MH 128 Liver

Periodic Acid-Schiff (PAS) Reaction

The Periodic Acid-Schiff (PAS) Reaction is a histochemical staining technique developed in the 1940s that detects polysaccharides, glycoproteins, and other carbohydrate-containing compounds in cells and tissues. It was instrumental in demonstrating the distribution and storage of glycogen, particularly in metabolically active tissues like the liver.

The Liver (fed, fasted) is an ideal model for studying glycogen distribution due to its importance in maintaining glucose homeostasis. Hepatocytes, the primary cells of the liver, are responsible for storing excess glucose as glycogen following meals and releasing it during periods of fasting. It is made up of lobules (fed, fasted) of radiating plates of hepatocytes surrounding a central vein.

The following pages will examine hepatic tissue from fed and fasted animals, stained using the PAS reaction to demonstrate glycogen storage and metabolism.

Liver (Fed)

This fed liver comes from animals that were fed to enhance glycogen storage through normal metabolic processes, simulating the post-meal metabolic state in which glucose is stored as long-chain glycogen polymers.

• Hepatocytes:
  • Cell Boundaries: Clearly defined due to the dense cytoplasmic staining
  • Nuclei: Appear dark blue to purple due to counterstaining with hematoxylin to provide contrast to the PAS staining
    • Binucleated Cells (1 (), 2 (), 3 ()): Common in the adult liver (up to 25%), which arise through nuclear division without cytokinesis
    • Cell Division: Dividing cells, prophase () and metaphase (), are easily identified due to their low glycogen content
  • Cytoplasm: Glycogen () appears as intense, coarse magenta-stained granular material in nearly all hepatocytes
    • 2,000 to 60,000 glucose residues per glycogen molecule provide numerous sites for staining
• Blood Cells:
  • Red blood cells appear unstained, as they lack significant carbohydrate content

This is consistent with the liver's role as the body's primary glycogen storage organ during the fed state. The human liver can store about 100-1200 grams of glycogen, which can provide around 400 calories.

Liver (Fasted)

This Liver is from animals that were food-restricted to deplete glycogen stores through normal metabolic processes. The stored glycogen is broken down to glucose and released into the circulation to maintain blood glucose levels.

Hepatocytes show dramatically reduced PAS staining compared to the fed animals.

• Nuclei: Remain dark blue to purple due to counterstaining with hematoxylin, unchanged from the fed state
• Cytoplasm: Sparse staining reflects depleted glycogen reserves
  • Scattered, light pink to magenta particles () appear sporadically
  • Staining intensity varies between cells from minimal (center) to completely absent (left)

These results demonstrate the PAS reaction's ability to detect physiological changes in carbohydrate storage and validate the liver's dynamic role in glucose homeostasis.

This serves as a physiological control to demonstrate the dynamic nature of glycogen storage.

Periodic Acid - Schiff (PAS) Reaction

While understanding the underlying mechanism isn't essential for basic histology practice, the staining process is detailed below for those interested in the biochemical principles.

• Periodic Acid-Induced Oxidation: Tissue sections are treated with periodic acid (HIO₄) at room temperature
  • Periodic acid selectively oxidizes compounds containing adjacent carbon atoms with hydroxyl groups (-OH)
  • Oxidation cleaves the carbon-carbon bond, creating aldehyde groups
• Schiff's Reagent Staining:
  • Tissue is treated with Schiff's Reagent, which reacts specifically with the newly formed aldehyde groups
    • Schiff's reagent is prepared by treating Fuchsin dye (a magenta dye) with sulfur dioxide gas (SO₂) or sodium bisulfite (NaHSO₃)
    • Fuchsin is reduced to form a colorless compound
  • Produces a characteristic magenta (purple-red) color wherever carbohydrates are present
    • Aldehyde groups react with the colorless Schiff's reagent, regenerating the colored form of fuchsin
  • Color intensity is directly proportional to the concentration of reactive aldehyde groups

This reaction is highly specific for polysaccharides and glycoproteins because the periodic acid oxidation requires adjacent carbon atoms with hydroxyl groups, which are present in all carbohydrates.