TRACER TECHNIQUES & UTILIZATION IN BIOGENETIC STUDIES

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: Phytohormoneauxin 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) flavinmonooxygenase-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 14CO2 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 + 14CO2 CH314COOHMgBr + 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., 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. 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 15- 20 days of germination • Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura stramonium • This method is applied to the biogenesis of morphine & ergot alkaloids