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Carbohydrate metabolism in thiamine deficiency

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CARBOHYDRATE METABOLISM IN THIAMINE DEFICIENCY
A Dissertation
Presented to
the Department of Biochemistry
School of Medicine
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
by
Harold A. Harper
April 194-1
UMI Number: DP21532
All rights reserved
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D isssM ort Ptibl Nag
UMI DP21532
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^
This dissertation, w ri tt en by
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on S tudies, a n d a p p r o v e d by a l l its m em bers, has
been p resen ted to a n d accepted by the C o u n c il
on G ra d u a te S tu d y a n d R esearch, in p a r t i a l f u l ­
f i l l m e n t o f r e q u ire m e n ts f o r ‘the degree o f
D O C T O R O F P H IL O S O P H Y
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Com m ittee on Studies
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TABLE OF CONTENTS
PAGE
INTRODUCTION . ..........
1
REVIEW OF THE LITERATURE ............................
5
Absorption ........................................
5
Liver G l y c o g e n .......................
6
Blood S u g a r ..................................
12
L a c t a t e ........................................ .
Pyruvate and Related Ketone Derivatives
14-
.........
16
E X P E R I M E N T A L ..................................
PROCEDURE AND RESULTS
21
............................
Adequacy of the Depletion D i e t ...............
Excretion of Pyruvate
22
............................
The Bisulfite Binding Power (B.B.S.) of Allantoin
Procedure for Determination of Urinary Pyruvate
21
25
26
.
29
The Absorption of Glucose from the Intestine . . .
56
....................................
58
Procedure
Glycogenesis, Glyeogenolysis and Blood Sugar
L e v e l s ....................................
68
P r o c e d u r e ...........................
68
Glycogenesis After Lactate or Pyruvate ...........
DISCUSSION .........
. . . . . . .
SUMMARY AND CONCLUSIONS
82
.................
91
............................
103
BIBLIOGRAPHY ........................................
105
INTRODUCTION
A scientific concept of deficiencies in the diet was
probably first noted by Lunin (1881) of B u n g e ’s school at
Basle.
The term ’’accessory food factor” was introduced by
Hopkins in England about 1906 and the term ’’vitamine” was
coined by Funk in his series of papers on the etiology of
beriberi published during 1911 from the Lister Institute in
London.
Vitamin B^ or thiamine is therefore unique since
the vitamin theory was conceived and developed from studies
of the deficiency attendant upon the lack of this factor.
Even as early as 1897, important contributions were
made by a group of Dutch medical officers stationed in the
Dutch East Indies - notably Eijkman, who noted that the dis­
ease could be produced experimentally in fowls, and Grijns
who opposed the toxin or infection theory and proposed a
nutritional deficiency as the cause of the syndrome.
In the course of his classical studies on the etiology
of beriberi, Funk published a series of papers dealing with
the isolation of a substance active in the cure of beriberi
from rice polishings.
In 1912 he published an article en­
titled "The Etiology of the Deficiency Diseases”, in the
course of which the facts concerning beriberi, scurvy, rick­
ets and pellagra were reviewed.
He postulated that other
vitamins similar to his anti-beriberi vitamine must exist
and when discovered, would prove capable of curing these
deficiency diseases.
These ideas were also found in his
2
book, MDie Vitamine” published in Germany in 1914-•
Then followed a period of investigation which revealed
the existence of an increasing number of vitamins.
The de­
lineation of the many factors associated with the vitamin B
complex originally thought to consist of a single entity,
has been particularly difficult.
The end of this problem
is, even today, not yet in view.
Much of the early work in vitaminology was devoted to
a demonstration of the characteristics of the deficiency
states, methods of biological assay and the determination of
the natural sources of the factor.
But until the chemical
structure of these substances became known, one could work
only with natural concentrates whose purity was speculative.
Multiple deficiencies in the diet, especially those associated
with the members of the B complex, makes interpretation of the
result of such work somewhat difficult.
Gradually, the period of cataloguing new vitamins
yielded to a period of analysis and synthesis.
This permit­
ted a more precise investigation of the physiology of these
various factors.
Thus the history of vitamin research
divides itself into these distinct phases and even today the
discovery of new factors is characterized by such a gradual
process.
The relationship of vitamin B^ to carbohydrate meta­
bolism was one of the earliest physiological functions of a
vitamin to be observed.
Funk (1914) noted that polyneuritis
in pigeons was more rapidly produced the greater the amount
3
of carbohydrate ingested by the birds, a fact which was con­
firmed by Braddon and Cooper in 1914*
This led Funk and v.
Schonborn (1914) to investigate the blood sugar and glycogen
content of the liver in pigeons fed on different vitaminfree diets.
They concluded that the sugar of the blood
greatly increased and the glycogen diminished especially on
diets presumably free of vitamin B.
These fundamental observations initiated extensive in­
vestigation of the problem which studies have continued unin­
terruptedly to the present time.
The evidence is contradic­
tory although the majority of it favors the suggestion that
in avitaminosis B-j_ there is a disturbance in glycogen storage,
hyperglycemia and associated errors in carbohydrate metabolism.
A study of previous work on the problem indicates that
the majority of the work is not comparable.
In the earlier
days, before 1932, the crystalline B factors were unavailable.
In fact the existence of most of them was unknown.
Hence
the basal diets used for the production of the symptoms were
inadequate in more than one respect.
Many workers merely
permitted the various experimental animals to take food ad
libitum.
As the deficiency of the vitamin became more acute,
a severe anorexia supervened leading to simple starvation.
Hence the observed symptoms were undoubtedly due to inanition.
Multiple, unrecognized deficiencies often reduced an experi­
mental animal to a moribund condition at which time certainly
no valid conclusions could be drawn.
The use of the pigeon
in many of the earlier experiments and the rat in later work
introduced a species difference which is now recognized as a
major point of conflict in a comparison of experimental results.
The rapid progress in the elucidation of the nature of
certain of the factors of the vitamin B complex together with
their synthesis and availability in crystalline form has made
it possible to prepare a diet which is deficient in thiamine
only.
It is thus only recently that one can avoid the possi­
bility that symptoms observed in animals reared on this diet
are to be ascribed to other than the lack of one dietary ele­
ment.
The present investigation was designed to study cer­
tain classical aspects of carbohydrate metabolism in animals
suffering from inadequate intakes of thiamine.
To avoid the
effect of gross inanition or of a moribund state, sub-optimal
amounts of thiamine were administered to maintain a sub-acute
deficiency syndrome characterized by a relatively constant
weight and food intake but a pronounced disturbance in inter­
mediary carbohydrate metabolism as evidenced by studies of
the urinary pyruvate.
Under these conditions, studies of the rate of absorp­
tion of glucose from the intestine and the rate and extent of
glycogenesis and glycogenolysis correlated with the blood
sugar level were carried out.
In addition, a study of the
ability of the avitaminotic organism to convert ingested lac­
tate and pyruvate into glycogen, was undertaken.
These meta­
bolites are of particular interest since they are character­
istically concerned with avitaminosis Bj.
For reasons to be
5
amplified, the relationship of the urinary pyruvate to the
degree of thiamine deficiency was also studied preliminary
to the work on other aspects of carbohydrate metabolism.
REVIEW OF THE LITERATURE
Absorption
A diminished function of the alimentary tract has long
been suggested in connection with B avitaminosis.
Osborne
and Mendel (1917) first reported that the food consumption of
rats was directly dependent on the amount of vitamin B in the
food.
Cowgill, Deuel and Smith (1925), in the third of a
series of papers on the quantitative aspects of the relations
between vitamin B and appetite in the dog, used as a criterion
of the vitamin B requirement the restoration of appetite in
the experimental animals.
Cowgill, Deuel, Plummer and Messer
(1926) measured gastric atony in dogs.
The concomitant anor­
exia has been attributed to this gastric or intestinal atony
(Harris, Clay, Hargreaves and Ward, 1933).
Gal (1930) has reported the absorption of glucose to be
only one-third of normal.
An interesting paper by Groen
(193S) deals with the absorption of carbohydrates in humans
suffering from alcoholic polyneuritis with pellagra.
The
diminished absorption was returned to normal by treatment with
yeast.
By the nature of the technic, the glucose solution
was maintained in contact with the same portion of the upper
small intestine.
The author concludes that diminished ab­
sorption was due to a defect of the absorptive capacity of the
wall and not to unusually rapid passage of the intestinal con­
tents.
It seems pertinent to point out at this juncture that
one must distinguish between deficient absorption and deficient
rate of absorption.
Westenbrink and Overbeek (1933) studied
intestinal absorption in vitamin
deficiency, thermostable
B-deficiency or B-complex deficiency and noted a reduction.
These reports are interesting in view of the various
studies on the decrease in enzymatic and other gastro-intestinal secretions observed in B deficiency.
Webster and Ar­
mour (1933) noted a marked drop in the secretion of gastric
juice in dogs on B deficient diets.
Babkin (1933) has made
a study of the effect of vitamin deficiency on the nervous
control of gastric secretion.
In avitaminosis B there was
found a definite impairment in the usual secretion after
sham feeding, subcutaneous injection of histamine, or intro­
duction of food or alcohol into the small intestine.
When
yeast was administered the response was normalized.
Cow­
gill and Gilman (1934) have reported a diminution of gastric
secretion in dogs deprived of vitamin B, although in an ear­
lier paper (Cowgill, Deuel and Smith, 1925) the restoration
of the appetite of a vitamin B deficient dog produced by the
administration of a vitamin B concentrate was shown not to be
due to a stimulation of the flow of gastric juice.
Studies
of the effect of thiamine on the intestinal motility in vitro
have not indicated any specific effect on peristalsis.
This
is not in harmony with experimental and clinical observations
in vivo.
Liver Glycogen
Indications of derangement in various aspects of carbo­
hydrate metabolism were noted by some of the earliest workers
in vitamin research.
The relationship between the carbohy­
7
drate content of the diet and the severity or rate of onset of
the polyneuritic symptoms first mentioned by Funk (1914) an<i
Braddon and Cooper (1914) was further studied by Collazo
(1923)
(l) who reported a toxic effect when carbohydrate was
introduced into the crop of an avitaminotic bird.
This toxi­
city. was attributed to the accumulation of intermediary pro­
ducts of the metabolism of sugars.
This same worker (Collazo,
1923) (2) performed experiments on pigeons, chickens, and dogs
wherein there was a lowering of the liver glycogen in avita­
minotic as well as starved animals.
The results have been
criticized on the ground that forced feeding was not resorted
to and hence inanition might have produced the phenomena ob­
served.
Rubino and Collazo (1923) reported studies in glyco-
genesis on avitaminotic pigeons compared with normal birds.
When glucose was administered and observations were made after
2 , 4 and 6 hours, the authors concluded that the laying down
of glycogen and utilization of the sugar was not impaired al­
though an inspection of their data indicates markedly lowered
glycogen values as depletion proceeds.
Sure and Smith (1930) showed that the glycogen content
of the livers of polyneuritic nursling rats fell to a small
fraction of normal and in 1931, Roche pointed out that in the
absence of vitamin B there was an increase in the carbon to
nitrogen ratio of the blood, partly caused by hyperglycemia
and partly by the accumulation of intermediate products of
carbohydrate metabolism.
Cowgill has repeatedly emphasized the importance of
8
guarded conclusions when dealing with specific effects of vit­
amin B deficiency.
Thus in 1932 Burrack and Cowgill agreed
that in the avitaminotic state there is a decrease in carbo­
hydrate tolerance when measured by blood sugar curves but they
maintained that this is the result of inanition and at that
time they rejected the theory that vitamin B^_ plays any speci­
fic role in carbohydrate metabolism.
Such a criticism had
been elicited previously from Header and Drummond in 1926.
A series of papers from Sure’s laboratory (Sure and
Smith, 1931, 1932; Sure, Kik, Smith and Walker, 1931; Sure,
Kik and Smith, 1932) discussed the question of a specific
relationship between vitamin B and carbohydrate metabolism
and the influence of underfeeding on the experimental observa­
tions.
Using the technic of paired feeding controls, their
conclusions support the view that vitamin B has a direct
specific effect, unrelated to food intake, on growth, lacta­
tion, water metabolism and glycogen formation.
Several pro­
vocative papers dealing with this question appeared from
AbderhaldenTs laboratory in 1932 and 1933 (Abderhalden and
Wertheimer, 1932, 1933).
In studies carried out in pigeons,
very high glycogen values were observed even at death.
In
fact an increasing glycogen content characteristically paral­
leled the progress of depletion.
Although the animals ex­
hibited symptoms of gross carbohydrate starvation the liver
presented a paradoxical contrast by its abnormally elevated
glycogen stores.
Carbohydrate appeared to evoke the convul­
sion state whereas a high fat diet tended to prevent it.
In
9
fact, the ability of a given substance to induce this syndrome
was suggested by the authors as a test for convertibility to
carbohydrate.
The administration of vitamin
not only cur­
ed the paralytic symptoms but also "reduced" (mobilized?) the
liver glycogen.
Permanently elevated glycogen values are certainly the
exception in avitaminosis B.
One wonders whether a species
difference might not explain partially these observations.
The recent work of Bobbitt and Deuel (194-0) on the rate of
glycogenolysis in the isolated livers of various animals sup­
ports this suggestion.
They reported that the glycogen of
the excised rat liver broke down very rapidly whereas the
pigeon exhibited the slowest rate of all the animals studied
nearly 75 per cent of the original being recovered after an
incubation period of 72 hours.
An experimental approach which lends Itself to better
control of various factors of the problem, is exemplified by
the work of Westenbrink (1933).
Rats, 150 to 200 gms. in
weight, were maintained for 5 to 8 weeks on normal, B^-deficient, thermostable B-deficient, or B complex-deficient diets.
After a 24 hour fast, 2 cc. of a 50 per cent glucose solution
was given per os and the animals were sacrificed 4 hours
later.
The results showed no difference between the normals
and those on the various avitaminotic diets.
