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1、The experiment principle The measurement of the S-Chol is an important indicator of the prevention and treatment，clinical diagnosis and nutrition investigation of the ASCD - atherosclerotic cardioascular disease.the average persons contents of the S-Chol range from 2.6 mmol/L to 6.5 mmol/L (100250mg
2、/L)。 Cholestenone is cyclopentanoperhydrophenanthrene ring derivative.not only does it participate in the component of the Plasma protein，but also be the essential composition of the cell structure，and be capable of translating into BR 25g Kayon，adrenocortical hormone and vitamin D，etc.cholestenone，
3、which is called by a joint name total cholesterol，exists as free cholesterol and cholersteryl ester.the measuring method of cholestenone consisit of chemical colorimetry and methods in enzymology.this experiment adopt the firt one.cholesterol and its lipid derivative react with o-phthaldialdehyde ，
4、using sulfuric acid as a catalyst ，produce amaranth material that absorbed light at 550 nm。Hence，colorimetry can be used for quantitative determination.when the content of the Cholestenone within the limits of 10.3mmol/L(400mg/1dL)，it will persent a positive relation with light absorption. It is not
5、 necessary to centrifugation in this method.Experiment material and the experimentation1Experiment materia:o-phthaldialdehyde reagent:Using 50mg of to-phthaldialdehyde and dissolving in 50ml absolute ethyl alcohol,after that keep in cold storage for no longer than one and a half months.mixed acid: 1
6、00ml of glacial acetic acid mixed with 100ml of concentrated sulfuric acidStandard stock solution of Cholestenone(1mg/ml):Using 100mg of the Cholestenone and dissolving in 100ml glacial acetic acid.Standard treatment fluid of Cholestenone(0.1mg/ml):dilute the stock solution 100times with glacial ace
7、tic acid 2test sampleDilute 0.1 ml human serum with glacial acetic acid into 4.00ml.3experimentationTest tube 1.5cm*15cm(*12);suction pipet 0.5ml(*5) ,10ml(*1),0.1ml(*1);SpectrophotometerOperation methods1make standard curveNumber 9 test tubes,after that adding reagent refer to the list followed: nu
8、mber Standard treatment fluid glacial acetic acid o-phthaldialdehyde reagent mixed acidamount to the content of total cholesterol in blood serum unknown /mgA550After that，mixing them uniformly and blandly，and stewing them for 10 minutes at the temporary range from 20 to 37 。Choose content of total c
9、holesterol(mg) as x- coordinate,and A550 as y-coordinate to make the standard curve.2sample test Number 9 test tubes,after that adding reagent refer to the list followed:Number 1(Comparison) 2(sample) 3(sample) human serum diluted/mlo-phthaldialdehyde reagent/ml glacial acetic acid /mlMixed acid /ml
10、A550 After that，mixing them uniformly and blandly，and stewing them for 10 minutes at the temporary range from 20 to 37 。After that determining the light absorbed at 550nm。Then，check the the content of the total cholesterol on the standard curve。Experiment dataAccording to the data we got from this e
11、xperiment，the average A550 =【（样品） +A550（样品） 】/2=（0.291+0.298）/2=0.295 make standard curve with origin8.0 with the forth and fifth point shielded and fitting。The equation of the fitting straight line followed：Y= 0.00166x-0.00935 （r2=0.99069） Substitute y with o.o295，we got x=（0.00935+0.0295）/0.00166=
12、183.343mgHence，the total cholesterol in the sample is 183.343mgResult analysis 1、the standard curve is not perfect linearly dependent 。error analysis as followed： 1 there is some residuals in the preparation of standard solution with different concentration 2the different order of adding reagent in
13、test tubes make the reactant react for different time.because of that,extent of reaction has some differences when measure the absorbance. 3the solution maybe not mixed perfectly because the the mixed acid is stickiness，which cause some error in the absorbance measure. 4The most major error is from
14、that the sample solution is not adequate for washing the cuvettes.hence the absorbance will be influenced by last sample solution. 5We did not blend the solution again after water bath2、the sample test has a little bit discrepancy,it means that the sample mixed homogeneouslyReflection Questions1、Wha
15、t should be taken care of in the operation of this experiment?why?Answer : after the concentration gradient solution madeup,it should be mixed homogeneous and stewing them for 10minutes in the temperation range from 20 to 37 。Because only in that way the reaction will react adequately，leading the ac
16、curate of the absorbance we got。 2、lipid is hard to be dissolved in water 。Dispersing them ing water ，emulsion will be get。Why normal person blood plasma and blood serum are limpid in sight with so much lipid？Answer ：the lipid in blood plasma and blood serum is not free。 most of them combine with the protein in blood plasma and blood serum，and transport and exist in form of lipoprotein，and part of the cholesterol h
