1. TRACER TECHNIQUES & UTILIZATION IN BIOGENETIC STUDIES
ShriRam College of Pharmacy
Dr. Ajay Sharma

2. INTRODUCTION
Living plants considered as biosynthetic laboratory primary as well as secondary metabolite.
Different biosynthetic pathway:
» Shikmic acid pathway: Aromatic amino acids
» Mevalonic acid pathway: Terpenes
» Acetate pathway: Fatty acids

3. Definitions:
Biosynthesis: Formation of a chemical compound by a living organisms
Biogenesis: Production or generation of living organisms from other living organisms 
Primary metabolites: Required for growth & normal physiological activity e.g., carbohydrates, fatty acids, amino acids, nucleic acids, proteins
Secondary metabolites: Biosynthetically derived from primary metabolites. They represent chemical adaptations to environmental stresses, or act as defensive or protective against microorganisms, insect & higher herbivores e.g., alkaloids, glycosides, resins, tannins

4. Various intermediate and steps are involved in biosynthetic pathway in plants can be investigated by means of following techniques: -
» Use of isolated organ
» Grafting methods
» Use of mutant strain
» Tracer technique
» Enzymatic studies

5. Isolated organ/tissue: This method is based on using isolated parts of plant e.g., stem, roots. This technique is useful in the determination of site of biosynthesis of particular compounds.
ex. Roots and leaves for the study of Nicotiana and Datura, petal disc for the study of rose oil, tropane alkaloids in the root of solanaceae family.
Grafting methods: This method is used fore the study of alkaloid formation by grafted plants.
ex. Tomato scions grafted on Datura produce alkaloids, while Datura scion grafted on Tomato produce less quantity of alkaloids.
This shows that main site of alkaloid biosynthesis is root.

6. Use of mutant strains: In this mutant strains of microorganisms are produced with the lack of certain enzymes.
ex. Gibberella mutant is used to produce isoprenoid compounds, Lactobacillus acidophillus is used for mevalonic acid pathway for isoprenoid biosynthesis
Enzymatic study: Phytohormone auxin plays critical roles in the regulation of plant growth and development.
Indole-3-acetic acid (IAA) has been recognized as the major auxin which is synthesized in plants is still unclear.
Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development.
TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana).

7. Tracer technique:
It can be defined as technique which utilizes a labelled compound to find out or to trace the different intermediates and various steps in biosynthetic pathways in plants, at a given rate & time. OR
In this technique different isotope, mainly the radioactive isotopes which are incorporated into presumed precursor of plant metabolites and are used as marker in biogenic experiments.

8. The labelled compound can be prepared by use of two types of isotopes.
» Radioactive isotopes:
[e.g. 1H, 14C, 24Na, 42K, 35S, 35P, 131I decay with emission of radiation]
For biological investigation – carbon & hydrogen.
For metabolic studies – S, P, and alkali and alkaline earth metals are used.
For studies on protein, alkaloids, and amino acid – labelled nitrogen atom give more specific information.
3H compound is commercially available.

9. » Stable isotopes:
[e.g. 2H, 13C, 15N, 18O]
Used for labeling compounds as possible intermediates in biosynthetic pathways.
Usual method of detection are: – Mass spectroscopy [15N, 18O]
NMR spectroscopy [2H, 13C]

10. SIGNIFICANCE OF TRACER TECHNIQUE
Tracing of biosynthetic pathway: e.g. by incorporation of radioactive isotope of 14C into phenylalanine, the biosynthetic cyanogenetic glycoside prunasin, can be detected.
• Location & quantity of compound containing tracer: 14C labelled glucose is used for determination of glucose in biological system
• Different tracers for different studies: For studies on alkaloids, proteins nitrogen and amino acid (Labelled nitrogen give specific information than carbon). For terpenoids O atom and glycosides O, N, S & C atom used
• Convenient and suitable technique

11. CRITERIA FOR TRACER TECHNIQUE
The starting concentration of tracer must be sufficient withstand resistance with dilution in course of metabolism
• Proper labeling: For proper labeling physical & chemical nature of compound must be known
• Labelled compound should involve in the synthesis reaction
• Labelled should not damage the system to which it is used

12. ADVANTAGES High sensitivity. Applicable o all living organism.
Wide ranges of isotopes are available.
More reliable, easily administration & isolation procedure.
Gives accurate result, if proper metabolic time & technique applied.

