Lipids and Lipoprotein Analysis PDF

INTRODUCTION - Those who walk for about 4 hours each week have an average cholesterol conc. 5% Disorders of lipids is of immense importance to...

INTRODUCTION - Those who walk for about 4 hours each week have an average cholesterol conc. 5% Disorders of lipids is of immense importance to lower and HDL-C conc 3.4% higher than medical practice owing to its strong relations to inactive persons Arteriosclerosis and thus obesity, HTN, DM and other Menstrual cycle: abnormalities - The plasma cholesterol and TG conc tend to Good prognosis in the mgt of these conditions are be highest at midcycle, the time of predicated on early detection of deranged blood lipid maximum estrogen secretion profile - The cyclical variation in cholesterol is not Indications for Lipid Profile include: observed with anovulatory cycles - Screening for primary & secondary Diet: hyperlipidemias - A high fat diet increases serum TGs. - Monitoring for risk of atherosclerosis - Ingestion of monounsaturated fat reduces - Monitoring treatment of hyperlipidemias cholesterol. CHOLESTEROL and TRIGLYCERIDE are the plasma - Plasma triglyceride conc is reduced when lipids of most interest in the diagnosis and sucrose intake is reduced. management of lipoprotein disorders - A high carbohydrate diet decreases the PREANALYTICAL CONSIDERATIONS serum conc. of VLDL-C, TG, cholesterol and protein. It is important to standardize conditions under which - Individuals who eat many small meals blood specimen are drawn & prepared for analysis. throughout the day tend to have conc. of total LDL and HDL-C that are lower than Biological Variations: when same type and amount of food is Age: cholesterol levels increase with age. eaten in three meals. Sex: women have lower level than men except in - Large protein meals at lunch or in the childhood & after early 50's. evening also increase the serum cholesterol Season: cholesterol levels are slightly higher in cold for at least 1 hour after a meal. periods. - In vegetarian individuals, conc. Of LDL & Food intake: daily intake of fat increases cholesterol VLDL-C are reduced by 37 % and 12%. levels. Smoking: Patients should be on their usual diet for 2 weeks - The plasma cholesterol, triglyceride and and are neither gaining nor losing weight. LDL cholesterol conc. are higher by about Medical conditions: thyroid, liver, and kidney 3%, 9.1% and 1.7% respectively in smokers diseases than in nonsmokers. Acute illness: It is recommended that lipoproteins - HDL cholesterol is lower in smokers than in measurement should be made no sooner than non-smokers 8weeks after any form of trauma or acute Alcohol ingestion: bacterial/viral infection and 3 - 4 months after - When moderate amount of alcohol is childbirth ingested for 1wk, the serum TG conc is Lifestyle: higher in sedentary and poor diet habits increased by more than 20mg/dL. - Prolonged moderate ingestion may increase SPECIMEN COLLECTION AND STORAGE: HDL-C conc, which is associated with reduced plasma conc of cholesterol ester Patient should fast for 12hours before sampling. transfer protein (CETP). - The concentration of LDL-C/HDL-C Plasma vs Serum: declines after eating - Either can be used when TC, TG and HDL-C - Chylomicrons are cleared within 6-9hrs and are measured. their presence after 12hrs fast is abnormal. - Plasma is preferred when lipoprotein is Posture: measured by ultra-centrifugation and - Decreases of as much 10% in conc. of TC, electrophoretic methods. LDL-C, HDL-C and apo-A-l and B, have been - Serum can be used when it is necessary to observed after 20 minutes recumbence store samples for weeks or months. - patient to be seated for 5min prior to Venous vs Capillary samples: sampling to prevent hemo-concentration. - Measurements in the capillary samples Prolonged venous occlusion leads to increase in seem to be little lower than venous cholesterol conc by 10-15% samples. - Torniquet should not be for more than Anticoagulants: minutes or two. - Some anticoagulants such as citrate exert Exercise: large osmotic effect resulting in falsely low - Mild exercise produces a slight decrease in plasma lipid and lipoprotein concentration. conc of cholesterol and TG that may - Heparin: because of its high M.W can alter persists for several days. electrophoretic mobility of lipoproteins.  - EDTA is preferred anticoagulant even Bloors Method: though TC and TG conc in EDTA plasma are - Principle: 2 step 3% lower than in serum. Cholesterol is extracted using an alcohol ether - EDTA inhibits oxidation of lipids and mixture proteolysis of apolipoprotein. Measured using the L-B Reaction - TC values of EDTA plasma should be multiplied by 1.03 to make it comparable to ENZYMATIC METHOD: serum values. Cholesterol Oxidase Method (Routine Lab - Assay of Storage: Choice): - TC, TG, HDL-C can be satisfactorily Principle: Cholesterol ester + H20 cholesterol analyzed in frozen samples. esterase.> Free cholesterol - Apolipoproteins can also be measured in Free Cholesterol oxidase.> 4 cholestene-3-one + frozen samples. H202 - Serum or plasma must be stored at - 70°C if - Trinders Reaction: stored for long time. H202 + 4-aminophenazone peroxidase -> - For short time storage (up to a month or Quinoneimine dye (red) + H20 two) the sample can be kept at - 2°. - Read at 500nm wavelength - Linear up to 600 - 700mg/dL (15.54 - ANALYTICAL METHODOLOGY 18.13mmol/L) TOTAL CHOLESTEROL: Advantages (in comparison to the Chemical Method): Chemical Method: via Liebermann-Burchardt - Precise and accurate Reaction - Lesser interferences - bilirubin, ascorbic - Modified Abell Kendall Method acid, Hb - Bloors Method - Smaller sample quantity Enzymatic Method: - Rapid; does not require preliminary - Cholesterol oxidase (Routine Lab) extraction step GC-MS Method (Reference Method) - Can be used to measure unesterified Isotope Dilution Mass Spectrometry (Definitive cholesterol by omitting de-esterification Method) step TRIGLYCERIDE: - Mild reagents; better suited for automated analyzers Chemical Method - Van Handel and Zilversmith Disadvantages: Enzymatic Method - They are not specific for cholesterol. - Glycerol Kinase Cholesterol oxidase reacts with other sterols e.g GC-MS Method (Reference Method) plant sterol LIPOPROTEINS: - Ascorbic acid and Bilirubin interfere by consuming H202 Polyanion Precipitation Bilirubin interference can produce falsely high or low Electrophoresis values Ultra-centrifugation Significant only at conc >5mg/dL decreasing Chol Immunochemistry values by 5-15% OTHER METHODS: GC-MS METHOD (Reference Method): - Mass Spectrometry Specifically measures cholesterol and does not - Chromatography detect related sterols - Homogenous assay Shows good agreement with the Definitive Method – CHOLESTEROL ESTIMATION Isotope Dilution Mass Spectrometry Cholesterol Desirable level: < 200mg/dL (< CHEMICAL METHODS: 5.2mmol/L); Conversion factor = 0.026 Abell Kendall Method (Former Reference Method): Estimation of Lipoproteins - Principle: 3 step Cholesterol is hydrolyzed with alcoholic KOH POLYANION PRECIPITATION: Unesterified cholesterol is extracted with petroleum Lipoproteins are precipitated with polyanions ( jelly heparin sulfate, dextran sulfate and Measured using the L-B Reaction phosphotungstate) - Liebermann-Burchardt Reaction (L-B Reaction should be in the presence of divalent Reaction): cations Mg, Ca and Mn Cholesterol + Sulfuric acid + Acetic anhydride => Most commonly for HDL and is reasonably specific bluish green solution LIPOPROTEIN ELECTROPHORESIS: It allows the supernatant to be analyzed without the need to remove it from the sedimented complex. Used to identify rare familial disorders (e.g Type I, III, V Hyperlipidemia) HOMOGENOUS ASSAY (Direct HDL-C Assay): Indications: - serum TG > 300 mg/dL Enzymatic method: - fasting serum is lipemic - First reagent - "blocks" non-HDLs - significant hyperglycemia, impaired glucose Use of Antibodies or Polymers or complexing agent intolerance e.g Cyclodextrin - serum uric acid > 8.5 mg/dL Modification of cholesterol esterase and oxidase - clinical evidence of CHD or atherosclerosis enzymes which makes them selective for HDL-C in patient < 40 years of age. Use of blanking step that selectively consumes Provides visual display useful in detecting unusual or cholesterol from non-HDL species variant patterns - Second reagent - quantifies accessible Agarose gel - most common; provides a clear HDL-C background and convenient in use Highly precise and reasonably accurate but lacks While PAGE gives a more detailed separation – specificity for HDL in unusual specimens e.g liver or fractionate LDL subclasses kidney disease More useful in qualitative analysis Does not require pretreatment Not desirable in LP quantitation due to poor precision and large systemic biases The "Three-step Procedure" (Reference method for HDL-C estimation): ULTRA-CENTRIFUGATION: - Ultracentrifugation to remove VLDL Preparative Ultracentrifugation - Heparin manganese precipitation to - Uses sequential density adjustments of remove LDL serum to fractionate major and minor - Analysis of supernatant cholesterol by the classes of LP Abell Kendall assay Density gradient methods (non-equilibrium or It is tedious and expensive equilibrium techniques) permit fractionation or several or all classes of LPs in a single run LDL-C Estimation Workhorse for separation of LPs for quantitative INDIRECT METHODS: purposes and preparative isolations Used in the reference methods for LP quantitation Fridewald Equation (Calculation Method) - Routine. because LPs are classically defined in terms of - LDL-Chol(mmol/L) = [TC - HDL-Chol] - hydrated density Plasma TG/2.175. or, Tedious, expensive and technically demanding - LDL-Chol(mg/dL) = [TC - HDL-Chol] - Plasma TG/5 IMMUNOCHEMICAL METHODS: VLDL (mg/dL) = [TAG]/5 or VLDL (mmol/L) = Use antibody-coated plates specific for epitopes on [TAG]/2.175 apolipoproteins both in routine and research lab The factor [TAG]/5 is an estimate of the VLDL cholesterol and is based on the average ratio of HDL-C Estimation: triglyceride to cholesterol in VLDL PRECIPITATION METHOD: Equation assumes patient fasted and plasma (TAG] does not exceed 5.0mmol/L - Precipitating reagents such as divalent - Limitations: not appropriate in cations and polyanions are used to remove Samples with TG > 400mg/dL all lipoproteins except HDL Patients with suspected Dysbetalipoproteinaemia - Enzymatic method for total cholesterol - Other limitations: (Cholesterol Oxidase) is used to quantitate Does not account for cholesterol associated with IDL HDL-C and Lp(a) - Demerit: Underestimate LDL-C in chronic alcoholics Interference from elevated TG levels causing Unsuitable for monitoring incomplete sedimentation after centrifuging which Mis-classifies 15 - 40% of patients when TG levels results in over estimation of HDL-C are between 200 to 400 mg/dL MAGNETIC METHOD Beta-Quantification (Reference method): Like the HDL-C precipitation method but uses a - Tedious - reserved for samples where precipitant that is complexed to magnetic particle Fridewald equation is inappropriate These sediments and does not require centrifugation - 2 steps Has been adapted for use in automated clinical Ultracentrifugation to remove VLDL leaving behind chemistry analyzers because, LDL and HDL as well as IDL and Lp(a)  Chemical Precipitation of HDL-C from either the Glycerol + ATP glycerol kinase_> Glycerophosphate + whole serum or the infranate obtained from the ADP ultracentrifugation Glycerophosphate Glycerophosphate Oxidase_> - LDL-C is calculated as difference btw Dihydroxyacetone + H202 Cholesterol measured in infranate and in Trinder's reaction: the HDL fraction o H202 + Chromogen peroxidase.> Pink - VLDL-C is usually calculated as the compound + H20 difference btw that in whole serum and the Read absorbance at 500m wavelength, and linear up amount in the infranate fraction to 700mg/dL VLDL-C/Plasma TG ratio: Merits: - may be useful in evaluation of type Ill - Fairly specific hyperlipoproteinemia Demerits: - Expressed in mol/mol or mass/mass - Glycerol kinase reacts with endogenous - Ranges 0.230-0.575 in samples without free glycerol causing interference which is beta VLDL clinically insignificant except in - Type Ill subjects have ratio > 0.689, usually DM, in range of 0.689 - 0.0919 emotional stress, IV admin of glycerol containing drugs or nutrients, DIRECT METHODS: contamination of blood collecting devices, and Methods based on selective Precipitation: prolonged storage under non-refrigerated conditions - Uses polyvinyl sulfate or heparin at low pH, - Can be corrected for by "double-cuvette OR blank" or the "single-cuvette blank" or by - Pretreatment using a mixture of polyclonal "Designated calibration blanking" antibodies to apo A-1 and po E linked to a - Or by enzymatically consuming glycerol in a resin to bind and remove VLDL, IDL, and pre-reaction step before measuring TG HDL Triglycerides Desirable Level: Formaldehyde Nephelometer and so not commonly used Formaldehyde + Chromotropic acid.----- Blue These light scattering assays are subject to solution interferences from larger TG-rich lipoproteins and Demerit: VLDL - Tedious, poorly characterized Other immunochemical methods available include: - Enzyme-linked Immunosorbent Assay GC-MS METHOD (Reference Method): (ELISA) - Radial Immunodiffusion (RID) Hydrolysis of fatty acids on TGs and measurement of - Radioimmunoassay (RIA) Glycerol Antibodies used may be either monoclonal or NB: Accuracy in TG is less relevant than that for polyclonal Chol, due to very large physiologic variation with CV of 25 - 30%, thus, contribution of analytical variation is insignificant ENZYMATIC METHOD: Glycerol Kinase Method: Principle: TAG + 3H20 lipase.> Glycerol + 3fatty acids