TRACER TECHN iOUES
1.
Ha
HEMDEASON
The understanding of metabolic processes has advanced a t a
f a n t a s t i c r a t e since the advent of t r a c e r methods. Prior t o t h e discovery
isotopes of the elements of concern t o biochemists, scone crude t r a c e r
experiments were done. Oleic acid, f o r example, was converted t o 9,lOdichlorostearic acid by adding chlorine across t h e double bond. T h i s crude
type of l a b e l could only t r a c e t h e general f a t e of t h e f a t t y acid molecule
by measuring chlorine b u t could not lead t o information regarding t h e
molecular b a s i s f o r t h e metabolic processes i n which this f a t t y a c i d i s
involved.
The discovery by Hevesy i n 19U. of an unstable isotope of lead
(Pb2l0) which had been assumed t o be contaminating radium, marked t h e beginning of t a g s or t r a c e r s within t h e nucleus of an atom. The chemical prop e r t i e s of the tagged atom a r e not s i g n i f i c a n t l y a l t e r e d i n these isotopic
t r a c e r s . F i r s t elements such a s nitrogen and hydrogen, which occur in
nature a s mixtures of two or more isotopes, were exploited by separating
t h e s t a b l e isotopes of these elements. The s t a b l e isotopes usually differ
from t h e predominaat isotope by one mass unit. While the permissible
d i l u t i o n of such i s a t o e s i s f a r below t h a t f o r radioactive isotopes,
deuterium, (a2) and N9, detectable by mass spectrometry, were s u f f i c i e n t l y
useful t o provide data which revolutionized our concepts of metabolism.
Schoenheber and coworkers a t Columbia presented an hypothesis which s t a t e d
t h a t body constituents were in a dynamic s t a t e of equilibrium with each
other and with the n u t r i e n t s provided t o the organism.
With the production of unstable isotopes of t h e l i g h t e r elements,
l a b e l s f o r most of the elements involved i n biological processes became
available. The necessity of having an accelerator or a neutron source t o
produce isotopes, limited t h e number of laboratories using radioisotopes
u n t i l t h e close of World War 11. A t t h a t time, nuclear reactors began t o
produce t h e unstable isotopes of biologically important elements in s u f f i c i e n t amounts and a t a cost low enough t o bring these precious tagged atoms
t o t h e a i d of s c i e n t i s t s i n most laboratories in t h i s country. With t h i s
increased a v a i l a b i l i t y of radioisotopes, great improvement i n instrumentat i o n a l s o came. Today a vast array of mass produced instruments f o r measuring various isotopes present i n many com ounds i s on the market. Because of
t h e ease of measuring Isotopic carbon (Cf4), t h i s method has frequently replaced more conventional methods of following t h e course of a reaction in
the laboratory. The a v a i l a b i l i t y of most common n u t r i e n t s and metabolites
labeled i n s p e c i f i c carbon atoms with $4 has a l s o enhanced t h e usefulness
of t h i s technique.
While the s p e c i f i c biochemical reaction can be studied without
isotopically labeled substrate in v i t r o , t h e r o l e of t h i s reaction i n t h e
i n t a c t organism can only be evaluated by in vivo experiments and usually this
requires isotopic substrate. Sane examples of t h i s application of isotopes
will be c i t e d i n t h e remarks which follow. B many cases t h e understanding
206.
of t h e metabolism of a compound has came from enzyme experiments designed
a f t e r isotopic studies i n t h e I n t a c t organism had indicated t h e general
nature of t h e metabolic processes involved.
I.
GENERAL PRIJ!?CIPLFS
-
A , Chemical properties and hence t h e name we give an element a r e
determined by t h e num3er of extranuclear electrons. Chemical reactions are
largely independent of t h e mass of t h e nucleus.
B. The rays o r p a r t i c l e s emitted by unstable isotopes a r e (a)
electrons, (b) positrons, ( c ) d - p a r t i c l e s or (d) Y -rays, each with i t s own
s p e c i f i c properties.
The r a t e of decay of unstable isotopes i s independent O f
temperature, pressure, etc., and of t h e chemical combination of t h e element.
Thus t h e half' l i f e of C14 i s 5,700 years regardless of t h e conditions or the
chemical form In which it i s found.
C.
D. The isotope must be incorporated i n t o t h e ccmpound which you
wish t o study, usually i n t o a specific position i n t h e molecule.
