Chapter 10Isotope Techniques

10.1 Radioisotopes

Radioactivity is a phenomenon that can be detected with high sensitivity whichcan be matched only by chemiluminescence and mass spectrometry. This makesradio-labelling a favourite technique in biochemistry. For this reason the requiredequipment is almost universally available.

However, there are also some disadvantages to this method which relate to thepossible dangers inherent in the use of radioactive material. Although the amountsof radioactivity handled in biochemical experiments is very small (in the order of102–103 Bq per sample, 107 Bq for a large synthesis), legal complexities are con-siderable. Since radioactive isotopes can not be destroyed, a considerable part of thecosts for each experiment is for disposal. A conscious effort has to be made to keepthe radiation dose to the experimenter and to the environment as low as reason-ably achievable (ALARA-principle), even within the legally permissible values.Each institution has its own safety rules for handling radioisotopes based on local,national, and international regulations. A radiation safety officer will train you onthose procedures before you are allowed to work with radioactive materials. Thefollowing rules form the basics of safe working practices:

� Lab coats and disposable gloves should be worn whenever handling radioactivematerials.

� Place appropriate shielding between yourself and the radioactive material. Forlow (3H) and medium (14C) energy “-radiation air provides sufficient shield-ing, for high energy “-radiation (32P) 10 mm Perspex is effective to shield the“-radiation, however, some Bremsstrahlung (X-rays) is produced. ”- and X-radiation can be shielded with lead bricks, low energy radiation also with “yellowperspex” which contains chemically bound lead.

� No mouth pipetting, use mechanical pipetting aids.� No food or drink are allowed in the lab, nor should cosmetics be applied. No

smoking either.� To contain spillages, all work is performed in a tray which should be lined with

absorbent tissue.

E. Buxbaum, Biophysical Chemistry of Proteins: An Introductionto Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 10,© Springer Science+Business Media, LLC 2011

123

124 10 Isotope Techniques

� Volatile radioactive material (iodine, sulphur) is handled in fume cupboardswhich are monitored regularly for flow rate.

� Collect all waste that is radioactive. Separate liquid waste, incinerable solidwaste, and non-incinerable solid waste. Dispose according to regulations.

� Before and after working with isotopes, check the workplace and yourself forcontamination. A GEIGER-counter may be used for ”-radiation and high energy“, but low energy “-radiation can only be detected by swipe tests. Do not forgetthe soles of your shoes!

� Pregnant females should not work with radioisotopes, nor in labs where suchisotopes are handled, to protect the embryo which is particularly sensitive toradiation.

� Any accidents or contamination must be reported immediately. The trouble youget after reporting it is nothing compared to what would happen if you do not!

10.1.1 The Nature of Radioactivity

The nuclei of atoms consist of positively charged protons and neutrons which, asthe name would suggest, are electrically neutral. Neutrons are required to preventthe breakdown of nuclei due to the electrostatic repulsion of protons.

Nuclei with the same number of protons (called atomic number Z) are said tobelong to the same chemical element. If they have different numbers of neutrons N ,they are said to be isotopes (from Gr. equal place, namely in the periodic system).The atomic mass is determined by the sum of protons and neutrons because themass of electrons is so small that it can be ignored. Nuclei with the same mass, butdifferent atomic number, are called isobars (from Gr. equal weight). Nuclei withthe same number of neutrons are called isotones (derived from isotope by replac-ing the p(roton) with n(eutron)). You can order nuclei in such a way that isotopesare in horizontal rows and isotones in vertical rows, the isobars are on diagonals.This is called a Karlsruhe Nuclide Chart1 [327], also available as a program forWindows www.iaea.or.at/programmes/ripc/physics/faznic/nuchart.zip or directly onthe Internet (www-nds.iaea.org/relnsd/vchart/index.html). In the nuclide chart iso-topes are given different colours depending on whether they are stable or whichmode of breakdown they have (see Fig. 10.1).

