The effect of neoplastic tissue on the metabolism of formic acid by the host

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content THE EFFECT OF NEOPLASTIC TISSUE ON THE
METABOLISM OF FORMIC ACID BY THE HOST
by
Abraham Morton Stein
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biochemistry and Nutrition)
June 1957
UMI Number: DP21578
All rights reserved
INFORMATION TO ALL USERS
The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
Dissertation Publishing
UMI DP21578
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code
ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6
7>b. O' ftio '57 5 81*}
This dissertation, w ritte n by
........Abr£^^;..Morton..Ste .........
under the direction o/his G uidance Com m ittee,
and approved by a ll its members, has been p re
sented to and accepted by the F a c u lty of the
G raduate School, in p a rtia l fu lfillm e n t of re
quirements fo r the degree of
D O C T O R O F P H I L O S O P F I Y
Date...
Guidance Committee
Chairiwan
ACKNOWLEDGEMENT
The author wishes to express his gratitude to the
staff of the Department of Biochemistry and Nutrition of
the University of Southern California for the privileges
and opportunities accorded to him during his stay at the
university.
The author wishes to thank Dr. Harold E. Pearson,
\
Department of Microbiology, for the courtesy extended to
him in the use of laboratory facilities at the Cancer
Research Building of The Los Angeles General Hospital.
In particular, the author wishes to acknowledge his
great indebtedness to Dr. John W. Mehl for scientific
guidance, sage counsel and understanding given to him and
for the intellectual freedom which he enjoyed in the
course of this work.
TABLE OF CONTENTS
CHAPTER PAGE
I. HISTORICAL INTRODUCTION ................... 1
The Effect of Tumor on Liver Catalase . . 1
Inducible Enzymes in Mammals ............ 8
The Effect of Tumor on Host Metabolism . . 12
Some Aspects of the Metabolism of Formic
Acid and Compounds Interrelated by One
Carbon Shifts............ 19
Synthesis of Methionine Methyl ........ 25
Oxidation of the Methyl Group and One
Carbon Compound Precursors to
Formaldehyde and Formate ............ 29
The Oxidation of Formaldehyde.... 3**-
Oxidation of Formic Acid in Mammalian
Liver........................... 36
II. STATEMENT OF PROBLEM AND. METHOD OF ATTACK . ' kl
III. MATERIALS AND METHODS...................... ^3
Chemicals........................ ^3
Animals.................... ............
Radioisotope Equipment ..................
Analytical and Metabolic Procedures . . . ¥+
Carbon dioxide titration .......... ..
Combustion of formic acid to carbon
dioxide.......... . . b6
V
CHAPTER PAGE
Steam distillation of formic acid . . . by.
Iodometric determination of formic acid *+8
Radioassay of BaC-^C^ ........ 50
Crystallization and plating of radio
active compounds ............ 51
Nitrogen determination and radioassay
on protein samples................ 5^
Catalase assay 55
Xanthine oxidase assay ........ 59
Recovery of respiratory CO2 and
urinary formate ............ 60
Incubation procedures. ........ 62
Formic oxidase ........... 62
Methionine synthesis and oxidation . 63
IV. EXPERIMENTAL RESULTS ...... V i ........... 67
Preliminary ................ 67
The Oxidation of Formate by Mouse Liver
Homogenates ...................... 6 7
The Effect of Tumor on the Oxidation of the
Methyl Group of Methionine to Formate . . 90
V. DISCUSSION....................... 113
VI. SUMMARY AND CONCLUSIONS . ................. 121
BIBLIOGRAPHY ................................... 123
LIST OF TABLES
TABLE PAGE
I. Composition of Kreb's-Ringer^Phosphate
Buffer .......................... 65
II. The Effect of Tumor on Liver Catalase
Activity................................. 68
III. The Effect of Xanthine Plus Hypoxanthine on
Formate Oxidation in Undialyzed Mouse
Liver Homogenates.......... 70
IY. The Absence of D-Amino Acid Oxidase in Mouse
Liver Homogenates ..... .............. 71
V. Xanthine Oxidase in Mouse Liver Homogenates . 72
VI. The Effect of Acetaldehyde on Formate-Cll+
Oxidation ............................... 75
VII. The Effect of Formate-Cll+ Concentration on
Oxidation ................ 86
VIII. The Effeict of Tumor and Added Catalase on
Formate Oxidation by Dialyzed Mouse Liver
Homogenates............................... 89
IX. The Effect of Tumor and Added Catalase on
Formate Oxidation by Dialyzed Mouse Liver
Homogenates............ 92
X. Recovery of Formate-C^ in Normal and
Sarcoma-180 Bearing Mice ................ 93
XI. Transfer of injected Carbonate to Respiratory
vii
TABLE PAGE
Carton Dioxide as Affected by Tumor .... 98
XII. Oxidation of L-Methionine-C11*!^ Vivo . . . 99
XIII. Oxidation of L-Methionine-C1* 4!^ In Vivo . . . 100
XIV. The Oxidation of the Methyl Group of
Methionine to Formate in Mouse Liver
Prisms ............................ 10^
XV. The Effect of Methionine Concentration on
the Oxidation of the Methyl Group to
l
Formate in Liver Prisms of Normal and
Tumor-Bearing Mice .................... 107
XVI. The Synthesis of Methionine from One-Carbon
Precursors in Liver Prisms of Normal and
Tumor-Bearing Mice ...... .......... 108
XVII. The Effect of Tumor on the Rate of Oxidation
of the Methyl Group of Methionine in
Mouse Liver Prisms....................... 110
LIST OF FIGURES
FIGURE PAGE
1. Steam Distillation of 1.0 m mole HCOOH .... *+9
2. Self-Absorption of Barium Carbonate ...... 52
3. Manometric Assay of Catalase Activity ..... 58
*+. The Effect of Aeetaldehyde on the Rate of
Oxidation of Formate-Cl^ .................. 7^
5. The Effect of Hypoxanthine and Hypoxanthine
Plus Aeetaldehyde on the Oxidation of
Formate-C-*-1 * ................................. 78
6. The Effect of Inosine and Inosine Plus
Aeetaldehyde on the Oxidation of Formate-C^*. 80
7. The Effect of Inosine and Formate Concentra
tion on the Oxidation of Formate-C^ .... 81
8. The Effect of Inosine on the Rate of Oxidation
of Formate-Cll+ . ......................... 83
9. The Effect of Formate-C^ Concentration on
Oxidation................................... 85
10. The Effect of Inosine Concentration on Oxida
tion of 0.1 M Formate in Liver Homogenates
of Normal and Tumor-Bearing M i c e .......... 88
11. The Effect of Inosine Concentration on Oxida
tion of 0.12 M Formate in Liver Homogenates
of Normal and Tumor-Bearing M i c e .......... 91
ix
FIGURE PAGE
12. Rate of Recovery of Graded Doses of Formate-C^
in Respiratory Carbon Dioxide . .......... 95
13. The Rate of Expiration of C-^C^ from
Formate-C^ ............ 96
1H-. The Effect of Methionine Concentration on
Oxidation................................ 106
15. The Rate of Methionine Oxidation in Liver . . . 109
16. The Rate of Labelling of Liver Protein by
, Methionine.............................. 112
CHAPTER I
HISTORICAL INTRODUCTION
A marked inhibitory effect by neoplastic tissue
on the catalase activity of the liver of the host
organism has been known for over forty years. The asso
ciation of the enzyme catalase with the oxidation of
formic acid has led to a study of the relationship of
formic acid metabolism to catalase in livers of tumor-
bearing mice. The historical background of the specific
inhibition of liver catalase by tumor, metabolic control
of enzyme activity in mammals, tumor-host relationships
and the metabolism of formic acid and related compounds
will be considered in turn.
I. THE EFFECT OF TUMOR ON LIVER CATALASE
In 1916, Brahn (1) reported studies demonstrating
a systemic effect of cancer in the inhibition of liver
catalase activity of the host in situations where the
liver tissue was not involved in the. tumor growth. In
19*f2, a series of studies on this effect were commenced
at the National Cancer Institute of the National Insti
tutes of Health by J. P. Greenstein and associates. An
attempt was made in these investigations to analyze the
2
nature of this inhibition and to determine whether this
effect was specifically related to the tumor-bearing
situation. The findings of this group weres
1. There is a decrease in livpr catalase activity
with progressive tumor growth to a level of 5 per cent to
10 per cent of normal activity. (2)
2. Upon extirpation of the tumor, liver catalase
values return to normal. (2,3) Under conditions of
tumor regression, the same relation is obtained. (3)
3. Kidney and erythrocyte catalase activities are
little affected, if at all. ()+)
1 +. Pregnancy and the growth of embryonic implants
do not affect liver catalase activity. (5) No depres
sion of catalase activity is found in regenerating rat
liver, nor does inanition produce an effect. (2)
5. No activators or inhibitors of liver catalase
activity can be extracted from either tumor tissue or the
livers of tumor-bearing animals. This is the case
whether the extracts are tested in vitro or injected
directly into animals. (6)
6. The hepatic activities of other readily
assayed enzymes studied by this group are found to be
less affected by tumor, although definite inhibitions
were obtained. Thus, a 25 per cent inhibition in liver
3
arginase activity is produced by a transplanted hepatoma
(7)» while little or no effect was found on liver
xanthine oxidase activity. (8)
The conclusions of this group have not been
entirely supported by later workers whose interest in
this problem was stimulated by this early work. The
specificity of this effect for tumor can be questioned on
the basis of the findings of Miller (9) that inanition
depresses liver catalase activity in rats as well as
those of Appleman et ajL. (10) who demonstrated that the
effect of feeding protein-free diets is identical to the
effect'of tumor on the same enzyme. Dounce (11) showed
that murine leprosy in rats produces lower catalase
values than in control animals. That dietary protein is
not the limiting factor in liver catalase levels was
shown by Weil-Malherbe (12) who used tumor-bearing rats
force fed a high protein diet and by Appleman et al. (13)
t ■
who used tumor-bearing rats fed a 100 per cent casein
diet. It is of interest that protein-free diets have
little or no effect on kidney and erythrocyte catalase
(10), whereas high protein diets markedly increase
kidney catalase activity. (13)
In a study of rat liver catalase activity during
liver regeneration after partial hepatectomy,
if
Stein et al. (I1 *) found a marked inhibition of catalase
activity on a nitrogen basis during the early phase of
regeneration, although the “inhibition” may be attributed
to a more rapid replenishment of liver nitrogen than of
\
liver catalase.
Several groups of investigators have demonstrated
that the growth of implanted tumors does not lead in
variably to depression of liver catalase and indeed, in
some instances, stimulation has been recorded. Klatt (15)
found that out of four mouse tumors tested, two produced
no ^effect, one produced a 30 per cent stimulation, and
one a *f0 per cent inhibition. Growth of three tumors on
the yolk sac of embryonated chicken eggs produced a
variable effect, ranging from 20 per cent inhibition to
30 per cent stimulation. Quantitatively similar results
were obtained by Skavinski (16) with the Jensen Sarcoma
and the Brown-Pearce Carcinoma growing on the chorio
allantoic membrane of embryonated chicken eggs. Begg (17)
has reported significant temporary increases in rat liver
catalase levels during the early phase of tumor growth.
A similar effect is seen in some of Greenstein's data (2)
showing a 100 per cent increase in liver catalase activity
seven days after implantation of Hepatoma-31 in rats. It
should be understood that the data of the greater part of
5'
the reports in the literature support the finding of
depressed liver catalase activity in the tumor-bearing
organism. (18)
The magnitude of the inhibition of liver catalase
by tumor reported by the above investigators with the
exception of Weil-Malherbe, is considerably less than
that reported by Greenstein and associates. An elegant
correlation of the growth of the Ehrlich aseites carcinema
with depression of catalase activity was' reported by
Lucke*. (19) Using a new method for allowing the •
i
catalysis of hydrogen peroxide splitting by catalase,
« # ’
Price and Greenfield (20) of the National Cancer Insti
tute have recently reported liver catalase values of
tumor-bearing animals comparable with those found by
other investigators.,
In 19^9, Nakahara (21) found an active fraction
isolated from human tumors which, when injected into
normal mice, produced marked depressions of liver cata
lase activity; The inhibitory activity of the fractions
was resistant to heating and to boiling 6 N hydrochloric
acid. Fractions of mouse mammary tumor were found to
inhibit mouse liver catalase in vivo by Greenfield and
Meister. (22) A similar effect was found by Adams (23)
to be produced by the injection of mouse tumor homogenates
into mice. Hargreaves and Deutsch (2*f) found heat stable
extracts of tumor tissues to inhibit the catalase activ
ity of rat liver homogenates as well as crystalline
erythrocyte catalase. However, the effect was not
specific for the source of the extract or the enzyme
inhibited. The extracts produced inhibition of other
porphyrin enzymes, and extracts of normal tissues pro
duced the same effect, although to a lesser extent.
Although the effect of tumor extracts on liver catalase
activity seems to be well confirmed, it is doubtful that
this effect will explain the effect by tumor in tumor
growth per se. Thus, it can be seen from the data of
Greenfield and Meister (22) that fractions of normal
liver are able to inhibit catalase in vivo. Since the
data are not given in terms of the weight of source
tissue, it is difficult to compare the absolute activ
ities of liver and tumor. Inhibition of liver catalase
t
activity in mice was produced by the injection of whole
and lyophilized spleen homogenates. (25) The depres
sions obtained were comparable to those produced by the
injection of tumor homogenates. Endo et al. (26) found
that "kochsafts1 1 as prepared by Hargreaves and Deutsch
(2*+) of both normal and tumor tissues possess equivalent
activity in the inhibition of liver catalase in vitro.
7
Fractions of tumor which were active in vivo showed no
activity in vitro. An interesting finding was that while
native egg albumin had no effect, boiled egg albumin was
extremely active in the In vitro test. Similar results
were obtained by Ceriotti and Spandrio (27) in a study of
the effective concentration range of tumor and normal
tissue extracts. They conclude that an activator as well
as an inhibitor might be present in their extracts.
In the fractionation and purification of liver
catalase from livers of normal and tumor-bearing rats,
Greenfield and Price (20) found that the amount of
catalase recovered from the livers of the tumor-bearing
rats corresponded to the inhibition obtained in the total
homogenate; a result that would not be expected on the
basis of a mechanism of reversible inhibition.
To the author’s knowledge, there is no review of
this field following Greenstein’s (18) treatment pub-
. T
lished in 195*+. On the basis of the information cited, it
would appear that a humoral mechanism of tumor inhibition
of liver catalase has been found wanting. It appears
that new types of data and new approaches are required
to bring understanding to this problem.
II. INDUCIBLE ENZYMES IN MAJMMA.LS
8
The problem of the inhibition of liver catalase
activity in the tumor-bearing situation involves the more
general problem concerning the significance of enzyme
activity in tissue preparations. The concept of ’ 'enzyme
activity" implies that under certain reproducible,
although possibly highly unphysiological conditions,
preparations of tissue or purified enzymes will catalyze
a well defined chemical reaction. The rate of the reac
tion is assumed to be proportional to the concentration
of enzyme protein mediating the reaction. In the inter
pretation of this type of data, it ‘ is assumed that the
chemical event studied is indeed homologous to a chemical
event catalyzed by the enzyme in vivo and that the
observed rate of reaction is a measure of the extent to
which this reaction proceeds in the intact animal.
The interpretation of the tumor effect on catalase
has been complicated by the lack of an understanding of
the metabolic function of catalase and of the regulation
of enzyme concentration in mammalian tissues. Possible
correlations of catalase and metabolic processes will be
discussed later.
The topic of physiological control of enzyme
activity in mammalian tissue has been recently reviewed
9
by Knox. (28) There is at present some information
lending support to a role for substrates in the regula
tion of enzyme activities in tissues of higher organisms.
Metabolic control of mammalian enzymes has been shown by
the administration.of substrates and by manipulation of
* ■
endocrine factors. The term inducible enzyme is chosen
to distinguish the phenomenon in metazoan tissue from the
possibly unrelated process of enzyme adaptation in
microorganisms.
Dietrich (29) has shown an elevation of xanthine
oxidase activity in mouse liver following prolonged
parenteral administration of xanthine. It is possible
that this may not be a direct effect of substrate on the
enzyme, since similar effects were produced by the
administration of histamine and cortisone, implicating
the pituitary-adrenal system. Sheffner (30) found that
the oxidation of D and L amino acids by rat kidney was
?
increased by supplementation of diets with methionine and
phenylalanine. The level of protein in the diet was a
factor in the response. Lardy (31) found a direct
relation between liver D-amino acid oxidase and the level
of protein or amino acids in the diet. Rector et al.
(32) found a linear correlation between kidney glutam-
inase and ammonia excretion following administration of
10
ammonium chloride. At 5 meq. of ammonium chloride per
day, glutaminase activity increased to twice the control
values in a period of six days. Knox (33) has shown a
tenfold increase in liver tryptophan peroxidase five
hours following the injection of tryptophan. Formyl
kynurenine formylase was unaltered. The response to
tryptophan was not dependent on the pituitary-adrenal
system, although cortisone produced a small increase in
activity. Elevation of tryptophan peroxidase activity in
response to administration of histamine was abolished by
adrenalectomy. Lee (3*+) has shown that ethionine inhib
its this process. This was interpreted to indicate
inhibition of the synthesis of the enzyme protein in
question. Gordon (35) found that chicken embryos
elaborate adenosine deaminase in response to injections
of adenosine. The rate of enzyme synthesis was twice
that in control embryos. Mandelstam and Yudkin (36) have
found that the activity of rat liver arginase was directly
proportional .to the, protein concentration in the diet.
This relation was valid whether enzyme activity is based
on liver weight, liver nitrogen or body weight. When the
animals were returned to- a lower protein diet, liver
arginase decreased to control values. A dependence of
liver arginase on dietary protein was also shown by
11
Lightbody. (37) Mandelstam (38) has formulated a
theory of enzyme adaptation for mammals in which the
substrate is presumed to lower free enzyme concentration
by binding enzyme, thereby stimulating the synthesis of
new enzyme by mass action. The theory predicts the
linear relation of arginase and dietary protein obtained.
Support for a concept of the modification of enzyme
activity by a mechanism involving protein-substrate
binding was obtained from studies of Dubnoff (39) in the
reversible adaptation of formyl hydrogenlyase in Escheri
chia coll. Formate was shown to exert a stabilizing
action on the enzyme in resting cells. Greenberg and co
workers (ifO) found increases in threonine dehydrase in
rat liver following intraperitoneal administration of
L-threonine. Induction was very rapid, with maximum
values obtained at five hours, as in the case of trypto
phan peroxidase. Perfusion of isolated rat livers with
threonine led to threefold increases in enzyme activity;
this process was inhibited by ethionine.
