6 Radiation Biophysics ErnsT-Geora NIEMANN, 6.1 Introduction ‘Asa science, radiation biophysics is concerned with the action of ionizing radiation on biological systems. In ‘ontrast to radiobiology, which deals with the biologi- al and medical consequences of radiation, radiation biophysics concerns itself mainly with the physical and chemical primary processes, and biological effects are attributed to them, Further neighboring fields are radiation chemistry and photobiology: the former because radiation-induced chemical alterations gener- ally initiate the biological-metabolic radiation effects, the latter because visible and especially ultraviolet light is often able to generate effects similar to ionizing radiation, The scientific interest in radiation biophysics results, first ofall, from the need to recognize and quantify the primary processes which start a biological reaction chain, and which lead finally to a radiation effect that is medically recognized and used. On the other hand, there is the possibility of gaining information about ‘metabolic processes through the agent radiation and learning about their variability under external in- fluences. In addition to this, the possibility of radiation damage always has to be considered and measures for effective protection taken. Interesting from a physical and biological point of view is also the fact that here ‘minimal amounts of energy, through complicated action chains, may release’ substantial macroscopic effects. 6.2 Radiation and Its Measurement 6.2.1 Types of Radi ion A given type of radiation is called “ionizing”, if its quantum energy is high enough (£>10 eV) to release electrons from an atomic or molecular structure. The radiation particles themselves may be charged (directly ionizing) or not charged (indirectly ionizing) (Table 6.1). Their energy is generally given in electron volts (CV) or multiples thereof (1 keV= 10" eV, 1 MeV=10° eV). 1 eV is the kinetic energy which a single charged Patil resives by pasing through a voltage diference oft 1 eV=1.602 107! J During nuclear decay, and also in particle accelerators, radiation is produced with energies which are usually Table 64, Tonizing radiation ‘Symbol Type ‘Mass Charge Production Directly ionizing radiation a the 4 2+ Nuclear decay oe O.00ss9 Nuclear decay Bet o.000s49 "Nuclear decay > iH 1 + Accelerator eel 2 + ‘Accelerator Indirectly ionizing radiation y— Elmagnrad. 0 ° Nuclear decay x Elmgnnd 0 ° Xray tube nok 1 0 ‘Nuclear decay ‘Nuclear fission ‘many times the ionization energy. During their pene- tration of matter, these types of radiation produce along their tracks a large number of ionizations and excitations, all of which could lead to chemical and biological consequences, In radiation biophysics, aside from the effect of external irradiation, the action of incorporated radio- nuclides is also of interest, ie, that of radionuclides which are directly built into the system. In addition to the effect of the radiation particle emitted during nuclear decay, in such cases transmutation — which is the transformation of the decaying nucleus into another chemical element — and the recoil energy acting on the emitting nucleus may have further consequences. 6.2.2 Interaction Between Radiation and Matter ‘The release of energy by radiation through ionization orexcitation is the first physical step in a long sequence of secondary reactions which may finally lead to a biological radiation effect. This primary interaction differs quantitatively and qualitatively for various types and energies of radiation. 6.2.21 Directly Ionizing Radiation The energy dissipation per unit pathway, the “stopping power”, is described for all heavy charged particles by ‘Bethe’s formula: 4nN, &z* Zz deem arate da2906 Radiation Biophysics Avogadro's number rest mass of the electron atomic number of the particle atomic number of the absorbing matter atomic weight of the absorbing matter @=density of the absorbing matter bein ( 2ma? -#) ~ "Ar 0B) I= ionization potential of the absorbing matter For electrons, because of their lower mass (and thus stronger deflection) a slightly modified equation is valid. While the first term of this equation is a constant, the second term expresses the dependence of energy dissi- pation along the pathway on the particle properties: high charge and high atomic number increase, high velocity decreases the energy loss of a particle along its As 7/A may be considered constant over a wide range, the energy dissipation is in a first approximation proportional to the mass per unit area, —dE~odx. The LET-value, often used in radiation biophysics (see Sect. 6.2.3), substantially corresponds to the mass stopping power, but considers only ionizations and excitationsin the vicinity of the primary track, not those of secondary particles. In addition to the direct ionization process, the production of bremsstrahlung plays an important role in the energy loss of f-particles in matter. A moving, electron is slowed down in the field of a (heavy) nucleus and the energy of deceleration is cmitted as a - quantum. The probability for this process increases strongly with particle velocity and atomic number of the absorbing matter. 6.2.2.2 Indirectly Ionizing Radiation a) X Rays and y Rays The absorption of these energetic forms of electromag- netic radiation in matter is described by a simple exponential expression; T= he". Here fy and J, are the radiation intensities before and after penetration of the material layer x, and jis the absorption coefficient. This 1 is composed of three components, describing three different interaction pro- cesses: +047 x= photoclectric-absorption coefficient o=Compton-absorption coefficient 17> pair-formation coefficient. In the photoelectric effect a photon delivers all of its energy to a bound electron in the absorbing matter. This process prevails in the region of low-energy 7 rays; its probability increases with the fifth power of the ‘material’s atomic number: zw moe ‘The energy balance of the photoelectric process is E,=hv-B, B.=binding energy of the electron ejected kinetic energy of the electron ejected energy of primary y-quantum. ‘The Compton effect can be considered as a collision process between a )-quantum and an electron, during which the photon transfers a part of its energy to the electron: Eth =h-B, This process predominates for medium energy rays. Its occurrence depends on the electron density of the absorber material; 4 one FG During pair formation a photon “materializes” in the field of a nucleus and forms an electron-positron pair. This process therefore can occur only if the quantum, energy corresponds to at least twice the electron mass (1.02 MeV). Only at much higher energies does it become the prevailing process, its probability increases with the square of atomic number: 2 Electron and positron lose their kinetic energies through ionization and excitation. Finally, the positron recom- bines with an electron and disintegrates together with it under emission of two photons of 0.51 MeV each (annihilation radiation), Figure 6.1 indicates in which regions of photon energy and atomic number the three different processes predominate.Photoolectic effect Pir formation ‘Compton effect ‘Atomic numberof absorber Me i % “Peneray Men) Fig. 6.1. Regions of predominance of photoelectric effect, Compton fle, and pair formation, Too ) Neutrons Neutrons also dissipate their kinetic energy in biologi- cal matter through three processes: elastic scattering, inelastic scattering, and neutron capture. Elastic scattering is a collision process between a neutron and an atomic nucleus, during which all kinetic energy is conserved as such. According to the collision laws of mechanics, the energy transfer is @ maximum if the nucleus and ‘the colliding neutron have equal masses, which is the case with the hydrogen nucleus. The recoil protons produced dissipate their energy to matter within a short range through ionization and excitation. In inelastic scattering, part of the energy transferred uring collision is used for the excitation of a nucleus. ‘The mucleus relaxes within a short time by emission of a 7-quantum, At high neutron energies a second neutron may also be emitted [(n, 2n) process]. By such collision processes the neutrons lose kinetic ‘energy until they are in thermal equilibrium with their ‘environment (thermal neutrons). Their energy is then Eq=tkT with k= Boltzmann constant = 1.38 x 10-9 J/K T= Absolute temperature [K] At 293 K environmental temperature this results in E Tn the case of neutron capture a slow neutron is absorbed into the nucleus of an atom, Under prompt emission of a 7-quantum this process produces an isotope (in most cases radioactive) of the absorbing atom [(n,))-process]. Sometimes (7, p) processes also ‘oceur, which lead to an element transformation. The probability of these processes increases with the period the neutron remains in the field of the nucleus, so it generally follows a 1/v function upon which, however, resonances may often be superimposed. Of great importance for biological aspects are the following reactions in which ['*C] and deuterium are Formed in the high atmosphere: 623 Dose and Dose Rate 291 CAN] (n,2) [2C]+0.66 MeV GH] (n,9) GH]+2.2 MeV The excess energy in the first case is carried by the proton, in the second by the y-ray. 6.2.3 Dose and Dose Rate In order to be able to quantify the biological effect of radiation one needs information on the amount of energy absorbed from the radiation per unit mass. The unit of this “energy dose” is the Gray [Gy], 1 Gy=1 J kg"? The radiation effect often depends not only on the dose, but also on the period during which the dose is applied. Therefore, in addition to dose the concept of dose rate is introduced [Gy s~']. The direct calorimetric deter- mination of energy dose is very difficult, as the amounts of energy are very small, A dose of 1 Gy would heat water by only 2.4% 10-*°C, Moreover, the radiation energy may to some extent be bound in the form of ‘chemical energy, which does not contribute to tempera- ture increase, For dose measurement, therefore, the ability of radiation to ionize a gas is used in most cases and an “ion dose” is defined which is given in [C kg” ']. Since the average energy which is necessary to produce an ion pair in air (34 eV) is known, one can give the relation 1 C kg"! 234 Gy. ‘And for water and tissue: 1C kg"! 2 36-38 Gy. These data are valid, however, only for X rays and y radiation; they may differ strongly for heavy charged particles. In the literature the older units rad [rd] and Roentgen {R] are often found, which can easily be transformed into the SI units 1 rd=100 ergs/g=10-? Gy 1 R=2.58% 107 C kg? 1. R =0.877 rd in air 1 R =0,93-0.98 rd in water and tissue. Tdentical energy doses from different types of radiation ‘may cause differing effects in biological systems. The ratio between a dose of a standard radiation (200 kV X rays) and that dose of any other radiation which causes an identical biological effect is called the “rela- tive biological effectiveness” (RBE) of the latter radia- tion, Dose of standard radiation D’ [Gy] REED ose of other radiation D [Gy] Of course itis necessary to define exactly the biological effect as well as all experimental conditions (dose rate,2926 Radiation Biophyis temperature, oxygen concentration, etc.). The dose of standard radiation mentioned above is often given as “equivalent dose” in Sieverts [Sv]. D’ [Sv]=RBE - D [Gy] The older unit of equivalent dose is rem (Roentgen- equivalent-man): 1 rem=0.01 Sv. The relative biological effectiveness of radiation de- pends strongly on its LET-value (linear energy trans- fer), which describes the energy dissipation of the radiation particle along its track [KeV/1] (Fig. 6.2) Roe Fig. 62. Relation between the relative biological effectiveness (RBE) of ionizing radiation and its LET value in tissue 6.2.4 Dosimetry For the measurement of dose and dose rate in the field of biological radiation effects the following techniques are used: 1. The ionization chamber. The ion pairs produced in 1a defined volume of air (or gas) by the radiation are collected at electrodes by an electric voltage applied to them, The current measured is directly proportional to the ion-dose rate [C kg" s~'], providing that the collecting field is strong enough to prevent the recombi- nation of ions, and that electron equilibrium prevails at the borders of the measuring volume. This can be achieved by the choice of suitable wall materials, which should have the same average atomic number as the gas filling. In this way air-equivalent as well as tissue- equivalent ionization chambers can be constructed 2. The Fricke dosimeter. In this type of chemical dosimeter the oxidation of Fe* to Fe’* in aqueous solution is utilized for dosimetry. Fe* +hv=Fe'* +e~ The iron (Ill) ion concentration is determined in a spectrophotometer at 304 nm. Advantages of this system are its good tissue equivalence, its indepen- dence of dose rate over a wide range, and the pos- sibility of applying it to any convenient form of measuring volume. The dose range covered is between 10 and 100 Gy. ‘Numerous other types of chemical dosimeters are known, but they are not used in the biological field to any great extent, 3. Film dosimeter. The blackening of a photograp! emulsion by ionizing radiation can also be used for dose measurement. It is utilized on a very wide scale in personal dose monitoring because the sensitivity is quite good (D400 sGy), Careful standardization is essential, as the results depend strongly on the emulsion properties and on the procedure of development. 