13. LIMITATIONS Kinetic effect Chemical effect Radiation effect
Radiochemical purity
High concentration distorting the result, expensive, short half life, hazardous

14. REQUIREMENT FOR TRACER TECHNIQUE
Preparation of labelled compound
The labelled compound produce by growing chlorella in atmosphere of 14CO All carbon compounds 14C labelled.
The 3H (tritium) labelled compound are commercially available. Tritium labeling is effected by catalytic exchange in aqueous media by hydrogenation of unsaturated compound with tritium gas. Tritium is pure β- emitter of low intensity & its radiation energy is lower than 14C.

15. By the use of organic synthesis
CH3MgBr + 14CO CH3 14COOHMgBr + H2O
CH314COOH + Mg(OH)Br
2. Introduction of labelled compounds
Precautions:
• The precursor should react at necessary site of synthesis in plant.
• Plant at the experiment time should synthesize the compound under investigation
• The dose given is for short period.

16. Root feeding: The plant in which roots are the biosynthetic sites e. g
Root feeding: The plant in which roots are the biosynthetic sites e.g., Tobacco. The plants are hydroponically cultivated to avoid microbial contamination.
Stem feeding: Substrate can be administered through cut ends of stem immersed in solution. For latex containing plant this method is not suitable.
Direct injection: Suitable for plant with hollow stem e.g., umbelliferous and opium.
Infiltration: Suitable for plant rooted in soil or other support without disturbing the roots (Wick feeding).
Floating method: When small amount of material is available this method is used. This technique is used in conjugation with vacuum infiltration to remove gases.
Spray technique: Compound has been absorbed after being sprayed on leaves in aqueous solution e.g., Steroids

17. Choice of solvent for extraction
3. Separation of compounds
Soft & fresh tissue: Maceration, infusion
Hard tissue: Decoction, hot percolation
Unorganized drug: Maceration with adjustment
Choice of solvent for extraction
Fat & oil: Non-polar solvents
Alkaloids, glycosides, flavonoids: Slightly polar solvents
Phenols: Polar solvents

18. 4. Detection of compounds
Geiger – Muller counter
Liquid Scintillation counter
Gas ionization chamber
Bernstein – Bellentine counter
Mass spectroscopy
NMR eletrodemeter
Autoradiography
Radio paper chromatography
5. Methods in tracer technique

19. Gas-filled detectors consist of a volume of gas between two electrodes, with an electrical potential difference (voltage) applied between the electrodes e.g., ionization chamber, proportional counter, GM counter.
In scintillation detectors, the interaction of ionizing radiation produces UV and/or visible light
Semiconductor detectors are especially pure crystals of silicon, germanium, or other materials to which trace amounts of impurity atoms have been added so that they act as diodes.

20. Detectors may also be classified by the type of information produced:
Detectors, such as Geiger-Mueller (GM) detectors, that indicate the number of interactions occurring in the detector are called counters
Detectors that yield information about the energy distribution of the incident radiation, such as NaI scintillation detectors, are called spectrometers
Detectors that indicate the net amount of energy deposited in the detector by multiple interactions are called dosimeters

21. Geiger – Muller counter
A Geiger counter (Geiger-Muller tube) is a device used for the detection and measurement of all types of radiation: alpha, beta and gamma radiation.
Basically it consists of a pair of electrodes surrounded by a gas. The electrodes have a high voltage across them. The gas used is usually Helium or Argon. When radiation enters the tube it can ionize the gas. The ions (and electrons) are attracted to the electrodes and an electric current is produced.
A scaler counts the current pulses, and one obtains a "count" whenever radiation ionizes the gas.
Advantages
They are relatively inexpensive, durable, easily portable, detect all types of radiations
Disadvantages
a)They cannot differentiate which type of radiation is being detected.
b)They cannot be used to determine the exact energy of the detected radiation & have a very low efficiency

24. Ionization Chamber
These detector collect all the charges created by direct ionization within the gas through the application of electric field.
If gas is air and walls of chamber are of a material whose effective atomic number is similar to air, the amount of current produced is proportional to the exposure rate
Air-filled ion chambers are used in portable survey meters, for performing QA testing of diagnostic and therapeutic x-ray machines, and are the detectors in most x-ray machine phototimers
Low intrinsic efficiencies because of low densities of gases and low atomic numbers of most gases

26. Proportional Counter
The proportional counter is a type of gaseous ionization detector device used to count particles of ionizing radiation.
While proportional counters are most commonly used for quantifying alpha and beta activity, they are also used for neutron detection, and to some extent for x-ray spectroscopy.
The pulses produced by a proportional counter are larger than those produced by an ion chamber. This means that the proportional counter is more conveniently operated in the pulse mode (ion chambers usually operate in the current mode).
Unlike the situation in a GM detector, the pulse size reflects the energy deposited by the incident radiation in the detector gas. As such, it is possible to distinguish the larger pulses produced by alpha particles from the smaller pulses produced by betas or gamma rays.

