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Ι
The Use of Isotopes in Biochemistry
1. Introduction.
During the past decade the chemistry of isotopes has gained
unexpected importance in the field of biochemistry. If one replaces
by isotopes the carbon, hydrogen, oxygen, nitrogen and other
elements present in the foods ingested or in intermediary products
of metabolism one can trace these substances in the animal organism
and obtain information about their deposition in the organs, their
transformation into other substances, and their excretion. The
great importance of isotopes for biochemistry justifies a brief
review of the structure of atoms and isotopes.
2. Atoms and Isotopes.
1 τιτοεκευτsτητ
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It is a well-known fact that the nucleus of the atom consists of
positively charged protons, and the shell of the same number of
negatively charged electrons. The chemical properties of an
element depend on the number of its electrons, for only the electrons
take part in chemical reactions. Elements which have the same
number of electrons but different atomic weights are called isotopes.
Thus the isotopes C11, C12, C13, and C14 are known. Each has
6 protons in its nucleus and 6 electrons in the shell. However,
there is a difference in their atomic weights, due to the fact that
their nuclei contain, apart from the 6 protons, 5, 6, 7 or 8 neutrons,
respectively. The mass of a proton is about the same as that of
a neutron and is approximately equal to the mass of a hydrogen
atom, í. e. 1. The mass of an electron is about 1/1800 of this mass.
The atomic weight of an element is, therefore, almost equal to the
sum of its protons and its neutrons.
2
I. Isotopes
3. Stable Isotopes.
It has been known for some time that many elements are mixtures
of several isotopes. Thus hydrogen, H, contains about 0.01 %
of the isotope deuterium (atomic weight 2), carbon (atomic weight
12.01) about 1.1 % of the isotope C13, nitrogen (atomic weight
14.008) about 0.37 % of 115, oxygen (atomic weight 16) about 0.2 %
of Ols in addition to 0.04 % of 017, and iron (atomic weight 55.84)
about 1.28 % of Fe58. The elements magnesium, sulfur, chlorine,
potassium, calcium, copper and barium also are mixtures of isotopes.
The isolation of D, C13, N1b, and 018 is achieved by fractional
distillation, by use of diffusion methods, electrolysis, mass spectroscopy, and other physical methods in which atomic weights play
a part. The rate of diffusion of heavy isotopes is always smaller
than that of light isotopes. In this way C13 has been separated
from C12 as methane or as cyanide. Ν'5 can be obtained as
115H3. The natural isotopes Κ38, Κ", and Κ41 are separated
from one another by electromagnetic methods (mass spectroscopy) (5).
4. Radíoaetßve Isotopes.
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Nuclear reactions are used for the production of radioactive
isotopes. The enormous quantities of energy required can be
released only by rapidly moving elementary particles, such as
protons, deuterons, neutrons, or α-particles. Neutrons are particularly suitable projectiles ; for protons, deuterons, and α-particles,
owing to their positive charge, are repelled by the positively
charged nuclei, so that most of these particles are deflected from
their path and do not hit the nuclei. Neutrons were first produced
through the action of radium on beryllium. One can obtain much
greater quantities of neutrons through the actions of protons or
deuterons produced in the cyclotron or by the uranium pile. If
deuterium, lithium, beryllium or carbon is bombarded with these
particles, neutrons are obtained which have an energy content of
approximately 106 electron volts. These can bring about nuclear
reactions. Thus one can obtain isotopic carbon, C14, through
the action of neutrons on nitrogen according to the equation:
1
14 -{- η — C14 - - p
Ι. 4
3
One usually writes this equation in its simplified form, 114(n, p)
C14, where n = neutron, p = proton.
The most important property of the radioisotopes is their
disintegration accompanied by radiation. Radiation is measured
by the half-life, i.e., the time in which the intensity of the radiation
decreases to half its initial value. The formation of those radioisotopes which are most important from the biological point of
view and their half-lives are presented in Table I. In addition
to the elements in Table I, Cυ61, CυB4, Mn 52 and Ζn°5 also have
been used in biological experiments.
