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THE INFLUENCE OF STRUCTURAL CONFIGURATION ON THE DEAMINATION OF AMINO ACIDS IN THE NORMAL RAT

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DOCTORAL DISSERTATION SERIES
TITLE
AUTHOR
UNIVERSITY
DEGREE
fa wT
I
PUBLICATION NO.
1' 2 3
y UNIVERSITY MICROFILMS
M ANN ARBOR ■ MICHIGAN
THE INFLUENCE OF STRUCTURAL CONFIGURATION
ON THE DEAMINATION OF AMINO ACIDS
IN THE NORMAL RAT
Morris Franklin Milligan — Thesis (Ph.D.)
Purdue University, 1942
Optically active amino acids were administered orally to albino
rats excreting constant amounts of urinary nitrogen; the distribution of the
extra urinary nitrogen was determined.
The nitrogen of 1(/) isoleucine,
1(/) alloisoleucine, d(/)leucine, l(-)leucine, d(-)norleucine, l(-/)norleucine,
1(/) valine, 1(/) ac-amino-phenylacetic acid, and d(-)<r-aminophenylacetic acid appeared in the urine as extra urea and ammonia.
That of
d(-)isoleucine, d(-)alloisoleucine, d(-)valine, d(-)pseudoleucine, and
l(/)pseudoleucine appeared in the urine largely as amino acid nitrogen.
It is concluded that:
a.) the presence of a substituent methyl
group on the beta carbon atom interferes with the deamination of amino acids
of the d- series but has no effect on the deamination of their enantiomorphs;
b.) the presence of two substituent methyl groups on the beta carbon inter­
feres with the deamination of amino acids of both series; and c.)<-aminophenylacetic acid does not conform to the previous generalizations, either
form being metabolized by the albino rat.
P U R D U E U N IV E R S IT Y
THIS IS T O CERTIFY T H A T T H E THESIS P R E P A R E D I’N D E R M Y SI'PER VISION
5X
Morris Franklin Milligan
ENTITLED
TIC INFLUENCE OF STRUCTURAL CONFIGURATION ON THE
DEAMTNATTOH .
OF oi.T U U ACTi;S TN UErfr. MOttf/AI. HnT
C O M P L I E S W I T H T H E UNIVERSITY R E G U L A T I O N S O N G R A D U A T I O N T H E S E S
A N D IS A P P R O V E D B Y M E A S FUL FILLING THIS P A R T O F T H E R E Q U I R E M E N T S
FOR THE DEGREE OF
Doctor o£ Philosophy-
it. e ,
e
P h o f k s s o r ix C h a r g e o f T h e s is
v t-y
H ead of S chool oh D epartm ent
■august 17.________ i« 42
T O T H E L I B RARIAN
-i
THIS THESIS IS N O T T O B E R E G A R D E D A S CONFIDENTIAL.
Tr*. C
I'R O K K K S O M
K K G ISTR A Il
FOHM
10-4-IO -1M
IN
iK
THE INFLUENCE OF STRUCTURAL CONFIGURATION
ON THL. DEa I'SNATION OF AilINO ^CIDS
IN THE NORMAL RAT
A Thesis
Submitted to the Faculty
of
Purdue University
by
Morris Franklin Milligan
in partial fulfillment of the
requirements for the Degree
of
Doctor of Philosophy
August, 1942
The writer gladly acknowledges the guidance and encouragement
of Dr* R* C. Corley during the carrying out of this investigation
and the preparation of the manuscript*
Eis thanks are also extended
to his compeers for their aid and enthusiasm.
Table of Contents
Page
Introduction
1
Historical
2
Experimental
Materials
13
Diets
14
Methods
lu
Synthesis ofdl-Pseudoleucine
15
Resolution ofoc-Aminophenylacetic acid
16
Procedure
18
Discussion
20
Summary and Conclusions
25
Bibliography
26
Tables
INTRODUCTION
The intermediary metabolism of amino acids is a problem which has
long intrigued biochemists and others associated with general problems
of metabolism*
As yet, we await even a moderately complete picture of
the catabolic fate of these substances.
Amino acids such as tryptophane, histidine, and tyrosine contain,
as part of their chemical structures, components which render possible
identification of certain of their catabolic intermediates.
The chem­
ically "undistinguished” amino acids, aliphatic in nature, present no
such convenient grouping: information concerning their metabolic fates
must necessarily be gathered by less direct means.
The picture is further complicated by the fact that all amino
acids of biological interest, with the exception of glycine or aminoacetic acid, exist in at least two stereoisomeric forms.
It is an
accepted fact that one enantiomorph may follow an entirely different
metabolic pathway than does its mate.
It has been the purpose of this investigation to study the metabo­
lism of a representative group of aliphatic amino acids, in the hope
that by comparing the metabolism of compounds demonstrating both posi­
tional and steric isomerism, some light may be thrown on the means by
which the intact animal handles amino acids.
2.
Historical
Amino acids, liberated from food proteins during the digestive pro­
cesses of the animal organism, enter the blood stream, are carried to the
liver, and are at that point burned as a source of energy, or carried in
the systemic circulation to various parts of the body where they are used
for tissue building or replacement.
We are here concerned with the burning of the amino acids, or their
catabolism; in this process, the amino acid is first deaminated to liber­
ate the nitrogen, leaving the carbon chain to be degraded, presumably to
COp and H 2O.
The liberated nitrogen, in the mammalian organism, appears
in the urine as urea or ammonia.
Friedman (1908), using dogs, fed several amino acids and determined
the carbon:nitrogen ratio in the urine.
The results of his experiments
led him to conclude that glycine, dl-alanine, and dl-<x-aminobutyric acid
were completely metabolized.
Under the conditions of his experiments,
N-methylation of the amino acids rendered them resistant to oxidation.
In 1913» Ringer and co-workers, using phlorhizinized dogs, determined
that isobutyric and isocaproic acids were capable of forming glucose,
while isovaleric acid heightened the production of acetone bodies.
On the
basis of this work, they postulated that the "iso-” acids, as the first
step in their degradation, underwent demethylation, presumably forming
methyl alcohol and the normal fatty acid containing one carbon atom less.
They further postulated that the ae-amino acids such as valine and leucine
are first oxidatively deaminated; after ensuing decarboxylation the "iso-"
fatty acid is degraded by demethylation as previously mentioned.
Ringer's statement concerning valine has been substantiated by Butts
and Sinnhuber (I9l4l)» these investigators found that valine may give rise
to significant quantities of liver glycogen.
This is of interest with
regard to the following generalizations laid down by Dakin.
Dakin (1913)» from experiments on phlorhizinized dogs drew'the fol­
lowing conclusions:
1.) Every amino acid having five or fewer carbon
atoms (except valine) is capable of forming glucose.
2.) All straight-
chain amino acids, with the exception of lysine, are sugar formers.
3»)
Amino acids having branched chains produce no glucose.
Knoop and Oesterlin (1925), on the basis of model experiments on the
reduction of keto acids in the presence of ammonia, suggested the follow­
ing theory of amino acid degradation:
R-CH-COOH
v
R -C -C O O H
i’Hg
RC -C O O H
im
0
Their work enhanced the previously held belief that ketonic acids were
intermediates in the metabolism of amino acids.
In 1926, Dakin presented a theory involving the existence of an inter­
mediate unsaturated amino acid as follows:
R -C H g-C H -C O O H
m 2
_____►
R-CH - OCOOE
----- *.
ne2
RCHg'C-CCOH
nh
R-CHg-C-COOH
0
Experiments which he carried out on pseudoleucine served to substantiate
his theory since administration of this compound, containing no hydrogen
atoms on the
mixture.
carbon atom, resulted in excretion of the unchanged racemic
It had previously been reported (Knoop and Okada (1 9 2 3 )) that
administration of dl-pseudoleucine to dogs resulted in the excretion in
the urine of the dextrorotatory form.