E. There must be a s u i t a b l e detecting system f o r measuring t h e
isotope concentrations under t h e conditions of t h e experiment. For s t a b l e
isotopes t h e maximum d i l u t i o n tolerance is about 100 fold. For radioisotopes d i l u t i o n of approximately 100,000 I s permissible. Radioisotopes can
be determined on material i n t h e gaseous, l i q u i d or s o l i d s t a t e .
A l i s t of the isotopes most frequently
used t o study biochemical
processes is shown i n Table 1;
TABLE I
SOME CHARACTEKETICS OFTHE ISUI'OFES
Element
and Mass
Number
USED I N STUDYIYYG LIVING PROCESSES
Type
of
Radiation
Half-
Life
-
Comments or Energy of Radiation
Part i c l e s
None
-----
0.02% i n Nature
E3
B-
12.1 yr.
0.017 mev
c13
None
Cl4
B-
-----
1.1% i n Nature
H2
(deu t e r i um)
(t r it i u n )
N E
018
Na22
NaZ4
P32
s35
None
None
af, 7
B-
88-
5,700 yr.
-----
Y -Rays
0.154 mev
0.38% i n Nature
-----
0.20%
2.6 yr.
0.58 mev
1.3 mev
1.38, 2.7 mev
14.3 days
1.4 mev
1.7 mev
14.8
hr.
87.1 days
i n Nature
0.17 mev
207
TABLE I (Continued)
Element
and Mass
Number
Type
of
Radiation
Half-
Life
-
4.5 x 108yr.
Comments or Energy of Radiation
Y -Rays
Part i c l e s
-I_
- 1.9
mev
K4'
88-
12.4 hr,
~a45
8-
180 days
0.26 mev (50%)
0.46 mev (50%)
Fe5'
8-
45.1 days
0.257, 0.460 mev
5.3 yr.
0.31 rnev
19.9 yr4
0.61 mev
K4O
c 060
B-, Y
zn65
Y
~r90
0-
1131
8-, Y
1.3
2.0 mev (25$)
3.6 mev (75$)
250 days
8 days
1.46 mev
0.6 mev
1.16,
1.32 mev
1.120 mev
0.080
- 0.722
Tracer techniques as t h e name implies permits following t h e f a t e
of atoms o r molecules by t h e mass or radioactivity of the isotopes. Because
of t h e great s e n s i t i v i t y of t h e methods of measuring radioisotopes they a r e
much preferred when available. Whereas s t a b l e isotopes a r e prepared by
separation from n a t u r a l sources t h e radioisotopes a r e prepared with a reactor or an accelerator, The simple procedure of tracing an element such
a s sodium or iron while useful, does not present t h e elegant power of t r a c ing a s p e c i f i c carbon or hydrogen atom through t h e complexities of metabolic
reactions. Examples of t h e various types of application or' isotopes t o
physiology and biochemistry w i l l be presented, attempting t o classify these
applicstions.
11. APPLICATICNS OF ISCrrOPES TO THE STUDY OF LIVING P R O C E S e
A.
Requirements and Limitations of the Tracer TechEique
The t r a c e r technique impinges upon biology chiefly a t t h e biochemical and physiological level. Its g r e a t e s t contribution has been i n t h e
area of intermediary metabolism, an understanding of which is e s s e n t i a l t o
t h e development of a l l areas of biological science. The advent of isotopic
t r a c e r s has made it possible t o follow t h e wanderings of a s p e c i f i c atomic
grouping wlthin t h e organism.
Before an isotope can be used a s a t r a c e r , one must make c e r t a i n
t h a t t h e i n i t i a l concentration of isotope i s high enough t o t o l e r a t e t h e
d i l u t i o n which occurs during metabolism and s t i l l remain detectable i n t h e
products separated a t t h e close of t h e experiment. For radioisotopes, t h i s
condition can usually be met, but f o r s t a b l e isotopes, it often presents a
problem. The labeled atom must remain attached t o t h e molecule or portion
of a molecule during t h e metabolic processes under study. The presence of
t h e isotope must not a f f e c t t h e metabolic process. T h i s becomes of some
mev
208
importance f o r isotopes of hydrogen where "isotope e f f e c t s " a r e observed.
The h a l f - l i f e of t h e radioisotope must be long enough t o permit t h e experiment t o be done. Unstable isotopes of o q g e n and nitrogen have h a l f - l i v e s
too short f o r most purposes, but fortunately t h e s t a b l e isotopes $8 and N15
a r e available in nature and a r e separated and used f o r t r a c e r experiments,
B.