Hormonal control of mammalian enzyme activity is
well established and has been demonstrated for several
enzymes and hormones. Some instances will be cited.
Drabkin (^+1) found that thyroxine increased cytochrome C
in rat tissues, while thyroidectomy resulted in marked,
12
decrease in concentration. Hexokinase in rat skeletal
muscle was increased about 70 per cent by thyroxine (*+2),
and adrenalectomy effected a similar increase in the
hexokinase rat brain grey matter. 0*3) Adrenalectomy
depressed mouse and rat liver catalase. (Mf) Fraenkel-
Conrat et al. (**-5) found that cortisone stimulated and
adrenalectomy inhibited rat liver arginase. Growth
hormone was found to oppose the effect of the adrenal
secretion. IJmbreit and Tonhazy found that adrenalectomy
depressed D-amino acid oxidase (MS) and proline oxidase.
0*7)
III. THE EFFECT OF TUMOR OH HOST METABOLISM
The concept of tumor-host relationships is well
recognized in the literature of cancer research, as it is
also recognized that tumor growth represents a stress on
the host organism. The mechanism of adaptation to this
stress is not known, although there is evidence of a
pituitary-adrenal effect which will be discussed below.
It is assumed tacitly that the tumor somehow competes
successfully for metabolites, with an accompanying
mobilization of the biosynthetic mechanisms of the host
to meet the demands of the growing tumor for metabolites.
The studies of Christensen and Riggs (MS) document
13
the intense concentrative uptake of amino acids by
Ehrlich ascites carcinoma cells. In general, the in
corporation of labelled metabolites by the growing cells
compares with that of the most active normal tissues.
Greenberg and collaborators have studied the incorpora
tion of Clk labelled glycine and tyrosine into several
normal tissues and tumor tissues of tumor-bearing rats.
The proteins of tumor tissue incorporated radioactivity
at a very rapid rate. Further, the proteins of non
neoplastic tissues of tumor-bearing rats incorporated
more radioactivity than the tissues of control animals.
Skipper, Rhoads and their collaborators (50,51) have
ILl
studied the uptake of G labelled formate, glycine and
purine bases into the purines of mouse and human tumors
growing as heterologous implants and npn-neoplastic
tissues. Many of the papers cited below compare the
uptake of labelled metabolites into tumor with that of
non-neoplastic tissues. However, beyond the fact that
tumors incorporate radioactivity at a rate of the same
order as that of liver, intestine and kidney, little
information has been provided by these studies on
qualitative metabolic alteration in tumor or on a
consistent set of increases in host metabolic rates as
might be inferred from the fact of rapid tumor growth.
lb
The effect of tumor growth on carcass and liver
weight and nitrogen has "been reviewed by Mider. (52)
Mider and collaborators (53) found that carcasses of rats
bearing transplanted tumors lost weight as the tumor grew.
Liver nitrogen increased markedly, followed by a pre
mortal return to normal values. There was no effect on
spleen and kidney nitrogen. A correlation was found (5^)
between the loss in carcass lipid and tumor growth,
indicating an increased caloric requirement by the tumor-
bearing animal. Further (55), in pair feeding experi
ments, the carcasses of tumor-bearing rats lost more
calories than those of control animals. . This difference
was accounted for by the calculated caloric value of the
lipid lost. In these experiments, the total body nitrogen
of the control animals was equal to the sum of the carcassj
and the tumor nitrogen of the tumorous animals. Begg and
Dickinson (56) found that tumor-bearing rats which were
force fed a high fat diet retained more nitrogen, sodium
and chloride and did not lose carcass weight. Systemic
responses of the tumor-bearing animal (anemia, enlarged
adrenals and depressed liver catalase) were not modified
by the diet. Stewart and Begg (57) have studied systemic
effect in tumor-bearing rats and found adrenal hyper
trophy (60-100 per cent), thymus involution (50-30 per
15
cent), sixfold increases in spleen weight and an increase
in liver weight (50-100 per cent). Since adrenal
ascorbic acid and cholesterol were found to decrease (17)*
these authors postulated involvement of the pituitary-
adrenal system in the response of the host to tumor
growth. The enrichment of tumor nitrogen at the expense
of the host has led Mider to propose that the tumor is a
nitrogen trap. (52)
LePage et al. (58) found that rats bearing the
Flexner-Jobling carcinoma injected with a single dose of
glycine-2-C**-1+ rapidly accumulated radioactivity in the
tumor. The total radioactivity in the tumor increased
$
with time, whereas that of other tissues tended to
decrease. Babson and Winniek (59) showed the nitrogen
trap of tumor to be relative since tumor implants
labelled with C -tyrosine and leucine lost radioactivity
with time. Similarly, Greenlees and LePage (60) found
1U-
that implants of glycine-2-Cx^ labelled Ehrlich ascites
carcinoma cells lost approximately. 9 per cent of their
protein radioactivity in twenty-four hours.
The increase of liver nitrogen in the tumor-
bearing animal (10,13,53»57) is of interest from the
point of view of the tumor-host relationship, and con
trasts markedly with the depressions of some liver
16
enzymes in cancer. (18) In this connection, Norberg
ll§-
and Greenberg (61) have found that glycine-2-C is in
corporated at a greater rate into proteins of liver,
plasma and spleen of tumor-bearing animals than into the
proteins of the normal controls. Alterations in the free
amino acid composition of the “tumor-bearing liver*’ were
reported by Cerecedo et al. (62) who found significant
increases in methionine, arginine, histidine, glycine and
cystine. Sassenrath and Greenberg (63) found increases
in free alanine, histidine and threonine, and decreases
in isoleucine, leucine, lysine and valine. The levels of
free amino acids in the livers of fasting animals did not
correspond to those in the livers of tumorrbearing
animals. White et al. (6*f) found a higher aspartic-
glutamic transaminase activity in the livers of tumor-
bearing rats.
The increase of metabolic activity of the liver
and other organs in neoplasia is substantiated by a
number of other studies. Zamecnik and Frantz (65)
studied the incorporation of C-^-carboxyl labelled
alanine into slices of normal liver, fetal liver,
hepatoma and liver of tumor-bearing animals. The
specific activity of the proteins in counts per minute
per milligram were, respectively, 30, 180, 211 and 8*f.
Marked increase in specific activity of liver and spleen
deoxyribonucleic acid of tumor-bearing mice using
phosphorus-32 (66) and C - * - 1 * labelled glycine-2 and formic
acid (67) was found by Kelly and associates. Increases
ranged from two to twelvefold. The biosynthesis of
purine and pyrimidine bases was found to be more rapid in
livers of tumor-bearing animals. Conzelman et al. (68)
have shown a *K) per cent increase in ribonucleic acid
guanine and two and threefold increases in deoxyribo-i
nucleic acid guanine specific activity, using C^-^-amino-
5-imidazolecarboxamide. Using ureido-C^lf labelled
carbamyl aspartic acid, similar findings were reported by
the same group in the incorporation in uracil and cyto
sine of ribonucleic acid. Furlong et al. (70) have
shown increases in total deoxyribonucleic acid radio-
/
activity in liver and spleen of tumor-bearing animals
following the injection of adenine-8-C-*-1* " .
Dinning et al. (71) have studied the effect of
leukemia on the incorporation of from methyl labelled
betaine and formic acid into urinary creatinine and
allantoin. The excretion of these substances was
increased in leukemia. The specific activities of both
compounds increased significantly in the urine of
leukemic mice injected with formic acid, but no changes
18"
were observed when betaine was used. Reid et al, (72)
have studied the urinary metabolites of histidine-2-C^1 *-
imidazole in normal and hepatoma-bearing rats. Marked
depression of incorporation into the urea of the
tumor-bearing mice was observed. Since urea carbon is
representative of body carbon dioxide, this finding
probably reflects a decreased oxidation of the one carbon
fragment derived from histidine in the tumor-bearing
animal. This point was not dealt with by the authors,
who deferred the discussion to a later (unpublished?)
article. Although Reid et al. felt there was no effect
by the tumor on the incorporation of radioactivity in
* •
uric acid and allantoin, their data show small but
consistent increases.in-the samples from the tumor-
bearing animals which are in line with the data of
Dinning et al. (71)
A simple interpretation, to account for the in-
?
creased metabolic activity of tissues of the tumor-
bearing host would be a type of “demand'* hypothesis as
discussed earlier. That this may not be a completely
adequate hypothesis is suggested by the data of Furlong
(70), who found that the supernatants from high
speed centrifugates of tumor homogenates stimulated in
vivo the uptake of adenine-8-C^ in deoxyribonucleic acid
19
of lung, muscle and thymus. Extracts of normal tissues
were less effective than those of tumor. These data
suggest that at least part of the increase of deoxyribo
nucleic acid synthesis in the host may be due to the
secretion of activating substances by the tumor.
IV. SOME ASPECTS OF THE METABOLISM OF FORMIC
ACID AND COMPOUNDS INTERRELATED BY ONE CARBON SHIFTS
Although the current interest in one carbon
compound biochemistry dates to Sakami's work in 19^8,
many observations in the older literature pointed to the
existence of reactions in intermediary metabolism involv
ing formic acid, formaldehyde and one carbon fragments
not necessarily related to carbon dioxide.
As early as 1897, Pohl (73) found that formic acid
was formed when he incubated formaldehyde with an extract
of rabbit liver. Incubating the extract of 500 g. of
.t
liver with 800 mg. of formaldehyde yielded 8.7 mg. of
formic acid in twenty-four hours. In 1911, Dakin and
Wakeman (7*0 found that the administration of sodium
salts of fatty acids led to ten to thirtyfold increases
in the excretion of formic acid, and McGuigan (75) found
that the oral administration of formaldehyde led to a
marked excretion of formic acid in the urine. Benedict
20
and Harrop (76) found the formic acid excretion in
*
diabetes to be two to three times that of normal subjects.
The administration of methanol to dogs led to a twenty
fold increase in formic acid in the urine.
In 1939> Abbot and Lewis (77) found that sarcosine
administration increased hippuric acid excretion in
sodium benzoate fed rabbits, and they postulated the de
gradation of sarcosine to glycine. Two years later
Bernheim and collaborators (78) studied the dissimilation
of sarcosine in “broken cell" preparations of liver.
They found 1 g. atom oxygen uptake per mole of substrate,
and colorimetric tests revealed the presence of glycine
and formaldehyde in the incubate. In 19^6, Shemin (79)
studied the metabolism of serine labelled with and
C13. The isotopes were found in the glycine moiety of
hippuric acid in essentially the same' ratio as the pre
cursor compound. Shemin postulated the cleavage of
1 >
serine to form glycine and formic acid.
At this time the studies concerning the metabolism
of the labile methyl group were beginning to correlate
with the studies on formate metabolism. In his study of
the labile methyl group as an essential dietary component,
du Vigneaud (80) in 1939 found that some rats would grow
on a diet considered free of this factor. This observation
21
was confirmed and extended by Bennett (81), who found
that rats would grow if. homocystine plus choline or liver
fractions were added to labile methyl free diets. Gillis
and Norris (82) found that the "animal protein factor"
spared methionine and choline in the diet. Stekol and
Weiss (83) found crystalline vitamin B^ an(* homocystine
spared methionine in the diet of rats, and du Vigneaud
(8*f) proved the biosynthesis of labile methyl groups in
bacteria-free rats whose body water had been labelled
with deuterium. This work established the partial in
dependence of the chick and rat from exogenous labile
methyl groups, and pointed to the existence of biosyn
thetic reactions in higher organisms leading to the
formation of the methyl group from oxidized precursors.
In 19*+8, Sakami (85) established the synthesis of
serine in the intact rat from glycine-C^OOH and formate-
Clk. He found protein serine labelled with in the
carboxyl group and in the beta carbon. In 19*+9j he
reported (86) that glycine-2-Cllf was incorporated in rat
protein serine labelled both in the alpha and beta
carbons. This implied that the alpha carbon of glycine
was a source of formic acid or a formic acid-like
intermediate. These reactions were studied in liver
slices by Siekevitz and Greenberg (8?) with glycine-2-Cll+
22
and formate-C^; acid soluble serine isolated by carrier
recrystallization was found labelled in the appropriate
positions. Sakami (88) found protein serine labelled in
the beta carbon using methyl labelled choline as a pre
cursor, thus establishing a link between the one carbon
fragment and the labile methyl group.
At this point, a third line of investigation con
verged with the study of one carbon compounds and the
biosynthesis of the labile methyl group. In l^S,
Sauberlieh and Baumann (89) found that the growth of
Leuconostoc citrovorum. which failed to grow on synthetic
media, was stimulated by liver concentrates of yeast
extracts. Some correlation was found between growth
response and anti-pernicious anemia hematological response.
High levels of folic acid in the media could replace the
liver extracts for maximal growth. In 19^9> Sauberlieh
(90) found that rats fed 1-10 jug. of folic acid per gram
f
of diet excreted large quantities of the Leuconostoc
citrovorum growth factor— afterwards termed the
“citrovorum factor.1 1 The' relationship of citrovorum
factor activity to folic acid was shown by Shive and
associates (91) in the synthesis of thymidine by
L. citrovorum. Acid hydrolysis of the citrovorum factor
destroyed its activity, but a compound was formed with
23
Lactobacillus casei activity similar to folic acid.
Furthermore, a hog liver concentrate, equivalent to folic
acid in the L. casei growth assay, reversed the inhibi-
bition of growth produced by methyl folic acid, a folic
acid inhibitor. The liver concentrate was, in this
respect, fifteen times more active than folic acid itself.
(92) Previously, Shive (93) had found that in sulfanil
amide bacteriostasis of Escherichia coli the diazotizable
amine which accumulates is *t-amino-5-imidazolecarboxamide.
(The compound was identified by synthesis and mixed
melting points of the picrates.) Shive postulated a one
carbon addition to the amine with ring closure to form
the purine bases. Folic acid involvement in this process
was shown by Wooley and Pringle (91 +)} who found that in
aminopterin (4-amino-pteroylglutamic acid) inhibited
bacterial cultures, a diazotizable amine accumulated
which was identified as N-amino-^-imidazolecarboxamide by
paper chromatography.
Studies of the chemistry of the citrovorum factor
between 1950 and 1952 have emphasized the relationship of
compounds with citrovorum factor activity to the metabo
lism of one carbon compounds. Jukes and associates (95)
found that a synthetic compound resulting from the
formylation and reduction of folic acid possessed
2b
citrovorum factor activity. Keresztesy and Silverman
(96) isolated a crystalline barium salt-from horse liver
which was twice as active as the synthetic compound of
Jukes. Shive and co-workers (97) prepared a crystalline
reduced and formylated folic acid with citrovorum factor
activity. Pohland et al. (98) proposed the structure
5-formyl-5»6,7,8-tetrahydropteroylglutamic acid as the
structure of the synthetic factor. Cosulich et al. (99)
found additional evidence for this structure, obtained
evidence for the formation of N-lQ-formyl-
tetrahydropteroylglutamic acid in the course of the re
action and prepared an anhydro compound of
tetrahydroformylpteroylglutamic acid with an imidazolium
bridge between and K^.
The relationship of folic acid to formate and one
carbon compound-linked metabolism in higher animals was
shown by Plaut, Betheil and Lardy. (100) These authors
found that in folic acid deficient rats, there is little
1 h
fixation of formate-CA radioactivity in serine and that
the oxidation of formic acid is considerably slower.
Elwyn and Sprinson (101) found that in folic acid
deficient rats the conversion of serine- to hippuric
acid proceeds at a slower rate than in normal rats.
Nichol and Welch (102) showed the conversion of folic
25
acid to citrovorum factor in normal and folic acid
deficient rat liver slices. The reaction was promoted by
ascorbic acid, presumably acting as a biological reducing
agent. Stekol et al. (103) showed an effect of folic
1 L
acid on the incorporation of glycine-2- o r serine-3-
1L,
Cx in the methyl group of choline.
The studies cited above have laid the basis for
the recent very rapid expansion of the study of one
carbon compounds and their co-factors in the metabolism
of purines, pyrimidines and amino acids. Significant for
the present investigation are the studies in the
synthesis and oxidation of the methyl, group with par
ticular reference to methionine and the formation of
carbon dioxide as the terminal oxidation product. This
work will be reviewed below.
Synthesis of Methionine Methyl
The biosynthesis of the methionine methyl group in
the intact animal and in tissue preparations has been
documented by many authors. Sakami and Welch (10*+)
' 1 i f
demonstrated in 1950 the synthesis of CA -methyl
labelled methionine and choline in the rat and in rat
liver slices. In 1951» du Vigneaud et al. (105) sur-
1 if
veyed the incorporation of' Cx labelled bicarbonate,
26
formate, formaldehyde and methanol into choline and
ereatine methyl groups in the tissues of rats. Bicarbon
ate was not utilized, while methanol, doubly labelled with
deuterium and cl l f , was utilized only after oxidation, as
judged by the rati o'of dilution of labels. Formate,
which was doubly labelled with and deuterium, lost no
deuterium during incorporation into the trimethyl amine
moiety of choline in the intact rat. (106) Berg (107),
in a study of methionine synthesis in guinea pig liver
slices, showed that formate is only slightly diluted by
non-labelled formaldehyde in incorporation into
methionine. Similar data were obtained by Mitoma (108),
who showed in addition that the incorporation of
formaldehyde-C^ in the methyl group of methionine by rat
liver slices was greatly decreased by unlabelled formate.
The significance of these observations is not yet clear.
Further, Berg (109,) found that a soluble pigeon liver
preparation incorporated formate carbon in methionine.
The addition of homocysteine stimulated incorporation in
serine and purines as well as methionine. He proposed an
intermediary compound of formate and homocysteine as a
common precursor of these compounds.
The study of Sprinson (110) employing 3-C -3-D-
serine fed to intact rats showed that in the synthesis of
‘ . 27
choline and thymine methyl the one carbon compound was
not oxidized to the level of formic acid. He proposed
that formylated derivatives of folic acid would be
■ *
excluded as directly participating in this reaction.
Nakao and Greenberg (111) have demonstrated
directly the requirement for leucovorin and pyridoxal
phosphate in the synthesis of methionine from formalde-
hyde-C^ and serine-3-C^ by a cell-free sheep liver
preparation.
In a study of the utilization of labelled
serine, glycine and formate, Arnstein and Neuberger (112)
demonstrated directly the effect of vitamin Bj2 on
methionine methyl synthesis previously inferred from
nutritional studies. They also showed that the specific
activity of the methyl group of methionine exceeded that
of the choline methyl in all cases, indicating the
relative position,of these compounds in the biosynthetic
pathways of the labile methyl group.
