M.Pharm class

1. TRACER TECHNIQUE

2. INTRODUCTION
• Tracer technique now widely employed in all branches
of science, had its origin in early part of last century,
when it was realized that elements existed with
identical chemical properties but with different
atomic weights.
• Such isotopes may be stable(13
C, 2
H)

3. or nucleus may be unstable.(14
C,3
H) and decay
with emission of radiation.
• If it is possible to detect these isotopes by suitable
means then they can be incorporated into presumed
precursor of plant constituents and used as markers in
biogenetic experiments.

4. DEFINATION
• It is a technique which utilizes a labeled compound to
trace or find out the different intermediates and
various steps involved in biosynthetic process in plant
at given rate and given time.
• When these labeled compounds are administered into
the plants they become a part of general metabolic
pool and undergo reactions characteristics to the
metabolism of that particular plant.

5. RADIOACTIVE TRACER
A radioactive tracer, or radioactive label, is a
chemical compound in which one or more atoms have
been replaced by a radioisotope. By virtue of its
radioactivity it can be used to explore the mechanism
of chemical reactions by tracing the path that the
radioisotope follows from reactants to products.
Radioisotopes of hydrogen, carbon, phosphorus,
sulphur, and iodine have been used extensively to trace
the path of biochemical reactions.

6. Significance of tracer techniques
• It is used for tracing of biosynthetic pathway by
incorporating radioactive isotopes into precursor or
starting material.
eg:By incorporation of 14
C to phenylalanine,the
biosynthesis of cyanogenetic glycoside prunacin can
be traced.

7. • Location and quantity of compound can be
determined within biological system.
• Radioactive tracer methods are used both in research
and routine analytical method.

8. Different tracer element for different
studies
• For studies on Proteins ,alkaloid and amino acids:
Nitrogen atom gives more specific information than
carbon atom.
• For studies on glycosidic linkage: O, N, S and C
atoms.
• For studies on Terpenoids: O atom

9. Basic steps involved in tracer
techniques
• Preparation of labelled compound.
• Incorporation of labelled compound to tissue system.
• Separation or isolation of labelled from tissue system.
• .Determination of nature of metabolites in various
biochemical fraction

10. Preparation of labelled
compound
• The labelled compounds may be prepared by use of
radioactive isotopes and stable isotopes.
• Radioactive isotopes14
C, 3
H,35
S,32
P,131
I, 60Co.

11. Criteria for selection of trace
elements
• The starting concentration of trace element must be
sufficient enough to withstand dilution in the course of
metabolism.
• For proper labelling the physical and chemical nature of
the compound must be known.
• Half life of the tracer isotope should be sufficiently long
• The labeled compound should not be damage to the
tissue system.

12. Introduction of labelled compound to
tissue system
• Root feeding
• Stem feeding
• Direct injection
• Infiltration
• Floating method
• Spraying technique

13. Root feeding
• The plant in which roots are biosynthetic sites;
this method is preferred e.g. Tobacco.
• In this type of experiment the plant are cultivated
hydroponically to avoid microbial contamination.

14. Stem feeding
• In this method substrate can be administered
through the cut ends of stem immersed in a solution.
• For latex containing plant; this method is not
suitable

15. Direct injection
• This method is applicable to the plant with hollow
stem. e.g. Umbelliferae and capsules bearing
plant(Opium poppy).

16. Infiltration : Wick feeding
• When it is desired to carryout feeding on plant rooted
in soil or other support without disturbing the
root ,wick feeding is applicable.

17. Floating method
• When small amount of material is available, floating
method is used. In this method leaf disc or chopped
leaves are floated on the substrate solution.
• This technique is also used in conjugation with
vacuum infiltration to remove gases.

18. Spray technique
• In this method compound are absorbed after being
sprayed on leaves in aqueous solution. e.g. Steroids

19. Separation or isolation of labelled
compound or metabolite
Depending on the nature of drug and its source different method of
extraction is employed:
• Soft and fresh tissue: Infusion, Maceration.
• Hard tissue: Decoction, Hot percolation.
• Unorganized drug: Maceration

20. Determination of nature of
metabolites
• Depending on nature of isotopes various instrumentation
techniques are used for determination of chemical nature of
intermediate and final product.
For radioactive isotopes
• GM-Counter
• Scintillation or liquid scintillation counter
• Ionization chamber

21. • These entire instruments characterized the nature of
radiation. Basically it depends upon the conversion of
kinetic energy of particle into fleeting pulse of light
as a result of its penetration into a suitable
luminescent medium

22. For stable isotopes
• Mass spectroscopy: gives molecular peaks depending
on mass/charge ratio.
• NMR: gives nature of carbon and proton.

23. Methods of tracer techniques
• Precursor-product sequence.
• Competitive feeding.
• Sequential analysis.

24. Precursor-product sequence
• In this method presumed precursor of the constituent
as under investigation in a labeled form is fed to
plant and after a particular time the constituent is
isolated, purified and its radioactivity is determined.
This method is used for biogenesis of morphine and
ergot alkaloid.

25. Competitive feeding
• This method is normally used to determine two
possible intermediates in the plants.
• Competitive feeding can distinguish whether B or B’
is the normal intermediate in the formation of C from A

26. B
A C
B’

27. • E.g. Biosynthesis of Hemlock alkaloids.
Connine and conhydrine.

28. Sequential Analysis
• The principle of this method of investigation is to
grow plants in the atmosphere of14
CO2 and
by analysis of plants at given intervals ,to obtain the
sequence in which various related compounds becomes
labelled .

29. • 14
CO2 and sequential analysis has been very
successfully used in the elucidation of the path
of carbon in photosynthesis.
• Determination of sequential formation of opium and
tobacco alkaloid

30. APPLICATIONS
• The application of radioactive tracers for the study of
biological processes originated with the hungarian-
bornphysical chemist George de Hevesy, who
performed his basic research in this area during the
1930'ies in Copenhagen

31. • Phosphorous-metabolism in mammalian bone tissue
• Transport of amino acids through small-intestinal
epithelium
• The dark reactions of photosynthesis
• Turnover and degradation of toxic compounds in the
liver

32. • Receptor-ligand binding studies
• Circadian variation in DNA-synthesis in rodent
mammary glands
• Biosynthesis of glycin-betain and its role in
osmoregulation in mussel

33. Tracers Used in Nuclear Medicine
Pharmaceutical Source Activity
(MBq)
Medical Use
Pertechnetate 99m
Tc 550 - 1200 Brain Imaging
Pyrophosphate 99m
Tc 400 - 600 Acute Cardiac Infarct
Imaging
Diethylene Triamine
Pentaacetic Acid (DTPA)
99m
Tc 20 - 40 Lung Ventilation
Imaging
Benzoylmercaptoacetyltri
glycerine (MAG3)
99m
Tc 50 - 400 Renogram Imaging
Methylene Diphosphonate
(MDP)
99m
Tc 350 - 750 Bone Scans

34. THANK YOU