Physiological Applications of Isotopes
1. Permeability, Transport, Cellular Uptake, and Distribution
Before t h e use of isotopes, physiological processes were often
studied using "unphysio3.ogical concentrations" of various rner;a'-lolites,
Radioactive metabolites can often be used a t normal or less < h m normal
physiological concentrations t o measure permeability under conditions where
t h e r e i s no net t r a n s f e r of a metabolite. For example, t h e passage of Na+
back and f o r t h across a c e l l membrane can be measured coiive~Lcni;ly using
Na24, Transgort of compounds i n t o axones, across t h e i n t e s t i r 3 1 wall and
placenta and aptake of compounds by b a c t e r i a l c e l l s have a l l been studied
effectively using isotopes. The k i n e t i c s of such processes can be readily
evaluated by frequent analysis of t h e medium f o r t h e isotopic t r a c e r .
2.
B t r a c e l l u l a r and Extracellular Space Determination by
Isotope Dilution
Frequently t h e f l u i d volume, or space available for solution
of' e l e c t r o l y t e s , both inside and outside t h e c e l l s of an organism must be
measured. Isotopes a r e useful f o r such measurements because t h e indicator
can be a noma1 coapnent of the space being measured, For t o t a l body water,
HgO and $0 have been used. For e x t r a - c e l l u l a r space Na+, C1' and Br' may
be used. For plasma volume, serum albumin, iodinated with IU1, and more
recently P32 d i -is opopylshosphorof luor i d a t e, which combines with plasma
proteins and with c e l l u l a r components of blood, have been used, The water
space of t h e brain has been measured using S35-sulfate. Cardiac output
and t h e p a t t e r n of blood flow through t h e heart a r e examples of similar
applications of isotopes in the c l i n i c a l f i e l d ,
3.
Hormone Studies
Because of the minute quantities required, hormones have been
d i f f i c u l t t o study. Without isotopes, t h e a n a l y t i c a l methods demanded t h a t
massive doses of hormone be given. This l e f t considerable doubt a s t o
whether t h e findings were r e l a t e d t o the t r u e metabolic f a t e of the hormone
i n question. The selective uptake of hormones by t h e t a r g e t organ can be
investigated using i s o t o p i c a l l y labeled hormone a t physiological concentrations. A good example of such experiments has been presented by Glascock
(1)who observed t h a t hexoesterol was concentrated more in t h e reproductive
organs than i n any other organ except the kidney, in 5.5 hours af'ter being
given a t a l e v e l of 1 %/kg of body weight.
Iodine-131 has been very useful i n labeling the thyroid hormone
and r e l a t e d compounds a t a s p e c i f i c a c t i v i t y s u f f i c i e n t l y high f o r experiments i n t h e whole animal using near physiological concentrations of t h e
hormone.
209
4.
.
Mineral Metabolism
The metabolism of t r a c e minerals, a s i n t h e case of hormones, can
be studied most readily a s radioisotopes, t o make t h e t r a c e amounts susceptible t o a n a l y t i c a l attack. Fe55 and FeS9 have been used t o e s t a b l i s h
t h e r a t e of i r o n absorption and incorporation i n t o heme. Isotopic studies
have shuwn t h a t red blood c e l l s l i v e about four months and p l a t e l e t s about
l w e e k , Isotopes of cobalt and copper have been u s e f u l i n metabolism
studies of these t r a c e metals (l), C a g and $2 have been extensively used
t o study t h e deposition and mobilization of bone ash.
C
Biochemical Applications
The e a r l i e s t experiments with isotopes revolutionized t h e e x i s t i n g
views regarding the general nature of metabolic processes. The p a r t i t i o n i n g
between exogenous and endogenous metabolism, a concept developed by Folin on
t h e b a s i s of t h e e f f e c t of dietary components on t h e concentration urinary
constituents, was abandoned when t h e deuterium and NE experiments of
Schoenheimer and coworkers demonstrated the dynamic nature of metabolic
processes. The more exciting contributions with isotopes have been those
i n which e n t i r e metabolic sequences and cycles have come t o l i g h t .
briefly.
research.