The work of Krebs (1933J has shown that amino acids of the d- series
are often more rapidly deaminated than their naturally-occurring enantiomorphs, at least by tissue slices or by extracts.
He has been able to
demonstrate in these experiments the existence of a system which, he states,
may be an artifact or a fragment of the original system deaminating the
<
1-amino acids.
He was further able to show (1935) that the system oper­
ating on the d-amino acids and that operating on the 1-amino acids deaminated
their substrates by different pathways; in these investigations, it was
found that octyl alcohol and cyanide inhibited the deamination of 1-amino
acids, while the d-deaminase was unaffected by these reagents.
work has been verified by a number of workers:
Krebs*
Bernheim (1935)» Kirsch
(1935, 1 9 3 6 ); and Neber (1936).
In 1936, Keilin and Hartree, using a preparation of d-amino acid
oxidase, found that d(-)N-monomethylalanine is deaminated, while d(-)Udimethy 1alanine and cc-aminoisobutyric acid are not.
This indicates that
there must be one free hydrogen atom on the nitrogen and that the oc- car­
bon atom must be unsubstituted save for the amino group.
The work of these
investigators indicated that peroxides might be formed during the process
of deamination; Bergel and Bolz (1933, 193U) have found, indeed, that
per­
oxides are formed when methylated amino acids are oxidized in modelsystems.
Considering these observations, Krebs (1936) postulated the following
scheme for the action of d-amino acid oxidase:
R-CH-COOH __ „
NHp
R-CH-COOH
HNH
__ _
O'-O
R-C-COOH _____
NH
R-C-COOH
0
4- HpOg
Klein and Handler (l9Ul), however, have reported that oxygen consumption
in the above system is only 15 to 30 %
that indicated by the equation;
this observation throws some doubt upon the scheme indicated above.
Recent work by du Vigneaud, Wood, and Binkley (19 )41) has led to the
observation of acetylation in vivo of a d-amino acid; p-bromophenyl-dcysteine, administered to a dog, appeared in the urine as the optically
active acetyl derivative.
This is the only observation of the kind, and
indicates that some provision may be existent in the animal organism for
the detoxication of d-amino acids.
From a teleological point of view, it seems strange that such a
system should exist when its substrate appears to be absent in nature.
Thus the stage was set for the discovery by K&gl and Erxleben (1939) that
tumor tissues contained what they described as significant quantities of
d-amino acids, d-glutamic acid being particularly notable by its presence.
This announcement had been presaged by the discovery of Fr&nkel and coworkers (l92li) that d-amino acids were formed by the prolonged action of
trypsin on casein, a piece of work vh ich was not held in high esteem by
other workers in the field.
For some time after the original work by KBgl
and Erxleben, some controversy existed over the validity of their claims;
one school of thought held that the d-amino acids were artifactual, but
recent work in other connections by Hotchkiss (l9ill) and Christensen,
Edwards and Piersma (19I4I) has demonstrated beyond reasonable doubt that
these substances may exist in nature, at least in significant concentrations.
Be this question as it may, both d- and 1-amino acids may be deaminated
by the animal under suitable experimental conditions.
Krebs (193&) stated
that "ample evidence has been accumulated showing that both stereoisomerides
of the amino acids can be attacked by the intact organism."
The work of the late Rudolph Schoenheimer, being of an outstanding
nature, deserves careful consideration.
In 1938 (Foster, Rittenberg, and
Schoenheimer), it was announced that lysine was the only amino acid re­
sistant to the introduction of deuterium and isotopic nitrogen from body
fluids.
In 19ij.O, Schoenheimer and Rittenberg announced that the most ex­
tensive interchange of normal nitrogen for isotopic nitrogen occurred in
the nitrogen of aspartic acid and glutamic acid, whether the isotopic
nitrogen -was introduced as ammonia or as a single amino acid.
On the basis
of these observations, they pictured the body as possessing a pool of
aminoid nitrogen, this nitrogen being passed around to suitable acceptors
by the intermediation of the dicarboxylic amino acids.
Lysine, it would
appear, does not participate in the communal metabolic scheme; this theory
has been advanced by Rose (1938) to account for the indispensability of
threonine in the diet, that amino acid supposedly being resistant to ex­
change of nitrogen in the body.
In view of the metabolic stability of
lysine, it is not surprising that natural lysine cannot be replaced in the
diet by d(-)lysine (Berg, 1936), ec-aminocaproic acid (Lewis and Root, 1920),
flt-hydroxy-t-aminocaproic acid, <*-hydroxycaproic acid, fc-hydroxvcaproic acid,
or £-aminocaproic acid (McGinty, Lewis, and Marvel, 192i|.).
The central
position in nitrogen distribution occupied by the dicarboxylic amino acids
is further emphasized by the discovery that proline, hydroxyproline, and
ornithine are readily converted by the body into glutamic acid:
Roloff,
Ratner, and Schoenheimer (19I4O); Weil-Malherbe and Krebs (1935) J anc* Neber
(1936).
These dicarboxylic amino acids serve to link amino acid metabolism
to carbohydrate metabolism, since aspartic acid is supposedly in equilib­
rium with oxaloacetic acid, and glutamic acid with «<-ketoglutaric acid,
the ketonic acids being accepted intermediates in carbohydrate metabolism.
Borsook and Dubnoff (19i^l) have further demonstrated the essential
role played by the dicarboxylic amino acids in the conversion of citrulline
to arginine; this is supposedly one of the steps in the formation of urea
(Krebs and Henseleit, 193^) from the nitrogen of catabolized amino acids.
Some light was thrown upon the means by which the body effects the
transfer of amino nitrogen by Braunstein and Kritzm&nn (1937). who reported
that a system existed in practically all tissues enabling en^araino acids to
react with «-ketonic acids.
As a result of their investigations (1937, 1939),
they reported that an equilibrium is reached between any ketonic acid and
any amino acid as long as one of these is dicarboxylic.
Braunstein and
Bychkov (1939) postulated, on the basis of experiments using muscle ex­
tracts, that deamination of amino acids in the animal body may occur by
the transamination reaction.
They indicated that oc-ketoglutaric acid may
react with alanine to form pyruvic and glutamic acids; the glutamic acid
is converted by Krebs' glutamic dehydrogenase (1935) "k0 ammonia and
dC-ketoglutaric acid.
Thus, they state, any amino acid may be deaminated
in the tissues.
Cohen (1939* 19)$» 19)(l)> however, has duplicated some of the work
of Braunstein and Kritzmann using refined analytical technics, and has
cluded
thatthe reaction is not
a general one.
con­
He has stated that previously
drawn conclusions were based on results obtained by grossly inaccurate
measurements, and has revised the generalizations promulgated by Braunstein
and Kritzmann.
Cohen has found l(^/)alanine,
l(/)glutamic acid, l(-)aspar-
tic acid, and l(-)cysteic acid are the only amino acids active in trans­
amination; the activity of the latter compound is due to its similarity,
in an electronic or polar sense, to the dicarboxylic acids.
The only equi­
libria functioning in his experiments were the following:
l(/)glutamic acid
l(-)aspartic acid
-«—
^-—
l(/)alanine-------- ^--..
<*-ketoglutaric acid
oxaloacetic acid
pyruvic acid
Thus we see that the original general theory proposed by Braunstein and
Kritzmann loses much of its utility in the light of Cohen's work.
Cohen's work also disposes of the general claim of Braunstein and
Kritzmann that amino acids of the d- series are deaminated at a lower
though significant rate by the transamination reaction.
Krebs (1935) stated that the 1-amino acid oxidase depends for its
activity upon the integrity of the cell structure and deteriorates rapidly
8.
on standing; this fact led him to postulate that the d-amino acid oxidase
previously discussed might be an artifact, or a fragment of the original
enzyme.