Nuclei are stable only at a certain ratio of protons to neutrons. If a nucleus has toomany neutrons, the excess neutrons will disintegrate into a proton (increasing atomicnumber by 1) and an electron, which is sent out at high velocity as “�-radiation:n ! pC C “�. Very few nuclides send out two electrons while converting two neu-trons into two protons (double “�-decay). In some neutron rich isotopes the neutronis sent out by spontaneous emission, reducing the atomic mass by one, the atomicnumber stays constant.

1 Karlsruhe is a city in Germany with a large nuclear research facility.

10.1 Radioisotopes 125

Fig. 10.1 Nuclides producedby various modes ofradioactive decay. For detailssee text. Isotopes are inhorizontal, isotones in verticalrows. Isobars are on diagonals

b-

If a nucleus has too many protons, the excess protons can be converted intoneutrons, the positive electrical charges are sent out as positrons (“C-radiation):pC ! n C “C. This process decreases the atomic number, the atomic mass staysconstant. Spontaneous emission of protons occurs in a few isotopes under reductionof both atomic number and mass. The other alternative is to send out two neutronsand two protons together, that is a helium nucleus (’-radiation ). This process de-creases atomic number by two and mass by 4. If larger nuclei than He are produced,we call this cluster decay.

Some nuclei also convert a proton into a neutron by electron capture, –-decay,which is the reversal of “-decay: pC C “� ! n. Double electron capture, wheretwo protons are converted into two neutrons at the same time, has only recentlybeen observed in some long-lived nuclides.

Very heavy nuclei may also break apart by spontaneous fission, creating twodaughter nuclei of approximately half the atomic mass of the parent nucleus andsome excess neutrons. Technically, isotopes undergoing spontaneous fission areused as neutron sources (e.g., 252Cf).

In any of these processes excess energy in the nucleus is emitted in the form ofhigh-energy photons (”-radiation, isomeric transition) . Note that ”- and X-rays(also known as RONTGEN-rays) are both high energy photons, but have differentorigin: ”-rays come from processes in the nucleus, X-rays from processes in theelectron cloud of an atom. Excess energy may also be used to propel an electronfrom its orbit (internal conversion). In contrast to “�-decay, the resulting electronsare mono-energetic.

Radioactive breakdown of unstable isotopes is a process of first order: the speedof breakdown depends on the amount of isotopes present:

v D dn

dtD �kn (10.1)

nt D n0e�kt (10.2)

126 10 Isotope Techniques

0

500

1000

1500

2000

2500

3000

3500

0 50 100 150 200

cpm

energy (keV)

β-spectrum

3H14C

Fig. 10.2 “-spectrum of 3H and 14C. Note the higher energy associated with radiation from 14Cand the broad energy distribution of the radiation from both isotopes

k is a constant for a given isotope. More common is to specify the half life periodt1=2 D ln.2/=k.

In biochemistry you will mostly work with “- and ”-radiators, the instrumen-tation required to measure ’-radiation, positrons and neutrons is usually availableonly in specialised centres. An example application would be positron emissiontomography (PET) to measure metabolic activity in various tissues, for example tolocate a tumor.

Each nuclear process, just like a chemical reaction, is accompanied by the releaseof a specific amount of energy. In ’- and ”-radiation this energy is entirely in theserays, therefore a spectrum has distinct lines characteristic for each isotope. In “-radiation on the other hand, only part of the energy is released with the electron, therest as ”-radiation. If the spectrum of “-decay is measured, a broad distribution isfound, up to a characteristic maximal value (see Fig. 10.2).

10.1.2 Measuring “-Radiation

10.1.2.1 The “-Counter

The most sensitive way to detect and quantitate “-radiation is to measure theluminescence that is created when “-radiation interacts with certain chemicals

10.1 Radioisotopes 127

Table 10.1 Properties of some “-emitting isotopes impor-tant to biological research. The photon yield is the averagenumber of photons produced by a “-ray as it passes throughthe scintillator, and in turn is proportional to the signal pro-duced by the photomultiplier. The penetration depth is anapproximate value for photographic emulsions

Isotope 3H 14C 32P

Maximum energy (keV) 18.6 156 1 710Photon yield 30 250 3 300Penetration depth (�m) 2 100 3 200