A number of examples of these contributions w i l l be presented
Where appropriate, I w i l l draw these examples from my own area of
1. Isotope Dilution
A fundamental concept, e s s e n t i a l t o the understanding of many
applications of isotopes, is the isotope d i l u t i o n principle. Analytically,
t h i s method is precise t o about I$. It depends upon t h e f a c t t h a t when a
knuwn quantity (xl) of a substance, S , labeled with an isotope, e.g,, c14,
is added t o a complex mixture containing an unknown amount (xz) of S, the
s p e c i f i c a c t i v i t y (A1) of S w i l l be reduced t o a new figure (%) t o t h e
degree t h a t t h e radioactive S i s d i l u t e d with unlabeled S. The following
equation expresses t h i s relationship:
A1Xl
+
Since A and xl a r e known, i f a small sample of S from t h e mixed,
labeled and unlabeled S can be i s o l a t e d and i t s s p e c i f i c a c t i v i t y , AZ,
determined t h i s equation can be solved f o r
t h e quantity desired. Many
variations in the application of this principle a r e used. When a t r a c e of
a highly labeled compound i s present i n a biological material, it can be
recovered and the t o t a l quantity of isotope ascertained by adding unlabeled
compound (cold c a r r i e r ) in a known amount and i s o l a t i n g a portion of t h e
compound. If a micro-assay is available f o r t h e substance, t h e s p e c i f i c
a c t i v i t y of the labeled compound a r i s i n g from a metabolic process can be
calculated
*,
.
210.
2.
Precursor-Product Relaticnship
One of t h e simplest questions often asked i s whether compound A i s
converted t o compound B. To answer t h i s question, frequently only qualitat i v e r e s u l t s a r e needed. For example: t h e conversion of phenylalanine t o
tyrosine (2)
.
*
CHz -CH -COOH
Cil
and tryptophan t o N1-methyl nicotinamide (3).
C% -CH -COOH
NH2
____.___p
H
0
WCONHz
CH3
I n cases where isotope from A i s found i n substance B, but with SO much
a s t o c a s t doubt on t h e directness of t h e relationship, other approaches w i l l
be needed t o e s t a b l i s h a precursor-product relationship, Examples of t h i s
type of application are: (1)the formation of t h e s t e r o i d nucleus of
cholesterol from a c e t a t e and isopentanyl pyrophosphate, (2) C% f i x a t i o n
i n t o c e r t a i n simple molecules during photosynthesis, (3) t h e formation of
porphyrin from succinate and glycine, (4) the formation of purines from COz,
formate, glycine, a s p a r t a t e N and glutamine N.
3.
Method of Isotope Competition
T h i s method i s applicable s p e c i f i c a l l y t o b a c t e r i a l systems. If
a metabolic sequence, represented by A>-B 4 C ----3D, e x i s t s i n a
c e l l then isotopically labeled A w i l l lead t o labeling of D. Addition of
unlabeled B or C would then be expected t o suppress t h e labeling of D.
4.
Metabolite Gverloadin(S
-
Ln animals many metabolic processes cannot be studied with isotopes
because t h e end product of one sequence of reactions (C) becomes the reactant
of another sequence of reactions:
A-
B
C-Cl-
Cz-C3
+co2*
If it i s suspected t h a t "C" i s involved i n the conversion of "A" t o CO
hypothesis might be tested by administering a small dose of isotopic&
the
211.
labeled "A" and simultaneously a dose of "C" of such magnitude t h a t it
appears in t h e urine. If t h e exogenous "C" equilibrates with t h e "C" formed
frcm "A''b it should be labeled and t h e l a b e l should be present i n t h e
urinary C" which i s isolated, A positive result implicates "C" a s an
intermediate i n the degradation of "A"; but a negative r e s u l t does not
eliminate "C" a s an intermediate.
5.