4. Thermoluminescence dosimetry. This technique uses the capability of some crystals (¢.g., Ca F,) to store radiation-produced electrons in crystal defects over a long period. When the crystal is heated up, the electrons return to the ground state via light emission. As the number of “excitons” is proportional to dose, the total ‘amount of light emitted during heatinggisalso a measure of the dose applied. Thermoluminescence dosimeters can be made very small and (for instance, mounted in Teflon) tissue equivalent. The measurement range extends from about 10° to 10? Gy and the measure- ‘ment is highly independent of dose rate (up to 10° Gy x). 5. Calorimetric techniques. According to the defini- tion of the Gray the measurement of temperature increase in material of known specific heat is an ideal dosimetry method, if chemical reactions can be ex- cluded (for instance for graphite). Because of the small amounts of energy transferred by radiation, it can be used only for high doses and is applied as a primary standard exclusively. 6. Other dosimetry techniques. Further dosimetric techniques are based on turbidity formation in glasses or organic polymers, on the discoloration of crystals, conductivity alteration in semiconductors, or even the mortality rate of bacteria due to ionizing radiation. These systems are used, however, only to a small extent in biophysical radiation dusimetyy, 6.3 Description and Interpretation of Radiation Action 63.1 Dose-effect Graphs and Target Theory In order to describe biological radiation effects, dose- effect graphs are generally used. A prerequisite for this, type of diagram is that the radiation effect can be given in a quantitative form, like the survival rate of viruses, bacteria, or tissue cultures, the residual activity of enzymes, of the yield of crop plants. Ifthe logarithms of such data are plotted versus dose, one of four typical curves are usually obtained (Fig. 6.3). 1. Exponential “single-hit” curve. In this, the simplest case, which is often observed in the inactivation ofbd +00: 10) 1 Single-hiteune — O%° eu 00 10. y Dose Muttple-it cuve Relative biological eect tm) 100. Stmulatoncuve OP Fig. 63, Dose-ffect curves viruses or enzymes, the degree of injury is described by an exponential law. In the semilogarithmic plot the dose-effect curve is linear and can be described by the equation Ngo" ‘The target theory tries to find a relation between the stochastic process of radiation absorption and the different types of dose-effect curves. This theory pos- 63.2 Direct and Inditect Action 298 tulates the existence of radiation-sensitive volumes (Cargets) within the cell or the molecule. Inside each of these targets one or more ionizations or excitations have to occur in order to inactivate the cell or the molecule. ‘Accordingly, a pure exponential graph is obtained if within one existing target only one hit is necessary for inactivation or killing (single-hit curve) 2, Multiple hit curves. After irradiation of bacteria or higher organisms dose-effect curves are often obtained which showa “shoulder”. Atlow doses no or only small effects are observed, while at higher doses the curve approaches the exponential form. According to the target theory, the interpretation of this type of curve is that within the organism either more than one target has to be hit, or that within one target several its have to occur in order to obtain the radiation effect observed. ‘The experimental error. however. is in most cases too large to distinguish between the two possibilities. The extrapolation of the exponential part of the shoulder curve to the dose zero gives an indication of the number of hits required or the number of sensitive targets, 3, Biphasic curves. “Sagging” dose-effect curves are always observed if individuals of different radiation sensitivity are present in an irradiated population, ‘These may be, for instance, bacterial strains of different radiation resistance, or cells in varying cell cycle stages, At low doses the more sensitive part of the population is preferably damaged, at higher doses also the less sensitive. The relative radiation sensitivities may be obtained in simple cases for both parts of the popu- lation by extrapolating the two exponential parts of the curve. 4. Stimulation curves. Upon irradiation of higher plants, and also with bacteria, fungi and animals, “positive” effects are sometimes observed at low doses, Characteristics such as yield, plant height or RNA synthesis rate in these ‘cases may be enhanced in comparison to the unirradiated control. Thus the dose- effect curve rises above the 100 % line before it drops at higher doses, usually again in an exponential form. The interpretation with the target theory is difficult; One has to assume that by the inactivation of one or a small number of targets biological processes are initiated which are able to repair or even overcompensate for the primary lesion, 6.3.2 Direct and Indirect Radiation Action The target theory is based on the assumption that within a cell a defined region, the target, has to be hit directly in order to damage the cell. Because of the statistical distribution of ionizations and excitations in matter, however, most of the primary events of energy absorption will occur outside of these regions and may form chemically active intermediates there. These are able to diffuse inside the cell and may, secondarily, damage macromolecules or structures ‘of biological importance. The probability of such indirect radiation action of course is strongly dependent on the concen-294 6 Ratiation Biophysics tration conditions within the cell. Moreover, it depends on the presence of tertiary reaction partners which may react with the intermediates and thus compete with the effect of biological importance. To which degree radia- tion damage in vivo is based on direct action is difficult to establish. The effect of “radioprotective agents”, which reduce the radiation effect if they are present, suggests an indirect action by competition reactions; but also for direct radiation effects such protective reactions — by repair or restitution —are conceivable. Jn vitro this differentiation usually is easier: For direct radiation effects at a constant dose the number of damaged molecules in solution must be proportional to concentration, while it should be independent of con- centration for indirect effects. 6.3.3 Energy-transfer Processes, Reaction Rates, Pulse Radiolysis and Flash Photolysis Independently of knowledge about the course of a reaction it is possible to define the energy yield G: G—Rumber of molecules formed or altered 100 eV of absorbed energy Particularly in the case of indirect radiation action this value may depend strongly on the presence and concen- tration of tertiary solution partners, ‘An exact description of a reaction chain is only possible if all the individual reaction steps are known and can be presented in reaction equations. The formation rate of a product x depends on the concen- tration of one or more reaction partners: ‘The constant & appearing here is the reaction rate constant. In simple cases reactions can be described by differential equations of low order (Table 6.2). The molecularity of a reaction (c., the number of primary ‘able 62. Reaction equations participating molecules) corresponds to the order of the ky (ky rate-determining) total reaction bimolecular, first order equation. 2. ka hy (Ky rate-determining) total reaction bimolecular, second order equation. 3. kak; (both k rate-determining) total reaction bimolecular, order of equation not integral. For the determination of rate constants in radiation- induced reactions the techniques of pulse radiolysis and flash photolysis are very useful. In both of these methods a short (10->f>10-® s) intense pulse of ionizing or optical radiation is applied to the system under investigation (in most cases a solution) and produces primary products in comparatively high concentrations. These concentrations as well as the rise and fall in concentration of secondary products can be observed by means of fast analysis methods; kinetic absorption spectrometry and kinetic conductivity measurements play a very important role in this field (Fig. 6.4). From the resulting curves the rate constants of the reactions can be calculated, 6.4 Molecular Effects of Radiation 6.4.1 Radiation Chemistry of Water Because of the high water content of most biological systems, water radiolysis and its resultant products have particular importance in the indirect action of radiation. Following the primary ionization HO +hv-+H,0* +7, ‘Order Differential form of Integral form of reaction equation Dimension of k reaction equation| oe ° anh hot mol s = 1 1 Fk a-») s > 7 Gables) & 2 Gn ksla-3) 6-%) Pseudo 1 a an F-Ks(a-9 6-0) « x Fa k(a—x mol”? s-# a ae CEG ‘x=concentration ofa reaction product, ‘a)b= concentrations of reactants ey reaction rate constantsFig. 64, Principle of arrangement for Mash photolysis or pulse radiolysis various secondary reactions may take place (Fig. 6.5). ‘Samuel and Magee postulated that the free electron loses its energy already in the close vicinity of the mother ion and recombines: H,0* +e" -+H,0*. The excited water molecule HO* then dissociates, forming two radicals, H,0*+H'+OH’, which possess enough kinetic energy to diffuse freely and react with other molecules. According to Platzman and Frahlich, on the other hand, the electron dissipates its energy in small steps only and will be thermalized at a distance of about 15 nm from the mother ion; hence the recombination Pinay pea nner or —— Ea, Masada ec 64.2 Radicals and Molecular Products 295 probability is small, It will then be surrounded by an array of oriented water dipoles, which strongly reduce the reactivity of the “hydrated electron”, eq +nH,O65, ‘The mother ion dissociates into H,0* Ht +O, while the hydrated electron in acid media reacts to ejq+H' +H 40,0. Following this reasoning also, the main products are H’ and OH" radicals, but their spatial distribution differs from those generated directly according to Samuel and Maggee. Back reactions produce molecular products: H'+OH'+H,0, H4+H' +H, OH’ +0H'+H,02, All of these processes are terminated after about 1077s, so that in pure water the following main products are available for further reactions Hy OY, ej, Ha, H2O2- 6.42 Radicals and Molecular Products Radicals are characterized by an unpaired electron in their outer shells and therefore usually are very reactive. ‘Back reactions —————+ Reactions wih other solves [ }— omsion +cr ver [0% Saws 4 ! dy: on Hy —+0H' oe W 3 —+ 0H" yO) — + HY 10 + OH | He Dittusion He, OH* ils { Recombination — Hy +H TeOr uy . = +00, L+H, _{hO* O ; bition sig —*H20 H+ OF ¢-—fhermsaton” — la 0* 0? 107 0? Time ater primary event ——— Fig. 65. The mechanisms involved in water radiolysis296 6 Radiation Biophysics ‘The radical character is indicated by an elevated point. Hi and OH’ are radicals, as is the hydrated electron regarded as H,0”. Typical examples of the reactions of | these radicals with organic molecules of the type RH are: RH | OH’ RH+H RH+ »R'1 HO, oR +H, RH” +nH,0, By direct radiation action, on the other hand, the main reaction occurring is RH S R+H Among the molecular products, HO, in particular is very reactive: RH+H0,-ROH +30. ‘Thus, in the course of water radiolysis, oxidation reactions predominate. Table 6.3 gives the rate con- stants for the reaction of water radicals with some important reaction partners. ‘Table 63.Rate constants k; for the reaction of water radials with some important reactants (1 mol” s~'] (Hart, 1971) Reactant Radical a ¥ on oa 6 xI® 25x10" 30510 i 25 x10 — 20x10 2 x10 on 30 x10" 2 x10" 6 x10 H.0 160 = - o 19 x10% 22x10" 10, 123210 9" x10" 45x07 co, 1) ae <10° 30108 CHOH =< 10 x108 16x10 Sox10 Thymine 17 x10 23x 108 46x10? Thymidine 25x10" 39310 Uracil i 45x10 Cytosine 80x10 42x10 Cytdine 42 x10 35x10 Adenine Bi x10" T9x10° 3x10 ‘Adenosine 10 x10 Lato 36x10" 6.4.3 Modification of Radiation Effects 6.4.3.1 Oxygen Effect and Back Reactions Oxygen is usually present in biological systems in ‘comparatively high concentrations and has a consider- able effect on further reactions of water radicals, This is due to the formation of reactive and long-lived molecu- Jar and radical products, H'+0;4HO} HO} +H'-+H,02 +0;+0; +nH,0, while at the same time organic peroxides are formed, R'+0,+RO3, which together with further organic molecules may initiate a chain reaction RO; +RH>ROH+R. ‘These reactions bring about biological effects following ‘on with X rays or 7 rays which are considerably enhanced by the presence of O,. The oxygen enhance- ment ratio (OER) is usually between 2 and 3; with high LET radiation itis substantially smatter. Oxygen also plays an important role in the Fricke dosimeter mentioned above. The overall reaction hereis 2 H,0+0, +4 Fe?