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Cholesterol practical pdf
- 1. Estimation of Serum Total Cholesterol by CHOD-PAP method
- 2. • Cholesterol: – Most abundant animal sterol. – Crystalline yellow solid. – Distributed in all animal cells. – Major component of cell memb. • Maintain memb. Fluidity • Determinant of membrane permeability. – Solid alcohol from bile
- 3. • Occur in two form: – Free cholesterol ( ~ 30 %) – Cholesterol ester ( ~ 70%) • Cholesterol esterified with FA at C3.
- 4. – Structure: – cppp ( phenanthrene nucleus and cyclopentano) • One –OH group at C3. [esterifies with FA] • One double bond between C5 & C6. • An 8-carbon aliphatic side chain attached to C17. • Contain 5 methyl groups. [C20,C13,C10,C25]
- 5. • Properties and reaction – Yellow crystalline solid – Under microscope, show a notched appearance – Insoluble in water and soluble in organic solvent such as chloroform, benzene , etheretc – several reaction given by cholesterol are – SALKOWSKI’S TEST – LIEBERMANN-BURCHARD REACTION – ZAK’S TEST.
- 6. • FUNCTION – Cell membrane: modulating effect on fluid state of the membrane – Nerve conduction: insulate nerve tissue – Bile salts and acids – Steroid hormones – Vitamin D
- 7. • Principle – Cholesterol esterase hydrolyzes Cholesterol esters in serum to give Free cholesterol & Fatty acid. – Cholesterol oxidase oxidises 3-OH group of Free cholesterol to liberate Cholest-4-ene-3-one & H2O2. – H2O2 is then converted to H2O & [O] by Peroxidase. – 4-Amino Antipyrine takes up the [O] & together with phenol forms a pink coloured quinoneimine dye, which is measured at 520nm. – Absorbance ∞ Total Cholesterol in sample.
- 8. Cholesterol ester Cholesterol + Fatty Acid Cholesterol Cholest-4-en-3-one + H2O2 Cholesterol Esterase Cholesterol Oxidase quinoneimine
- 9. • Specimen – Serum sample is used. – Fasting blood sample is preferred.
- 10. • Procedure • Mix well. Incubate at 37°C in a water bath for 10 minutes or at RT (25- 35°C) for 15 minutes. • Remove from water-bath & cool to RT. • Set colorimeter to zero using blank at 520 nm & measure the absorbance of standard, test. Blank (B) Standard (S) Test (T) Working cholesterol reagent 1.0 ml 1.0 ml 1.0 ml Serum - - 10 μml Standard (200 mg/dl) - 10 μml -
- 11. • Hazardous materials – This procedure uses phenol, which is caustic. • Avoid mouth pipetting. • Avoid contact with skin & mucous memb.
- 12. • HDL cholesterol estimation – LDL,VLDL, Chylomicron (ApoB containing lipoprotein) are removed by precipitating them using polyanion- divalent cation. – Example of polyanion-divalent cation: • Heparin-Mn2+ • Dextran sulphate-Mg2+ • Sodium phosphotungstate-Mg2+
- 13. • Polyanions react with +vely charged groups on lipoproteins (facilitated in +nce of divalent cations) causing aggregation & a cloudy precipitate. • Precipitation is usually complete within 10-15 min at RT.
- 14. • Precipitate is then sedimented by centrifugation. • Centrifugation at higher forces, accelerates sedimentation & improve complete precipitation of apo-B containing particles. • HDl-cholesterol in clear supernatant is estimated by CHOD- PAP method.
- 15. • LDL cholesterol estimation – Estimated by indirect method using friedwald equation Total cholesterol = HDLc + LDLc + VLDL LDL = T. Cholesterol – (HDLc + VLDL) [VLDL = TG/5] • Thus equation becomes, LDL = T. Cholesterol – (HDLc + TG/5)
- 16. • Limitations of friedwald equation – It can’t be used when: I. Serum Triglyceride > 400 mg/dl II. Type III hyprelipoproteinemia – [elevated VLDL, CM, IDL thus high TG level] – False high value of VLDL is obtained. – Thus false low value of LDLc is obtained.
- 18. Tests included in Lipid profile test 1. Serum Total cholesterol 2. Serum Triglyceride 3. VLDL 4. LDLc 5. HDLc
- 19. • Normal range: – Total cholesterol • Desirable: <200 mg/dl • Borderline: 200-239 mg/dl • High risk: ≥ 240 mg/dl – HDLc • Low risk: ≥ 60 mg/dl • High risk: ≤ 40 mg/dl – LDLc • Desirable: < 130 mg/dl • Borderline: 130-159 mg/dl • High risk: ≥ 160 mg/dl
- 20. • Causes of Hypercholesterolemia » Nephrotic syndrome » Diabetes Mellitus » Obstructive Jaundice » Hypothyroidism » Chronic alcoholism » Type IIa Hyperlipoproteinemia
- 21. Biochemical basis of hypercholesterolemia in Nephrotic syndrome Massive proteinuria [> 3.5 gm per day] ↓ Loss of albumin ↓ Hypoalbuminemia ↓ As compensation liver synthesizes globulins [apoB100] ↓ ↑ed synthesis of VLDL & LDL ↓ Hypercholesterolemia
- 22. Biochemical basis of hypercholesterolemia in Diabetes Mellitus ↓ Insulin : Glucagon ratio ↓ ↑ed activity of Hormone sensitive Lipase ↓ ↑ed Fat mobilisation ↓ ↑ed FFA ↓ ↑ed β- oxidation ↓ ↑ed Acetyl CoA ↓ Channeled to Cholesterol Biosynthesis ↓ Hypercholesterolemia
- 23. Biochemical basis of hypercholesterolemia in Obstructive Jaundice Obstruction in bile duct ↓ ↓ed bile flow ↓ ↓ed bile acid & bile salt excretion ↓ ↓ed bile acid (salt) excretion ↓ ↓ed cholesterol utilization for bile acid (salt) synthesis ↓ Hypercholesterolemia
- 24. Biochemical basis of hypercholesterolemia in Hypothyroidism ↓ T3 & T4 ↓ ↓ed expression (downregulation) of gene for LDL receptor ↓ ↓ed LDL receptor ↓ ↓ed uptake of LDL ↓ ↑ed LDL in blood ↓ Hypercholesterolemia
- 25. Biochemical basis of hypercholesterolemia in Chronic Alcoholism Over consumption of Alcohol ↓ Ethanol → Acetaldehyde → Acetate → Acetyl CoA ↓ ↑ed NADH : NAD+ ratio ↓ ↓ed oxidation of Acetyl CoA via TCA cycle ↓ ↑ed Acetyl CoA ↓ Channeled to Cholesterol Biosynthesis ↓ Hypercholesterolemia
- 26. • Type IIa Hyperlipoproteinemia [Primary Familial hypercholesterolemia] – Defect in LDL receptor: • LDL receptor deficiency • Defective binding of apoB100 to LDL receptor • [LDL-Receptor] complex isn’t internalized – Thus elevated plasma LDL level leading to hypercholesterolemia.
- 27. • Causes of Hypocholesterolemia I. Malabsorption II. Pernicious anemia III. Hyperthyroidism IV. Drugs like Nicotinamide, Clofibrate