Two interesting reports by Schrader (1931-1933) deal
with other aspects of the problem.
The determination of gly­
cogen and blood sugar was carried out on 200 rats.
The ani-
10
mals were either B-^-deficient or given a fuller’s earth adsorbate to supply B^.
Glucose or dl lactic acid (|r neutral­
ized with NaOH) were administered.
Starvation caused a de­
pletion of liver and body glycogen in the deficient as well,
as the supplemented group.
Both d glucose and the dl lactate
induced glycogen formation in both groups although quantita­
tive differences may exist.
The author concludes, ” ....
we can only say that the major part of the evidence thus far
obtained indicated no serious defect in the glycogen formation
or glycogen liberation stages on the vitamin B-deficient ani­
mals.
The evidence however, is not yet convincing."
The
1933 paper deals with the respiratory quotient after glycogen
or lactic acid.
Although the heat production was somewhat
lowered in the deficient animals, there was a rise in R.Q. in
both cases.
Several other observations, some of clinical
origin, are of interest.
(1935)
Vorhaus, Williams and Waterman
noted instances of increased utilization of carbohy­
drate in diabetic individuals treated with large doses of
crystalline vitamin B^.
Drill (1937) observed that liver
glycogen is no longer maintained in thyroxin-injected rats
unless increased amounts of thiamine and riboflavin are admin­
istered.
Schroeder (1937) states that the liver glycogen of
rats rises and falls with the presence or absence of vitamin
B}_ in the diet but that muscle glycogen is unaffected.
Lajos (1936) administered glucose to 4 day fasted rats and
then sacrificed the animals at intervals from 20 minutes to
240 minutes.
When the glucose was accompanied by insulin the
11
rise of liver and muscle glycogen was much more marked than
without insulin.
Vitamin
administered with glucose had
an effect similar to insulin although slower in action.
These findings have excited much comment.
Williams and
Spies (193S) criticize them on the ground that by the technic
used in the preparation most of the thiamine would be lost.
They feel that the results were due to some substance other
than thiamine.
Further work on this relationship is certain­
ly indicated especially in view of several reports of adrenal
and islet hypertrophy in thiamine deficiency (Ogata, 1920$
Bierry and Kollmann, 1928$ Wolbach, 1925).
During ether
anaesthesia the glycogen content of the liver is apt to fall.
If thiamine was administered three days previous to the anaes­
thesia this drop was decreased according to Lauber and Bersin
(1939).
Thiamine administration immediately before anaes­
thesia was ineffective.
Lipschitz, Potter and Elvehjem
(1938) have concluded that polyneuritis is a disease in which
there is a general disturbance of carbohydrate metabolism
rather than a condition that is characterized primarily by
lesions in the nervous system.
Thus it appears that the picture with respect to glyco­
gen metabolism is by no means clear.
Further work directed
towards its clarification is definitely indicated.
Williams
and Spies in their recent monograph (1938) summarize the sit­
uation very adequately when they state
There is convincing evidence of frequent disturb­
ance of glycogen storage and blood sugar In Bj avita­
minosis.
Further indications of this are found in
clinical experience.
However the direction and ex­
tent of the deviations from normal are such as to
12
make it improbable that there is a specific, uncompli­
cated and primary relationship between thiamine and
these functions.
Marked irregularities in the ex­
perimental evidence appear to be associated in part
with species differences, in part with the complicat­
ing effects of attendant inanition or subsidiary defi­
ciencies and in part with variations in the character
of damage to the central nervous system.
A dual defi­
ciency of thiamine and flavin is suggested as the most
probable cause of the disturbance.
Blood Sugar
In the course of our previous references to the litera­
ture the relation of thiamine deficiency to the blood sugar
has been mentioned.
Several other reports seem worthy of
inclusion at this point.
In 1930, Lepkovsky, Wood and Evans
felt that glucose tolerance was normal in beriberi rats un­
less they were moribund.
Bell (1931) studied the blood sugar
levels of rats and pigeons in B-^ deficiency.
There was some
species difference but no increase in the "true” sugar of the
blood in the B-^-deficient rats;
in fact there was a fall in
the terminal stages.
But Nitzescu and Benetato (1931) came to the conclusion
that glucose tolerance, as measured by the blood sugar curve,
is less in avitaminotic than in normal birds.
Other examples
of diminished glucose tolerance with hyperglycemia are to be
found in the reports of Burrack and Cowgill (1932), Lajos
(1936), Funk and v. Schonborn (1914-) > Abderhalden and Werth­
eimer (1933), Levinson (1937) and Kauffmann - Cosla, Vasilco
and Oeriu (1932).
More recently, clinical reports of similar observations
in humans are to be found.
For example Vorhaus, Williams and
13
Waterman (1935)
amin
(2) conclude that in experimental human vit­
deficiency there is
a disturbance of carbohydrate
metabolism characterized by
a rise in the blood sugar and
glycogen content of liver and muscle.
in B-^ deficiency.
This is most marked
Further they state that the clinical syn­
drome of diabetes mellitus is suggestive of a nutritional dis­
turbance and there is reason to think that a deficiency of B
may be a factor in the production and clinical course of this
condition.
Scattered observations relating to endocrine relation­
ships support the concept of diminished sugar tolerance as
characteristic of the deficiency state.
hypertrophy have already been
mentioned.
Adrenal and islet
In fact in the very
early stages of the work onvitamin B, Funk and v. Schonborn
(1914) called attention to a possible relationship between
vitamin B, carbohydrate metabolism and the endocrines.
This
was stimulated by the observation that adrenal hypertrophy ac­
companies avian beriberi.
However the pigeons receiving
adrenalin died sooner although the time of onset of beriberi
was the same as in the controls.
According to Martin (1937)
the usual responses to insulin are lost in depancreatized
dogs maintained on a diet free of the vitamin B complex.
This, together with Lajo’s work, if confirmed, might suggest
thiamine as potentiating insulin.
McIntyre and Burke (1937)
state that vitamin B-deficient rats (no B^ and very small
amounts of the other B vitamins) showed a diminished toler­
ance to insulin.
M
Molnar and Petranyi (1939) have described a case of
trigeminal neuralgia in which the patient was unable to util­
ize injected thiamine, most of it being excreted.
However,
the administration of cortin along with the thiamine permitted
retention of most of the injected thiamine and cure of the
neuralgia was effected.
Laszt (1938) has reported that
thiamine produced its effect only in the presence of adreno­
cortical hormone and vice versa.
Lactate
The occurrence in the body fluids of the thiamine defi­
cient animal of abnormal quantities of various metabolites
lends further support to the relationship between vitamin Bj_
and carbohydrate metabolism.
Most observers refer to an in­
crease of lactate and/or pyruvate but a few have reported the
occurrence of the aldehyde of pyruvic acid, methyl glyoxal,
and there is one reference to the isolation of alpha ketoglutaric acid.
Since 1930 more attention has been focused on these
factors.
Hayasaka (1930) in a series of papers on his obser­
vations of lactic acid metabolism in beriberi pointed out that
the recovery process after muscular fatigue is more prolonged
in beriberi than it is in normal individuals and the carbon
dioxide output is lowered.
Even light exercise is followed
by an elevated blood lactic acid which is prolonged, thus indi­
cating inability to resynthesize lactate to glycogen.
Kinnersley and Peters (1930) reported similar observa­
tions in the pigeons.
This was the forerunner of a series of
15
investigations by these workers which were destined to clarify
the enzymatic role of thiamine.
In this early work they re­
ported that symptoms of incipient opisthotonus are accompanied
by an increase of lactic acid in the lower part of the brain
stem.
Such an accumulation of lactic acid in the heart was
suggested by Drury, Harris and Maudsley (1931) as an explana­
tion of the bradycardia which they reported as occurring in
thiamine deficient rats.
In 1932 Kauffmann - Cosla, Vasilc.o
and Oeriu confirmed this increase in blood lactic acid in
avitaminotic dogs indicating an increased excretion of the
metabolite as well.
Many in vitro studies of cellular metabolism in vit­
amin B deficiency have emanated from Peters’ laboratory.
In
1932 (Meikeljohn, Passmore and Peters) it was reported that
the brain of avitaminotic pigeons exhibited a diminished oxy­
gen uptake when minced and mixed with lactic acid, in compari­
son to normal brain tissues.
The respiration could be im­
proved by the addition of vitamin B-^ concentrates.
This fact
was later made the basis of the so-called catatorulin test for
vitamin B-^.
But in 1933 Meikeljohn showed that the actual
amount of lactate removed during the oxidation is not influ­
enced by the presence of the vitamin since the avitaminotic
brain itself, without any added vitamin B-^, can already re­
move the lactate in vitro.
Harris in his review of the water-soluble vitamins
published in the Annual Review of Biochemistry for 1933, ad­
vanced the suggestion that the vitamin is somehow involved in
16
an enzyme system, possibly the coenzyme for the lactic acid
dehydrogenase of Banga and Szent-Gy5rgyi.
He again points
out the occurrence of bradycardia in avitaminosis
and the
relationship of this to an increased concentration of lactic
acid accompanied by improvement in the heart rate following
its removal.
In 1934 Thompson showed that the kidney behaves
similarly to brain in exhibiting an impaired oxygen uptake
when lactate is added to the avitaminotic tissue and according
to Sinclair (1933), the low respiratory quotient of brain tis­
sue from a deficient pigeon is raised on the addition of vit­
amin Bj_.
Pyruvate and Related Ketone Derivatives
Shortly after the observations on lactate were recorded,
reports of a similar increase in pyruvate were forthcoming.
Peters and Thompson (1934) stated.that pyruvic acid is formed
by the brain of a deficient pigeon but not by normal birds
when the tissue respires in lactate solution.
When vitamin
B^ was added this acid largely disappeared.
Other workers had approached the problem by a study of
the so-called bisulfite binding substances (B.B.S.) in the body
fluids.
In beriberi it was frequently observed that the
ability of the plasma to bind sodium bisulfite was markedly in­
creased.
From the. chemical viewpoint this indicates the pre­
sence of substances possessing a free carbonyl group.
For
clinical purposes the bisulfite-binding poxver was assumed to
represent mainly pyruvic acid.
Thompson and Johnson (1935)
reported abnormally large amounts of bisulfite-binding sub­
17
stances in the blood of pigeons and rats when the animals sub­
sisted on a vitamin Bj-free diet.
That the accumulation of
pyruvic acid (which was assumed to represent the B.B.S.) was
specifically related to the nutritional deficiency was proved
by a decrease in this metabolite following the administration
of vitamin B^.
Similar investigations have been extended to human beri­
beri.
Thus Platt (1938) reported a rise in the pyruvate con­
tent of blood, urine and spinal fluid in these cases.
An
intravenous injection of 5 mg. of thiamine restored the values
to normal in 10 to 15 hours.
Shindo (1938) actually isolated
the pyruvate from the blood as the 2 , 4. dinitrophenylhydrazine,
obtaining 2 mg. of the hydrazone from 150 gm. of blood taken
from a beriberi patient.
A specific colorimetric procedure for determining small
amounts of pyruvate was developed by Lu (1939)
her (1939)
(l) and used by
(2) to show that in thiamine-deficient rats, the
concentration of blood pyruvate was increased as bradycardia
developed.
Lu and Platt (1939) studied the effect of light
muscular exercise on thiamine-deficient humans.
High values
for blood pyruvate were found in both normal and deficient
subjects but as had previously been noted for lactate (Hayasaka, 1930), while one-half hour sufficed for recovery in nor­
mal subjects, the period was much prolonged in beriberi cases.
Bollmann and Flock (1939) reported very similar results in
studies of the pyruvate content of working muscles in normal
and thiamine-deficient rats.
Guzman-Barron and Lyman (1940)
18
made an in vitro study of the ability of kidney slices to syn­
thesize carbohydrate in the presence of pyruvate.
Tissues
from B^-deficient rats were quite unable to accomplish this
transformation when compared to normal controls.
Similar
experiments had been undertaken by Lipschitz, Potter and
Elvehjem (1938).
Liver and kidney tissue from both fasting
and B-l-deficient birds showed a diminished capacity to remove
pyruvate.
The brain tissue was also incapable of removing
pyruvate when deficient in vitamin
alone.
but not from inanition
When glucose was administered to polyneuritic birds,
the ability of the liver tissue to remove pyruvate was res­
tored.
From these experiments, the authors conclude that
polyneuritis is not primarily a disease of the central nervous
system but rather is characterized by a general disturbance of
carbohydrate metabolism.
The literature contains several references to the oc­
currence of methyl glyoxal in vitamin B^ deficiency.
Geiger
and Rosenberg (1933) mentioned its presence in the urine of
polyneuritic dogs and rats and in the urine and cerebrospinal
fluid of infants with symptoms of Bj_ deficiency.
Several
Japanese workers have also recorded its presence even record­
ing a case in which the milk of a beriberi patient was highly
toxic because of the large quantities of methyl glyoxal excreted
therein (Arakawa, 1930; Chiba, 1932; Uga, 1935).
Platt and Lu (1936) suggested a determination of the
B.B.S. in the blood and urine as a method of diagnosis of vit­
amin B]_ deficiency.
From other sources ample evidence appears
19
to suggest the use of caution in interpreting this procedure.
Wilkins, Taylor and Weiss (1937) pointed out that an increase
in the B.B.S. of the blood occurs in conditions probably unre­
lated to thiamine deficiency.
Simola (1939) stated that the
B.B.S. of blood or urine is by no means synonymous with the
pyruvic acid content.
Other keto acids in the urine included
alpha keto-glutaric acid and in 1939 Platt and Lu noted that
in beriberi the increase in B.B.S. is greater than can be ac­
counted for by pyruvic acid.
In fact B.B.S. could be reduc­
ed but slightly even though pyruvic acid was returned to nor­
mal.
Taylor, Vifeiss and Wilkins (1937) concluded from an ex­
tensive study of the factors influencing the B.B.S. of the
blood in health and disease that it is a quantitative measure
of metabolic processes involving formation and accumulation
of carbonyl compounds in the blood but that no distinction of
the various substances is possible.
