Radioactive Tracers | Zoology Optional Notes for UPSC PDF Download

Introduction

The living plant can be likened to a biosynthetic laboratory, producing not only primary metabolites essential for general growth but also a diverse array of secondary products with pharmaceutical significance. Primary metabolites, such as sugars, amino acids, and fatty acids, are universally distributed in nature and play a crucial role in basic cell metabolism. On the other hand, secondary metabolites, including glycosides, alkaloids, flavonoids, and volatile oils, are biosynthetically derived from primary metabolites and exhibit a more restricted distribution, often confined to specific taxonomic groups. These secondary products serve as chemical adaptations to environmental stresses, acting as defensive, protective, or offensive chemicals against various threats.

Cellular Economy of Secondary Metabolites

Secondary products, in terms of cellular economy, are generally expensive to produce and accumulate. They represent a strategic investment for the plant, contributing to its survival and adaptation to the surrounding environment.

Biosynthesis

Biosynthesis is the process through which complex chemical compounds are built up from simpler ones by a series of enzymatic reactions within the cells of a living organism. The key participants in biosynthesis are enzymes, and the process requires precursors such as ATP, NADH, NADPH, FADPH, and FADH. The compounds generated through biosynthesis encompass a wide range, including carbohydrates, proteins, vitamins, antibiotics, fats, alkaloids, and gums.

Biogenesis

Biogenesis, defined as the development of life from preexisting life, underscores the continuous nature of living processes.

Biosynthetic Pathways in Plants

Plants employ various biosynthetic pathways to produce an array of compounds. Three major pathways include:

• Shikmic Acid Pathway
• Mevalonic Acid Pathway
• Acetate Pathway

Each pathway involves numerous steps and intermediates, contributing to the diversity of plant metabolites.

Investigation Techniques for Biosynthetic Pathways

Understanding biosynthetic pathways requires sophisticated investigation techniques. Several methods are employed, including:

1. Tracer Techniques

Tracer techniques involve the use of labeled compounds to trace intermediates and steps in biosynthetic pathways. Labeled compounds can be prepared using two types of isotopes:

a. Radioactive Isotopes

• Examples: C¹⁴, P³², H¹, Na²⁴, S³², P³⁵
• Applications:
  • Biological investigations: Carbon and hydrogen
  • Metabolic studies: Sulphur, phosphorus, alkali, alkaline earth metals
  • Studies on proteins and amino acids: Labeled nitrogen

b. Stable Isotopes

• Examples: H²², C¹³, N¹⁵, O¹⁸
• Applications:
  • Labeling compounds as possible intermediates in biosynthetic pathways
  • Detection through mass spectroscopy and NMR spectroscopy

2. Other Investigative Approaches

Other methods include:

• Use of Mutant Strain
• Use of Isolated Organs
• Grafting Method

Each technique contributes to a comprehensive understanding of the intricate processes involved in plant biosynthesis.

Tracer Techniques in Biosynthetic Studies

Significance of Tracer Techniques

1. Tracing of Biosynthetic Pathway:

   • Incorporation of radioactive isotopes into precursors allows the tracing of entire biosynthetic pathways.
   • Example: Using C¹⁴-labeled phenylalanine to trace the biosynthesis of cyanogenetic glycoside prunasine.

2. Location and Quantity Determination:

   • C¹⁴-labeled glucose, chemically indistinguishable from native glucose, helps determine both the location and quantity of glucose in a biological system.
   • Radioactive assay aids in quantifying glucose present in tissues.

3. Different Tracers for Specific Studies:

   • Labeled N₂ atoms provide more specific information for studies on proteins, alkaloids, and amino acids than labeled carbon.

Criteria for Tracer Techniques

• Starting concentration must be sufficient to withstand dilution during metabolism.
• The labeled compound must be involved in synthesis reactions.
• The labeled compound must be harmless to the system.
• Proper labeling requires knowledge of the physical and chemical nature of the compound.
• The tracer should be highly pure.
• Radioactive isotopes with longer half-life periods are preferred (e.g., ¹⁴C with about 5000 years).

Steps for Tracer Techniques

1. Preparation of Labeled Compound:

   • Compounds are conveniently prepared from natural sources.
   • Example: Growing chlorella in an atmosphere containing ¹⁴CO₂ labels all carbon compounds in the organism as ¹⁴C.
   • Tritium labeling is commercially available and involves catalytic exchange or organic synthesis.