(fluors). Toluene, xylene, and other aromatic hydrocarbons give off ultraviolet lightwhen hit by “-rays. This far-UV light is used to generate near-UV fluorescentlight in 2,5-Diphenyloxazole (PPO), which in turn induces blue fluorescence in1,4-Bis-2-(5-phenyl-2-oxazolyl)-benzene (POPOP). This blue light is detected withphotomultipliers or solid-state detectors. Since each photomultiplier produces somenoise signal, “-counters use 2 photomultipliers both facing the sample vial. The sig-nals from these tubes are sent through a coincidence detector, a count is registeredonly if both tubes produce a signal at the same time. This lowers noise to about30–50 events per minute.

The number of photons produced and hence the signal from the photomultiplieris proportional to the energy of the “-ray (see Fig. 10.2 and Table 10.1). The signalis fed into an analyser, and from there into a computer. Isotopes with very different“-spectra, like 3H and 14C, can be measured in parallel in the same sample. Thisis, however, not quite as easy as with ”-radiation, as “-radiation does not occur indiscrete lines. Thus some of the events from the high-energy isotope have the sameenergy as those coming from the low energy one. The computer programs that comewith modern counters can correct this, provided that the ratio of high to low energyactivity is within a certain range. This range needs to be determined for each isotopepair and each counter type (as different manufacturers use different programs).

The sample is mixed directly with the scintillator as low-energy “-radiation isscreened easily, even by a sheet of paper. If the sample contains water, deter-gents like Triton-X100 must be added to create a stable emulsion of the samplein the scintillator. Modern scintillator cocktails offered by supply companies arebiodegradable, a considerable advantage compared to the messy chemicals used inthe home-made cocktails of yesteryear. Solid samples are digested in suitable chem-icals, like quaternary ammonium bases, before they are neutralised and mixed withthe scintillator. Samples should be of neutral pH, as both high and low pH can in-duce chemiluminescence in scintillation cocktails, which is difficult to distinguishfrom the signal caused by “-radiation. Chemiluminescence is also reduced by lowtemperatures (about 10 °C). Phosphorescence is another possible problem, samplesshould be stored under reduced light for about 1 h before counting.

128 10 Isotope Techniques

Counting efficiency depends on the energy of the “-radiation, medium and highenergy radiation from isotopes like 14C and 32P can be counted with efficienciesof 90 % and better, while for low energy radiation, for example produced by 3H,counting efficiency may be less than 30 %.

Radioactivity is a stochastic phenomenon, the number of events per unit time isgiven by a POISSON-distribution. This means that for a counting error of less than2 %, the counter has to detect 10 000 events. Counting time required depends on theactivity in the sample, becoming shorter as activity increases. On the other hand, forreasons of cost and possible exposure risks, one would like to keep the activity aslow as possible. Thus activities between several hundred and several 10 000 countsper minute (cpm) are a useful compromise (counting time 1–30 min per sample).The counter should be programmed to reject samples with less than 100 cpm, asthese cannot be measured in a reasonable time.

The luminescence can be quenched by certain chemicals, which may be presentin the sample. Quenching leads to both a reduction of counting efficiency and a shiftof the detected spectrum to lower energy (see Fig. 10.3). This shift (sample channelratio (SCR) can be used to correct for the reduced counting efficiency.

A second method to detect quenching is to use an external standard. This isa small amount of americium or a similar ”-radiator, which is placed next to thesample. The ”-radiation produces COMPTON-electrons in the sample, which canbe detected by the counter. So in the absence of quenching, the count rate induced

0

5000

10000

15000

20000

25000

30000

35000

40000

0 50 100 150 200

cpm

energy (keV)

Quenched 14C

8%10%16%42%

Fig. 10.3 Quenching of 14C radiation by addition of chloroform to the sample. The peak becomesnarrower, the maximum is shifted to lower energies, and the integral under the curve (total counts)becomes smaller

10.1 Radioisotopes 129

by that standard should be identical for all samples, reduction indicates quenching.“-counters will list this reduction as quench-identification parameter (QIP).