The blood B.B.S. are in­
creased in diseases other than thiamine deficiency and in
diabetes there may be an attendant ketonemia.
However, the
administration of thiamine definitely lowered the blood B.B.S.
in their experiments.
Once more taking up the question under more controlled
conditions, Banerji and Harris (1939) proposed a carbohydrate
tolerance test for vitamin B-^.
By studies of the urinary
B.B.S. as rats became more deficient in the vitamin, these
authors observed a progressive rise in the excretion of these
substances which was increased by the administration of carbo­
hydrate of lactate and decreased when thiamine was administer-
20
ed.
Further studies of factors influencing the urinary B.B.S.
were reported by Shils, Day and McCollum (194-0) .
EXPERIMENTAL
PROCEDURE AND RESULTS
Animals for these experiments were selected from the
stock colony of the Department of Biochemistry.
Males were
used for all of the experiments with the exception of the
pyruvate experiments when both sexes were used.
In general
it was attempted to select animals between 120 and 160 grams
in weight although in a fdw cases these limits are extended
because of inability to secure a sufficient number of ani­
mals to constitute an experimental group.
The experimental plan involved a comparison of animals
maintained on a diet deficient in thiamine with those adequate­
ly nourished with respect to this factor.
Such normal ani­
mals were always maintained on the deficient diet supplemented
with 100 gamma thiamine, 100 gamma riboflavin and 100 gamma
pyridoxine per week^, for several days prior to the experi­
ment.
For depletion of thiamine reserves the animals were
maintained on the following diet, deficient in thiamine:
casein (alcohol extracted)
corn-starch
butter (washed)
autoclaved yeast
(Harris)
cod liver oil
salt mixture (OsborneMendel 1919)
18.0
56.0
5.0
15.0
2.0
4-0
In addition, after the 5th or 6th week when food con­
sumption diminished, each animal was fed 2 or 3 times weekly
^Crystalline products obtained from Merck and Company
22
0.1 cc. of a mixture containing 30 gamma of pyridoxine, 30
gamma of riboflavin and 10 gamma of thiamine.
This latter
supplement not only serves to maintain the appetite of the
animal, avoiding any sign of the gross inanition, which has
elicited criticism of many of the previous experiments in
thiamine deficiency, but it also produces a sub-acute state
of deficiency.
In this case one has an opportunity to ob­
serve physiological aberrations emanating from a relatively
uncomplicated syndrome, unvitiated by the multiplicity of fac­
tors attendant upon an acute, pre-mortal crisis.
Adequacy of the Depletion Diet
Recent vitamin literature abounds with the introduction
of new factors, many of the B complex, requisite for the nutri­
tion of the rat as well as other species.
It will be recalled
that earlier work on the physiology of vitamin
was compli­
cated by the lack of other factors now known to be essential.
With the availability of the more important factors in crys­
talline form one can devise a more complete diet.
But it is
not yet possible to supply a synthetic vitamin mixture which
gives assurance of adequacy.
In the diet used in these ex­
periments autoclaved yeast has been used as a source of all of
the factors of the B complex including choline, with the ex­
ception of thiamine.
This is a time-honored procedure and is
routine in biological assays for vitamin B-^.
However, to
guarantee the production of an uncomplicated deficiency state
it was decided to test the growth-promoting properties of the
diet when supplemented with thiamine.
23
Young rats, -40 to 50 grams in weight, were placed on
the diet supplemented with 30
gamma of thiamine per week for
a period of about 40 days (April 1 to May 9,
1940).
In the
main, satisfactory although retarded growth was obtained.
On May 9, these animals were divided into two groups, one
group being maintained on the diet supplemented only with 30
gamma thiamine weekly but the
members of the
other group re­
ceived 100 gamma thiamine per
week.
30 symptoms of
On May
riboflavin deficiency were observed in certain of the members
of both groups so that 100 gamma riboflavin were then added
to the thiamine supplements.
Since, in a few cases allevia­
tion of these symptoms did not occur as rapidly as desired
and some evidence of acrodynia seemed apparent, vitamin B^,
(100 gamma per week) was also made a part of the synthetic
supplements.
Thus two sets of supplements were used vary­
ing only in their thiamine content.
Under these conditions,
as the growth records indicate (Table I and Figure 1), the
rate of growth was stimulated so that only thiamine was the
limiting factor.
The adequacy of the diet so supplemented
thus seemed assured.
These experiments also indicated that
autoclaved yeast may not be a safe source of the so-called
heat stable factors of the B complex.
In ordinary assay
procedures where animals are not observed for more than 7 to
8 weeks these subsidiary symptoms may not manifest themselves
but in these experiments where it was desired to maintain the
animals in a sub-acute, thiamine-deficient state for indefin­
ite periods, these considerations are of major importance.
TABLE I
THE ADEQUACY OF THE THIAMINE-DEFICIENT DIET
Group
Animal No.
Date of
Observation
A
30 gamma
thiamine
weekly
12
13
14
15
17
19
20
21
Body Weight in Grams
5/9
5/23
1
5/30
87
100
63
90
80
73
76
68
93
76
90
85
87
&4
79
63
84
107
85
101
64
74
85
82
96
77
74
1
2
4
5
7
10
60
66
80
62
73
64
73
76
74
72
71
78
Group A
Group B
AverageiS
80
68
77
74
Group A
Weight gains
5/9 to 6/24
B
100 gamma
thiamine
weekly
Group B
104
98
102
104
93
6/3
6/7
6/14
6/24
89
79
85
85
81
85
85
85
86
86
92
96
92
98
103
92
110
105
100
105
106
104
128
78
116
114
112
118
117
130
117
85
115
112
114
99
83
100
138
131
138
118
73
122
I 64
160
165
132
802
150
183
163
174
135
105
165
84
93
120
111
142
113
154
104
33 gms.
86 gms.
-^-Added 100 gamma riboflavin at this point
^Symptoms of acrodynia.
supplement.
Added 100 gamma pyridoxine to weekly
StCE
is.
H
No. 6303, U n iv e rsity B ookstore, L os A ngeles
25
Excretion of Pyruvate
It is often difficult to make a comparative appraisal
of various experiments patterned on nutritional deficiencies
since one is hard pressed to determine the relative degrees
of deficiency which existed at the time the interpretations
were made.
Thus one can distinguish a progressive series of
symptoms in vitamin deficiency according to the presence of
mild, sub-acute or acute deficiency states and in rat beri­
beri one rarely observes typical polyneuritic convulsions un­
less the deficiency is prolonged by the presence of sub-opti­
mal amounts of thiamine in the diet.
A rapid depletion of
the vitamin reserves is always accompanied by a sudden, acute
onset of symptoms followed in a very short time by death.
It was important, therefore, to devise at least a semiquantitative criterion of the deficiency state - a criterion
which would not only be a specific indicator of the sub-acute
syndrome but in addition would be sufficiently reproducible to
permit the reduction of all experimental animals to a similarly
deficient state.
Growth curves were not suitable because of
the age of the animals used.
The increase in the pyruvate
content of the body fluids seemed a feasible method for our
purpose.
The quantitative comparison of the relationship
between thiamine deficiency as measured by the bradycardia
technic, and the blood pyruvate in beriberi rats as reported
by Lu (1939)
(2) lends validity to the use of this procedure
for our end.
However, routine tests of the blood present
some difficulty in rats due not only to inability to secure
26
large samples at frequent intervals but also as Bueding and
Wortis (194<0) have reported, blood pyruvate is not stable and
hence accurate analysis requires some modification of the pro­
cedure.
The observations of Banerji and Harris (1939) and
later, those of Shils, Day and McCollum (194-C) on the in­
crease of B.B.S. in the urine of thiamine-deficient rats,
suggested the urine as a better source for the quantitative
data.
The Bisulfite Binding Power (B.B.S.) of Allantoin
However, as has been pointed out, the determination of
the total bisulfite binding substances of the urine lacks the
specificity of a direct determination of pyruvate.
Clift
and Cook (1932) published a detailed study of this procedure
and Banerji and Harris (1939) used it in their study of the
problem, suggesting a reduction of the pH to 3, under which
conditions glucose was not bound.
Preliminary experiments
with this method indicated that the urinary B.B.S. did not
always vary in the same direction as the pyruvate.
An ex­
periment on the bisulfite-binding power of allantoin indicat­
ed that this substance also binds bisulfite.
TABLE II
BISULFITE BINDING POWER OF ALLANTOIN
50 mg. of allantoin-*- made up to 100 cc.
5 cc. samples taken for assay (2.5 mg.)
Sample No.
cc. 0.01 N Iodine to
titrate bound B.B.S.
1
2
3
^"Eastman Kodak Company
3.0
3.0
3.0
Mg. Bisulfite
bound
3 x 0.52=1.56
28
If one assumes that 1 mol. of. allantoin binds 1 mol.
of bisulfite which is liberated by alkali, according to the
following reactions:
0
0
II
NIL - C
2
OH
1 /
C — NH
/
\N-
SOoNa
NaHSO.
-> NH,
NH-CH
0
C — NH
II
/
C -NH-CH
3
\
C— 0
NH
H
0
+ NaHC03 (sat.)
Release of bound
bisulfite
0
II
0
n h 2—
II
c
C -NH
/
N H — CH
\
+ NaHS03 +• NaHC03
N- - C = 0
H
Titration of bisulfite released
NaHS03 + I2 + H 20 =
I
2 HI + NaHSO^
= NaHSO^/2
1 cc 0.01 N iodine « 0.52 mg. NaHS03
Molar weight allantoin = 1 5 8
Molar weight NaHS03 = 104
158 x 1.56/104 = 2.37 mg. allantoin
2.50 mg. allantoin (Theoretical)
95*0 per cent recovery
then one obtains good agreement with the amount of allantoin
29
theoretically present.
This fact assumes that but one car­
bonyl group is able to bind bisulfite, possibly as illustrated
in the above reactions.
Since allantoin presumably follows a metabolic pathway
similar to that of uric acid in man, one would predicate
changes in this fraction arising from increased endogenous
protein catabolism.
The latter stages of thiamine deficiency
might well contribute to an increased allantoin output by such
mechanisms.
Thus the determination of the urinary B.B.S. in­
troduces the possibility of erroneous interpretation.
Very
recently, Shils^ has utilized certain adsorbing agents by
which he expects to eliminate these interfering factors and
thus account for most of the B.B.S. as pyruvic acid.
In the absence of suitable assurance as to the specifi­
city of the B.B.S. procedure a direct determination of the
urinary pyruvate was decided upon.
This proved to be a much
more sensitive method of assaying the deficiency state.
The
samples are easy to collect and the excreted pyruvate appears
to be quite stable thus obviating the difficulties attendant
upon the use of blood for the purpose.
In the course of
these experiments there were defined certain factors profoundly influencing the pyruvate output in thiamine deficiency.
Procedure for Determination of Urinary Pyruvate
Rats from our stock colony, 120 to 160 grams in weight,
•^Personal communication, January, 194-1 •
*
30
were used for these experiments.
The animals were fed the
thiamine-free diet previously described supplemented with 20
gamma thiamine, 60 gamma pyridoxine and 60 gamma riboflavin
weekly.
vals.
Groups of 10 animals were studied at weekly inter­
These were placed in individual metabolism cages with
facilities for collection of the urine.
A measured quantity
of the depletion diet was allowed with water ad libitum.
At
the end of 24- hours the food was removed and the consumption
recorded.
Urine collections were made by dilution to 20 cc.
representing a 24- hour.sample.
The animals were then fasted
for 24- hours and the urine collection made as before.
The urinary pyruvate was determined by a modification
of the specific hydrazine procedure of Lu (1939)
(l)•
To a
15 cc. cone point centrifuge tube there were added 1.5 cc. of
10 per cent trichloracetic acid.
A 0.5 cc. sample of urine
was added and the contents mixed by rotation.
One cc. of a
0.1 per cent solution of 2, 4- dinitrophenylhydrazine in 2N
HC1 was then added and mixed.
Extraction of the hydrazones
and unchanged hydrazine with ethyl acetate was carried out as
in the original method, the ethyl acetate extracts being
transferred to a second centrifuge tube.
Rapid separation
of the ethyl acetate layer was accomplished by centrifuga­
tion.
The pyruvic hydrazone was extracted as in the original
method with three successive 2 cc. portions of 10 per cent
sodium carbonate.
The combined carbonate extracts (6 cc.)
were then poured into a graduated colorimeter tube and 2N
sodium hydroxide was added to bring the volume to 10 cc.
31
The contents were then mixed by inversion and readings made
after 10 minutes in a Klett-Summerson photoelectric colori­
meter (Summerson, 1939) with a No. 54 filter.
The colori­
meter was previously calibrated for pyruvic acid by the use
of a lithium pyruvate standard prepared according to Wendel
(1931-32).
As an index of adequate excretion as well as of the
validity of the urine dilutions, simultaneous determinations
of 24 hour creatinine excretions were made.
Pyruvate values
were considered valid only when accompanied by a normal urin­
ary output as determined by this method.
In the determina­
tion of creatinine, a modification of the micro method of
Shaffer (1914) was employed; a Klett-Summerson photoelectric
colorimeter with the No. 54 filter was used.
Tables III to VIII for the male rats, and IX to XV
for the female rats, record the pyruvate excretion when the
animals subsisted on the thiamine-deficient diet, together
with other data pertinent to the experiment.
Tables XVI and XVII are summary tables for these ex­
periments showing the average values and a statistical apprais­
al of the variations observed.
32
THE URINARY PYRUVATE IN THIAMINE DEFICIENCY
Tables III to VIII
Male rats placed on thiamine-deficient diet
May 29, 194-0
Tables IX to XV
Female rats placed on thiamine-deficient diet
June 7, 194-0
Tables XVI and XVII
Summary tables
Tables XVIII to XXI
Response of pyruvate excretion to thiamine feeding
Table XXII
Summary table
TABLE III
EXCRETION OF URINARY PYRUVATE
Male Rats: June 3, 4* 1940
6th and 7th Days of Depletion
Number
1
2
3
4
5
6
7
8
9
10
Averages
Body Weight
Food Intake
on Test Day
Grams
Grams
Urinary Pyruvic Acid*
Food ad lib. During First
for 24 hrs.
