Determination of the total cholesterol in serum

1. Determination of the total cholesterol in serum Dept.of Biochemistry

2. What is Cholesterol? • Cholesterol is a fatty substance (lipid), which is essential to healthy life. • It is Amphipathic • it has both a water-soluble region (a polar -OH group) and a fat-soluble region (a non-polar steroid ring structure and hydrocarbon tail).

3. Cholesterol Functions • Cholesterol is in the brain, nervous tissue, skin and adrenal glands. • Membrane component • Precurser to • Bile acids • Vitamin D • Steroid hormones

4. Bile acids • Bile acids are polar derivatives of cholesterol and aid in: • lipid digestion • lipid absorption • cholesterol excretion

5. Steroids derived from cholesterol

6. Cholesterol(Ch) and Cholesteryl Ester(CE) • Cholesterolis transported in the bloodstream of all animals. • A large portion of the cholesterol in blood is in the form of cholesteryl esters. Cholesterol Cholesteryl Ester

7. What are lipoproteins? • Lipoproteins areMolecular complexes that consist of lipids and proteins. • mainly transport lipids in blood plasma. • Hydrophilic lipids (FC, PL) on surface. • Hydrophobic lipids (TG, CE) in core.

8. Classification of plasma lipoproteins • according to their density • Chylomicron (CM) • Very low density lipoprotein (VLDL) • Low density lipoprotein (LDL) • High density lipoprotein (HDL)

9. LDL and HDL • LDL: carries cholesterol to the peripheral tissues • it can be deposited and increase the risk of arteriosclerotic heart and peripheral vascular disease. • high levels of LDL are atherogenic. “Bad cholesterol”. • HDL transports cholesterol from the peripheral tissues to the liver for excretion, • hence HDL has a protective effect. “Good cholesterol”. Normal Artery Cholesterol Clogged Artery

10. Clinical significance • Risk evaluation • Normal: 3.0～5.20mmol/L • Borderline: 5.20～6.20mmol/L • High blood cholesterol: ≥6.20mmol/L • Hypercholesterolemia • Hypocholesterolemia

11. Hypercholesterolemia • High blood cholesterol (≥6.20mmol/L) • Usually a result of high LDL/low HDL cholesterol levels Leads to • atherosclerosis: narrowing of artery walls • decreased blood and oxygen supply to heart • coronary heart disease(CHD) • Observed in many disorders • nephrotic syndrome, hypothyroidism, Diabetes mellitus, extrahepatic biliary obstruction

12. Hypocholesterolemia • A decrease in the plasma cholesterol: less common • Observed in: severe liver injury, hyperthyroidism, pernicious anemia, malabsorption syndrome.

13. Lipoprotein Profile • Lipid profile is a measure of the lipid contents of the blood. Includes: • Total Cholesterol • LDL Cholesterol • HDL Cholesterol • Triglycerides

14. Who needs to have their Cholesterol checked? Cholesterol screening is used as part of assessing someone's risk of heart disease. It is necessary to check your cholesterol if you fit any of these groups: • If you have evidence of heart disease • If you have a family history of high cholesterol • If you are a diabetic • If you have high blood pressure • If you have a family history of heart disease

15. What Are Some Ways To Control Blood Cholesterol and Triglycerides? • Healthy Eating • Physical Activity • Weight Loss • Medication

16. Principle (CHOD-POD method) Cholesterolesterase(CHE) Cholesterolester + H2O Cholesterol+Fatty acid Cholesterol oxidase(CHOD) Cholesterol+O2 Cholestenone+H2O2 Peorxidase(POD) H2O2 + 4-Aminoantipyrine + Phenol Quinoneimine + 4 H2O • This kit uses a coupled enzymatic reaction scheme: • By the catalysis of CHE and CHOD, Cholesterol ester is catalyzed to yield H2O2, which oxidates 4-Aminoantipyrine (4-AAP) with phenol to form a colored dye of quinoneimine. • The absorbency increase is directly proportional to the concentration of cholesterol.

17. Specimens & Materials • Specimen: serum（血清） • Reagent: • R1: Phosphate buffer (pH 7.4)，Phenol(酚) • R2: 4-AAP, Cholesterolesterase(CHE), Cholesterol oxidase(CHOD), Peroxidase (POD) • Working reagent: • R1 (10ml) + R2 (1ml) • Standard: cholesterol；CS=5.2mmol/L • Water bath, Test tubes, Pipettes • Spectrophotometer

18. Method • Mix well, measure the absorbance of T and S setting zero with B, λ=510nm.

19. Calculation • CT (mmol/L)=AT /AS x CS • Normal value of T-cho: 3.1~5.7mmol/L

20. Notice • If you want to detect cholesterol only, omit the Cholesterol Esterase. • The assay is not influenced by hemoglobin values up to 200mg/dl or by bilirubin levels up to 10mg/dl. • Interference from grossly hemolyzed specimens is correctable by use of a serum/plasma blank.

21. Operating steps of Spectrophotometry • Switch on , for 20 min before using. • Select Wave length of Maximal Absorption • Prepare test sample, blank sample, standard sample . put them into Spectrophotometry. • To “Blank”, mode “Tor A” , Set T =100orA=0 • Pull the pole once time • Change mode to “T”, Set T =0 • Change mode to “A”. • pull the pole second time, record A1;third time ,record A2, forth time ,record A3.

22. Test2 Test2 Test2 Test2 Test2 Test1 Test1 Test1 Test1 Test1 Standard Standard Standard Standard Standard Blank Blank Blank Blank Blank Photometric Mode: Transmittance, T0% ~ 100% Absorbance, A0 ~ 1 Concentration, C （3）Determine T handle 0setT100%：Blank T handle 1setT 0%：A=1.----- Ahandle2、3、4assay A：Standard、test1、2 Rest state：handle 1A=1.-----