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Edit Comment Close Premium member Presentation Transcript LIPID PROFILE : LIPID PROFILE INTRODUCTION : INTRODUCTION DEFINITION : Lipids are group of organic substances of fatty nature which are insoluble in water, soluble in fat solvents & which are related to fatty acids. IMPORTANCE of LIPIDS : IMPORTANCE of LIPIDS Reservoir of energy Constituents of cell membrane Constituents of neural tissue As a insulator & protective coating As vitamins CLASSIFICATION OF LIPIDS : CLASSIFICATION OF LIPIDS : LIPOPROTEINS : LIPOPROTEINS Spherical complexes of lipids & proteins. Transport various lipids & fat soluble vitamins to & from tissues. Contain core of hydrophobic lipids surrounded by hydrophilic lipids & proteins. LIPOPROTEINS-CLASSIFICATION : LIPOPROTEINS-CLASSIFICATION PLASMA LIPOPROTEINS : PLASMA LIPOPROTEINS PLASMA LIPOPROTEINS : PLASMA LIPOPROTEINS PLASMA LIPOPROTEINS : PLASMA LIPOPROTEINS APOLIPOPROTEINS : APOLIPOPROTEINS Required for assembly & structure of lipoproteins. Serve to activate enzymes important in lipoprotein metabolism. Mediate binding of lipoprotein to cell surface receptor. MAJOR APOLIPOPROTEINS : MAJOR APOLIPOPROTEINS MAJOR APOLIPOPROTEINS : MAJOR APOLIPOPROTEINS MAJOR APOLIPOPROTEINS : MAJOR APOLIPOPROTEINS LIPID PROFILE TESTS : LIPID PROFILE TESTS TOTAL LIPIDS SERUM TOTAL CHOLESTEROL SERUM H.D.L – C TC / H.D.L – C SERUM TRIGLYCERIDES SERUM PHOSPHOLIPIDS ELECTROPHORESIS INDICATIONS : INDICATIONS Screening for primary & secondary hyperlipidemias Monitoring for risk of atherosclerosis Monitoring treatment of hyperlipidemias. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE Variation & error can be introduced before or during venepuncture or when samples are handled & stored before analysis. It is important to standardize conditions under which blood specimen are drawn & prepared for analysis. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE Biological variation: Age = cholesterol levels increase with age. Sex = women have lower level than men except in childhood & after early 50’s. Season = cholesterol levels are slightly higher in winter. Food intake = daily intake of fat increases cholesterol levels. Therefore it is important that patient should be on their usual diet for 2 wks & are neither gaining nor losing weight. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE Biological variation: 5. Medical disorders = thyroid , hepatic & kidney disease. 6. Life style factors = Fasting Posture Venous occlusion Anticoagulants Recent M.I Stroke Cardiac catheterization Trauma Acute infection pregnancy BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE Biological variation: It is recommended that lipoproteins measurement should be made no sooner than 8 weeks after any form of trauma or acute bacterial / viral infection & 3 – 4 months after child birth. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE FASTING : Ideally patient should fast for 12 hours before sampling. The concentration of LDL-C/HDL-C declines after eating Chylomicrons are cleared with in 6 – 9 hours & their presence after 12 hours fast is abnormal. TC/HDL-C levels can be measured in non fasting individuals. NCEP recommended that patient fast for at least 9 hour before blood specimen are taken for lipid/lipoprotein analysis. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE POSTURE: Decreases of as much 10 % in conc. of TC,LDL-C,HDL-C & apo-a I & B, have been observed after 20 minutes recumbence. Postural changes are reversible when patient resumes original position. NCEP guidelines = patient to be seated for 5 min. prior to sampling to prevent hemo concentration. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE POSTURE: 4. If recumbent position is used, this position should be used each time patient is sampled. 5. Prolonged venous occlusion leads to increase in cholesterol conc. By 10 – 15 %. 6. Torniquet should not be for more than minutes or two. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE EXERCISE: Mild exercise produces a slight decrease in conc. Of cholesterol & triglyceride that may persists for several days. Those who walk for about 4 hours each week have an average cholesterol conc. 5 % lower & HDL-C conc. 3.4 % higher than inactive persons. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE MENSTRUAL CYCLE: The plasma cholesterol & triglyceride conc. Tend to be highest at midcycle, the time of maximum estrogen secretion The cyclical variation in cholesterol is not observed with anovulatory cycles. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE DIET: A high fat diet increases serum triglycerides. Ingestion of monounsaturated fat reduces cholesterol & LDL cholesterol. Plasma triglyceride conc. Is reduced when sucrose intake is reduced. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE DIET: A high carbohydrate diet decreases the serum conc. of VLDL-C , TG, cholesterol & protein. Individuals who eat many small meals throughout the day tend to have conc. of total LDL & HDL-C that are lower than when same type & amount of food is eaten in three meals. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE DIET: Large protein meals at lunch or in the evening also increase the serum cholesterol for atleast 1 hour after a meal. In vegeterian individuals, conc. Of LDL & VLDL-C are reduced by 37 % & 12%. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE SMOKING: The plasma cholesterol , triglyceride & LDL cholesterol conc. are higher by about 3 % 9.1 % & 1.7 % respectively in smokers than in non smokers. HDL cholesterol is lower in smokers than in non smokers.s BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE ALCOHOL INGESTION: When moderate amount of alcohol is ingested for 1 week, the serum TG conc. Is increased by more than 20 mg/dl. Prolonged moderate ingestion may increase HDL-C conc., which is assoc. with reduced plasma conc. Of cholesterol ester transfer protein.( CETP ) BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE VENOUS Vs CAPILLARY SAMPLES: Measurements in the capillary samples seem to be little lower than venous samples. 2. Measurements in the fingerpick samples tends to be more variable than in venous samples. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE PLASMA Vs SERUM: Either can be used when TC,TG & HDL-C are measured. Plasma is preferred when lipoprotein are measured by ultra centrifugal / electrophoretic methods. Serum can be used when it is necessary to store samples for weeks or months. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE ANTICOAGULANTS: Some anticoagulants such as citrate exert large osmotic effect. This results in falsely low plasma lipid & lipoprotein concentration. Heparin because of high M.W can alter electrophoretic mobilities of lipoprotein. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE ANTICOAGULANTS: EDTA is preferred anticoagulant even though TC & TG conc. In EDTA plasma are 3 % lower than in serum. EDTA retards certain oxidative & enzymatic alterations that occur in lipoproteins during storage. TC values of EDTA plasma should be multiplied by 1.03 to make it comparable to serum values. BLOOD SAMPLING & STORAGE : BLOOD SAMPLING & STORAGE STORAGE: TC,TG,HDL-C can be satisfactorily analyzed in frozen samples. Apolipoproteins can also be measured in frozen samples. Serum / plasma must be stored at – 70 deg if stored for long time. For short time storage ( upto a month or two ) the sample can be kept at – 20 deg. ESTIMATION OF PLASMA LIPIDS : ESTIMATION OF PLASMA LIPIDS Cholesterol & triglycerides are plasma lipids of most interest in diagnosis & management of lipoprotein disorders CHOLESTEROL – chemical method : CHOLESTEROL – chemical method Standard reference method. Modified ABELL KENDALL method. PRINCIPLE : 1)Cholesteryl esters hydrolyzed with alcoholic KOH 2) Unesterfied cholesterols are extracted with petroleum ether. 3) Then they are measured with LIEBERMANN BURCHARD REAGENT. CHOLESTEROL – enzymatic method : CHOLESTEROL – enzymatic method PRINCIPLE : These measure total cholesterol directly in plasma/serum through a series of reaction in which cholesterol esters are hydrolyzed , the 3- OH group of cholesterol is oxidized & H2O2 is liberated which is measured . CHOLESTEROL – enzymatic method : CHOLESTEROL – enzymatic method Cholesteryl esters + h2o ---------- cholesterol + free fatty acids Cholesterol + o2 --------------------------------------- ---cholesten-4 en-3one + h2o2 H2o2 + phenol + 4-aminoantipyrine -------- quinoenimine +2h2o The absorbance of quinoenimine produced is measured at 500 nm. CHOLESTEROL – enzymatic method : CHOLESTEROL – enzymatic method ADVANTAGES : Less subject to interference by non-sterol substances that react in chemical method. Consume only micro liters quantity of sample. Do not require preliminary extraction step. Rapid method. If cholesterol ester hydrolase step is omitted, they can be used to measure Unesterfied cholesterol. CHOLESTEROL – enzymatic method : CHOLESTEROL – enzymatic method DISADVANTAGES : They are not absolute specific for cholesterol Cholesterol oxidase reacts with sterols other than cholesterol & also with plant sterols. Ascorbic acid & bilirubin interfere with enzymatic methods by consuming h2o2. CHOLESTEROL – enzymatic method : CHOLESTEROL – enzymatic method DISADVANTAGES : 4. Interference of bilirubin is complex & can produce falsely high or low values. 5. Interference by bilirubin seems to be significant only at conc. > 5 mg/dl. 6. At this level, it has been reported to decrease cholesterol values by 5-15 %. SERUM CHOLESTEROL: INCREASED IN : SERUM CHOLESTEROL: INCREASED IN PRIMARY HYPER LIPOPROTEINEMIA SECONDARY HYPER LIPOPROTEINEMIA Diabetes mellitus Hypothyroidism Nephrotic syndrome Chronic uremia Hemodialysis Hepatic glycogenoses. Obstru. Liver disease. Chronic alcoholism SERUM CHOLESTEROL: DECREASED IN : SERUM CHOLESTEROL: DECREASED IN Severe liver damage. Hyperthyroidism Malnutrition Myelo proliferative disorders Chronic anemia Infection Drugs Hypo beta lipoproteinemia A beta lipoproteinemia Tangier’s disease TRIGLYCERIDE-ENZYMATIC METHOD : TRIGLYCERIDE-ENZYMATIC METHOD PRINCIPLE : Serum TG are hydrolysed to glycerol & free fatty acids by lipase. In presence of ATP & glycerokinase , glycerol is converted to Gly – PO4 which is then oxidised to yield h2o2. H2o2 reacts with ESPAS to form colored complex, the intensity of which is measured at 546 nm ( 530-570 nm ) TRIGLYCERIDE:INCREASED IN : TRIGLYCERIDE:INCREASED IN Familiar hypertriglyceridemia Von gierke disease Diabetes mellitus Hypothyroidism Nephrosis Chronic renal failure Diet Drugs TRIGLYCERIDE:DECREASED IN : TRIGLYCERIDE:DECREASED