24 hr,. Fast
mg.
mg.
142
140
125
132
161
149
128
153
143
144
15
15
15
13
14
16
14
15
17
18
1.81
1.81
1.72
1.72
1.64
2.11
1.88
1.96
1.81
2.17
0.65
0.82
0.78
0.53
0.82
0.65
0.78
0.69
0.94
0.61
141.7
15.2
1.84 ± 0.05
0.70 ±0.05
^Including standard error of the mean for acid values calculated
as follows:
S.E. = ‘v 5 3 F
n
VrT~
TABLE IV
EXCRETION OF URINARY PYRUVATE
Male Rats: June 12, 13, 194-0
15th and 16th Days of Depletion
Number
1
2
3
4
5
6
Body Weight
Food Intake
on Test Day
Grams
Grams
118
130
130
151
114
131
13
17
19
15
12
7
122
8
128
9
122
121
15
14
13
16
14
136.7
14.8
10
Averages
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hr. Fast
mg.
2.20
2.05
2.13
2.13
1.80
mg.
0.77
0.77
1.39
1.56
2.54
2.34
0.94
0.77
0.65
0.70
0.98
0.94
2.26±0.08
0.95 t o . 09
2.20
2.79
2.42
TABLE V
EXCRETION OF URINARY PYRUVATE
Male Rats: June 19, 20, 194-0
22nd and 23rd Days of Depletion
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
Food Intake
on Test Day
mg.
mg.
124
114131
121
126
13-4
115
119
1412
12
12
12
9
13
15
13
13
2.5-4
2.68
3.742.31
2.31
1.95
2.54
2.54
3.65
1.78
1.35
1.47
1.11
0.82
0.70
0.82
1.02
1.19
1.80
0.90
135.6
12.5
2.68 iO. 14
1.13 ± O.Oi
126
14-6
Grams
Urinary Pyruvic Acid
Food ad lib. During First
24 hr. Fast
for 24- hr s.
TABLE VI
EXCRETION OF URINARY PYRUVATE
Male Rats: June 26, 27, 1940
29th and 30th Days of Depletion
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
138
127
139
132
119
167
138
Food Intake
on Test Day
Grams
132
147
13
12
15
15
8
15
13
13
14
14
133.9
13.2
140
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hr. Fast
mg.
mg.
2.13
2.82
2.95
2.80
2.34
4.43
2.90
*1.85
4.67
3.77
1.39
1.39
1.31
1.39
1.39
1.97
1.31
*1.23
1.27
1.80
3.20 ± 0.27
1.45 ± 0 .0'
*Not considered in the averages because of low creatinine output
TABLE VII
EXCRETION OF URINARY PYRUVATE
Male Rats: July 3, Ay 1940s
36th and 37th Days of Depletion*
Number
Body Weight
.Grams
1
2
3
A
5
6
7
8
9
10
Averages
135
134
135
Food Intake
on Test Day
Grams
15
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 h r . Fast
mg.
mg.
2.95
2.95
2.95
1.06
121
121
10
12
10
12
142
126
121
143
119
10
6
10
14
11
3.45
1.60
2.67
4.35
1.88
1.15
1.02
1.02
0.70
1.52
0.90
0.90
129.7
11.0
2.76* 0.26
1.02 * 0.06
3.20
I .64
*Thiamine feeding during previous seven day period
0.82
1.11
TABLE VIII
EXCRETION OF URINARY PYRUVATE
Male Rats: July 10, 11, 194-0
43rd and 44th Days of Depletion*
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
Food Intake
on Test Day
Grams
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hr. Fast
mg.
mg.
165
125
136
118
133
135
143
139
110
128
8
7
9
8
9
7
8
7
8
6
5.50
3.444.51
4.51
2.95
2.38
3.78
3.441.88
3.28
133.2
7.7
3.57 i 0.34
*No thiamine feeding during previous seven day period
1.50
1«44
1.32
1.59
1.25
1.47
1.07
0.82
0.95
0.99
1.24 ± 0.08
TABLE IX
EXCRETION OF URINARY PYRUVATE
Female Rats; June 10, 11, 194-0
3rd and 4th Days of Depletion
Number
Body Weight
Grams
Food Intake
on Test Day
Grams
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
1
2
138
118
12
12
1.64
1.64
0.61
0.53
3
4
5
139
145
9
13
0.86
0.62
1.35
121
136
136
10
9
123
132
17
17
15
14
10
140
10
1.85
1.15
0.45
0.45
0.53
0.53
0.49
0.57
0.65
132.8
12.9
1.50 ± 0.11
0 . 5 4 ± 0.04
6
7
8
Averages
1.11
1.88
1.88
I .64
TABLE X
EXCRETION OF URINARY PYRUVATE
Female Rats: June 17, 18, 194-0
10th and 11th Days of Depletion
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
Food Intake
on Test Day
Grams
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
1.76
1.99
2.35
1.82
1.96
1.88
1.56
1.62
120
.144
118
135
126
146
130
127
121
136
18
13
17
12
18
22
10
18
16
18
2.46
I .84
0.86
1.11
0.86
0.74
0.78
0.78
0.86
0.98
0.74
0.69
130.3
16.2
1.92 ± 0.10
0.84 ± 0.04
TABLE XI
EXCRETION OF URINARY PYRUVATE
Female Rats: June 24> 25, 194-0
17th and 18th Days of Depletion
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
Food Intake
on Test Day
Grams
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
3.52
3.52
3.52
134
146
112
137
117
131
134
117
14
16
11
16
11
19
16
13
13
13
3.76
2.54
2.95
4.60
1.89
5.90
1.18
1.18
0 *86
1.36
0.98
1.39
0.98
0.82
1.10
0.98
128.5
15.2
3.90 ± 0.42
1.08 ±0.06
6.40
TABLE XII
EXCRETION OF URINARY PYRUVATE
Female Rats: July 1, 2, 194-0
24th and 25th Days of Depletion
Number
Body Weight
Grams
1
2
3
45
6
7
8
9
10
Averages
Food Intake
on Test Day
Grams
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
1.64
145
134
132
125
131
108
115
116
130
112
17
12
12
15
13
13
11
14
15
13
2.87
1.97
2.95
2.30
2.95
2.80
2.05
1.31
2.38
2.87
0.78
0.78
1.19
0.86
0.70
0.74
0.86
1.03
0.70
124.8
13.5
2.44 t o .16
0.93 ± 0.08
TABLE XIII
EXCRETION OF URINARY PYRUVATE
Female Rats: July 8, 9, 1940
31st and 32nd Days of Depletion
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
118
148
138
125
115
135
133
125
139
119
129.5
Food Intake
on Test Day
Grams
9
9
11
9
8
7
11
12
10
11
9.7
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
3.04
3.04
3.12
2.58
1.56
2.54
1.95
1.88
3.07
2.38
1.11
0.94
1.35
1.06
1.47
1.23
1.23
0.98
1.06
1.19
2.52 i 0.18
1.16 ± 0.04
TABLE XIV
EXCRETION OF URINARY PYRUVATE
Female Rats: July 15, 16, 194-0
38th and 39th Days of Depletion*
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
142
118
120
126
135
130
146
122
127
136
130.2
Food Intake
on Test Day
Grams
8
5
9
4
7
10
9
7
8
10
7.7
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
3.16
1.93
1.74
1.80
1.74
1.93
2.35
2.91
2.22
2.38
0.65
0.65
1.15
0.70
0.65
0.57
0.70
0.78
0.70
0.86
2.22 ± 0.15
0.74 i 0 . 05
*Thiamine feeding during previous seven day period
TABLE XV
EXCRETION OF URINARY PYRUVATE
Female Rats: July 22, 23, 19X0
45th and 46th Days of Depletion*
Number
Body Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
110
117
125
135
135
128
115
147
135
105
125.2
Food Intake
on Test Day
Grams
7
4
10
8
9
10
5
7
8
7
7.5
Urinary Pyruvic Acid
Food ad lib. During First
for 24 hrs.
24 hrs. Fast
mg.
mg.
2.50
2.95
2.95
2.62
3.77
3.93
I .64
2.95
2.87
*-#
0.94
1.08
1.08
0.94
1.13
1.18
0.65
0.95
0.78
2.901 0.21
0.9710.06
*No thiamine feeding during previous seven day period
■^Insufficient output of creatinine.
Not included in average
TABLE XVI
EXCRETION OF PYRUVATE BY RATS DURING
PROLONGED THIAMINE DEFICIENCY
SUMMARY
Male Rats
Days on
Diet
6- 7
15-16
22-23
29-30
36-37**
4 ,3 - 4 4 * * *
Average
Body
Weight
Average Food
Intake on
Test Day
Grams
Grams
141.7
136.7
135.6
133.9
129.7
133.2
15.2
14.8
12.5
13.2
11.0
7.7
M.D.:S.E.M.D.*#*#
(6-7 and 43-44- days)
Urinary Pyruvic Acid in mg.#
Food ad lib. for
During First
24 hr. Fast
24 hr. Period
1.84
2.26
2.68
3.20
2.76
3.57
± 0.05
± 0.08
± 0.14
±0.27
±0.26
±0.34
0.70
0.95
1.13
1.45
1.02
±0.05
± 0.09
±0.08
±0.07
±0.06
1.24 ±0.08
5.00
5.75
Each value is the average of ten experimental animals
■^Including Standard Error of Mean calculated as follows:
S.E. of Meani
**Thiamine feeding during previous seven day period
#*#No thiamine feeding during previous seven day period
**##Mean Difference: Standard Error of Mean Difference
When this value exceeds 3.00 the results are considered
significant
TABLE XVII
EXCRETION OF PYRUVATE BY RATS DURING
PROLONGED THIAMINE DEFICIENCY
SUMMARY
Female Rats
Days on
Diet
3- 4
10-11
17-18
24-25
31-32
38-39*"*
45-46***
Average
Body
Weight
Average Food
Intake on
Test Day
Grams
Grams
132.8
130.3
128.5
124.8
129.5
130.2
125.2
12.9
16.2
15.2
13.5
9.7
7.7
7.5
M.D.:S.E.M.D.****
(3-4 and 45-46 days)
Urinary Pyruvic Acid in mg.*
Food ad lib. for
During Firs24 hr. Period
24 hr. Fast
1.50 ± 0.11
1.92 ± 0.10
3.90 ±0.42
2.4-4 ± 0.16
2.52 ± 0 . 1 8
2.22 ±0.15
2.90 ±0.21
0.54 ± 0.04
O .84 ± 0.04
1.08 ±0.06
0.93 ± 0.08
1.16 ± 0.04
0.74 ±0.05
0.97 ±0.06
5.94
5.95
Each value is the average of ten experimental animals
*Including Standard Error of Mean calculated as follows:
S.E. of Mean*
**Thiamine feeding during previous seven day period
***No thiamine feeding during previous seven day period
-*-*-fr#Mean Difference: Standard Error of Mean Difference
When this value exceeds 3*00 the results are considered
significant
48
From these data it will be noted that a definite in­
crease in pyruvate has occurred - for the male animals, from
a mean of 1. 84 + 0.05 m.g. per day
at the 6th
to 3.57 + 0.34 mg* Per day at the43rd day;
increase from 1.50 + 0.11 mg. per
for
day at the
0.21 mg. per day at the 45th day.
The
day of depletion
females, an
3rdday to 2.90 +
values for the quotient
of the mean differences to the standard error of the mean dif­
ferences exceed 3.00, indicating statistical validity.
In order to evoke this excretion it is necessary that
the animal be well nourished.
As the lowered values for the
fasting period indicate, the state of alimentation of the ani­
mal markedly affects the pyruvate output.
This fact was also
reported for the urinary bisulfite-binding substances by Shils,
Day and McCollum (1940).
Presumably the level of liver and
muscle glycogen is an important factor.
The relation to
food intake is not, however, strictly quantitative, for al­
though a markedly diminished intake (less than 5 gm. in 24
hrs.) will lower the expected output, a normal intake (10 gm.
or more) does not produce a proportionate increase.
For the
fasting period the increased excretion is in the same direc­
tion as that observed for the collection period during which
food was allowed.
According to the needs of the animal thiamine feeding
even in small dosages produced a temporary lowering of the
output of pyruvate.
This was observed for the males between
the 29th and 36th days and similarly in the females between
the 32nd and 39th days.
Suspension of the supplement was re-
49
fleeted by a return to elevated levels.
As recommended by Banerji and Harris (1939) for eval­
uation of the urinary bisulfite-binding substances as a re­
flection of the thiamine status of the animal, elevated
values should respond to dosage with thiamine.
As the re­
sults detailed in Tables XVIII to XXI and summarized in Table
XXII indicate, this has been observed with urinary pyruvate.
TABLE XVIII
RESPONSE OF PYRUVATE EXCRETION TO THIAMINE FEEDING
63-64 Days on Diet
June 14, 15, 1940
Number
i
Sex
Weight
Grams
1
2
3
4
5
6
7
8
9
10
F
F
F
F
F
M
M
M
M
M
120
120
127
111
137
129
125
138
125
111
Thiamine Administered
During Week Previous
to Collection
Food Intake
on Test Day
gamma
Grams
30
30
30
30
30
30
30
30
30
30
16
13
14
16
19
16
14
18
20
16
Averages M
125.6
16.8
3.76
4*27
3.76
3.44
4.77
2.88
3.44
6.72
7.30
5.41
0.87
0.70
1.71
0.94
1.16
0.94
0.61
1.80
1.25
1.64
21
1.07 ±0.16
5.-15 ± 0. 79
1.25 * 0.24
•
15.6
mg.
0
123.0
mg.
