Vikram Shrestha

Assistant professor of Biochemistry

CONTENTS

 Introduction
 Radioactive and stable isotopes
 Types of radioactive decay
 Activity and Half-life
 Units of Radioactivity
 Measurement and detection of radioactivity
 Applications
 Radiation hazards
 Radiation safety and protection
Radioactive and stable Isotopes
Radioactivity
 The spontaneous degradation of
nucleus and transmutation of one
element to another with consequent
emission of rays or particles is known
as radioactivity.

 It was first discovered by Henri

Becquerel in 1896.

 Nobel prize was awarded to him jointly

with Marie Curie and Pierre Curie in
1902.
 The unstable isotopes undergo radioactive decay e.g
tritium

 Elements capable of under going radioactive decay are

called radionuclide

 Radioactivity is unaffected by Temperature, pressure,

concentration or by any physical or chemical conditions
Types of Radioactive decay

 Alpha decay
 Beta decay
 Gamma decay
Alpha decay

 Elements having atomic number greater than 70 undergo this type of decay
 This type of decay produces 2 proton and 2 neutrons identifiable as 2 He
 Atomic number decreases by 2 and atomic mass decreases by 4
 (2p + 2n) in α- particle are more ionising and so are harmful to tissue
 They have less penetrating power.
Beta decay

 Beta decay (β decay) is a type of radioactive decay in which a beta particle

(an electron or a positron) is emitted from an atomic nucleus.
 There are two types: beta minus and beta plus. In the case of beta decay
that produces an electron emission, it is referred to as beta minus.
 In the case of beta decay that produces an positron emission, it is referred
to as beta plus.
Gamma radiation

 It refers to electromagnetic radiation of extremely high frequency and

therefore high energy per photon
 They are classically produced by the decay from high energy states of
atomic nuclei.
 Emission of ϒ –rays in itself leads to change in atomic number or mass.
 Gamma rays are low ionizing but highly penetrating.
Activity and Half-life

 The rate of decay of a radioactive source is called its

activity and is simply the rate at which radioactive parent
atoms decay to daughter atoms.
 Half life is defined as the time taken for a radioactive
isotope to become half of its original activity. It is given by
the formula : t⅟2 = 0.693
λ
 Radioactive elements continuously lose it radioactivity.
 Rate or the half life is a constant for a particular isotope.
Some radioactive isotope with Half-life
Units of radioactivity
 Becquerel (Bq) is the SI unit of radioactivity and is defined as one
decay per second (dps)

 Curie is the older conventional unit and is defined as 3.7 × 1010

decays per second. (1 Ci = 3.7 × 1010 Bq).

 Roentgen (r) is the amount of ϒ or X-ray which deposits 8.33 × 10-6 J

of energy per gram of air to produce 1.61 × 1012 ions pairs /gm.

 Radiation absorbed dose (rad) is the quantity of ϒ or X –ray that can

deposit 100 ergs or 1 × 10-5 J/gm in matter including biological
tissue.
Relative biological equivalence (RBE) is the ratio between the doses
(rad) of radiation under investigaton and of ϒ rays producing
identical biological effect. RBE is 1 for ϒ rays.

Roentgen equivalent mammals (REM) measures a radiation in terms

of its effects on mammalian tissues. REM = rad × RBE
Measurement of radioactivity

 Autoradiography
 Gas filled detector
 Scintillation counting
-Crystal scintillation detector
- Liquid scintillation detector
Autoradiography

 In this process a photographic emulsion is used to visualize molecule

labelled with radioactive element.
 This method is used to visualise nucleic acids and fragments that
have been hybridised with nucleic acid probes labelled with P32
 After hybridization hydrolysis and separation of fragments by gel
electrophoresis.
 A photographic film is applied to cover gel and is allowed to
incubate.
 The emulsion or film contains silver halide crystals.
 As energy from the radioactive material is dissipated
the silver halide becomes negatively charged and is
reduced to metallic silver, thus forming a particulate
latent image.
 Photographic developers show these silver grains as a
blackening of the film, then fixers are used to remove
any remaining silver halide and a permanent image
results
Suitable isotopes
 In general, weak beta -emitting isotopes (e.g. 3H, 14C
and 35S) are most suitable for autoradiography,
particularly for cell and tissue localisation
experiments.