It is possible that the work on transmanination, carried out as
it has been on minced tissue and extracts, has resulted in the study of
an artifactual system; future work will indubitably serve to clear up the
questions surrounding this field of investigation.
Work on the intact animal is highly desirable from several points of
view; in the first place, the study of isolated systems, as we have seen,
may lead to much ado about a material or a phenomenon later found to be
artifactual.
Furthermore, no observations can be as valid as
those made
on an intact, normal animal, where the natural systems of metabilism are
at work undisturbed by chemical or physical manipulation.
In studying amino acid metabolism with the intact, normal animal, ad­
vantage may be taken of the fact that the nitrogen excreted as the result
of deamination appears chiefly either as urea or ammonia, depending upon
the acidity of the urine.
If an amino acid be administered to an animal
in nitrogen balance, it is likely that the nitrogen resulting from deam­
ination will appear in this fraction; if, on the other hand, the compound
is not deaminated, we may logically expect that it will be excreted as the
free amino acid, and appear as such.
Stekol and Schmidt (1933) have used this technic with dogs; they fed
amino acids, and concluded from urine analyses that glycine and l(/)glutamic acid are converted to urea.
Leighty and Corley (193&), using the same method, found that dlalanine, dl-oc-aminobutyric acid, and dl-oc-aminovaleric acid are catabolized by the normal dog.
The administration of dl-valine caused an increased
excretion both of urea and amino acid nitrogen; this indicated that one
form was catabolized while the other was not.
Snyder and Corley (193®)* in
an ensuing study on dogs, found that the d- forms of amino acids are not
significantly deaminated unless the (5-carbon atom holds 2 hydrogen atoms.
They found that the 1- forms are deaminated if the <x- and
carbon atoms
bear 1 hydrogen atom each, and further noted that dl-pseudoleucine is not
significantly deaminated.
It is considered that an extension of this work would be of value;
therefore, the present investigation deals with the metabolism of the in­
dividual stereoisomeric forms of valine, leucine, isoleucine, norleucine,
pseudoleucine, and phenylglycine, utilizing the adult white rat as the
experimental animal.
A great deal of work has been done on the relation between the
structural configuration of the amino acids and their actual rotatory
powers.
Since in many cases there appears to be no absolute relation existent
between an amino acid fs configuration and its rotation, it is customary
to indicate both in writing the names of the individual stereoisomer.
For
example, that leucine which is ordinarily dextro-rotatory has been found
to belong, indeed, to the dextro series; it is written, therefore, as
d(/)leucine.
On the other hand, that isoleucine which is ordinarily dextro­
rotatory has been found to belong to the levo series; it is written, there­
fore, as l(/)isoleucine.
These examples serve to illustrate not only the
method of indicating the structural and physical properties of the amino
acids, but also the difficulties attendant upon any attempt to correlate
rotation and structure.
Determination of the rotatory properties of an amino acid is carried
out simply by finding the effect upon plane polarized light of a solution
of the amino acid.
The only difficulty encountered here is that occasion­
ally a change in pK or temperature will effect the direction of rotation
10.
and consequently the sign.
For example, it is known that the direction
of rotation of l(/)alanine in water changes sign with an increase in
temperature (Clough, 1918)*
"If it is assumed that the sign of the pre­
fix is determined by the direction of the free amino acid in water at
25 °, the rule would be in accord with the usual designations of sign."
(Dunn, 19)-d-)•
Determination of the structural configuration of an amino acid pre­
sents a problem which is much more difficult and which can be attacked
only by indirect means.
There are three general methods for handling
this problem; these methods will be discussed one by one.
The most direct method is that of relating the amino acids to a
reference standard, much in the same general way as electrode potentials
are related to the normal hydrogen electrode.
The reference standard in
this case is taken as lactic acid; there is evidence that the dextro­
rotatory lactic acid belongs to the levo series.
By converting natural alanine, therefore, to l(/)lactic acid without
the possibility of Walden inversion, we have demonstrated that this natural
alanine, which is dextro-rotatory, belongs to the levo series (Freudenberg
and Rhino, 192b) •
Further, since natural serine can similarly be con­
verted to l(/)alanine (Fischer and Raske, 1907), or to natural cystine
(Fischer and Raske, 1908), it is seen that cystine and serine exist
naturally as members of the levo series.
This relation has been extended
to many of the amino acids (Dunn, 191+1 )•
A second method is to note similarities in the optical properties of
amino acids and their derivatives to those of compounds whose configura­
tion is already known or established.
Let us cite two examples.
First,
amides or esters of the acetyl, benzoyl, and other N-derivatives of
natural alanine and of derivatives of l(/)lactic acid show analogous
11.
changes in optical rotation (Freudenberg and Meister, 1935)> thus we have
demonstrated again that natural alanine belongs to the levo series.
In
the second place, l(/)aspartic acid may be converted without Walden in­
version to an *,w-diamino acid derivative which shows changes in optical
rotation analogous to the corresponding derivative of natural ornithine
(Karrer, Escher, and Widmer, 1926); since l(/)aspartic acid may be simi­
larly converted to l(-)serine (Schneider, 1937)* which we have seen is
related to l(/)alanine, the position of ornithine in the levo series is
established.
The third method mentioned is a biological one: it was noted early
that the natural amino acids were, as a rule, readily metabolized by an
experimental animal or by microorganisms, while the unnatural isomer was
often untouched.
This phenomenon has been exploited by Ehrlich (1906)
in the preparation of d(/)leucine, d(-)alanine, and d(-)valine; in each
case the synthetic racemic mixture was exposed to the action of growing
yeast cultures, which destroyed the natural or levo form.
The method was
extended by Ehrlich and Wendell (1908) to phenylalanine, serine, ^-aminobutyric acid, and a-aminophenylacetic acid.
The biological resolution thus accomplished indicates that the isomer
attacked presumably belongs to the levo series; conclusions based on such
experiments are apt to be invalid unless supported by further evidence.
Abderhalden, Faust, and Haase (193U) studied the metabolism of the levorotatory pseudoleucine; since the compound was not deaminated by the
normal dog, they concluded that the compound was a member of the dextro
series.
That this is an unwarranted conclusion is indicated by the fact
that neither isomer is attacked, either in the dog (Snyder and Corley,
1938 )> oi*j as found in the present investigation, in the albino rat.
12.
The present paper is concerned with the biological destruction of
the pure stereoisomers of the amino acids.
The exact limitations of
this phenomenon are not known, although Snyder and Corley (1938) have
indicated generalizations correlating the structural configuration of
the amino acid and its susceptibility to destruction in the body of the
normal dog.
The present study constitutes an extension of the work of
Snyder and Corley as applied to the albino rat.
Experimental
Materials:
The amino acids used in this study were either synthesized in this
laboratory or were taken from lots previously found in this laboratory
to be of sufficient purity.
Dl-leucine was synthesized by the method of Marvel (19^1*) and re­
solved by the method of Fischer and Warburg (1905).
Dl-isoleucine was synthesized by the method of Marvel (l9iAl>) and
resolved by the method of Locquin (190?)•
D(-)alloisoleucine and l(/)alloisoleucine were prepared by the
method of Carter (1937) from the previously mentioned dl-isoleucine.
D(-)nor leucine and 1(/) nor leucine were materials kindly turned over
to me by Dr. R* C. Corley.
Dr. M. L. Lyons (I9I4J.) had obtained them by
the resolution with brucine of an Eastman Kodak product.
D(-)phenylglycine and l(/)phenylglycine were prepared by resolution
with brucine of an Eastman Kodak product.
The procedure, being a new
one, is given in detail.
D(-)valine and l(/)valine were products of Hoffman-LaRoche and
Company; they were from a lot used previously by Snyder and Corley (1938)
and found at that time to be of the requisite purity.
Dl-pseudoleucins was prepared from tertiary butylacetic acid by an
application of the method mentioned above for the synthesis of leuoine.
The tertiary butylacetic acid was kindly furnished by the Mallinckrodt
Chemical Works.