A possible insight into the mechanism of
methionine synthesis and degradation is afforded by the
studies of Cantoni on the structure of the transmethylat-
ing compound of methionine. “Active methionine” has been
characterized by this author as S-adenosylmethioninea
product of the enzymatic reaction of adenosine
28
triphosphate and methionine. (113) This compound has
been shown to transmethylate directly to creatine (ll1 *-),
and one of the products of the reaction is S-
adenosylhomocysteine. (115) Dubnoff has stated (116)
that in the synthesis of methionine from formate-C^ in
rat liver slices the "methylthioladenosine fraction”
showed five times the total activity of the methionine
fraction. Unfortunately, no details are available. The
occurrence and significance of adenosinethiomethyl ribo
side has been investigated by Schlenk and collaborators
(117), who propose the concept of , , transthiomethylationl ,
to explain the occurrence of the compound.. Schwartz and
Shapiro (118) have shown that in Aerobacter aerogenes
methionine can be formed from adenosinethiomethyl ribo
side, and suggested that this compound may be formed from
active methionine in the isolation process of Smith and
Schlenk involving^ as it does, boiling the cells at 100°.
Canellakis and Tarver (12Q) have shown the production of
methyl mercaptan from labelled methionine in rat liver
mitochondria. These authors also showed (121) the rapid
oxidation of or S?5 labelled methyl mercaptan to
carbon dioxide and sulfate, respectively. The methyl
group radioactivity was incorporated into the beta carbon
of serine and the methyl groups of methionine, choline
29
and creatine.
A hypothetical role in one carbon compound metabo
lism has been considered for S-methyl methionine, a
compound found by Challenger and Hayward (122) in
asparagus and by Shive and co-workers (123) in cabbage
juice. S-methyl methionine has been shown (12k) to react
enzymatically with homocysteine to produce methionine in
cell-free extracts of Aerobacter aeroeenes. and to produce
adenine thiomethyl riboside in yeast more efficiently than
methionine itself.. (125) Shive and collaborators (126)
found this compound more effective than methionine in
supporting growth of methionineless mutants of Escherichia
coli and in reversal of sulfanilamide inhibition.
Further, S-methyl methionine competitively reversed the
inhibition produced by S-ethylethionine, whereas
methionine reversed the inhibition non-competitively.
No clear picture of the mechanism of methionine
synthesis emerges from these data. However, the pre
liminary evidence points to a possible significant role
for the sulfonium derivatives of methionine.
Oxidation of the Methyl Group and One Carbon Compound
Precursors to Formaldehyde and Formate
The study of the oxidation of isotopically
labelled, methyl, group of methionine was initiated and
30
largely carried out in du Vigneaud’s laboratory. Later
aspects of this work were continued by C. G. Mackenzie at
the University of Colorado. This work has led to the
»
recognition of formaldehyde and formic acid as inter
mediates in the terminal oxidation of the one carbon
compounds.
Reference has been made to the early work of
Dakin (71 *) and of Benedict and Harrop (76) in the excre
tion of formic acid, and that of Bernheim (78) on the
oxidation of the methyl group of sarcosine in vitro.
* Ling and Tung (127) in 19^8 demonstrated an enzyme
in a fractionated saline extract of an acetone powder of
rabbit kidney capable of oxidative demethylation of
alpha-N-methyl amino acids with the formation of formal
dehyde. The enzyme was inert toward sarcosine but active
with respect to nine out of sixteen N-methyl amino acids
tested. Oxygen uptakes in slight excess of the theoreti
cal for observed formaldehyde production were obtained.
The following year Mackenzie et al. (128)
administered C^-methyl labelled methionine by stomach to
a rat and obtained 32.b per cent of the activity in the
respiratory carbon dioxide at the end of twenty-four
hours. In another study (129), these authors showed that
the rate of oxidation of methionine methyl to respiratory
31
carbon dioxide was highly dependent on dosage; at 0.5 per
cent dietary level the maximum rate of excretion was 0.9
per cent per hour, while at 1.2 per cent dietary level
the excretion was 5.7 per cent per hour.
1U.
In a study of the oxidation of Cx -methyl sarcosine
in rat liver slices and washed homogenates, Mackenzie
(130) demonstrated the formation of labelled formaldehyde,
formate and carbon dioxide. Non-isotopic formaldehyde was
isolated from large scale incubates with non-labelled
sarcosine by blowing air through the medium and trapping
the aldehyde in a methyl cellosolve-dry ice bath, thus
lending support to the occurrence of free, formaldehyde in
tissues. Data were presented showing that the oxidation
of sarcosine to formaldehyde was mediated by a sedimen
table fraction of the liver homogenate, and oxidation of
formaldehyde to formate occurred in the supernatant.
Further, excretion of labelled formic acid in the urine
7 t
of rats which had been injected with the labelled pre
cursor was shown.
Siekevitz and Greenberg (131) demonstrated the
formation of radioactive formate in rat liver slices from
Ik
methyl labelled methionine and choline, glycine-2-C and
serine-3-C^lf. The latter was the most efficient reaction.
In a study of the incorporation of the methyl group of
32
sarcosine into serine, Mitoma and Greenberg (132) reported
data showing that the formaldehyde resulting from the
oxidation of sarcosine is in an activated state and
probably in equilibrium with non-activated, free formal
dehyde in the medium.
Mackenzie and du Vigneaud (133) showed that dietary
choline increases the oxidation of the methyl group of
methionine to respiratory carbon dioxide in the intact
rat. This result was interpreted to indicate that the
availability of methyl groups controlled the rate of
oxidation.
The first evidence for sarcosine as a mammalian
metabolite was obtained by Horner and Mackenzie (13*0 in
the administration of methyl labelled methionine or
betaine to rats made sareosinuric by feeding excess
sarcosine. Sarcosine isolated from the urine was found
to contain radioactivity in the methyl group.
J
Mackenzie et al. (1353 demonstrated the production
of formaldehyde, isolated as non-isotopic formaldimedon
and estimated gravimetrically, from dimethylethanolamine,
dimethylglycine and sarcosine in rat liver homogenates.
The latter two compounds were oxidized by washed sediment
alone, whereas dimethylethanolamine required the re
constituted homogenate, a fact considered by the authors
33
to be in harmony with the requirement of the choline
cycle for the oxidation of the alcohol group prior to the
demethylation of ethanolamine bases. Since Muntz (136)
had demonstrated the production of dimethylglycine rather
than dimethylethanolamine as a product of. the trans
methylation of choline, this appears to be a likely
interpretation, but it would be desirable to demonstrate
the failure of dimethylathariolamine to demethylate
p
directly. Production of formaldehyde directly from
dimethylglycine was demonstrated (135) by the use of
methoxyacetate, which inhibited the production of formal
dehyde from sarcosine, but had no effect on demethylation
of dimethylglycine/ On the basis of these data,
J
Mackenzie proposes (137) a methyl oxidation cycle based
on the choline cycle, where two of the three methyls of
choline are obtained as one carbon compounds. Formate
and formaldehyde production from the methyl group has
been recently reviewed by Mackenzie. (137)
The role of tryptophan in formate synthesis has
been studied by Knox and Mehler (138), who demonstrated
the peroxidation of this amino acid in rat liver fractions,
followed by a subsequent oxidation to form formyl-
kynurenin. Hydrolysis of this compound yields kynurenine
and formic acid. (139)
31 * -
The source of formic acid produced in histidine
catabolism was shown by Tabor et ajLo(l^O) to be the
amidine carbon. These authors followed the dissimilation
ik
of ring labelled histidine-2-C in cell-free extracts of
Pseudomonas fluorescens and recovered radioactive formate
from the incubate. The mechanism of this*reaction, now
elaborated in-considerable detailj is beyond the scope
of this review.
The Oxidation of Formaldehyde
» The early work of MeGuigan (75) on the in vivo
oxidation of formaldehyde to urinary formate has been
quoted. Strittmatter and Ball (1*H) in discussing this
problem in 1955* stated that most of the work concerning
aldehyde oxidation to that time was executed in reference
to aldehydes other than formaldehyde. In this year, a
paper by these authors (1^1) and a summary of unpublished
work by Mackenzie (137) appeared on the mechanism of
formaldehyde oxidation by mammalian liver.
Strittmatter and Ball (l*fl) described the oxida
tion of formaldehyde by a fractionated acetone powder of
the non-sedimentable fraction of a beef liver homogenate.
Diphosphopyridine nucleotide and glutathione stimulated
the reaction, while cysteine and other sulfhydryl
35
compounds and triphosphopyridine nucleotide were inactive.
The reaction was followed speetrophotometrically by the
reduction of DPN, and formate production was shown by-
specific tests. Acetaldehyde was oxidized by the prepara
tion, but glutathione was not required. A kinetic
analysis of data relating formaldehyde and glutathione
concentration to activity supported the existence of a
formaldehyde-glutathione compound as the active substrate
for the dehydrogenase in question. It is of interest that
their enzyme preparation possessed high thiolesterase
activity.
Working with a rat liver mitochondrial system,
Mackenzie (137) showed that cysteine stimulated an in
crease in the rate of oxygen uptake by formaldehyde.
Homocysteine inhibited the uptake completely and gluta
thione was not tested. The rate of oxidation of formal
dehyde supplemented with cysteine was found to be
equivalent to that of thiazolidine carboxylic acid
synthesized as the addition compound of cysteine and
formaldehyde. It is not stated whether formic acid
accumulates in the medium as would be expected from the
author*s data on the oxidation of radiosarcosine in liver
“particles.'1 Mackenzie noted that while thiazolidine
carboxylic acid could be oxidized by meta-periodate to
36
form free formaldehyde, the oxidation product of cysteine
and formaldehyde, tested in the acid soluble extract of
the incubate, did not release formaldehyde upon such
treatment. It remains to be seen whether free formic acid
can be formed hydrolytically from this presumptive inter
mediate by some liver enzyme not in the mitochondrial
fraction.
Oxidation of Formic Acid in Mammalian Liver
The oxidation of formic acid in plants and bacteria
is toeyond the scope of this review. In this section, the
eatalase dependence of formate oxidation in mammalian
liver will be considered.
The studies of Chance (1^2) in 19*+8 and 19^9,
utilizing his rapid speetrophotometric method, indicated
a mechanism for the peroxidation of formic acid to carbon
dioxide and water by a hydrogen peroxide complex of
eatalase. Evidence was presented for the peroxidation of
methanol and formaldehyde as well. His information is
based on the effect of formic acid in returning the
spectrum of a catalase-hydrogen peroxide complex to that
of free eatalase.: Formic acid is simultaneously consumed
in the process. A study of the effect of pH on the reac
tion was interpreted to indicate that undissociated formic
37
acid was the true electron donor in the system. The same
evidence was obtained using living Micrococcus lysodeik-
ticus cells (1^3), where the spectra of various hemo-
proteins have been measured by difference under various
physiological conditions.
Earlier work by Keilin and Hartree (lMf) had
indicated a peroxidative action of eatalase in the coupled
oxidation of ethanol to acetaldehyde in the presence of a
source of hydrogen peroxide provided by.the notatin
system (glucose oxidase).
, Heppel and Porterfield (1^5) have reported the
coupled oxidation of nitrite in the presence of eatalase.
This was confirmed by Keilin and Hartree (lW-6), and
evidence was presented as to the coupled oxidation of
aldehydes and phenols using manometric methods. Further,
they described a system for the oxidation of ethanol to
acetic acid (confirming earlier work (l1 ^)) using crystal-
»
line xanthine oxidase and eatalase in the presence of
small amounts of acetaldehyde. The hydrogen peroxide
produced in the xanthine oxidase reaction with acetalde
hyde is utilized in the eatalase mediated oxidation of
ethanol to acetaldehyde, the latter generating more
hydrogen peroxide to complete the cycle. The efficiency
of the catalase-coupled oxidation of alcohol is, according
, 38
to Keilin and Hartree, 12,500 times less efficient.than
that of the classical reaction of eatalase on hydrogen
peroxide itself.
A direct demonstration of the role of hydrogen
peroxide in the oxidation of alcohol by eatalase was
given by Laser. (1^7) By allowing hydrogen peroxide to
diffuse through a cellophane membrane into the reaction
mixture within the manometer flask, he was able to obtain
as much as 80 per cent utilization of the hydrogen
peroxide for the oxidation of alcohol.
> The significance of the eatalase coupled oxidation '
of alcohol in mammalian liver is questionable because of
the presence of a powerful hepatic dehydrogenase-DPN
system, but it serves as a valid enzymatic model for
eatalase peroxidation.
Studies of the oxidation of formic acid in
mammalian systems have confirmed the peroxidation mecha-
»
nism discussed above. Weinhouse and Friedmann (1M3)
reported a very rapid oxidation of formate-C^ in the
intact rat. In the first hour, 60 per cent was oxidized;
and by three hours, essentially all the radioactivity was
recovered in the respiratory carbon dioxide. Mathews and
Vennesland (1* 4 - 9), in a study of the oxidation of formate
in animal tissues, were unable to demonstrate a DPN
39
specific dehydrogenase for formate, but they did observe
an enzyme which oxidized formic acid in dialyzed extracts
of liver and kidney in the presence of adenosine triphos
phate. Previously, Kruhjrfffer (150) had reported that the
oxidation of formate in dialyzed rat liver preparations
was stimulated by DPN and inhibited by cyanide. The
author concluded on the basis of this evidence that the
dehydrogenation of formate was DPN linked and dependent
on the electron transport system.
Nakada and Weinhouse (151) studied the oxidation
of 'formate in dialyzed rat liver extracts. Reasoning
from the findings of Chance (l*+2) that hydrogen peroxide
and eatalase were implicated in formate oxidation, they
interpreted the findings of Mathews and Vennesland *(1^-9)
to mean that adenosine triphosphate was acting as a
source of hydrogen peroxide. They tested and obtained
stimulation of oxidation of formic acid with adenosine
mono and triphosphate, xanthine, hypoxanthine and
butyraldehyde. Further confirmation of this mechanism
was obtained by Rappoport et al. (152) These authors
found that dialyzed extracts of rabbit liver oxidized
formate aerobically in the presence of substrate amounts
of hypoxanthine, inosine, adenylic acid, DPN and alkali
hydrolyzed DPN. With dialyzed pigeon liver extracts,
1*0
deficient in xanthine oxidase, no formate oxidation was
obtained with or without these compounds. Anaerobically,
dialyzed rat liver extracts with hypoxanthine or DPN, and
methylene blue failed to oxidize formate. Oxidation was
inhibited by nitrite which is known, as previously
mentioned (lH5)} to decompose the catalase-hydrogen
peroxide complex.
In a study of the oxidation of formic acid in
folic acid deficient rats, Friedmann, Nakada and Wein
house (153) observed a parallelism between the decrease'
in liver eatalase activity and oxidation of formate in
liver preparations. In intact folic acid deficient rats,
less radioactivity was recovered in the respiratory carbon
1U-
dioxide from a test dose of formate-Gx^ and more in
urinary formate. A lower rate of expiration of radio
activity was observed in the deficient animals. The
r
findings on formate oxidation confirm the earlier work of
Plaut (100) which has been cited above. Friedmann et al.
(153) interpreted their data, to signify that folic acid
deficiency produces a non-specific decrease in liver
eatalase activity in line with the folate deficiency
anemia, but this is by no means the only possible
explanation.
CHAPTER I I
STATEMENT OF PROBLEM AND METHOD OF ATTACK
This dissertation presents the results of an
investigation into the functional association of catalase
and formic acid metabolism in mouse liver and the effect
of cancer on this relation- . The experiments reported here
were designed in an attempt to demonstrate a systemic
effect of tumor on the oxidation of the one carbon
compound. It was hoped to provide a basis for an inter-
pretation of the depression in liver catalase activity of
tumor-bearing animals previously discussed. The emphasis
of this study is on the effect of implanted tumors on the
peroxidation of formic acid in dialyzed whole mouse liver
homogenates, and on the oxidation of methionine to formic
acid in whole cell preparations of mouse liver.
f
The oxidation of formic acid was followed by
measuring the radioactivity of formate-C incorporated
into carbon dioxide. After a preliminary finding of
impaired formic acid oxidation in mouse liver homogenates
was obtained, a series of studies varying the concentra
tion of labelled substrate and kind and concentration of
co-oxidant were planned to quantitate the effect and to
determine whether the hydrogen peroxide producing systems
or the system peroxidizing the substrate was limiting.
The nature of the enzymatic deficiency in liver homoge
nates of tumor-bearing mice was tested directly by adding
crystalline catalase to the incubates. The findings in
/
this system were corroborated by catalase and xanthine
oxidase measurements on the liver homogenates.
In an effort to provide a physiological interpre
tation for these results, a study was made of the
production of formic acid in intact mice using methyl
labelled methionine as a model compound. Ambiguity in
the findings then led to an investigation of this
reaction in surviving whole liver cell preparations.
Preliminary observation of decreased methionine oxidation
in livers of tumor-bearing mice was followed by several
experiments to quantitate the results and to determine
within the limits of a whole cell preparation whether the
effect of tumor on methionine oxidation was real.
Parallel experiments were designed to detect an effect of
tumor on the synthesis of methionine from labelled one
carbon precursors.
CHAPTER I I I
MATERIALS AND METHODS
I. CHEMICALS
The chemicals used in this investigation were of
Chemically Pure or Analytical Reagent grade. Sources of
special preparations and fine chemicals are listed below.
Xanthine, Wyeth Corporation, distributed by
General Biochemicals Inc., Chagrin Falls, Ohio.
Hypoxanthine. Dougherty Chemical Co.
L-Methionine« D.L-homocysteine thiolactone,
inosine. California Foundation for Biochemical Research,
Los Angeles,. California.
Catalase, beef liver, crystalline, Worthington
Biochemical Corporation, Freehold, New Jersey.
i i+ " ' /
Sodium Formate-C . specific activity 1 jiC/juM,
Tracerlab Inc.
L-Methionine-C^-^-methyl. specific activity 0.75
jiC/juM, D.L-Serine- V Cllf. specific activity 1 jiC/jaM,
Glycine-2-C ^ . specific activity 1.32 juC/jaM, California
Foundation for„Biochemical Research, Los Angeles,.
California.
Mfr
II. ANIMALS
Male white Swiss mice of the Webster strain were
used throughout this study.
Ill. RADIOISOTOPE EQUIPMENT
In the earlier part of this work, radioactivity
was assayed with a Tracerlab Autoscaler and a Nuclear
internal gas flow tube, model D-^f6. Most of the samples
were assayed with a Nuclear model 182-A scaler with
automatic sample changer and “micromil“ end window and
Clary time recorder. The efficiency of the micromil end
window was found to be roughly half of that of the
internal gas flow tube, but this was readily sacrificed
to obviate the spurious counting obtained with the former
tube.