 This is because the energy of the radiation is low

 Radiation with higher energy (e.g. 32P) give faster

results but poorer resolution because the higher
energy negatrons produce much longer track lengths,
exposing a greater surface area of the film, and result
in less discrete images
Choice of emulsion and film

 Autoradiography emulsions are solutions of silver

halide that can be made to set solid by the inclusion
of materials such as gelatin- used for microscope
slides

 X-ray film is the alternative and is used for gels

Direct autoradiography

 In direct autoradiography, the X-ray film or emulsion is

placed as close as possible to the sample and exposed
at any convenient temperature.
 Quantitative images are produced until saturation is
reached.
Fluorography
 If low-energy b-emitters are used it is possible to
enhance the sensitivity several orders of magnitude by
using fluorography
 A fluor (e.g. sodium silicate) can be used to enhance
the image.
 The b-particles emitted from the isotope will cause the
fluor to become excited and emit light, which will
react with the film.
 This has been used for example for detecting
radioactive nucleic acids in gels.
 The fluor is infiltrated into the gel following
electrophoresis; the gel is dried and then placed in
contact with a preflashed film
Intensifying screens

 Intensifying screens are used when obtaining a fast

result is more important than high resolution.

 It is useful for example in gel electrophoresis or

analysis of membrane filters where high-energy b-
emitters (e.g.32P-labelled DNA) or ϒ-emitting isotopes
(e.g. 125I-labelled protein) are used.
 The intensifying screen consists of a solid phosphor,
and it is placed on the other side of the film from the
sample.

 High-energy radiation passes through the film, causes

the phosphor to fluoresce and emit light, which in turn
superimposes its image on the film
Low-temperature exposure

 When intensifying screens or fluorography are used

the exposure should be done at low temperature.

 The light is of low intensity and a back reaction occurs

that cancels the latent image.

 Exposure at low temperature(-70C)slows this back

reaction and will therefore provide higher sensitivity.
Preflashing
 The response of a photographic emulsion to radiation
is not linear and usually involves a slow initial phase
(lag) followed by a linear phase.

 Sensitivity of films may be increased by preflashing.

 This involves a millisecond light flash prior to the

sample being brought into juxtaposition with the film
and is often used where high sensitivity is required or
if results are to be quantified
Gas filled Detector

 It is filled with gases designed to capture and measure

ions produced by radiation.
 Ionisation chamber, proportional counter and Gieger
counter are different gas filled detectors.
 The Geiger counter is an instrument used for detecting
and measuring ionizing radiation used widely in
applications such as radiological protection,
experimental physics and the nuclear industry.
 It detects ionizing radiation such as alpha particles,
beta particles and gamma rays using the ionization
effect produced in a Geiger–Müller tube; which gives
its name to the instrument
 A Geiger counter consists of a Geiger-Müller tube, the
sensing element which detects the radiation, and the
processing electronics, which displays the result.
Scintillation counting

 The process of scintillation is one in which flash of light of a characteristic

spectrum is emitted following the absorption of radiation.
 The emitted radiation is usually less energetic than that absorbed.
 In clinical laboratory sodium iodide crystal scintillation detector and organic
liquid scintillation detector are commonly used.
 Radioactive isotopes interact with matter in two ways,
ionization and excitation.

 The latter effect leads an excited atom or compound

(known as a fluor) to emit photons of light. The
process is known as scintillation.

 Essentially, a photomultiplier converts the energy of

radiation into an electrical signal, and the strength of
the electric pulse that results is directly proportional to
the energy of the original radioactive event.
Crystal scintillation

 It is also called solid scintillation counting

 The sample is placed adjacent to a crystal of fluorescent
material .
 For ϒ- isotopes –sodium iodide, for α- emitter zinc
sulphate, and for β-emitter –organic scintillation eg
anthracene
 Crystals are placed in photomultiplier that is attached to
high voltage.
 This method is useful for ϒ- emitting isotopes since their
atoms rarely collide and cause ionisation.
Liquid scintillation