The procedure, being a new one, is given in detail.
Resolution of the dl-pseudoleucine was carried out by the method of
Abderhalden, Faust, end Haase (193U)*
1k.
Diets:
The basal diet upon which the experimental animals were maintained
consisted of finely ground Friskies, a commercial animal food prepara­
tion.
The food as it came frcm the bag was placed in a ball mill and
ground to an impalpable powder.
The homogenization thus achieved insured
a uniformity of nitrogen content; Kjeldahl analysis indicated that the
powder contained
Methods:
Adult female albino rats were maintained in cages of the Hendrix
circular type.
The cages were turned upside down and covered with a
solid circular piece of tin which could be wired in place to prevent es­
cape of the animals.
Each cage rested upon a number 16 mesh wire screen,
which in turn rested upon a metal funnel.
The latter led to the 2 ounce
urine collection bottle, which contained 3 drops of toluene.
Ordinary
drinking fountains fastened on the sides of the cages contained distilled
water at all times.
The powdered animal food was weighed into a food cup.
In order to
prevent undue loss of the material by spilling, the food cups were
placed inside tubes made
of number 26galvanized sheetiron.
fitted over the food cup
and extendedfrom the bottom to the top of the
cage, being fastened to the cage wall by a wing screw.
terior
Each tube
Facing the in­
of the cage, and about cue inch fr :m the floor, there was a hole
in the tube large enough
to allow theadult- rat* s headto enter.
This
allowed the animal to eat at will without the possibility of spilling
substantial amounts of the ration.
Analysis of the urine samples was carried out as follows:
Total
nitrogen was determined by the method of Bonner (19142), this method being
15.
a modification of the original Kjeldahl (1883) method.
A new indicator,
modified bromo-cresol green, as suggested by Ma and Zuazaga (19i|2), was
used.
Urea and ammonium nitrogen was determined by the method of Sobel,
Yuska, and Cohen (1937); in the final titration of the ammonia, the
indicator mentioned above was used.
Amino acid nitrogen was determined
by the method of Sahyun (1939); the depth of color developed was meas­
ured on the Fhotelometer.
Creatinine was determined by the microchemical
modification of Folin* s picric acid colorimetric method (19110; in
accordance with the recommendation of Hoffman (I9I+I), measurements were
made upon the Photelometer.
Synthesis of dl-pseudoleuoine (q-amino-fi,fi-dimethylbutyric acid):
One hundred and twenty grains of tertiary butylacetic acid was brominated by the procedure of Homeyer, 'Whitmore, and Wallingford (1933).
The yield of tx-bromo-tertiary butylacetic acid was ll|.l grams, or 70^ of
the theoretical amount.
One hundred and twenty grams of the bromoacid was added slowly with
stirring to 3*5 liters of C. P. aqueous ammonia (28^i NHj) in a 5 liter
round bottom flask.
A rubber stopper was wired in place, and the solu­
tion was allowed to stand for one week; at the end of this period, the
stopper was removed and the solution was heated on the steam cone for
four hours to get rid of the excess ammonia.
The solution was then con­
centrated to dryness at reduced pressure.
An attempt was made to purify the pseudoleucine by extracting the
chief contaminant, ammonium bromide, with absolute methanol.
It was
noted, however, that the amino acid was almost as soluble in the metha­
nol as was the ammonium bromide.
It was therefore necessary to extract
the pseudoleucine from the reaction mixture with hot glacial acetic
acid, in which ammonium bromide is only sparingly soluble.
16.
Repeated evaporation of the acetic acid solution at reduced pres­
sure with intermittent dilution with distilled water resulted in the
isolation of a white powdery material which gave a very weak qualitative
test for bromide ion.
This material was formylated by the method of Fruton and Clarke
(193h)•
From the reaction mixture was isolated 28*5 grams of formyl-
pseudoleucine; the yield, based on a-bromo-tertiary butylacetic acid,
was 31% of the theoretical amount.
The purity of the material was deter­
mined by means of its melting point, 208-210°C. (uncorrected), and its
neutral equivalent, 160 ± 2 (theoretical 159)*
These values are in
agreement with those given by Abderhaldan, Faust, and Haase (193W»
It is believed that further work on the amination of cc-bromotertiary butylacetic acid will lead to the formation of pseudoleucine in
considerably higher yields; this is indicated by the minimal tendency
of the bromoacid to lose hydrogen bromide, this latter reaction being
one of the most troublesome side reactions in preparations of amino
acids by this method.
Resolution of ofc-Amino-oc-Phenylaoetic Acid?
Fischer and Weichhold (1908) have described the resolution of
dl-<*-amino-CT-phenylacetic acid; they used cinchonine to precipitate the
formyl derivative of the 1- form, after which quinine was used to
precipitate the d- form.
The resolution has also been described using
^-bromo-(Jf-camphor-)sulfonic acid (Betti and Mayer, 1908).
These methods are not convenient and it was decided to attempt the
resolution in the most common manner, with brucine and absolute alcohol.
Accordingly, 10 grams of farmyl <*-emino-oc-phenylacetic acid, prepared by
the method of Fruton and Clarke (193^U)»
dissolved in I50 ml. of warm
absolute alcohol; this solution was poured slowly into a warm solution of
1722 grams of brucine in 1400 ml. of absolute alcohol.
Upon standing in the
refrigerator, 16 grams of a brucine salt precipitated from solution.
The precipitate was filtered with suction, washed with 100 ml. of abso­
lute alcohol, and sucked dry.
The dry material was dissolved in one
liter of distilled water and treated with 25 ml. of C.P. aqueous ammo­
nia (2B$ NH^).
In one minute the brucine had precipitated; it was
filtered off and washed with 100 ml. of cold distilled water.
The fil­
trate and washings were combined, extracted twice with 50 ml. portions
of chloroform and once with 50 ml. of ether, and concentrated to 500 ml.
at reduced pressure, -this step serving to remove traces of ether and
chloroform and also some part of -the excess ammonia.
The concentrated
solution was rendered acid to Congo red by the addition of 10$ HBr solu­
tion, following which the solution was concentrated to dryness at re­
duced pressure.
The dry residue was taken up in 200 ml. of 10$ HBr so­
lution; refluxing for thirty minutes removed the formyl groups quantita­
tively.
The solution now containing the amino acid hydrobromide, ammo­
nium bromide, and formic acid was concentrated to dryness at reduced
pressure.
Dissolution in warm 50$ alcohol followed by neutralization
with C.P. ammonia water resulted in the precipitation of the free d(-)-oCamino-ot-phenylacetic acid.
The yield of d(-)-ct-amino-oc-pheaylaoetic acid was 3.5 grams, corre­
sponding to 86$ of the theoretical amount, based on racemio formylated
amino acids.
The filtrate and alcoholic washings from the insoluble brucine salt
were combined and concentrated to dryness at reduced pressure.
The pro­
cedure thereafter was exactly the same as for the insoluble brucine salt.
The yield of l(/)-oc-amino-oc-phenylacetic acid was 3*7 grams, correspond­
ing to 90$ of the theoretical amount, based on racemie formylated amino
18.
acids.
Physical constants of all amino acids prepared in this investiga­
tion are to be found in Table 3»
Literature values for all amino acids
utilized in this investigation are to be found in Table 2.
Procedure:
It had previously been determined that the rats differed in their
ability (or desire) to eat the powdered basic ration.
Accordingly,
rats 1, 2 and 5 received 1+.00 grams of the ration daily, while rata 2,
3 and 6 received 5*00 grams; these amounts correspond respectively to
172 and 215 milligrams of nitrogen.
The indicated quantities of the ra­
tion were weighed into food cups; on the days when an amino acid was to
be fed (see Tables Is. to 9 inclusive), a quantity of 1he amino acid corre­
sponding to 25 milligrams of nitrogen was mixed thoroughly with the
basic ration.