* 4 Fe** +4 OH” Other inorganic reaction partners, on the other hand, are able to reduce the effects of water radicals con- siderably cl +OH'=Cl' +0H- cr+cr +Cl, Cr+H 34H’, Apparently a chain reaction is of major importance here also. GA3.2 Repair Processes and Sensitizers Modifications of biological radiation effects are pos- sible on three different levels: At the radical stage, on the level of macromolecules, and in the realm of metabolic processes. Just as O, and Cl” act on the radical level and are able to enhance ot reduce their efficiency considerably, there are other “radical scav- engers” like histidin, which by their high reactivity with water radicals suppress the biological effects of the latter to a high degree, ‘A number of organic compounds, especially those ‘with -SH groups (such aseystein or cysteamin), have the capability to “cure” organic radicals by hydrogen transfer, and in so doing switch over the dimer form R'+RSHRH+RS RS'+RS'+RS—SR. This process is only possible, however, if the bioradical has not already been transformed by oxygen into a peroxiradical of the form RO} On the other hand, it is possible to increase the radiation sensitivity of a cell by a factor of up to 4 by substituting tne base thysuine in the DNA of this cell by a halogen-substituted pyrimidin base, such as S-bromo- uracil. All of these modifications of the radiation effect on the radical or molecular level are effective only if the “radiation protectors” or the “sensitizers” are already present in the cell during irradiation. Biological effects of radiation are generally depen dent on dose rate in the sense that lower dose rates orfractionated (interrupted) irradiations are less efficient. This indicates that comparatively slow metabolic pro- cesses are also able to repair radiation damages. This holds true, however, only for radiation effects which can be described by a shouldered dose-effect curvesi.e.. for the case that enzymatic processes are capable of repairing “sublethal” lesions. Biological effects pro- duced by high LET radiation therefore generally are not reparable by such mechanisms. In addition to radiation, cells or organisms are often simultaneously exposed to other physical or chemical burdens which already by themselves may produce lesions. Combined effects of radiation and other harm- ful influences have not yet been investigated to any extent but there are indications that such burdens do not always act independently, adding their effects, but that there may be synergisms which lead either to an amplification or a reduction of the combined effect Especially in the region of low radiation doses and low concentrations of pollutants, such nonlinear combined effects would require new concepts in the radiobiologi- cal assessment of radiation burden. 6.5 Radiation Effects on Biomolecules and Molecular Structures 6.5.1 Radiation Effects on Pro Proteins are present in the cell either in the form of structure elements or as enzymes which control the complete cell metabolism. They are polypeptides of ‘molecular weights between 10° and 10°, fixed by means of hydrogen bonds in defined secondary and tertiary structures which are important for their function. Damaging radiation doses in vitro are in the range of some 10 Gy; indirect effects play a major role, The OH radical reacts very fast with the aromatic amino acids and with the sulfur-containing amino acids methionine, ceystein, and cystine. The latter are also strongly attack- cd by the hydrated electron. Also, in the case of direct radiation action, when the protein itself is ionized, energy transfer occurs; the damage is manifested in most cases at the a-carbon of a glycine unit or at the sulfur of a cystein or cystine residue. Invitro the damaging doses for enzyme functions are also in the range of some 10 Gy, about one order of magnitude above that for compiete cells. Enzymes are present in the cell in great multiplicity and with very different concentration, Moreover, they are conti- nuously resynthesized, so that the deficiency of certain enzymes could be comparatively irrelevant for the cell function. On the other hand, it cannot be excluded that special enzymes of high radiation sensitivity might be present in the cell in very small concentrations only; such enzyme damage could lead to a complete break- down of metabolism. However, such proteins have as yet been neither isolated nor identified. ‘The radiation sensitivity of enzymes in vivo usually is higher than in vitro. This leads to the conclusion that 6.5.3 Radiation Effects on Membrane Structure 297 inside the cell additional mechanisms may contribute to the loss of enzyme functions. These actions could be caused by radiation effects on cell structure elements such as membranes. Nothing is known, however, about the prevailing mechanisms. 6.5.2 Radiation Effects on Nucleic Acids Nucleic acids often are considered to be the determin- ing element for radiation effects in biological systems, because (as DNA) they are carriers of the genetic code and (as RNA) have important transfer functions in protein synthesis. The good correlation between the radiation sensitivity of different plant varieties and the nucleic acid content of their nuclei (the interphase chromosome volume, ICV) supports this view. One has toconsider that the alteration or destruction of one base among ten thousand may already be of biological importance, mainly if important information is avail- able only once in a cell, Of course it will not be possible to identify such small alterations; a chemical detection of radiation effects in nucleic acid molecules is possible only after high radiation dose treatment; chain breaks and base alterationsare the most importanteffects here. Breaks of single DNA strandsare due in most cases to & destruction of the phosphate-ester bond; they can be detected by methods of physical chemistry (the ‘measurement of viscosity, sedimentation, light scatter- ing, or flow biretringence). Ihe cleavage of a double- stranded DNA occurs if such a bond is destroyed in each strand within the space of a few bases. The probability of this defect is much lower than for the single chaitr break; while the latter can be interpreted as a single-hit event, the double-strand break follows the pattern of a double-hit process In vivo direct radiation action prevails, apparently due to shielding of DNA against radical action by proteins (histones), In aqueous solution the radiation sensitivity of DNA is considerably increased, because here radicals may produce an indirect effect, which may in turn be reduced by radical scavengers (such as histidin). Damage to bases can be caused by direct as well as indirect radiation action (base release, cleavage of the imidazol ring or of the heterocyclic hexagonal ring). The pyrimidine bases thymine, uracil, and cyto- sine are about 100 times more sensitive than the purine bases guanine and adenine. UV irradiation of pyrimi- dine bases —mainly of thymine —causes the formation of dimers, which dissociate again upon irradiation with visible light 6.5.3 Radiation Effects on Membrane Structures Membranes control the material transport in. and between the cells and as structure elements they play a ‘major role in bioenergetic processes. Radiation effects ‘on membrane systems are detectable overa broad range of radiation doses: Protein synthesis, located at the endoplasmatic reticulum, and photosynthesis, which298 6 Rauiation Biophysics occurs at the chloroplast membranes, are very insen- sitive to radiation. An alteration in the Na* and K* permeability of outer cell membranes and of neurons is detectable only after an irradiation dose of some 100 Gy, whereas functional defects of the central nervous system happen already after 0.5 Gy. Lysosomes, small membrane bags containing cata- bolic enzymes, might cause heavy metabolic damage by the uncontrolled release of their enzymes due to radiation. Indeed, an increased concentration of lyso- somal enzymes is found in irradiated cells; whether they are responsible for cell damage, however, still needs substantiation, Oxidative phosphorylation, located at the mitochon- drial membrane system, is comparatively radiation sensitive. Liver mitochondria break up after about 10 Gy, and a drastic reduction of oxidative phosphory- lation occurs in the thymus an analogous reduction has already been observed after 0.25 Gy. 6.6 Radiation Effects on Cells and Organisms 6.6.1 Radiation Effect on the Cell After irradiation with very high doses, in the 100 Gy range, all cells suffer the “interphase death”, a break- down of the complete cell metabolism, which cannot be related to a defined initial process In reproducing cells damage is observed after much lower irradiation doses. Bergonie and Tribondeau realized already in 1906 that X-irradiation had the strongest effect on cells having the highest reproductive activity, the longest mitosis’ phase, and the lowest morphological and functional differentiation. This “reproductive” death is defined as the loss of unlimited reproductibility: Cells are considered as survivors, if they have passed at least five-six division steps, ice., produced 32-64 daughter cells. Radiation sensitivity during the cell cycle varies very much; usually @ decrease in the direction 02,7 _Caliphase iy Time Fig. 66. Variation of radiation sensitivity during the cell eycle (Sinclair). Chinese hamster, D=7.1 Gy 0 2 @ 6 8 Ww of 8 © 20 Tine between two radiations mi Fig. 6:7. Influence ofthe length of interval between two iradiation ‘doses of 1.5 Gy each on the survival rate of cel. (After Elkind) M>G,>G;>Sis observed (Fig. 6.6). Since the dose- effect curves foritradiation at various stages ofthe cycle are of different types, different mechanisms of action have to be assumed. Already at sublethal radiation doses a prolongation of the cell cycle and a retardation of mitosis appear. These effects are also dependent on the cell phase. If the total irradiation dose is split into two or more fractions, applied at intervals, the effect of this “frac- tionated” irradiation is much lower than that of the single irradiation. This can be explained by the presence of repair mechanisms which ate able to eliminate sublethal lesions. The duration of the interval between the two irradiation phases determines the magnitude of | this effect (Fig. 6.7). ‘After treatment with densely ionizing radiation (high LET), sensitivity variation in the cell cycle as well as repair processes occur to a much lower degree. 6.6.2 Genetic Radiation Effects Asa result of the radiation effects on DNA mentioned above, chromatid and chromosomes breaks may occur, ‘while at the same time alteration of bases may produce localized changes of genetic information. Both pheno- ‘mena could be either lethal already in the mother cell, or lead to a mutation, a sudden modification of a gene. Most of these mutations are recessive, which means that they become expressed only if ina later generation two genes, mutated in the same way, encounter each other Investigations of the dose-effect curves for the occur- rence of mutations in the fruit fly Drosophila have demonstrated that the mutation rate showed a linear increase with dose and was independent of dose rate. This means that even the smallest radiation doses contribute to an increase in mutation rate and that this radiation effect is accumulated over long periods. The doubling dose for the natural mutation frequency of human cells is estimated to be 0.5 Gy.Recent investigations with mice, however, have dem- onstrated that upon a reduction of dose rate by a factor of 104, about three-four times less mutations occur than are expected; therefore a repair system also for genetic radiation effects has to be assumed. 6.6.3 Radiation Stimulation Dose-effect curves describing a quantitative parameter like plant yield often show, instead of a simple shoulder, a positive trend at low ‘radiation doses, sometimes considerably exceeding the 100% line. The maximum of this positive “stimulation” region usually lies at about one-tenth of the 50°% damaging dose. Although this phenomenon is sometimes utilized in agricultural practice to obtain yield improvements, its occurrence is strongly and unpredictably dependent on environmen- tal parameters and is difficult to reproduce. The stimulation effect may be caused by the fact that low radiation doses initiate repair processes which over- compensate for the radiation damage present and therefore lead to an increased net metabolism. Little is known about the nature of this effect; in some systems investigated, an increased RNA synthesis rate and a shift of the Redox potential seem to contribute to it 6.7 Radiation Hazards and Radiation Protection 6.7.1 Natural and Man-made Radiation Burden ‘The main basis for the estimation of radiation risk is the knowledge of the natural radiation burden and its relation to man-made radiation sources. Natural radiation sources act externally as well as internally (by ingestion or inhalation of radionuclides) on the human body. They consist of two components: Cos- ‘mic radiation originates from the sun and from space and contains primarily X rays and very energetic pro- tons. By interactions with atoms in the outer atmo- sphere numerous secondary products are generated, (e°,e°,7,n.. .) which are partly absorbed in the atmo” sphere; other parts reach the earth’s surface or even penetrate to great depths. By nuclear reactions with ‘components of the air the radionuclides ['*C] and PH] are also produced by cosmic radiation. Terrestrial radiation results from natural radionuclides in, the earth's surface. These radionuclides ((*K], UJ, F2°Th) possess half-lives of 10° years and more. The last two are starting points of radioactive decay serics which contain short-lived solid and gaseous elements that are also found in the air and as aerosols. ‘Natural radionuctides and their decay products may bbe taken up by the body either via the nutrition chain or through the lung and cause an additional “internal” radiation burden. Table 6.4 gives some data on the natural radiation burden. These figures are standard values only, how- 6412 Radiation Protection 299 ‘Table 64, Natural radiation burden [nSv/year] Reproductive ‘Bone marrow organs (1) Cosmie radiation Tonizing component 280 280 (2) Terrestrial radiation including air 00 00 @) Internal radiation duet incorporated 216 175 radionuclides — = 1003 962 ever; they vary strongly with the elevation above sea level and with the geological formation: At anelevation ‘of 2000 m above sea level the cosmic radiation contri- utes about 1.2 mSv/year. In monazite areas in India and in Brazil, where the soils possess a high thorium content, the terrestrial radiation burden is increased to a value of up to 100 mSv/year. An additional “artificial” radiation burden results from the medical use of ionizing radiation and radio- nuclides, from the professional use of radioactive material in science and industry, and from nuclear ‘weapons tests. Inquiries in the United Kingdom have shown that the medical application of radiation, aver- aged over the total population, results in an annual dose of 340 jSv for the bone marrow and of 190 Sv for the reproductive organs, In most countries an additional dose in the vicinity of nuclear power plants is prohibited by law from exceeding 300 ySv/year. The average radiation burden caused by the occupational use of radiation and radionuclides amounts to about 5 mSviyear, but only for less than 1/1000 of the population. The nuclear weapons tests performed up to now will add a total radiation burden of about 1.2 mSv for the world population until the year 2000. From these data it follows that as yet the radiation dose caused by man-made applications of radiation and radionuclides is clearly below the natural radiation burden 6.7.2 Radiation Protection Based on the knowledge on the natural radiation burden and on the assumption that no threshold dose for genetic radiation effects exists, the ICRP (Inter- national Commission for Radiation Protection) has recommended maximum tolerable doses for the exter- nal irradiation of individuals in the gencral population and for various categories of radiation workers (Table 6.5). These recommended maximum values take into consideration the differing radiation sensitivities. of various organs and also the fact that the genetic risk for a small group (the radiation workers) is lower than for the whole population. However, even within the range of tolerable doses the actual radiation burdens have to be kept as low as possible. The radiation protection3006 Radiation Biophysics ‘Table 68. Recommended maximum radiation doses [for individuals} (ICRP recommendation) [mSv year] General Radiation workers population CA CHB Total body, gonads 5 30 15 red bone marrow Skin, bone, 30 300100 thyroid gland Hands, forearms, 8 70250 feet, lower legs Other single organs 15 100 ‘Table 66. Maximum annual intake of radioactivity for radiation workers of Category A (ICRP recommendation) In ie Tn water and food (ahalation) (ingestion) [MBq year) [MBjyear] oH Dae 3x10? =p 3x10! 2x108 Mn 6x10 210? SMa 3x10! 1x10 "C0 bx? 210! vse 710" 1x10 ny Deg 1310? BCs x10! 4108 PR 2107? 7x10? laws and regulations valid in the different countries usually follow closely the ICRP recommendations, For the definition of the maximum amounts of radioactivity which may annually be inhaled (through the lung) on ingested (through water or food) some additional aspects have to be considered: the different enrichment of the individual radionuclides in different ‘organs, the varying radiation sensitivity of these organs, and the biological half-lives of the radionuclides (j.e., the period after which 50% of the activity taken up is lost by physical decay and by excretion). The values given in Table 6.6 are related to professional radiation workers of Category A. Lhey are calculated in such a ‘way that no organ will be exposed to an annual dose of ‘more than 50 mSy. The values accepted for the general population are lower by factors of up to 333 compared with those given here. Selected References Bacg,Z.M., Alexander, P.: Fundamentals of Radiobiology, Oxford, London, New York, Pars: Pergamon Press 196 Copgle JE" Biological effects of radiation. London: Wykeham 197i Dertinger, H., Jung, H.: Motekulare Srablenbologie. Berlin-Heidel- berg’New York: Springer 1969. Dessiuer,F.: Quantenbiologie (Ergin von. K. Sommermeyer, Hers), Berlin-Gittingen-Heidelberg-New York: Springer 1964, Errera,M., Forsberg, A.: Mechanisms in radiobology. New York- London: Academic Press 1960 Frit-Nigli,H.:.Strahlenbologie, Grundlagen und Ergebnisse ‘Stutigar: Theme 1959, Hiug.0,, Kellere,A.M.: Stochastik der Strablenwirkung. Becln Heidlberg-New York: Springer 1966, 1ea,D.E.: Action of radiaion on INing cell, 2nd ed. Cambridge: University Press 1956, Matheson, M.S,, Dorfman,LM.: Pulse radiolysis, Cambridge (Mass): MIT-Press 1969, Phillips, G.. (Ed) Energetics and mechanisms in radiation biology. London-New York: Academic Press 1968 Sureffer,C.: Strablen-Biochemi, Heidelberger Taschenblicher, Bd. 59,60 Herin-Hedelberg-New York: Springer 1969. Timoeff-Ressowsky,N.V., Ivanov, V1, Korogodi,V.J.: Die An- ‘wendung des Trelferprinzips in der Strablenbiologc. Jena: G. Fischer 19727 Isotope Methods Applied in Biology HeLmur Simon 7.1 Introduction ‘The rapid development of biology in the molecular field and in a quantitative sense would not have been possible without the extensive application of stable and radioactive isotopes. Apart from radioactive isotopes as radiation sources most isotope applications can be classified into one of the three main groups: 1. Isotopes and isotope-labeled compounds as ana- lytic acids, 2. Isotopes for marking the course of an atom, a group of atoms or a molecule in in vivo and in in vitro systems. 3. Analyses that apply isotope effects. In many cases, especially those involving analytic work, the process can be quite simplified and the detection limits for any substance can often be im- proved by orders of magnitude when labeled with isotopes. More important, however, isthe fact that with the isotopes and isotope-labeled compounds insight into the course of reactions can be gained, which often, for fundamental reasons, would otherwise not be possible ‘The main difference between applications { and 2 — to be termed “analytic” or “kinetic” methods for short — can already clearly be seen from the first tracer experiments in history. In 1913 Paneth and Hevesy determined the solubility oflead sulfide with the help of a natural radioactive lead isotope. They iden- tified a lead ion concentration of roughly 10" gil. The ‘method is very sensitive: it does not. however, achieve anything that could not be achieved with the help of other methods. The kinetic method, on the other hand, allows observations of exchange, metabolic, and trans- port processes that are only possible because in one and the same element various types of nuclei can be distinguished. Thus, in 1915 Hevesy observed how lead froma lead sheet exchanges with lead ions in a solution. In 1923 he analyzed the uptake and exchange of lead by plants. For this experiment, plants that had previously absorbed radioactive lead ions froma solution were put into. nutritive solution containing nonradioactive lead ions. After a while the plants no longer contained radioactive lead. An especially good example showing the simul- taneous application of all three possible methods mentioned above is the identification of the path of carbon during photosynthesis of green plants. Ra active carbon led to the establishment of the Calvin cycle (cf. Chap. 13.1). The plants differ, however, according to the path of the carbon dioxide before itis fixed in the carbohydrates and a varying isotope fractionation takes place. This means that the photo- synthesis products differ due to minimal differences in the isotope effects (cf. Chap. 13.1) in the ratio of the two naturally occurring carbon isotopes [!C] and [°C] (cf. Chap. 13.1). Which of the paths the carbon dioxide takes in a plant until its fixed in the carbohydrates can be deduced from the ["°C]/['°C] ati. 7.2 Stable and Radioactive Isotopes 7.2.1 Comparative Study The various isotopes of an element characterized by its atomic number only vary by the number of neutrons in the nucleus. Because of the equal number of protons, the number and grouping of the electrons are the same in the various isotopes of acertain element, and thus the chemical properties, too, are the same. For the minor influence ofthe differences of mass, .g.,on the reaction velocity of the various isotopes, see’ Sect. 7.3. The influences of the varying number of neutrons on the ‘mass and other magnitudes depending on it, as well as magnetic and nucleochemical properties, are shown in Tables 7.1 and 7.2. Naturally, corresponding differ- ences are to be found also in the radioactive isotopes of an element. However, radioactivity is a magnitude so easily measured that as.a rile no use is made of the differences based upon mass and magnetism for mea- suring purposes. In addition, the proportion between radioactive and stable nuclides is so small,e.g., <10~*, in most tracer methods with radioactive isotopes that spectroscopic and other methods, which do not mea- sure radioactivity, ae not sensitive enough to be able to identify the low concentration of radioactive isotopes. Some properties of important stable isotopes are shown in Table 7.2.and in Table 7.3 those of radioactive isotopes are shown, There exist no radioactive isotopes with a long enough life-time of the important elements nitrogen and oxygen. If stable as well as radioactive isotopes of one element are available, the application of one or the ‘other sort is determined by the nature of the problem to be solved. A number of aspects in connection with the application of stable and radioactive isotopes are summarized in Table 7.4,302 7 Isotope Methods Applied in Biology ‘Table7.1. The different number of neutrons in the variousisotopes ofan element influence is mass and magnetism. From this, differences inthe isotope properties that can be applied as measuring units can be derived Property ofthe Depending magnitude Measuring unit with ‘Measuring method Information of| nucleus inatoms and molecules isotope effect measurement Mass ‘Atomic and Specific charge, ‘Mass spectrometry, Quantitative and smoloculae weight Velocity of sltracentifupation positional sedimentation, Semiquantitative Densimetry: quantitative chromatography Semiquantitative Energy and Absorption, emission Infrared spectrom. Positional and polarization and polarization", eurys Semiquantitative emission spect ‘quantitative analysis, Polarimetry, optical semiquantitative rotatory dispersion, ‘croular diciroism Magnetism Magnetism and is [Noclear magnetic [Nuclear magnetic positional and reciprocal effet resonance, esonance spectroscopy, Semiquantitative on other cia Electron spin resonance lectron spin resonance Snd eleetrone (eierostructre), sectroncopy, Eyperfinesructare Mssbavet spectroscopy of absorption (Missbauer efor) Absorption of Absorption of [Nuclear process Determination of quantitative corpuscular corpuscular emitted radiation radiation radiation + Is also influenced by the magnetism ‘Table 72. The properties of some stable jentopes Flement Symbol Relative natural Nuclear mass Nuclear magnetism Chemical sit {Daton (opm) Spin quantom number Momentum Us) ydeoaen 939885 hones 12 0 270268 unis youu ‘0 dass carbon Bomoonn 0 a ° Bomsss 12 200 026 Niteosen ieoosoms sco dian3s7 13663 iSovost 12 800 38304 omen on387 Bows 0 e ° dias ieooitss Sa 0 —1s900 113039 isis 0 = ° Phosphorus NP 100.000 josie 2 wo {130s sole BS 35018 pignoe o 8s 0.780 sono Sp oars 8 ais Boone ° 8 on soon 5 Chore C1 87708 soos 32 082088 val nines 366566 32 ‘68308 7.2.2 Stable Isotopes and the present in 100 nuclei of the isotopic mixture. This Principles of Isotope Measuring relative natural abundance (ct. Table 7.2)is expressed in atomic percentage. Applying correspondingly exact When working with stable isotopes the following measuring methods, e.g., mass spectrometry, it be- parameters and quantities to be measured are of comes apparent, however, that natural abundance is importance: relative natural abundance, atomic per not a natural constant, but that deviations in the per centage, and atomic percentage excess. Atomic pet- thousand range occur as, e.g,, with ["C] (cf. Sect. centage signifies the number of nuclei of the isotope 7.3.2). Besides this, the atomic percentage excessis often‘Table 73. Properties of some radioactive isotopes 7.223 Radioactive Isotopes 303. Element Symbol—‘Half-life Max. energy 7 eneray Mass (10° kg) Range of. indx10- dx per GB Brradiation respectively type inthe water (m=) of radiation ‘iT 12260, 007 = exo Sel st60a 025 = 594 Sodium Na 15h 22 44; 221 305 x10-* Magnesium — Mg 24h 087 O0x-1358% KE 54 x10" Phosphorus =P. iad 2n 973x107 Phosphorus SP. 24 040 = 1 7010-* Sulfur a 6356 0267 = 62710" Potasium 8K 24h 573; 3.26 243; 0.51 43. <10-¢ Calum Ca 1654 040 = 678% 10-* ron Fe 27 EC L210 Pe 454 021; 25019 549 x10-+ 0.418% 10? Cobalt Co. 5.26a ) 238x107 0.10% 10? oS 00%) 2.13,100%%) Zine “20 masa Fostitto 083 von f 329x107? 