Radioisotope Techniques

Mak Oi Tong Department of Life Sciences National Cheng Kung University

Radioisotope
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Atoms with unstable nuclei transform into other stable atoms. Results : Release of energetic particles or radiant energy,

Atoms, isotopes and radioisotopes

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Atomic number, mass (mass number or atomic weight) Carbon : 12C 6

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atomic number = 6 and atomic mass = 12 Contains 6 protons, 6 neutrons and 6 electrons.

Isotopes : atoms have identical chemical properties but different atomic weight.
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e.g. 11C, 12C, 13C and 14C 14C > ] + 14N + oe- (antineutrino) 6 7 11C > ] + + 11B + oe (neutrino) 6 5 Lighter atoms 1:1 and 1.5:1 at high atomic number

Ratio of neutrons and protons

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Types of radioactive decay

Negative-Beta (electron ] ) emission

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Ejected electrons emit from the nucleus which are identical with the orbital electron in mass and charge (neutron to proton).
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> 3He2 + ] + oe- (antineutrino) 14C > 14N + ] + oe- (antineutrino) 6 7 32P > 32S16 + ] + oe- (antineutrino) 15 35S > 35Cl + ] + oe- (antineutrino) 16 17

Positive-Beta (positron, ] +) emission

+ n ]

is same mass as electron but having opposite charge, and it will emit as gamma (^) radiation.
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+ ] +

e- > ^ (1.02 MeV) 11C > 11B + ] + + oe (neutrino) 6 5 13N > 13C + ] + + oe (neutrino) 7 6

Decay by electron capture

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Conversion of a nuclear proton into a neutron by capture one of the two electrons in the innermost K-shell, ie. K-capture (KC) and emission of a positron ] +.

Decay by ^-radiation
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emission occurs after ] or ] + emission.

Decay by \ - particle emission

Heavy atoms with more than 82 protons will emit an \ -particle which is a helium nucleus of atomic mass number of 4 (2 protons and 2 neutrons) with a charge of +2. 226Ra , 235U etc. are examples for \ 88 92 particle emitters and are not for biochemical studies but for radiological toxicity.

Occurrence and artificial production of radioactive isotopes

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Pierre and Marie Curie introduced the term radioactivity to designate the process of spontaneous emission of radioisotope. Artificial production of radioisotopes is normally done by neutron bombardment.

Other methods: \ -particle, proton and deuterons

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To-day, over 1500 isotopes are produced artificial produced. On earth, radioisotopes are produced by neutral bombardment, and 14C and 3H (tritium) are in equilibrium. Radioisotopes can be used for radiocarbon (14C) dating.

Properties of radioactive emissions

Energy of ] -particle emissions

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] + and e- travel near the speed of light, 3 x 1010 cm/s or 3 x 108 m/s. Emax is the maximum energy value, and the Eave Average) is equal to 1/3 of Emax.

Interaction of ] -particular with their environment

n ] will

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cause electron excitation and emit UV or visible light. In air, about 34 ev is required to produce one ion pair. An ejected electron, with an energy of 1.5 MeV, will travel about 2 meters in air, and about 1 cm in tissue. Eventually the ejected electron will be captured by an empty electron orbit of a positive ion. When a positron encounters an its electron, the two particles annihilate (combine) each other and their masses are converted into electromagnetic ^radiation which is I the form of two 0.51-Mev

Gamma radiation and its interaction with matter

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Gamma rays are high energy photons or light particles that are electrically neutral with zero mass and consequently can penetrate matter readily. ^ -Ray is emitted from the nucleus with discrete energy range between 10 KeV to 3 MeV. Many isotopes emit of ^- ray of several energies in discrete steps (Fig. 6.2). Photoelectric effect is a ^- ray of relatively low energy (<0.1 MeV) interacts with an orbital electron, transferring all its energy to the electron and the electron is displaced from the atom.
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This electron interacts with other atoms leading to further ionization.

The Compton effect results when ^- rays of medium energy (>0.1 MeV) undergo elastic collisions with loosely bound orbital electron and a portion of the ^- ray energy is transferred to the ejected electron. The photon is deflected in a new direction with reduced energy and undergo several more similar collisions before losing all its energy. Compton electrons have a wide range of energies and these electrons will produce secondary ionization as they travel through the medium, dissipating their energy.

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Pair production results when a relatively energetic (>1.02 MeV) ^- ray interacts with a nuclear force field. The photon has all its energy converted into a positron and an electron. This is essentially the conversion of energy into mass. Both the ejected positron and electron may cause ionization of atoms in their paths, and collies together.

Kinetics of radioactive decay

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The process of decay of a radioactive nucleus into a stable, nonradioactive nucleus is irreversible. Rate of decay (particle emission) is equal to the rate of disintegraton of the nuclei, which is proportional to the number of radioactive nuclei present. Rate of decay decreases exponentially with time. It is independent of pressure, temperature and other chemical and physical changes. The rate is first order reaction.