Again, the programs that come with modern instruments can do quench cor-rection for you automatically, but a healthy scepticism towards this form of datamanipulation is in order. In particular, at high quenching the multiplication withhigh correction factors can seriously affect the statistical error of the results. Makesure that your samples quench as little as possible, and that all samples of an exper-iment quench to about the same degree. In particular, samples should be clear andcolourless (yellow is most harmful as it is complementary to the blue light counted).Also, make sure that your standards have a similar composition to your samples.Calibration of the quench correction is possible by making a series of samples withthe same amount of radioactivity, but different concentrations of chloroform, whichis a very effective quencher.

10.1.2.2 Cherenkov-Counting

High energy “-radiation causes oxygen molecules in air to dissociate, the resultingradicals recombine under emission of blue light (CHERENKOV-radiation). Isotopeslike 32P can be counted in a “-counter without the use of scintillation liquid. Count-ing efficiency is about 25 %, the spectrum is similar to 3H. This option is attractivefrom both the economical and ecological point of view and also because the sampleis not destroyed but may be recovered for other measurements.

10.1.2.3 Autoradiography and Autofluorography

Radioactivity was detected by HENRI BECQUEREL by its ability to blacken a photo-graphic plate in the absence of light. This property can be used to locate radioactivityin a sample. Typical applications include the location of bands in an electrophoresisgel or the location of binding sites on a histological section. For gels and the likeX-ray film is most often used. The gel is stained and dried as usual, then placedagainst the film in a light-tight box and incubated at �20 °C (to reduce background)for several hours or days, depending on activity. Then the film is developed. Thereare pens with radioactive or phosphorescent ink available to place marks on the gelwhich afterwards allow alignment of gel and film.

The blackening of photographic film is proportional to the radiation intensity,but only if the intensity is high enough. At low intensities the sensitivity is reduced(SCHWARZSCHILD-effect2). This can be counteracted by flashing the film with asmall amount of light. Pre-flashed films are commercially available.

With low-energy “-radiation gels can screen much of the radiation from the film.This reduced sensitivity can be overcome by incubating the gel with a fluor like PPO

2 Named after the photographer KARL SCHWARZSCHILD, 1873–1916.

130 10 Isotope Techniques

or sodium salicylate before drying. The film then registers the light coming from thefluor rather than the “-radiation directly (autofluorography).

High energy “-radiation may penetrate the film without blackening it. In this casean intensifying screen may be placed behind the film where the radiation induceslight flashes in heavy-metal based fluors. This light is registered on the film.

For histological sections, the photographic emulsion is poured onto the slide anddried. Thus the silver-grains develop directly on the slide and can be viewed togetherwith the tissue.

10.1.2.4 Phosphor-Imaging

The use of X-ray film for autoradiography involves a tedious procedure, andlinearity is limited to about 1 order of magnitude. This makes quantitative assaysdifficult.

Certain ceramic crystals react to radiation by displacement of their components inthe crystal lattice. When the atoms return to their proper position, energy is releasedin the form of light. However, because the mobility of atoms in a solid is limited, theenergy-rich state is meta-stable, activation energy in the form of heat or intense lightis required to induce recombination. These physical principles have been used fora long time for the dating of ceramics from archaeological sites by thermolumines-cence. In the last couple of years instruments have come on the market where a platecovered with very small ceramic crystals is exposed to radioactivity on blots, gels,histological sections and the like. Afterwards, the plate is scanned by a laser beamto induce recombination. The resulting phosphorescence is measured quantitatively.The procedure is more sensitive than autoradiography on X-ray film, and the re-sponse of signal intensity as a function of radiation dose is linear over 4–5 ordersof magnitude. The required instruments are very expensive, but because the ceramicplates can be reused, no consumables are needed which makes phosphor-imagingcost-effective if large numbers of autoradiographies are needed.