178 49%) Bromium Br 3598 088-237 0.47%107? Iodine say oa 0.036 Iodine my oud oueias 0.25% 10? © Abbreviations: EC= Eleccon capture KE=Conversion electrons, Further explanations see p. xxx 1 MeV (Millon electron volt)=1.60 x 10°" J ‘Table 74. Aspects for the application of radioactive isotopes or stable isotopes ‘Radioactive notopes or ‘Stable atopes Detection sensitivity usually higher by orders of magnitude. Mea- Degree of relative measuring accuracy, e.g. by means of mass suring often possible in the presence of large amounts of other materials ‘Studies of the distribution by means of radioautography can be carried out right into the inracslular range. Easy isolation of imponderable quantities by means of chromatographic procedures ‘Complete replacement of one molecule postion by a radioactive nuclide is possible, however substances are then prone to rapid Autoradiolsisand can only be manipulate in very sll amounts, ‘due to dangerous radiation. Application outside special isotope laboratories soften not possible. ‘Thedetection ofan isotope position ina molecule requires chemical degradation, ‘With the Fourier-NMR spectroscopy tritium can be detected in a rmolectle postion, i the activity is roughly 1-5 107 Bq, applied, This represents the difference between the atomic percentage of a given preparation and the atomic percentage of natural abundance. The differences in the mass of nuclei are the basis for a number of analytic methods (Table 7.1). By mass spectrometry and by possibly ultracentrifugation, as well as by gas chromatagraphy, labeled ancl nonlabeled molecules are separated. Another group of mass- dependent methods is based on the measurement of & quantity that is proportional to the isotopic composi- tion. Such procedures (e.g,, densimetry) require ex- tremely pure preparations for measuring. Optical methods can often be applied to mixtures, since they lead to separate measuring signals. Spin quantum number and nuclear magnetic momentum are spectrometry substantially higher. Thereby exact determination of isotope effects possible Dilution Fimited due to natural abundance. For measuring purposes the substances must usually beuvailablein pure form or extensively enriched. ue to the possible high degree of substitution in single molecule positions, nuclear properties that are influenced by the chemical bond and the surroundings of the isotope can be measured. From this the intramolecular labeling position can be detected. However, important intramolecular correlating forces ean also be detected BBy means of NMR measurements the turnover of cell material or interactions inside the ell structures (membrane) can be observed, Iniramolecar doubl-abeling possible No security measures need to be taken, especially important for isotopes of the elements of which the mainly naturally occurring element shows no values other than zero. This is especially the case with [°C] and [°C] (ef. Table 7.2), 7.2.3 Radioactive Isotopes A preparation has the radioactivity of 1 Beequerel (Bq) when one nuclear decay takes place per second (dps decays per second). The unit Curie, still often applied, is equivalent to 3.70- 10" Bq, Since { min is a more suitable time unit for measur- ing. dpm (decays per minute) is usually applied; 60 dpm 1 Ba.304 7 Isotope Methods Applic in Biology Specific radioactivity stands for the number of Bq per gram or per mol (or subunit) in a. substance. Specifications such as dpm/mol are usually applied. As shown in Table 7.3, the energies of the emitted radiation is stated in million or kilo electron. volts (MeV, keV), Here f~ and f*-radiation (negative or positive electrons) and y-radiation (electromagnetic radiation emitted from an atomic nucleus) are of interest. Electron emission can also be caused by conversion electrons, The energy difference between two nuclear states is not emitted in form of j-radiation, but transferred to an electron of the atomic shell, which is thus released. Contrary to f-radiation (see below) these electrons are ‘monoenergetic. As symbol, e~ isused. The conversion ratio means the ratio: number of conversion elec- trons/number of y quanta. Further electromagnetic radiation can be caused by electron capture of the nucleus from the shell. It is not rare for this process to compete with f*-radiation, The falling in of electrons from the outer shells into the gap of the lower shell (mostly the K shell) leads to a characteristic X-radiation. In its place an electron can also be emitted (inner photoelectric effect), the kinetic energy of which is equal to the energy of the character- istic X-radiation minus the binding energy of the electron. Such electrons are called Auger electrons, ‘The number of atomic nuclei disintegrating per unit of time is proportional to the nuclei available in each aN a N stands for the available number of nuclei, for the quantity that decays in the time interval dt. The disintegration constant is 2 Integrated from 0 to 1 the result is N=Noe™ AN. (1) a) If f equals the time after which the radioactivity of a preparation has becn reduced to hall (half-life period) (¢ = T;,2), then Np is twice as much as N, and it follows that (7.3) From the half-life, and according to Eq. (7.1), itis possible to compute how many atoms of a radioactive isotope make up, e.g., 1 GBq. Carbon-14 has a halflife of roughly 5760 years=1.28 x 10" s, The result is an dt In2N Tin” N=2.62x 107° atoms of (§4C] are 1 GBq, Taking Avogadro's constant (6.02 10°*) and the ‘atomic weight into consideration, the result for 1 GBq [C] is 5.94 x 10°° kg (ef. Table 7.3). Emitters with a maximum energy of more than 1.6 x10" J are considered hard emitters (as, e.g, [°*P] 0.693 «N 10% = 0.693 x VITO with 2.7 x 10-3 J or Na] with 2.2% 10 J energy of B-radiation). Emitters of average energy are considered as such with energies of 0.3-1.6%10 "J (e.g. [J] with 1x10"? J Soft emitters are emitters with maximum energies of less than 0.3 x 10-" J ((!8C) with 0.24 10-8 J, 5S] with 0.2710 ® 4, or PH]=7 with 0.029 x 10"! J). Electrons of 0.6 x 10°? J already have 94% of light velocity. Within a certain range the penetration capacity of electrons can be described half-quantitatively by means of an exponential function: A= Age™, (5) ‘Ag stands for the activity measured after the radiation hhas penetrated the thickness of a layer d of an absorber. p stands for the activity per unit of time and area without the absorber, 4 stands for the absorption coefficient. As a rule, the thickness of the layer is not stated in units of length, but rather in 10° kg/10~* m? with B emitters, since the chemical nature of the absorbing material has only little influence on the numeric value of 4. 7.2.4 The Most Important Measuring Methods for Radioactive Isotopes ‘The physical bases of the measuring methods are ionization (ionization chambers, proportional counter, Gciger-Miiller counting tube, semiconductor detectors), scintillation, and radioautography. The “Cerenkov counters” play a role for f-emitters with maximum, energies = 1.0 MeV. Only a fraction of the disintegra- tion rate (dpm) of a preparation is registered by the detector. For the counting rate cpm (counts per minute) is applied: cpm =CE x dpm. The countingeefficiency CE < 1.0isinfluenced among others by the following factors: geometry of the emitter to the detector, absorption of radiation in the prepara- tion, in the detector wall, ctc., and counting efficiency of the detector. In addition, every counting rate cpmg, no ‘matter how it has been registered, is made up of two constituents: the counting rate of the sample itself (cpm,), and background radiation (cpmy,). Back- ground radiation is the counting rate due to cosmic radiation and the always present ambient radiation ‘The error of an observed counting rate is computed as follows: Sea= W/epm x (7.5) Here S.4 stands for the relative standard deviation (68% confidence interval), r stands for the measuri time.’ The ratio is obtained from the Poisson distri bution to which the radioactive disintegration obeys as ‘purely statistical process. Itis obvious that the relative error becomes smaller with an increasing counting rate and/or longer measuring times. In case the background radiation is not negligible in comparison to the sample counting rate (cpm»=cpmg —cpmye), Eq. (7.5) has tobe extended: (76) Here fg and tye stand for the time needed to determine emp and cpm, ‘The fact that counters never register absolute values free of background radiation is usually insignificant for tracer technique, since relative measurements are suffi- cient. To be able to carry these out, measurements have to be done under exactly the same conditions or with precisely known counting efficiency. 7.2.4.1 Counting Tubes ‘A counting tube consists of an appropriate tube filled ‘with gas, into which, isolated, a thin wire is inserted that shows an electric high voltage against the tube. When a Aiparticle enters the counting tube, .g., through a thin- walled window, it collides with the electrons of the gas molecules. Thereby electrons are kicked out from the atomic shell. In the high voltage electric field, these are accelerated on their way toward the wire. Their energy may become so high, that in turn they kick out electrons from gas molecules. The constant repetition of this effect leads to an electron avalanche, and finally to a voltage pulse on the wire, that can be registered. The experimental result of a solid radioactive preparation situated outside a counting tube is a complex function of the preparation’s layer thickness, its position in relation to the counting tube, the thickness of the window, etc, The substances that are to be measured can also be converted into gas and this can then be filled into the counting tube. This method is expensive but very exact and sensitive, 7.2.4.2 Scimillation Counters When electrically loaded elementary particles hit phos- phorus light flashes (scintillation) develop. On photo- ‘cathodes of secondary electron multipliers (photomul- tipliers) the scintillation flashes produce electrons that hit other electrodes and release secondary electrons. ‘The avalanche-like process intensifies the initial pulse by 10°- to 10°-fold so that it can be processed further electronically. An analog pulse amplitude produced by the photomultiplier is proportional to the clectron energy absorbed in the scintillator. A differentiation is made hetween solid and liquid scintillator systems, Solid scintillators are mainly used for y emitters. Theit energy is too high to be able to directly excite scintilla- tors. During the absorption of the 7 quanta fast electrons develop that cause light quanta in the phos- phorus. Since with electrons produced by 7-radiation, scintillation is only one of the possible slowing-down processes, only part of their energy is transformed into light quanta. With thallium-doped sodium iodide 7.2.42 Scintillation Counters. 305 (Nal(TD) one calculates with an effectiveness of 12%-14% With energies >50 keV the number of released quanta is proportional to the electron energy. With low energies an increase of extinguishing effects is noticed. Nal(Tl) and CsI(T1) can be applied as solid scintillators. Organic scintillators, like anthracene, trans-stilbene, and therphenyl, are only applied when better resolving times are aimed at with the help of their short decay period. The intensity of scintillation is proportional to the energy that the 7 quanta deliver to the scintillator. For identifying an impulse height distribution that is analogous to the spectrum it is possible to form a channel (window) with two discriminators and then examine which impulses fall into this channel. By continuously shifting this channel and registering the impulses, the required impulse height distribution can be found. Such measurements can, however, be made much faster in multichannel analyzer in which the appropriate energy sector is divided into a number of channels (e.g., 512). In these channels the impulses are sorted out and registered according to magnitude, The impulses stored in the individual channels can be printed out or registered. The procedure of liquid-scintillization counting is, simply speaking, based on the fact that during disintegration of a substance dissolved in an appro- priate solvent, the disintegration energy to a large extent is transferred to the molecules of the solvent. If, in addition, the solvent also contains scintillators, the excitation energy of the molecules of the solvent can be transferred to these. During their transition into the ground state, light isemitted which can be registered by a photomultiplier. In the end, the result is a photon impulse height spectrum that is analogous to the disintegration spectrum (Fig. 7.1). Disturbances of the energy transmission between molecules of the solvent, or absorption of the emitted light, are called quenching. It means a decrease of Background radiation AL Channel window) \ vies _ \ a ali al 3|\ \ \ reer Fi. 74. A photon pe ht pecan sons othe 8 disintegration ofthe isotopes PH), ("C]. and ["P] in dependency of| the eneray (logarithmic). In the energy region (channel, window) between the discriminators A and Ball impulses of tritium, but also such of (*C] and (*P] are registered. In the channel B-C impulses of [Cand (P} and above the discriminator Conly those of (*P] are registered. Th discriminator A mainly eliminates noise impulses, an ‘expecially large Faction of which i to be found inthe extremely iow nergy rte306 7 Isotope Methods Applied in Biology the cpm values. If the sample inhibits the transfer of energy at the level of the solvent, this is called chemical quenching. If the emitted fluorescent radiation is absorbed by dyed material this is called colour quench- ing. In practice every sample shows more or less quenching, In Fig. 7.1 the ordinate corresponds to the fre- quency of the impulses 4.V/.V of a certain energy. Back- ground radiation is kept as low as possible. This is achieved by means of impulse discrimination, whereby the spectral sector of the sample, optimized against the background radiation, is faded out by means of two discriminators. Figure 7.1 shows how, with the help of scintillization counting, multiple-labeled samples can be measured when the isotopes vary by more than a factor of S in their maximum energy. The liquid scintillation counting is actually the most frequently applied measuring technique in the analytics of f emitters. 