IV. ANALYTICAL AND METABOLIC PROCEDURES
Carbon dioxide titration. Primary standards,
potassium acid phthalate and sodium carbonate, were
prepared according to Koltoff and Sandell. (15^) The
two standards agreed within the limits of the titrimetric
method employed (0.5 per cent). Carbon dioxide-free
water was prepared by boiling distilled water for about
h5
forty-five minutes. Occasional batches of this water
were tested by the procedure recommended by Koltoff and
Sandell (1^), titrating large volumes to the phenol-
<
phthalein end point, and found to be virtually free of
acid. “Carbon dioxide free'* solutions of sodium
hydroxide were prepared by allowing sodium hydroxide to
saturate in water in a closed pyrex flask, filtered with
suction through a medium grade fritted glass filter and
suitably diluted with carbon dioxide-free water.
Carbon dioxide was titrated as a monobasic acid
between the phenolphthalein and methyl orange end points.
Since in titration of carbonic acid the bicarbonate end
point is reached before the phenolphthalein end point,
phenolphthalein color standards were prepared by adding
one half of an equivalent of acid to a solution of
standard sodium carbonate, and the end point color
determined by comparison. For convenience, a methyl
orange color standard was prepared by, adding 0.08 to 0.12
ml. standard HC1 to COg-^free water; the end point was
determined by comparison and the amount was subtracted
from the titer. Control of the volumes of the solutions
titrated at the end points and of the amounts of
indicator added was found essential for accuracy in this
procedure. The amounts of CO2 titrated generally ranged
*+6
from 0.05 to 0.1 ra mole.
The accuracy of‘this procedure was checked by a
gravimetric determination as barium carbonate. Ex.:
BaCO^, calculated by titration, 138.8 mg., found by
weight, 139. mg.
Combustion of formic acid to carbon dioxide.
Method I. The reagent of Pirie (155) which was
used, contained 8 per cent HgC^, 2 per cent sodium
acetate *3^0 and 2 per cent glacial acetic acid. The
sample, 0.2 to 0.8 mM in 2 to 3 ml. volume, acid to
methyl orange, was gassed for fifteen minutes with air
drawn through an Ascarite trap. Excess reagent was
admitted and the sample was heated at 100° for one hour.
The resulting earbon dioxide was trapped ^in two NaOH bead
towers in series, each containing 15 ml. of I NaOH. The
samples were titrated and corrected for blanks. The
values' agreed with HCOOH determined by weighing the
Hg2Cl2 precipitate and direct titration of formic acid.
Sample data: assay of 0.5 ml. aliquots of
approximately M HCOOH: -
HgpClo Titration Titration
m moles m moles HCOOH m moles CO2
mg* HCOOH
1. 268.*+ 0.576 0.585 ---
2 . 267.1 0.573 0.581
3. 271.0_______0.581 0.570
1
b7
Method II. The reagent of Grant (156) was used,
\
containing 20 per cent HgCl2, 30 per cent sodium acetate*
3H20, 8 per cent NaCI, w/w. The samples were combusted
in 50 ml. Erlenmeyer flasks with a center well as
described by Katz et al. (157) The sample, 0.1 to 0.5
m moles HCOOH in a minimal volume of water and 3 ml.
reagent were placed in the main compartment of the flask,
the edge of the center well was greased with vaseline and
about 0.2 ml. of 5 N NaOH was pipetted in it. The flask
was stoppered with a serum bottle stopper, evacuated
through a small bore hypodermic needle with shaking at
the water pump and stored overnight at 70 to 75°. The
contents of the center well were siphoned out (157) and
precipitated with Ba(0H)2. BaCO^ and Hg2Cl2 were deter
mined gravimetrically.
Sample data:
mM HCOOH Hg2Cl2 BaC03
determined
0.5
0.5
mg.
m moles
mg.
m moles
HCOOH co2
236.2 0.500 102. M - Q.k9k
237.2 0.502
101.5
0.^90
- Steam distillation of HCOOH. Formic acid was
distilled in a glass still with the steaming chamber
separately heated with a micro-burner. The volume of the
‘ ■ — ■ If8
liquid phase in the steaming chamber was kept at 3 to
b ml. Distillation was carried out in the presence of
about eight drops of 85 per cent phosphoric acid. The
recovery of HCOOH on steam distillation, determined
iodometrically (vide infra), averaged 99*5 per cent in
two determinations. A curve of the steam distillation of
1.0 m moles HCOOH carried out in the presence of 0.2 ml.
85 per cent phosphoric acid is shown in Fig. 1.
Iodometric determinaticn of HCOOH. The procedure
of Fleury et al. (158) was used with slight modification.
Standard iodine was prepared according to Koltoff and
Sandell (15*0 and was used to standardize approximately
0.02 N sodium thiosulfate. Standard thiosulfate was used
to standardize about 0.2 N potassium iodate and checked
against the normality of the potassium iodate as deter
mined gravimetrically; the value agreed within less than
O A per cent.
The Hg2Cl2 precipitate was transferred to test
tubes, 0.5 ml.- of 20 per cent celite was added and washed
twice with water. To the precipitate in 50 ml. Erlenmeyer
flasks were added ^ ml. standard iodate solution and 5 ml.
of freshly prepared 20 per cent KI. One to two minutes
were allowed to permit the liberated iodine to react with
the Hg2Cl2. Excess iodine was titrated with thiosulfate
h9
0.8
O
O o.e
0.4
0.2
SO
100
ML STEAM DISTILLATE
Figure 1. Steam distillation of 1.0 m mole HCOOH.
50
solution to the starch end point, and formate equivalence
was calculated by difference. This procedure was used
primarily with the samples for formate combustion by
Method II.
Sample data:
m mole HCOOH
combusted
e
ml. 0.238
I KIO^ '
ml. 0.0198
Na2S20^
m mole
HCOOH
O.1 * b 8.06 0.398
o.h b 8.32
0.395
o.h b
7.83 0.**00
o.h b 8.00
0.399
Radioassay of BaC-^O^. In general, the procedure .
reported by Weiss (159) was used. The contents of
alkali traps were made up to 100 ml. with C02-free water
after the addition of 200 to 300 mg. of NaCl. About a
20 per cent excess of saturated Ba(0H)2 was added and the
precipitate was allowed to settle. The supernatant was
decanted, the precipitate transferred to a small test
tube, and washed five times with C02“free water and twice
with methanol. The samples were made up in methanol,
using 100 to 120 mg. in 2 ml. of methanol per planchet
for infinite thickness and lesser amounts for finite
thicknesses. Samples were transferred to 1 inch aluminum
planchets with lips (PL-2, Technical Associates,
Glendale, California) mounted on brass or aluminum
sleeves with wide rubber bands, and centrifuged twenty-
minutes. The methanol was poured off, the barium
carbonate dried, weighed if less than infinite thickness
and counted. Counting rates were corrected for self
absorption and back-scattering by the use of empirical
curves as shown in Fig. 2. This curve was used to
determine the weight of barium carbonate corresponding
to infinite thickness.
Crystallization and plating of radioactive
compounds. Methionine and barium formate samples were
plated on aluminum planchets in aqueous solution and
dried on an aluminum heating block at 60°.
1. Crystallization of methionine. The acid
soluble fraction of incubates containing methiqnine
carrier (vide infra) were extracted three times with two
volumes of ether. Three volumes of.95 per cent ethanol
were added and crystallization was allowed to proceed in
the cold room. The crystals were centrifuged off, taken
4
up in a minimal volume of water on a boiling water bath
and recrystallized five to seven times from ethanol-water,
If necessary, insoluble impurities were removed by
centrifugation. Crystals from the third and following
recrystallizations were plated, 5 to 15 mg. per planchet,
52
ISO
100
o
f O
so
80 20 1 0 0 40 60
PERCENT
Figure 2. Self-absorption of barium carbonate.
Curve 1, per cent of specific activity at 7 mg. per
planchet. Curve 2, per cent of maximum counting rate.
and counted.
Sample data: the uptake of formate-C^ into
methionine in mouse liver slices:
Recrystalli
zation No.
Weight
mg.
c.p.m. Factor”" * * c.p.m.
3
5.6
355 0.57 109
b 8.2 **08 0A95 101
5 5A 399 0.57
110
2. Crystallization of harium formate. (160)
The acid soluble fraction of incubates containing carrier
formic acid were steam distilled as described above. The
distillate was carefully neutralized to the phenol
phthalein end point with about 0.15 N BaCOKQg. The
sample was concentrated on a hot plate, crystallized with
acetone-alcohol, and recrystallized as described for
methionine. Insoluble impurities were removed by
centrifugation. The specific activity of most samples
did not change after the third recrystallization.
Sample data: the oxidation of methyl labelled
methionine to.formic acid in mouse liver prisms.
Recrystalli
zation No.
Weight
mg.
c.p.m. Factor”' * ’ c.p.m
3
29.6 1530 0.27 191
k
11.5
990
0.^3 199
5
11.0 876
0 M 5 19**
9+
Nitrogen determination and radioassay on protein
samples. The acid insoluble fraction of incubates (vide
infra) was washed three times with 7 per cent trichloro
acetic acid, twice with methanol and twice with ether.
The powder was air dried and 5 ml. of N. NaOH were added.
1. Protein plating. The samples were allowed to
solubilize in the alkali overnight. A 1 ml. aliquot was
precipitated with 25 ml. of 7 per cent trichloroacetic
acid and allowed to stand overnight. The precipitate was
centrifuged, washed two times with 7 per cent trichloro
acetic acid, ethanol and ether as" before, air dried, and
then made up in about 1 ml. of concentrated formic acid
with slight warming. The formic acid solutions were
plated on tared aluminum planchets, dried on the
aluminum block, weighed and counted.
2. Nitrogen determination. In 10 ml. Pyrex
digestion tubes, 0.2 ml. aliquots of the protein solution
in alkali were digested with 0.5 ml* of 7*2 N sulfuric
acid containing 0.2 per cent mercuric sulfate. Two drops
of saturated potassium persulfate and one drop 30 per
cent hydrogen peroxide were used to clear the digestion
mixture. Stock Nessler*s reagent was made up by dissolv
ing 100 g. mercuric iodide and 76 g. potassium iodide in
about 150 ml. water, filtering and making up to 250 ml.
55
The working solution was made by adding 5 ml. to 1G0 ml.
of 8 per cent NaOH. The digests were diluted with 10 ml.
of water, a 1 ml. aliquot was taken, 5 ml. of water were
added, and 2 ml. of working Nessler's solution were added
immediately before reading in the Klett-Summerson
colorimeter. The samples were read against a digestion
blank with the 5*K) mju filter. Values were calculated by
use of a standard curve. About 0.1 mg. N per tube was
assayed. The color yield was. 68 Klett units per 0.1 mg.
Catalase assay. Liver catalase activity was
assayed by measuring the oxygen evolution produced by
adding properly diluted homogenates to standardized
hydrogen peroxide solutions. Two methods were employed.
Method 1. The method of Appleman (161)-^ was used,,
The reaction vessel consists of an inverted Y-tube; 1.0
ml. of the enzyme solution is introduced in one,arm of
the tube and 5.0 ml. of 0.6 M H2O2 buffered at pH 7.0 in
the other arm. The reaction was recorded as described by
1
Appleman for a period of time such as to insure that the
rate of reaction had decayed. The maximum rate of reac
tion was estimated graphically and taken as a measure of
-A/e are indebted to Dr. D. Appleman of the Depart-'
ment of Agriculture at,the University of California at
Los Angeles for permission to use his equipment.
56
enzyme activity.
Method 2. The method of Price and Greenfield (162)
was used with slight modification in instrumentation and
conditions. Hydrogen peroxide was titrated iodometrically
as recommended by Koltoff and Sandell. (15*+) Merck
’ ’Superoxol,*' 30 per cent hydrogen peroxide was used. An
aliquot of a suitable dilution containing about 0.2 meq.
of H2O2 (2 eq./mole) was titrated in 10 ml. of ^ N H2SOI4 . ,
1 g. KI and 3 drops of 3 per cent ammonium molybdate to
the starch end point. The concentration of the reagent
as purchased was about 23 N. The substrate was then
diluted to 0.1 M with M/15 pH phosphate buffer of
Sorensen. The buffer was prepared by dissolving 29.2 g.
of Na2HP0if*12H20 and 7*05 g. of K^POij. in distilled water
to a final volume of 2 liters. Suitable dilutions of the
liver homogenates, usually 1:25 of the 1:5 homogenates
(vide infra). were made with the „same buffer.
The reaction vessel consisted of. a 100 ml. round
bottom flask, containing a short Teflon covered Alnico
bar, and placed over a magnetic mixer to provide rapid
stirring to the reaction mixture. Pressure was recorded
in the closed system with a P6a ± 2.5 p.s.i. differential
pressure transducer (Statham Laboratories, Los Angeles,
California). The transducer was calibrated with a
' 57
mercury manometer. The volume of the system was measured
by injecting.water into the reaction flask and measuring
the resultant pressure change. The output of the pres
sure transducer was led into a Leeds and Northrup
Speedomax pen-writing recorder (catalog #69800). Nominal
scale speed was one second full scale deflection with 10
millivolts sensitivity full scale. The sensitivity was
modified in operation so that the full scale corresponded ,
to 100 mm. mercury.
The reaction vessel was immersed in a water bath '
thermostated at 30 ± 0.3° with a Fenwal Thermoswitch. In
the reaction vessel, 25 ml. of diluted substrate were
allowed to equilibrate to bath temperature with stirring;
the sensitivity was calibrated with a standard resistance
and the enzyme was injected into the substrate while
stirring was maintained at the maximum rate obtainable
with the magnetic stirrer. The reaction was recorded
until the reaction began to decay (twenty to sixty
seconds). The curves were analyzed as for Method 1.
Fig. 3 shows the rate of! oxygen evolution plotted
i
against concentration of crystalline beef liver catalase.
Deviation from the straight line relation may be due
either to limiting diffusion of oxygen into the gas phase
at higher reaction rates, or, more likely, limiting rate
?8
60
_ l
eo
ML. I:2500 CATALASE
Figure 3. The effect of catalase concentration
on reaction rate. The effective concentration of added
cystalline beef liver preparation is indicated on the
abscissa.
59
of response of the potentiometric recorder. Determina
tions of activity were carried5out at rates less than 50
ml. oxygen per minute.
Xanthine oxidase assay. The method of Litwack
et al. (163) was employed for the determination of
xanthine oxidase. In this procedure, activity is deter
mined by measuring the disappearance of xanthine colori-
metrically in deproteinized aliquots of the reaction
mixture.
Five ml. of mouse liver homogenate (1:5) buffered
at pH 7M were preincubated for forty minutes in 20 ml.
beakers in a Dubnoff metabolic shaker (Precision Instru
ments Company) in air at 37.5°. To each beaker were
added at zero time 0.3 ml. of pH 7 buffer and 0.6 ml.
of 0.038 M xanthine or water. At 0, 30, 60, 90 and 120
minutes, 1.0 aliquots were pipetted into test tubes
containing 1 ml. of bo per cent sodium tungstate, 5 ml*
of water and 1 ml. of 2 N ^SOi*. The samples were
allowed to stand with occasional shaking, centrifuged
and the supernatant was used as such.
Xanthine was determined colorimetrically as
follows: To 0.3 ml. of the supernatant were added 2.5
ml. of water and 5 ml. of saturated sodium carbonate.
Color was developed with 1 ml. of 1:1 dilution of
60
Folin-Ciocolteau reagent (Steriproducts Laboratories,
Whittier, California). The samples were read on the
Klett-Summerson colorimeter with the 660 mju filter against
a reagent blank. A standard curve was prepared by dilut
ing the 0.038 M xanthine solution to 50 jug*/ml.
(0.865sl00) and color was developed in a mock acid
soluble mixture. A straight line was obtained in the
range from 0 to 100 jug •» the color yield for 50 jug. was
295 Klett units.
The color yields at the various times were cor-
' >
rected with the corresponding incubate blanks. The data
were plotted against time and, following Litwack et al.
(163), the maximum rate of xanthine disappearance was
taken as a measure of xanthine oxidase activity.
Recovery of respiratory COg and urinary formate.
Rate studies. The rate of expiration of radio
activity in the respiratory carbon dioxide from intra-
1L.
peritoneally injected sodium formate-C was measured in
the equipment of W e i s s . (157)
The dosage employed was *+5 per 20 g. body
weight. The specific activity was 3000 to *K)00 c.p.m./juM.
_ _
We are indebted to Dr. W. Marx and Dr. S. Weiss
of this department for permission to use their equipment.
61
The dose was administered at zero time, the mouse was
placed in the glass cage, and a stream of C02-free air
was swept through the cage into fritted glass gas scrub
bers containing 15 ml. of N NaOH, C02-free. The samples
were diluted to 100 ml., titrated and assayed as
described above.
Total recovery studies. The equipment used for
these studies was a bank of six metabolic cages such as
those described by Mackenzie et al. (129) except that the
urine traps consisted of 50 ml. round bottom flasks
fitted with standard taper ground glass joints to the
bottom of the cages. After injection of the labelled
compounds, the mice were kept in the cages overnight
(about eighteen hours) with no food but with access to
water. Respiratory COg was collected in fritted glass
gas scrubbers with 150 ml. of 2 N NaOH; the samples were
diluted to 250 ml., aliquots were titrated with 0.2 N
standard HC1 and assayed at infinite thickness as
described above.
To recover the urine, the cages were washed, and
the washings added to the trap contents. The samples
were made alkaline to phenolphthalein with dilute NaOH
and concentrated on the hot plate. The formic acid was
distilled as described above and oxidized to CO2 by
62
Method 2. Mercurous chloride was determined iodometri-
cally as described above. /
Incubation procedures.
Formic oxidase. The mice were sacrificed by
cervical rupture, the liver was rapidly removed and the
gall bladder dissected out. The liver was then weighed
and homogenized in ice cold, pH 7.4- Sorensen’s phosphate
' buffer, M/15, prepared by dissolving 3 8 . g. Na2HPQ4 . * 12H20
and 3*56 g. of KH2P0ltf to a final volume of 2 liters. The
A. H. Thomas 10 ml. tissue grinder with a Teflon pestle
was used. A maximum of 30 to HO ml. of whole homogenate
was dialyzed overnight (twenty to twenty-four hours)
against 2 liters of the same buffer. To each flask were
added 2 ml. of homogenate (333 mg. tissue fresh weight).