IN A beta lipoproteinemia Malnutrition Recent weight loss Vigorous exercise Drugs NOTE: TG levels are inversely related to HDL-C levels. TRIGLYCERIDE : TRIGLYCERIDE HDL-CHOLESTEROL: HOMOGENOUS ASSAY : HDL-CHOLESTEROL: HOMOGENOUS ASSAY PRINCIPLE : The method depends on the properties of detergent which solubilizes only the HDL so that HDL-C is released to react with the cholesterol esterase & cholesterol oxidase & chromogen to give color. The intensity of color is formed proportional to conc. Of HDL in sample, the absorbance of which is measured at 600 nm( 600- 670 nm ) HDL-CHOLESTEROL: INCREASED IN : HDL-CHOLESTEROL: INCREASED IN Moderate consumption of alcohol, insulin & estrogen. Hyper alpha lipoproteinemia Hypo beta lipoproteinemia HDL-CHOLESTEROL: DECREASED IN : HDL-CHOLESTEROL: DECREASED IN Stress Acute M.I Stroke Surgery Starvation Diabetes mellitus Hypothyroidism Liver disease Nephrosis Uremia Chronic anemia LDL-CHOLESTEROL : LDL-CHOLESTEROL FRIDEWALD CALCULATION : LDL-C (MMOL/L) = (TC – HDL CHOLESTEROL) – PLASMA TG / 2.175 LDL-C (MG/DL) = (TC – HDL CHOLESTEROL) – PLASMA TG / 5 LDL-CHOLESTEROL : LDL-CHOLESTEROL FRIDEWALD CALCULATION : DISADVANTAGES : Underestimate LDL-C in chronic alcholics Unsuitable for monitoring Misclassifies 15 – 40 % of patients when TG levels are between 200 to 400 mg/dl Fails if fasting TG levels > 400 mg/dl. Not reliable if suspect type III dyslipidemia LDL-CHOLESTEROL: INCREASED IN : LDL-CHOLESTEROL: INCREASED IN Familiar hypercholesterolemia Familiar combined hyperlipidemia Diabetes mellitus Hypothyroidism Nephrosis Chronic renal failure LDL-CHOLESTEROL: DECREASED IN : LDL-CHOLESTEROL: DECREASED IN Severe illness Certain drugs A beta lipoproteinemia VLDL-C/ PLASMA TG ratio : VLDL-C/ PLASMA TG ratio VLDL-C/PLASMA TG ratio may be useful in evaluation of type III hyperlipoproteinemia Expressed in mol/mol or mass/mass Ranges 0.230-0.575 in samples without beta VLDL Type III subjects have ratio > 0.689, usually in range of 0.689 – 0.0919 NORMAL LEVELS : NORMAL LEVELS SERUM CHOLESTEROL & TG RISK CATEGORIES : SERUM CHOLESTEROL & TG RISK CATEGORIES CARDIAC RISK RATIO : CARDIAC RISK RATIO Cardiac risk ratio = LDL-C/HDL-C Normal = 0.5 – 3.0 Moderate risk = 3.0 – 6.0 High risk = > 6.0 MAJOR RISK FACTORS THAT MODIFY LDL GOALS : MAJOR RISK FACTORS THAT MODIFY LDL GOALS Cigarette smoking Hypertension Low HDL cholesterol ( < 40 mg/dl ) Family history of premature CHD Age ( men > 45 ; women > 55 ) Diabetes mellitus Pre existing CHD ATHEROGENIC INDEX : ATHEROGENIC INDEX ATHEROGENIC INDEX = combination of ratio of LDL-C to HDL-C X apo-B with ratio of apo-B to apo-AI Atherogenic index = (TC-HDL-C) x apo-B/ (apo AI X HDL-C) ESTIMATION OF LIPOPROTEINS : ESTIMATION OF LIPOPROTEINS ULTRACENTRIFUGAL METHODS ELECTROPHORETIC METHOD POLY ANION PRECIPITATION METHOD HOMOGENOUS ASSAY LIPOPROTEIN ELECTROPHORESIS : LIPOPROTEIN ELECTROPHORESIS USE : identify rare familiar disorders ( e.g TYPE I, III, V HYPERLIPIDEMIA ) INDICATION : serum TG > 300 mg/dl fasting serum is lipemic significant hyperglycemia, impaired glucose intolerance serum uric acid > 8.5 mg/dl clinical evidence of CHD or atherosclerosis in patient < 40 years of age. COMPLICATIONS OF HYPERLIPIDEMIA : COMPLICATIONS OF HYPERLIPIDEMIA ACUTE MYOCARDIAL INFARCTION STROKE DIABETES MELLITUS GALL STONES METABOLIC SYNDROME PANCREATITIS REFERENCES : REFERENCES HENRY’S clinical diagnosis & management by lab. methods 21/e TIETZ’S fundamentals of clinical chemistry 6/e Slide 66: THANKS You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.