In Table 1 is indicated the weight of each amino acid
equivalent to 25 milligrams of nitrogen.
The food (or food and amino
acid) was made available at the time of urine collection.
Urine was collected regularly at 2l>-hour intervals; the fecal ma­
terial (retained on the screen beneath the cage) was discarded.
Com­
plete collection of the urine sample was insured by washing the screen
and metal funnel with a fine stream of distilled water until the collec­
tion bottle was full.
The animals usually cooperated by urinating in
the pan in which they were placed during the process of washing the
screen and funnel.
The sample was then placed in a 100-ml. volumetric flask, being
transferred quantitatively, and diluted to the mark.
After thorough mix­
ing, the diluted sample was filtered through dry "Whatman No. 1 filter
paper, the first portion of the filtrate being discarded.
The filtration
procedure is necessary when Photelometric determinations axe to be
carried out on the sample.
Aliquots of this filtered material were
used in determining total nitrogen, urea and ammonium nitrogen, amino
acid nitrogen, and creatinine.
20.
Discussion
In Tables I4. to 9 are presented the results obtained with six ani­
mals; it is to be noted that with the exception of l(/)alloisoleucine,
no amino acid was administered to the same animal twice.
constancy for the creatinine figures furnishevidence of
The reasonable
satisfactory
collection of the samples of urine.
In each instance following the administration of a compound, there
was a sufficient increase in the total urinary nitrogen to account for
the bulk of the extra nitrogen administered.
A comparable increase in
the urea and ammonium nitrogen, recorded as urea nitrogen, is to be in­
terpreted to indicate deamination of the amino acid ingested.
An in­
crease in amino acid nitrogen (recorded as amino nitrogen), with no
significant increase in urea nitrogen, denies deamination, as it presum­
ably indicates excretion of the unchanged compound.
Typical results of the administration of resistant amino acids are
shown in Table l±, where the ingestion of d(- )pseudoleucine, d(-)valine,
and d(-)alloisoleucine failed to occasion any significant increase in the
urea and ammonium fraction.
The amino acid, it will be noted, was not
excreted in its entirety on the following day; this might be expected,
since amino acids are known to be threshold substances while urea is not.
This, however, does not explain fully
thefailure of the unmetabolized
amino acids promptly to be excreted.
Both forms of norleucine were readily metabolized (Tables I4 to 7 in­
clusive); Snyder and Corley (1938) found that either form of an amino
acid may be deaminated if unsubstituted on the «c- and
carbon atoms.
The same tables show that the leucines are similarly handled, substanti­
ating the claims of Snyder and Corley.
21.
In Tables I; to 9 are presented data indicating that the 1 - forms of
both isoleucine and alloisoleucine are deaminated, while the d- forms
are not.
This is in accord with the previously recorded experiments of
Snyder and Corley on d(-)isoleucine, d(- )alloisoleucine, and l(/)isoleucine; if we represent by the following formulas the structural rela­
tionship of the four compounds, inspection reveals that both d- forms
should undergo the same metabolic fate by virtue of their similar con­
figurations about the c^- carbon atom.
CH?
CHj
Et - C - H
E - C - Et
H - C - NH2
KH2- C - H
COOE
COOH
d(- )isoleucine
1 (/)isoleucine
ch3
CH3
H - C - Et
Et - C - H
E - C - NHp
NH2 - C - H
COOE
COOH
d(-)alloisoleucine
l(/)alloisoleucine
The formulas above are not intended to represent the actual configurations
of the various molecules, and are merely relative.
The experiments on the pseudoleucines (Tables 1; to 7 inclusive) in­
dicate that neither form of this amino acid is significantly deaminated
in the animal body.
This is in accord with the observations of Dakin
(1926) and Snyder and Corley (1938)*
Knoop and Okada (1923), using dogs,
found that the 1 - form was excreted in the urine after the administration
of dl-pseudoleucine.
Some caution should be used in viewing the values for amino acid
nitrogen, particularly with regard to the pseudoleucines; the method for
22.
analysis was that of Sahyun (1939)* in which the amino acid is caused to
react with ^-naphthoquinone-sulfonic acid.
The resultant color was
measured on the Photelometer, using a green filter.
The standard curve
had been constructed using pure alanine solutions and it was considered
that perhaps some of the amino acids used in this study might not have
their nitrogen measured quantitatively in such a manner.
Accordingly,
solutions of several of the amino acids (d(-)isoleucine, l(/)phenylgly­
cine, d(-)pseudoleucine, d(-)alloisoleucine, and l(-)leucine) were ana­
lyzed; all except pseudoleucine were found to give true values.
The
pseudoleucine values were approximately Q0fo of the actual, this being due
in all probability to a steric effect.
The experiments on the valines (Tables ij. to 7 inclusive) indicate
that, in agreement with the work of Snyder and Corley (1938)» the 1- form
is significantly deaminated while the d- form escapes the fate of its
enantiomorph.
The results obtained in this study with the enantiomorphic o*-aminophenylacetic acids indicate that the white rat is capable of deaminating
both forms.
These compounds have been the subject of earlier study;
Neubauer (1909)* Dakin and Dudley (1911+), and Snyder and Corley (1938)
have indicated that the dog is capable of resolving dl-«*-aminophenylacetic acid, the levo-rotatory form being excreted unchanged.
The dis­
crepancy may be explained on a basis of difference in species, although
one would not conclude a priori that the metabolism of the rat and the
metabolism of the dog should thus differ.
Let us postulate that amino acids of the d- series are broken down
in the intact animal by being first inverted to their respective epimers.
Objections may logically be raised that the d-amino acid oxidase con­
stitutes a separate system for the handling of d-amino acids.
Indeed,
23all the experimental evidence gathered by Krebs (1933), Bernheim (1935),
and others has been cited as proof of the existence of a separate system.
Keeping in mind the fact that all work on the d-amino acid oxidase has
been carried out with minced tissues or extracts, it is possible to
interpret such work as indicating that the d-amino acid oxidase is truly
artifactual.
For instance, Krebs (1935) found that octyl alcohol in­
hibits the deamination of 1 -amino acids and not that of d-amino acids;
he found that an aqueous extract of ground hog kidney attacked the dforms of valine and isoleucine while failing utterly to affect the 1 forms.
These facts point, according to many, to a separate system for
the deamination of amino acids of the d- series; however, since the intact
animal, as we have seen, deaminates only the 1 - forms of valine and iso­
leucine, it is even more probable that the d-amino acid oxidase does not
function in metabolism, and that its existence is purely artifactual.
On
this basis, our postulate is justified.
It is possible to combine the views of Dakin (1926) and of Bergmann
and co-workers (1935) into one essentially logical scheme.
Let us presume
that the following equilibria exist in the organism:
RCHpCHCOOH
IJHg
RCH= CCOOH
RHp
^
RCHpCCOOH
NH
As a part of Dakin's theory, this is a tenable view; such a series of
equilibria could explain conversion of certain amino acids of the d- series
into their epimers.
An extension of this view can offer a reason for the failure of cer­
tain d-amino acids to be metabolized.
Bergmann and co-workers (1935)» in
their polyaffinity theory, state that the spatial configuration of the
amino acid may affect the ease with which the appropriate enzyme can effect
combination.
Accordingly, we may postulate that a methyl group of the
2hp- carbon atom of amino acids of the d- series interferes with the pre­
liminary enzymatic inversion.
That this may be true is indicated by the
failure of d(-)valine, d(-)alloisoleucine, and d(-)isoleucine to be
deaminated; further, amino acids of either series, if unsubstituted on
the (1- carbon atom, are deaminated.
The resistance of the pseudoleucines
to deamination is also explained adequately on this basis.
The deamination of both forms of <x-aminophenylaeetic acid is a phe­
nomenon not entirely explained by any existing theory.
As previously
mentioned, the present observation is not in accord with earlier work,
and the promulgation of a probable mechanism is therefore difficult.
