Introduction

This document outlines a historical laboratory method for extracting insulin from bovine or porcine pancreas. The process involves several steps, each critical for isolating insulin from the pancreas tissue. This method was crucial before the advent of recombinant DNA technology, which now allows for the production of human insulin in laboratories.

Pancreas Preparation

The first step in the insulin extraction process is the preparation of the pancreas. The pancreas must be finely minced to increase the surface area for chemical reactions and to facilitate the extraction process. This step is crucial as it ensures that the insulin within the pancreas cells is accessible for extraction.

Step-by-Step Instructions

  1. Obtain fresh bovine or porcine pancreas.
  2. Clean the pancreas thoroughly to remove any fat or connective tissue.
  3. Mince the pancreas into small pieces, aiming for a consistency that maximizes surface area.
  4. Place the minced pancreas in a sterile container.

Acid Treatment

The next step involves treating the minced pancreas with diluted sulfuric acid. This acid treatment is necessary to break down the cellular structure of the pancreas and release the insulin.

Step-by-Step Instructions

  1. Measure 5cc of diluted sulfuric acid per pound of pancreas.
  2. Add the sulfuric acid to the minced pancreas and stir gently.
  3. Allow the mixture to sit and stir continuously for 3-4 hours to ensure thorough acid treatment.
  4. After the treatment period, the mixture should be observed for any changes in consistency or color.

Alcohol Extraction

Following the acid treatment, the next step is to extract the insulin using alcohol. This process helps to separate the insulin from other components of the pancreas.

Step-by-Step Instructions

  1. Add 60-70% alcohol to the acid-treated pancreas mixture.
  2. Stir the mixture thoroughly to ensure the alcohol mixes well with the pancreas tissue.
  3. Centrifuge the mixture to remove any solid particles.
  4. Filter the liquid to remove any remaining solids.

Neutralization

The filtrate obtained from the alcohol extraction needs to be neutralized to remove any residual acid and prepare it for further processing.

Step-by-Step Instructions

  1. Measure the filtrate and add an appropriate amount of sodium hydroxide to neutralize the acid.
  2. Stir the mixture gently to ensure complete neutralization.
  3. Concentrate the neutralized filtrate under vacuum to reduce its volume and increase the concentration of insulin.

Protein Separation

The concentrated filtrate is then heated to remove lipids and other non-insulin proteins. This step is crucial for isolating insulin from other proteins.

Step-by-Step Instructions

  1. Heat the concentrated filtrate to 50°C to remove lipids.
  2. Add ammonium sulfate (37g/100cc) to the filtrate to precipitate insulin.
  3. Stir the mixture gently to ensure the ammonium sulfate mixes well with the filtrate.
  4. Allow the mixture to sit for a period to allow the insulin to precipitate.

Precipitation & Purification

After the insulin has precipitated, it needs to be separated from the other components of the filtrate.

Step-by-Step Instructions

  1. Skim the protein layer from the top of the mixture.
  2. Dissolve the skimmed protein layer in hot acid alcohol.
  3. Neutralize the solution with sodium hydroxide.
  4. Refrigerate the solution to allow any remaining impurities to settle.

Isoelectric Precipitation

The final step in the purification process involves adjusting the pH of the solution to precipitate insulin.

Step-by-Step Instructions

  1. Adjust the pH of the solution to 5.0 using appropriate chemicals.
  2. Add tricresol (0.3%) to the solution to aid in the precipitation process.
  3. Centrifuge the solution to remove any remaining impurities.

Final Processing

The precipitated insulin needs to be dissolved and prepared for storage.

Step-by-Step Instructions

  1. Dissolve the precipitate in acid water.
  2. Adjust the pH of the solution to 2.5 using appropriate chemicals.
  3. Filter the solution through a Mandler filter to remove any remaining impurities.

Preservation

The final step in the insulin extraction process is to preserve the insulin for storage.

Step-by-Step Instructions

  1. Add tricresol (0.1%) to the filtered insulin solution to act as a preservative.
  2. Add sodium chloride to the solution to achieve isotonicity.
  3. Store the insulin in sterile vials under appropriate conditions.

Common Mistakes

  • Not thoroughly mincing the pancreas can lead to incomplete extraction of insulin.
  • Overheating the filtrate can denature the insulin, rendering it ineffective.
  • Failing to properly neutralize the filtrate can result in impurities in the final product.

Practical Tips

  • Use sterile equipment and containers throughout the process to prevent contamination.
  • Carefully measure all chemicals and solutions to ensure accurate proportions.
  • Monitor the pH levels closely during the purification process to avoid denaturing the insulin.

Getting Started

Before attempting this process, ensure you have access to a laboratory setting with the necessary equipment and chemicals. This process is not suitable for survival scenarios due to the complexity and equipment required. However, understanding the historical method can provide valuable insights into the challenges faced in insulin production before modern techniques were developed.

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