10.1.2.5 Geiger-Muller Counters

For detection of contamination, the classic GEIGER-MULLER-counter is used, butthis works only for radiation with high enough energy to penetrate the window of thecounting tube. For low-energy “-radiation a methane flow-through counter may beused, this is effectively a GEIGER-MULLER-tube without window. However, wipetests are more common, the surface to be checked is wiped with a filter paper soakedin ethanol (or another solvent suitable for your sample), the paper is then placed intoa counting vial with scintillation fluid and counted.

10.2 Stable Isotopes 131

10.1.3 Measuring ”-Radiation

10.1.3.1 The Na.Tl/I Scintillation Counter

”-Radiation interacts less with matter than “, thus it is not necessary to mix thesample with scintillator. Instead, the sample is brought close to a NaI-crystal dopedwith Tl which emits light-flashes when hit by ”-radiation. Those are detected by aphotomultiplier. The resulting signal is proportional to the energy of the ”-quant.Since ”-spectra consist of discrete lines, determination of different isotopes in par-allel is easier than with “-radiation.

10.1.3.2 Other ”-Detectors

Ge-based solid state ”-detectors are usually found only in specialised centres, theywork only at cryogenic temperatures.

GEIGER-MULLER counters work very well with ”-radiation, but exact quantita-tion is not possible with them.

It is possible to use a “-scintillation counter to count ”-radiation. For this pur-pose the scintillation cocktail is mixed with a solution of 10 % ZnCl2 in water.”-radiation interacts with the Zn2C-ions, producing COMPTON-electrons, whichthen cause light flashes in the fluor. For low energy ”-radiation like 125I counting ef-ficiency is about 80 %, for high energy isotopes like 22Na efficiency is lower, about20 %. The sample need not be mixed with the scintillation fluid but can be placedinside an Eppendorf vial or the like, hence the method is very economical.

10.1.3.3 Photon Activation

High energy X-rays can kick protons or neutrons out of a nucleus, thus creating ra-dioactive elements. The intense X-rays required are produced in a linear acceleratorby directing fast electrons onto a water-cooled gold or platinum target. Within a fewhours after irradiation the daughter-nuclides of light elements (first two rows of theperiodic system) have decayed, then the samples can be placed into a ”-spectrometerfor analysis. This allows the sensitive determination of elemental composition evenin complex samples, e.g., for environmental monitoring.

10.2 Stable Isotopes

Many biologically relevant elements have not only one, but several stable (non-radioactive) isotopes, for example hydrogen (atomic mass 1 and 2), carbon (12and 13), nitrogen (14 and 15), and oxygen (16 and 18). Compounds enriched ina rare stable isotope can be used to follow metabolic pathways, the products are

132 10 Isotope Techniques

detected in a mass spectrometer (see Chap. 39 on p. 383). This has uses in routineclinical diagnosis. For example, if patients are given deuterium oxide (heavy water),it will distribute throughout the body. If then a urine or saliva sample is analysed,the amount of water in the body can be calculated from the volume of deuteriumoxide given, the deuterium concentration in that volume and the deuterium concen-tration in the sample obtained. Since the lean body mass consists of 73.3% water,lean body mass can be determined and the fat content of the body can be calculatedas difference between total and lean body mass.

The specificity of enzymes is so high that they can distinguish between differ-ent isotopes of the same element. For example, the carbon fixing enzymes in C3-(ribulose bisphosphate carboxylase (Rubisco) ) and C4-plants (phosphoenolpyru-vate carboxylase) both prefer 12CO2 over 13CO2, so that the carbon in all livingorganisms contains less 13C than the carbon dioxide in air. This depletion of heavycarbon is quantified by the �13C-value which is several �. Interestingly, it is some-what higher in C3- than in C4-plants. Thus, although sugar produced by sugar beet(a C3-plant) and by sugar cane (a C4-plant) are chemically identical, they can bedistinguished in a mass spectrometer (see Sect. 30.2 on p. 274 for details). The useof stable isotopes to determine the structure of proteins in NMR-experiments is de-scribed on p. 316, their use for determination of reaction mechanisms on p. 383.In geochemistry and ecology, stable isotopes are even more important than in thebiomedical sciences. However, that is beyond the scope of this book.