The
explanation that the phenyl group does not interfere with the enzyme-amino
acid combination is not compatible with observations made on experimental
animals other than the albino rat.
Further studies on amino acids bearing a variety of aliphatic con­
stituents on the
carbon atom should furnish evidence regarding the view
mentioned above.
Similarly, since the effect appears to be a steric one,
the effect of aliphatic substituents of various degrees of complexity on
the
£-, or even t- carbon atoms should provide a basis for a more com­
plete mechanism.
Summary and Conclusions
Optically active amino acids were administered orally to albino
rats excreting constant amounts of urinary nitrogen; the distribution
of the extra urinary nitrogen was determined.
The nitrogen of l(/)iso-
leucine, l(/)alloisoleucine, d(/)leucine, l(-)leucine, d(-)norleucine,
l(/)norleucine, l(/)valine,
amino-phenylacetic acid, and d(-)<x-
aminophenylacetic acid appeared in the urine as extra urea and ammonia.
That of d(-)isoleucine, d(-)alloisoleucine, d(-)valine, d(-)pseudoleucine, and l(/)pseudoleucine appeared in the urine largely as amino
acid nitrogen.
It is concluded that:
a.) the presence of a substituent methyl
group on the beta carbon atom interferes with the deamination of amino
acids of the d- series but has no effect on the deamination of their
enantiomorphs; b.) the presence of two substituent methyl groups on the
beta carbon interferes with the deamination of amino acids of both series
and c.) dfc-aminophenylacetic acid does not conform to the previous general
izations, either form being metabolized by the albino rat.
Bibliography
Abderhalden, E., Faust, W . , and Haase, E.
Berg, C. P.
2. physiol. Chem. 228, 187 (1934)
J. Nutrition 12, 671 (1936)
Bergel, F., and Bolz, K.
Z. physiol. Chem. 220 , 201 (1933); 223, 66 (1934)
Bergmann, M., Zervas, L., Fruton, J. S., Schneider, F., and Schleich, H. J.
J. Biol. Chem. 109, 325 (1935)
Bernheim, F.
Betti,
J. Biol. Chem. Ill, 217 (1935)
and Nayer, M.
Bonner, W. A.
Ber. 4l, 2071 (1908)
Thesis, Purdue University (1942)
Borsook, H.» and Dubnoff, J. W.
J. Biol. Chem. l4l» 717 (l94l)
Braunstein, A. E., and Bychkov, S. M.
Nature 144, 751 (1939)
Braunstein, A* E., and Kritzman, ¥■• G.
(1939)
Butts, J. S., and Sinnhuber, R. C.
Carter, H. E.
Nature 140, 5^3 (1937); l43» 609
'
J. Biol. Chem. 139, 9&3 (I94l)
Personal communication (1937)
Christensen, H. N., Edwards, R. R., and Piersma, H. D.
141, 187 (1941)
Clough, G. Y.r.
J. Biol. Chem.
J. Chem. Soc. 113, 526 (1918)
Cohen, P. P. Biochem. J. 33, 147& (1939); J. Biol. Chem. 13 6 , 565 (1940);
140, 711 (1941)
Dakin, H. D.
J. Biol. Chem. 14, 321 (1913); £7, 341 (1926)
Dakin, H. D-, and Dudley, H. W.
J. Biol. Chem. 18, 48 (1914)
Dunn, M. S. Ann. Rev. Eiochem. K), 91 (1941)
DuVigneaud, V., Wood, J. L., and Binkley, F.
Ehrlich, F.
Biochem. Z. 1_, 8 (1906)
Ehrlich, F., and Wendell, A.
Fischer, E«
J. Biol. Chem. 138 , 3^9 (1941)
Biochem. Z. j3,438 (1908)
Ber. 39, 2322 (1906)
Fischer, E-, and Raske, K.
Fischer, E., and Warburg, 0.
Ber. 40,3717
(1907); 4l> 893 (1908)
Ber. 38 , 3997 (1905)
.
27
Fischer, E-, and Weichhold, 0.
Folin, 0.
Ber. 4l, 1288 (1908)
J. Biol. Chem. 17, I469 (1914)
Foster, G. L., Rittenberg, D., and Schoenheimer, R. J. Biol. Chem. 125,
13 (1938)------------------------------------------------- --Fr&nkel, S., Gallia, H., Liebster, A., and Rosen, S.
225 (1924)
Freudenberg, K., and Meister, M.
Freudenberg, K., and Rhino, F.
Friedmann, E.
Biochem. Z. l45,
---
Ann. 518, 86 (1935)
Ber. 5 7 , 1547 (1924)
Beitr. chem. Physiol, u. Path. 11, I5 I (1908)
Fruton, J. S., and Clarke, H. T.
J. Biol. Chem. 106, 667 (1934)
Hoffman, W. S. Photelometric Clinical Chemistry, William Morrow & Co.
New York (1941)
Homeyer, A. H., Whitmore, F. C., and Wallingford, V. H.
5 5 , 4209 (1933)
Hotchkiss, R. D.
J. Biol. Chem. l4l, 171 (1940
Karrer, P., Escher, K., and Widmer, R.
Keilin, D., and Hartree, E. F.
Kirsch, B.
Helv. Chim. Acta 9, 3^1 (1926)
Proc. Roy. Soc.
(London) B, 119, ll4 (1936)
Klin. Wochschr. 15_, 170 (1936 ); Biochem. Z. 280 , 4l (1935)
Kjeldahl. J.
Z. anal. Chem. 22, 366 (I88 3 )
Klein, J. R., and Handler, P.
Knoop, F., and Oesterlin, H.
Knoop, F., and Okada, N.
J. Biol. Chem. 139, 103 (1940
Z. physiol. Chem. 148, 294 (1925)
Arch. ges. physiol. (Pflugers) 201, 3 (1923)
K8gl, F., and Erxleben, H.
Krebs, H.
J. Am. Chem. Soc.
Z. physiol. Chem. 2p8, 57 (1939)
Z. physiol. Che. 217, 191 (I933)i Biochem. J. 29, 1620 (1935);
Ann. Rev. Biochem. 5., 248 (193&)
Krebs, H. A., and Henseleit, K.
Z. physiol. Chem. 210, 33 (1932)
Leighty, J. A», and Corley, R. C.
Lewis, H. B., and Root, L. E.
Locquin, R.
Lyons, M. L.
J. Biol. Chem. 120, 331 (1937)
J. Biol. Chem. 43, 79 (1920)
Bull. soc. chim. (4) _1» 595 (1907)
Thesis, Purdue University (1941)
Ma, T. S., and Zuazaga, G.
Ind. Eng. Chem. (Anal. Ed.) 14, 280 (1942)
28.
Marko, D.
Ann. 3 6 2 , 333 (1908)
Marvel, C. S.,
Org. Syn. 21, 60 (l94la);21, 74 (l94lb)
McGinty, D. A., Lewis, H. B., and Marvel,
75 (1921.)
Keber, M.
C. S. J. Biol. Chem. 62,
”
Z. physiol. Chem. 2ij0, 59 (1936 )
Neubauer, 0-
Deut. Arch. klin. Med. 95, 211 (1909)
Ringer, A. L., Frankel, E. M., and Jonas,
L. J. Biol. Chem.
14, 5^5 (1913)
Roloff, M., Ratner, S., and Schoenheimer,
R. J. Biol. Chem.
13 6 , 561 (1940)
Rose, W. C.
Sahyun, M.
Physiol. Rev. ^18, 109 (193^)
J. Lab. Clin. Med. 24,
Schneider, F.
5I4.8
(1939)
Ann. 529, 1 (1937)
Schoenheimer, R., and Rittenberg, D.
Snyder, F. H., and Corley, R. C.
Physiol. Rev. 20, 218 (1940)
J. Biol. Chem. 122, 491 (193*3)
Sobel, A- S., Yuska, H., and Cohen, J.
