TRACER TECHNIQUES & UTILIZATION
IN BIOGENETIC STUDIES
ShriRam College of Pharmacy
Dr. Ajay Sharma
INTRODUCTION
1. Living plants considered as biosynthetic laboratory
primary as well as secondary metabolite.
2. Different biosynthetic pathway:
» Shikmic acid pathway: Aromatic amino acids
» Mevalonic acid pathway: Terpenes
» Acetate pathway: Fatty acids
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
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
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.
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: Phytohormone auxin 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) flavin monooxygenase-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).
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.
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.
» 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]
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
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
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.
LIMITATIONS
•
Kinetic effect
•
Chemical effect
•
Radiation effect
•
Radiochemical purity
•
High concentration distorting the result, expensive,
short half life, hazardous
REQUIREMENT FOR TRACER TECHNIQUE
1. 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 βemitter of low intensity & its radiation energy is lower than
14C.
•
By the use of organic synthesis
CH3MgBr + 14CO2
CH3 14COOHMgBr + 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.
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
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
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
• 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.
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
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
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 xray 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
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.
• Operates at higher voltage than ionization chamber
• Must contain a gas with specific properties
• Commonly used in standards laboratories,
physics laboratories, and for physics research
health
• Seldom used in medical centers
Types of Proportional Counters
1. Gas flow proportional:
- with window (e.g., laboratory alpha-beta counters)
- windowless (e.g., tritium measurements)
2. Air Proportional (alpha counting only)
3.
Sealed proportional (e.g., BF3, He-3 neutron detectors)
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
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
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
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)
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
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)
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
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
develop
film
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.
METHODS IN TRACER TECHNIQUE
1.
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.
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
2. DOUBLE & MULTIPLE LABELLING:
This method give the evidence for nature of biochemical
incorporation of precursor arises, double & triple labeling. In
this method specifically labelled precursor and their
subsequent degradation of recover product are more
employed.
Applications:
•
This method is extensively applied to study the biogenesis of
plant secondary metabolite.
•
Used for study of morphine alkaloid. e.g., Leete, use Doubly
labeled lysine used to determine which hydrogen of lysine
molecule was involved in formation of piperidine ring of
anabasine in Nicotina glauca.
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 & B’ is normal intermediate in the formation of C
from A.
Applications:
•
This method is used for elucidation of biogenesis of propane
alkaloids.
•
Biosynthesis of hemlock alkaloids (conline, conhydrine etc)
e.g.
biosynthesis
of
alkaloids
of
Conium
(hemlock) using 14C labelled compounds..
maculactum
4. ISOTOPE INCORPORATION:
This method provides information about the position of bond
cleavage & their formation during reaction.
e.g. Glucose-1-phosphatase cleavage as catalyzed by alkaline
phosphatase this reaction occur with cleavage of either C – O
bond or P – O bond.
5. SEQUENTIAL ANALYSIS:
The principle of this method of investigation is to grow plant
in atmosphere of
14CO
2
& then analyze the plant at given time
interval to obtain the sequence in which various correlated
compound become labelled.
Application:
•
14CO
2
& sequential analysis has been very successfully used
in elucidation of carbon in photosynthesis
•
Determination of sequential formation of opium hemlock and
tobacco alkaloids
•
Exposure as less as 5 min 14CO2, is used in detecting
biosynthetic sequence as –
Piperitone
---------
Mentha piperita.
(-)
Menthone
----------
(-)
Menthol
in
APPLICATIONS OF TRACER TECHNIQUE
1.
Study of squalene cyclization by use of
14C,
3H
labelled
mevalonic acid.
2.
Interrelationship among 4 – methyl sterols & 4, 4-dimethyl
sterols, by use of 14C acetate.
3.
Terpenoid biosynthesis by chloroplast isolated in organic
solvent, by use of 2-14C mevalonate.
4.
Study the formation of cinnamic acid in pathway of coumarin
from labelled coumarin.
5.
Origin of carbon & nitrogen atoms of purine ring system by
use of 14C or 15N labelled precursor
6.
Study of
formation
of
scopoletin
by use
of
labelled
phenylalanine
7.
By use of
45Ca
as tracer, found that the uptake of calcium by
plants from the soil (CaO & CaCO2)
8.
By adding ammonium phosphate labelled with
32P
of known
specific activity the uptake of phosphorus is followed by
measuring the radioactivity as label reaches first in lower part
of plant, than the upper part i.e. branches, leaves etc.
Thanks