Rate of decay:

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N = amount of radioactive nuclei present f= decay constant

Activity is the quantitative measurement of radioactive and if the activity of sample is A (by detector), it becomes the counting rate of the sample. Efficient of detector

Half-life (t )

Units used in radioactive measurements

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The electron volt

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One electron volt (1 eV) is the kinetic energy developed by an electron when it is accelerated through a potential difference of 1 volt in a vacuum. 1 eV is equal to 3.85 x 10-20 calories or 1.602 x 10-19 joule. It is normally expressed in KeV or MeV. The electron emitted from 14C has an average energy of 4500 eV per nucleus (atom).

The curie: the unit of radioactive disintegration

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One curie is equal to the number of disintegration per second in 1 gram of pure radium 226.

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It is also equal to 2.22 x 1012 disintegration per minute (dpm). One becquerel (one disintegration per second) is equal to 3.70 x 1010 becquerels.

Safety in handling radioisotopes

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First to know whether it is ] - or ] + -ray isotopes. Prevent injection, inhalation or absorption of radioisotopes in human body. Laboratory coat, plastic gloves etc. Should be worn for body protection. Restricted areas for handling radioisotopes must be labeled, clean and neat. Covered with plastic absorbent paper with a tray line. Have special containers for any radioisotope disposal such as glasses, plastic, paper, water and organic solvent.

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Monitor the working place routinely for any contamination of radioactivity by using GeigerMuler survey monitor for strong ] and ^ ray. Wear a personal dosimeter for high energy radioisotopes. Wash immediately if any contamination is present. If skin has contacted with radioisotope, wash with immediately for at least 2 minutes. Special care is required for anyone handling 125I or 131I because of the effects on thyroid gland in human body.

Statistics of counting radioisotopes

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Compare Fig. 6.1 and Fig. 6.5. Standard deviation s:

A more common situation is:

Confidence of the true value.

Percent relative standard deviation (% RSD)

For sample comparison, % RSD is generally required.

Measurement of radioactivity
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By gas ionization. By scintillation counting

By gas ionization
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When a energetically charged particle, such as ] - particle, passes through a gas, its electrostatic field displaces orbital electron fro the gas molecules close to its path. This displacement produce a positive ion from the other part of the molecule, resulting an electron pair.

By gas ionization If an electric field is applied in the chamber, the displaced electrons are accelerated toward the anode and the positive ions move toward cathode slowly (because of their larger mass). n If higher electric fields are applied, the electron displaced by the first ] - particle collision is accelerated and collides with other gas molecules to produce additional ion pairs. n This result is an amplification of the number of electrons from the first electron, giving a shower of electrons.
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monitor
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A type of radioactive detector. It is a gas-filled tube with cylindrical cathode and a fine wire anode. A common gas mixture is the so called Q-gas which is 1.3 % isobutane in helium. One end of the tube has a window through which the ] - particles from the radioactive sample.

monitor
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A voltage potential is applied between the electrodes of the tube and produces shower The electrons are collected at the anode within a microsecond, and a strong electrical pulse is formed. The number of electrons collected is proportional to the number of initial ionization, and is proportional to the number of ] - particles entering the chamber. The region is called proportional region. Efficient, for 14C about 1 to 5 % and for 3H is nearly zero.

By scintillation counting
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Use scintillator or fluors for ] - and ^- ray counting. Scintillator can absorb radiant energy to form excited atoms or molecules and return rapidly to ground state, releasing excitation energy, to form photon and heat. Amount of photon released directly related to the amount of radiant energy. Liquid scintillation counting is that the scintillators are dissolved in a solvent called cocktail.

By scintillation counting
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Steps for photon formation:

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Radiant energy is absorbed by solvent and become excited. The solvent transfers the the excited energy to the scintillator to cause excitation. The scintillators release photon which is detected by the phototubes. The phototube converts photons into amplified numbers of electron, and then becomes voltage pulse by a pulse height analyzer, and this is called ] - and ^- scintillation counter.

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] -

energy spectrum refers to Fig. 6.12. Dual-labeled mixture activity determination refers to Fig. 6.13.

Cocktail composition and sample preparation

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Primary fluor is 2,5-diphenyloxazole (PPO). Secondary fluor is 1,4-bis-2-(5-phenyloxazolyl) benzene (POPOP). Common solvents: toluene and dioxane
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Toluene is used for organic compounds. Dioxane is used for aqueous samples.

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Heterogeneous counting is that the radioactive samples are in solid support. Homogeneous counting is that the radioactive samples are in dissolved form. Background counting is caused by electronic noise, cosmic radiation and other natural radioisotopes present. Cerenkov counting is used for direct ] - particle counting without fluor, only for high energy ] - emittor, e.g. 32P.