27. Operates at higher voltage than ionization chamber
Must contain a gas with specific properties
Commonly used in standards laboratories, health physics laboratories, and for physics research
Seldom used in medical centers
Types of Proportional Counters
Gas flow proportional:
- with window (e.g., laboratory alpha-beta counters) windowless  (e.g., tritium measurements)
Air Proportional (alpha counting only)
3. Sealed proportional  (e.g., BF3, He-3 neutron detectors)

29. Scintillation Counter
Scintillators are used in conventional film-screen radiography, many digital radiographic receptors, fluoroscopy, scintillation cameras, most CT scanners, and PET scanners
Scintillation detectors consist of a scintillator and a device, such as a PMT, that converts the light into an electrical signal
More sensitive than Geiger-Muller counter.
Wide spread detection.
Principal
Incident radiation interact with material
Atoms are raised to excited states
Excited states emit visible light: fluorescence
Light strikes photosensitive a surface
Release of a photoelectron Multiplication

30. Desirable properties of Scintillators:
High conversion efficiency
Decay times of excited states should be short
Material transparent to its own emissions
Color of emitted light should match spectral sensitivity of the light receptor
For x-ray and gamma-ray detectors, µ should be large – high detection efficiencies
Rugged, unaffected by moisture, and inexpensive to manufacture
Amount of light emitted after an interaction increases with energy deposited by the interaction
May be operated in pulse mode as spectrometers
High conversion efficiency produces superior energy resolution

31. Materials:
There are organic (toluene, xylene) and inorganic scintillators (NaI), CsI
Sodium iodide, CsI, CaI activated with thallium [NaI(Tl)], coupled to PMTs and operated in pulse mode, is used for most nuclear medicine applications
Fragile and hygroscopic
Bismuth germanate (BGO) is coupled to PMTs and used in pulse mode as detectors in most positron emission tomography (PET) scanners
PMTs perform two functions:
Conversion of ultraviolet and visible light photons into an electrical signal
Signal amplification, on the order of millions to billions
Consists of an evacuated glass tube containing a photocathode, typically 10 to 12 electrodes called dynodes, and an anode

32. Organic VS Inorganic scintillators
Inorganic scintillators have greater:
light output
longer delayed light emission
higher atomic numbers
than organic scintillators
Inorganic scintillators also linear energy response (light output is ά energy absorbed)

36. Semiconductor Detectors
Operate essentially like ionization chambers. Ge, Si, NaI are solid semiconductor materials (they form solid crystals with valence-4 atoms). They can be “doped” to control electrical conductions
With valence 3 or 5 atoms introduced into the lattice
Donor states: n-type semiconductor
Acceptor states: p-type semiconductor
Works on same principle as gas-filled detectors (i.e., production of electron-hole pairs in semiconductor material)
• Only ~3 eV required for ionization (~34 eV, air), Usually needs to be cooled (thermal noise)
• Usually requires very high purity materials or introduction of “compensating” impurities that donate electrons to fill electron traps
caused by other impurities

39. AUTORADIOGRAPHY
Detecting radioactive compounds with a photographic emulsion (x-ray film)
Types:
In-vivo autoradiography - receptors are labelled in intact living tissue by systemic administration of the radioligand (PET)
In-vitro autoradiography - slide-mounted tissue sections are incubated with radioligand so that receptors are labelled under very controlled conditions
Uses :
Map anatomical location of radiolabelled ligands to visualize and quantify receptors in tissue
Trace neurons by axonal transport of radioactively labelled amino acids, certain sugars, or transmitter substances
Measure DNA production (e.g., 3H-thymidine)

