A STUDY OF THE METABOLISM OF HISTIDINE AND RELATED IMIDAZOLES IN THE BODYOF THE MOUSE AND OF THE RAT AND OF THE AVAILABILITY OF D-TRYPTOPHAN IN THE MOUSE

86 Pages·2.938 MB·English

by  CELANDER, DAVID ROBERT

A STUDY OP THE METABOLISM OP HISTIDINE AND RELATED
IMIDAZOLES IN THE BODY OF THE MOUSE AND OF THE RAT
AND OF THE AVAILABILITY OF D-TRYPTOFHAN
IN THE MOUSE
by
David Robert Celander
Chairman
Professor Clarence P# Berg
A dissertation submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy
in the Department of Biochemistry in the Graduate
College of the State University of Iowa
August, 1952
ACKNOWLEDGEMENT
The author wishes to express his
deep appreciation to Dr. Clarence P. Berg
whose cooperation, interest and helpful
suggestions have been invaluable through­
out the course of these investigations.
ii
TABLE OP CONTENTS
Page
Acknowledgement ................................ 11
Introduction ............................... 1
Chapter I ............................. 10
The Availability of D-Histidine and Related
Imidazoles and of D-7ryptophan in the Mouse
Introduction .................... 10
Experimental .......................12
Discussion ........................... 25
Summary ........................ 29
Charts ................................. 31
Tables .......................... 36
Chapter II ................................. 55
A Study of Certain Aspects of the Metabolism
of D- and L-Histidine and Related Imidazoles
in the Rat”
Introduction........ 55
Experimental • • • ..... ............. 56
Discussion ...................... 61
Summary ........ 69
Chart............ 71
Tables ........................ 72
Bibliography ................... 77
Biographical Items................... 83
ill
1
INTRODUCTION
Although histidine was first isolated over 50
i
years ago (41, 49) and characterized as®f-amino*j^4(5)-
imidazolepropionic acid by 1911 (65), its role in metabo­
lism has not yet been completely elucidated. It has been
established as essential for growth in a number of species,
among them the cockroach nymph (44), the chick (48), the
mouse (87) and the rat (72). It is necessary for nitrogen
balance in the rat (94) and in the adult dog (75) but is
dispensable in the adult human subject (73). An important
constituent of animal protein, it is present, e.g., to the
extent of 7 - 8 per cent in hemoglobin; it exists in muscle
in the form of two dipeptides, carnosine (^-alanyl-J.-
histidine) and anserine (y^-alanyl-l-Me-L-histidine); and
the betaine of thiolhistidine, ergothioneine, is found to
the extent of 10 mg. per 100 ml. in circulating blood.
Studies carried out by Ackroyd and Hopkins (1)
and later by Rose and Cook (71) afforded presumptive
evidence that histidine might bear some precursorial rela­
tionship to the purines. This idea persisted until Tesar
and Rittenberg (85), who fed rats histidine labeled with
in the nitrogen attached to the ^T-carbon atom, found
amounts of the isotope in the tissue purines which were too
2
small to support the conclusion that the Imidazole ring
could serve as a specific precursor of purine nitrogen.
They concluded that the nitrogen of the imidazole ring was
not specifically reutilized, but found its way into the
general nitrogen pool.
Remmert and Butts (69) reported that {.-histidine
is converted to glycogen when it is administered orally to
fasting rats. Their work was confirmed by Featherstone and
Berg (34) who also showed that, per mole, histidine and
glutamic acid are about equal in their ability to promote
glycogen formation in the fasting rat.
Recently, experiments have been reported by
Bouthillier in which carboxyl-labeled DL-hlstidlne (19)
and L-hlstidine-2-C14 (82) were administered parenterally
to young male rats. With the former compound, 30 per cent
of the administered radioactivity appeared as respiratory
carbon dioxide in four hours. Appreciable activity was
found in the excreted urea and about 20 per cent was
excreted in the urine as histidine. The authors claimed
4
that the amounts of found in proline and hydroxyproline,
4
isolated from carcass proteins, would indicate a signifi­
cant metabolic relationship between histidine and these
4
amino acids, and that the low activity of glutamic acid
precludes its being a direct product of histidine
degradation. L-Histidme-2- C14 led to the excretion of
appreciable amounts of C14 in both respiratory carbon
dioxide and urinary urea. Analysis of liver serine showed
that 25 to 33 per cent of the radioactivity in the liver
was due to this substance. The authors considered this
good evidence that carbon 2 of the imidazole ring of
histidine is converted to formate in the intact animal.
The excretion of urocanic (imidazoleacrylic) acid
following the administration of large amounts of histidine
orally and parenterally to dogs (53) and parenterally to
rabbits (47) led Kotake to suggest (51) that this substance
was a normal intermediate in histidine metabolism. Darby
and Lewis (10) observed urinary urocanic acid excretion in
five of eight rabbits given large amounts of histidine
orally. All of the five showed symptoms of severe intoxi­
cation and four of the five died. These workers were unable
to demonstrate the excretion of urocanic acid after paren­
teral administration of histidine. They concluded that the
urocanic acid probably represented an abnormal pathway of
histidine metabolism.
The in vitro work which has been done with histi­