The samples were incubated in 15 ml. Warburg flasks
with two side arms. The gas phase was air, the temper
ature 37*5°, the final volume of the incubation mixture
was 3.0 ml. In one side arm was placed 0.2 to 0.5 ml. of
formate-C^, in the other 0.3 ml. of 50 per cent trichloro
acetic acid. The i*im of the center well was greased with
f
vaseline and in it were placed a 1" x 7/8" filter paper
wick, Whatman #1, folded lengthwise and 0.2 ml. of
0.8 N NaOH, C02-free. The liver homogenate and other
additions were pipetted into the main compartment.
63
Additions other than the homogenate and alkali
were made at room temperature. The flasks were allowed
to chill in the 5° cold room and the homogenate and
alkali were added in that order. The flasks were pre
incubated five minutes in the Warburg bath and the
substrate tipped in at zero time. The reaction was
stopped by tipping in the trichloroacetic acid, after
which the flasks were shaken for an additional hour.
The alkali wick was transferred with a forceps to
a test tube containing 0.6 or 1.2 m moles of Na2C0^
carrier. The center well contents were siphoned (157)
into the test tube with C02-free water containing about
10 drops 1 per cent phenolphthalein in ethanol per 100 ml.
of water. The contents of the tube were mixed and the
radioactivity was allowed to diffuse out of the filter
paper wick overnight. The samples were diluted to about
100 ml. with C02~free water, 200 to 300 mg. NaCl were
added and precipitated with saturated Ba(0H)2 and counted
as described.
The recovery of Cll+02 by this procedure was tested
by measuring the transfer of NaHC1^©^ to the center well
contents as shown below.
6k
Assay of
solution added
Trial Recovery
‘c.p.m.
1107
1155
11^6
c.p.m
%
3.
1
2
' llkO 100.k
1128. 99.2
1121 98.
ave. 1136 t 2k
% 100 + 2.1 99.5 t 0.26
Methionine synthesis and oxidation. The mouse
livers were obtained as before. Liver prisms were cut on
a Mcllwain and Buddie tissue chopper (16N-) with the
detailed by these authors, first chopping the liver in
one direction, and then rotating the stage 90° and
repeating the chopping operation. The prisms were washed
twice with ice cold Krebs-Ringer-phosphate .buffer, pH
7.^, with the composition shown in Table I, prepared
essentially according to Umbreit et al., (165), but with
the indicated change in buffer composition. Excess fluid
was sucked away from the surface of the liver prisms
which had been allowed to settle in chilled test tubes.
Aliquots of the remaining thick suspension were pipetted
into the 20 ml. beakers with a 1 ml. blowout pipette
instrument set at 0.2 mm.i/ The procedure used was as
-^We are indebted to Dr. J. Katz of this depart
ment for permission to use his equipment.
TABLE I
COMPOSITION OF KREB‘S-RINGER-PHOSPHATE BUFFER
100 parts 0.9$ NaCl
b parts 1.15$ KC1
3 parts 1.22$ CaCl2
1 part '2.11$ K^POi*.
1 part 3.82$ MgS0v7H20
12 parts M/15 Sorensen's buffer, pH 7.*+
66
with a truncated tip. The tissue was added to pre-chilled
beakers in the cold room. The beakers were preincubated
five minutes in the Dubnoff metabolic shaker in oxygen
and the labelled substrates were added at zero time to
give a final volume of 2.0 or 3*0 ml.
The reactions were stopped by adding 2.0 ml. of
acid protein precipitant. For the oxidation reaction,
2.0 ml. of 10 per cent HFO^ containing 0.75 mM HCOOH per
ml. were added. (166) For the synthesizing reactions,
2.0 ml. of 17 per cent trichloroacetic acid containing
50 or 75 mg* D,L-methionine per ml. were added. The
flasks were shaked an additional twenty to thirty minutes,
the contents transferred to small centrifuge tubes and
homogenized by hand with the A. H. Thomas Teflon pestle.
The samples were centrifuged and the supernatants used as
such. The carriers were isolated by the procedures
indicated above.
CHAPTER IV
EXPERIMENTAL RESULTS
I. PRELIMINARY
' f
In the course of preliminary studies on the
oxidation of formic acid in the intact mouse, the finding
of a depression*of liver catalase in tumor-bearing mice
has been confirmed and 'extended to leukemic mice (Table
II). These data are also of interest since the weight of
the implanted Sarcoma-180 is considerably less than the
5 per cent estimated by Greenstein (18) to be required to
produce detectable liver catalase depression. These
results are presented here to establish the occurrence of
this depression in a population of mice bearing trans
planted tumors under the present experimental conditions.
Pertinent information on formate oxidation in vivo
will be presented in conjunction with the studies on
oxidation of the methyl group of methionine in vivo.
II. THE OXIDATION OF FORMATE BY
MOUSE LIVER HOMOGENATES
In early studies on formate oxidation in mouse
liver homogenates, a rapid reaction was obtained in
6 8
TABLE II
THE EFFECT OF TUMOR ON LIVER CATALASE ACTIVITY
Mice
Liver Tumor
weight % weight %
Liver catalase
units/20 g.
V
Sarcoma-180
Control (7)-^ 5.32 ± 0.97*^ --- 80.3 ± 15
Tumor (10) 5.19 i O.63 3.86 t 0.63
U/
Leukemia-'
Control (9) ^.27 t 0.29 --- 79.8 * 13
Tumor (10) *+.91 t 0.^5 --- 5/- 56.1 ± 13
' •
1/
~ By the method of Appleman (161). The unit is
defined a 1 ml. of oxygen per second at 0° and atmospheric
pressure from 0.5 M hydrogen peroxide.
-^The numbers in parenthesis indicate the group size.
■^/standard deviation.
^Lymphatic leukemia, P-1532, obtained from the
Jackson Memorial Laboratories, Bar Harbor, Maine. Grown in
DBA/2 mice.
^The mass at the site of inoculation
index of the extent of the neoplastic process
is not an
•
69
agreement with Nakada and Weinhouse. (151) Furthermore,
in undialyzed homogenates incubated for one hour, a
marked stimulation was obtained upon the addition of
xanthine and hypoxanthine (Table III). This finding
prompted a study of potential co-oxidants in this.system.
D-Methionine was investigated as a hydrogen
peroxide source with the hope of detecting D-amino acid
oxidase in mouse liver homogenates. Table IV reports the
negative results in agreement with the findings of Shack
(167) who reported the absence of this enzyme in mouse
liver when tested with D-alanine.
Table V presents manometric data on xanthine
oxidase in mouse liver homogenates. The interpretation
of these data is complicated by the fact that the addition
of hypoxanthine prolongs but does *not alter the initial
Qq2, a fact which suggests that xanthine oxidase activity
is responsible for the greater part of endogenous oxygen
uptake. It will be noted that the volume of this system
is one half that used in most of the experiments, and
that in Table III the unsupplemented homogenate oxidized
about b u moles of formate at a formate concentration of
0.01 M. In accordance with the equations,
70
TABLE III
THE EFFECT OF XANTHINE PLUS HYPOXANTHINE ON FORMATE
OXIDATION IN UNDIALYZED MOUSE LIVER HOMOGENATES
Two ml. of liver homogenate in a final volume of
3.0 ml. were incubated for one hour. Tfftiere indicated,
1 4 -.I p . moles of hypoxanthine and 6.2 pM of xanthine were
added to the flasks.
Sodium ,
formate-C1*
M
Xanthine +
hypoxanthine
p moles C^-^Op/
flask
0.01 -
1.75
0.01
+
h.5l
0.10 - 3.92
0.10
+
10.0
71
TABLE IV
THE ABSENCE GF D-AMINO ACID OXIDASE IN
MOUSE LIVER HOMOGENATES
Two ml. of mouse liver (1:5) homogenate in a final
volume of 3.0 ml. were incubated with the indicated con
centrations of D-methioninei/ with alkali in the center
well. The .values are in jal. oxygen uptake.
1
Time
(mins.)
Oxygen uptake
—
Endogenous 0.001 M
0.005 M
0.01 M
30 131 131 132
135
120 125
128
115
60 202
207 207 225
186
19*+
210 190
-^This preparation produced net uptakes of
oxygen of 3*+ and 79 pi. at 0.01 M at thirty and sixty
minutes, respectively, in rat liver homogenate.
72
TABLE V
XANTHINE OXIDASE IN MOUSE, LIVER HOMOGENATES
One ml. of liver homogenate (1:5) in a final *
volume of 1.5 ml. was incubated with 3 p moles of
hypoxanthine with alkali in the center well. Values are
in oxygen uptake.
i Oxygen uptake
Time
(mins.) Endogenous Hypoxanthine -Endogenous
pi. p atoms pi. p atoms pi. p atoms
30 70 6.2
73 6.5 3
60 108 9.6 15k 13.8 **6 *f.l
90
13fc
11.9 - 216 19.3 82 7.3
120 150 13
23*f
20.9 80.b 7.5
73
2 hypoxanthine + 2 02 *- 2 uric acid + 2 H202
2 H202 — ► 2 H20 + 02
2 hypoxanthine + 02 2 H20 + 2 uric acid
i
and
2 H2C^+ 2 HG00H ---> 2 C02 + k H20 ,
about 9«5 A moles of hydrogen peroxide are available for
the peroxidation of formate if it is assumed that
xanthine oxidase activity accounts for all the oxygen
uptake. Similar considerations hold for the value of
10 moles of formate oxidized in the supplemented
homogenate, utilizing the corresponding value at sixty
minutes for oxygen uptake in Table V. The two experi
ments cannot be compared directly since the levels of co
oxidants are different in the supplemented flasks, but it
would appear that the utilization of apparent hydrogen
peroxide is less than 50 per cent efficient in both
supplemented and unsupplemented flasks.
The effect of acetaldehyde as a, co-oxidant was
tested. It was found that while this compound produces
stimulation of formate oxidation, at maximal concentra
tions its effect is less than that of hypoxanthine. An
experiment in dialyzed liver homogenates is shown in Fig.
^ and details are presented in Table VI as an example of
7b
3
ce
x
CO
<
d
N
2
CM
g
40 80 1 2 0 1 6 0
SO
40
30
>
20
1 6 0 1 2 0 40 80
UM CH.CHO / FLASK (S)
Figure b. The effect of acetaldehyde on the rate
of oxidation of formate-C^ in dialyzed mouse liver
homogenates. Incubation time, one hour; formate con
centration, 0.01 M.
TABLE VI
75
THE EFFECT OF ACETALDEHYDE OH FORMATE-Cll+ OXIDATION
Two ml. of dialyzed mouse liver homogenate were in
cuhated for one hour in a final volume of 3.0 ml.;
concentration of formate, 0.01 M, 1605 c.p.m./;i mole.
Flask
CH3CHO
M
mg. BaCOo
per
planchet
c.p.m.l/
per
planchet
c.p.m.^
total
Cllf02
p. moles
1 0.0061 3^.8
567
3090
25.8
525 3050 1.91
2 0.0061
3^.5
598
V .
3210
31.8 587 3190
1.99
3
0.013*f 33.0 689 3730
31.7
722 39*+0 2.39
*+ 0.013*f
35.7 715
3830
31.5 707
3870 2.1+0
5
0.020
37.1 827
kb20
32.7 789
1+290
2.71
6 0.020 37.2 788 1+210
28.7
7*+l HO70 2.58
7
0.0267 35.6 882 1+720
3fc.l 865
1+660 2.92
8
0.0267 35.5
870 1+720
3^.3 867
1+660
2.91
9 0.0333 37.9 917
1+860
33.7 909
1+780 3.01+
10
0.0333
38.0 901 1+780
33.7 869
1+700
2.95
11 0.04-16
36.3
9¥f 5020
32.8 918 5180 3.18
12 0.0^16
3*+.7 925
1+970
33.0
908 1+930 3.08
76
TABLE VI (continued)
Flask
CH^CHO
M
mg. BaCOo
per
planchet
c.p.m.i^
per
planchet
c.p.m.-^
total
c^o2
)i moles
13
0.050 3*+.9
978 52^0
31.8 892 i+870
3.1^
1*+ 0.050
35.3
936 5020
3^.2 932 5020
3.1^
^Corrected for background, 20 c.p.m. Total of
10,2*+0 counts taken.
2/
- Corrected for self absorption and calculated to
total amount of carbon dioxide.
77
the calculation involved, counting barium carbonate at
finite thicknesses. It can be seen that under these
conditions the oxidation of acetaldehyde is limiting.
From the Linevreaver-Burk (168) plot of the data, a
maximum rate of '3.5^ p moles of Cllf02 and Kg value of
0.006 M are obtained. The latter is quantitatively
similar to the value of 0.007 M obtained by Gordon, Green
and Subrahmanyan (169) using a purified pig liver alde
hyde oxidase with crotonaldehyde as the substrate.
Although this experiment is of interest in adding further
evidence to the mechanism of formate oxidation in
mammalian liver, acetaldehyde per se is not adequate as a
co-oxidant where it is attempted to render the rate of
formate oxidation limiting.
All subsequent tests in formate oxidation by liver
\
homogenates were performed in dialyzed preparations.
The experiment shown in Fig. 5 was performed with
/
the hope of demonstrating that the hydrogen peroxide
production in this system was not limiting. The linear
response obtained in this experiment will be discussed
below.
Because of the difficulties of working with hypo
xanthine in sufficiently concentrated solutions to be
conveniently added to incubation media, the effect of
78
t c .
X
\
C /5
<
l i _
\
< u
P
O
5
XL
0 - CONTROL
t-92 PM CH3CH0 / FLASK
2 3
PM HYPOXANTHINE / FLASK
Figure 5« The effect of hypoxanthine and hypo
xanthine Plus acetaldehyde on the oxidation of
formate-C1^ in dialyzdd mouse liver homogenates.
Incubation time, one hour; formate concentration, G.01 M.
7 9
inosine (hypoxanthine riboside) was tested as shown in
Fig. 6, repeating the experiment of Fig. 5-
At equivalent concentrations inosine is as effec
tive a co-oxidant as is hypoxanthine. The curves of
Figs. 5 and 6 are approximately superimposable at the
same concentrations of inosine and hypoxanthine. It can
be seen from Fig. 6 that acetaldehyde has no detectable
effect on activity in the presence of optimal concentra
tions of inosine. This may be interpreted as indicating
that inosine and acetaldehyde are oxidized by different
enzymes, and therefore saturation of formate peroxidation .
would have been demonstrated. Alternately, a one enzyme
hypothesis is possible, consistent with the observed
failure of acetaldehyde to inhibit the enzyme, providing
the affinity of inosine for the enzyme is sufficiently
great. In this case, the argument for saturation of
formate peroxidation by peroxide producing systems in
this experiment would be untenable.
Evidence that the hydrogen peroxide' producing
systems are not limiting is obtained in the experiment
*
shown in Fig. 7. When the formate concentration is
raised tenfold, the yield of increases greatly, a
finding which indicated clearly that sufficient hydrogen
peroxide is available at the 0.01 M level to saturate the
80
Lu
CM
0 - CONTROL
• - 9 2 PM CH3CH0 / FLASK
4 8
PM INOSINE / FLASK
1 2 1 6
Figure 6. The effect of inosine and inosine plus
acetaldehyde on the oxidation of formate-C^ in dialyzed
modse liver homogenates. Incubation time, one hour;
formate concentration, 0.01 M.
1 0
0.10
8
cc R
X 6
Lu
N *
0.01 M
o
2
O
5
2 6 4 8 1 0
0.10 M HC^OO'
0.01 M HC,400“
PM INOSINE / FLASK
Figure 7. The effect of formate concentration on
inosine oxidation coupled formate oxidase in dialyzed
mouse liver homogenates. Incubation time, one hour.
------------------------------------------ : g2
system. The inhibition of activity at high inosine and
formate concentrations which was obtained in this and
other experiments is of some interest. A possible inter
pretation, not verified in the present investigation,
involves the well known inhibition of xanthine oxidase
activity by excess substrate. (170) In any case, this
result suggests that at higher formate concentrations the
hydrogen peroxide generating system may become limiting,
and thereby the interesting kinetic situation would exist
wherein the rate of peroxide formation required to
saturate formate peroxidation is itself a function of
formate concentration.
The linear function of activity with .inosine and
hypoxanthine concentration obtained in this and other
experiments may be interpreted by the data shown in Fig.
8. It will be seen that the initial rate of formate
oxidation is not affected by inosine concentration.
Obviously, those portions of the curves of Figs. 5» 6 and
7 at submaximal concentrations of co-oxidant do not,
therefore, reflect rates of oxidation but rather yields
of oxidation based on the total amount of hydrogen
peroxide available to the system in the time studied.
Further, the lack of effect of substrate.on reaction rate
in the concentration range studied implies that the
83
0.002 M INOSINE
U . 6
0.001 M INOSINE
so
40 60
TIME (MINUTES)
80 1 0 0
Figure 8. The effect of inosine on the rate of
oxidation of formate-C^ in dialyzed mouse liver
homogenates. Formate concentration, 0.08 M.
— --------------------------------------- _ _ gjj-
affinity of Inosine for its oxidizing enzyme is very
great. The persistence of a linear reaction for eighty
minutes with appropriate inosine concentration indicates
that under these conditions a one point assay at sixty
minutes is a valid measure of the reaction rate.
It thus appears that in this system the rate of
hydrogen peroxide production is limiting. However, it may
not be the sole rate determining factor, viz., it is pos
sible that given a fixed rate of peroxide production,
formate oxidation at lower formate concentrations may be
a function of formate and catalase concentration.
To obtain precise information on this point, the
effect of formate concentration on activity was tested
and the results are shown in Fig. 9 and detailed in Table
VII as an example of data obtained in plating barium
carbonate at infinite thickness. Conventional enzyme
kinetics are observed with a maximum velocity of 9.15
ji moles per hour and Ks of 0.0123 M. The solid point
_ curve in Fig. 9 is calculated from these rate constants.
Taken together, the findings of these experiments
suggest that formate andi by implication, catalase are
rate determining if not limiting in this system. With
this information available, the effect of tumor on the
t
oxidation of formate in dialyzed mouse liver homogenates
85
8
40
X .
<
_l 6 30
U.
0 J
o
2 5
O
20
6 8 10
Figure 9. The effect of formate-concentration
on rate of oxidation in dialyzed mouse liver homogenates.
Incubation time, one hour; inosine^concentration 0.002 M.
Solid circles represent p . moles open circles, the
Lineweaver-fiurk function, (S)/V.
86
TABLE VII
THE EFFECT OF FORMATE-C1** CONCENTRATION ON OXIDATION
Data of Fig. 9» Values for duplicate flasks are
indicated.
Sodium formate Barium carbonate
Concentration
M
Specific activity
c.p.m./ja mole
c.p.m. at
infinite
thickness
ja moles
ave.
