Unit 1.

2
Study of utilization of radioactive isotopes in the
investigation of Biogenetic studies.

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 Introduction
• Isotopes: Iso (equal) + topes (place): Occupy same place in the periodic table.
• Elements with same atomic number but different atomic weight.
• Same number
12 of proton
13 14 but differ in neutron.
• Examples: 6C C , 6C Atomic mass= No. of Proton + No. of Neutron
6
1 2 3

(1P)
1H
H 1N) 1H(1P, 2N)
(1P,1H,
Hydrogen Deuterium Tritium

• Radioactive atom: Radiation is emitted from atoms when an unstable atom decays to become more
stable. When an atom has extra neutrons or protons, it causes the element to become unstable.
• There are 3 types of radiation given off by radioactive atoms:
• Alpha particles
• Beta particles
• Gamma rays
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 Introduction
• Two types of isotopes:
1. Radioactive isotopes (Radioisotopes):
• Radio (radiation)+ isoptopes-unstable isotopes
• Decay with the emission of radiation α,β,γ form
• Examples: 3H, 14C, 35S, 131I, 24Na
2. Stable isotopes:
• Do not emit radiation
• Examples: 2H, 13C, 15N

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 Techniques used for the investigation of biosynthetic pathway

• There are 5 techniques used for the investigation of biosynthetic

pathway of primary and secondary metabolites.
1. Tracer technique
2. Use of isolated organ and tissues
3. Grafting method
4. Use of Mutant strains
5. Enzymatic studies
• Out of the above 5 methods, in Tracer technique method
radioactive isotopes are used for the investigation of biogenetic
studies.

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 Tracer technique
Radiolabelled tracers/ Radioactive labelled
compounds:
• When 2 or more atom of the compound is
replaced by Radioisotopes used for the study of
biosynthetic pathway known as radiotracers.
• Used as a marker in biosynthetic studies
Radiolabelled
• The technique which utilizes radioactive labelled Glucose at Carbon -2
compound to find out or to trace the various
precursors and intermediates involved at different CH3-14*COOH
Radiolabelled acetic
stages of biosynthetic pathways at given rate and acid
time, this technique is called as Radiotracer 14*
technique.
• A* B* C* D*
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 Tracer technique
Significance of tracer techniques-
• Applicable for living systems. Wide ranges of isotopes are available.
• High sensitivity
• More effective
• Simple administration and isolation.
• Shows accurate results when enough metabolic time & technique is used.
• Position & Quantity of compound containing tracer isotope 14C marked glucose is used for
glucose determination in the biological system.
• For different studies, different tracers can be used. For studies on nitrogen and amino acid,
Labelled nitrogen gives specific information than carbon.
• Biosynthetic pathway can be traced by incorporating radioactive isotopes into the precursor or
starting material.
• e.g- By incorporation of 14C to phenyl alanine, the biosynthesis of cyanogenetic glycosides,
prunacin can be traced. Location and quantity can be determined in biological system.

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 Tracer technique
• Different trace elements used for different studies-
1. For studies on protein, alkaloids and amino acid, nitrogen atom gives
more specific information than carbon.
2. For studies on glycosidic linkage- O, N, S and C atom.
3. For studies on terpenoids- O atom.

Steps involved in tracer techniques-

4. Preparation of labelled compound
5. Incorporation of labelled compound
6. Separation and isolation of labelled compound
7. Determination of nature of metabolites in various biochemical
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 Tracer technique
Steps involved in tracer techniques-
1. Preparation of labelled compound:
• In biological investigation, the use of bioactive isotopes enables the
metabolism of compounds to be followed in living organisms for detection
and estimation of soft and easily absorbed radiation from labelled
compound.
• Labelled compounds may be prepared by use of radioactive isotopes and
stable isotopes
• E.g- Radioactive isotopes- 14C, 3H, 32P, 131I
• Stable isotopes- 2H, 15N, 13C, 18O
• Radioactive carbon and hydrogen are mostly used in biological investigation.
• Radioactive isotopes having long half-life are used.
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 Tracer technique
Steps involved in tracer techniques-
1. Preparation of labelled compound:
Criteria for selection of trace elements-
• Starting concentration of trace element must be sufficient to withstand dilution in the
course of metabolism.
• Physical and chemical nature of compound must be known.
• Half-life should be sufficiently long (Radioactive isotopes of carbon have mass
numbers of 10, 11 and 14. 10C has a half-life of 8.8 s and 11C a half-life of 20 min.
which limits their usefulness in biological research. However, 14C has an estimated
half-life of over 5000 years).
• Should not damage the tissue system
• Should have low radiation energy.
• Instruments used to detect properties of metabolites are Scintillation chamber, GM
(Geiger-Muller)counter, Autoradiography,
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9
 Tracer technique
Steps involved in tracer techniques-
1. Preparation of labelled compound:
Methods of preparation:
a. Many compounds which are most conveniently prepared from natural sources
(e.g. certain amino acids by the hydrolysis of proteins) are produced by growing
Chlorella in an atmosphere containing 14CO2. All the carbon compounds of the
organism thus become labelled, each compound possessing a uniform labelling
of its carbon atoms.
b. Many tritium (3H)-labelled compounds are commercially available.
c. Tritium labelling is effected by catalytic exchange (platinum catalyst) in aqueous
media, by irradiation of organic compounds with tritium gas and by
hydrogenation of unsaturated compounds with tritium gas. Tritium is a pure β-
emitter of low toxicity, half-life 12.43 years, with a radiation energy lower than
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 Tracer technique
Steps involved in tracer techniques-
2. Incorporation of labelled compound to tissue system