-H
O
O
•
Averages F
Urinary Pyruvic Acid
Food ad lib. During First
During 24 hr. 24 hr. Fast
Period
TABLE XIX
RESPONSE OF PYRUVATE EXCRETION TO THIAMINE FEEDING
70-71 Days on Diet
June 21, 22, 194-0
Number
Sex
Weight
Grams
1
2
3
4
5
6
7
8
9
10
F
F
F
F
F
M
M
M
M
M
120
12 8
125
128
118
132
128
132
133
117
Thiamine Administered
During Week Previous
to Collection
gamma
30
30
30
30
30
30
30
30
30
30
Urinary Pyruvic Acid
Food Intake Food ad lib. During First
on Test Day During 24 hr. 24 hr. Fast
Period
mg.
mg.
13
13
11
12
9
10
13
13
14
10
3.60
4 *443.28
2.71
3*28
4*85
4.10
8.70
0.86
1.23
0.86
1.31
1.72
1.64
1.06
2.20
1.64
0.98
Grams
4.61
4 .44
Averages F
124.2
11.6
3.46 ±0.25
1.20 ± 0.14
Averages M
128.4
12.0
5.34 ±0.85
1.50 ± 0.02
TABLE XX
RESPONSE OF PYRUVATE EXCRETION TO THIAMINE FEEDING
77-78 Days on Diet
June 28, 29, 194-0
Number
Sex
Weight
Grams
1
2
3
4
5
6
7
8
9
10
F
F
F
F
F
M
M
M
I
M
136
125
115
155
135
132
141
134143
111
Thiamine Administered
During Week Previous
to Collection
Food Intake
on Test Day
gamma
Grams
30
30
30
30
30
30
30
30
30
30
5
k
6
5
7
10
8
Urinary Pyruvic Acid
Food ad lib. During First
During 24 hr. 24 hr. Fast
Period
mg.
mg.
2.62
0.98
3.28
2.94
2.87
2.46
3.28
1.11
0.95
1.02
0.90
9
1.98
5.00
10
6
5.90
3.60
1.23
0.90
0.90
0.90
1.80
Averages F
133.2
5.4
2.83 ± 0 .12
1.00 t 0.01
Averages M
132.2
8.6
3.95 ± 0 .61
1.15 t O .01
TABLE XXI
RESPONSE OF PXRUVATE EXCRETION TO THIAMINE FEEDING
98-99 Days on Diet
July 19, 20, 1940
Number
1
2
3
4
5
6
7
8
9
10
i
Sex
F
F
F
F
F
M
M
M
M
M
Weight
Thiamine Administered
During Week Previous
to Collection
Food Intake
on Test Day
Grams
gamma
Grams
143
139
144
600
600
600
15
13
141
600
600
600
600
600
600
600
137
136
141
138
137
140
Urinary Pyruvic Acid
Food ad lib. During First
During 24 hr. 24 hr. Fast
Period
mg.
mg.
2.95
15
17
18
13
12
13
*
2.46
2.79
2.82
2.01
2.70
2.75
*
0.95
0.61
*
1.11
0.86
1.31
0.82
0.74
1.11
*
2.02
Averages F
140.7
15.0
2.55 *0.14
0.88*0.10
Averages M
138.4
14.0
2.57*0. 26
1.00±0.11
•^Insufficient creatinine excretion
TABLE XXII
RESPONSE OF PYRUVATE EXCRETION TO THIAMINE FEEDING*
SUMMARY
Days on
Diet
Sex
Average
Weight
Grams
Thiamine Administered
per Week Previous to
Collection
gamma
Average Food
Intake on
Test Day
Grams
Urinary Pyruvic Acid**
Food ad lib. During First
During 24 hr. 24 hrs Fast
Period
mg.
mg.
63-64
F
M
123.0
125.6
30
30
15.6
16.8
4.00 ± 0.21
5.15 * 0.79
1.07* 0.16
1.25 *0.24
70-71
F
M
124.2
128.4
30
30
11.6
12.0
3.46 * 0.25
5.34 *0.85
1.20 ± 0.14
1.50 ±0.02
77-78
F
M
133.2
132.2
30
30
5.4
8.6
2.83 * 0.12
3.95 * 0.61
1.00 ±0.01
1.15 ± 0.01
98-99
F
M
140.7
138.4
600
600
15.0
14.0
2.55 * O.I4
2.57 ±0.26
0.88± 0.10
1.00 ±0.11
F
3.18
1.86
M
3.13
4.47
M.D.s
S.E.M.D.
70th99th Day
*Each value is the. average of five experimental animals
**Including standard error of the mean
55
In these experiments a group of male and female animals
was depleted for long periods during which elevated pyruvate
levels were maintained.
Small dosages of thiamine (20-30
gamma per week) did not produce extensive diminution in these
levels.
From the 78th day of depletion to the 98th day, 600
gamma per week were fed to each animal.
This produced a sig­
nificant drop in the pyruvate outputs in the males, from 5.34*
0.85 on the 70th day to 2 . 5 7 * 0 . 2 6 on the 98th day, and in
the females, from 3*4-6*0.25 to 2 .55 * 0 .14- for the correspond­
ing period.
A consistent sex difference in the pyruvate excretion
appears to prevail in the twenty series of experiments summar­
ized in Tables XVI, XVII and XXII in all cases except two (fe­
males, 17-18 days, Table XVII), which is considered an aber­
rant result.
This variation appears both during alimenta­
tion and following fasting.
These results indicate that in animals on a thiaminedeficient diet some estimate of the nutritional status
with
respect to thiamine is reflected by the level of urinary
pyruvate.
The relation to the state of alimentation is not
surprising and it is of interest to note that the sex differ­
ences observed coincide with those reported for liver glycogen
by Deuel et al.
(Deuel, Gulick, Grunewald and Cutler (193-4)5
Deuel, Hallman and Murray (1937); Deuel, Butts, Hallman, Mur­
ray and Blunden (1937).
Thiamine supplements adequate for minimal growth are
not sufficient to abolish completely the heightened pyruvate
56
output.
In fact, one obtains more constantly elevated values
when animals are maintained in this sub-acute avitaminotic
state rather than in an acute, and often moribund, condition.
In the remaining experiments dealing with thiaminedeficient rats, the depletion technic which has been described
was used.
All animals were used between the 6th and 7th week
of depletion when an assay of their pyruvate excretion reveal­
ed excretions averaging 3.02 mg. up to 4»26 mg. for the 24
hours during which food was taken.
These values are in the
range which the previous study has indicated to signify a de­
pleted state although by virtue of small thiamine supplements
the animals usually presented no external signs of gross mor­
bidity and complete inanition was avoided.
One might there­
fore predicate that the studies to follow are characteristic
of the metabolic dysfunction attendant upon the mild, rather
than the acute, avitaminotic state.
The Absorption of Glucose from the Intestine
Preparatory to studies of the rate and extent of glycogenesis it was necessary to ascertain the effect of a defi­
ciency in thiamine on the rate of absorption of glucose from
the intestine.
For if this were markedly diminished, it .
might in itself be sufficient to account for lowered glycogen
values which might be observed.
The literature contains little or no strictly quantita­
tive data on glucose absorption in thiamine deficiency although
impaired absorption has been assumed because of the many exper­
iments which prove the existence of a definite gastro-intes-
57
tinal dysfunction associated with anorexia, diminished peri­
stalsis and deficient enzymatic secretion and activity.
A review of the various studies on absorption of
sugars has been published by Pierce (1935) in which are re­
ported the various experiments on the influence of the method
of administering of sugars, their concentration, the length
of time over which the observations are made, etc.
Relative­
ly large variations exist in the absolute absorption rates
for glucose when the results of different workers are compar­
ed.
To a great extent this is attributable to differences
in technic.
In the experiments here reported it was of interest to
determine the absorption rate under the particular conditions
to be employed in the liver glycogen experiments.
Since the
interpretations were to be made by comparing the values obtain­
ed on deficient animals v/ith those on normal animals studied
under identical conditions, these technical objections are of
no consequence.
Extensive studies of the absorption of sugars have been
carried out by Cori who is responsible for the absorption meth­
od which bears his name.
In experiments on rats in which the
gastro-intestinal tract functioned as a unit, Cori (1925) con­
cluded that the rate of absorption is, within wide limits, in­
dependent of the concentration and absolute amount of glucose
present in the intestine.
Substantially similar conclusions
were reached in the work of Deuel et al. (Deuel, Hallman, Mur­
ray and Samuels, 1937) obtained in the course of studies on
58
normal, compared to adrenalectomized, rats.
These authors
confirmed Cori’s observations that there was no consistent
falling off of absorption rates as time proceeds.
Procedure. - For the determination of the rate of ab­
sorption of glucose from the intestine the following procedure
was used on both normal and thiamine-deficient animals.
Fol­
lowing a 24- hour fast during which only water was allowed, the
animals were fed by stomach tube, 1 cc. of a 32 per cent solu­
tion of glucose per 100 grams body weight, measured by pipette
into the stomach tube.syringe.
The. feedings were spaced 5
minutes apart to permit accurate assay of the gastro-intestinal
contents at the expiration of the desired time interval.
Ab­
sorption was allowed to proceed for 1 or 2 hours when the ani­
mal was anaesthetized by the intraperitoneal injection of 1
cc. of a 1 per cent solution of sodium amytal per 100 grams of
body weight.
The entire intestinal tract from duodenum to
rectum was then dissected out to permit unimpeded washing of
the contents although the gut remained attached at the duo­
denum to the stomach and esophagus still in situ.
The animal
body was then elevated and with the rectal end of the gut plac­
ed in a collection flask a stomach tube was passed into the
stomach via the esophagus and, by means of a syringe attached
to the tube, 50 cc. of warm water was forced through the en­
tire tract and the contents thus collected.
The anaesthesia
was so administered that these operations would be completed and
the lavage commenced at the expiration of the selected 1 or 2
hours interval.
59
The intestinal contents were then treated with 5 cc.
of a 10 per cent solution of zinc sulfate followed by 5 cc.
of a 0.5 N solution of sodium hydroxide, allowed to stand
for 5 minutes, made up to 100 cc. and centrifuged.
The re­
sulting water clear supernatant was used for assay of the
glucose recovered.
Glucose assays were carried out by the
method of Shaffer and Hartmann (1920) the reagent having been
calibrated against Bureau of Standards glucose.
Assay of
the glucose solution fed was also made in order to determine
the actual amount of glucose admitted to the gastro-intestinal tract.
Cori has introduced the term "coefficient of absorp­
tion" (Cori, 1925) as a means of recording data on the com­
parative rates of absorption of the sugars.
This is defined
as the milligrams of sugar absorbed per hour per 100 gram
rat.
The data here reported are based on this coefficient.
The glucose recovered has been corrected to conform to
the limitations of the technical procedure as determined by
the experiments reported in Table XXIII.
These are essen­
tially positive controls in which glucose was fed after all
anatomical manipulations had been completed.
It was then
immediately washed through so that the percentage recovery
could be ascertained.
As the experimental data indicate,
95 per cent of the glucose fed could be recovered by this
procedure and the corrections are made accordingly.
Tables XXIV and XXV record the results on the absorp­
tion of glucose in normal and deficient rats observed at the
60
end of the 1 hour period of absorption;
Tables XXVI and
XXVII, similar data obtained after a 2 hour absorption per­
iod.
Table XXVIII summarizes these experiments, together
with a statistical appraisal of the differences.
The aver­
age absorption coefficient for the normal animals observed
at the end of a 1 hour period is 183±14*«4- mg*
This com­
pares to a coefficient of 161± 9.2 mg. for the deficients
after a similar period;
for the 2 hour periods 1 5 9 * 3 . 3 mg.
for the normals corresponds to 132 ±3.06 for the deficient
animals.
The difference, 27 mg., represents a decrease of
17 per cent in the rate of absorption.
The estimation of absorption rates at the end of 1
hour is not reliable on account of marked differences in the
rate of gastric motility.
Since little or no glucose is ab­
sorbed from the stomach (MacLeod, Magee and Purves (1930);
Maddock, Trimble and Carey (1933), absorption cannot take
place until the sugar enters the intestine.
Thus one ob­
serves markedly varying absorption rates during this period.
As Pierce (1935) has also mentioned in his review, the error
of the mean is rather high and a statistical appraisal of
the apparent differences in rate of absorption fails to prove
them significant.
A comparison of the 2 hour period obser­
vations is much more valuable.
Here the error is small and
highly significant differences obtain.
Most workers have
therefore limited their observations to no less than 2 hour
intervals, feeding additional doses of glucose for periods
exceeding it.
61
These results do not agree with those of Gal (1930)
who stated that absorption was reduced to one-third of nor­
mal,
One suspects that his experimental animals wrere in a
highly deficient state or possibly a multiple avitaminosis
prevailed.
The relatively mild but definitely decreased
absorption which was observed in these experiments would
appear to further substantiate the belief that in this case,
one is dealing with a controlled sub-acute deficiency state.
To the list of gastro-intestinal dysfunctions previously re­
ported as characteristically associated with thiamine defi­
ciency one must now add this quantitative evidence of the
diminished absorption of glucose.
TABLE XXIII
CONTROL EXPERIMENTS ON RECOVERY
OF GLUCOSE FROM THE INTESTINE
Number
Weight
Sex
Grams
1
2
3
4
5
6
7
174
154
202
146
170
141
176
Averages
166
M
M
M
M
M
M
M
Glucose Glucose
Fed
Recovered
mg.
mg.
486
468
486
486
454
486
454
486
468
Recovered
Per
cent
468
468/486 x 100 = 96.3
4.68/486 x 100 = 96.3
468/486 x 100 = 96.3
428
456
440
440
>428/454
456/454
440/454
440/486
x
x
x
x
100
100
100
100
= 94.5
= 94.0
=97.0
=92.5
95.3
TABLE XXIV
THE ABSORPTION OF GLUCOSE FROM THE INTESTINE
Period of Absorption - 1 Hour
Normal Male Rats
Number
Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
134
124143
142
112
130
142
116
144
128
Glucose
Fed
mg.