 In this process sample is mixed with a scintillation cocktail

containing a primary solvent and one or more fluors.
 Primary solvent is usually aromatic solvents like xylene,
toluene or pseudocumene
 Primary scintillator absorbs energy from primary solvent
and convert it into light.
 2,5 –diphenyloxazole(PPO) in a concentration 3 to 6 g/L is
commonly used material. It acts as primary fluor.
 Sometime the light emitted by PPO is not detected by
photomultiplier. In such case 1,4-bis(5-phenyloxazol-2-yl)
benzene (POPOP), a secondary flour is used
Applications
A. Dilution Studies C. Therapeutic Uses
B. Diagnostic Tests D. Research Uses
Dilution Studies Diagnostic Uses Therapeutic uses Research Uses
1. Volume and 1. Thyroid Teletherapy Carbon Dating
space Function Test
2.Estimation of GI 2. Study of Beads, Needles Autoradiography
protein loss in Regional Blood Application
Protein-losing Flow
Enteropathy
3. To measure GI 3. Radioactive Iron Extra corporeal Food and
Blood loss Studies irradiation of Packaging
Blood Industries
4. To study life 4. Vitamin B12 and Regional
span of RBC Fat Absorption Application
Studies
5.RIA IV Application
6. Scanning of Systemic
organs Applications
A. Dilution Studies
1. Volume and space:-This method is used for estimation of
plasma volume, RBC Volume, Total water Volume, ECF
volume.
Plasma volume I 131
labelled human serum
albumin via IV
RB cells volume Cr 51 labelled RB cells Via IV
Total body water H 3 titrated water given orally
ECF Volume Br 82 orally or IV
Total exchangeable Na24 orally or IV
volume

 It depend on principle that when a known amount of radioactive

tracer is introduced into unknown volume and if after mixing,
the concentration of radioactive tracer is estimated then total
volume of tracer in which tracer is diluted is calculated by V=
N/n.
2. Estimation of GI protein loss in Protein –Losing
Enteropathy:-
 I 131 labelled human serum albumin is given via IV. Tracer
will remain in circulating compartment and will not appear in
stool in significant quantity unless there is GI protein loss.
 In normal person > 0.1% of injected radioactivity appear in
24-hr stool.< 2% radioactivity in stool indicates excessive
protein loss

3. To measure GI Blood loss:- Cr 51 labelled RBC given IV will

appear in stool in loss of Blood through GIT. Faecal
radioactivty over 72hrs is compared with blood radioactivty
 By this method minimum 5ml blood loss can be estimated
4. Study of RB cells life span:- Cr 51 labelled RBC given
IV and RBC volume is estimated.
 Blood samples are collected daily or on alternate days
which cause reduction of radioactivity
 In healthy subjects 50% radioactivty will be still
present in circulating blood at the end of month.
 In haemolytic anaemia, t1/2 (half life ) of
chromium will be reduced to below 24 days(mild
cases).
 T1/2 will be reduced to 8 to 10 days in severe
anaemia.
B.Diagnostic Uses
1.Thyroid radioactive uptake studies :-I 131Dose of 10μci orally
given and its accumulation is measured externally over the gland
 Radio-iodine uptake of gland indicates the iodine “trapping” ability
 Thyroid uptake of 24 hr I 131 is routinely carried.
 4 hr and 48 hr uptake are done in case of rapid turnover or delayed
uptake .
 Normal range 20- 40%
 High RAI uptake is usually consistent with hyperthyroid state.
 Low RAI uptake is a characteristic feature of hypothyrodism

 Renal excretion of I 131 is an indirect evidence of thyroid function.

Proportion of administrated dose is inversely proportional to thyroid
uptake.
 24hr urine is collected and radioactivity is measured
 Normal range is 30-60% of the administered dose
2.Radioactive Iron Studies:-
Fe59 is used for determination of :-
 Rate of absorption from GIT
 The rate of disappearance of Fe59 from plasma
 The rate of turnover of plasma Fe indicates the activity of
erythropoietic marrow
 The incorporation of Fe59 into RBC indicate the extent of
effective erythropoiesis
 In normal subject, t1/2 of Fe is 60-120 min
 Most of Fe accumulates in erythropoietic bone marrow
 In aplastic anaemia , t1/2 of Fe is greater than 120 mins.
 Liver and spleen count rapidly increases showing that Fe is going
to RE cells rather than being used for erythropoiesis.
3. Vitamin B12 Absorption Studies:-
Oral dose of 0.5 to 1.0μg of labelled B12 is used
Vit B12 can be assessed by
 Measurement of faecal radioactivity
 Measurement of accumulation of B12 in liver
 Measurement of 8 hr plasma activity
 Schilling test (commonly used)
Method:-
 1.o μg of labelled B12 is given orally.
 For labelling B12 Co60 , Co58 , Co57 are used.
 1000 μg non-radioactive stable B12 is given IM
 Urine is collected over 24 hr period.
 Radioactivity of urine is then and of standard is calculated by
 %dose excreted in urine = total count/min in 24hr urine sample ₓ100
counts /min in standard(test dose)
 Normal subjects :- urinary excretion is greater than 15% of test dose.
 In pernicious anaemia, or vit B12 deficiency subjects, excretion is less than
5%.
4. Fat Absorption Studies:-
 Use of I131 ,labelled triolein and oleic acids.
 Measurement of blood radioactivity 3-4hrs after oral ingestion

