lipid profile

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Slide 1: 

LIPID PROFILE Dr.Pankaj Gupta MBBS, DCP Std. Gandhi medical college, Bhopal

LIPIDS : 

LIPIDS These are a group of organic substances which are : Insoluble in water Soluble in fat solvents. (ether, alc) Related to fatty acids Important in biological metabolisms.

Classification : 

Classification

Chemical Properties of Lipids : 

Chemical Properties of Lipids Enzymatic hydrolysis (lipase, alkaline pH) Saponification (KOH, NaOH) Libermann Burchard Reaction glacial acetic acid Choloestrol + acetic anhydride green colored compound H2SO4 Triglycerides cholestrol

Slide 5: 

Precipitation - Phosphotungstic acid + MgCl2 or Heparin + MnCl2 are used to precipitate chylomicrons, LDL and VLDL in the serum. Extraction by ether alcohol and isopropanol Electrophoretic fractionation of Lipoprotines(beta, Prebeta and Alpha ) HDL

LIPOPROETINS : 

LIPOPROETINS Complex of lipid and proteins. Important for transport of Cholesterol , triglycerides and Fat soluble vitamins. Made up of triglyceride cholesterol Apoprotein phospholipids hydrophilic hydrophobic

Types of Lipoproteins : 

Types of Lipoproteins Chylomicrons ( Exogenous TGs) VLDL ( Endogenous TGs ) LDL ( Cholesterol to periphery) HDL (Centripetal transporter) Lp(a) (CE)

Apolipoproteins : 

Apolipoproteins

Digestion : 

Digestion

Enzyme participating in Lipoprotein metabolism : 

Enzyme participating in Lipoprotein metabolism

Important lipid profile tests : 

Important lipid profile tests Total lipids Serum total cholesterol Serum HDL cholesterol Total cholesterol/HDL ratio Serum triglycerides Serum phopholipids Electrophoretic fractionation of lipoproteins

Pre-analytical considerations (NCEP) : 

Pre-analytical considerations (NCEP) Steady metabolic state with usual diet. 12hr fasting sample. Pt should be seated for 5 mins before sample collection. Tourniquet to be released within 1min. Multiple determination within 2 mnths (1 week apart).

Determination of serum total cholesterol : 

Determination of serum total cholesterol Reference Range: ≤ 200 mg/dl (5.2 mmol/L) Desirable 200 – 240 mg/dl (5.2-6.2mmol/l) Borderline > 240 mg/dl (>6.2mmol/L) High

Methods : 

Methods

Enzymatic Method : 

Enzymatic Method PRINCIPLE:- CH Esterase Cholesterol ester Cholesterol + Fatty Acids CH Oxidase Cholesterol + O2 Cholesterol-4en-3one +H2O2 Peroxidase 2H2O2 + 4AAP+phenol Quinonimine + 4H2O (end point) RED

Procedure : 

Procedure Take Three Test tubes Label them as Test , Standard and Blank To each test tube Add 1000ul of reagent R1 Add 10ul sample to test, 10 ul Std to Std and 10ul distilled water to Blank. Mix well and incubate for 5 min at 37deg C or 15 min at room temperature. Take readings at 530 nm filter

Calculation : 

Calculation Total Cholesterol = AU x 200 ÷ AS

Interfering Factors : 

Interfering Factors Anticoagulants Hemolysis Hyperlipemic samples No interference by Bilirubin upto 10mg/dl Acsorbic acid upto 7.5 mg/dl Uric acid upto 20mg/dl Hemoglobin upto 200mg/dl

Determination of HDL : 

Determination of HDL Reference range: >60 mg/dl Desirable 35 – 60 mg/dl Boderline < 35 mg/dl High risk

Method : 

Method Principle:- Chylomicrons. LDL. VLDL are precipitated in presence of phosphotungustic acid and magnesium chloride. Reagents :- Precipitate reagent . Std – 25mg/dl.

Procedure : 

Procedure 0.5ml serum + 0.5 ml HDL reagent mix well and allow to stand for 10 mins. Centrifuge for 20mins at 2000rpm or 10 mins at 4000rpm Cholesterol reagent 1000ul in three test tubes and blank, std and sample (50ul of test , blank and std.) Mix and incubate at 37 degC for 5 min or room temp 15 mins Read Absorbance at 530 nm CALCULATION= test / std x 25 x 2

Interfering Factors : 

Interfering Factors Increased Estrogen therapy Insulin therapy Steroids Moderate intake of alcohol. Decreased Stress and acute illness Obesity Lack of exercise Starvation and anorexia Smoking Hypertriglyceridemia >400mg/dl Drugs- antihypertensive and diuretics

Determination of triglycerides : 

Determination of triglycerides Reference range: < 200 mg/dl Desirable 200-400mg/dl Borderline >400 mg/dl High

Method : 

Method Principle: Lipoprotein lipase Triglycerides + H2O2 Glycerol + Fatty acid Glycerol Kinase Glycerol + ATP Glycerol -3- phosphate + ADP GPO Glycerol-3-po4 + O2 DHAP + H2O2 POD H2O2 + 4AAP +p-Chloropheol Red quinoneimine

Slide 28: 

Proceudre:- 1000 ul + 10 ul ( Mix and incubate for 5min at 37 deg C). Linearity :- 1000mg/dl. Calculation :- test /std x 200.