O.Ol 967 3000
967 2950
3.99
0.02
1+83 2120
1+83 2170
5.77
0.0*+ 322 1708
322
----
6.98
0.06
215 1167
215
1322
7.53
0.08 201
1275
201
123*+
8.11
0.10
193 1151
193
1188
7.87
87
was investigated.
Fig. 10 shows the results of,an experiment with
control liver homogenates and liver homogenates of tumor-
hearing mice. Formate-C^ oxidation, at 0.10 M concen
tration, was studied with varying inosine concentrations.
Maximum activity in both homogenates occurs at about the
same concentration of co-oxidant. While the depression
of activity in the liver homogenates of tumor-bearing
mice is doubtless real, it does not correspond in
magnitude to the catalase effect obtainable with the
Ehrlich ascites carcinoma. (19) The data in Table VIII
confirm this effect. In addition, measurement of
xanthine oxidase activity failed to show a tumor effect
on this enzyme, as discussed by Greenstein (18), thus
eliminating xanthine oxidase as a factor in the depres
sion. The effect of catalase addition is interesting in
that no significant effect is obtained in the normal
liver homogenates, while a distinct stimulation is
obtained in the samples from tumor-bearing animals. This
constitutes further evidence for the contention that at
high formate concentrations, peroxide generation in this
system may be limiting with respect to high catalase
concentration, but not rate determining at lower catalase
concentrations.
88
U_4
• - CONTROL
0 - TUMOR
o
dM INOSINE / FLASK
i
Figure 10. The effect of inosine concentration
on oxidation of formate-C14" in dialyzed liver homogenates
of normal and tumor-hearing mice. Incubation time, one
hour; formate concentration, 0.10 M. Tumor used was the
Ehrlich ascites carcinoma.
8 9
TABLE VIII
THE EFFECT OF TUMOB AND ADDED CATAIASE ON FORMATE
OXIDATION BY DIALYZED MOUSE LIVER HOMOGENATES
Final formate concentration, 0.1 M, specific
activity, 153 e.p.m./ mole, 5 P mole inosine per flask
was incubated for one hour. Crystalline catalase, 0.1
ml., was added as indicated. Tumor was the Ehrlich
ascites carcinoma.
Homogenate
>
Catalase
Cllf02
production
p moles/flask
Xanthine
Average oxidase
(/iM xanthine/
hour/g^.
liver)!'
Normal -
8.17
-
9.55
8.88 13.0
- 8.90
+
9.50
+
10.U-
9.83
+
9.65
Tumor -
6.55
•
- 6.68 6.58 12.6
- 6.83
+
9.18
+
9-27 . 9.55
+
10.3
3/Method of Litwack et a^. (163)
90
To provide further evidence on this point, the
last two experiments were repeated at 0.012 M concentra
tion in formate as shown in Fig. 11 and Table IX. It can
be seen that the depression of formate oxidation is in
the order of magnitude of that reported for liver
catalase. In Table IX, the depression in formate oxida
tion is closely matched by the catalase values obtained
at the same time formate oxidation was assayed. In this
case, appreciable stimulation is obtained on adding
catalase to the liver homogenates of normal as well as
those of tumor-bearing mice.
III. THE EFFECT OF TUMOR ON THE OXIDATION OF
THE METHYL GROUP OF METHIONINE TO FORMATE
Studies on the excretion of formic acid and
carbon dioxide in intact normal and tumor-bearing mice
are of aid in the interpretation of the data obtained on
the in vivo catabolism of the methyl group of methionine.
The data in Table X show the recovery of-bj p . moles
sodium formate-C*^ administered per 20 g. body weight to
normal and Sarcoma-180 bearing mice. Although the total
recovery in tumor-bearing mice is significantly lower
than in normal mice, the difference is very small and
probably cannot be accounted for by differences in the
91
CONTROL
: TUMOR
gM INOSINE / FLASK
Figure 11. The effect of inosine concentration
on oxidation of formate-Cl^ in dialyzed homogenates of
normal and Ehrlich ascites careinoma bearing mice.
Incubation time, one hour; formate concentration, 0.012 M,
92
TABLE IX
THE EFFECT OF TUMOR AND ADDED CATALASE ON FORMATE
OXIDATION BY DIALYZED MOUSE LIVER HOMOGENATES
Final formate concentration, 0.012 M, specific
activity 521 c.p.m./p mole, 5 p. mole inosine per flask
was incubated for one hour. Crystalline catalase, 0.1
ml., was added as indicated. Tumor was the Ehrlich
ascites carcinoma.
Homogenate
V
Catalase
c^o2
produced
p mole/
flask
Average
Catalase activ
ity (ml. O2/
min./ml.
homogenate)!/
Normal - 6.20
- 5.90 5.82 730
-
5.39
+
7.52
+
7.16 7.26
4* '
7.00
Tumor . -
---
-
3.27 3-30
331
-
3.3^
+■
5.90
+
5.16 5.59
+
5.72
-^Method of Price and Greenfield. (162)
93
TABLE X
,1b
RECOVERY OF SODIUM FORMATE-C IN NORMAL
AND SARCOMA-180 BEARING MICE
Sodium formate-Cl1 *, specific activity lf86 c.p.m./
pi mole was injected intraperitoneally, k5 pi moles/20 g.
mouse weight. The mice were kept in metabolism cages
eighteen hours.
cl1 * Recovery
■
Body
weight
S*
Tumor
weight
%
Respiratory
COo
pi moles
Urinary
HCOOH
p. moles
Total %
1/
33.5 ---
31.9 ---
3^.7 ---
32.9 ---
29.8 ----
35.0 ---
Ave. 33.0±1.
36.0
38.5
36.9
39.9
35.2
33.5
36.712.8
6.27
M-.ll
6.M-9
2.10
5.2**
8.20
5.^812.2
9b.0
9^.5
97.1
93.3
90.5
92.7
93.712.2
2b. Q
27.8
28.7
30.8
2*f.l
25.0
Ave. 26.9±2
7.02 33.7
3.7b 32.1
5.19 37.1
5.16 3^.3
,5.68 31.6
3.52 3^.6
.6 5.oto. 3 33.912.0
6.19
6.M+
3.7^
b.52
3.62
5.b0
*f.98*1.2
88.6
85.3
90.8
86.3
78.3
89.O
86.313.2
- Total BaCOo per 20 g. recovered in normal and
tumor-bearing mice was 5690 1 260 mg. and 5500 ± 310 : mg.,
respectively.
2/
Standard deviation.
9»fr
partition of formate between excretion in the urine and
oxidation to carbon dioxide in the two groups. The dif
ference may reflect the greater utilization of formate
for synthesis in the tumor-bearing animal. No interpre
tation is available for the failure to observe inhibition
of formate oxidation in the group of tumor-bearing
animals analogous to that observed in liver homogenates.
Possibly the parenterally administered dose is not
primarily oxidized in the liver, or it may reach the
catalytic sites in the liver in a manner such that the
rate of oxidation is never limiting at the dose level
employed. It is difficult to decide whether this experi
ment yields information on the rate of oxidation of
endogenous formate as affected by tumor.
Fig. 12 shows the rate of recovery of radioac
tivity in respiratory carbon dioxide from graded doses of
l U-
sodium formate-Cx . Although not shown in the graph, a
linear response is also obtained in the 0.1 p mole to
1 p mole dose. It would appear that the percentage con
version of formate to carbon dioxide in the intact animal
is essentially insensitive to dose administered in the
0.1 to 50 p. mole range.
Fig. 13 shows the rate of oxidation of a *+5 jiM/20 g.
test dose of sodium formate-C^ in an intact mouse. The
95
LU 6
U J
20
J J M SODIUM FORMATE-C1 4 INJECTED/20 G .
40 6 0 80
1 4
100
INJECTED/20
Figure 12.. Rate of recovery of graded doses of
sodium formate-Cl^ in respiratory CC^.
96
30
I
|
i
i
■ I
!
i
i
i
1
100 20 80 40
TIME (MINUTES)
60
Figure 13. The rate of expiration of C-^C^ froni
an intraperitoneal dose of b5 jx moles sodium formate-Cl^/
20 g.
97
rapid rate of oxidation is in agreement with the findings
of Weinhouse and Friedmann. (l*+8) Preliminary attempts
to assess the rate of oxidation of formate in normal and
tumor-bearing mice as measured by the initial rate of
1U-
Cx h)2 expiration in intact animals failed as judged by
findings on the expiration of C-^02 from test doses of
sodium carbonate-C^. Table XI shows typical findings in
a normal and Sarcoma-180 mouse. It will be seen that the
initial higher rate of expiration of radioactivity in the
normal mouse is related to a greater expiration of carbon
dioxide. Of significance are the facts that the specific
activities of the BaCO^ samples in the first time interval
are the same for both animals, and that the total carbon
dioxide excretion per unit time tends to plateau out to
the same value. In note 1/ to Table X, it will be seen
that there is no difference in the total C02 expiration
in normal and tumor-bearing mice over long periods of
I
time. This result has been obtained several times.
With this information as a background, the
recovery of from methyl labelled methionine in
respiratory carbon dioxide and urinary formate was
studied in mice given an accompanying intraperitoneal
dose of unlabelled sodium formate. The results are
presented in Tables XII and XIII at a 1 and 30 p. mole
98
TABLE XI
TRANSFER OF INJECTED CARBONATE TO RESPIRATORY CARBON
DIOXIDE AS AFFECTED BY TUMOR
Forty-five ju moles sodium carbonate, specific
activity, 551 * c.p.m./;i mole were injected per 20 g.
mouse weight.
Time
(mins.)
p . mole C-^02/
20 g.y
Barium carbonate
Specific Mg./20 g./
activity W 10 min.
Normal mouse
10 27.1 11,980
125
20
36.3
^ O O
115
3°
39.6 l,!+60 130
bo fo.7 627 97.1
60 bl.6 276 85.2-
Tumor mouse
10 17.8 10,500
9^.1
20 28.6 5,930 100
30 33.9
3,060 95.8
bO
36.9
1,680
9^.7
60
39.9 817
82 ,b
-^Cumulative values.
“ C.p.m. per planchet of barium carbonate at
infinite thickness.
99
TABLE XII
OXIDATION OF L-METHIONINE-C1^ IN VIVO
One ju mole methionine and 0.** m moles sodium
formate were injected intraperitoneally per 20 g. body
weight. Mice were kept in metabolism cages for eighteen
hours. Tumor was the Sarcoma-180.
Tumor
Resp. CO2
Urinary formate
weight
%
s
c .p.m./
20 g. x 10-2
m moles/
20 g. body
weight
G.p.m./
m mole
x 10“2
Total c.p.m./
20 g. body
weight
x 10-2
-------
1680
0.093 1080 100
----
1590 O.lllf 1560
179;
---- .
1070 1070
2010 0.05^ . 13^ 73
1^00
1550 t 35Q-S
1.28 h86 0.123
933 115
0.63 1600
953
—
1.10 1090 0.123 755 91
0.58 2120 815
0.68
605
1180 ± 680
0.156 1120 17b
^See Table XIII.
2/
-Standard deviation.
10G
TABLE XIII
OXIDATION OF L-METHIONINE-G1^ IN VIVO
Thirty mole methionine and 0.4 m mole sodium
formate were injected intraperitoneally per 20 g. body
weight. Mice were kept in metabolism cages eighteen
hours. Tumor was the Sarcoma-180.
Urinary formate
Tumor
weight
%
Resp. CO2
c.p.m./
20 g. x 10-2
m moles/
20 g. body
weight
C.p.m./
m mole,,
x K T 2
Total c.p.m./
20 g. body
weighty
x 10“2
4480 0.103 4180 432
4150
\
*+370
------------
3950 0.112 7600 847
— — — —
3650 0.066 3870 258
4730
4222 t 388-'
0.095
6l4o 586
1.37
3120
0.095
5060 486
8.18 3680
. . . .
3.22 2720 0.138 4580 632
3.05
2450 0.165 3390
6430
558
3.30 3370 0.116
745
4.81 3230 0.148
3095 t 4l9~/’^/
7280 1080
-^Pooled data (with Table XII) on total excretion
of formate: Control mice 0.091 ± 0.009 m mole/20 g.
Tumor mice 0.131 t 0.009 m mole/20 g.
"p*1 value by * * tM test is less than 0.001.
2/
- Standard deviation.
2/f l p" value by "tn test is less than 0.001.
101
dose level of L-methionine per 20 g. body weight in
normal and Sarcoma-180 bearing mice. Unfortunately, a
reliable degradation of the labelled methionine sample is
not available, so that the results cannot be expressed in
terms of gram atoms of carbon oxidized. However, the
total radioactivity administered at both dose levels was
the same (0.25 /iC/20 g. body weight) so that the two
groups of animals.may be compared on the basis of re
covery of radioactivity.
In agreement with du Vigneaud and collaborators
(129)j it was found that the fraction of the methyl group
of methionine excreted in respiratory CO2 increases with
the dosage, and this finding is extended to include
recovery in urinary formate. The data on the total radio
activity in respiratory C02 with the 30 p mole dose
(Table XIII) show a highly significant inhibition of
methionine, oxidatitpn in the tumor-bearing group of the
order of 27 per cent. This finding is also suggested with
the 1 p mole dose, but is probably not significant here
because of the large variation in response. Surprisingly,
the excretion of radioactivity in urinary formate is
highly irregular in both groups, and it is difficult to
obtain information from these data. However, pooled data
on total formate excretion (Table XIII, note 1/) show a
University df Southern -California Library
102
highly significant increase in the tumor-bearing animals,
of the order of Mf per cent, a result that would be
expected from the previous experiments on formate oxida
tion in mouse liver. It should be noted that the dose of
carrier formate administered was O.b m mole per 20 g.
body weight, much higher than in previous experiments,
and was chosen to render these tests comparable to some
experiments performed on in vivo oxidation of the methyl
group of methionine by Weinhouse and Friedmann. (171)
The uniformity of the data on formate excretion, when
r
contrasted with the variability of incorporation of
radioactivity into urinary formate (as well as the uni
form effect of tumor on incorporation of radioactivity
into respiratory C02)j suggests that there is incomplete
equilibration in the in vivo situation between the pool
of administered formate and the biosynthetic formate
arising from the methyl group of methionine. It is not
possible, therefore, to assess from this experiment the
site or sites of decreased methionine oxidation leading
to a lower recovery of radioactivity in respiratory C02
in the tumor group.
In order to obtain information on this point, the
oxidation of methyl labelled methionine was studied in
whole mouse liver cell preparations. Mackenzie (130) has
103
shown that methionine is not oxidized in liver homogenates.
Liver prisms were cut according to the procedure of
Mcllwain and Buddie (16^-j in order to obtain reasonably
homogeneous samples from the several pooled mouse livers
required to furnish sufficient material for the experi
ments. Mcllwain and Buddie report excellent corres
pondence in various metabolic indices between tissue
prisms and slices. This preparation has the further
advantage that,-like homogenates, it may be dispensed by
pipetting. Protein nitrogen was estimated in.the various
beakers to normalize the results. As' judged by this
measure, the amount of tissue in each beaker was rather
uniform. The tests were run in the presence of 20
p moles of sodium formate as. a trap to obtain a more
quantitative recovery of radioactivity. At this level
(0.01 M), only 2 to 2.5 p moles of formate would be
expected to be oxidized if whole cell preparations are
assumed to be as efficient as homogenates. The tumor
effect on formate oxidation would tend to increase some
what the recovery of radioactivity in formic acid as
compared to that obtained in preparations from normal
mice.
In a preliminary experiment shown in Table XIV, a
per cent decrease in methionine oxidation in liver
10*f
TABLE XIV
THE OXIDATION OF THE METHYL GROUP OF METHIONINE
TO FORMATE IN MOUSE LIVER PRISMS
.» One ml. prism suspension, 1 pi mole L-methionine-
specific activity 96,500 c.p.m./ p. mole and 20
p moles sodium formate were incubated in a final volume
of 3.2 ml. for thirty minutes; gas phase, oxygen; Krebs-
Ringer phosphate buffer, pH 171 mg. barium formate
carrier. Tumor was the Ehrlich ascites carcinoma.
C.p.m./mg. barium formate
Recrystallization No. Average
3 ^ 5
Total
c.p.m.
Formate pro
duced p. moles
per g.
protein N
Control
9.7
11.0 10.2 10.3 1760 0.590
10.5
12.1
11.5
11. M- 1950 0.566
.
?
Tumor
5.5
5.6 5.8 5.6 967 0.317
5.8 6.0 6.2 6.0 1030 0.327
105
prisms of Ehrlich ascites carcinoma bearing mice was
obtained. This effect was confirmed in subsequent
experiments.
The effect of methionine concentration on the rate
of oxidation is shown in Fig. 1*+ and detailed in Table XV.
The reason for the apparently linear response of activity
with methionine concentration, also obtained if the data
are plotted on a nitrogen basis, is not known, but the
data suggest that the Ks of the system lies considerably
above 5 p moles methionine per 2 ml.
The synthesis of methionine from one-carbon pre
cursors was tested with the same tissue pools used in the
previous experiment. Table XVI records the lack of a
significant tumor effect on the synthesis of methionine
from serine-S-C^, glycine-2-C^ and formate-C^1 * ' .
The rate of oxidation of methionine methyl at the
0.002 M level is shown in Fig. 15 and detailed in Table
XVII. These data, showing sustained oxidation throughout
the incubation period of the previous tests, support the
contention that the effect observed is due to a real
difference in rate and not to a decay in activity of the
liver preparation from tumor-bearing mice. Although the
data shown in Fig. 15 are approximately linear, when
calculated on a nitrogen basis, the curves show an initial
106
50
40
NORMAL
TUMOR
I 2 3
m M L-METHONINE-C1 4 ^ / BEAKER
5
Figure 1*+. The oxidation of methionine to formic
acid in liver prisms from normal and tumor-bearing mice
as a function of methionine concentration. Incubation
time, thirty minutes. Sodium formate trap, 20 pM per
beaker, 2 ml. final volume.
107
TABLE XV
THE EFFECT OF METHIONINE CONCENTRATION ON THE
OXIDATION OF THE METHYL GROUP TO FORMATE IN
LIVER PRISMS OF NORMAL AND TUMOR-BEARING MICE
One ml* prism suspension, L-methionine-C-^Sio,
specific activity, 200,000 e.p.m./M mole, and 20 p. mole
sodium formate were incubated under oxygen in Krebs-
Ringer phosphate buffer, pH 7.k in a final volume of
2.0 ml.; 171 mg. barium formate carrier added. Tumor
was the Ehrlich ascites carcinoma.