5. Renal clearance Studies :-

 Cr 51 labelled EDTA has been used for estimation of renal clearance and GFR

6. Study of pulmonary function :-

 Radioactive CO2(cyclotron produced ) , Xe133 is used for such studies.
 It helps to identify regional abnormalities in lung before total lung function
is impaired.

7. Scanning of organs :-
 Sacnning of Organs
ORGAN SCANNING RADIOACTIVE ADVANTAGES
ISOTOPES
1. Thyroid Scintiscan I131 or 99m •Distinguish diffuse and
glandular pattern seen in
orally given Nodular Goitres.
•Hot or Cold Nodules can be
separated easily
•Shape, size and position of
gland can be detected
2. Renal Scintiscan Hg 203 labelled •Renal shape, size and
chlormerodrin position can be determined .
Via IV •Detect Segmental lesions
•Renal infarcts can be better
identified.

3. Brain Scanning Tc 99m or In 133m Safe and informative

procedure in neurological
investigation.
4. Lung Scanning Human serum albumin Identification of pulmonary
labelled with I131 via IV infarction in early stage when
X-ray is normal
 8. Radioimmuno Assay

 Developed for the first time by Berson and Yalow –Nobel prize in 1977.
 A technique to measure concentration of antigens.
 Hormones, growth factors tumour markers, bacterial antigens etc are
quantified.
 The radioisotope used for labelling antigen is I125
C. Therapeutic uses of Isotopes
 Dose of ionising radiation is concentrated in tumour tissue without
harming adjacent healthy tissue

 The tumour tissue can be attacked by radiation in two ways

- external sources
-internal sources

 Radioactive material may be applied locally over the surface or may be

implanted into tumour tissue or may be injected.

 Intra-arterially as microspheres

 Intralymphatically

 Most commonly it is given orally or IV injection.eg P32 is concentrated

more in malignant cells than in normal cell
1. External sources
1.Teletherapy:-
 Co60 offer powerful radiation for treatment of malignancies.
 Compared to radium it is cheap and has ϒ-rays that can penetrate deep
into tissue enabling treatment of deep cancer.
 it doesn't cause skin reactions.
2. Beads Needles and Aspiration:-
 Co60 can be encapsulated in gold or silver needles, wires, rods or
cylinders and thus can be used in treatment of cancer of cervix.
 In dermatology P32 is applied to paper or polythene sheets for
treatment of squamous cell carcinomas, superficial angiomas, Mycosis
and keratosis
 In ophthalmology Sr90 is used for lesions of cornea, conjunctiva and
sclera.
3. Heavy Particles:-
 Heavy paricles have greater capacity to produce ionisation in tissue.
 Heavy particle Proton irradiation of pituitary gland is used in Diabetic
Retinopathy.
4. Extra-corporeal Irradiation of Blood:-
 This method is useful in treatment of patients with leukaemia.
 In this case, blood is taken out from the patient via forearm artery and is
circulated around a Cs137
 Cs137 emits powerful ϒ-rays and the blood is returned to the same patient via
forearm vein.

5. Boron 10 neutron Irradiation :-

 B10 has been recently used in treatment of inoperable fatal brain tumour like
glioblastoma multiforme.
 When injected IV into the patient B10 will be rapidly taken up by tumour tissue in
brain.
 After 10 mins the head of patient is placed in a beam of slow neutrons. B10 in
tumour tissue absorbs neutron and transformed to B11 which disintegrates
immediately into α-particle and lithium isotope.
 α-particle are highly ionising hence it destroys the tumour cells without affecting
harm to adjacent brain cells
2. Internal Sources
1. Regional Application:-
 Gold 48 a non toxic and chemically inert in the body is very useful
radioisotope as it requires no coating.
 It is introduced into pleural or peritoneal cavities forms colloidal suspensions
and remain within the cavities without getting absorbed .
 Its Beta and gamma radiations are absorbed by the tissue
 Hence it is used in treatment of malignant pleural and peritoneal effussions.