Labeling Patterns i n T i s s u e Components
When any compound, labeled i n a s p e c i f i c carbon atom i s degraded
through simple 2, 3 and 4 carbon compounds, these small molecules a r e
labeled i n a single carbon atom and lead t o c h a r a c t e r i s t i c labeling p a t t e r n s
i n t h e glucose of l i v e r and muscle glycogen, i n f a t t y acids and/or i n none s s e n t i a l amino acids. Perhaps t h e most useful indicator of the i d e n t i t y
of degradation products a r e t h e amino acids alanine, a s p a r t i c acid, glutamic
acid, glycine and s e r i n e , Compounds leading t o labeling in any position i n
pyruvate or a c e t a t e give c h a r a c t e r i s t i c labeling patterns especially i n
glutamic acid and alanine. The experiment i s thus very simple. The compound of i n t e r e s t , uniquely labeled, i s fed or injected i n t o an animal and
t h e animal i s s a c r i f i c e d about twelve hours l a t e r . The l i v e r glycogen and
l i v e r or carcass p r o t e i n a r e isolated, hydrolyzed and t h e fragments degraded, carbon by carbon, t o C02 which i s examined quantitatively f o r C 1 4
a c t i v i t y . Table I1 i l l u s t r a t e s t h e l a b e l i n p a t t e r n i n the most indicative
amino acids when acetate-l-C1*, acetate-2-C f4 a r e fed t o r a t s . From Table
I11 (4) it is evident t h a t t h e labeling p a t t e r n i s Glutamfp and alanine
from l i v e r and carcass of a r a t receiving tryptophan-7a-C
i s essentially
t h e same a s t h a t observed when a ~ e t a t e - 1 - Ci ~s ~administered (5). Likewise
t h e p a t t e r n i n these two amino acids i s similar t o t h a t from a ~ e t a t e - 2 4 ~ 4
when t r y p t 0 p h a n - 5 - C ~was
~ given (6). These r e s u l t s c l e a r l y indicate t h a t
C-5 of tryptophan
> C-2 of a c e t a t e
C-7a o f tryptophan
3
6
C - 1 of a c e t a t e
Trapping Techniques
A small molecule suspected t o be a product of degradation might
e x i s t i n t h e metabolic pool i n quantities too small t o detect and it might
not be excreted in t h e urine normally. If a sample of t h e fragment can be
withdrawn from the pool i n t o t h e urine by detoxification of some foreign
compound, it can be degraded and its labeling p a t t e r n determined. Acetate
(7) and glycine a r e good examples of molecules which can be trapped i n t h i s
way, glycine a s hippuric acid and a c e t a t e a s an acetylated amine,
When t r y p t ~ p h a n - Cwas
~ ~ injected i n t o t h e r a t together with
cyclohexylalanine, t h e acetylcyclohewlalanine was excreted. It was i s o l a t e d
from t h e urine, hydrolyzed and t h e r e s u l t i n g a c e t a t e degraded stepwise t o
reveal a labeling p a t t e r n in a c e t a t e consistent with t h e amino acid labeling
pattern. Table IV shows t h e r e s u l t s obtained (6, 8 ) .
212.
7.
Evaluation of the Extent t o Which Alternate Pathways
Operate
Glucose metabolism i s recognized t o proceed by two pathways;
glycolysis t o 2 molecules of pyruvate and via the pentose pathway by which
C4, C 5 and c 6 a l s o give rise t o pyruvate while C1 yields CO2. Thus, t h e
findine of C14 from C - 1 of glucose i n l a c t a t e r e f l e c t s glycolysis and a
comparison of t h e labeling i n l a c t a t e from C - 1 of glucose i n one experiment
and C - 6 of glucose i n another experiment has been used t o determine the
extent t o which glucose metabolism in t h e bovine, corneal epithelium
proceeds by glycolysis (9), Another method proposed by Blmenthal e t a l e
(1O)is based on t h e f a i l u r e of C-1 of glucose t o l a b e l acetate when the
pentose pathway prevails.
8
0
Reaction Mechanisms
Both b organic chemistry and biochemistry, isotopes present a
powerful t o o l for determining the mechanism by which a reaction proceeds,
Perhaps the best knom example of the solution of a reaction mechanism
problem with isotopes was the elucidation of the stereospecific addition to,
and removal of hydrogen frcm, carbon-4 of the pyridine nucleus of nicotinamide-adenine dinucleotide (NAD) (11)
H
H
H
I
1
R -P
R-P
Time w i l l not permit t h e detailed discussion of these experiments, but it
was shown t h a t most NAD requiring enzymes a r e specific for one of t h e
s p a t i a l l y d i s t i n c t hydrogen atoms and t h a t the atom i s transferred d i r e c t l y
(not via a proton i n the medium) t o t h e reduced product.
Another aspect of t h e mechanism of reactions brought t o l i g h t by
isotope experiments was the concept of a meso-carbon atom such a s t h a t
found i n glycerol or c i t r i c acid: (12, 13)
c%a
I*
HOCH
I
CHZOH
(2% -COOH
I
I*
HO-C -COOH
I
C% -COOH
The carbon atom marked * has been considered a s a syaetric carbon atom by
organic chemists. These molecules have a plane of symetry f o r they a r e
b
a -C-a type structure.
d
A closer examination shows t h a t they a r e in r e a l i t y
213
b
al-C-aZ type molecules, where a 1 and a2 a r e r e l a t e d t o each other a s a r e
d
our r i g h t and l e f t hands, It i s not surprising i n retrospect t h a t an enzyme
or some other optically a c t i v e reagent should r e a c t a t a d i f f e r e n t r a t e with
a 1 than with a2. Thus the two "a" groups a r e d i f f e r e n t t o the enzyme and
only one group r e a c t s a s hers been c l e a r l y established with s p e c i f i c a l l y
labeled c i t r a t e and glycerol.
