid soln) + Saponification + Extraction + Colorimetry COLORIMETRIC NON-ENZYMATIC METHOD • • CHOLESTEROL METHODOLOGIES • Variables: o Hemolysis: falsely increased o Icteric: 5mg to 6mg increase in cholesterol per mg of bilirubin ▪ Bilirubin interferes with TC measurement (absorbs light @ 500nm) ▪ Icteric - dark yellow to orange discoloration of plasma/serum due to high bilirubin End-product do not use enzymes Liebermann Burchardt Reaction o Acetic anhydride test o Most commonly used chemical method/colorimetric non-enzymatic method (colorimetric assay) o • Sodium sulfate: end color stabilizer ▪ Used because end color may fluctuate Salkowski Reaction COLORIMETRIC ENZYMATIC METHODS NOTES TO REMEMBER CHOLESTEROL OXIDASE METHOD • • • • • Total cholesterol is measured (CE + FC/HDL-C, LDL-C) o Total cholesterol including lipoproteins Sample: 12-hour fasting serum or plasma (not necessary) o Fasting has little effect o EDTA: preferred anticoagulant Increases with age: 2 mg/dL/year (45-65 years old) Two weeks prior to testing: patient is on usual diet, no restrictions in diet & activity Recommendation: o Initial screening for lipid profile: age 20 or above ▪ Should be repeated at least once every 5 years • • • • • CHEMICAL METHODS • Dehydration and oxidation of cholesterol to form a colored compound (colorimetric method) o The presence of double bonds and hydroxyl group in the sterol’s structure makes it possible for cholesterol to carry out a colorimetric assay. ▪ Binds chromogenic agents to form color • H2O2 produced is proportional to amount of cholesterol. Measures free cholesterol (Hydrolyzation of CE to cholesterol is needed to measure CE) Advantages: o Microliter amount of sample o Do no require preliminary extraction step; short time False increase: gross hemolysis False decrease: o Hemoglobin – pseudoperoxidase activity may consume H2O2 o Bilirubin – oxidized by H2O2 losing its absorbance ▪ 5 mg/dL ↑ bilirubin = ↓ cholesterol (5-15%) Chromogenic Agents o Provide color to H2O2 o Used agents ▪ Phenol ▪ 4-aminoantipyrine o • Interferences: o Bilirubin – interferes both spectrally and chemically o Hemolysis – may cause dilution of lipid (false ↓) o Ascorbic acid CHEMICAL METHODS • Van Handel & Zilversmith – Colorimetric method o Uses spectrophotometry • Hantzsch Condensation – Fluorometric method o Fluorochromes are added for fluorescence CDC REFERENCE METHOD • Abell, Levy and Brodie Method o Modification of Abell-Kendal Assay (3-step method) o Chemical method o Uses hexane extraction after hydrolysis with alcoholic KOH followed by reaction with Liebermann Burchardt color reagent. o Saponification (hydrolysis) → extraction (hexane) → colorimetry (Libermann-Burchardt) ENZYMATIC METHOD GLYCEROL KINASE METHOD • • CLINICAL SIGNIFICANCE • • Increased Cholesterol (hypercholesterolemia) o Hyperlipoproteinemia II, III, V o Biliary cirrhosis o Nephrotic syndrome o Poorly controlled diabetes mellitus o Alcoholism o Primary hypothyroidism ▪ Thyroid hormones metabolize cholesterol ▪ Reduced thyroid hormone = decreased cholesterol metabolism = ↑ cholesterol Decreased Cholesterol (hypocholesterolemia) o Severe hepatocellular disease (most common cause) ▪ Hepatic cirrhosis - total malfunction of liver ▪ Deficient synthesis of cholesterol in liver o Malnutrition o Severe burns o Hyperthyroidism ▪ Increased thyroid hormone = increased cholesterol metabolism o Malabsorption syndrome ▪ Improper absorption of dietary cholesterol • • • Universally used for TAG measurement Involves hydrolysis of TAG to free fatty acids and glycerol, followed by phosphorylation of glycerol to glycerophosphate o glycerophosphate = glycerol-3-phosphate o glycerophosphate is proportional to TAG levels Major interference: o Glycerol (free glycerol) ▪ Perform TAG blank to correct TAG values. ▪ Can interfere or undergo phosphorylation ▪ False increase in TAG measurement Common step: o Hydrolyze → phosphorylate Reaction A o The amount of NADH proportional to TAG o Measured at 500-600nm, spectrophotometrically • Reaction B o The amount of H2O2 is proportional to TAG o Measured at 500 nm, spectrophotometrically • Reaction C o Involves ADP rather than glycerophosphate o NADH consumption is measured at 340 nm. TRIGLYCERIDE METHODOLOGIES NOTES TO REMEMBER • • • • Sample: 10-12 hr fasting serum or plasma (necessary) o If stored at -20 C ̊ – warm sample to 37 C ̊ before testing. o Fasting - no food intake, only water is allowed ▪ TAG is mainly derived from diet ▪ Recent food intake may increase TAG levels Lipemia: >400 mg/dL TAG Increasing with age: 2mg/dL/year (45-65 years old) Most commonly used method is based on hydrolysis of TAG and measurement of glycerol o Glycerol measured is directly proportional to TAG TRIGLYCERIDE BLANKS • • • Free glycerol in serum may interfere w/ enzymatic methods Not routinely performed (little practical importance) Glycerol are normally present in serum (<1.5mg/dL) • • • <1.5 mg/dL free serum glycerol = ≈14mg/dL TAG ↑ free glycerol: strenuous exercise, uncontrolled diabetes. o Diabetes - Inefficient metabolism of glucose o After exercise, non-carbohydrate energy source (TAG → Glycerol + fatty acids) are used Blank Assay: o Removal of lipase in the reaction series to detect only free glycerol in the serum o Without lipase, triglyceride will not be hydrolyzed into glycerol and fatty acid o Therefore, only the free glycerol in the sample will be measured. The measured free glycerol is subtracted from the triglyceride measured. (corrected method) • PATIENT PREPARATION • • • CDC REFERENCE METHOD • Modified Van Handel and Zilversmith o Colorimetric chemical method o Cannot be automated (time-consuming) o Involves alkaline hydrolysis (saponification) using alcoholic KOH, solvent extraction with chloroform and the extract is treated with sicilic acid (chromatography) to isolate TAG, and a color reaction with chromotrophic acid gives rise to pink end color o Interfering substances are removed during extraction with chloroform. ▪ Interfering subs. - bilirubin, Hgb, ascorbic acid o Sicilic acid – remove phospholipids from the chloroform extract. • • • • • • Increased Triglyceride (hypertriglyceridemia) o Hyperlipoproteinemia ▪ I, IIb, III, IV, V o Alcoholism o Nephrotic syndrome o Hypothyroidism o Pancreatitis o Diabetes mellitus Decreased Triglyceride (hyportriglyceridemia) o Malabsorption o Malnutrition o Hyperthyroidism o Brain infarction LIPOPROTEIN METHODOLOGIES • Lipoprotein o Lipid transporters o Concentration assumes concentration of lipids • • • Preferred sample: 10-12 hr fasting serum (SST) o SST - serum separator tube o Fasting is unnecessary (Cholesterol & HDL) EDTA plasma can be used o Cholesterol and TAG concentration are 3% lower than serum Cholesterol, TAG and HDL can be used using frozen sample. o Sample should be thawed first before analysis Lipemia in non-fasting serum: hyperlipidemia, patient under total parenteral nutrition therapy. Reference method for quantitation of lipoproteins Basis of the classical term for different lipoproteins Fractionation of lipoproteins based on density. o Density of lipoprotein is based on their protein and TAG content. Reagent: Potassium bromide solution (1.063 density) Frozen sample should not be used! o TAG-rich LPP do not withstand freezing ELECTROPHORESIS • • • NOTES TO REMEMBER • Fasting: 10-12 hours o Fasting state: TAG is present in VLDL o Non-fasting state: TAG is present in chylomicrons ▪ Only Cholesterol and HDL can be measured using non-fasting sample. Diet: o HDL and LDL – declined