Isotopes: TWO TYPES OF ISOTOPES,Radioactive isotopes. Stable isotopes, Radiolabelled Tracers ( Radiolabelled compounds), Radiotracer Technique, Steps in Tracer Technique, Selection of Radioisotopes. Preparation of Radioisotopes. Introduction/Insertion of Radiolabelled compound in biological system (Plant part) Seperation and determination of labelled compound in various biochemical reaction, Preparation of labelled compounds : Insertion of Radiolabelled compound in plant part, Root feeding, Stem feeding, Direct Injection, Floating Methods, Spray technique, Separation or Isolation of Radiolabelled compound and detection of radioisotope labelled compound. Detection and assay of Radioactive labelled compound, Detector system used (Analysis of Isotopic content). Method in Tracer Technique, Precursor – Product sequence Double and Multiple Labelling . Competitive Feeding,Sequential Analysis Applications of Tracer Technique

1. 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

2.  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

3. • Hydrogen Isotopes :
H1 H1 H1
( 1P ) (1P, 1N) (1P, 2N )
Hydrogen Deuterium Tritium
TWO TYPES OF ISOTOPES
1. Radioactive isotopes.
2. Stable isotopes
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4. 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
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5. 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

6.  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.

7.  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

8. •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:
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9.  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

10. 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

11. 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

12. 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

13.  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.

14. 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.

16. 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.
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17.  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
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14
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19. • 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
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20. Thank you!