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?

Acetic acid, acetic anhydride, and sulfuric acid Signup and view all the answers

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

A direct reaction resulting in a chromophore Signup and view all the answers

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

$620$ nm Signup and view all the answers

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

Frozen Signup and view all the answers

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

To prevent clotting before analysis Signup and view all the answers

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

It lacks coagulation proteins. Signup and view all the answers

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

It reflects the risk of cardiovascular diseases. Signup and view all the answers

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

To prevent browning of the solution Signup and view all the answers

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

Cool the sulfuric acid in an ice bath Signup and view all the answers

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

By dissolving 250 mg of cholesterol in 100 ml of glacial acetic acid Signup and view all the answers

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

37°C Signup and view all the answers

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

To avoid interference with the color reaction Signup and view all the answers

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

386.65 g/mol Signup and view all the answers

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

0.0 ml Signup and view all the answers

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

10 minutes Signup and view all the answers

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

5.5 ml Signup and view all the answers

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

Should remain clear Signup and view all the answers

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

To stop the reaction and allow the components to cool 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?

To prevent water from interfering with the light path Signup and view all the answers

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

To compare patient serum concentration against known values Signup and view all the answers

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

From mg/ml to mmoles/L Signup and view all the answers

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

An absorbance reading corresponding to a cholesterol concentration above 5.2 mmol/L Signup and view all the answers

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

3.2 - 5.2 mmol/L Signup and view all the answers

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

Removing any bubbles present in the test tube Signup and view all the answers

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

The absorbance reading of the blank Signup and view all the answers

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

It indicates the risk of heart disease Signup and view all the answers

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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