Stekol, J. A. and Schmidt, C. A.
J. Biol. Chem. 118, 443 (1937)
Univ. Cal. Pub. in Physiol- 8 , 31 (1933)
Weil-Malherbe, H., and Krebs, H. A.
Biochem. J., 29 , 2077 (1935)
Weight
of Amino Acids Equivalent
to 25 Milligrams
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dl-oc-aminophenylacetio
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Physical
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Table 1*
Disposal in Urine of Nitrogen of Amino Acids Ingested by the Rat.
Day of
Experiment
Total N
Urea N
mg.
Amino N
mg.
Kr
*g«
6
129
105
2.1;
9.U
7
li;0
112
3*1
9.6
8
138
105
1.7
10.5
9
135
110
U.3
10.2
10
lh5
103
2.2
9.7
11
133
107
2.5
10.3
12
158
128
U.5
10.3
13
135
1114.
3.3
9.5
lh
128
Ill
2.8
10.1;
15
151
13U
3.1
10.1
16
lijO
112
1.6
10.1
17
138
115
2.1?.
10.0
18
161;
110
11+.9
9.U
19
137
111+
8.1;
9.9
20
150
105
2.5
10.3
21
151
110
12.5
10.0
22
129
116
8.3
10.1;
23
130
106
3.U
9*3
2b
152
130
lp.1
9.6
25
128
111
2.3
10.1
26
133
117
3.0
9.9
27
155
110
18.7
9.5
28
130
108
12.8
9.8
Rat 1.
Amino Acid
d(-)nor leucine
d(/) leucine
d(-)pseudoleucine
d(-)valine
1(/)-oc-aminophenylacetic acid
d(-)alloisoleucine
Bach amino a-cid -was supplied in the amount to contain
25 milligrams of nitrogen (see Table 1).
Table 5
Disposal in Urine of Nitrogen of Amino Acids Ingested by the Rat.
Day of
Experiment
Total N
mg.
Urea N
6
ll+O
7
Amino N
mg.
Kr
mg.
115
1.9
7.2
ll+5
122
2.8
6.8
8
ua
109
3.1
6.9
9
li|8
120
2.1+
7.6
10
11+0
115
2.3
7.1
11
li+1
117
3.1+
7.U
12
I65
11+8
2.2
6.9
13
150
130
3.2
6.7
11+
11+5
126
3*1+
6.2
15
171
150
3.6
7.1
16
H+1+
122
2.1+
7.0
17
11+6
129
3.1
7.3
18
IS
120
16.1+
6.8
19
II48
131
7.2
6.5
20
11+3
117
1+.0
7.1;
21
162
122
H+.1+
6.6
22
H+3
127
6.3
7.0
23
12+6
128
2.1+
7.1;
21+
170
151
1+.8
7.6
25
151
118
3.3
6.8
26
152
122
2.9
7.5
27
173
120
19.1
7.5
28
150
115
8.3
6.9
Rat 2.
Amino Aoid
d(-)nor leucine
d(/) leucine
d(-)pseudoleucine
d(-)valine
d (- )-coaminopheny1acetic acid
d(-)alloisoleucine
Each amino acid was supplied in the iamount to contain
25 milligrams of nitrogen (see Table 1).
Table 6
Disposal in Urine of Nitrogen of Amino Acids Ingested by the Rat.
Day of
Experiment
Total N
mg.
6
171
7
Urea N
mg.
Amino N
mg.
Kr
mg.
130
2.6
9.6
i6e
125
1.5
10.1
8
159
118
2.1+
10.3
9
I65
128
3.2
9.1+
10
161
121+
1.1+
9.8
11
158
129
1.8
9.8
12
178
11+8
1+.1
10.0
13
160
120
3.1
9.5
11+
151+
127
3*1+
9.9
15
180
15k
2.1
9*1+
16
159
131
1.9
10.1
17
160
133
2.1+
10.2
18
178
129
18.2
10.5
19
161
125
7.6
9.2
20
155
130
3.1;
9.1+
21
177
151
2.1
9*9
22
15U
128
1.7
10.2
23
159
129
3.2
9.7
21+
181
133
16.7
10.1
25
15U
127
7.2
9.5
26
158
133
3.6
9.9
27
179
133
19.0
10.0
28
160
130
10.0
10.1
Rat 3.
Amino Acid
l(/)nor leucine
d(-)-<*-aminophenylacetic acid
l(/)pseudoleucine
l(/)valine
d(-)pseudoleucine
d(-)alloisoleucine
Bach amino acid was supplied in the amount to contain
25 milligrams of nitrogen (see Table 1).
Table 7
Disposal in Urine of Nitrogen of Amino AcidB Ingested by the Rat.
Total N
mg.
Urea N
mg.
Ami n o N
mg.
SEi
Kr
160
119
3.3
8.3
7
137
110
3.9
8.5
8
15k
115
k*l
8.7
9
152
116
3.0
6.1
10
158
108
2.2
7.7
11
152
111
2.2
7.9
12
176
133
3.5
7.8
13
156
Ilk
3.1
8 .1+
1k
155
118
1+.0
8 .1+
15
173
135
3*3
7.9
16
160
121
3.7
8.5
17
150
117
3.0
7.6
18
178
110
21.3
8.1
19
15B
118
9.5
8.0
20
Ui8
109
1+.1
7.6
21
171
135
3.0
8.5
22
153
105
3*2
23
155
107
1+.0
8.8
22+
176
110
17.1
8.2
25
150
116
6.1+
7.9
26
153
117
3.1+
8.1+
27
180
111+
18.7
8.6
28
Rat I)..
Amino Acid
l(/)nor leucine
d (- )-o-aminopheny1acetic acid
l(/)pseudoleucine
l(/)valine
•
6
00
■y of
irimemt
l(/)ps eudoleucine
d(-)alloisoleucine
3.8
5.1*
91
119
rat
escaped
from cage
♦Sample incomplete as
Each amino acid was supplied in the amount to contain
25 milligrams of nitrogen (see Table l).
Table 8
Disposal in Urine of Nitrogen of Amino Acids Ingested by the Rat.
Day of
Experiment
Total N
®g.
6
155
7
Urea N
mg.
Amino N
_ “g.
Kr
m£.
121
1+.6
9 .0
H+7
112
3 .6
9 .5
8
150
119
3 .7
1 0 .1
9
li+9
120
2 .0
9 .6
10
11+3
111
1 . 1+
9 .7
11
H+7
116
2 .3
9 .8
12
170
136
2 .8
8 .8
13
151
120
2 .7
8 .9
11+
H+7
111+
1+.0
9.1+
15
178
1 L+0
2 .0
8 .6
16
li+5
123
2 .7
8 .9
17
131
118
3 .5
8 .9
18
177
138
1+.1
9 .3
19
153
121
3 .3
9.1+
20
12+3
116
2 .9
8 .5
21
168
118
17.1+
8 .9
22
156
125
6 .6
9 .0
23
152
120
1+.0
9 .3
21+
173
12+1
3 .1
9 .6
25
151
116
2 .8
8 .6
26
H+9
118
1+.1
8 .8
27
171+
li+0
2 .7
9.1+
28
152
120
3 .3
9 .0
Rat 5.
Amino Acid
l(-)leucine
1 (/) -ct-aminophenylacetic acid
l(/)isoleuoine
d(-)isoleucine
l(/)alloisoleucine
l(/)alloisoleucine
Each amino acid was supplied in the amount to contain
25 milligrams of nitrogen (see Table 1).
Table 9
Disposal in Urine of Nitrogen of Amino Acide Ingested by the Rat.
Day of
Experiment
Total N
mg.
Urea N
mg.
Amino N
...mg.
Hr
mg.