after eating Posture: o Patient should be seated for 5 minutes prior to collection o Standing to seating: extravascular fluid enters the vascular system causing hemodilution ▪ 10% decrease (TC, HDL, LDL, Apo-A1, Apo-B) is observed after 20-minute period of seating ULTRACENTRIFUGATION CLINICAL SIGNIFICANCE • Lipoproteins are differentiated based on electrophoresis and buoyant density. o Basis of nomenclature • • Separation of lipoprotein based on their net charge. Basis of the nomenclature due to electrophoretic pattern Electrophoretic pattern: o Origin (point of application): Cathode (-) o HDL - α-band – most anodal (+) o VLDL - between α2 and β band ; 2nd most anodal o LDL - β band; least anodal o Chylomicrons - remains in the origin ▪ No electrophoretic pattern o IDL - may migrate to beta or pre-beta region o Lp(a) - migrate toward pre-beta region o Lipoprotein X - only migrate toward the cathode o β-VLDL - migrate with LDL Support Medium: o Agarose Gel Staining dyes: o Oil Red O, Fat Red 7B, Sudan Black B ▪ Reacts with the ester bonds in TAG and CE HOMOGENOUS DIRECT LDL METHOD • • Useful when TAG is elevated (600 mg/dL) Uses two reagents: o First Reagent: selective removal of non-LDL lpp o Second Reagent: release of cholesterol from LDL then measured enzymatically. CALCULATION OF LDL • • • POLYANION PRECIPITATION METHOD • • • Only measures one type of lipoprotein Some lipoproteins are precipitated with polyanions and divalent ions o Polyanions: Heparin sulfate, dextran sulfate, phosphotungstate o Divalent Ions: Mg2+, Ca2+, Mn2+ Most useful lipoprotein test: Enzymatic method + Precipitation method • • Indirect method of LDL quantitation Widely used method in clinical laboratory o Incorporated in most fully automated analyzers Two Formula: o Friedewald Formula – most commonly used o De Long Formula Limitations: o TAG >400 mg/dL o Presence of CM and β-VLDL Friedewald Formula o General Formula: HDL • • • • Uses dextran sulfate (synthetic heparin) with magnesium (precipitant). o Precipitate Apo-B containing lpp (CM, LDL, VLDL) Semi-automated analyzers: Sample are pre-treated with precipitant to remove other lipoproteins. o Most consistent analytical error in HDL assay is the presence of Apo-B containing lipoproteins CDC Reference Method: 3-step method 1. Ultracentrifugation – removes VLDL & chylomicrons 2. Precipitation (heparin + Mg2+) – removed remaining Apo-B containing lipoprotein (LDL) 3. Abell-Kendal Assay – method for cholesterol analysis Homogenous assay – most popular method for HDL analysis o Utilized by most fully-automated analyzers o Do not require pretreatment with precipitant. o First Reaction: reagent forms complex with non-HDL lipoprotein o Second Reaction: releases HDL to be measured enzymatically. LDL • Carry cholesterol from liver to peripheral tissue BETA QUANTIFICATION METHOD • • • • • • Combination of ultracentrifugation and chemical precipitation. Cholesterol is contained in the three major lipoproteins (HDL, VLDL, LDL) o Total Cholesterol = HDL + LDL + VLDL 1st Step: ultracentrifugation to remove VLDL and CM 2nd Step: measure total cholesterol (HDL + LDL) 3rd Step: precipitation of LDL then measure HDL LDL = TC - HDL • De Long Formula o General Formula: OTHER METHODS • • • Chromatography o Uses either Gel Chromatography or Affinity Chromatography. Immunochemical methods o Uses antibodies specific to epitopes present on the lipoproteins. Immunoassay/Immunonephelometry o Used for Apolipoprotein Assay o Utilizes antibody specific against specific apolipoprotein o An antibody-apolipoprotein complex in measured using nephelometry. o Studies have shown that Apo A1 and Apo B are better indicators of atherosclerotic disease than lipoprotein assay o Lp (a) is measured using immunoturbidimetry