Glucose Recovered
(corrected)
mg.
442
408
476
476
374
442
476
408
476
442
189
164
246 .
103
269
19S
265
139
238
204
131.5
Coefficient of
Absorption
mg./lOO gm./hr.
189
200
161
263
94
187
149
232
165
186
183 ± 1 4 . 4
•^Averages include Standard Error of the Mean calculated as
follows:
S.E. =
V—
r
i~x
TABLE XXV
THE ABSORPTION OF GLUCOSE FROM THE INTESTINE
Period of Absorption - 1 Hour
Thiamine-deficient Male Rats
Number
1
2
3
4
5
Grams
Glucose
Fed
mg.
125
130
134
392
424
424
Weight
100
120
6
138
7
111
8
107
124
130
119
9
10
11
Averages
121.6
326
391
456
345
345
408
408
376
Glucose Recovered Coefficient of
(corrected)
Absorption
mg.
mg./lOO gm./hr.
248
253
204
143
171
269
145
139
177
210
200
115
132
I 64
183
183
136
180
193 •
186
152
148
161 ± 9.2
TABLE XXVI
THE ABSORPTION OE GLUCOSE FROM THE INTESTINE
Period of Absorption - 2 Hours
Normal Male Rats
Number
Weight
Grams
1
2
3
4
5
6
7
8
9
10
Averages
138
148
133
144
115
138
150
140
130
150
138.6
Glucose
Fed
mg.
476
510
442
476
374
476
510
476
442
510
Glucose Recovered Coefficient of
(corrected)
Absorption
mg./lOO
gm./hr.
mg.
84.2
0.0
74.0
10.0
0.0
6.3
31.4
29.0
8.6
25.7
142
172
13 8
161
162
170
158
159
166
161
159 ±3. 3
TABLE XXVII
THE ABSORPTION OF GLUCOSE FROM THE INTESTINE
Period of Absorption - 2 Hours
Thiamine-deficient Male Rats
Number
1
2
3
4
5
6
7
8
9
10
Averages
Weight
Glucose
Fed
Glucose Recovered
(corrected)
Grams
mg.
mg.
110
134
120
150
128
128
110
130
115
118
358
424
392
489
55.0
70.5
84.O
123.0
65.3
112.0
95.0
90.5
34.0
32.0
124.3
424
424
358
424
376
376
Coefficient of
Absorption
mg./lOO gm./hr.
137
132
128
122
140
122
119
128
148
146
132 ± 3.06
TABLE XXVIII
COMPARISON OF GLUCOSE ABSORPTION IN NORMAL AND
THIAMINE-DEFICIENT MALE RATS
SUMMARY
Period of
Number of
Absorption Experiments
Hours
Average
Weight
Thiamine
Status
Grams
1
1
2
2
10
11
10
10
131.5
121.6
138.6
12^.3
Coefficient of
Absorption
1
n2
t =. x-x1
t*
t*#
Actual Theoretical
mg./lOO gm./hr.
Normal
Deficient
Normal
Deficient
183.0
161.0
159.0
132.0
^•Calculation of t value (Fisher, 1933)
s2 -
Diff.
{s(x-x)2+ S(x-x>)2}
(nl+1) (n2+l)
n-j+H 2+2
n = n-^+n^
where:
n]_= number of cases in Exp. I
n2 = number of cases in Exp. II
SCx-xJ^-sum of differences from the mean (Sd^)
x = arithmetical mean of Exp. I
x f= arithmetical mean of Exp. II
± 14.4
± 9.2
i 3.3
± 3.1
22
1.24
2.86
27
5.66
2.88
theoretical" is the value of t
when P = 0 . 0 1 for the number of cases
included in the experiment,
(cf.
Table of t, Fisher, p. 177.)
68
Glycogenesis. Glvcogenolvsis and Blood Sugar Levels
Procedure. - Male rats, normal or' thiamine-deficient,
were fasted for. 48 hours with only water ad lib. allowed dur­
ing this period.
Certain of the animals were then anaesthe­
tized by the intra-peritoneal injection of 1 cc. of 1 per cent
sodium amytal per 100 grams body weight and the liver glycogen
and blood sugar determined to obtain control values for these
factors.
Others were fed glucose by stomach tube — 1 cc. of
a 35 per cent solution per 100 grams body weight - and sacri­
ficed 3, 6 or 12 hours after the feeding in order to deter­
mine the liver glycogen and blood sugar at these various in­
tervals .
The determination of the blood sugar was made by remov­
al of blood by means of a needle introduced directly into the
ventricle exposed after anaesthesia had supervened.
The
blood was then discharged into the paraffin lined well of a
spot test plate and exactly 0.1 cc. samples were secured by
the use of a special Folin blood sugar pipette.
This sample
was discharged into a 15 cc. cone point centrifuge tube con­
taining 10 cc. sodium tungstate, to precipitate the blood pro­
teins.
By centrifugation a clear supernatant was obtained
from which 4 cc. samples were taken for assay by the colori­
metric micro method of Folin and Malmros (1929).
The Klett-
Summerson photoelectric-colorimeter (Summerson, 1939) using
a number 54 filter was used.
Blood sugar values were deter­
mined by comparison against a standard prepared by dilution
from a 1 per cent stock solution of glucose in saturated ben­
69
zoic acid solution. ■
Immediately following the obtaining of the blood and
the discharge of the 0.1 cc. sample into the tungstate - a
period not exceeding 30 seconds - the liver was dissected
out and plunged into a mixture of solid carbon dioxide and
ether.
These operations were planned to coincide with the
feeding periods so that the removal of the organ would be
accomplished exactly at the expiration of the proper experi­
mental period.
The frozen tissue was then weighed to the second place
on an analytical balance and then placed in a round bottom,
50 cc. centrifuge tube to which 1 cc. of a 40 per cent solu­
tion of potassium hydroxide per gram liver weight was then
added.
Complete dissolution of the tissue was accomplished
by boiling with the alkali in a water bath.
When this had
occurred as evidenced by the absence of any solid particles
when the solution is viewed by transmitted light, the tubes
were allowed to cool.
The glycogen in solution was precipitated out by the
addition of 2.2 volumes of ethyl alcohol, returning the mix­
ture to the water bath for a
moment to allow the alcohol to
just reach the boiling point
before removing the tubes to
cool before centrifugation.
The precipitated glycogen remained in the bottom of
the tube following centrifugation and the decanting of the
supernatant.
The tube was allowed to
mouth wiped dry with a clean towel and
drain for a time, the
5 cc.of distilled
70
water was added with heating to redissolve the glycogen.
Re­
precipitation was accomplished by the addition of 10 cc. of
alcohol followed by heating, cooling and centrifugation as be­
fore.
By this latter procedure a superior white product was
obtained since much of the foreign material was not precipi­
tated and could be poured off.
The tubes were now allowed
to drain and dry before proceeding with the assay.
The precipitated glycogen was hydrolyzed to glucose by
the addition of 25 cc. of 0.6 N HC1 followed by heating in a
boiling water bath for 2 and one-half to 3 hours.
The
hydrolysate was treated with the Somogyi precipitants (ZnSO^
and NaOH), made alkaline to phenolphthalein and brought up to
100 cc. in a volumetric flask.
Five cc. aliquots were assay­
ed by the procedure of Shaffer and Hartmann in the case of
livers obtained from fasting rats but for fed rats, further
dilution was usually necessary to obtain glucose samples
which fall within the range of sensitivity of the ShafferHartmann reagent.
From this assay one obtains the amount
of glucose in the sample by comparison of the titration fig­
ures with those of a calibration table prepared for the re­
agent by assay of Bureau of Standards glucose.
Using the
factor 0.927 the per cent glycogen was calculated by the fol­
lowing expression:
Mg. glucose x 0.927 x dilution from original sample
Weight of liver in mg.
X
Per cent glycogen
The results of these experiments are detailed in Tables XXIX
71
to XXXVI and summarized together with a statistical appraisal
of the apparent differences between normal and thiamine-defi­
cient animals in Table XXXVII.
TABLE XXIX
THE LIVER GLYCOGEN AND BLOOD SUGAR
Normal Male Rats After a 4.8 Hour Fast
Number
Weight
Grams
1
2
116
101
3
4
5
134
108
142
128
6
7
131
8
126
9
10
11
164
176
163
12
14-6
Averages
Liver Weight
Grams
5.28
4.32
5.81
4*65
5.74
4*54
4 .64
4 .84
7.21
5.05
6.23
5.47
Glycogen
Per cent
Blood Sugar
mg./lOO cc.
0.13
0.45
0.08
0.19
128
138
128
0.16
O .48
0.86
114
139
0.35
0.39
152
113
0.26
122
0.41
0.47
93
118
0.35 ±0.06*
126 ±3.0-
121
152
•^Including Standard Error of the Mean calculated as follows:
S.E. =
TABLE XXX
THE LIVER GLYCOGEN AND BLOOD SUGAR
Thiamine-deficient Rats After a 48 Hour Fast
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Averages
Weight
Liver Weight
Glycogen
Blood Sugar
Grams
Grams
Per cent
mg./lOO cc.
122
90
102
100
120
108
100
120
87
83
107
105
99
100
136
4-29
3.16
2.90
3.83
0.51
0.18
0.28
0.90
0.65
0.18
0.12
0.10
0.12
0.17
0.10
0.13
0.11
0.24
0.16
118
92
122
160
180
130
135
105
0 .2 6 ± 0 .06
138*7.1
4.08
5.08
3.83
4.08
3.30
3.20
4.36
3.63
3.85
3.30
5.23
140
160
180
TABLE XXXI
THE LIVER GLYCOGEN AND BLOOD SUGAR
Normal Male Rats
Fasted 48 hours, fed glucose (l cc. of a 35 per cent solution
per 100 gms. body weight), sacrificed 3 hours after feeding.
Number
1
2
3
4
5
6
7
8
9
10
Averages
Weight
Liver Weight
Glycogen
Blood Sugar
Grams
Grams
Per cent
mg./lOO cc.
123
5.01
5.69
4.34
5.40
4-51
5.77
8.37
5.27
5.74
5.18
2.64
2.22
148
160
88
141
93
127
176
109
130
113
2.30
1.59
2.02
2.64
2.60
139
131
191
174
200
2.46
187
183
2.44
2.56
148
122
2.34* 0 .10
162 ± 8.1
TABLE XXXII
THE LIVER GLYCOGEN AND BLOOD SUGAR
Thiamine-deficient Male Rats
Fasted 48 hours, fed glucose (l cc. of a 35 per. cent solution
per 100 gms. body weight), sacrificed 3 hours after feeding.
Number
Weight
Grams
1
2
8
125
142
107
79
98
92
133
142
9
160
3
4
5
6
7
Averages
Liver Weight
Grams
5.27
5.87
4.25
2.99
3.79
3.26
4.95
4.98
5.28
Glycogen
Per cent
Blood Sugar
mg./lOO cc.
1.72
1.73
1.71
180
190
170
145
165
1.86
1.75
1.36
1.63
2.16
200
2.21
165
190
1 .801: 0 .08
176 i 6*1
TABLE XXXIII
THE LIVER GLYCOGEN AND BLOOD SUGAR
Normal Male Rats
Fasted 48 hours, fed glucose (l cc. of a 35 per cent solution
per 100 gms. body weight), sacrificed 6 hours after feeding.
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Averages
Weight
Liver Weight
Glycogen
Blood Sugar
Grams
Grams
Per cent
mg./lOO cc.
120
118
116
82
124
96
119
126
127
124
149
135
124
102
96
110
127
91
102
5.90
5.63
5.83
4.05
5.70
4-51
5.63
5.71
5.41
5.25
6.24
5.52
5.02
4.12
4*49
X .44
5.76
4.50
4.67
2.50
1.71
2.38
2.08
2.25
2.26
2.62
1.41
1.81
1.58
2.40
2.67
2.18
1.55
2.03
1.42
1.10
2.28
1.93
220
200
190
186
198
179
186
170
179
173
165
165
178
183
187
183139
183
130
2.01 ± 0 .10
178 ± 4 .5
TABLE XXXIV
THE LIVER GLYCOGEN AND BLOOD SUGAR
Thiamine-deficient Male Rats
Fasted 4-8 hours, fed glucose (1 cc. of a 35 per cent solution
per 100 gms. body weight), sacrificed 6 hours after feeding.
Number
Weight
Grams
1
2
113
110
3
4
5
107
6
88
7
9
97
104
125
10
126
8
Averages
105
126
Liver Weight
Grams
4-35
4-31
4-19
6.74
3.83
3.93
5.90
3.80
5.80
5.27
Glycogen
Blood Sugar
Per cent
mg./lOO cc.
1.56
196
1.43
1.50
1.10
170
195
190
0.85
160
1.00
1.60
170
155
190
1.54
1.77
2.27
1.46 ± 0 .12
210
261
190 ± 6.0
TABLE XXXV
THE LIVER GLYCOGEN AND BLOOD SUGAR
Normal Male Rats
Fasted 48 hours, fed glucose (l cc. of a 35 per cent solution
per 100 gms. body weight), sacrificed 12 hours after feeding.
Number
Weight
Grams
1
2
100
102
3
4
5
102
133
7
130
14-2
115
8
128
6
9
106
10
11
12
128
126
13
Averages
117
180
Liver Weight
Grams
4*34
4-36
5.86
4 *66
5.10
5.34
4.92
4*73
3.91
5.90
4.80
4-54
5.95
Glycogen
Per cent
Blood Sugar
mg./lOO cc.
1.77
1.46
165
174
191
178
157
139
1.64
0.91
1.41
1.58
1.43
1.76
1.11
0.63
1.54
0.92
148
178
160
260
1.04
155
155
160
1.32 ± 0 .09
169 ± 8.0
TABLE XXXVI
THE LIVER GLYCOGEN AND BLOOD SUGAR
Thiamine-deficient Male Rats
Fasted 4-8 hours, fed glucose (l cc. of a 35 per cent solution
per 100 gms. body weight), sacrificed 12 hours after feeding.