40. Advantages:
Highly specific tool to pharmacologically characterize receptors in
tissue
Provides location of receptor (etc) in tissue
Enables characterization of receptors in different tissues
Technically easy
Disadvantages: -
Everything binds to everything (easy to misinterpret results)
There are no biochemical or physiological criteria to assess the
binding specificity (i.e., to determine whether the binding site really
corresponds to an actual receptor)
The presence of a high-affinity radiolabelled receptor does not
necessarily imply that the receptor has physiological significance
Ligands are not always very specific

41. Radiation will hit silver grains in emulsion and expose them
Expose to film
or emulsion
Isotope will emit
radiation (usually beta)
Incubate tissue with
radioactive ligand

42. develop
film

43. Mass Spectrophotometer:
Mass spectrometry (MS) is an analytical technique that measures the
mass-to-charge ratio of charged particles.
It is used for determining masses of particles, for determining the
elemental composition of a sample or molecule , and for elucidating
the chemical structures of molecules, such as peptides and other
chemical compounds.
NMR Spectrophotometer:
NMR spectroscopy, is a research technique that exploits the magnetic
properties of certain atomic nuclei to determine physical and chemical
properties of atoms or the molecules in which they are contained.
It relies on the phenomenon of nuclear magnetic resonance and can
provide detailed information about the structure, dynamics, reaction
state, and chemical environment of molecules. 

49. METHODS IN TRACER TECHNIQUE
PRECURSOR PRODUCT SEQUENCE:
In this technique, the presumed precursor of the constituent under investigation on a labelled form is fed into the plant and after a suitable time the constituent is isolated, purified and radioactivity is determined.
Disadvantages:
The radioactivity of isolated compound alone is not usually sufficient evidence that the particular compound fed is direct precursor, because substance may enter the general metabolic pathway and from there may become randomly distributed through a whole range of product.

50. Applications:
Stopping of hordenine production in barley seedling after days of germination
Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura stramonium
This method is applied to the biogenesis of morphine & ergot alkaloids

51. 2. DOUBLE & MULTIPLE LABELLING:
This method give the evidence for nature of biochemical incorporation of precursor arises, double & triple labeling. In this method specifically labelled precursor and their subsequent degradation of recover product are more employed.
Applications:
This method is extensively applied to study the biogenesis of plant secondary metabolite.
Used for study of morphine alkaloid. e.g., Leete, use Doubly labeled lysine used to determine which hydrogen of lysine molecule was involved in formation of piperidine ring of anabasine in Nicotina glauca.

53. 3. COMPETITIVE FEEDING:
If incorporation is obtained it is necessary to consider whether this infact, the normal route of synthesis in plant not the subsidiary pathway. Competitive feeding can distinguish whether B & B’ is normal intermediate in the formation of C from A.

54. Applications:
This method is used for elucidation of biogenesis of propane alkaloids.
Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis of alkaloids of Conium maculactum (hemlock) using 14C labelled compounds..

55. 4. ISOTOPE INCORPORATION:
This method provides information about the position of bond cleavage & their formation during reaction.
e.g. Glucose-1-phosphatase cleavage as catalyzed by alkaline phosphatase this reaction occur with cleavage of either C – O bond or P – O bond.

56. 5. SEQUENTIAL ANALYSIS:
The principle of this method of investigation is to grow plant in atmosphere of 14CO2 & then analyze the plant at given time interval to obtain the sequence in which various correlated compound become labelled.
Application:
14CO2 & sequential analysis has been very successfully used
in elucidation of carbon in photosynthesis
Determination of sequential formation of opium hemlock and
tobacco alkaloids
Exposure as less as 5 min 14CO2, is used in detecting
biosynthetic sequence as –
Piperitone (-) Menthone (-) Menthol in Mentha piperita.

57. APPLICATIONS OF TRACER TECHNIQUE
Study of squalene cyclization by use of 14C, 3H labelled mevalonic acid.
Interrelationship among 4 – methyl sterols & 4, 4-dimethyl sterols, by use of 14C acetate.
Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of 2-14C mevalonate.
4. Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin.

58. Origin of carbon & nitrogen atoms of purine ring system by use of 14C or 15N labelled precursor
Study of formation of scopoletin by use of labelled phenylalanine
By use of 45Ca as tracer, found that the uptake of calcium by plants from the soil (CaO & CaCO2)
8. By adding ammonium phosphate labelled with 32P of known specific activity the uptake of phosphorus is followed by measuring the radioactivity as label reaches first in lower part of plant, than the upper part i.e. branches, leaves etc.

59. Thanks