1. Root feeding
2. Stem feeding
3. Direct injection
4. Infiltration
5. Floating method
6. Spraying technique

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 Tracer technique
Steps involved in tracer techniques-
2. Incorporation of labelled compound to tissue system
1. Root feeding- In case roots are biosynthetic sites e. g- Tobacco. The plants are cultivated
hydroponically to avoid microbial contamination.
2. Stem feeding- Labelled compounds are administered through the cut ends of
stem immersed in a solution. For latex containing plants this method is not
suitable.
3. Direct injection- This method is used in plants with hollow stem. e.g-
Umbelliferae and capsule plants (opium poppy). Micro-syringe is used to inject
labelled compound solution.
4. Infiltration (wick feeding)- A thread is drawn through the stem which is dipped
into radioactive solution or a flap can be cut in stem and this dipped in the
solution.
5. Floating method- When a small amount of material is available, this method is
used. Leaf disc/chopped leaves are floated on labelled compound solution.
6. Spraying technique- Compounds have been absorbed after being sprayed on
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 Tracer technique
Steps involved in tracer techniques-
3. Separation and isolation of labelled compound:
• Different methods are used depending on nature of drug and its source.
1. Soft tissue (Fresh)- Infusion, Maceration
2. Hard tissue- Decoction and hot percolation
3. Unorganized drug- Maceration with solvent
4. Fat and oil- Non-polar solvent
5. Alkaloids, Glycosides, Flavonoids- Slightly polar solvent
6. Plant phenol- Polar solvent

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 Tracer technique
Steps involved in tracer techniques-
3. Separation and isolation of labelled compound:
Detection and assay of radioactivity labelled compound-
When radioactive tracers are used in biogenetic studies, adequate methods for the detection and
estimation of the label are essential.
• For soft and easily absorbed radiation from 3H, 14
C labelled compounds- Liquid scintillation
counter.
• Modern instruments are used for mixed radiation like 3H and 14
C. This is possible because both
are β-emitters and different radiation energy.
• With all counters, the instrument is connected to a suitable rate meter which records the count
over a given time.
• Different instruments are used to determine nature of metabolites. e.g- GM Counter,
Scintillation or liquid scintillation counter and ionisation chamber.

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 Tracer technique
Steps involved in tracer techniques-
3. Separation and isolation of labelled compound:
For stable isotopes-
1. MS gives molecular peak depending on mass/charge ration.
2. NMR gives nature of carbon or proton
3. Autoradiography
Advantages-
• Whole morphological parts like leaves or section, for which the resulting
negative is viewed under microscope.
• Radioactive compounds on paper/thin layer chromatography can be
detected and amount of radioactivity in different spots determined by
density measurement.
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 Tracer technique
Steps involved in tracer techniques-
4.Determination of nature of metabolites in various biochemical fractions-
• Depending on the nature of isotopes, various instruments are used to
determine the chemical nature of intermediate and final product for
radioactive isotopes.
1. Geiger-Muller counter
2. Scintillation or liquid scintillation counter
3. Ionization chamber
4. Mass Spectrophotometer
5. NMR Spectrophotometer
6. Autoradiography
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 Tracer technique
Steps involved in tracer techniques-
4.Determination of nature of metabolites in
various biochemical fractions-
1. Geiger-Muller counter:
• It is a type of particle detector that
measures ionizing radiation.
• e.g. alpha, beta particles or gamma rays,
by ionization produced in low-pressure gas.
• Usually helium, neon or argon with
halogens added in the Geiger-Muller tube,
which conducts electrical charges briefly
when a particle or photon of radiation
makes the gas conductive by ionization.
• This indictment has been detected in form
of current pulse.
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 Tracer technique
Steps involved in tracer techniques-
4.Determination of nature of metabolites in
various biochemical fractions-
2. Scintillation or liquid scintillation counter
• scintillation detector or scintillation counter is
produced when the scintillation detector is
coupled to an electronic light sensor such as a
photomultiplier tube (PMT) or a photodiode.
• A scintillator is a material that exhibits
scintillation- a luminescence property that is
stimulated by ionizing radiation.
• Samples shall be dissolved or suspended in a
"cocktail" containing a solvent(aromatic
organics such as benzene or toluene), typically
some form of a surfactant, and small amounts
of scintillators.
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 Tracer technique
Steps involved in tracer techniques-
4.Determination of nature of metabolites in
various biochemical fractions-
3. Ionization chamber
• The ionization chamber is the simplest of all
gas-filled radiation detectors and is
commonly used for ionizing radiation,
including x-rays, gamma rays and beta
particles.
• Conventionally, the term "ionization
chamber" is used solely to describe those
detectors that collect all the charges caused
by direct ionization of the gas using an
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 Tracer technique
Steps involved in tracer techniques-
4.Determination of nature of metabolites in various biochemical fractions-
5. NMR Spectrophotometer:
• NMR spectroscopy is a research technique that exploits the magnetic
properties of certain atomic nuclei to determine the physical and chemical
properties of the atoms or molecules they contain.
• It relies on the phenomenon of 14 nuclear magnetic resonance and can
provide detailed information on the structure, dynamics, reaction status and
chemical environment of the molecules.