dine has led to considerable confusion. In 1926, Edlbacher
(23) and GySrgy and RBthler (39), working independently,
discovered that liver possessed the property of
4
hydrolytically destroying histidine. Edlbacher observed
that the agent responsible was most active at pH 9 and was
inactivated by heating at 90° C. for 10 minutes. He con­
cluded that the action was enzymatic and named the enzyme
histidase. Subsequent work (29, 30) showed that an inter­
mediate compound was formed which, upon treatment with
sodium hydroxide, yielded two moles of ammonia, one mole of
L-glutamic acid, and one mole of formic acid per mole of
L-histidine. Two mechanisms by which these products might
be formed have since been proposed. Edlbacher and Neber
(31) presented a scheme for histidase action which involved
initial rupture of the ring with retention of the °f»amino
group* They contended that this e<-earbon-nitrogen bond
was unbroken and was responsible for the optical activity
of the glutamic acid isolated as a product of alkaline
4
hydrolysis. On the other hand, Kotake (51) is of the
opinion that histidine is degraded enzymatically first to
urocanic acid, thence to isoglutamine and finally to
4
glutamic acid. In 1942, Edlbacher and Viollier (32) sep­
arated crude histidase into two active components: histi­
dase which acted upon L-histidine and urocanase which
degraded urocanic acid. Their purified histidase was
inhibited by urocanic acid. In a recent preliminary report,
Mehler and Tabor (58) presented the results of a trapping
5
study which led them to conclude that urocanic acid is the
primary product of histidase action.
In vitro techniques have also been used to eluci­
date certain oxidative pathways of histidine metabolism.
In a series of papers describing D amino acid oxidase and
£, amino acid deaminase, Krebs (54) presented data which
indicated that both liver and kidney were able to deaminate
histidine oxidatively and that the rate of ammonia produc­
tion was greater from D-histidine than from the L isomer,
Featherstone and Berg (35) were unable to show appreciably
greater oxygen uptake by liver and kidney slices in the
*
presence of histidine, and hence concluded that the primary
pathway of histidine catabolism is probably hydrolytic. In
1943, a IL-hlstldine oxidase was reported (27) present in
liver. It deaminated both D- and L-histidine and required
4 *
yeast adenine nucleotide as a co-factor. In 1944, Blanchard,
4
Green, Nocito and Ratner (9) prepared an L amino acid oxi­
dase from rat liver and kidney which was capable of con­
verting L-histidine slowly to the keto acid and ammonia,
Enzymes capable of oxidatively deaminating L-histidine are
also reported to have been isolated from rat skin (10, 84)
and to be present in the venom of Vinera asnis L. (95).
A survey of the literature would indicate that
the hydrolytic pathway of histidine catabolism is probably
6
the most important insofar as the complete destruction of
histidine is concerned, but that a number of systems exist
which operate oxidatively on this compound. It is impor­
tant to note that most of the latter appear to be
relatively weak.
Not to be overlooked is histidine decarboxylase.
This enzyme, which is present in kidney and other tissues,
brings about the reductive decarboxylation of histidine to
histamine (98).
Valuable information concerning histidine metabo­
lism has also been garnered from growth studies in rats
involving the supplementation of histidine-deficient diets
with compounds related to L-histidine, In the early
studies, advantage was taken of the fact that a casein
hydrolysate could be rendered deficient in histidine by
s /
precipitation with silver oxide, or silver sulfate, and
4
barium hydroxide (50, 72). Employing a diet in which this
hydrolysate, supplemented with tryptophan and cystine,
served as nitrogen source, Cox and Rose (16) and Harrow and
Sherwin (40) reported that imidazolelactic acid would
support slow to moderate growth in rats. These authors
also tested urocanic acid. The former investigators con­
cluded that it would not replace histidine for growth, but
the latter felt that it possessed some beneficial effects.
7
Harrow and Sherwin also reported imidazolepyruvic acid to
be effective as a substitute for histidine though less
active than the hydroxy acid. Since compounds of this
nature are presumed to bring about growth by virtue of the
animal's ability to convert them to histidine, these
references are often cited as early evidence for the
reversible deamination of amino acids. In 1934, Cox and
Berg (13) demonstrated that growth would occur in rats
when D-histidine was substituted in the diet for the
L modification.
In 1937, Conrad and Berg (11) were able to show
the inversion of D-histidine to the L isomer in growing
rats. The L-histidine content of animals fed a D-histidine
supplemented diet, in which the L isomer had been rigor­
ously reduced to a known minimum, was shown to be higher
than the amounts of L-histidine present in the bodies of
the animals at the outset as estimated from analysis of
experimental controls. The increment of L-histidine in
the bodies of the experimental animals could not be
accounted for on the basis of that ingested in the diet.
None of the additional histidine was found as the D modifi­
cation. This was the first proof of the actual inversion
of the D isomer of an essential amino acid in the animal
body.