Oxygen-18 has been used t o oxidize various substrates and a
number of oxygenases shown t o incorporate molecular oxygen not oxygen from
water i n t o the product.
9.
Isotopes i n Enzyme Assays
Rapid methods of following an enzyme catalyzed reaction a r e often
based upon t h e use of radioisotopes, For example, D r . Ghblson and coworkers
(6) have been studying t h e conversion of quinolinic acid t o n i c o t i n i c acid
rnonoucleotA.de by following t h e release of C1402 from t h e 2-carboxyl group of
t h e substrate
\
R i b ose -P
-t co2
When isotope methods can be develoved, they a r e frequently more precise and
rapid because of t h e ease of estimating radioactivity.
W
e have f e l t t h a t isotopic techniques a r e j u s t one more t o o l which
should be understood and used by a l l who work i n biochemistry, All of our
students i n laboratory courses receive sane experience with C14 and our
graduate courses provide repeated contact with t h i s technique.
T h i s hasty exposure has been intended only t o t e l l you of some
p o s s i b i l i t i e s and limitations of the isotopic methods i n solving biochemical
problems. It i s manifestly unsuitable f o r problems not involving dynamic
properties of matter. For s t a t i c or r e l a t i v e l y s t a b l e s i t u a t i o n s , t h e
application of isotopes i s r e s t r i c t e d largely t o t h e i r a n a l y t i c a l use. I
should l i k e t o close by discussing briefly a use of isotopes which has been
t h e subject of discussions i n these conferences i n p a s t years. I h e s i t a t e
t o discuss t h i s application because many of you a r e working a c t i v e l y i n t h i s
f i e l d , I r e f e r t o t h e use of K4O measurements f o r estimating t h e composition
of l i v e animals or cuts of meat.
D. K40 Methods f o r Carcass Composition
There i s great need f o r a method which would permit one t o
ascertain t h e body composition of a meat animal a t i n t e r v a l s during the
growth period without s a c r i f i c i n g t h e animal. Even a rapid measure of t h e
mass of muscle i n a cut of meat would be useful. Body composition of human
214
subjects and experimental animals has concerned the physiologist f o r over
100 years and a great deal of e f f o r t has been devoted t o t h i s problem
recently. Skin fold thickness WId height-weight data, used t o assess the
n u t r i t i o n a l s t a t u s of human populations, i s of no value f o r other species.
More precise methods a r e needed i f the energy or protein stored during a
period of metabolic study is t o be determined without replicating t h e
animals so t h a t samples can be taken a t the desired intervals. Even if a
sample of the population can be sacrificed, t h e methods available a r e not
ideal, chiefly because of t h e d i f f i c u l t y of obtaining a representative
small sample of a carcass o r a large cut of meat.
Potassium analysis by t h e usual means a s an indication of t h e
mass of muscle would not be considered seriously when samples of the tissue
a r e available f o r l i p i d o r protein determination, The recognition of K40,
aY-ray emitter, a s a normal constituent of natural potassium and t h e
development of very sensitive and e f f i c i e n t liquid s c i n t i l l a t i o n methods of
detecting radiation suggested t h a t whole animals or cuts of meat could be
analyzed f o r K 4 0 which r e f l e c t s the potassium content, which i n turn r e f l e c t s
the lean body mass.
The use of K U measurements t o assess t h e composition of l i v e
animals, h c l u d i n g man, a s w e l l a s cuts of meat has been described by many
workers since the measurement of potassium i n t h e body by i t s radioactivity
was f i r s t described (15). A brief review of t h i s work up t o 1961 was published by Anderson and Langham (16), and the problem was discussed by
Andrews and Christian (17) a t t h e Mexico City Conference l a t e in 1961. The
method agrees well with other methods of determining lean body weights of
man (18)
.