6
158
12i+
3-1
7-6
7
166
130
1*5
7.2
8
160
127
2.8
8.3
9
168
13i+
2.7
8.1
10
160
128
3-1;
7.5
11
158
131
3-1
7.2
12
179
159
2.6
7.7
13
166
137
1*8
7.7
Ih
159
130
2.9
7.9
15
180
151
1*3
8.1
16
163
133
2.8
7.5
17
159
129
2.1
7.6
18
I8I4
155
3-0
7.9
19
162
133
3.0
8.0
20
158
125
3-9
7.2
21
171;
128
18.U
7.2
22
168
122
8.8
7.1
23
160
129
2.3
7.5
2k
181
152
3-8
8.0
23
158
133
1*1;
7.2
26
162
127
2.1
7.7
27
178
153
1-9
7.5
28
155
13U
3-2
8.1
Rat 6.
Amino Acid
l(-) leucine
1(/)-a-aminopheny1acetic acid
1(/)isoleucine
d(-)isoleucine
l(/)alloisoleucine
l(/)alloisoleucine
Each amino acid was supplied in the amount to contain
25 milligrams of nitrogen (see Table l).
THE INFLUENCE OF STRUCTURAL CONFIGURATION
CM THE DEAMINATION OF AMINO ACIDS
IN THE NORMAL RAT
An Abstract of a Thesis
Submitted to the Faculty
of
Purdue University
fcy
Morris Franklin Milligan
in partial fulfillment of the
requirements for the Degree
of
Doctor of Philosophy
in
Biochemistry
August, 19^42
It has been demonstrated (Snyder and Corley, 1958) that the body of
the normal dog exhibits a high degree of specificity in the deamination
of subcutaneously administered amino acids.
These investigators have in­
dicated that configuration on the <*.- carbon atom and substitution on the
carbon atom are the factors determining whether the animal body can deaminate the amino acid under consideration.
To extend our knowledge of
deamination, the metabolic fate of the separate isomers of a number of
aliphatic amino acids has been followed in the normal rat.
Adult female albino rats were maintained in metabolism cages on a
diet of finely ground Friskies (a commercial animal food) until the values
for urinary nitrogen became reasonably constant.
stance to be studied was added to the stock diet.
At this time, the sub­
Evidence as to its
fate was furnished by the change or lack of change in the urinary total
nitrogen (Kjeldahl), urea and ammonium nitrogen (Sobel, Yuska, and
Cohen), and amino acid nitrogen (Sahyun).
Adequacy of urine collection
was indicated by the constancy of creatinine values.
That the changes
are caused by the substance given is indicated by the return of urinary
values afterward to those prevailing before.
All amino acids utilized in this study have been synthesized and re­
solved in this laboratory, with the exception of dl-or-aminophenylacetic
acid, d(-)valine and l(/)valine.
Dl-oc-aminophenylacetic acid was ob­
tained from the Eastman Kodak Company and has been resolved in this lab­
oratory.
The isomeric valines were products of Hoffmann-LaRoche, Inc.
The specific rotation of the amino acids was measured to indicate their
purity.
At least two experiments were conducted on separate animals
with each amino acid.
Table 1.
A summary of the results obtained is presented in
As may be noted, the bulk of the nitrogen administered was al-
2.
ways recovered in "the urine, either in the urea and ammonium fraction,
or in the amino acid nitrogen fraction.
Our results with the valines, the isoleucines, the pseudoleucines,
the leucines, and 1(/Oalloisoleucine are in accord with the results of
Snyder and Corley.
We have noted that the norleucines, in accordance
with the generalizations of Snyder and Corley, are readily deaminated.
D(-)alloisoleucine, as might be expected from its configurational rela­
tionship to d(-)isoleucine, escaped deamination.
We have found that
both enantiomorphic cc-aminophenylacetic acids, contrary to previous re­
ports, are readily deaminated; since earlier work was carried out on
dogs, the discrepancy must be ascribed to a difference in species.
Discussion
As in the report of Snyder and Corley (1938)#
have found the
amino acids of the 1- series have been deaminated readily if the *-
and
carbon atoms bear one hydrogen atom each, while most of the d- amino
acids studied have not been significantly deaminated unless the
atom holds two hydrogen atoms.
carbon
These facts indicate that deamination
proceeds by way of dehydrogenation of the <*• and p- carbon atoms; how­
ever, the deamination of a-aminophenylacetic acid and the known destruc­
tion of glycine in the body are not in accord with such a generalization.
The work of Friedmann (1906) and of Knoop and Oesterlin (I925) indi­
cates that deamination proceeds by the formation of an imino acid; the
observed resistance to deamination of pseudoleucine vitiates this view.
To explain the results with most of the amino acids studied, let us
assume that amino acids of the d- series are deaminated by being first
inverted to their respective epimers.
As noted in this study and in the
work of Snyder and Corley, the d- amino acid oxidase of Krebs (1935)
does not appear to function in the normal animal, at least under the ex­
perimental conditions employed.
The resistance of certain amino acids
of the d- series to deamination is then attributed to a steric inter­
ference with the preliminary inversion, which is believed to be enzy­
matic in nature.
Summary
D(-)norleucine, 1(/)nor leucine, d(/) leucine, l(-)leucine, l(/) isoleucine, l(/)alloisoleucine, l(/)valine, d(-)-c*-aminophenylacetic acid,
and 1(/)-o^aminophenylacetic acid were deaminated readily after inges­
tion by the normal rat,
D(-) isoleucine, d(-)alloisoleucine, d(-)valine,
d(-)pseudoleucine, and 1(/)pseudoleucine were not similarly deaminated.
The following conclusions are drawn for the amino acids studied in
the normal rat under the experimental conditions employed.
The presence
of a substituent methyl group on the beta carbon atom interferes with
the deamination of amino acids of the d- series but has no effect on the
deamination of their enantiomorphs.
The presence of two substituent
methyl groups on the beta carbon atom interferes with the deamination of
amino acids of both series.
An exception is <x-aminophenylacetic acid,
either stereoisomeric form of this amino acid being readily deaminated*
Bibliography
Friedmann, E.
Kjeldahl, J.
Beitr. Chera. physiol. (Hofmeister) 11, I5I (I9O8 )
Z. anal. Chem. 22, 366 (1883)
Knoop, F., and Oesterlin, H,
Krebs, H.
Sahyun, M.
Z. physiol. Chem. ligB, 29I4. (1925)
Biochem. J. 29, 1620 (1935)
J. Lab. Clin. Med. 2b» 5^3 (1939)
Snyder, F. H., and Corley, R. C.
J. Biol. Chenu 122, 2#1 (1938)
Sobel, A. E., Yu ska, H., and Cohen, J.
J. Biol. Chem. 118, 1*1+3 (1937)
Table 1
i
Amino Acid:
Nitrogen Recovered in Urine as:
Total N
Urea and
Ammonium N
Amino
Acid N
l(/)valine
/
/
-
d(-)valine
/
-
/
l(/)isoleueine
/
/
-
d(-)isoleucine
/
-
/
l(/)alloisoleucine
/
/
-
d(-)alloisoleucine
/
-
/
d(/)leucine
/
/
-
l(-)leucine
/
/
-
d(-)norleucine
/
/
-
1(/)nor leuc ine
/
/
-
d(-)pseudoleucine
/
-
/
l(/)pseudoleucine
/
-
/
d (- )-oc-arainophenylac etic acid
/
/
-
!(/)-(*-
/
/
-
VITA
Mori'is Frans:! in Lillie,an was born in Pine Village, Indiana,
November
11,
1914.
He attended the public scuools at ALgin, Illinois,
and graduated with the .a., B. degree from the University of Illinois
in 19B7.
He then enrolled in the Graduate School of Puroue University,
where he held an assistant snip in the Department of Ohemistx*y.
He
was married on July 10, 1940, to Opal Dewey Collins of best Lafayette,
Indiana.
He received the degree of Doctor of Philosophy from Purdue
University in 1942.
f
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