Lipid and steroid metabolism

Experiment: Lipid and Steroid Metabolism

Objective:

• To demonstrate the hydrolysis of lipids and steroids by enzymes.
• To investigate the role of bile salts in lipid digestion.

Materials:

• Olive oil
• Cholesterol
• Pancreatic lipase
• Cholesterol esterase
• Bile salts (e.g., sodium cholate)
• pH buffers (pH 7.4 and pH 8.0)
• Test tubes
• Water bath (capable of maintaining 37°C)
• Spectrophotometer
• Centrifuge
• Pipettes and other necessary lab equipment for accurate measurement and transfer of liquids

Procedure:

Part 1: Hydrolysis of Lipids

1. Prepare two test tubes containing:
   • Test tube 1: 1 mL olive oil, 1 mL pancreatic lipase, 1 mL pH 7.4 buffer
   • Test tube 2 (Control): 1 mL olive oil, 1 mL boiled pancreatic lipase (inactivated), 1 mL pH 7.4 buffer
2. Incubate both test tubes in a 37°C water bath for 30 minutes.
3. After incubation, centrifuge the test tubes at a suitable speed for a sufficient amount of time to separate the layers.
4. Observe and record the appearance of the supernatant in each test tube. Note any differences in clarity or turbidity.

Part 2: Hydrolysis of Steroids

1. Prepare two test tubes containing:
   • Test tube 3: 1 mg cholesterol, 1 mL cholesterol esterase, 1 mL pH 8.0 buffer
   • Test tube 4 (Control): 1 mg cholesterol, 1 mL boiled cholesterol esterase (inactivated), 1 mL pH 8.0 buffer
2. Incubate both test tubes in a 37°C water bath for 30 minutes.
3. After incubation, centrifuge the test tubes.
4. Observe and record the appearance (color) of the supernatant in each test tube.

Part 3: Role of Bile Salts

1. Prepare two test tubes containing:
   • Test tube 5: 1 mL olive oil, 1 mL pancreatic lipase, 1 mL sodium cholate, 1 mL pH 7.4 buffer
   • Test tube 6 (Control): 1 mL olive oil, 1 mL pancreatic lipase, 1 mL water, 1 mL pH 7.4 buffer
2. Incubate both test tubes in a 37°C water bath for 30 minutes.
3. After incubation, centrifuge the test tubes.
4. Measure the absorbance of the supernatant at 410 nm using a spectrophotometer. Ensure the spectrophotometer is blanked with an appropriate control (e.g., buffer).
5. Record the absorbance values for each test tube.

Results:

• In Part 1, the supernatant of the test tube containing active lipase will likely show increased turbidity compared to the control due to the presence of emulsified fatty acids resulting from lipid hydrolysis. The control (boiled lipase) should remain relatively clear.
• In Part 2, the supernatant of the test tube containing active cholesterol esterase might show a change in color (depending on the specific cholesterol esterase and its products), whereas the control should show minimal or no color change. Precise color observation requires a comparison against an appropriate blank.
• In Part 3, the supernatant of the test tube containing bile salts (sodium cholate) should exhibit a higher absorbance at 410 nm than the control (water). This indicates enhanced lipid hydrolysis and the resulting increase in free fatty acids or other spectrophotometrically detectable products. The exact absorbance values will depend on the specific experimental conditions.

Significance:

• This experiment demonstrates the crucial role of enzymes (pancreatic lipase and cholesterol esterase) in the digestion and metabolism of lipids and steroids.
• It highlights the importance of bile salts in facilitating lipid digestion by emulsifying lipids, increasing the surface area available for enzymatic action.
• The findings are relevant to understanding conditions such as hyperlipidemia and other metabolic disorders involving lipid metabolism.