7.2.43 Radioautographic Procedures Radioautographic procedures allow a qualitative, and at times also a half-quantitative, but inany case an exact localization of radioactive areas on an object, e.g., ona thin section of a test animal, an immobilized cell suspension or a chromatogram. Classic radioauto- graphy takes advantage of a blackening of a photo- emulsion through radioactive radiation by bringing the film onto the object. For an optimal blackening of X-ray films 10%10F electrons pe em? of film, and for a just visible blackening 10°-10° electrons have to it the film, depending on the energy of the f emitter. With tritium thenecessary product “radioactivity x time” for visible blackening of a film is roughly 60 times higher than with ('*C]. In the case of microradioautograms, ¢.g., of histo- logical preparations, a quantitative evaluation is pos- sible through silver grain counting, or in the case of ‘energy rich emitters through track counting. This kind of evaluation is often applied. However, the in most ceases not ideal contact between the film and the object will have to be substantially improved. Therefore the object to be examined is coated with a special nuclear emulsion. Compared to the frequently applied X-ray films, these have a higher concentration of silver bromide and in addition the grains are substantially smaller and of uniform size. Thus an essentially higher degree of resolution is reached, and the sensitivity toward soft f emitters is increased. After exposure the film is developed, the silver grains precipitating directly nto the object. Then both are examined under the microscope. Very strong magnifications are obtained with electron microscopic radioautography. Here film emulsions with grains of 0.03-0.01 um are applied. Resolutions of 0.10.3 jm are obtained. In photograph- ic emulsions the mean range of f emitters amounts to roughly 1 um in the case of [°H), 10 ym in the case of (C] and [S], and 800 ym in the ease of [P]. When applying ['H]-labeling, physical parameters of macro- molecules can be made visible. With molecular radio- autography it was for instance possible to observe the replication of a E. coli chromosome. 7.3 Isotope Effects 7.3.1 Main Causes of Isotope Effects When applying isotopes itis generally assumed that the isotopes of an element, which vary in weight, react uniformly, i.e., that in the process of any physical or chemical reaction no discrimination of labeled and nonlabeled molecules takes place. However, such dis- criminations can be observed more of less clearly in the case of quantitative measurements, These isotope ef- fects mainly depend on mass differences of the isotopes and the thus resulting differences of the zero-point energies of the bonds (cf. Fig. 7.2) || [Soe seats cme “TPT, aston st) z € é emaarce \ es Nuclear stance — Fig. 72. Potential curve ofa molecule HX and of the transition ate ‘The asymmetry of the potential energy curve extremely exaggerated {for better distinction) isthe reason forthe smaller X-*H distance in ‘amparison tothe X-H bond. The varying zero point energies Band 2B cause different activation energies fora reaction (arrONS), and are ‘the main cause of the Kinetic isotope effect For a first rough approximation it can be said that the zero-point energy of & chemical bond is propor tional to the oscillation frequency. The following applies to the frequency 1: ve a . an Here k stands for the force constant and =~" for the reduced mass. mm, In the case of a C-['H]-bond and a C-PH]-bond the force constant k is the same. The reduced mass 11 ratio for both bonds is roughly 1:2. Consequently, vn2¥oy=2)/2:1 ~1.421 applies for the ratio, This can for instance be very clearly seen in IR-spectra When taking a look at, c.g, the IR spectra of chloroform CHCl; and CPH)Ch, it can be observed that the C-Hestretching frequency appears at 3050 em=1, and the C-PH]stretching frequency at 2260 m=", respectively. This corresponds to a ratio of 1.35:1. As ean be seen from this, the isotope effects arethe smaller, the less the mass-difference percentage of two isotopes. They are already negligible in the case of carbon isotopes. Contrary to this, the influence of isotope effects must always be accounted for during the application of hydrogen isotopes, since experimental results can often only then be interpreted. This is especially the case when, in the course of a reaction, bonds of an isotope are fractioned in the rate determin- ig step. Basically, a differentiation is made between netic” and equilibrium isotope effects. Equilibrium isotope effects can be observed, ¢.g., in ‘the case of phase transitions or exchange reactions after establishment of equilibrium, For the exchange equilib- rium between water and deuterium containing hydro- gen sulfide H.0+ PHS & PHIHO+H,S at 25°C the following value applies, &;/k This is an extreme value. ‘The fact that acidsare substantially weaker in [?H],0 than in HO is also a consequence of equilibrium isotope effects. The difference is the bigger, the weaker is the acid. Thus the coefficient of dissociation of chloracetie acid in HO and PH],0 is Ky/Koy=2.74. For acetic acid the ratio is 3.33, and for the hydrogen carbonate anion it is 3.95. For heavy nuclides the effects are again much smaller. For the following two reactions the equilibrium constants are 1.034 and 1.017 respectively (NJ + ENTE [NDE + NINE (PC]Os +EC]O,8(C]O3" +(7C]0, ‘The ions are in water and NH; and COs in the gas phase Generally speaking, it can be said that the distribu- tion of an isotope between two molecules A and B during the reaction A+BISA'+B ‘equals the product of the quotients of the distribution function Q in the ground state of labeled molecules A’ and B’ respectively, and the nonlabeled A respectively B and the zero-point energies of the ground state. Herew=hxv/kT,h normal oscillation, k- solute temperature, 1: lanck constant, v= frequency of joltzmann’s constant, T=ab- jumber of degrees of freedom. 7.3.2 Kinetic Isotope Kifects and Their Determination ‘A kinetic isotope effect is computed from the quotients of the reaction rates of the labeled and non-labeled compounds. The determination of kinetic isotope effects allows insight into the reaction mechanism, This determines 1.32 Kinetic Isotope Effects end Theie Determination 307 their importance, They are different from 1.0, when the bond taken into consideration is broken during the rate-determining step of a reaction sequence. A differentiation is made between the following isotope effects (cf. reaction scheme below, the higher index number stands for the heavier isotope): A\CA,+B 8s A\C+A.B A:CA, +B #45 A\C+A.B intramolecular or primary isotope effect secondary isotope effect ky (Dhar ¥ haz Kinetic isotope effect, also called intermolecu- lar isotope effect, Here n stands for, e.g., the number of hydrogen atoms or other identical groups on a carbon or other atom. If no other positions of equal valences are to be found on a molecule, i... n=1 and the isotope bond is broken during’ reaction, k2, becomes inapplicable, and the result is a kinetic or intermolecular isotope The splitting off of a deuterium from carbon, nitrogen, or oxygen can be two to ten times slower than the splitting off of protium, meaning that the isotope effect ky/kay=2-10. The following applies tothe relation between the rate constant for the splitting of XH, X—(?H] and X—PH] bonds: iat craton feu) Row The maximum isotope effects during the rupture of bonds between carbon-carbon, etc., are given in Table 7.5. Table7.5. Maximum kinetic isotope effects duving the rupturing of bond between heavy atoms. The loss of a stretching oseltion at 25°C in the ground sate is assumed Bond orem") ike Cte up CN 4 CO 3 + kalhe=19kulky 09. For the determination of kinetic isotope effects two methods are mainly applied: 1. Separate measuring of the reaction velocity of the labeled and nonlabeled compound (noncompetitive method). 2. Simultaneous competitive measuring of the reac- tion velocities3087 Isotope Methods Applied in Biology For method 1 the availability of sufficient quantities of isotopically pure compounds sa precondition, and it requires an experimental procedure that is absolutely ‘comparable, which is usually difficult to achieve. If the labeled molecules are only available in trace quantities, as is the case in tracer experiments with actively labeled compounds, the following equa tions are valid for the calculation of the isotope effect IE: na-/) (79) (7.10) Here R=specific radioactivity and the indices, a=re- actant, b=product, O=beginning of the reaction, ‘f=the extent of the reaction at the time 1 With mass spectrometry, isotope effects and isotope content of stable isotopes can be determined relatively with great accuracy. Since the exact isotope content of every material depends upon its previous fate, the values are referred to internationally accepted standard substances and the deviations are stated in per thou- sand. Thus, e.g., for the ['°C] content of a material the following is valid "CIP Coampte = PCI Caanaed 1999, (7.11) 8° O%9 =: 7.4 Analytic Isotope Application The possibilities of applying labeled materials for analytic problems are almost unlimited. Here, only a few principles can be mentioned. 74.1 Activation Analysis If neutrons, charged elementary particles, or high- voltage photons are fired onto stable atoms, these can be transformed into radioactive nuclides. By measuring the radiation being emitted by the sample, it is often possible to quantitatively determine the presence of certain elements. This method is used for the deter- mination of trace elements. Since most elements with atomic numbers > 10 practically have effective cross sections for the thermic neutrons (ef. P. 224) m1 reactions are often used. The four main elements of biological materials, H, C, N, and O, are hardly activated through thermic neutrons, Therefore, thermic neutrons are very useful for determining trace elements in tissues, The activation analysis was often applied to the trace analysis of Zn, Cu, Mn, Mo, and Co. These metals are essential constituents of a number of enzymes. For toxicological studies, too, the activation analysis is of interest. As an example, the determination of arsenic in a sample of Napoleon’s hair come to be known. A length of 1-2 mm of hair was sufficient to prove the presence of ['°As] by means of the [’“As](n, 7) reaction, and to prove the fact that Napoleon had taken an unusually high amount of arsenic during the last years of his life, The activation analysis can also be applied in tivo. The amounts of calcium, sodium, and chloride ions were determined through total body radiation of human beings and animals by fast neutrons with varying energies, ‘A comparison of the isotope composition of chemi- cally absolutely identical materials, formed in different ways, as a rule shows slight but significant differences Higher plants show differences in the first step of the fixation of carbon dioxide. As a consequence the 3C/2C ratios of their organic material differ. 7.4.2 Isotope Dilution Analysis There are a number of varieties in dilution analysis ‘They are mainly based on the determination of specific radioactivity or atomic percentage excess. It is applied especially when the separation of a substance from a mixture can be reached in pure form but not quantitatively. In the case of the direct isotope dilution analysis, the substance to be determined is added in a labeled form, in an exactly determined quantity and specific radioactivity. After blending, a small amount of the substance that is to be determined is isolated in absolutely pure form, and the specific radioactivity determined. Ifa quantity M, with the specific radioactivity S, was added and the substance with the specific radioactivity S, was isolated, it must follow that My * S)=53(Mi+M,) and ‘Si-S2 (ES In this context it can be seen that the determination of M, is unreliable when the difference $,-S; becomes too small and thereby inexact. S,-S, becomes small when the quantity of added M, is substantially higher than ‘M,. With an extremely high ratio M,/M, a determina- tion of M, becomes impossible. In this case a labeled reagent with a known specific radioactivity is added to the substance mixture, whereby the substance to be determined, and perhaps other substances too, are quantitatively transformed into a labeled derivative (derivative dilution analysis). The excess of the labeled reagent is removed and later the nonlabeled derivative is added and a reverse dilution analysis is carried out. This procedure is very suitable for the determination of even the smallest quantities. The derivative dilution analysis is, however, based upon the precondition that the substance to be deter- mined and the labeled reagent form a uniform deriva- My (7.12)tive as to quantity. I this is not to be achieved, another variation is possible. The substance to be determined is added to the substance mixture in labeled form (e.g., with ['*C)), and later a reagent is added which is labeled with another isotope (¢.g.