 Yttrium 90 synovectomy- Yttrium 90 is used in arthritis and haemophiliac

patients.
 The affected joint is injected with Yttrium 90 radioisotope under X-ray
control and joint is immobilised for 36hrs.
 Yttrium isotope emits β-particle. It has low penetration and rapid decay that
makes suitable isotope for joint space to dissolve and destroy inflammed
synovium
2. IV Application
 Tiny ceramic microspheres of radioactive gold or yttrium are used for
treatment of tumours of lung, prostate, and bone.
 These particles are stopped at arterial end of capillary because of their
size.
3. Systemic Uses :-
 P32 is used in treatment of
-chronic myeloid leukaemia
- polycythemia vera
-multiple myeloma
- Primary haemmorrhagic thrombocytosis
- Carcinoma of breast and prostate

 I 131 is used in thyroid cancer : uptake of I 131 by thyroid cancer tissue is less.

 Thyrotoxicosis : I 131 has been also been used for the treatment of primary
thyrotoxicosis.
 I 131 is also used for controlling ectopic arrhythmias :- When usual therapy
fails, sometimes I 131 is useful.
D.Research Uses

 Carbon Dating
Nobel prize in 1960 –Willard Libby
Radioisotopes like C12 and C14 is used in Paleobiology
 Autoradiography
Radio labelling of nucleic acid using P32
 Food and packaging industries
Gamma radiation using radioisotopes Co60 and Cs 137
Radiation Hazards

 Exposure to radioactivity is hazardous to living organisms.

 In general the effects of radiation can be divided into three

categories
# Immediate Effect
# Delayed Effect
# Genetic Effect
 1 Immediate effect
Bone marrow GIT syndrome CNS syndrome
syndrome
1. Radiation 200-1000 rads to 1000- 5000 rads 5000- 10,000 rads
exposure (in rads)
2. Immediate effect •Severe damage in •leads severe Blood brain barrier is
hematopoietic damage to mucosal lost leading to
system epithelium, fluid cerebral vasculitis,
•Depletion of loss, electrolyte meningitis, and
leukocytes and imbalance and choroid plexitis
myelocytes leads to haemorrhage in
gross GIT
immunosuppressio •nausea vomiting
n and increased and acute
susceptibility to diarrhoea
infection.

3.Death in 10-20 days 3-5 days. 8 – 48 hrs

days/hrs
2. Delayed effects of radiation
a) Carcinogenesis:-
 Different tissue have different susceptibility to cancer.
 Bone marrow and other rapidly dividing cells are prone to cancer
 Persons exposed to <10 rads at Hiroshima and Nagasaki in 1945 are still
showing higher rate of leukaemia mortality rate

b) In utero radiation exposure

 embryonic stage is most susceptible in life of organism to radioactivity
 In utero exposure leads to :
Growth retardation , congenital malformation and foetal or neonate
death.

c) Shortening of life span:

 Moderate dose of X-rays and ϒ-rays is found to decrease life span
(experimentally done in mice and rats)
3. Genetic effects of radiation

 According to the target theory of Lea, each interaction of radiation with a

target molecule can lead to molecular damage of target molecule.

 If target molecule is a DNA, it will undergo a damage mutagenesis.

 Also mutation depends upon efficiency of DNA repair mechanism.

 The magnitude of damage depends on

# The stage of germ cell development
#Dose rate
#Interval between exposure and conception
Radiation Safety and Protection

 Recommended level of maximum radiation exposure for

occupational worker is 5.0 rem/year and for public is 0.5
rem/year.
 The most popular triad of radiation protection is time, distance
and shield. (TDS)
 Minimum possible time should be spent near radiation zone.
 Handling of radioactive material should be done from maximum
possible distance.
 Person should be shielded by lead.
References

 Teitz,; A Text book of Clinical Chemistry;5th edition; page

no217-220
 Wilson and Walker; Practical biochemistry; 5th edition; page
688-702
 DM Vasudevan; Shreekumari S; Text book of Biochemistry;
6th edition; Page 592-597
 MN Chatterjea; Rana Shinde; Textbook of Medical
Biochemistry; 7th edition; Page 686-690
 U. Satyanarayana; U Chakrapani; Biochemistry; third edition;
page 717-718
 RN Roy; Textbook of biophysics;2nd edition; page 173-178