Jh 1959, the value of 73 milliequivalents of potassium per kg of
lean body weight was adopted in t h e place of a value of 63 used i n the
e a r l i e r studies. Anderson and coworkers a t Los Alamos have worked on t h i s
method f o r approximately t e n years, It has been applied t o l i v e hogs (19),
t o hams (20), and t o l i v e sheep (21)
Because t h e r e s u l t s indicated t h a t
t h e e r r o r s of prediction of body composition were t o o hiGh f o r satisfactory
use in most animal breeding programs, the K40 method was checked against the
flame photcmetry method by Kirton and Pearson (22). They observed t h a t there
was a significant correlation between carcass composition of lambs and the K
content a s measured by flame photometry, but not when estimated by K40 cont e n t . Gn separable lean and f a t the two methods agreed. On ground lamb and
g ~ o u n dpork t h e K40 measurements agreed with t h e flame photometry method,
though t h e latter method gave values for K which were much more closely rel a t e d t o $ water, $ f a t , and $ protein. This relationship was closer f o r
pork than lamb. They concluded t h a t "A degree of precision a t l e a s t comparable t o t h a t obtained by flame photosnetry is needed f o r a non-destructive
method (such a s
before the accuracy i s great enough t o be useful."
.
E.
e)
Some Recent Developments
Isotopes can be expected t o make many more important contributions
t o t h e field of biological sciences. Three exciting developments of recent
years w i l l be mentioned briefly here. The development of density gradient
centrifugation has made it possible t o separate macromolecules containing
215
heavier isotopes from t h e i r normal counterparts. For example, deoxyribonucleic acid frcm b a c t e r i a l c e l l s grown with a N15 nitrogen source can be
separated from N14 DNA using a cesium chloride solution a s a medium (23).
T h i s technique makes it possible t o t r a c e DNA O r a m each parent of b a c t e r i a l
c e l l s among progeny molecules.
A similar application of radioisotopes i s exemplified in t h e use
of t r i t i a t e d thymidine t o locate within chromosomes the DNA formed during
a given period j u s t a f t e r administration of the labeled thymidine (24).
T r i t i u m has a d i s t i n c t advantage in such experiments i n t h a t i t s '
B i s so
low i n energy t h a t the s i l v e r grains i n a photographic emulsion activated
by tritium l i e clustered within one micron of the labeled locus.
FinaUy, mass spectrometry i s finding new use in establishing t h e
i d e n t i t y of ccmpounds separated by the rapid, gas chromatographic technique.
Since flavor compounds a r e frequently v o l a t i l e , t h e i r separation by gas
chromatography has proved f e a s i b l e , Drs. Waller and Mason in our laboratories have separated t h e v o l a t i l e flavor components from peanuts i n t h i s way
and a r e examining t h e separated compounds by mass spectrometry.
Biemann (25) has described the use of mass spectrometry f o r
establishing the s t r u c t u r e of substances which were not previously considered v o l a t i l e enough, The substances are sublimed d i r e c t l y i n t o t h e
ionizing electron beam. The method is very s e n s i t i v e and rapid, and requires no chemical pretreatment.
The i l l u s t r a t i o n s given may serve i n a small way t o indicate the
wide variety of applications of isotopes, If t r a c i n g an atcm or group of
atoms w i l l provide a better understanding of a dynamic process or if you
need precise a n a l y t i c a l data, you might f i n d isotopes useful.
1. Glascock, R . F., Use of Radioisotopes in Animal Biology and the Medical
Sciences, Vol. 2, pp. 49-67, Intern. Atcmic Energy Agency, Academic
Press, New York, 1962,
2. BlOCh,
3.
4.
5,
K.9
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BiOl.
Ch-.,
157,
661 (1945).
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Heidelberger, C., E , P. Abraham and S. Lepkovsky,
J. Biol. Chem., 179
151 (19491, -
Gholson, R. K., D. R. Rao, L. M. Renderson, R. J. H i l l and R. E. Koeppe,
J. Biol. Chem., 2 3 , 179 (1958).
.
H i l l , R. J., D. C. Hobbs, and R. E. Koeppe, J. Biol. Chem.,
(1958)
230,
169
216
Mathur, G. P. Ph.D. thesis, Oklahoma S t a t e University, 1961,
Phillips, M. B . and H. S. Anker, J. Biol. Chem.,
227,
465 (1957).
Gholson, R, K. and L. M. Henderson, Biochim. Bioploys. Acta,
(1958)
9.
10
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30,
424
Masurat, J. HI and M. Helfant, Science, 122, 72 (1955).
Blumenthal, H. J., K. F. Lewis and S. Weinhouse, J. Am. Chem. Soc,,
76, 6093 (1954).
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ll. Vennesland Birgit, J. Cell. and Comp. Physiol., 47_, Suppl. 1, 201
(19563
12
.
13
14
Ogston, A. G., Nature, 162, 963 (1948). Potter, V. R.,
Heidelberger, Nature 164, LBO (1949).
and C.
Schambye, P., H. G. Wood and G. Popjak, J, Biol. Chem., 206, 875 (1954).