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 Tracer technique
Steps involved in tracer techniques-
4.Determination of nature of metabolites in various biochemical fractions-
6. Autoradiography:
• It is a tool for examining the distribution of radioactive material in a plant
object, e.g. histological tissue, chromatography sheet.
• This method uses a photographic film or emulsion as an ionizing radiation
detector. The specimen is in close contact with the emulsion for a period
(exposure duration).
• In this technique, a sample containing a radiolabelled metabolite is placed in
direct contact with suitable photosensitive material such as x-ray
(photographic) film for a specific period.
• The pattern of delivery of radioactive substances can be elucidated with the
aid of the autograph collected.
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 Tracer technique
Methods of tracer techniques
1. Precursor Product sequence
2. Competitive feeding
3. Sequential analysis

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 Tracer technique
Methods of tracer techniques
1.Precursor Product sequence:
• 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.
• Radioactivity of the isolated compound alone is not
sufficient evidence that the particular compound fed is a
direct precursor, because the compound may enter the
general metabolic pathways and distributed randomly
through a whole range of products.
• Further evidence can be made by double and triple
labelling experiments by using either different isotopes of
specific labelling by one isotope at two or more positions
in the molecule.
• Application- Restricted synthesis of hyoscine, distinct
from hyoscyamine in Datura stramonium. This method is
applied to the biogenesis of morphine and ergot
alkaloids.
• Leete in his experiment used two doubly labelled lysines
to determine which nitrogen of the lysine molecule was
involved in the formation of the piperidine
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 Tracer technique
Methods of tracer techniques
2. Competitive feeding:
• If incorporation is obtained, it is necessary to consider
whether this is the normal route of synthesis in plant
not the subsidiary pathway.
• Competitive feeding experiments can be of value in
determining which of two possible intermediates is normally
used by the plant.
• Competitive feeding can distinguish whether B or B' is
the normal intermediate in the formation of C from A
(Fig.2.2).
• Inactive B and B' are fed with labelled A to separate
groups of plants and a control is performed by feeding
labelled A only to another group.
• If the product C obtained is inactive in the plant receiving B and
the product is active in the plant receiving B' indicates the
intermediate is B and not B’, then we may conclude that
the pathway from A to C probably proceeds via B.
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 Tracer technique
Methods of tracer techniques
2. Competitive feeding:
• In comparative studies on the rates of demethylation of codeine and unnatural codeine
derivatives in Papaver orientale, Kirkby and colleagues in 1972 overcame this problem by
using the same plant simultaneously as the control and as the test plant.
• They did this by administering to the plant a mixture of 3H-labelled codeine and 14C-
labelled unnatural codeine derivative.
• The products of the conversion of both could be independently followed by their
characteristic radiations, even when the metabolites produced were chemically identical.

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 Tracer technique
Methods of tracer techniques
2. Competitive feeding:
• Tyrosine is the precursor for 3,4-
dihydroxyphenylpyruvic acid and 3,4-
dihydroxyphenylethylamine for the synthesis of
Norlaudanosoline which produces morphine
through reticuline.
• 3,4- dihydroxyphenylethylamine is produced from
tyrosine via 3,4-dihydroxyphenylalanine (DOPA).
• It was considered that another precursor i.e 3,4-
dihydroxyphenylpyruvic acid would also be
synthesized through DOPA but by labelling
experiments and competitive feeding it has been
confirmed that tyrosine directly gives 3,4-
dihydroxyphenylpyruvic acid without any
intermediate.
• Application- Used for elucidation of biogenesis of
tropane alkaloid; biosynthesis of alkaloids of Conium 14C
labelled compound.
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 Tracer technique
Methods of tracer techniques
3. Sequential analysis:
• Principle of this method is to grow a plant in an
atmosphere of 14CO2, by analysis of the plants at given
time intervals, to obtain the sequence in which various
related compounds become labelled.
• From the results obtained, certain biosynthetic routes may
become apparent and others rejected.
• Here, again degradation of the isolated radioactive
compounds is important, because some units of the
molecule may become labelled more rapidly than others.
• This method has been very successfully used in the
elucidation of the path of carbon in photosynthesis and
also for determining the sequential formation of the
opium, hemlock and tobacco alkaloids.
• Exposure periods to 14CO2 as short as 5 min have been used
to obtain evidence of the biosynthetic sequence
piperitone- + (-)-menthone -+ (-)-menthol in Mentha
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