Number
1
2
3
4
5
6
7
8
9
10
11
12
Averages
Weight
Liver Weight
Glycogen
Blood Sugar
Grams
Grams
Per cent
mg./lOO cc.
112
112
128
124
117
129
98
116
131
12 8
94111
4.68
4-37
5.11
4.85
4.21
4-96
3.78
3.98
4-91
4-49
3.96
4..
I. 64.
1.11
2.14
1.49
1.14
1.45
1.13
1.58
1.30
I .46
1.45
2.10
118
126
152
135
200
130
135
135
135
178
152
1.49 ± 0 .09
145 ± 6 . 0
TABLE XXXVII
LIVER GLYCOGEN AND BLOOD SUGAR
SUMMARY
Period after
Feeding
Number of
Exper.
Thiamine Average Liver Diff.
t
Diff.
t
Average
t
t
Glycogen
Status
Actual Theoretical Blood Sugar
Actual Theoretical
mg./
Per
Per cent
mg./lOO cc. 100
cc.
cent
G
S
12
12
Normal
15
11
Deficient 0.261 0.06
3
3
10
9
10
8
6
6
19
10
12
12
13
12
Hours
Controls
No sugar
Controls
No sugar
0.3510.06
1261 3.0
0.09
0.95
2.79
Normal
2.34 10.10
Deficient 1.80±0.08
0.54
3.86
2.90
19
10
Normal
2.011 0.10
Deficient 1.4610.12
0.55
3.22
13
11
Normal
1.3210.09
Deficient 1.4910.09
0.17
1.21
12
1.24
2.83
16218.0
176 ±6.0
14
1.15
2.92
2.77
17814.5
190 1 6.0
12
1.53
2.77
2.81
169 ±8.0
145 ±6.0
24
2.10
2.81
138 ±7.1
G = Glycogen
S = Blood Sugar
t theoretical is the t value necessary for a P value of 0.01 according to the Table of t (Fisher p. 177).
81
It will be observed that a definitely diminished quan­
tity of glycogen was found in the liver of the thiamine-defi­
cient rats observed 3 or 6 hours after the feeding of glucose
when a comparison with similarly treated normal rats was made.
The diminished absorption of glucose in the thiamine-deficient
animals should account for a certain amount of this effect but
the difference seems too great to suggest this delayed absorp­
tion as being entirely responsible.
It was not possible to show a significant difference
between the blood sugar levels of the two experimental groups
observed at the 3 and 6 hour periods.
Actually the averages
for the deficient animals are higher than those of the normals
suggesting a diminished sugar tolerance.
This is in harmony
with the opposite situation observed in connection with the
hepatic glycogen.
However the relatively large variations
in the individual blood sugar values for these periods did not
permit the obtaining of a statistically valid difference.
The results for the 12 hour period are particularly
noteworthy.
For, although as has been pointed out, the
thiamine-deficient animals exhibited significantly lowered
hepatic glycogen, the comparison made 12 hours after the ad­
ministration of glucose reveals no significant difference
between the normal and deficient animals.
In fact, the defi­
cient animals have maintained their liver glycogen at the
same level as had been observed at the end of 6 hours while
the glycogen of the normal liver has decreased at the usual
more or less regular rate in answer to the metabolic demands
82
of the organism.
This seems to indicate an abnormal reten­
tion of the hepatic glycogen in the thiamine-deficient ani­
mals, a conclusion which is substantiated by an inspection
of the blood sugar level at this same 12 hour period.
In
this case a statistically significant difference between the
normal and deficient animals was shown.
The lower blood
sugar values in the case of the deficient animals would then
be interpreted as signifying a diminished ability to mobil­
ize the hepatic reserves in support of which hypothesis rela­
tively elevated blood sugar concentrations will serve.
At this point one recalls the observation of Abderhalden and Wertheimer (1932, 1933) on the abnormal retention
of hepatic glycogen reserves by polyneuritic pigeons.
Pos­
sibly the observations here reported for rats are manifesta­
tions of a similar phenomenon.
not strictly comparable.
However the retention is
If it were, it should be confirmed
by the comparison of the 4-8 hour fasted controls.
However
this is not the casd since neither the liver glycogen nor
blood sugar levels are significantly different in the two
experimental groups.
Glvcogenesis After Lactate or Pyruvate
Having investigated the factors concerned with glycogenesis In thiamine-deficient animals it was of interest to
study the ability of the avitaminotic organism to convert In­
gested lactate or pyruvate into glycogen.
As has been point­
ed out both in the survey of the literature and in connection
83
with our own experiments on urinary pyruvate, a definite im­
pairment of the ability of the organism to remove these car­
bohydrate metabolites is characteristic of avitaminosis B-^.
But does this impairment extend to the conversion of these
substances into hepatic glycogen?
The following experiments
were designed to answer this question.
The ability of the rat to convert orally administered
lactate or pyruvate to liver glycogen was well established by
Shapiro (1935).
The natural isomer oflactic
acid, the 1
(+) form, was found to be much superior to the
unnatural d
(-) antipode, or the racemic dl form.
In these experiments the natural 1 (+) isomer was
used.
It was prepared as a 15 per cent solution of the
sodium salt from the ammonium zinc double salt.'*’
The de­
tails for the preparation of the sodium salt of 1 (+) lactate
from the ammonium zinc double salt as well as those concern­
ing the preparation of a solution of sodium pyruvate from
pyruvic acid were obtained by reference to the
Shapiro (1934)•
work of
Polaroscopic examination of the sodium
1
(+) lactate solution gave an observed rotation of (-) 3.750.
^This salt was available in the Department of Biochem­
istry having been prepared by Lucien A. Bavetta to whom our
gratitude is herewith expressed.
^The salts of the optically active lactic acid exhibit
rotation in a direction opposite to that of the free acid.
The designation 1 (+) refers to the free acid.
84
By virtue of the quantity of double salt originally used the
final concentration of the sodium salt is 15 per cent accord­
ing to Shapiro.
Thus the specific rotation is:
20°
_ (-) 3.75
' 15.0 x 2
=
12.5
D5880A0
Where
20°
&
D5880A°
s Specific rotation in degrees at 20°
Centigrade using a sodium lamp at wave­
length 5880 Angstrom units
(-) 3.75
15.0
* Observed rotation in degrees
= Concentration of solution under examina­
tion in per cent
2
= Length of polarimeter tube in decimeters,
a value which
is exactly that reported by Shapiro for one pre­
paration used
inher studies.
A 6 hour period after the feeding of the salts was
chosen for removal of the liver since Shapiro had reported
maximum glycogen
formation at this interval.
48 hour fast,both normal and
Following a
deficient test animals were
fed 1 cc. of the 15 per cent solution of sodium 1 (+) lactate
per 100 grams body weight or 2 cc. per 100 grams body weight,
of the pyruvate solution containing 100 mg. (calculated as
pyruvic acid) sodium pyruvate per cc.
In these dosages no
toxicity was observed and no marked intestinal disturbances
were noted.
TABLE XXXVIII
THE LIVER GLYCOGEN FORMED AFTER 1 (+-) SODIUM LACTATE
Normal male rats fasted
hours, fed 1 cc. of a 15 per cent
solution of 1 (+•) sodium lactate per 100 gms. body weight,
sacrificed after 6 hours.
Number
1
2
3
4
5
6
7
8
9
10
11
Weight
Grams
Liver Weight
Grams
4.98
4 •24
4.23
X .46
4. BO
7.29
5.81
4.60
5.20
4.99
3.74
107
93
92
95
101
136
93
103
106
114
90
Glycogen
Per cent
1.75
2.32
1.77
1.62
1.74
0.24
0.71
1.51
2.08
1.37
1.19
1.48 ±0
Average
■^Including Standard Error of the Mean calculated as follows:
S.E. =
^ £ da
i~r
TABLE XXXIX
THE LIVER GLYCOGEN FORMED AFTER 1 (+) SODIOM LACTATE
Thiamine-deficient male rats fasted 4,8 hours, fed 1 cc. of
a 15 per cent solution of 1 (+) sodium lactate per 100 gms.
body weight, sacrificed after 6 hours.
Number
1
2
3
4
5
6
7
8
9
10
Average
Weight
Liver Weight
Grams
Grams
112
68
86
108
13 8
12 8
4-85
2.41
3.65
4-89
5.81
5.11
4.06
4 .44
4.63
4.73
104
118
116
125
Glycogen
Per cent
2.00
0.31
1.47
0.49
1.39
1.27
0.46
1.65
0.96
1.78
1.18±0.18
TABLE XL
THE LIVER GLYCOGEN FORMED AFTER SODIUM PYRUVATE
Normal male rats fasted 48 hours, fed 200 mg. (calculated as
pyruvic acid) sodium pyruvate per 100 gins, body weight,
sacrificed after 6 hours.
Number
1
2
3
■4
5
6
7
8
9
10
11
Average
Weight
Grams
122
132
102
90
112
146
133
125
109
83
82
Liver Weight
Grams
4.90
5.34
4 •24
3.66
4.24
5.63
5.48
5.57
4-92
3.17
3.84
Glycogen
Per cent
1.29
0.80
1.10
0.22
1.14
1.08
1.44
1.12
0.87
0.63
1.01
1.00
±
0.10
TABLE XLI
THE LIVER GLYCOGEN FORMED AFTER SODIUM PYRUVATE
Thiamine-deficient male rats fasted 48 hours, fed 200 mg.
(calculated as pyruvic acid) sodium pyruvate per 100 gms.
body weight, sacrificed after 6 hours.
Number
Vi/eight
Grams
Liver Weight
Grams
1
2
3
4
5
6
7
8
9
95
138
146
101
124
123
92
100
111
4.07
4.93
5.02
3.84
4-76
4.60
3.62
3.70
10
11
101
116
3.95
4.56
Average
4.06
Glycogen
Per cent
1.41
0.24
0.15
1.06
1.40
1.53
1.61
1.15
1.07
1.55
1.46
1.15 ±.0.18
TABLE XLII
A COMPARISON OF THE LIVER GLYCOGEN FORMED 6 HOURS AFTER THE FEEDING OF
1 (+) SODIUM LACTATE OR SODIUM PYRUVATE TO NORMAL AND
THIAMINE-DEFICIENT MALE RATS
SUMMARY
Substance Fed
Thiamine
Status
Number of
Experiments
t*
Theoretical
0.30
1.18
2.86
0.15
0.68
2.84.
Difference
Per cent
Per cent
1 (+) sodium
lactate
1 (+•) sodium
lactate
Normal
11
1.48± 0.16
Deficient
10
1.18 ±0.18
sodium pyruvate
sodium pyruvate
Normal
11
11
1.00± 0.10
1.15 ± 0.18
Deficient
t
Actual
Average
Glycogen
*t Theoretical is the t value necessary for a P value of O.Ol according to the Table of
t (Fisher, p. 177)
Both of these metabolites proved to be excellent glyco
gen formers not only in the normal but also in the thiaminedeficient animals.
A statistical appraisal of the differ­
ence in the average glycogen formed in the two groups does
not reveal any real difference notwithstanding the fact that
in the case of glucose there is a significant difference at
this period.
Thus it would appear that the inability of the
organism to dispose of lactate or pyruvate does not extend to
this phase of the cycle.
One might suggest that the explana
tion for the increased pyruvate and lactate in thiamine-deficiency lies rather in that fraction (1/5 of the lactate pro­
duced according to the Cori cycle) which is ultimately com­
pletely broken down in the tissues for energy purposes.
DISCUSSION
The fact that the tissues of a thiamine-deficient
animal exhibit a diminished oxygen uptake suggested to sever­
al workers that there was either a deficiency of oxidases or
an interference with their function (Dutcher (1918); Hess
(1921, 1922).
The idea went unchallenged until 1926 when
Marrian attributed these effects to inanition and in 1932,
Westenbrink, in the course of a critical review of the role
of vitamin
as an oxidative catalyst, concluded that there
was no real difference between the respiratory powers of
polyneuritic and normal tissues.
More recent studies from Peters1 laboratory have re­
vived the idea and, together with evidence from other sources,
clarified the exact function of thiamine in these enzymatic
processes.
In 1929 Kinnersley and Peters had reported that
lactic acid in abnormal quantities was present in the brain
of avitaminotic pigeons and its presence was thought to ac­
count for the acute symptoms of pigeon polyneuritis.
In
fact a delicate in vitro test for thiamine was devised by
measuring the effect of this substance in increasing the
oxygen uptake of polyneuritic pigeon brain respiring in
Ringer-phosphate solution with lactate, pyruvate or glucose
as substrate.
Normal brain tissue did not exhibit this so-
called "catatorulin effect1' (Passmore, Peters and Sinclair,
1933).
In 1937 Lohmann and Schuster contributed the important
finding that a pyrophosphate of thiamine isolated from yeast
92
functioned as a co-carboxylase, that is a co-enzyme for the
carboxylase which acts on pyruvic acid whereby in yeast fer­
mentation acetaldehyde and carbon dioxide- are produced.
Stern and Hofer (1937) were successful in synthesizing co­
carboxylase from pure thiamine.
This co-enzyme has also
been shown to increase the in vitro oxygen uptake of brain
tissue obtained from thiamine-deficient animals (Peters et al.
1939) although to a lesser extent than vitamin
itself.
The oxidative removal of pyruvic acid is catalyzed by thiamine
pyrophosphate or thiamine itself which is easily phosphorylated by the organism.
Lactate, which was considered the
more important in this connection in the earlier experiments,
is only indirectly concerned since an elevated concentration
of pyruvate tends to inactivate the lactate oxidase.
According to Lipmann (1937) all of the thiamine in
mammalian tissues is in the form of co-carboxylase.
(1938)
Tauber
by exposing duodenal mucosae to the action of thiamine
in phosphate solution produced co-carboxylase as evidenced by
the ability of the compound to evolve CO 2 in a pyruvate
medium to which washed yeast to supply carboxylase, had been
added.
