Lab 2 - cholesterol
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Questions and Answers

What is the significance of determining serum cholesterol levels?

  • It determines blood glucose concentration.
  • It measures the oxygen levels in the blood.
  • It is crucial for evaluating the risk of atherosclerosis and heart disease. (correct)
  • It helps in diagnosing liver diseases.

Which of the following is NOT a method for quantitative analysis of blood constituents?

  • Whole blood sample
  • Hemoglobin concentration (correct)
  • Serum analysis
  • Protein-free filtrate

What distinguishes serum from plasma in blood analysis?

  • Plasma is obtained after coagulation of blood.
  • Serum contains higher protein levels.
  • Serum is obtained after blood coagulation, while plasma contains coagulation proteins. (correct)
  • Plasma cannot be frozen for storage.

What is the chemical composition of the Lieberman-Burchard reagent used for cholesterol determination?

<p>Acetic acid, acetic anhydride, and sulfuric acid (D)</p> Signup and view all the answers

What type of reaction occurs between cholesterol and the Lieberman-Burchard reagent?

<p>A direct reaction resulting in a chromophore (D)</p> Signup and view all the answers

At what wavelength is the chromophore produced by the reaction of cholesterol with the Lieberman-Burchard reagent measured?

<p>$620$ nm (A)</p> Signup and view all the answers

How should serum and plasma be stored if not used immediately?

<p>Frozen (C)</p> Signup and view all the answers

Which of the following best describes why whole blood is treated with an anticoagulant during collection?

<p>To prevent clotting before analysis (B)</p> Signup and view all the answers

What is the consequence of serum being obtained after coagulation of blood?

<p>It lacks coagulation proteins. (C)</p> Signup and view all the answers

Why is the determination of serum cholesterol levels essential in clinical diagnostics?

<p>It reflects the risk of cardiovascular diseases. (B)</p> Signup and view all the answers

What is the purpose of adding anhydrous sodium sulfate to the cholesterol color reagent?

<p>To prevent browning of the solution (D)</p> Signup and view all the answers

Which step must be completed before mixing the concentrated sulfuric acid with the acetic acid-acetic anhydride solution?

<p>Cool the sulfuric acid in an ice bath (B)</p> Signup and view all the answers

How is a cholesterol standard solution prepared in the laboratory procedure?

<p>By dissolving 250 mg of cholesterol in 100 ml of glacial acetic acid (A)</p> Signup and view all the answers

What temperature should the test tubes be placed in for the color reaction?

<p>37°C (A)</p> Signup and view all the answers

Why must the test tubes be clean and dry before starting the measurement?

<p>To avoid interference with the color reaction (B)</p> Signup and view all the answers

What is the molar mass of cholesterol used to prepare the standard solution?

<p>386.65 g/mol (A)</p> Signup and view all the answers

What volume of glacial acetic acid would be used in test tube number 5?

<p>0.0 ml (A)</p> Signup and view all the answers

How long should the test tubes remain in the water bath to ensure proper reaction?

<p>10 minutes (D)</p> Signup and view all the answers

What is the total volume for each reaction mixture in the test tubes?

<p>5.5 ml (C)</p> Signup and view all the answers

What final state should the cholesterol color reagent be in before use?

<p>Should remain clear (A)</p> Signup and view all the answers

What is the purpose of placing the tubes in an ice bath after the reaction?

<p>To stop the reaction and allow the components to cool (A)</p> Signup and view all the answers

What is the significance of wiping off the water from the outside of the test tubes before reading absorbance?

<p>To prevent water from interfering with the light path (A)</p> Signup and view all the answers

Why is it important to establish a standard curve of cholesterol?

<p>To compare patient serum concentration against known values (B)</p> Signup and view all the answers

How should the cholesterol concentration of patient serum be converted after determination?

<p>From mg/ml to mmoles/L (D)</p> Signup and view all the answers

Which absorbance reading indicates that a patient may have high cholesterol levels?

<p>An absorbance reading corresponding to a cholesterol concentration above 5.2 mmol/L (B)</p> Signup and view all the answers

What is the desirable cholesterol level range in mmol/L according to the mentioned criteria?

<p>3.2 - 5.2 mmol/L (B)</p> Signup and view all the answers

What is a crucial preparation step before transferring tube contents to cuvettes?

<p>Removing any bubbles present in the test tube (A)</p> Signup and view all the answers

Which reading must be used as a reference when measuring absorbance at 620 nm?

<p>The absorbance reading of the blank (A)</p> Signup and view all the answers

What does a concentration reading directly translate to regarding cholesterol levels?

<p>It indicates the risk of heart disease (D)</p> Signup and view all the answers

Flashcards

Serum Cholesterol

The amount of cholesterol present in serum (liquid part of blood after clotting).

Lieberman-Burchard reagent

A chemical reagent used to measure cholesterol in serum.

Atherosclerosis

A disease where plaque builds up inside arteries, narrowing them.

Serum vs Plasma

Serum is obtained after blood clotting, lacking clotting proteins; plasma is the liquid part of unclotted blood, containing clotting proteins.