Table I
(p = proton, d = deuteron, n = neutron, α = alpha particle,
γ = gamma radiation)
Radioisotope
Η 8(Triiium)
Carbon, C11
Carbon, C14
Sodium, Na"
Phosphorus, Pa
Sulfur, 585
Chlorine, C188
Potassium, 1{49
Calcium, Ca 46
Iron, Fe"
Iron, Febe
Bromine, Brθ e
Iodine, I131
Nuclear Reaction
Η 2(d,ρ)Η 3
B'o(p,y)C11
C1e(d,ρ)C14 or 114(n,ρ)C14
ΝaS8(d,p)Νa74
Ρ 81(n, .)1"8 or Se2(n,ρ)P88
or C186(n,α)Ρ '8
C18θ(n,p)S85
C187(n,y)C138
Κ '1(n,y) Κ 42
Sc"(n,p)Ca"
Μnbb(d,2n)FeeB
Feóe(d,p)Feδe
Brßι(η,γ)Br88
Τe130(d,n) Ι "s1
Half-life
31 years
20.35 minutes
10,000 years
14.8 hours
14.3 days
87.1 days
37 minutes
12.4 hours
180 days
4 years
47 days
34 hours
8 days
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For the quantitative determination of radioactive isotopes,
radiation is measured by means of an electroscope or by a GeigerMüller counter. The unit of radiation is the curie, which is the
amount of radiation emitted per second by one gram of radium.
It corresponds to 3.7 x 101° disintegrations per second. With
sensitive counters radiations as weak as 10-5 microcurie can be
measured (1 microcurie = 10-1 curie).
In biological experiments one never uses the pure radioisotopes
but always their mixtures with the natural elements, since it is
4
I. Isotopes
assumed that both atoms behave identically in the organism.
The radioisotope is used mainly as a tracer for the normal atom.
The amount of tracer used must be small in order to prevent
undesirable effects through excessive radiation. Thus the ratio
p32 : pal in the most powerful preparations of radiophosphorus
is approximately 1 : 106.
5. Use of Isotopes in Biology.
Both stable and radioactive isotopes are used in biological
experiments. Stable isotopes have the advantage of constant
physical properties ; some radioisotopes disintegrate so rapidly
that their radiation is no longer measurable after only a few hours.
Other radioisotopes with very long half-lives emit such weak
radiation that difficulties are encountered in their measurement.
These difficulties arise because some of the radiation is lost through
scattering and some is lost through absorption by the air ; on the
other hand, higher values might be obtained by the interference of
cosmic rays. Hence, all these factors must be taken into account.
The great advantage of radioisotopes is that extremely small
quantities can be measured and that the instruments for measuring
them are far simpler than the mass spectrometer required for
measuring stable isotopes.
It is easy to interpret experiments with isotopic sodium, chlorine,
and other inorganic elements, because these elements are always
in the ionic state in the food ingested, as well as in the organism.
On the other hand, the elements hydrogen, carbon, nitrogen, sulfur,
and phosphorus, which compose the organic molecules, cannot be
introduced as elements, because this is not their state in biological
material. In the last few years many organic substances labeled at
certain points of their molecules by isotopic elements have been
synthesized. Among these are carbon dioxide, methanol, acetic
acid, pyruvic acid, butyric acid, glucose, benzoic acid, tyrosine,
and methionine, in addition to such complicated substances as
the steroids (2) or the carcinogenic dibenzanthracenes (3). Thus
the synthesis of acetic acid containing two isotopic carbon atoms
takes place in the following way (4) :
H3C*Ζ — C*Η3.C*ΟΟΗ
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BaC*Ο3 —. BaC*3
Ι. 5
5
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Acetic acid which contains only one C* in the methyl group can
be obtained through the saponification of NaC*N (6). In some
cases use is made of biosynthesis ; thus gluthathione is prepared (8)
by means of yeast grown on a culture medium containing isotopic
sulfate ions, S*04".