Swick, R. W. and A. Nakao, J. Biol. Chem,, 206, 683 (1954).
Gholson, R. K.,
Ueda, I. and L. M. Henderson, Fed. Proc., 22, 651 (1963).
15
Burch, P. R. J. and F, W. Spiers, Nature, 177, 519 (1953).
R. M., Arkiv. Fysik, 3, 337 (1951).
16.
Anderson, E. C. and W. H. Langham, Science, -9
133 1917 (1961).
_c
Sievert,
17
Andrews, F. N. and J. E. Christian, Use of Radioisotopes in Animal
Biology and the Medical Sciences, Vol. 2, pp. 189-198, Intern.
Atomic Energy Agency, Academic Press, New York, 1962.
18
Allen, T.
19
Zobrislry, S. E., H. D. Naumann, A. J. Dyer and E. C. Anderson, J.
Animal Scf., 18, 1480 (1959).
.
.
.
.
20
21
22
23
24
25.
e.,J. Gerontol.,
15, 348 (1960).
Kulwich, R,, L. Feinstein, C. Golumbic, R. L. H i n e r , W. R. Seymour and
W. R, Kauffman, J. Animal Sci., 20, 497 (1961).
Kirton, A. H., E. M. Pearson, R. H. Nelson E. C. Anderson and R. L,
Schuch, J. Animal Sci., 20, 635 (1961j.
Kirton, A. E. and A. MI Pearson, J. Animal Sci.,
22, 125 (1963)
.
Meselson, M. and F, W. Stahl, Proc. Nat. Acad. Sci., 44, 671 (1958)
--
.
Hughes, W. L. in McElroy and G18s6, The Chemical Basis of Development,
The Johns Hopkhs Press, B a l t b G
Biemann, K., Angew. Chemie,
74. 102
(1962).
217.
TABLE I1
LABELING PATTERN I N NON-ESSEXTIAL AMINO
ACIDS FRCM CEETAIN METABOLDES
CCMPOUND
COMPOUND
ADMINISTERED
ISOLATED
CH$1400H
GLUTAMATE
PATTERN
c5
+ c1 > 95%
~ 5 / ~=1 2
ASPARTATE
c1
+ c4 > 95%
c1 = c4
c2 = c3
C1%,C
OOH
ALANINE
c1 =
SERINE
C1 = 9C$
GLUTAMATE
ALANINE
CH3CL40C OOH
GLUMMATE
ALANINE
90
and
-
95$
218
DISTRIBUTION OF CL4 IN GLVTAMIC A C I D
AND ALANIIE FRGM DL-TRYFTOPHAN-C14*
GLUI'AMIC A C I D
Sa muc/mole
Percent of t o t a l $4
C1
c2
in
Trypt ophan-5 -C
Carcass
Liver
Carcass
2847
16.9
23.8
6004
24.3
15.9
1246
23 45
24.5
38.8
2 -3
12.4
23 43
20.7
39 02
2.4
7.7
20 03
c3
2 00
m8
c4
74.1
8345
c5
SA muc/mmole
4 e2
4
Percent of t o t a l ~ 1 in
20.4
39.6
39 m7
38 e 5
6
c2
Liver
22.7
94
C1
38.6
c3
Wean values for two animals
DEERJBUI'ION
OF $4
FRCM POS~TTIONS7a AND 5 OF
TKYPTOPHAN I N TRAPPED ACEX'ATE
Tryptophan-7A-C14
Tryptophan-5-C
muc/mmole
rnuc/mmole
Acetate
22.1
25 .5
c -1
19.9
1.4
m3
24.5
C -2
14
Tryptophan-7a -C14
14
219
MR, PEARSON: It is almost nom, but we have time f o r
j u s t one or two questions
we don% have very much time,
--
If there a r e no questions now, i f you should have any l a t e r
t h a t you would personally like t o ask the speakers about, you may t a l k
t o them individually. I am sure most of them will be available for
a l i t t l e while, anyway.
D r . Kemp,
A t t h i s time I will turn t h e program back t o our Chairman,
CHAIRMAN KHP: Thank you, A I ,
Just one thing before we go. You see t h e ten names here, we have
put them back out there for you t o see i f you don't remember them. Please
put the name on a sheet of paper and pass them down, and, Harold Hedrick, if
you w i l l pick them up on t h i s side, and I ' U ask Carroll Schoonover t o pick
them up on the other side,
We w i l l now recess f o r lunch, and reconvene here a t 1:15 sharp.
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