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Utilization of radioactive isotopes in the investigation of biogenetic studies

The document discusses the utilization of radioactive isotopes in biogenetic studies, explaining the difference between radioactive and stable isotopes and their applications in various biological investigations. It details the radiotracer technique for studying biosynthetic pathways, including steps for isotope selection, preparation, insertion, and detection of radiolabelled compounds. Additionally, it outlines methods and applications of tracer techniques in studying various alkaloids and photosynthesis processes.

Education◦
Vth Semester B. pharmacy Pharmacognosy and Phytochemistry-II Unit-I Basic Metabolic Pathway(Part-6) (Utilization of radioactive isotopes in the investigation of biogenetic studies) Presented By : Miss. Pooja D. Bhandare (Assistant Professor) Kandhar College of Pharmacy, Nanded
 UTILIZATION OF RADIOACTIVEISOTOPES IN THE INVESTIGATION OF BIOGENETIC STUDIES • Isotopes: Iso = same (equal), Topes =Place. • They occupy same place in periodic table. “ Elements with same atomic number but different atomic weight ( Same number of protons but differ in neutrons). • Example- C6 C6 C6 • Atomic mass = No. of Protons + No of Neutrons • Atomic Number= No of protons 12 13 14
• Hydrogen Isotopes : H1 H1 H1 ( 1P ) (1P, 1N) (1P, 2N ) Hydrogen Deuterium Tritium TWO TYPES OF ISOTOPES 1. Radioactive isotopes. 2. Stable isotopes 1 32
1. Radioactive isotopes: (Radioisotopes)- Radio (Radiation)+ Isotopes Unstable isotopes. The isotopes which emit the radiation are called Radioactive isotopes. Decay with the emission of radiation (α, β, and γ radiation) Example – H, C, S, I, Na, K, S,  For biological Investigation- Carbon and hydrogen For metabolic studies- S,P and alkali and alkaline earth metals are used For studies on proteins , alkaloids and amino acid-labelled nitrogen atom give more specific information.  H compound is commercially available. 3 3514 131 24 42 35 3
2. Stable Isotopes : • Stable isotopes are non radioactive form of atoms ( they do not emit radiation). • Although they do not emit radiations, their unique properties enable them to be used in a broad variety of applications, including water and soil management , environmental studies, nutrition assessment studies and forensics. • Example- H, C, N, O Used for labelled compounds as possible intermediates in biosynthetic pathway. Usual method of determination are:- MASS Spectroscopy ( N, O ) NMR Spectroscopy ( H, Cl ) 2 13 15 18 15 18 2 13
 Radiolabelled Tracers ( Radiolabelled compounds) • When one or more atom of chemical compound replaced by radioisotopes used for the study of the biosynthetic pathway, is known as Radiotracer. Radiotracer Technique : The technique which utilises radioactive labelled compounds to find out or to trace various precursors and intermediate involved at different stages of biosynthetic pathway at given rate and time. In this technique, different isotopes, mainly the radioactive isotopes which are incorporated into the presumed precursor of plant metabolites and used as marker in the biogenetic studies.
 Steps in Tracer Technique 1. Selection of Radioisotopes. 2. Preparation of Radioisotopes. 3. Introduction/Insertion of Radiolabelled compound in biological system (Plant part) 4. Seperation and determination of labelled compound in various biochemical reaction
•Preparation of labelled compounds : a) Growing chlorella in atmosphere of CO2 b) Nuclear Reactor/ Acceralator N7 + n0 C6 + P1 a) Tritium gas : Tritium labelled compounds ( H )are commercially available. Tritium labelling is effected by catalytic exchange in aqueous media by hydrogenation of unsaturated compound with tritium gas. b) By the use of Organic Synthesis: 14 3 14 1 6
 Insertion of Radiolabelled compound in plant part Precaution: • The precursor should react at necessary site of synthesis in plants. • Plant at the experiment time should synthesis the compound under investigation. • The dose given is for short period. 1. Root feeding: When the root is biosynthetic site (Ex- Tobacco and radiolabelled compound. 2. Stem feeding: Cut end of stem immersed in water, nutrient and radiolabelled compounds
3. Direct Injection: Which have hollow stem ( Umbelliferous fruits), 4. Infilteration : It is also called Wick Feeding method. 5. Floating Methods: Substrate solution which contains Radioactive compound. 6. Spray technique
Separation or Isolation of Radiolabelled compound and detection of radioisotope labelled compound. Depends on Nature of drugs and sources of drugs- Soft tissue- Infusion, Maceration Hard tissue- Decoction, hot percolation Unorganised drugs- Maceration Alkaloid, Glycoside, Flavonoids- Slightly polar solvent
Detection and assay of Radioactive labelled compound Detector system used (Analysis of Isotopic content) a. Geiger- Muller Counter- Detection and measurement of all types of radiation. b. Liquid Scintillation Counter – Scintillators are used (For β emitting radioisotopes) c. Gas Ionization Chamber d. Bernstein- Bellentine Counter e. Mass spectoscopy f. NMR Electrodemeter g. Autoradiography- to trace the location of radioactive isotopes in biological system h. Radio paper chromatography
 Method in Tracer Technique 1. Precursor – Product sequence In this technique, the presumed precursor of the constituent under investigating 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 form there may become randomly distributed through a whole range product.
2. Double and Multiple Labelling This method give the evidence for nature of biochemical incorporation of precursor arises double and triple labelling. In this method specifically labelled precursor and their subsequent degradation of recover product are more employed. • Application: 1. This method is extensively applied to study the biogenesis of plant secondary metabolite. 2. Used for study of morphine alkaloid.
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 and B’ is the normal intermediate for the formation of C from A. B A C B’ • Application: This method is used for the biological of propane alkaloids. Biosynthesis of hemlock alkaloids ( conline , conhhydrine etc) e.g. Biosynthesis of alkaloids of Conium manuculatum (Hemlock) using C labelled compounds. 14
 Sequential Analysis The principal of this method of investigation is to grow plant in atmosphere of CO2 and then analyze the plant at given interval to obtain the sequence in which various correlated compound become labelled. • Application: 1. CO2 and sequential analysis has been very successful used in elucidation of carbon in photosynthesis. 2. Determination of sequential formation of opium hemlock and tobacco alkaloids. 3. Exposure as less as 5 min CO2 is used in detecting biosynthetic sequence as – Piperdine ------------(-) Menthone ------------- (-) Menthol in Mentha piperita 14 14 14
• Applications of Tracer Technique 1. Study of squalene cyclization by use of C, H labelled mevalonic acid. 2. Interrelationship among 4-methyl sterols and 4,4-dimethyl sterols, by use of C acetate. 3. Terpenoids biosynthesis by chloroplast isolated in organic solvent by use of 2- C mevalonate. 4. Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin. 5. Origin of carbon and nitrogen atoms of purine ring system by use of C or N labelled precursor. 6. Study of formation of scopoletin by use of labelled phenyalanine. 7. By use of Ca as tracer, found that the uptake of calcium by plants from the soil (CaO and CaCO2). 8. By adding ammonium phosphate labelled with P of know specific activity the uptake of phosphorus is followed by measuring the radioactivity as labelled reaches first in lower part of the plant, than the upper part i.e. branches, leaves, etc. 14 3 14 14 14 15 45 32
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