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    Experiment No 08 Cholesterol Estimation

    Uploaded by

    Ameen Shaikh
    This experiment estimates total cholesterol levels in blood serum or plasma through a colorimetric method. Cholesterol esterase and cholesterol oxidase enzymes react with cholesterol to produce hydrogen peroxide. This then reacts with a chromogen to produce a pink compound, with intensity directly proportional to cholesterol levels. Three test tubes are used - a blank, positive control with a known cholesterol level, and sample tube. Absorbance readings are taken and used to calculate the total cholesterol level in the unknown sample based on the standard curve. The experiment is performed before and after a meal to analyze any changes.

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    Experiment No 08 Cholesterol Estimation

    Uploaded by

    Ameen Shaikh
    169 views2 pages
    This experiment estimates total cholesterol levels in blood serum or plasma through a colorimetric method. Cholesterol esterase and cholesterol oxidase enzymes react with cholesterol to produce hydrogen peroxide. This then reacts with a chromogen to produce a pink compound, with intensity directly proportional to cholesterol levels. Three test tubes are used - a blank, positive control with a known cholesterol level, and sample tube. Absorbance readings are taken and used to calculate the total cholesterol level in the unknown sample based on the standard curve. The experiment is performed before and after a meal to analyze any changes.

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    This experiment estimates total cholesterol levels in blood serum or plasma through a colorimetric method. Cholesterol esterase and cholesterol oxidase enzymes react with cholesterol to produce hydrogen peroxide. This then reacts with a chromogen to produce a pink compound, with intensity directly proportional to cholesterol levels. Three test tubes are used - a blank, positive control with a known cholesterol level, and sample tube. Absorbance readings are taken and used to calculate the total cholesterol level in the unknown sample based on the standard curve. The experiment is performed before and after a meal to analyze any changes.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    169 views2 pages

    Experiment No 08 Cholesterol Estimation

    Uploaded by

    Ameen Shaikh
    This experiment estimates total cholesterol levels in blood serum or plasma through a colorimetric method. Cholesterol esterase and cholesterol oxidase enzymes react with cholesterol to produce hydrogen peroxide. This then reacts with a chromogen to produce a pink compound, with intensity directly proportional to cholesterol levels. Three test tubes are used - a blank, positive control with a known cholesterol level, and sample tube. Absorbance readings are taken and used to calculate the total cholesterol level in the unknown sample based on the standard curve. The experiment is performed before and after a meal to analyze any changes.

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    © All Rights Reserved
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    Experiment No-07

    Name of Experiment: Estimation of total cholesterol in blood serum/plasma


    Theory:
    Cholesterol (chole-bile, stereos solid and ol- alcohol) is an organic molecule (lipid). It is an
    essential structural component of animal cell membranes and serves as a precursor for the
    biosynthesis of steroid hormones, bile and vitamin D. In blood, cholesterol present in the form of
    cholesterol ester which gives to cholesterol and fatty acid in presence of cholesterol esterase.
    This free cholesterol reacts in presence of oxygen and cholesterol oxidase to give Cholest-4-en3-one and hydrogen peroxide. This generated hydrogen peroxide breaks down to water and
    nascent oxygen in presence of peroxidase. This nascent oxygen binds with chromogen and gives
    Quinoneimine a pink color product. The intensity of pink color is directly proportional to total
    cholesterol of blood serum/plasma. This total cholesterol is summation of HDL (high density
    lipoprotein- good cholesterol), LDL (Low density lipoprotein-bad cholesterol) and triglycerides.
    Hence total cholesterol = HDL + LDL + 20% Triglycerides.
    Cholesterol esterase

    Cholesterol ester + H2O ------------ Cholesterol + Fatty acids


    Cholesterol oxidase

    Cholesterol + O2-------------- Cholest-4-en-3-one + H2O2


    Peroxidase

    H2O2--------------------H20+ [0]
    [0] + 4-amino antipyrine+ phenol------------Quinoneimine (Pink color) + H20
    Requirement:
    Reagent-1: it has component of Goods buffer (phosphate buffer pH 7.0), cholesterol
    esterase, cholesterol oxidase, peroxidase, chromogen (4-amino antipyrine + phenol),
    stabilizer and preservative
    Reagent-2/Standard: it is blood serum with known concentration of total cholesterol of
    200mg/dl
    Blood serum/plasma

    I.

    II.
    III.

    Method:
    [A] Isolation of Blood plasma/serum: liquid part of uncoagulated blood is called plasma and
    coagulated blood is called serum. Isolation of Blood plasma is carried out by centrifugation.

    2ml of blood is withdraw from healthy volunteer (before meal and after meal condition)
    It is kept in vaccumtainer tube having some pinch of EDTA (Acts as anticoagulant)
    Now it is centrifuged at 4000 rpm for 10 minutes at room temperature and pellets is
    discarded and supernatant is collected which is plasma.

    [B] Development of Color:

    Three test tubes is taken and named as blank, positive control and unknown respectively.
    In blank test tube:2000l of reagent-1 is added
    In positive control test tube:2000l of reagent-1 and 20 l of reagent-2 are added
    In unknown test tube:2000l of reagent-1 and 20 l of blood plasma/serum (in which
    total cholesterol will be estimated) are added
    All test tubes are incubated for 10 minutes at 37C in dark.
    Afterwards, color is developed and absorbance/ OD are read at 490nm in
    spectrophotometer/colorimeter against blank.

    Tabulation:
    Test tubes

    Blood
    serum/plasma

    Reagent1

    Reagent2

    Blank
    Positive
    control
    Unknown
    Calculation:

    ----------

    2000l
    2000l

    -----20l

    20l

    2000l

    Incubation
    at 37C for
    10 minutes
    in dark
    ,,
    ,,

    OD
    490nm

    Amount of
    total
    cholesterol

    ---200mg/dl

    ,,

    ???

    Amount of total cholesterol in unknown sample (mg/dl):


    Amount of total cholesterol in positive control X OD of Unknown
    ------------------------------------------------------------------------------------OD of positive control
    Result:
    Case

    Before meal

    After meal
    Remarks:
    Mark Obtained:
    Signature:

    Total cholesterol
    Standard range
    Calculated
    (mg/dl)
    value
    (mg/dl)
    <200:normal
    110-160:very good
    170-200: good

    Condition
    (Normal/good/very
    good range)

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