[H] or (*S). After the reaction has taken place with the labeled reagent, a nonlabeled derivativeis added and a certain quantity needed for the de- termination of radioactivity is isolated, Later both isotopes are analyzed separately and from this the content of the substance to be determined is established in the mixture. ‘As was mentioned at the beginning, compounds labeled with stable isotopes can also be used for dilution analyses. The relative natural abundance limits the possible diluting factors. tis possible, however, e.g,,t0 determine the ["°C}/{*2C] ratio of a sample in relation to a standard sample with an accuracy of 0.1% There- fore, the addition of a compound labeled almost quan- titatively with [°C] in a carbon position can, compared to the nonlabeled compound with natural abundance, still be detected in a ratio of 1:10° 74.3 Radioimmunological Analysis The radioimmunological determination of hormones or of other biologically effective substances that occur in low concentrations is a very important variation of the isotope dilution analysis (radio-immunoassay) Here, use is made of the principle of competitive protein-binding analysis. If antibodies against a sub- stance that is to be determined, e.g., insulin or steroid hormones, are brought together in the presence of the a Ie 6a Reaction we we) Nod) y SOR Sa Reaction § ee Fig. 73. The principle of Radioimmunoassay (RIA), A A defined (quantity of aioactiveantigen a and defined quantity of antibodies Mare added to a solution containing the molecules that are to be determined (antigen) and any other molecules O. The antibodies ‘must have lessondingsites than and a topether. The quantity ratio Of a to be determined, and the added quantity a in the antigen- Sntibody complex andi, which isolated =1:3. Tt follows that the Amount ofthe (radioactive) antigen molecules that had been added, was thee times as much a6 the amount of non-abeled molecules brginaly in the solution tobe determined, B As under A. The ratio of the amount of & to be determined to the added amount 4 in the ‘complex =11, Tt follows that there were as many molecules to be determined present as were added in radioactive form 75. Studies of Distribution 309 radioactively labeled substance a competition develops between the radioactive and nonradioactive molecules over a limited number of bonding sites on the protein (antibody). The ratio of radioactive-labeled and non- labeled molecules on the protein corresponds to the quantity ratio in the analysis setup. If the bonding protein and the radioactive substance are kept constant, the fewer radioactive molecules will be bonded, the ‘more nonradioactive molecules exist. In such a way, €-g,, estrogens in the range of 10°° g or amphetamines in $0 ul of urine can be quantitatively determined (sce Fig. 7.3) 75 Some Examples for the Application of Isotopes ‘The manifold possibilities of isotope applications are best illustrated by a number of examples. It is amazing what different kinds of biological phenomena can be studied with the help of isotopes. The possibilities range from the determination of age, over studies of distribu- tion where radioactivity, e.z., serves only as indicator for the movement of an animal, to tests on a molec- ular level with analyses of complex. mechanisms, such as the path of carbon during photosynthesis or the protein biosynthesis, and finally to questions concern- ing stereochemistry and the determination of the veloity determining pace in in iow or in tro enzyme The following fact must, however, be stressed: isotope methods are extremely potent. This is valid for the possibility of achieving interesting “correct” results, as well as for the possibility of producing “nonsense” which will not immediately be recognized as such. Only very carefully planned, neatly carried out, and critically evaluated experiments give the “right” results. 7.5.1 Studies of Distribution In order to be able to trace the movements of, ¢.2., larvae of wireworms cobalt-60 was implanted in theit body cavity. The penetrating y rays were to be registered above the ground’s surface. Data about the number and movements of mosquitoes and other insects were found by feeding the larvae with [°2P)-labeled phos- phates, and then releasing the insects in the area that wwas to be examined. By putting up traps in certain areas and at certain times it was possible to carry out a dilu- tion analysis from the known “specific radioactivity” of the insects. Mention should also be made here of volume and space determinations. These again are based on the principle of the dilution analysis, To be mentioned are the determination of the water content in living human or animal bodies or the determination of “space” for special compounds or compound classes. The following example serves as an explanation: plasmaprotein is, e.g, labeled with iodine-131, (The aromatic rests of the310 7 Isotope Methods Applicd in Biology amino acids are thereby iodized through electrophilic substitution.) A quantity that is small in comparison to what might be expexted in a test animal, e.g., a rat, is injected. After equilibrium i established some plasma is taken and the radioactivity of the protein is determined. Assuming that 4 mg with 100,000 cpm/mg of protein hhad been injected, and that the isolated material shows 500 cpm/mg the protein plasma content is calculated to be 800 mg. If, at the same time, through chemical analysis, 70 mg protein were found per milliliter of plasma, the “space” of the plasmaprotein is calculated to be 11.4 ml In the field of pharmacology and medicine, studies about absorption, distribution, and secretion of chemo- therapeutica, and in more recent times all their decom- position products are becoming increasingly important Here, radioautography plays an important role. 7.5.2 Metabolism and Transport 75.21 Typical Cases and Results In every living system all constituents are in a dynamic state, While many materials, .g.,in the blood, are to be found in concentrations which, except in pathological states, only slightly vacillate, a single molecule has a characteristic life-span before it is resorbed or decom- posed, This efflux corresponds to an influx which leads tou steady state. Therefore, metabolism and transport must, asa rule, be considered together. Therefore, many phenomena concerning transport were studied with the help of labeled compounds. Studies of absorption by microorganisms, the transport of substances in plants, or the absorption of nutritive substances through the membranes of the digestive system in animals, are only some examples of important fields of research that often supplied fundamental scienti findings, Already in the 1930's rats were fed with [!°N]- labeled amino acids and it was found that these were unexpectedly fast incorporated in the proteins of the liver, though the total protein content of the liver was not changed. This means that there is a decomposition rate exactly corresponding to the synthesis rate. The halflife period of a rat’s liver proteins was determined as 5-6 days. The proteins of the muscle, on the other hand, have a half-life period of 30 days. In the meantime, much research data became available for ‘numerous substance groups in various organs of dif- ferent living organisms, With the general availability of carbon-14, tritium, and other isotopes (cf. Tables 7.2 and 7.3), and the development of methods, important metabolic path- ways and the biosynthesis of many low-molecular substances were clarified during the 1950's. The path of carbon during photosynthesis in green plants, the biosynthesis of pyrimidines, purines, porphyrines, amino acids, steroids, etc. belong in this category. The Krebs cycle was formulated without the application of isotopes, however it was only through isotope studies that the role of acetic acid became clear, as well as the fact that it is here a matter of synthesis without net synthesis. Then in the 1960's mainly the biosynthesis of macro- molecules, such as nucleic acids, proteins, and poly- saccharides, was clarified with the help of isotope techniques. The procedure for clarifying the biosynthesis of a Jow-molecular compound was usually as follows: a possible precursor, mostly labeled with [“C], was applied to the biological system (raw enzyme prepa- rations, whole or broken cells of microorganisms or organs, whole plants or animals). After a certain time, which often had to be found out by experimentation, the product was isolated and its radioactivity was determined. On the supposition that the precursor reaches the site of the product's synthesis, the product's radioactivity is a necessary but not sufficient precondi- tion for the existence of a direct biogenetic linkage between the applicated compound and the product. A comparison of various possible precursor compounds, shows that as a rule the biogenetic link with the product is the tighter, the higher the rate of incorporation, respectively the rate of specific incorporation is. Rate of incorporation stands for the ratio of the amount of isotopes found in the product to the total amount that had been applicated. Specific rate of incorporation stands for the ratio of the specific radioactivity of the precursor to that of the product. The isotope distri- bution was determined by the product’s decomposition. Because of this, hypotheses could be developed which were to be checked by means of further experiments. Until all detailed questions were answered such re- search, at times, could take a couple of decades. One example is the cholesterol biosynthesis. By applying deuterium-labeled acetic acid Bloch had already in the 1940"s demonstrated that there was a biogenetic link between acetic acid and cholesterol. Later, double labeled acetate '“CHy!"COOH was incubated with slices of liver, the synthesized cholesterol was isolated, and (after dilution with carrier material) the carbon isotope distribution was determined according to the following scheme. The quantitative evaluation sree tvooeieh Now Mevalonic acid 4 ow Ca vA ‘Scheme: Cholesterol biosynthesis from 1-{°C],2{C]aceti acid via mevalonic acid. [°C]=X, ["C]=0. The carboxyl group paren- thesized in the mevalonic acid formula is Tost, oncood ——— Acetic acid Cholesterolshowed that all carbon atoms stem from acetate, that both C-atoms of the acetate are used, and that a compound with six C-atoms is an intermediate, of which one C-atom is lost. Stereochemical questions concerning the biosyn- thesis of cholesterol were only answered a couple of years ago, especially by Corntorth with deuterium and iritium labelings. Questions concerning the regulation of the biosynthesis are still being worked at in various laboratories with the help of labeled compounds. For studies of biogenesis leading to sufficiently large product quantities, (!°C]-labeled precursors are pre- ferred nowadays, since the distribution of the [°C] in the product can often be detected by NMR spectros- copy, and the often very tiresome chemical decomposi- tion for the detection of the isotope distribution can be left out. In the elucidation of the path of carbon during photosynthesis, the first stable carbon fixation product, constituted an important question. When ['*C]O, was added to a light-exposed algae suspension that was in a steady state of photosynthesis, and the experiment was abruptly stopped after 20 s, it resulted from the algae extract, after two-dimensional paper chromatography and radioautography, that more than 20 compounds had already been labeled by carbon-14. If the experi- ‘mental time is shortened, the result, extrapolated to the time zero, was that only 3-phosphoglyceric acid was [C] labeled. The radioactive carbon was in the carboxyl group. 7.5.22 Some Definitions and Theoretical Considerations In order to complete the processes qualitatively de- scribed above, it can be said that in general every biological system contains intermediate products of ‘main reaction paths with more-or-less many side paths. When attempting to analyze the temporal changes of the specific radioactivity of substances, this results in integral equations that can only be solved by approxi- mation procedures However, Zilversmit developed criteria by which precursor-product relations can be identified when the system is in a steady state, As already mentioned, the velocity with which a substance is synthesized from its precursors, and with which it is decomposed is exactly equivalent in the steady state. (Instead of synthesis and decomposition, efflux and influx from and to other compartments, or both can take place.) The follow- ing is supposed for a reaction AB: p= the constant velocity with which the precursor A is converted into the product B, r= amount of B in the space under consideration (c.g., an organ or tissue), x =amount of labeled B in the space under con- sideration Sl = specific radioactivity of A as function of the time ¢ 1.522 Some Definitions and Theoretical Considerations 311 The amount of labeled material being converted into the product B per unit of time is p xf(O), and px applies to the amount of labeled material that disap- pears per unit of time. Accordingly, the changes of the specific radioactivity of B in time amounts to: ax F-—p* 743) Since r is constant it is possible to write +(«*[a)-p[r0-] 4) and the result is («2/a){(ao- as) Figure 7.4 shows the process of the specific radio- activity of A and B in dependency of time. The numerator of Eq. (7.15) reflects the slope of B’s specific radioactivity curve. The denominator is equivalent to the difference of the specific radioactivity of A and Bat a given time, As can be seen from Fig. 7.4, the specific radioactivity of A must be higher than that of B, as long as the slope of the B curve is positive. At the maximum of B’s specific radioactivity the slope is equal to zero, ive, the specific radioactivity of A and B must be the same. As svon as the specific radioactivity of D decreases, that of A is lower than that of B, i.e., the denominator in Eq, (7.15) is negative. In other words, if A is a precursor of B, the curve of the specific radioactivity of A must intersect that ofthe product Bat 4 maximum, under conditions of a steady state. The term turnover time can also be gathered from Fig, 7.4. This describes the time required for turning over quantity of substance that is to be found in the steady state in the pool under consideration. It can_be computed by dividing the area below the curves of the eaAef (t) Specie actvty —— las precursor A (Zilversmit etal) For further details see text3127 Isotope Methods Applied in Biology specific radioactivity between the times t, and ty through the increase m of the specific radioactivity of B between f and fy 7.8.23 Farther Examples and Important Experiments in the Field of Molecular Biology ‘The steady state described in the previous chapters does not necessarly apply to ll areas of an organism. This is demonstrated in a famous experiment made in 1946 by Shemin and Rittenberg, in which they studied the biosynthesis of heme with ['N}labeled glycine in human beings. Through a number of previous experi- ‘ments on animals, it was known