While carbon and nitrogen are so firmly bound to organic
molecules that they are not exchanged under the usual experimental
conditions, some of the hydrogen and oxygen atoms are very
loosely bound. A continuous exchange of hydrogen atoms of
ΟΗ, ΝΗ2í and other similar groups is always taking place with
those of the solvent (water), so that labeling these groups by
isotopic hydrogen is not practical. The hydrogen atoms of the
CH group, on the other hand, are bound so firmly that under the
usual experimental conditions no exchange takes place with water.
The hydrogen atoms bound to certain activated carbon atoms are
exchanged at an intermediate rate ; for example, the hydrogen
atoms linked to the ii-carbon atoms of amino acids can be exchanged
through treatment of these substances with heat or sulfuric acid,
but are stable under the usual experimental conditions at body
temperature (9). By boiling amino acids with heavy water and
strong alkali, deuterium atoms can be built into their molecules
and the amino acids thus labeled for metabolic experiments (10).
Glycine at 120° C. exchanges for deuterium the hydrogen atom
linked to the α-carbon ; at 135° it also exchanges the other hydrogen
atoms (11). Glutamic acid, cystine, and tyrosine also contain
hydrogen atoms which can be exchanged slowly ; in tyrosine the
unstable hydrogen atoms are those ortho to the hydroxyl group (12).
Amino acids containing deuterium atoms linked to their ß- and
y-carbon atoms are resistant even to boiling hydrochloric acid
and are, therefore, well-suited to metabolic experiments (13).
Oxygen, like hydrogen, also shows different tendencies for
exchange in different atomic groups. The oxygen atoms of
carboxyl groups are easily exchangeable with the isotopic oxygen
atoms of water. On the other hand, the oxygen atoms of the
peptide bonds or of the phenol groups of tyrosine are not exchangeable (14).
Problems of the distribution of inorganic ions, of their transport,
and of permeability can be solved easily by using isotopes. More
important is the use of isotopes in the search for metabolic
5
I. Isotopes
precursors or for catabolic products. The great advances achieved
by this method will be discussed in Chapters III, IV, VIII, X,
XI and XV.
REFERENCES
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(1) M. D. KAMEN: Radioactive Tracers in Biology (New York 1947).
(2) R. TURNER: J. Am. Chem. Soc., 69, 726 (1947).
(3) C. HEIDELBERGER, P. BREWER, W. DAUBEN: J. Am. Chem. Soc., 69,
1389 (1947).
(4) R. ABRπIS: Experientia, 3, 488 (1947).
(55 ) 0. WALKEII : Ann. Rep. Chem. Soc., 35, 136 (1939).
6) H. ANKER : J. Biol. Chem., 166, 219 (1946).
(7) G. HEVESY : Ann. Rev. Biochem., 9, 641 (1940).
(8) R. G. FRANKLIN : Science, 89, 298 (1939).
(9) R. SCHOENHEIMER, D. RITTE κ BERG, Α. KESTON : J. Am. Chem. Soc.,
59, 1765 (1936).
(10) GÜNTHER, K. ΕΟΝHÖFFΕ R : Zschft. physikal. Chem. A., 180, 185 (1937).
(11) Α. KROGH, H. UssING : Compt. rend. Carlsberg Lab., 22, 282 (1938).
(12) Α. KESTON, D. RITTENBERG : J. Biol. Chem., 123, LXVIII (1938).
(13) D. RITTENBERG, R. SCHOENHEIMER, A. KESTON, G. FORSIER : J. BIOT.
Chem., 125, I (1938).
(14) W. MEARS, H. SOBOTE.A : J. Am. Chem. Soc., 61, 880 (1939).
15) M. KAMEN : Ann. Rev. Biochem., 16, 631 (1947).
(16) J. SACRs : Chem. Reviews, 42, 411 (1948).
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