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Spectrophotometry

A method to measure the concentration of a substance by measuring the intensity of light absorbed or transmitted.

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Chromophore

A molecule that absorbs light at a specific wavelength. In this case, the colored product of the cholesterol reaction.

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Direct Reaction

A chemical reaction that doesn't require separating proteins (or other substances) from the sample before measuring.

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Cholesterol implication

High cholesterol can lead to heart disease and atherosclerosis.

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Blood sample types

Blood can be analyzed from whole blood, serum, or a protein-free filtrate.

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Blood storage

Serum and plasma samples should be stored frozen to maintain stability.

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Cholesterol Color Reagent Preparation

A multi-step procedure creating a reagent for cholesterol measurement. It involves mixing glacial acetic acid, acetic anhydride, and sulfuric acid, followed by adding anhydrous sodium sulfate.

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Cholesterol Standard Prep

Creating a specific concentration of cholesterol solution, usually 2.5 mg/mL, in glacial acetic acid for calibration and comparison in tests.

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Test Tube Cleanliness

Essential for accurate cholesterol measurement. Removing water, which interferes with the reaction, is critical.

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Color Reaction Interference

Minimal interference from other components in the serum sample (such as non-cholesterol substances).

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Glacial Acetic Acid

A highly concentrated form of acetic acid, used in the reagent preparation and as a solvent for the cholesterol.

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Acetic Anhydride

A chemical compound used in the preparation of the cholesterol color reagent.

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Sulfuric Acid

A strong acid added to the acetic acid and acetic anhydride mixture.

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Anhydrous Sodium Sulphate

A drying agent added to the reagent to remove any water that might interfere with the test.

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Cholesterol Stock Solution

A concentrated solution of cholesterol used to prepare test samples from serum(patient)

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Incubation Temperature

A reaction at a constant temperature (37°C) to perform the color reaction for a set amount of time (10 minutes).

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Water Level

Ensure the water in the bath is high enough to submerge the tubes' mixture.

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Cooling Tubes

Cool tubes in ice/cold water for 10 minutes.

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Transfer to Cuvettes

Move contents from tubes to cuvettes, removing outside water.

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Absorbance Reading

Measure absorbance at 620 nm compared to a blank.

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Standard Curve

Create a plot of cholesterol concentrations (mg/ml) versus absorbances.

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Cholesterol Concentration

Find patient serum cholesterol level using the standard curve.

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Conversion to mmoles/L

Change the determined cholesterol concentration from mg/ml to mmoles/L.

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Cholesterol Levels

Interpret patient cholesterol levels: high, normal, or low.

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Report Sheet

Record data on a table including test tube number, absorbance, concentration (mg/ml), and (mmoles/L).

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Patient Cholesterol Interpretation

Based on results, decide if the patient has high, normal, or low cholesterol.

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Study Notes

Serum Cholesterol Determination

  • Objective: Determine cholesterol concentration in serum samples using the Lieberman-Burchard reagent and assess if it's normal or abnormal.

Theory

  • Serum cholesterol measurement is critical for diagnosing atherosclerosis and heart disease.
  • Blood analysis for normal chemical constituents is crucial in many clinical diagnoses.
  • Cholesterol analysis can be performed on whole blood, serum, or protein-free filtrate.
  • Whole blood is prevented from clotting by anticoagulants (e.g., oxalate, citrate, EDTA)
  • Plasma is the portion of whole blood after centrifugation without clotting.
  • Serum is the liquid portion obtained after blood coagulation has occurred, lacking coagulation proteins
  • Both serum and plasma need to be stored frozen to preserve enzyme constituents.

Reagent Preparation (Lieberman-Burchard)

  • Glacial acetic acid (350 ml) is placed in a 2-liter flask.
  • Acetic anhydride (550 ml ) is slowly added to the acetic acid and mixed.
  • The flask is sealed to prevent moisture exposure and refrigerated overnight or cooled in ice.
  • 200 mL of concentrated sulfuric acid is cooled in an ice bath and added slowly to the acetic acid/acetic anhydride mixture while stirring.
  • Anhydrous sodium sulfate (20g) is added and stirred until dissolved.
  • The solution should not turn brown.

Cholesterol Standard Preparation

  • A 2.5 mg/mL stock cholesterol solution is prepared by dissolving 250 mg of cholesterol in 100 mL of glacial acetic acid in a volumetric flask.

Measurements

  • Clean and dry test tubes are used

  • Serum, cholesterol, glacial acetic acid, and color reagent are mixed in specific volumes (5.5 mL in total).

  • Mix sample mixtures on a vortex mixer.

  • The tubes are kept in a 37°C water bath for 10 minutes.

  • Tubes are cooled in an ice bath for 10 minutes.

  • Absorbance is measured at 620 nm against a blank to establish a standard curve

  • The Cholesterol concentration (mg/ml) of the patient's serum is determined from the curve

  • Convert the concentration from mg/mL to mmol/L.

  • Evaluate if cholesterol level is high, normal, or low (desirable range: 3.2–5.2 mmol/L).

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