2. Introduction of Labeled Compound into Biological System:

   • Methods include root feeding, stem feeding, direct injection, wick feeding, floating method, and spray method.
   • Selection of method depends on the plant part and the site of biosynthesis of the desired metabolites.

3. Separation and Determination of Labeled Compound:

   • Separation methods depend on the type of plant material (soft/fresh tissue, hard tissue, unorganized drugs).
   • Different solvents are used based on the type of plant material (nonpolar for fats, oils, alkaloids, glycosides; polar for phenols; slightly polar for flavonoids).
   • Determination methods include Geiger Muller counter, scintillation counter, autoradiography, gas ionization chamber, Bernstein Ballentine counter, mass spectrometer, and NMR spectrometer.

Methods of Tracer Techniques in Biosynthetic Studies

I. Competitive Feeding

• Principle:

  • Accurately determines the actual precursor involved in the biosynthesis of a specific metabolite.
  • Two precursors are introduced into separate groups of plants, and radioactivity observations determine the biosynthetic pathway.
    

• Example:

  • Competitive feeding experiment with tyrosine and DOPA showed that tyrosine directly gives 3,4-dihydroxy phenyl pyruvic acid.

• Applications:

  • Elucidation of biogenesis of propane alkaloids.
  • Study of alkaloids like connine and conhydrine (hemlock).

II. Precursor Product Sequence Method

• Principle:

  • The presumed precursor in labeled form is fed to plants.
  • Isolated and purified constituents are analyzed for radioactivity to determine the precursor's incorporation.

• Disadvantages:

  • Radioactivity alone may not confirm direct precursor involvement.
  • Further confirmation often requires double and triple labeling experiments.

• Example:

  • Labeled lysine (⁶C₁₄, ⁷N₁₅) fed to Nicotiana glauca confirmed the sequential formation of anabasine alkaloid.

• Applications:

  • Study of morphine and ergot alkaloids biosynthesis.

III. Sequential Analysis Method

• Principle:

  • Plants are grown in ¹⁴CO₂ atmosphere.
  • Plant metabolites are analyzed at intervals to determine the sequence of labeled compound formation.

• Example:

  • Menthapiperita exposed to ¹⁴CO₂ for 5 minutes provided evidence of biosynthetic sequences.

• Applications:

  • Elucidation of carbon incorporation in photosynthesis.
  • Determination of sequential formation in opium and tobacco alkaloids.

IV. Isotope Incorporation Method

• Principle:

  • Provides information about bond cleavage and formation during reactions.
  • Example: Cleavage of glucose-1-phosphotase catalyzed by alkaline phosphatase.

• Example:

  • Cleavage in the presence of ¹⁸O-enriched H₂O yields labeled glucose or phosphate.

• General Applications of Tracer Techniques:

  • Study of cyclization sequences using 1⁴C, 3H-labeled mevalonic acid.
  • Investigation of interrelationship among sterols using ¹⁴C acetate.
  • Terpenoid biosynthesis study by chloroplasts using two ¹⁴C mevalonate.
  • Formation studies of scopoletin using labeled phenylalanine.
  • Cinnamic acid pathway in coumarin biosynthesis from labeled coumarin.
  • Origin of carbon and nitrogen atoms in purine ring system using ¹⁴C or ¹⁵N labeled precursors.
  • Calcium uptake in plants using 45Ca tracer.

Application in Different Experimental Setting

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Isolated Organs, Tissues, and Cells

• Principle:

  • Cultured organs, tissues, or cells under aseptic conditions for feeding experiments.

• Applications:

  • Studies on petal discs for essential oil pathways.
  • Use of isolated shoots and leaves for tobacco alkaloids and tropane alkaloids study.
  • Isolated roots for tropane alkaloids pathways in solanaceous plants.

Grafting

• Principle:

  • Uniting cut surfaces of different plants (stock and scion) for growth.

• Applications:

  • Important in plant propagation.
  • Biosynthetic studies on tobacco alkaloids and tropane alkaloids in solanaceous plants.

Mutant Strains

• Principle:

  • Mutant strains of lower plants lacking specific enzymes, affecting normal metabolic pathways.

• Applications:

  • Gibberellins biosynthetic studies using mutant strains.
  • UV-induced strains of Claviceps purpurea for alkaloid pathway blockage studies.