Experiment Determination Of Cholesterol In HDL by MikeJenny


									       Experiment 6
Determination Of Cholesterol
          In HDL
1.Aim And request

 Comprehend     the method of determination of
  cholesterol in HDL
 Learn about with the importance of HDL in
  vivo and its clinical significance.

 lipids are normally transported together with
  protein components.
 lipoproteins are composed of the lipid
  components and a number of
  apolipoproteins; in these amphiphilic
  proteins, the hydrophobic residues face the
  lipid core of the lipoprotein, while the
  hydrophilic side is facing the aqueous
   Phosphotungstic acid and magnesium chloride can
    precipitate CM、VLDL and LDL of the serum, then remove
    them with centrifugation.
   Extract cholesterol with anhydrous alcohol and acetic ether
    to precipitate protein, and then centrifuge to remove
   Add development solvent (concentrated sulfuric acid- iron
    trichloride), which can react with cholesterol and produce
    stable purple compound. The shade of purple is in direct
    proportion to the amount of cholesterol.
   Using the standard cholesterol solution known
    concentration as control can determine the amount of
    cholesterol in HDL.
               Phosphotungstic acid,      HDL
 CM VLDL       magnesium chloride
 LDL HDL                                  VLDL
                  centrifugation          LDL

              anhydrous alcohol, acetic
              concentrated sulfuric
              acid- iron trichloride   Stable
                                       purple compound.

   ⑴ Prepare for the HDL extract tube(H extract tube): Put
    exact 0.5 ml serum and sodium phosphotungstic acid
    0.25ml into a centrifuge tube, mix, then put magnesium
    chloride 0.25ml, mix thoroughly, centrifugate 4000r/min for
    10min to get the upper liquid 0.2ml for use, as the HDL
    extract tube.
   ⑵ Prepare for the total cholesterol extract tube (T extract
    tube): Put serum and distilled water 0.1ml each.
⑶   Extract cholesterol:Put extract solution
  1ml into the T and H centrifuge tube
  respectively,shake it thoroughly,then
  centrifugate 4000r/min for 3min, get the
  supernatant to determine the cholesterol.
 ⑷ Development: Mark 4 dry tubes with
  number, perform according to the following
  table as below.
HDL extract tube(H extract tube)
                                                           Get the
               sodium phosphotungstic acid                 supernatant
               0.25ml; magnesium chloride 0.25ml           0.2ml

                  centrifugate 4000r/min for
Serum 0.5 ml      10min
                                                                 H extract tube
total cholesterol extract tube (T extract tube):

                    serum and distilled water 0.1ml each

               T extract tube
 Extract cholesterol:

                                             Get the supernatant
                    extract solution 1ml               0.6ml

                    centrifugate 4000r/min
  H extract tube    for 3min                            H tube

                   extract solution 1ml      Get the supernatant
                   centrifugate 4000r/min
                   for 3min
T extract tube
                                                         T tube
reagent amount: ml         blank tube(B) standard tube(S)    H tube       T tube
HDL upper liquid(extraction)                                  0.6
total cholesterol supernatant                                              0.6
extract solvent                 0.5
standard cholesterol solution                  0.5
distilled water                 0.1            0.1
development solvent              1.5           1.5             1.5         1.5
                                shake thoroughly
concentrated sulfuric acid      1.5            1.5             1.5         1.5

    Shake the tubes immediately, and then place them in room temperature for
10 minutes.
    Adjust the spectrophotometer to zero with blank tube. Determine the
absorbance of each tube under 550 nm wavelength.
 4.Result        and Calcuation
         ODH             ODT              ODS

      cholesterol of HDL(mg/dl) =                  × 200 ;
      total cholesterol (mg/dl) =     × 200
       (standard cholesterol solution 200 mg/dl)

    total cholesterol in serum :2.59-6.47mmol/L
     cholesterol in HDL: male 41.1-62.3mg/dl; female 41.7-67.3mg/ml

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