Quenching
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It is the process that causes a reduction in the amount of fluorescence produced by ] - emission in a liquid scintillation cocktail, reduction amount of light reaching the photomultipler tube. Chemical quenching is that the transfer of energy is interfered from solvent to fluor molecules (fluorescence reduction).
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By acid By dipole-dipole interaction of quenching agent Electron capture by quenching agent.

Color quenching is the presence of colored compounds which can absorb the photons emitted by the fluor, e.g. Pigment, blood.

Quench correction
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Internal standard method: n Add another non quenching radioactive standard and recounted. External standard method: n Use of a highly radioactive ^- radiation source, e.g. 226Ra or 137Cs, which is brought near to the test sample vial and the sample is recounted in a short time. Efficient by ES refers to 6-19.

Solid scintillation counting of radiation

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A crystalline inorganic fluor, sodium iodide (NaI), with small amount of thallium (Tl) as activator. ^ Rays interact with crystalline fluor to form ion pair, the Compton effect and induce photoelectric and excite crystalline fluor, finally produce photon to reach photomultiplier tube. The crystalline fluor is sealed in a metal cylinder from external light. The cylinder must be hermetically sealed because of hygroscopic of NaI. It is designed for the counting of 125I, 51Cr and 60Co.

Autoradiography
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A technique for the determination the location of radioisotopes in tissue, tissue sections, chromatograms and gels. It is a non-destructive method for radioactive compounds, and can be isolated and recounted. Results are shown on a X-ray film and developed. The ] - ray hits the silver bromide of the film and reducing to metallic silver from silver ions, and black spots will be appeared. For best darkening of the film, at least 1 x 106 (] - particle) passing through the film per cm2 per minute.

Autoradiography
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If sample contains 1 x 103 cpm/cm2, it will take about 1000 minutes or 17 hours to induce the black spot. For weak ray, enhancer ( fluorography, PPO) is need to enhance the sensitivity, particularly 3H. The photoelectric effect are more stable under -50 to -80C, about 4x more stable. The resolution can be increased by decreasing the thickness of the sample and the distance between sample and the film

Methods of labeling biochemical compounds

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Two general methods: n biosynthesis and n chemical synthesis.

Biosynthesis
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Used by either whole organisms (cells) or specific enzymes to synthesize radioactive compounds. The common used radioisotopes are 14C, 32P and 35S. Factors for the successful use of biosynthesis:
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By using whole organism, labeled compound(s) must be accumulated in large number from starting compound. For using a specific enzyme, the enzyme must be easily isolated and the substrate is readily available, and the labeled compound must be isolated and purified from the reaction mixtures. For using microorganism, labeled compound is isolated from the microorganism or the cell-free culture supernatant fluid.

Whole organism biosynthesis gives a relatively large number of labeled products but enzyme synthesis gives only one or specific labeled products.

Chemical synthesis
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Products can be labeled in one or few positions. Starting materials always be scarce and expensive. Results give low yield. Give a racemic mixture of optical isomers, e.g. D- and Lisomers of amino acids. Types of labeling of compounds:
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Labeling of proteins. Labeling of nucleic acids. Formation of 14C carbon-carbon bonds. Synthesis of tritium labeling [3H] compounds from NaB3H4. Synthesis of 35S-labeled L-cysteine

Types of radiochemical labeling

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Isotopically: one or more radioactive atoms have replaced stable atoms of the same element, e.g.
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3H 1

<=> 1H1, 14C6 <=> 12C6, 35S15 <=> 31P15 They are the same molecules.

Nonisotopically: a radioactive isotopes has been substituted for a stable atom of another element, e.g.
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125I

<=> for 1H1

Three forms of labeling positions:

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Specific: [1-14C]-D-glucose random: randomly distributed (G-). n *CH3-CH2COOH, or CH3-*CH2COOH or CH3-CH2*COOH Uniform: all related atoms are labeled, [U-], e.g. [U-14C]sucrose.

Radioimmunoassay
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Ab (antibody) + Ag (antigen) <=> Ab-Ag complex Present an abstract about the basic principle and application of radioimmunoassay.

Designing a radioisotope experiment

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First and foremost in the design of any experiment is the formulation of the question to be studied, the purpose and nature of the experiment. Next is what experimental techniques to answering the question. Is radioisotope necessary ? What type of radioisotope ? Which position and and which type of labeled compound. Availability of the labeled compound. How much specific activity and amount of radioactivity required.

Designing a radioisotope experiment

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What is the cost of the labeled compound ? Experimental methods. What type of detection system and its efficiency ? The dilution effects and the yield of the radioisotope. Control and correction factors needed. Expression and interpretation of the results, and finally The possible isotope effects and possible effects of radiation damage.