that glycine is incor- porated in the heme. For 3 days the test persons took in PSN|-glycine with their food. It was to be expected that the labeled substance would appear in the erythrocytes with a certain velocity, pass through a maximum, and then decrease again, as is shown in Fig. 7.4. However, the result was as follows. After the intake of ['°NJ- ‘glycine had been stopped, the incorporation of "NJ in the heme was not reduced but it increased for another 25 days. After that the labeling remained constant for roughly 100 days, and then abruptly started to decrease. From this the suprising conclusion must be drawn, that the hemoglobin and presumably all the erythrocytes are not synthesized and catabolized continuously. but rather in batches. After the application of ['°N]-glycine a certain amount of erythrocytes was synthesized, These existed for roughly 100 days and were then catabolized again. Thus, the enzymes leading to cata- bolism do not attack the erythrocytes at random, they “recognize” the old erythrocytes Tracers were and are especially important for re- search in the field of molecular biology. This fact will be demonstrated in a couple of examples. As we know, a series of three nucleotides in the desoxyribonucleic acid indicates an amino acid in the gene product protein. These triplets in the messenger ribonucleic acid (MRNA) had to be determined for the 20 amino acids that participate in the synthesis of proteins. Matthaei and Nierenberg achieved the experimental breakthrough in 1961 by adding the synthetic mRNA. poly-uridine to a mixture of transfer ribonucleic acids (RNA) and ribosomes loaded with amino acid. Out of the 20 possible amino acids, only phenylalanine was polymerized into a polypeptide. The series of three uracil bases (UUU) codes for phenylalanine. The quantities thus turned over were imponderable, they could only be separated by chromatographic methods and detected through radioactivity. The further identi- fication of the genetic code resulted through experi- ‘ments with synthetic triplets. If, e.g. the triplet contain- ing the bases uracil, cytosine, uracil (UCU) iscombined with ribosomes, the result is triplet ribosome complex. Ifa mixture of tRNA, loaded with their respective amino acids (aminoacyi tRNA) is added to the com- plex, only the aminoacyl tRNA adheres to the UCU- Toaded ribosomes, which has the pl sponding to the UCU. If there are 20 mixtures of the 20 different aminoacyl tRNA, in which in each single mix- ture only one sort of aminoacyl residue is radioactive, and to this some of the UCU-ribosome complex is added, and after a while the incubation is sucked off over a membrane filter, then in this example radio- activity ean be detected only on the filter in which the amino acid serine was radioactively labeled. This means that only seryl-tRNA adheres to the UCU-ribosome complex and cannot pass therefore through the filter. Consequently, the triplet UCU means serine. In another famous experiment [*P] and [**S] label- ings were applied by Hershey and Chase. It was demonstrated that only the deoxyribonucleic acid (DNA) of T phages, but not their protein, contain the information for the production of viruses. T4 phages were cultivated in a medium of labeled sulfur and phosphorus. Because of this, the proteins were labeled with F°S] due to their content of sulfur-containing amino acids, and the DNA was labeled with [“P). Since DNA does not contain sulfur, and virus proteins do not contain phosphorus, both polymers were labeled with only one sort of nuclide. Of these phages so labeled only [2P] penetrated into the host cells, while the [°°S] radioactivity remained on the surface of the cell and could be removed by shearing forces. The infected host cells produced new T4 phages. It was thus demon- strated that protein-free nucleic acid contains all the information needed for the production of viruses. ‘The experiment by Meselson and Stahl supplies information about the nature of DNA replication. Escherichia coli cells were cultivated in a medium in which all nitrogen salts contained almost only (N]. Thereby the heavy [*N-isotope was incorporated in the produced DNA. After a certain growth in the “heavy” medium, the cells were transferred to a “light” medium, i.e., one with ['*N}nitrogen salts. Before the change of media took place, and at_various times thereafter, cells were reprocessed onto DNA and these were then the DNA of the cells was isolated and a density-gradient centrifugation to equilibrium in cae- sium chloride conducted. The cells that had been cultivated in the “heavy” medium contained only “heavy” DNA. After one generation in the “light” 1. Generation in normal medium 2. Generation in normal madium Scheme: The semiconservative reduplication of deoxyribonucleic ‘cid. The fat printed lines indicate “heavy”, ie, [N}sontaning DNA. The shin lines are “light” strands, containing [“N]17.5.3 The Steric Process of Enzyme Reactions on Prochral Systems 313 medium the DNA took up a postion in the density fradient that vas in between the “heavy” and the “light™ DNA. After a second generation the “semi- heavy” and “light” ones were present in a ratio of 1:2 (eh scheme), From this, the so-called semiconservative DNA replication was confirmed ‘After the discovery of specific endonucleases forthe double-stranded DNA (restriction nucleases) it became possible, combined with the tracer technique, to se- gquence DNA. This means determining the sequence of bases in the DNA. What the diverse procedures have in common isthe fact that the varying long oligonucteo- tides can, duc to [2P}abeling and after electrophoretic separation in polyacrylamide gels, be made, visible through radiodutography. With less than 10"? kg of DNA, roughly 100 base sequences can be determined Sequences with more than 10,000 base pars were detect- ed, *PHiabeled phosphates having been applied whose specific radioactivity was roughly 4% 10" Bq/mmol The radioautography takes a couple of hours, The methods have yielded surprising new findings. Only wo Will be mentioned here. In phages there are cases in trhich the base sequences canbe readin two or even in all the three possible frames, These so-called overlapp- ing genes make it necessary to widen the definition ofa gene. The definition should nowadays be:""A gene isa section on the DNA, which sets a code for a certain polypeptide when read in a certain frame.” Genes in higher eels can be interrupted by noncoded DNA. The gene for egg white (ovalbumin) hs seven sections fintrons), that are not translated into the gene product. ‘The noncoded introns of this gene. are. fist co- transcribed into a premRNA and then cut out. Only the so-called exons are translated into the polypeptide. 1.5.3 The Steric Process of Enzyme Reactions on Prochiral Systems. Many compounds participating in the metabolic pro- cess are chiral. In most cases this chirality is caused by four different ligands (Cabed) on one or more carbon atoms. It has been known for a long time that enzymes, that are also all chiral, act in different ways toward the various enantiomer forms of a substance. Besides the numerous chiral compounds, almost all biological ‘molecules are prochiral, ie., they contain one or more carbon atoms of the Caabe type. Some examples are: citric acid with two ~CH»COOH groups, ora primary alcohol R—-CH,OH with its two hydrogen atoms on the carbon atom carrying the OH group. In prochiral molecule the two groups a do not act the same way in a chiral surrounding. This circumstance, known as the Ogston hypothesis, is very important for the interpre- tation of tracer experiments. In 1948 Ogston pointed out that the transition HOCH,CH—"COOH+ + +H,'"NCH,"3COOH ‘SNH, serine lyeine Fig. 75. If molecule with a prochiral group Caabd interacts witha chiral reagent at three diferent points, the two groups a are exposed tw differing influences one group (ais labeled, it canbe identified ‘then, under the influence of catalytic group (eg. A) 2 pliting off fora substitution ofa group a results could very well proceed over the symmetric inter- mediate product HOOC—CH—"COOH (amino malonic acid) SNH) even though the glycine shows the same *N/!*C ratioas does the serine, Ogston substantiated this view by means of Fig, 7.5. According to this, there are no two identical arrangements in which solely the combining site of the two carboxyl groups A and A’ would be exchanged for a chiral reagent (enzyme) interacting with the substrate. If, however, itis assumed that, e.2., due to the catalytic effect of the combining site A. only the carboxyl group a can be split off, then the result shown above is quite compatible with the symmetric intermediate product amino malonic acid. Such a circumstance can, in principle, be confirmed only by means of isotopic labeling. In the meantime hundreds of | examples exist. Such tests are especially interesting in connection with the evolution of the active centres of enzymes. Thus, there have been cases in which, ¢.g., reversibly from one compound a methylene group was converted into a methine group. if nie u This is, €.¢., the case with the group of pyridoxal- dependent enzyme reactions. At the moment seven quite different kinds of reactions that are catalyzed by these enzymes are known. It was observed that always the same steric hydrogen atom of pyridoxamin’s methy- lene groupis removed, respectively is fixed always to the same side of the pyridoxamin’s methylene group. Since in each of the seven tested reactions there are two possibilities, the probability of the steric identity being314 7 Tsoxope Methods Applied in Biology identical is 1/2" ~0.008. It is more likely that a family of | enzymes has one common origin, and that a successful steric arrangement was conserved during evolution. 1.54 Studies of Isotope Exchange By isotope exchange studies substantial kinetic and ‘mechanistic insights can be gained in systems that are in a steady state, One main reason is the fact that velocity measurements of completely or step-by-step reversible reaction chains are biologically relevant only when all the products of this reaction chain are in a steady state. This applies to chemical reactions as well as to transport phenomena through membranes, and to phase tran- sitions, etc, Often measurements of initial velocities are of no use at all. If, e.g., the entering velocity of a potassium ion into a cell iso be measured, no reliable results would be gained by making the cells completely potassium free, and then bringing them into a medium With potassium ions, and measuring their entrance into the cells. Even if this could be achieved under physio- logical conditions, the actual initial phase, occurring without a reverse reaction, is very short and not easy to follow in an experiment. A much more advantageous experiment is one in which, under physiological condi- tions in a medium, cells are mixed with such a small amount of radioactive potassium ions that the equilib- rium between the medium and the inside of the cel is undisturbed with regard to the potassium concentra- tion, and yet the entering of radioactivity into the cells can be measured. Measurements of enzyme reaction velocities in the initial phase, i.e., in the absence of products, often supply little information about the velocities under in vico conditions, since the products can havea regulating effect on the enzyme activities. But, even when measur- ing initial velocities of reactions as functions of the concentrations of substrates, produets, inhibitors, etc., ofien only net velocity ofthe chemical turnover can be measured (This hecomes the less exact, the more the equilibrium is approached! If for mechanic reasons the velocities of partial steps are of interest, this cannot be realized for such that lead to nonmeasurable inter- mediates. In such cases, the measuring of changes in concentration often make visible only the small dif- ferences of large velocity constants with similar magni tudes. Isotope exchange measurements can often lead to interesting information, One examples the so-called fumarase reaction, Here, the reversible splitting off of water from L-malate with the formation of fumarate is catalyzed (¢f. schematics). If a small quantity of sufficiently high [*C}-labeled malate or fumarate was added to an equilibrium mixture of malate and fuma- rate in the presence of fumarase, the velocity of the transition malate fumarate, respectively in the reverse direction, could be measured. Assuming the exchange velocity (C}malate = (C}-fumarate to be 1.0, it can be observed that in (**O -labeled water the exchange ["O}-malate = [O}-water is 4.0, and the exchange (11}-malate = [U}-water is 0.43, Without going into detail, the following can then be concluded From the enzyme substrate complex A, OH™ and Hare split off and B develops. The OH group is at ‘once released into the water. The tritium is bonded to the enzyme by the base B. The complex B can now react as follows: (1) Back to A or (2) hy setting free fumarate. The [!%O) exchange, which takes place four times faster in comparison with the transition malatefumarate, shows that, on the average, the complex B returns to A four times’ more often. The exchange of the tritium, bonded to the base B, with the protons of the water is a relatively slow process. Almost every second enzyme ‘molecule that transformes a fumarate molecule back into malate still has the tritium and again fixes it to the C33 of the malate. Selected References Bichmana,K.: Messung radioaktiver Nuklde. Weinheim /Bergst. ‘Verlag Chemie 1970 Bentley. .: Molecular asymmetry in biology, Bd. I, Il. New York London: Academic Pres 1969-1970, Birkenfeld,H., Haase,G., Zahn H.: Massenspektrometrische Is0t0- Penanalyse. Berlin: VEB Deutscher Verlag 4, Wissenschaften 1969.