Determination of the total cholesterol in serum Dept.of Biochemistry

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).

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

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

Steroids derived from cholesterol

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

What are lipoproteins? Lipoproteins are Molecular 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.

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

LDL and HDL Normal Artery Clogged Artery Cholesterol 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”.

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

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

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

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

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

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

Principle (CHOD-POD method) Cholesterol+O2 Cholestenone+H 2 O 2 Cholesterol oxidase(CHOD) Cholesterolester + H 2 O Cholesterol+Fatty acid Cholesterolesterase(CHE) H 2 O Aminoantipyrine + Phenol Quinoneimine + 4 H 2 O Peorxidase(POD) This kit uses a coupled enzymatic reaction scheme: By the catalysis of CHE and CHOD, Cholesterol ester is catalyzed to yield H 2 O 2, 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.

Specimens & Materials Specimen: serum （血清） Reagent: –R 1 : Phosphate buffer (pH 7.4) ， Phenol( 酚 ) –R 2 : 4-AAP, Cholesterolesterase(CHE), Cholesterol oxidase(CHOD), Peroxidase (POD) Working reagent: –R 1 (10ml) + R 2 (1ml) Standard: cholesterol ； C S =5.2mmol/L Water bath, Test tubes, Pipettes Spectrophotometer

Method BST Working reagent0.6ml dH 2 O 10  l -- Standard- 10  l - Serum-- 10  l Mix well, incubate for 15mins at 37  C dH 2 O1.0ml Mix well, measure the absorbance of T and S setting zero with B, λ=510nm.

Calculation C T (mmol/L)=A T /A S x C S Normal value of T-cho: 3.1~5.7 mmol/L

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.

1.Switch on, for 20 min before using. 2.Select Wave length of Maximal Absorption 3.Prepare test sample, blank sample, standard sample. put them into Spectrophotometry. 4.To “Blank”, mode “Tor A”, Set T =100 or A=0 5.Pull the pole once time 6.Change mode to “T”, Set T =0 7.Change mode to “A”. 8.pull the pole second time, record A1;third time,record A2, forth time,record A3. Operating steps of Spectrophotometry

Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Standard Test1 Test2 T handle 0 set T100% ： Blank T handle 1 set T 0% ： A= A handle2 、 3 、 4 assay A ： Standard 、 test1 、 2 Rest state ： handle 1 A= （ 3 ） Determine Photometric Mode: Transmittance, T 0% ~ 100% Absorbance, A 0 ~ 1 Concentration, C

WeekContent 3Volume transfer, accuracy and precision 4 Spectrophotometry & plotting of calibration curve 5Estimation of serum total protein & albumin 7Enzyme inhibition test 8Alkaline phophatase 9Glucose tolerance test 11Cholesterol(total) 12Final experimental exam