The vitamin is always excreted in the free form so
that the kidney is evidently able to hydrolyze the co-enzyme.
Any interference with the phosphorylating mechanisms of the
body should inhibit the function of thiamine.
This hypo­
thesis is supported by the observation of Lipschitz, Potter
and Elvehjem (1938) that iodoacetate inhibits the phosphory­
lation of thiamine.
Goodhart and Sinclair feel that co­
93
carboxylase is not found in the blood plasma or cerebrospinal
fluid but only in the blood cells.
This would support the
idea that thiamine diffuses through the cell wall and is phosphorylated only within the cell.
Thus it would appear that at least one function of
thiamine in carbohydrate metabolism is to dispose of pyruvic
acid, and indirectly, of lactic acid.
It apparently func­
tions as a co-enzyme for (l) pyruvate oxidase (Peters), (2)
carboxylase (Lohmann), and (3) a dehydrogenase-carboxylase
(Lipmann).
This latter reaction was observed with Bacterium
delbruckii and was considered to involve not only decarbo­
xylation but also a loosening of hydrogen atoms, as:
ch3-co-cqoh—
> c h 3cooh +
co2
This last reaction indicates that the term "co-carboxylase”
is not sufficient to characterize all aspects of the pyrophos­
phate of thiamine.
An isolated and unconfirmed observation on another
function of thiamine introduces a provocative aspect of the
question.
This is the suggestion of Zambotti and Ferrante
(1939), that vitamin Bi accomplishes the transformation of
pyruvic acid from the keto to the enol form.
The keto form
of pyruvic acid is very toxic but the enolic form is not and
can be easily metabolized, these authors point out.
They
report that the addition of vitamin B-^ to a keto solution of
pyruvic acid in vitro brought about a demonstrable diminution
of its toxicity.
This keto to enol transformation by the
94agency of vitamin
has been investigated by the use of the
polarograph (Zambotti and Ferrante, 194-0) •
The modern theory of enzyme action supposes that the
various enzymes are composed of two components.
This first is
a heat labile, non-dihlyzable portion, the enzyme proper which
is associated or identical with a protein acting as a carrier
(the "tr&ger11 of Willstatter), while the second portion is a
heat stable, dialyzable fraction, the co-enzyme which activ­
ates the enzyme.
When these components are separated neith­
er possesses activity but it can usually be restored by simply
mixing the two.
The term co-enzyme conveys the idea that
this component is something external to the enzyme but actual­
ly it is rather to be thought of as an integral part thereof.
The exact method by which the various co-enzymes per­
form their functions is not clear.
It is highly probable,
however, that in the case of the phosphorylated complexes
like adenylic acid and thiamine pyrophosphate they might act
as phosphate donators.
The minute amount of phosphate avail­
able from this source for the phosphorylation mechanisms as­
sociated with glucose absorption from the intestine makes it
unlikely that the delayed absorption in thiamine deficiency
is attributable to this fact.
Probably the hypotonicity of
the gastro-intestinal tract which always accompanies thiamine
deficiency would account for most of the slowed absorption on
a purely mechanical basis.
According to the Embden scheme, hexose diphosphate is
converted into lactic and pyruvic acid in the muscle during
95
the anaerobic phase of muscle contraction.
While the details
are not entirely clear the process is somewhat as follows:
(1) Hexose to diphosphoric ester (I) Harden-Young Ester
OH
CHoOH
/
\
CHp-O—P=0
I2
t
00
1
OH-C-H
I
H-C-O H
I
H-C-OH
I
c h 2o h
C=0
OH
t
OH-CH
|
H-C-OH
I
H-C-OH 0H
\
c h 2 o -p = o
+ 2HqP 0 ,
^ - 2 ------->
+ 2H?0
^OH
I
(2) Hydrolysis of the ester to two triose phosphoric
acids
j m
^OH
CH9-0-P=0
i
C=0
I
OH-C-OH
I
H-C-OH
H-C-OH
i
*
'
/
OH
CHo-0-P=0
\
I
CH9-0-P=0
\OH
OH
C=0
I
CH2OH
;
//°
C
iXH
H-C-OH
I
\OH
/
Dioxyacetone
phosphoric
acid
II
OH
CH2-0-P-0
\
OH
Glyceric
aldehyde phosphoric acid
III
96
(3) II and III interact by a sort of Cannizzaro re­
action whereby II is reduced at the keto group and III is
oxidized at the aldo group producing:
I I ------ *IV
I I I ------ >V
OH
/
CHo-0-P=0
COOH
I
I
\
HT
h
0H
/ 0H
CHz-0-P-0
\ oh
c h 2oh
IV
Glycerophosphoric acid
V
Phosphoglyceric acid
(4) Compound V forms pyruvic acid and phosphoric acid
COOH
I
H-C=OH
OH
I
/
CHp-0-P=0
\
OH
.
>
COOH
I
C*0
I
CHo
^
+ H 0 PO,
3 U
V
VI
Phosphoglyceric acid
Pyruvic acid
(5) Pyruvic acid is reduced by glycerophosphoric acid
to form lactic acid (VII) and glyceric aldehyde phosphoric
acid (ill) which can participate in Reaction (3)
97
OH
/
c h 9- o -p = o
COOH
I-
I 2
H-C-OH
c-o
I
CH3
\
OH
c h 2o h
OH
COOH
I
H-C-OH
ch3
/
CHo-0-P=0
I
\
+ H-C-OH
OH
//°
C-H
VI.
Pyruvic
acid
IV
Glycerophosphoric acid
VII
Lactic
acid
III
Glyceric aldehyde
phosphoric acid
All of these reactions are controlled by enzymes which
are more or less specific to the reaction in question.
Hence
the reactions are reversible and proceed in a direction con­
sonant with the relative concentration of the substrate and
products.
It is thus reasonable to assume that the presence
of an excess of any one reaction product would inhibit the
rate at which the reaction proceeds in the direction forming
this product.
The inability of the polyneuritic organism
to remove pyruvate and indirectly lactate, would thus act as
a factor to raise the concentration of the reaction products.
The rate at which the reactions proceed to the right would be
slower.
This might be exhibited by a slower breakdown of
liver glycogen since it acts as a reservoir for the blood
sugar and the muscle glycogen which is more directly concern­
ed with the enzymatic reactions we are discussing.
The existence of a relationship between the blood and
urine pyruvate and the liver glycogen is indicated by several
observations.
Most important is the direct connection
between the amount of pyruvate excreted and the nutritional
98
state of the animal.
A well nourished animal in which the
liver and muscle glycogen is maintained will excrete much more
pyruvate than a fasted animal wherein the precursor of pyruvate
is diminished by virtue of a lower glycogen store.
The universally accepted sex differences in carbohydrate
metabolism make their appearance in this connection also.
Since the glycogen store of the female is lower than that of
the male if the hypothesis just advanced is valid, one would
expect a lower output of pyruvate in the female than in the
male, under similar conditions of alimentation or fasting.
This was precisely what was observed in the urinary pyruvate
experiments.
Finally, direct proof of the relationship between
blood (and presumably urine) pyruvate and glucose is found in
the report of Bueding, Stein and Wortis (194-1) who determined
the blood pyruvate at intervals following the ingestion of
glucose by human subjects.
A series of changes in the blood
pyruvate resembling the curve of a sugar tolerance study were
observed.
There was a gradual rise from a fasting level of
1.04- mg./100 cc. to a peak of 1.4-3 mg./lOO cc. at the end of
one hour after the taking of the sugar.
This was followed
by a gradual return after 3 hours, to the normal level.
The apparently impaired ability of the thiamine-defi­
cient organism to store ingested glucose as glycogen is sup­
ported by somewhat elevated blood sugar levels during the
post-absorptive period.
If this deficient glycogen forma­
tion is real it would suggest that thiamine is concerned with
99
the enzyme systems operating in the conversion of glucose to
glycogen although its precise role is difficult to evaluate.
On the other hand, the diminished rate of absorption of glu­
cose certainly accounts for some of the apparent impairment
in glycogenesis observed in the thiamine-deficient animal.
But it is questionable, in the light of evidence from other
sources, that it is responsible for all of it.
It should be
pointed out, however, that in the case of the lactate and
pyruvate experiments where a difference in the rate of absorp­
tion observed in normal control, versus that of thiaminedeficient, animals was presumably not operating, substantially
identical glycogen formation was noted at a period (6 hours
after feeding) which had previously been shown by glucose
feeding to indicate significantly impaired ability to form
glycogen in the case of thiamine-deficient rats.
It is during the later periods after glucose feeding
that a more characteristic effect is apparent.
The blood
sugar is significantly lower in the deficient animals as com­
pared with the normal controls indicating that the organism
is utilizing sugar but is unable to maintain the blood level
constant because of a retarded rate of supply from the liver.
Whether this slower rate of glycogen liberation is to be at­
tributed to events in the metabolism of carbohydrate in the
muscle as has already been suggested, or is associated with
the endocrine control of glycogenolysis, cannot be determined
from these experiments.
There appears to be little question that lactate and
100
pyruvate are not properly metabolised in the muscle tissue of
the thiamine-deficient animal.
But this metabolic dysfunc­
tion does not extend to the conversion of these metabolites
into glycogen when they are administered by mouth.
Hence,
the process by which the lactate is removed from the muscle
and carried to the liver for the formation of glycogen, in
accordance with the Cori cycle, would be unaffected in
thiamine deficiency.
Presumably the pyruvate formed in excess might also be
in part removed by this conversion to glycogen.
An accumula­
tion of pyruvate characterizes the deficiency state but al­
though pyruvate is undoubtedly formed in normal quantities,
it is removed more slowly because of a retarded rate of con­
version to further breakdown products.
The blood manifests
this accumulation and in response, the kidney eliminates the
material.
If pyruvate is administered to the rat an imme­
diate excretion of the unutilized portion will occur.
When
one feeds sodium pyruvate to thiamine-deficient animals this
excretion is not augmented by virtue of the deficiency.
It
is therefore reasonable to assume that pyruvic acid which is
not converted by the organism into glycogen or lower combus­
tion products will be excreted immediately.
We are directly concerned, then, only with that frac­
tion of the tissue pyruvate which would ordinarily be com­
busted to lower breakdown products writh the consequent liber­
ation of energy.
In the thiamine-deficient animal a much
smaller proportion of the total pyruvate formed will have
101
this fate.
excreted.
More is unavailable to the organism and must be
As the thiamine deficiency progresses this pheno­
menon becomes more pronounced.
A greater proportion of the
pyruvate is wasted and the energy requirements of the tissue
cannot be met by carbohydrate.
The slower rate of glycogen
supply to the muscle contributes to the increased pyruvate
excretion which has been observed in thiamine deficiency.
This permits a more gradual formation of pyruvate which is to
a large extent excreted because of failure to be further meta­
bolized.
If a more rapid formation occurred, a larger frac­
tion of the total formed might be converted to liver glycogen
with a consequent smaller percentage excreted.
Brain tissue metabolizes carbohydrate almost exclusive­
ly, having a respiratory quotient close to 1.
Thus one would
suppose that in carbohydrate starvation which thiamine defi­
ciency tends to foster, the brain would be one of the first
tissues to manifest this deprivation since its requirement
for carbohydrate is extreme.
On this basis the marked effect
of thiamine deficiency on the respiration of brain tissue in
vitro or the polyneuritic convulsions in vivo. could be ex­
plained.
A similar carbohydrate starvation has been suggest­
ed as one basis for the hypoglycemic convulsions observed in
cases of insulin shock.
Such gross inefficiency in the economical use of the
energy-yielding metabolites of muscle as is manifested in
thiamine deficiency must be the basis of the characteristic
clinical picture in mild deficiency states.
102
One must not infer that only in thiamine deficiency
are these metabolic dysfunctions produced.
The role of
nicotinic acid amide as a constituent of cozymase, of ribo­
flavin as the prosthetic group of the Warburg Yellow Enzyme,
of ascorbic acid as a hydrogen acceptor and donator and pos­
sibly the function of pyridoxine in some system similar to
nicotinic acid amide - all these point to the fundamental
part which these vitamins play in the intermediary metabol­
ism of foodstuffs.
Thiamine is but one link in the chain
and even though present in adequate amounts, might fail in
its efforts by virtue of deficiencies of other factors.
When one considers, however, the very fundamental
processes which are influenced by thiamine, one can under­
stand why, as Williams and Spies have stated, thiamine appears
to be good for everything and at the same time good for nothing
in particular.
SUMMARY AND CONCLUSIONS
1.
A method of inducing and maintaining a sub-acute
state of thiamine deficiency in rats has been formulated.
The deficiency appears to be due to thiamine alone.
2.
An estimate of the existence and degree of defi­
ciency was obtained by a series of studies of the excretion
of pyruvate as depletion progressed, as well as the ability
of thiamine to reduce this elevated pyruvate output.
The
state of alimentation of the animal as well as the sex were
found to influence markedly the pyruvate excretion.
3.
The rate of absorption of glucose from the in­
testine was studied quantitatively and found to be decreased
on an average of 17 per cent in thiamine deficiency.
4.
The level of liver glycogen and blood sugar of
the deficient animals was found to differ from that of normal
controls fed identical amounts of glucose and observed 3 or
6 hours after the feeding.
The liver glycogen was lower and
the blood sugar was higher in the thiamine-deficient animals.
Although the impaired rate of absorption accounted for some
of this effect, it is questionable whether it explains satis­
factorily the total reduction actually observed.
5*
The liver glycogen and blood sugar observed 12
hours after the administration of glucose indicated abnormal
retention of the liver glycogen in thiamine deficiency.
6.
The thiamine-deficient animals were found to con­
vert orally administered sodium pyruvate and sodium 1 (+)
lactate into glycogen with a facility equal to that of normal
controls.
7.
The observations of the present study are dis­
cussed in relation to the role of thiamine in the enzyme
systems associated with the metabolism of carbohydrate.
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