Methionine
mole/
beaker
C.p.m./mg. barium formate Formate pro-
Recrystallization No. Average duce^ *?oles/
3 ^ 5 g‘ Pr° 610
1
8.5
Control
10.8 11.3 10.2 0.312
9.0
11.3 11.3
10.8 0.296
2 21.8 22.8
26.5 23.7 0.651
20.2 23.2 23.0 22.1 0.680
3 27 M 32.*+
35.5 31.7 0.880
28.8
33.1
32.1+
31. ' b 0.85*+
5 *+8.5. *+6.0 51.0 *+8.5 I.1+8
50.3 55.7
58.2
55 A 1.55
1 7.2
Tumor
7.2 7.6 7.2
0.23
, 6.9
7.0 8.0
7.3
0.20
2
13.7
12.6 15.2 13.8
0.37**
13.5 l*+.7
16.1 l*+.8
---
3
22.0 20.6
25.7
22.8
0.565
19.8 18.9 23.7
20.8
b.603
5 27.9
32.2 33.8
31.3
0.883
*+0.8 38.8 *+0.2 39.9 1.03
j
108
TABLE XVI
THE SYNTHESIS OF METHIONINE FROM ONE-CARBON PRECURSORS
IN LIVER PRISMS^/ OF NORMAL AND TUMOR-BEARING MICE
One ml. liver prisms, 10 p. moles L-methionine and
26 p . moles D,L-homocysteine thiolactone were incubated foi
thirty minutes in oxygen in Krebs-Ringer phosphate buffer,
pH 7.*+ in 2.0 ml. final volume. Two p . moles D,L-serine-
3-01*+, specific activity 235,000 c.p.m./^i mole; 1.5
/i moles glycine-2-C14-, specific activity 363,000 c.p.m./
ju moles; 2.0 p. moles formate-Cl*+, specific activity
17*4-,000 c.p.m./p mole were added as indicated. ,
D,L-methionine carrier, 150 mg., was added. Tumor was
the Ehrlich ascites carcinoma.
Prisms
C.p.m./mg. Averaee
methionine average
Recrystallization No.
if 6 7 Nos*6>7
Total
c.p.m.
Methionine
produced
p moles/g.
protein N
Serine
Control
29.5 19.5
19.8 19.6 29*40 0.272
17.2 15.0
12.5 13.7
2060 0.221
Tumor
2*4-. 5
19.8 22.2 21.0 3150
0.335
21.3 16.6 17.2 16.9 25*40 0.285
Glycine '
Control
1*4-. 3 7.,7
7.8 7.8 1170
0.075
17.0
7.3
8.0 7.6 11*40
0.075
Tumor
. 12.9
8.1
8.3 ■ • 8.2
1230 O.O87
16.6 10.1 .9.5 9.8 1*4-70 0.10*4-
Sodium formate
Control 1*4-.*4-
13.5
13.8' 13.6 20*40 0.6*4-3
11.9 11.9 11.2 11.5 1720
0.53$
Tumor 16.7
16.2
15.6 15.9
2380 0.588
9.8 7.8 10.1 8.9 1330 0.302
^Prisms pool same as that of experiment in
Table XV.
109
0.3
0.2
NORMAL
TUMOR
0.1
20
40
MINUTES
. ] , Figure 15. The rate of oxidation of methionine-
C1 H3 in liver prisms of normal and tumor-bearing mice.
b p. moles methionine, 20 p moles sodium formate per
beaker, 2.0 ml. final volume.
110
TABLE XVII
THE EFFECT OF TUMOR ON THE RATE OF OXIDATION OF THE
METHYL GROUP OF METHIONINE IN MOUSE LIVER PRISMS
One ml. liver prisms, 20 p . moles sodium formate
and b p . moles L-methionine-Cl^H^* specific activity
136,000 c.p.m./jti mole were incubated under oxygen in
Krebs-Ringer phosphate system in 2.0 ml. final volume;
171 mg. barium formate carrier added. Tumor was the
Ehrlich ascites carcinoma.
C1** Recovery of radioactive formate
lime in "protein" Barium formate ffioies/g.
(mins.) c.p.m./mg. c.p.m./mg. nrotpin I
Recrystallization No. Protein «
3 b 5
Control
20 l8.*f
m m * .
135 1^3
3.61
22.2 l¥f 1 b7 l*f2 3.82
bo 31.8 3ko 326 330
8.53
32.7 3 5b 312 306 8.28
Tumor
20 16., 78.^ 80.1
78.3 2.39
18.5 9^.9
9^.0 92.0 2.26
bO 38.6
185 171
178 5.26
3b.2
191 199 179
5.61
_ _ — lii
lag followed by a more rapid oxidation. This may be seen
in the last column of Table .XVII.
In order to obtain evidence that the liver cell
permeability was not limiting and thereby conceivably
responsible for the effect, the incorporation into total
protein was determined in this experiment. As can be
seen graphically in Fig. 16, no tumor effect was observed
in the rate of protein labelling.
112
40
>20
NORMAL
TUMOR
O io
4 0 20
MINUTES
, Figure 16. The incorporation of methionine-
radioactivity into the proteins of liver prisms of
normal and tumor-bearing mice. Methionine concentration,
h u mole per beaker. Data of Table XVII.
CHAPTER V
DISCUSSION
The mechanism of utilization of inosine as a co
oxidant in the formate oxidase system deserves mention.
Data given in Fig. 8 show that two atoms of oxygen per
mole of inosine are available for peroxidation of formate.
Inosine may undergo two oxidations while remaining at the
nucleoside level. The difficulties with this formulation
are that these reactions never have been demonstrated,
and that the riboside of uric acid is not known. A more
likely mechanism for the reaction is based on the phos-
phorolytic cleavage of inosine to hypoxanthine and
ribose-l-phosphate shown'by Kalckar (172) for a purified
liver enzyme. Inosine, according to this formulation,
would act as a hypoxanthine precursor. ' The possibility
that inosine is oxidized to xanthosine which is then
* ■
cleaved to xanthine and oxidized further is ruled out by
the findings of Friedkin (173) that xanthosine is a very
poor substrate for nucleoside phosphorylase. If a
mechanism of prior phosphorolysis of inosine followed by
oxidation is accepted, xanthine oxidase assays on the
liver homogenate become meaningful as measurement of
potential rates of hydrogen peroxide production.
11^
The experiments performed here on the mechanism of
formate peroxidation have been primarily kinetic, since
the object of the tests was to develop an assay in which
the peroxidation step per se could be demonstrated to be
the limiting step. Based primarily on earlier findings
(18), confirmed here (Table VIII), that xanthine oxidase
was not affected in the tumor-bearing situation, it was
decided to employ the endogenous purine catabolizing
enzymes for the coupled oxidation of formate. The
evidence that at certain formate concentrations the
assay measures oxidation of formate rather than formation
of hydrogen peroxide is as follows:
1. At maximal inosine and low formate concentra
tion, acetaldehyde has no effect on the reaction. This l i \
under conditions where acetaldehyde would be expected to
be oxidized by aldehyde oxidase and contribute additional
hydrogen peroxide t to the system. That the rate of hydro
gen peroxide production is not limiting is further
demonstrated by increasing the formate concentration to
obtain a higher yield of C-^Og.
2. At a fixed (maximal) inosine concentration,
the rate of reaction is a conventional enzymatic function
of formate concentration.
3. At low formate concentrations, the addition of
— — ---------------------------- . _______________
115
crystalline catalase to the incubation mixture stimulates
the rate of oxidation.
As discussed under Results, the data suggest that
the hydrogen peroxide producing systems are limiting in
the absolute sense, whereas at suitable formate and
catalase concentrations, the latter will determine the
rate of reaction within the limitations imposed by the
*
rate at which hydrogen peroxide is generated, which is
regarded as a fixed parameter of the system. A rational
basis for this interpretation is obtained by considering,
after Chance (1^3), the catalase-hydrogeh peroxide complex
as the active entity present in steady state concentra
tion. At low formate concentration, the reaction would
be expected to be a function of both formate and catalase.
Increasing the catalase concentration will increase the
rate of reaction within the limits of hydrogen peroxide
formation. Since at higher formate concentration, adding
catalase does not affect the rate of reaction, it must be
assumed that either formate tends to inhibit complex
formation, or that high formate concentrations utilize
with maximum efficiency possible to the system the total
hydrogen peroxide output. This can be restated by con
sidering the competition of hydrogen peroxide and formate
for the catalase-hydrogen peroxide complex. At low
116
formate concentrations, the presence of additional
catalase will.expedite the reaction by binding a larger
amount of hydrogen peroxide which would otherwise de
compose the complex leaving formate unreacted. At higher
formate concentrations, the complex is destroyed so
rapidly by formate that the production of hydrogen is now
limiting and the addition of catalase cannot stimulate
the reaction further.
The evidence for a tumor effect on formate per
oxidation is as follows;
i 1. Inhibition of oxidation is obtained in a range
of inosine concentration and under conditions where
formate oxidation is rate determining.
2. A systemic depression of catalase activity,
but not of xanthine oxidase, accounts for the effect in
the terms stated.
3. Addition of catalase to homogenates of livers
of tumor-bearing mice restores formate peroxidation to
normal values.
This is interpreted to mean that the potential for
formate peroxidation, in accordance with its hypothecated
relation to catalase activity, is depressed in the tumor-
bearing animal.
The failure to detect a tumor effect on the
117
excretion of the h ' j p moles dose of formic acid in the
intact mouse (Table X) complicates this interpretation.
As pointed out in the section oh Results, it is difficult
to interpret these findings since the nature of the
limiting process involved in this situation cannot be
ascertained. On the other hand, the significant increase
in formate excretion in tumor-bearing mice at the 0.*f
m mole dose (Table XIII) suggests that under these highly
unphysiological conditions, a depression in the rate of
oxidation of formate in vivo occurs.
t The data on the in vivo oxidation of the methyl
group of methionine to carbon dioxide parallel the formate
data somewhat, in that a significant tumor effect is
detected only at the higher methionine dose. As previously
discussed, it is impossible to determine from this experi
ment the site of tumor inhibition. The significant point
of this experimentfis, however, that an inhibition of the
process did occur in the tumor-bearing situation, warrant
ing further study of this process in an isolated system.
Of great interest in this experiment is the failure to
obtain evidence for the equilibration of endogenous and
administered formate. The regularity observed in C-^02
excretion as compared with the labelling of urinary
formate suggests that a large portion of biosynthetic
118
formate by-passed the exogenous pool
, . The evidence supporting a tumor-induced depression
of methionine oxidation to formate comes from the studies
of surviving liver prisms and may be summarized as
follows s
1. The effect is independent of methionine con
centration.
2. The rate of methionine oxidation is constant
in preparations of livers of both normal and tumor-bearing!
mice. The effect cannot be, therefore, ascribed to a
tumor effect on the functional integrity of the liver
prism during the course of incubation.
3. No tumor effect was obtained on the rate of
labelling of protein by methionine, a fact which suggests,
but does not prove, the absence of a tumor effect on cell
permeability.
A generalized depression of one-carbon compound
metabolism in the livers of tumor-bearing mice, at least
as methionine metabolism is concerned, is not indicated
since the biosynthesis of methionine from one-carbon
compound precursors was found to proceed at normal rates.
It is important to note that ,the latter experiments may
not have been conducted under optimal conditions for
methionine synthesis. In this connection, a preliminary
119
study of the glyeine-serine exchange in mouse liver
homogenates has shown no significant tumor effect on rate
of reaction.
The rate of methionine oxidation reported in Table
XVII differs' significantly from that shown in Table XV.
No explanation is offered beyond citing the possibility of
biological variation in the two groups. However, it is to
be noted that the magnitude of the tumor effect is
essentially the same in all the experiments performed.
The failure to saturate the oxidation mechanism
with increasing methionine concentration is in general
accord with the behavior of methionine in the in vivo
oxidation.
In interpreting these results with regard to the
whole animal, it is important to remember that quanti
tative enzyme assays do not measure the processes as they
occur physiologically, but rather the potential for these
processes to occur. Assuming the rate controlling steps
observed in vitro reflect the limiting processes which
obtain in vivo, the results may be interpreted to mean
that formate biosynthesis and oxidation are depressed in
the tumor-bearing animal. In this connection, the data of
Reid et al. (72) are of interest in that a systemic
depression of the oxidation of the amidine carbon of
120
histidine to carbon dioxide in rats bearing transplanted
hepatomas was detected* k physiological theory for
regulation of the in vivo rate limiting factor in these
reactions is proposed wherein control of the synthesis of
specific enzymes in the animal is a function of the
available substrates, following the proposals of
Mandelstam. (37) It would be expected that in the
tumor-bearing organism, with its greatly increased meta
bolic needs, more of the available methionine would be
used for synthesis and consequently less disposed of via
oxidation. If regulation of the factors involved proceeds
in the mode of inducible enzymes, as previously discussed,
the conclusion would follow that at lower steady state
concentration of substrates the pertinent enzymes would
decrease accordingly. This concept could be tested ex
perimentally by studying the factors involved in the
physiological control of the reaction rates as measured
in vitro.
The association of the catalase depression with a
tumor effect on oxidation of one-carbon compounds permits
a reinterpretation of this effect along an integrated
concept of the tumor-host relation.
CHAPTER VI
SUMMARY AND CONCLUSIONS
The peroxidation of formic acid and the oxidation
of methionine have been studied in the intact mouse and
in isolated mouse liver systems. An assay for formate
peroxidation has been developed in which catalase concen
tration has been shown to be a rate determining factor.
Studies in homogenates of normal and tumor-bearing mouse
liver have indicated a systemic depression of formate
\
oxidation by tumor. The evidence presented affords
identification of the tumor induced enzymatic lesion with
liver catalase depression.
Studies of the oxidation of methionine in vivo
have shown a decreased oxidation to carbon dioxide in the
\
tumor-bearing mouse. In whole liver cell preparations of
tumor-bearing mice, a decreased oxidation of methionine
to formic acid has been demonstrated.
It is concluded that in mice bearing transplanted
tumors an inhibition of the oxidation of the methyl group
of methionine to formate, as well as the oxidation of
formate to carbon dioxide, occurs, and that the depres
sant action of tumor on liver catalase activity bears a
relation to this process.
BIBLIOGRAPHY
BIBLIOGRAPHY
< 8
1. Brahn. B.. Sitzber., ksl. creuss. Akad. Wiss.. 20.
k-78 (191677 ” “ " --- ----- — " -------
2. Greenstein, J. P., Jenrette, W. V., and White, J.,
J. Hat *1. Cancer Inst.. 2, 283 (191 +1**^2).
3. Greenstein, J. P., and Andervont, H. B., J. Nat*1.
Cancer Inst.. 2, 3^5 (19^1^2).
b. Greenstein, J. P., Andervont, H. B., and Thompson,
J- J- Nat *1. Cancer Inst.. 2, 589 (19*H-te).
5. Greenstein, J. P., and Andervont, H. B. J. Nat*1.
Cancer Inst.. }+, 283 (19t +3-l +L 0 •
6. Greenstein, J. P., J. Nat*1. Cancer Inst.. 3» 397
(19^2-^).
7. Greenstein, J. P., Jenrette, W. V., and White, J.,
J. Nat*1. Cancer Inst., 1, 687 (19I +0“1 +1).
8. Greenstein, J. P., Jenrette, ¥. V., and White, J.,
J. Nat1!. Cancer Inst.. 2, 17 (19l H-1 +2).
9. Miller, L. L., J. Biol. Chem., 122, 113 (19H-8).
I
10. Appleman, D., Skavinski. E. R., and Stein, A. M., 1
Cancer Res.. 10. *+90 (1950).
11. Dounce, A. L., and Shanewise, R. P., Cancer Res.. 10.
103 (1950).
12. Weil-Malherbe, H., and Shade, R., Bio chem. J., M-3.
118 (19N-8). “ |
13. Appleman, D., Skavinski, E. R., and Stein, A. M.,
Cancer Res.. 11, 926 (1951). i
l1 *. Stein, A. M., Skavinski, E. R., Appleman, D., and j
Shugarman, P. M., Am. J. Physiol.. 167. 581 (195l)i
15. Klatt, 0. A., and Taylor, A.. Cancer Res.. 11. 76b J
(1951). !
16. Skavinski. E. R.. and Stein. A. M.. Cancer Res.. 11.
768 (1951).
12*+
17. Begg, R. ¥., and Dickinson. T. E., Cancer Res.. 11,
3*+l (1951).
18. Greenstein, J. P., The Biochemistry of Cancer, The
Academic Press, New York (195*0»
19. Lncke1, B., and Berwick, M., J. Nat fl. Cancer Inst..
15, 99 (195*+) -
20. Price, V. E., and Greenfield, R. E., J. Biol. Chem..
202, 363 (195*0.
21. Nakahara, ¥., and Fukuoka, F., Gann. *+0, *+5 (19*+9);
1+1, >7 (1950).
22. Greenfield, R. E., and Meister, A., J. Nat*1. Cancer
Inst.. 11. 997 (1951).
I 23. Adams, D. H., Brit. J. Cancer. 5. 115 (1951).
I 2*+. Hargreaves, H. B., and Deutsch, H. F., Cancer Res..
I 12, 720 (1951).
j
I 25. Day, E. G., Gabrielson, F. C., and Lipkind, J. C.,
J. Nat11. Cancer Inst.. 15, 239 (195*+).
26. Endo, H., Sugimura, T., and Ono, T., Gann. *+6. 51
(1955).
27. Ceriotti, G., and Spandrio, L., Tumori. 1+1, 538
(1955).
28. Knox, ¥. E., in Raeker, E., Cellular Metabolism and
j Infections. Academic Press, Inc., New York (195*0 •
| 29. Dietrich, L. S., J. Biol. Chem.. 211. 79 (195*0-
I '
j 30. Sheffner, A. L., and Bergeim, 0., J. Nutrition. 50.
j 1*+1 (1953).
i
! 31. Lardy, H. A., and Feldott, G., J. Biol. Chem.. 186.
| 85 (1950).
! 32. Rector, F. C., Jr., Seldin, D. ¥., and Copenhaver,
j J. H., J. Clin. Invest.. 3l+, 20 (1955).
33. Knox, ¥. E., and Mehler, A. H., Science. 113. 237
237 (1951).
125
31 *. Lee,-N. D., and Williams, R. H., Biochim. Biophys.
Acta.. 2, 698 (1952).
35. Gordon. M. W.. and Roder, M., J. Biol. Chem., 200.
855 (1953).
36. Mandelstam, J., and Yudkin, J., Biochem. J., 51, 681
(1952). “
37. Lightbody, H. D., and Kleinman,.A., J. Biol. Chem.,
122, 71 (1939).