Interfering factors : 

Interfering factors Increased Pregnancy and OCPs Acute illness Obesity Smoking Physical inactivity Drugs Heavy meal Alcohol ingestion Decreased Strenuous exercise Wt. loss

Determination of LDL : 

Determination of LDL Reference Range: <130 mg/dl Desirable 130-159mg/dl Borderline ≥ 160 mg/dl High

Methods : 

Methods Traditional Method: Friedewald Formula LDL (mg/dl) = Total Cholesterol- {HDL+TG/5} Limitation: Not suitable for sample with TG>400mg/dl. Not suitable for samples with Increased Chylomicrons.

Direct LDL estimation : 

Direct LDL estimation Two reagents are used. 1st reagent disrupts all lipoproteins except LDL and de-esterifies the released cholesterol. 2nd reagent releases the cholesterol from LDL and this cholesterol is oxidise by CH oxidase in H2O2 which gives a colored compund.

Interfering factors : 

Interfering factors Increased Pregnancy Steroids, progestin and androgens. Not fasting Decreased Oral estrogen therapy

DIANOSTIC INTERPRETATION OF TESTS : 

DIANOSTIC INTERPRETATION OF TESTS Hyperlipidemias are classified as:- Primary Secondary

Normal values : 

Normal values

Risk Ratio : 

Risk Ratio Total Cholesterol/HDL Low risk 3.3 to 4.4 Avg.risk 4.4 to 7.1 Moderate risk 7.1 to 11.0 High risk >11.0 Atherogenic index (TC-HDL) x ApoB Apo AI x HDL

Slide 37: 


Frederickson Classification of Hyperlipoproteinemias : 

Frederickson Classification of Hyperlipoproteinemias

Cholesterol : 

Cholesterol Increased Type II familial Hypercholesterolemia Hyperlipoprotenemia I, IV, V. Cholestasis Hepatocellular disease and biliary cirrhosis Nephrotic syndrome CRF Pancreatic and prostatic malignancy Hypothyroidism Alcoholism Poorly controlled DM Von geirkes Disease obesity Decreased Hypo alpha lipoproteinemia Severe hepatocellular disease Myloproliferative disease Hyperthyroidism Malabsorption syndrome Chronic anemia Severe burns COPD Mental retardation

LDL cholesterol : 

LDL cholesterol Elevated Familial type II hyperlipidemia DM Hypothyroidism Nephrotic syndrome Cholestasis AIP Anorexia Nervosa Hepatoma Drugs – Thiazides, Cyclosporin, tegretol Reduced Hypolipoprotenemia Tangeirs disease Type I hyperlipidemia Severe liver disease Reye’s syndrome Malabsorption Malnutrition Gaucher’s disease Hyperthyroidism Niacin , toxicity.

HDL : 

HDL Elevated Familial Hyper alpha lipoprotinemia Alcohol Exercise Exposure to chlorinated hydrocarbons Estrogen Reduced Tangier disease Familial hypertryglyceridemia Smoking DM type 2 Obesity Malnutrition Gaucher’s Anabolic steroids Beta blockers

VLDL : 

VLDL Obesity DM type 2 Glycogen storage disease Hepatitis Alcohol Renal failure Sepsis Stress Cushings syndrome Pregnancy Acromegaly

IDL : 

IDL Multiple myeloma Monoclonal gammopathy Autoimmune disease Hypothyroidism

Chylomicrons : 

Chylomicrons Autoimmune disease DM type 2

Lp(a) : 

Lp(a) Renal insufficiency Inflammation Menopause Orchidechtomy Hypothyroidism Acromegaly Nephrosis GH Isotretinoin

Special conditions : 

Special conditions Thyroid disease:- Elevated LDL – activity of LDL receptor in hypothyroidism. Some may show mild hypertriglyceridemia. Thyroid replacement therapy.

Diabetes Mellitus : 

Diabetes Mellitus Def. of insulin or insulin resistance is associated with:- Decreased LPL – Dec. in catabolism of VLDL and Chylomicrons. Increased Triglycerides. Increased release of free fatty acids from adipose tissue. Increased hepatic VLDL production.

Renal Disorders : 

Renal Disorders Nephrotic syndrome is associated with pronounced hyperlipoproteinemia (mixed type i.e Hypercholesterolemia and hypertriglyceridemia).(inc. hepatic VLDL, loss of apoC II, decreased clearance). ESRD is associated with hypertriglyceridemia. ( triglyceride lipolysis and remnant clearance reduced)

References : 

References Harrison’s Internal Medicine 17th edition. Godkar Text Book Of Medical Lab Technology. E. F. Goljan pathology Interpretation of Diagnostic Tests , Wallach A Manual Of Lab and Diagnostic Tests. Henrry’s lab medicine Practical Biochemistry Harold Varley

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