38. Mandelstam, J., Biochem. J.,-21> 67*+ (1952).
39. Dubnoff,JJ. W., in McElroy, W. D., and Glass, B.,
The Mechanism,of. Enzyme Action. 198, The Johns
Hopkins University Press, Baltimore (1955).
kQ. Sayre, F. W., Jensen, D.., and Greenberg, D. M.,
J. Biol. Chem., 219. Ill (1956).
*+1. Drabkin, D. >L., J. Biol. Chem., J. Biol. Chem.. 182.
335 (1950).
k2. Smith, R. H., and Williams-Ashman, H. G.. .Biochim.
Biophys. Acta. 2> 295 (1951)•
*+3« Reiss,.M., and Rees,. D. S., Endocrinology. *+1. ^37
kk. Adams, J.-H., Biochem. J., %0, *f86 (1952).
*+5. Fraenkel-Conrat, H., Simpson, M. E., and Evans, H. M.
J. Biol. Chem., Ik?. 99 (19^3). . ' ,
*+6. Umbreit, W. W., and Tonhazy, N. E., Arch. Biochem.
Biophys.. 32, 96 (1951).
^•7. Umbreit, W. W., and Tonhazy, N. E., J. Biol. Chem..
191, 2*f9 (1951).
i+8. Christensen, H. N., in McElroy, W. D., and Glass, B.,
Amino Acid Symposium. New York (1955).
*+9. Winnick, T., Friedberg, F.. and Greenberg, D. M.,
I- Biol. Chem.. 173. 189 (19*+8).
50. Bennett, L. L., Jr., Skipper, H. E., Stock, C. C..
and Rhoads, C. P., Cancer Res.. 15. ^85 (1955).
126
51.. Bennett, L. L., Jr., Toolan, H. W., and Rhoads, C.. P.,
Cancer Res., 2, 262 (1956).
52., Fenninger, L. D., and Mider,, G. B., Adv. Cancer Res.,
2, 229 (195^).
53. Sherman, C. D., Jr., Morton, J. J., and Mider, G. B.,
Cancer Res.. 10, 37k (1950).
5^. Mider, G. B., Sherman, C. D., Jr., and. Morton, J.J.,
Cancer Res.. % 222 (19^9).
55* Mider, G. B., Fenninger, L. D., Haven, F. L., and
Morton, J. J., Cancer Res., 11, 731 (1951).
56. Begg, R. W., and Dickinson, T. E., Cancer Res., 11.
k09 (1951).
57. Stewart, A. G., and Begg, R. W., Cancer Res.. 13. 556
(1953). .
58. LePage, G. A., Potter, V. R., Heidelberger, C., and
Hurlbert, R. B., Cancer Res.. 12. 153 (1952).
59. Babson, A. L., and Winnick, T., Cancer Res.. I1 *. 606
(195^).
60. Greenlees, J., and LePage, G. A., Cancer Res.. 15.
256 (1955).
61. Norberg, E., and Greenberg, D. M., Cancer. 383
(1951).
62. Cerecedo, L. R., Reddy, D. V. N., Lombardo, M. E.,
McCarthy, P. T., and Traver, J. J., Proe. Soc.
Exp.Biol. Med.. 80, 723 (1952).
63. Sassenrath, E. N., and Greenberg, D. M., Cancer Res..
lfc, 563 (195^).
6*f. . White, J. M., Ozawa, G., Ross, G. A. L., and j
McHenry, E. W., Cancer Res., l^f, 50o (195*+). j
65. Zamecnik, P. C., Frantz, I. D., Jr., Loftfield, R. B.J
and Stephenson, M. L., J. Biol. Chem.. 175. 299
(19^-8). ■ . ~
66. Payne, A. H., Kelly, L. S., and White, M. R.,
Cancer Res., 12, 65 (1952).
127
67. Payne, A. H., Kelly, L. S., Beach, G.. and Jones,
H. B., Cancer Res.. 12. 1+26 (1952).
68; Conzelman, M. G., Jr., Mandel, H. G., and Smith,
P. K., Cancer Res., li+, 100 (195*+).
69. Anderson, E. P., Yen, C. Y., Mandel, H. G., and
Smith, P. K., J. Biol. Chem.. 218. 625 (1955).
70. Furlong, N. B., Watson, E. J. P., Davis, W. E.. and
Griffin, A. C., Cancer Res.. 15. 710 (1955).
71. Dinning, J. S., Seager, L. D., Payne, L. D., and
Totter, J. R., Science. 116. 121 (1952).
i
72. Reid, J; C;, Landerfield, M. 0., and Simpson. J. L.,
J. Nat1!. Cancer Inst.. 12, 929 (1951-52). j
“* I
73. Pohl, J., Arch, f. exp. Path, u. Pharmakol.. 38. 65
(1897). ~ i
i
i 7*+. Dakin, H. D., and Wakeman, A. J., J. Biol. Chem.. 2, !
| 329 (1911) j
j 75* McGuigan, H., J. Am. Mefi* Ass»n.. ^2, 981+ (191*+). !
i i
i 76. Benedict, E. M., and Harrop, G. A., J. Biol. Chem.. !
| ¥f3 (1922). " I
l _ ;
j 77. Abbott, L. DeF., Jr., and Lewis, H. B., J. Biol. Chem. . i
I m , *+79 (1939). " j
78. Handler, P., Bernheim, M. L. C., and Klein, J. R.,
J. Biol. Chem.. 1^8, 211 (19^1).
i “ " "
! 79. Shemin, D., J. Biol. Chem.. 162. 297 (19*+6).
| 80. Du Vigneaud, V., Chandler, J. P., Moyer, A. W., and
I Keppel, D. M., J. Biol. Chem.. 131. 57 (1939).
J
! 81. Bennett, M. A., and Toennies, G., J. Biol. Chem..
163. 235 (19H-6).
82. Gillis, M. B., and Norris, L. C., J. Biol. Chem..
179. 81+7 (19^9).
83. Stekol, J. A., and Weiss, K., J. Biol. Chem.. 186.
3*»3 (1950).
81+. Du Vigneaud, V., Ressler, C., and Rachele, J. R.,
Science. 112, 267 (1950). , ______________
128
85. Sakami, ¥., J. Biol. Chem.. 176. 995 (19^8).
86. Sakami, W., J. Biol. Chem.. 178. 519 (19^-9).
87. Siekevitz, P., and Greenberg. D.-M.. J. Biol. Chem.,
180, ----
88. Sakami, W., J. Biol. Chem.. 179. **95 (19^9). j
!
89. Sauberlich, H. E., and Baumann, C. A.. J. Biol. 1
Chem.. 176. 165 (19^8). “ j
90. Sauberlich, H. E., J. Biol. Chem.. 181,. **76 (19^9). |
91. Bardos, T. J., Bond, T. J., Humphries, J., and !
Shive, ¥.., J. Am. Chem. Soc., 71. 3852 (19**9) • |
| 92. Bond, T. J., Bardos, T. J., Sibley, M., and Shive, ¥.,'
I J. Am. Chem. Soc.. Zl, 3852 (19^9).
■ 93* Shive, W., Ackerman, ¥, ¥., Gordon, M., Getziedander, >
1 M. E., and Eakin, R. E., J. Am. Chem. Soc.. 69.
j 725 (19^7).
I 9h. ¥ooley, D. ¥., and Pringle, R. B., J. Am. Chem. Soc.. |
j Zi, 63^ (1950). |
J 95. Brockman, J. A., Roth, B., Broquist, H. P., j
! Hultquist, M. E., Smith, J. M., Fahrenbach, M. J.*
1 Cosulich, D. B., Parker, R. P., Stokstad, E. L. R.,
and Jukes, T. H., J. Am. Chem. Soc., 72, **325 i
(1950). “ |
i
i 96. Keresztesy, J. C., and Silverman, M., J. Am. Chem. !
: Soc., Z3, 5510 (1951).
i 97. Flynn, E. H., Bond, T. J., Bardos, T. J., and Shive, j
i ¥., J. Am. Chem. Soc.. Z3, 1979 (1951). !
i
; 98. Pohland, A., Flynn, E. H., Jones, R. G., and Shive, 1
j ¥., J. Am. Chem. Soc.. Z3» 327^ (1951).
j :
i 99. Cosulich, D. B., Roth, B., Smith, J. M., Jr.,
! Hultquist, M. E., and Parker, R. P., J. Am. Chem.j
! Soc., Z|f, 3252 (1952). “ |
! i
1100. Plaut, G..¥. E., Betheil, J. J., and Lardy, H. A., J
| Biol. Chem.. 18**. 795 (1950). j
1
1101. Elwyn, D., and Sprinson, D. B., J. Biol. Chem.. 18J+, !
i __________ kZ5_(1950)_.________ I_____ . *
102.
103.
10*f.
105.
106.
107.
108.
109.
110.
111.
112.
113.
lib.
115.
116.
117.
129
Nichol, C. A., and Welch, A. D., Proc. Soc. Exp.
Biol. Med.. Zit, 52 (1950).
Stekol, J. A., Weiss, S., and Weiss, K. W., Arch.
Biochem. Biophys.. 3o. 5 (1952).
Sakami, W., and Welch, A. D., J. Biol. Chem.. 187.
379 (1950).
Du Vigneaud, V., Verly, W. G. L., Wilson, J. E.,
Rachele, J. R., Ressler, C., and Kenney, J. M., j
J* Am* Chem. Soc.. Zl» 2782 (1951). j
. ~ i
Ressler, C., Rachele, J. R., and du Vigneaud, V.,
J. Biol. Chem.. 197. 1 (1952).
Berg, P., J. Biol. Chem.. 1^0, 31 (195D
Mitoma, C., Thesis, University of California,
Berkeley (1951).
Berg, P., J. Biol. Chem.. 205, 1^5 (1953). !
I
Elwyn, D., Weissbach, A., and Sprinson, D. B., ;
J- M* Chem. Soc.. Z2> 5509.(1951).
Kakao, A., and Greenberg, D. M., J. Am. Chem. Soc..
ZZ, 6715 (1955).
Arnstein, H. R. V., and Neuberger, A., Biochem. J.,
ii, 259 (1953).
Cantoni, G. L., J. Am. Chem. Soc.. Zl±» 29*+2 (1952).
j
Cantoni, G. L., and Vignos, P. J., Jr., J. Biol.
Chem.. 209. 6»+7 (195*+).
Cantoni, G. L., and Scarano, E., J. Am. Chem. Soc..
Zl, W (195^).
1
Dubnoff,, J. W., in McElroy, W. D., and Glass, B.,
Symposium on Phosphorus Metabolism. Vol. II,
151, The Johns Hopkins University Press,
Baltimore (1952).
Smith, R. L., and Schlenk, F., Arch. Biochem.
Biophys.. 38, 167 (1952); Smith, R. L.,
Anderson, E. E., Jr., Overland, R. N., and
Schlenk, F., Arch. Biochem. Biophys., |+2, 72
(1953); Schlenk, F., and Smith, R. L., J. Biol.
Chem. . 20**, 27 (1953)._________________ “________
130
118. Schwartz, M., and Shapiro, S. K., J. Bacteriol.. 67.
98 (19W-.
119- Weygand, F-, Junk, R., and Leber, D., Zt^schr. f.
phvsiol. Chem.. 291. 191 (1952).
120. Canellakis, E. S., and Tarver. H..Arch. Biochem.
Biophys.. k2, 387 (1953).
121. Canellakis, E. S., and Tarver, E. H., Arch. Biochem.
Biophys.. j+2, kb6 (1953).
122. Challenger, F., and Hayward, B. J., Chemistry and
Industry. 729 (195*+) •
123. McRorie, R. A., Sutherland, G. L., Lewis, M. S.,
Barton, A. D., Glazener, M. R., and Shive, ¥., j
J. Am. Chem. Soc.. 76. 115 (195*+) • |
12^. Shapiro, S. K., Biochim. Biophys. Acta. 18. 13^
| (1955). |
i 125. Schlenk, F., and DePalma, R. E., Arch. Biochem. i
j Biophys.. 57. 266 (1955). j
I \
I 126. McRorie, R. A., Glazener, M. R., Skinner, C. G., and!
| Shive, ¥., J. Biol. Chem.. 211. ^89 (195^). j
! ' ' i
127. Ling, K.-H., and Tung, T.-C., J. Biol. Chem.. 17^.
6^3 (19^-8).
| 128. Mackenzie, C. G., Chandler, J. P., Keller, E. B.,
Rachele, J. R., Cross, N., and du,Vigneaud, V.,
J. Biol. Chem.. 180. 99 (19^9)•
129. Mackenzie, C. G., Rachele, J. R.,Cross, H.,
Chandler, J. P., and du Vigneaud, V., J. Biol.
Chem.. 183. 617 (1950).
130. Mackenzie, C. G., J. Biol. Chem.. 186. 351 (1950).
131. Siekevitz, P., and Greenberg, B. M., J. Biol. Chem.,
186. 275 (1950).
132. Mitoma, C., and Greenberg, D. M., J. Biol. Chem..
128, 599 (1952).
133. Mackenzie, C. G., and du Vigneaud, V., J. Biol.
Chem.. 195. *+87 (1952).
131
13*+. Horner, W. H., and Mackenzie, C. G., J. Biol. Chem..
187. 15 (1950).
135. Mackenzie, C. G., Johnston, J. M., and Frisell,
W. R., J. Biol. Chem.. 203. 7^3 (1953).
136. Muntz, J. A., J. Biol. Chem.. 182, *+89 (1950).
137♦ Mackenzie, C. G., in McElroy, W. D., and Glass, B.,
Symposium on Amino Acid Metabolism. 68^-,
The Johns Hopkins University Press, Baltimore
(1955).
138. Knox, W. E.,,and Mehler, A. H., J. Biol. Chem..
187. bl9 (1950).
139. Mehler, A. H., and Knox, W. E., J. Biol. Chem..
187. >*31.(1950). I
1^*0. Tabor, H., Mehler, A. H., Hayaishi, 0., and White,
J.. J. Biol. Chem.. 196. 121 (1951).
1*+1. Strittmatter, P., and Ball, E. G., J. Biol. Chem..
213. (1955).
lb2. Chance, B., J. Biol. Chem.. ISO. 9^7 (19^9); 182.
6b9 (195§).
1^3. Chance, B., in McElroy, W. D., and Glass, B.,
The Mechanism of Enzyme Action. 399, The Johns
Hopkins University Press, Baltimore (1951 *).
I lM+. Keilin, D., and Hartree, E. F., Proc. Roy. Soc.. B,
j 11^, 1^1 (1936); Biochem. J., 32, 293 (19^5).“
! 1^5. Heppel, L. A., and Porterfield, V. T., J. Biol,
j Chem.. 178. 5*+9 (19^9).
| 1^-6. Keilin, D., and Hartree, E. F., Biochem. J., 60,
| 310 (1955).
1^7. Laser, H., Biochem. J., 61, 122 (1955).
1**8. Weinhouse, S., and Friedmann, B., J. Biol. Chem..
121, 707 (1951).
l*+9» Mathews, M. B., and Vennesland, B., J. Biol. Chem..
186. 667 ( 1950) .
150. Kruhtfffer, P., Biochem. J., |±8, 601 * (1951).
151.
152.
153.
15*+.
155.
! 156.
j
| 157.
j 158.
| 159.
| 160.
!
| 161.
| 162.
i
I 163.
i
i
| I 6 * f .
I
| 165.
i
i
; 166.
!
5
(
I 167.
i
132
Nakada, H. I., and Weinhouse, S., Arch. Biochem.
Biophys.. *fr2. 257 (1953).
Rappoport, D. A., Oro, J., and Collins, V. P.,
Fed. Proc.. i>, 361 (1955).
Friedmann, B., Nakada, H. I., and Weinhouse, S.,
J. Biol. Chem.. 210. *fl3 (195*0-
Kolthoff, I. M., and Sandell, E. B., Textbook of
Quantitative Inorganic Analysis, The Macmillan
Co", "Ne'w ’ York'TITO':------- I
I
Pirie, N. W., Biochem. J., j+0, 100 (19*+6). j
Grant, W. M., Ind. Eng. Chem.. Analyt. Ed., 19. 206 j
(19*00. 1
i
Katz. J.. Abraham. S.. and Baker. N.. Analvt. Chem.. ;
26, 1503 (195*0. ” |
Fleury, P. F., Perles, R., and Le Dizet, L., Ann. j
Pharm. Franc., 11, 581 (1953)5 Chem. Abst.7~V8.
2523c (l9Ff7.
1
Weiss, S., Thesis, Univ. Southern California (195*0. |
Bassham, J. A., Benson, A. A., Kay, L. D., Harris,
A. Z., Wilson, A. T., and Calvin, M., J. Am.
Chem. Soc.. £6, 1760 (195*0.
Appleman, D., Analyt. Chem.. 23. 1672 (1951)•
Price, R. E., and Greenfield, V. E., J. Biol. Chem..
209. 355 (195^)-
Litwack, G., Bothwell, W., Williams, J. N., Jr., and|
Elvehjem, C. A., J. Biol. Chem.. 200. 303 (195*0 \
Mcllwain, H., and Buddie, H. L., Biochem. J., ,53,
k-12 (1953) .
Umbreit, W. W., Burris, R. H., and Stuaffer, J. F., j
Manometric Techniques and Tissue Metabolism, I
119, Burgess Publishing Co., Minneapolis (19**9) • j
Fujita, A.j and Numata, I., Biochem. Ztschr.. 299. j
2*1-9 (1938). v ;
Shack, J., J. Nat*1. Cancer Inst.. 3, 389 (19l +2-1 +3).'
168.
169.
170.
171.
172.
173.
133
Lineweaver, H., and Burk, D., J. Am. Chem. Soc.,
51, 658 (193^)•
Gordon, A. H., Green, D. E., and Subrahmanyan, V.,
Biochem. J., 76*+ (19^0).
Dixon, M., and Thurlow, S., Biochem. J., 18, 926
( 1921+).
Weinhouse, S., and Friedmann, B., J. Biol. Chem..
121, 733 (1952).
Kalckar, H. M., J. Biol. Chem.. 158. 723 (19^5).
Friedkin, M., J. Am. Chem. Soc.. £l±» (1952).

Asset Metadata

Creator Stein, Abraham Morton (author)

Core Title The effect of neoplastic tissue on the metabolism of formic acid by the host

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-604307

Unique identifier UC11354707

Identifier DP21578.pdf (filename),usctheses-c17-604307 (legacy record id)

Legacy Identifier DP21578.pdf

Dmrecord 604307

Document Type Dissertation

Rights Stein, Abraham Morton

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA