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The comparison of the metabolism of C¹⁴ carboxyl and methylene labeled glycine in the intact rat

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The comparison of the metabolism of C¹⁴ carboxyl and methylene labeled glycine in the intact rat

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Content THE COMPARISON OF THE METABOLISM OF C1^ CARBOXYL AND METHYLENE
LABELED GLYCINE IN THE INTACT RAT
A Thesis
Presented to
the Faculty of the Graduate School
University of Southern California
In Partial Fulfilment
of the Requirements for the Degree
Master of Science
Harry N» Barnet
August 19L9
Bio 's-o ! 5 < Z £ I
This thesis, written by
^ •1
under the guidance of h—%3. Faculty Committee,
and approved by a ll its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fu lfill
ment of the requirements fo r the degree of
MASTER OF SCIENCE
E^a^Bog-ar-dus-----
Dean
s'
IS
Faculty Committee
Chairman
TABLE OF CONTENTS
Introduction and Historical Review................ .1
Statement of Problem.................................. ..9
Materials............................................*10
Methods and Results,............................ *20
Discussion............................... ♦ .1*2
Summary.............................................. h9
Bibliography. .... • .f>0
LIST OF TABLES
TABLE PAGE
I. Control Experiment, I. Liver Glycogen Levels After
Varying Lengths of Fast.............................22
II* Control Experiment II. Liver Glycogen Values at
Various Times After Glycine Administration......... ...23
III. Purity of the Glycogen Isolated From the Feeding
Experiments* ...... ....27
IV. Liver Glycogen Levels for Control Animals of
Experiment II. ...............................29
V* Fraction Recoveries From Radioactive Glycine Feeding
Experiments....... 30
VI. Expired Carbon Dioxide Recoveries. ..................31
Vila. Radioactivity Data and Balance Sheet for the Carboxyl
Labeled Glycine Administration Experitnent ....... .3U
Vllb. Radioactivity Data and Balance Sheet for the Methyl
Labeled Glycine Administration Experiment.............35
VIIc. Radioactivity Data and Balance Sheet for the Bi
carbonate and Inert Glycine Administration Experiment...36
VIII. Radioactivity Data and Balance Sheet for the Expired
Carbon Dioxide Fraction.............. 37
IX. Degradation Experimental Data.........................Ill
LIST OF FIGURES
FIGURE PAGE
1. Acetic Acid Chlorination Apparatus. .....11
2. Apparatus for the Ammoniation of Chloroacetic Acid.....12
3. Respiration Chamber........................ .....13>
Lw Combustion Train CO2 Absorption Flask..............I?
5. BaC-^03 Self-Absorption Correction Curve........ .....32
CHAPTER I
INTRODUCTION AND HISTORICAL REVIEW
In glycine metabolism* the molecule undergoes many trans
formations. One of these pathways in the metabolic pattern* the
formation of glycogen* is irregular as compared to the action by the
other glycogenic amino acids since glycine requires more time to act in
forming glycogen. It is the purpose of this study to investigate the
mechanism of glycogen formation from glycine by comparing the metabolism
of glycine labeled with C^ in the carboxyl group with that of glycine
labeled in the methylene group in the intact rat. This conversion can
only be understood on the basis of the fate of the carbon atoms in the
glycine molecule since the glycogen is derived through some source of
carbon atoms whether directly or indirectly from the glycine administered.
Pfluger and Junkersdorf (1910) observed the formation of
glycogen after feeding amino acids to normal fasted rats and found that
the formation was slower in the case of glycine than some of the other
amino acids. Ringer and Lusk (1910) showed a similar conversion of
glycine to glucose in the phlorhizinized dog* this lending support to
the finding of Pfluger that glycine is a glycogen former. Luck (1928)
administered amino acids to rats per os and found only glycine caused an
appreciable rise in amino acid content of muscle while glycine and alanine
both gave an equal increase in the amino acid content of the systemic
blood. In the same experiment* Luck observed a great increase in the
2
amino nitrogen content of the liver due to glycine but not alanine*
Studying the rate of protein catabolism, Reid (1939) found a marked rise
after glycine feeding in dogs, MacKay, Wick and Carne (19U0) compared
the glycogenic activities of glucose, glycine and alanine with the
observation that glycine leads to the formation of hepatic glycogen in
an amount equal to that, caused by alanine but at a much slower rate,
the glycogen production reaching a peak sixteen hours after feeding to
rats. These findings, which are in agreement with the earlier work of
Pfluger and Junkersdorf (1910) and Ringer and Lusk (1910), do not agree
with the findings of Reid (1939), Greisheimer and Arny (1930) and Wilson
and Lewis (1929) who could not demonstrate a rise in liver glycogen due
to glycine. However, the disagreements in the latter experiments can for
the most part be explained on the basis of insufficient time for glycogen
production, i,e. experiments which were conducted for a period of from
four to six hours only,
Olsen, Hemingway and Nier (19U3) attempted to explain the
mechanism of glycogen formation by feeding glycine labeled in the
carboxyl group with the stable isotope, C^, They found a peak liver
glycogen level at sixteen hours after feeding but could not account for
the rise in glycogen by the conversion of the fed glycine, since the
isotope excess indicated that no more than one carbon atom from the fed
glycine in every twenty-nine was incorporated into glycogen* From the
data they suggested that glycine promotes the formation of glycogen
rather than itself being converted to glycogen, which would be in
agreement with the catabolism experiments of Reid (1939),
3
In the experiments cited above, with the exception of the work
by Olsen et al, there were no means of tracing the metabolism of the
molecule fed and consequently no means of establishing whether the
glycine was incorporated directly into the glycogen or whether glycogen
formation was stimulated indirectly. The work of Olsen et al was,
however, incomplete as there were no means of determining the fate of
the methylene carbon atom. It was not known whether this carbon
atom was carried along as a unit with the carboxyl fragment through the
various stages of metabolism or whether there was a complete decarboxyla
tion and deamination with an entirely separate fate for each atom (carboxyl
and methylene). Similarly there was no evidence presented to establish
a carbon dioxide fixation rate, i.e. by administering isotopic NaHC03
such that part of the glycogen isotope excess could be explained on a
carbon dioxide equilibrium basis# Wood, Lifson and Lorber (19U£)
administered labeled bicarbonate with lactate or glucose and studied
the fate of the bicarbonate (CO2) with respect to the incorporation into
rat liver glycogen formed as a result of the glucose administration and
were particularly interested in the position of the incorporated carbon
atoms in the glycogen molecule. Their results demonstrated that the
CO2 was readily incorporated into the glycogen molecule. Similarly
Armstrong, Schubert and Lindenbaum (19U8) have shown the incorporation
of CC> 2 administered as inorganic carbonate and bicarbonate into rat liver
glycogen using the radioactive isotope of carbon, C^# Similar experi
ments have been performed by Solomon et al (19U1), Wood, Venesland and
Evans (19U5) and Gould et al (19^9)• Work leading to an explanation of
h
glycogen formation by pathways other than carbon dioxide fixation has
been brought out by Lifson* Lorber, Sakami and Wood (19U8), studying the
incorporation of labeled acetic and butyric acids into rat liver
glycogen and attempts to explain the incorporation on the basis of the
ultimate position of the tagged carbon atoms in the glycogen molecule
using the tricarboxylic acid (Krebs) cycle as the principle pathway*
This work appears to be very important since it deals with the acetate
molecule and its incorporation into the tricarboxylic acid cycle and
hence to glycogen* Thus is afforded a possible explanation of the
formation of glycogen from glycine since it is known that the glycine
molecule can be deaminated and could* therefore* at some stage in its
metabolism form an acetate residue. Lifson et al (19U8) demonstrated
conclusively that the glycogen resulting could have been derived from
the entrance of the acetate (butyrate) residue directly into the tri
carboxylic acid cycle and hence through pyruvate to glycogen. The study
included the degradation of the glucose molecule revealing the positions
of the incorporated atoms* Buchanan* Hastings and Nesbett (19U3)> however,
could account for the incorporation of radioactive carbon from carboxyl
labeled acetate into liver glycogen after feeding acetate plus glucose
only by CO2 fixation* there being no activity due to direct incorporation
of the acetate residue into the carbohydrate cycle. The evidence was*
however* incomplete since these workers did not investigate the metabolism
of the methylene labeled compound* Additional limitations were placed
on the experiment by the short (five hour) half-life of the C^- used.
That glycine carbon atoms may be incorporated or transformed
5
into the acetic acid molecule has been most conclusively demonstrated
by Sprinson (19U9)> ‘ who, upon feeding methylene labeled glycine (alpha
carbon atom) to rats, has shown the utilization of the alpha carbon of
glycine for the formation of acetic and aspartic acids* By degradation
of the acetic acid isolated, Sprinson showed that the alpha carbon was
incorporated into both the alpha and the carboxyl atoms in equal amounts
as determined by the specific activities of the carbon of each fragment
of the molecule* Not to be overlooked is the similar incorporation of
the alpha carbon into the aspartic acid molecule since this amino acid
may also enter the tricarboxylic acid cycle through a transformation to
oxalacetic acid, an intermediate of the cycle# Using this evidence
together with that of Lifson et al (19U8), it would seem highly probable
that the glycine molecule is incorporated into glycogen by its conversion
to an acetate residue*
A unique approach with the aid of isotopes to the mechanism of
glycogen formation from glycine has been presented in evidence by Sakami
(19U8, 19U9)• He proposed a pathway to glycogen by the condensation of
a glycine molecule with a formic acid residue to yield serine which is
subsequently oxidatively deaminated to pyruvic acid following with the
formation of glycogen* The glycogen obtained as a result of administering
glycine labeled in the carboxyl group with and formic acid with the
radioactive isotope upon degradation definitely established his
original hypothesis as a probable pathway. Additional evidence for this
mechanism has been shown in the work by Winnick, Moring-Claesson and
Greenberg (19U8), who studied the distribution of amino acids in liver
6
homogenate protein following uptake experiments with Cpk labeled glycine
(carboxyl). They found approximately sixty per cent of the isotopic
carbon in the serine isolated. However, experiments by Greenberg and
Winnick ( 1 9 1+ 8) failed to show the incorporation of carboxyl labeled
glycine into the serine obtained from the protein hydrolysates of the
intact rat organs, which is in disagreement with the work of Sakami (1 9 1+ 8).
The more recent work of Sakami has shown the utilization of the methylene
carbon atom of the glycine molecule for the alpha and beta carbon atoms
of serine in the intact rat* He concludes from this evidence that glycine
may itself be a source of formic acid or formate derivative for the
conversion to serine and hence to glycogen. Since serine may form glycogen,
as shown by Butts, Blunden and Dunn ( 1938) and Schofield and Lewis ( 1 9 1+ 7),
and may give rise to pyruvate, as shown by Chargaff and Sprinson ( 1 91+ 3),
it would seem that the above work might provide an important step in the
pathway for the conversion of glycine to glycogen.
In a recent report by Todd, Barnes and Cunningham ( 191*7) it has
been shown that pre-feeding with a high glycine diet, unlike a high
alanine diet, leads to a marked increase in carbohydrate (liver glycogen)
storage during fasting. This action, having been termed by Mirski,
Rosenbaum, Stein and Wertheimer ( 1938) as the “protein effect”, is in
agreement with the results of their earlier work which consisted of
pre-feeding high protein diets to rats causing a similar retention of
carbohydrate stores (liver glycogen) upon a twenty-four hour fast or
exposure to cold. The “protein effect“ has been amply confirmed by other
workers and on the basis of these investigations, it would seem that the
7
effect might be concerned -with glyconeogenesis since the effect could not
be demonstrated by Todd et al (19U7) in adrenalectomized rats, and since
the work of Reid (1939)* previously mentioned, showed that glycine feeding
causes a definite increase in catabolism of protein in dogs using as an
index the nitrogen and inorganic sulfur excretion. Glycine produced a
marked rise in both N and inorganic sulfur as compared to alanine which
acted in a manner similar to glucose.
The classical works of Lusk (1913, 1913a), Dakin (1913) and
Van Slyke and Meyer (1913-lU) serve as the foundation for many phases of
our present day knowledge of the metabolism of amino acids, as does the
work of Pfluger (1910) and Ringer and Lusk (1910). The famous work on
animal calorimetry by Lusk (1913* 1913a) clearly demonstrated the specific
dynamic action of glycine and compared the action with that of alanine
and phenylalanine* Later work by Wilhelmj and Bollman (1928) confirmed
the earlier classical experiment by Lusk but was carried out under more
rigidly controlled conditions through the administration of the amino acid
intravenously* Keich and Luck (1931-32) presented evidence for the
mechanism of amino acid metabolism by administering amino acids in various
ways and studying the relative rates of amino acid disappearance and urea
formation, this work supplementing the earlier work of Luck (1928)
previously mentioned. The significance of this work with respect to the
formation of glycogen from glycine lies in the specific dynamic action of
the glycine as an indirect stimulus to formation by glyconeogenesis. This
is in agreement with the evidence for the "protein effect" but may be
looked on as a dynamic effect causing the excess breakdown and utilization
8
of protein for carbohydrate synthesis as compared to the latter action
which may be a protein sparing action, the glycine being converted to
carbohydrate without displacing the amino acids which are already
present. The previously cited work of Ringer and Lusk (1910), which
has since been confirmed by many workers including Janney (1915)>
Csonka (1915) and the recent report by Hawkins, McKee, Hawley and Kummer
(19U?) with phlorhizinized dogs, lends support to the latter mechanism
since the report by Hawkins et al (19U7) showed that the typical increase
in urinary glucose and nitrogen output in these treated animals is not
affected by the method of administration, i.e. oral versus parenteral*
CHAPTER II
STATEMENT OF PROBLEM
To study the metabolism of carboxyl and methylene
labeled glycine in the intact rat, in order to determine the extent
to which each of the carbon atoms of glycine is incorporated into
glycogen, and the position in the glycogen molecule in -which each of
the carbon atoms of glycine is found*
CHAPTER III
MATERIALS.
The present problem involving the feeding of isotopic compounds
which were not available for purchase* necessitated the synthesis of
these compounds from simpler starting materials which were available*
All C-^ radioisotopic compounds mentioned in subsequent paragraphs were
obtained through the permission of the Atomic Energy Commission by proper
application* The glycine labeled in the carboxyl and methylene positions
was synthesized by the method of Amatuzio and Armstrong (19L8) and is
essentially that described by Tolbert (19L8) and Ostwald (19U8) with
slight modification. The procedure was selected on the basis of the
available starting materials* yield* apparatus required and simplicity
of method which was of considerable importance due to the contamination
danger in working with radioactive carbon. The synthesis was effected
starting with the labeled sodium acetate which was converted to the
free acid using heat and dry HCl gas* the acetic acid subsequently being
halogenated to the raonochloro derivative by direct chlorination with a
final conversion of the monochloroacetic acid to glycine by ammoniation
using a large molar excess of ammonia. The apparatus (Figures I and II)
used in the synthesis is essentially the same as that described by
Amatuzio and Armstrong (19^8). It should be noted* however* that a
slight modification in the apparatus was required due to the high
volatility of the acetic acid. This modification* the insertion of an
Reaction vessel
1. Acetic Acid Chlorination Apparatus*
Fig* 2. Apparatus for the Anunoniation of Chloroacetic Acid*
13
extremely low temperature surface condenser at the top of the water
condenser (Figure I), was made to increase the efficiency of the condensing
system and cut the contamination hazard* The glycine so obtained re
presented an overall yield of 25-30 per cent based upon the weight of the
sodium acetate used. This yield checked by both counts recovered and
weight. The specific activity of the final product was 2100 and 2300
counts per minute per mg. of carbon for the carboxyl and methylene
labeled compounds respectively* The specific activity is based upon
the infinite thickness count-*- of a sample of the glycine which was diluted
with carrier and oxidized to CO2 a.nd counted as barium carbonate.
The C-^ labeled sodium bicarbonate used in the carbon dioxide
fixation control experiment was made fbom C - * - ^ labeled barium carbonate#
The method of preparation and apparatus used is essentially that described
by Alien* Gest and Kamen (19147)* The carbon dioxide liberated from the
barium carbonate by the action of lactic acid contained in the vial is
absorbed in an alkaline solution in the opposite arm of the apparatus*
The resulting solution* containing a calculated quantity of sodium
hydroxide such that the liberated CO2 forms principally bicarbonate, is
adjusted to the correct pH if necessary and the sodium bicarbonate
precipitated from the solution with acetone. The quantative and radio
activity analysis indicated a product in accordance with theoretical
values and having an activity of the desired amount.
The animals used in all experiments were normal male albino
rats ranging in weight from ninety to one hundred and fifty grams and
varying in age from thirty to ninety days. The animals were raised on
1. To be explained in Chapter IV on Methods,
lU
a stock diet of Purina Laboratory Chow and were chosen at random from
stock litters of the Scripps Metabolic Clinic Research Department
Experimental Animal Vivarium* All animals were derived from a strain at
least fifty generations old and were thoroughly examined for irregularities
or sickness before using. The fasted animals were placed on wire racks
within the cages at the onset of fasting. The food bins being removed,
the animal then had no source of food since the sawdust and excreta were
inaccessible. Water was given ad libitum during the fast. As a means
of checking the fast, the animals were weighed immediately before and
after the fasting period for determination of loss in weight.'
For the administration of glycine and the bicarbonate to the
animals, a syringe was equipped with a No. 17 needle approximately
tv/o inches in length, which had been made blunt by adding a small
quantity of solder around the periphery. This arrangement was found to
be far superior to the rubber catheter both with respect to time and
ease of handling necessitated by radioactivity contamination hazards.
With this arrangement it was an easy operation for one person to hold
the animal, insert the needle into the stomach and depress the syringe
plunger.
In the feeding experiments with the radioactive materials, it
was necessary to use a respiration chamber to collect the desired fractions
of carbon dioxide expired during the course of the experiment. For
this, the apparatus (Figure III) described by Armstrong et al (l?i|8)
was used. The air entering the respiration chamber is freed of carbon
dioxide by passage through 10 per cent sodium hydroxide in the first
15
Fig. 3. Respiration Chamber.
16
large flask (A) and the second smaller flask (B) of barium hydroxide,
and is then humidified by passage through the flask (C) of saturated
sodium chloride. Blank runs were made in order to test the efficiency of
the carbon dioxide absorbing system. Results of these runs showed the
amounts of carbon dioxide to be so low as to not appreciably effect the
results when collecting the expired carbon dioxide of the animal. The
animal during the course of the experiment is kept in the cage in the
chamber (D) having two outlets, one at the top of the chamber and the
other leading from the chamber via the funnel and filtering flask (E)
arm to the three-way stop cock (F) so that no expired carbon dioxide will
be trapped in the apparatus. The expired carbon dioxide mixed with air
then passes through the absorption flasks (G or Gf) where it is absorbed
in a two normal sodium hydroxide solution, the second flask of saturated
barium hydroxide acting as an indicator. The excreta, feces and urine,
are collected in the test tube in the filtering flask (E) via the funnel
in the chamber (D), The air flow through the apparatus is maintained by
application of a vacuum to the absorption flasks (G or G’),
Since the various end products of the metabolism of the glycine
were to be analyzed for radioactivity and a comparison made on the
activity of the barium carbonate samples resulting from oxidation, it was
necessary to modify the dry combustion apparatus. The modification (Figure
IV) consisted of the insertion of an absorption flask as shown, the
remainder of the apparatus being unchanged, i,e, a conventional dry
combustion train. This permitted the direct precipitation of the absorbed
carbon dioxide as barium carbonate which was centrifuged and weighed in
Fig* U. Combustion Train CO2 Absorption Flask.
18
the flask without the necessity of any transfer of the precipitate, thus
reducing losses and contamination hazard.
The measurement of radioactivity of the various samples was
made with a Geiger-Mueller Counter. The counter consisted of a bell
type end window Geiger-Mueller tube having a thin mica window (Tracerlab
Model TGC-2, density s 1.98 mg/cm^) encased in a protective lead housing
unit, a preamplifier and the counting and scaling circuit (Model GSU
National Technical Laboratories ). The planchetts, metal dishes for
holding the material to be measured, consisted of rectangular pieces of
aluminum sheet metal having a circular central depression or well of
sufficient depth that an ''infinitely thick"^ layer could be obtained*
This type of a device for holding the material to be counted was decided
upon after much experimentation with various types of planchetts since
it was found to be the most efficient, economical and easiest to handle
when preparing samples. Although this may seem an insignificant point,
it should be remembered that the entire problem depends upon the radio
activity measurement and any factors tending to alter it could be of
serious consequence. Similarly a more efficient counting device was
not available which could have improved the overall efficiency and
permitted the use of a less potent feeding material. The counting
efficiency of the apparatus described above was not greater than three
per cent which is in accordance with other investigators' reports
using the bell type end window external counter with C^, a weak be£a
emitter. This was determined by diluting a sample of barium carbonate
of known activity obtained from Oak Ridge* In all cases the counting
2. The meaning of the term "infinitely thick" will be discussed in
Chapter IV*
19
ms reproducible to within four per cent as the outter limits but in
the error
most cases/does not exceed one per cent even from one day to the next,
CHAPTER IV
METHODS AND RESULTS
Since the study is based upon the results obtained from
feeding experiments with glycine, which upon administration leads to
the production of glycogen at a peak level after a fasting period
(MacKay et al 19U0), it wgs necessary to run a series of control
experiments. The purpose of these experiments was to determine the
conditions such that a maximum amount of liver glycogen be produced
over the fasting level which had to be consistantly low. Similarly a
control experiment was needed to establish the length of time after the
glycine administration at which the glycogen reached a peak level. The
actual conditions of experimentation in these experiments, to be described
below, were for the most part those of MacKay et al (191*0) and for this
reason should in a sense be classified as confirmatory experiments.
However, the results were not in complete agreement with the previous
work and, therefore, a slightly different set of conditions was*': established.
In the first of the control experiments, three groups of
seven normal male albino rats weighing from 118 to 137 grams and ranging
in age from forty to fifty days old were fasted twenty-four, thirty-six
and forty-eight hours respectively plus an additional sixteen hour period
for each group to simulate the experimental feeding period. At the end
of each period the animals of the group were sacrificed, after nembutal
anesthesia, and the livers extirpated and dissolved in hot thirty per
21
cent potassium hydroxide. The glycogen in the resulting solution was
determined on an aliquot by the method, of Good, Kramer and Somogyi (1933).
The results (Table l) clearly demonstrate the far more consistent values
with the twenty-four plus sixteen hours fasted group which is to be
desired, as is the low level of 0.07 per cent glycogen obtained with
these animals. Neither the forty-eight or the thirty-six hour animals
were sufficiently consistent ar at a low enough level for consideration*
Having established the length of fast, it was then necessary
to run the second control experiment for the purpose of determining the
length of time for peak level glycogen. Three groups of six normal male
albino rats weighing from 10U to 129 grams and approximately forty days
old were fasted twenty-four hours and at the end of the period the animals
of each group were given one cubic cent Dimeter of a 3M solution of glycine
per square decimeter of body surface and placed in a cage on racks as
described above. The body surface area from gross body weight was cal
culated by the formula of Carman and Mitchell (1926). At the end of
twelve, fifteen and eighteen hours respectively the groups were sacri
ficed and the livers treated and glycogen determined as in the first
experiment described above. The data (Table II) for the experiment
clearly places the highest level with the eighteen hour group, the level
of 1.27 per cent being nearly twice as high as either of the other two
groups of animals. This level represents a net increase of 1.2 per cent
in liver glycogen over the fasting control value. It should be mentioned,
however, that the findings do not preclude the possibility of there being
a peak liver glycogen level at, for example, twenty or twenty-two hours
22
TABLE I
CONTROL EXPERIMENT I
Length of
Fast (Hrs.)
2li*
36'
1 *8*
CiYCOGEN LEVELS AFTER VARYING LENGTHS OF FAST
Liver Weight Glycogen Per Cent Liver
limal in Grams in Mgs* Glycogen
908 Lu2 0 0
25
5.2 2 o.ol*
923 5.0 2 o.ol*
937
U.2
h
0.10
878 u .u 0 0
98 7 u.2 11 0.26
Average m 0.07
986 U.ii
12
0.27
907 U.7
22
0.1*7
879 U.3 3k 0.79
995 3.9 9 0.23
883 u .u 1 0.02
932 3.9
20
0.51
968 U .l 2h 0.59
Average » 0.1*1
h 3.9 Ik 0.36
99k 3.7 7 0.19
936
3.7
l i O
1.03
993 3.9 16 0.1*8
971
3.2
15 0.1*7
909 3.6
h
0.11
981
3.9
16 0.1*2
Average = 0.1*3
■ i f
Plus an additional 16 hours simulated feeding period
TABLE II
23
CONTROL EXPERIMENT II
LIVER GLYCOGEN VALUES AT VARIOUS TIMES AFTER
GLYCINE ADMINISTRATION
No, of Hours
After Glycine
Administration* Animal
Liver Weight
in Grams
Glycogen
in Mgs.
Per Cent Liver
Glycogen
12 88 6*3 55 0.87
85 5.5 85
1.6
111 5.0 b2 0.8U
82 k.9
16
0.33
101 5.6 53 1.05
83 5.5 31 0.56
Average a 0.87
15 Qb
6.1 2 0
Ilk 5.3
80
1.5
78 5.9 Ul 0.7
100
5.1
U3
0.9b
92 5.0
33 0.66
10b
Iu6 36 1.0
Average * 0.80
18
95 5.1*
103 1.9
37 5.5 39 0.71
77 5.2
61 1.2
86
l * . i * 52 1.2
76 5.3 91 1.7
113
5.6 5o 0.9
Average a 1.27
*lcc. of 3M glycine per sq, dm, of body surface administered by stomach
tube to 2b hour fasted male rats*
2k
though this is probably not the case as indicated in the evidence by
MacKay et al (19U0),
The radioactive glycine feeding experiments were run after
establishing the above control glycogen levels. The first two experi
ments were carried out using the carboxyl labeled glycine and a
second set of two experiments using the methylene labeled compound under
identical conditions. The experiments consisted of feeding one cubic
centimeter of 3M radio glycine solution per square decimeter of body
surface to a normal male albino rat weighing approximately one hundred
grams ^uid approximately forty days old. The solution was administered
to the rat, which had been previously fasted for twenty-four hours, by
stomach tube using the arrangement described above. The animal was then
placed in the respiration chamber described (Figure III, page 15),
equipped with a water bowl for drinking and the air flow into the chamber
started. The expired carbon dioxide was absorbed in a ten-fold excess
of 2N sodium hydroxide catching any CO2 which might spill over from
excessive air flow in the second flask containing a saturated barium
hydroxide solution. The air flow was switched every two hours to a new
set of flasks and the previous two hour sample of expired carbon dioxide
in solution removed, the solution poured into a 250 ml, centrifuge flask
and the carbon dioxide precipitated as barium carbonate by the addition
of an equivalent of UN ammonium chloride solution and a two-fold excess
of 3N barium chloride solution rinsing the absorption flask with each of
these solutions. The precipitate was centrifuged after standing at least
twenty minutes, washed three times with C02*-free water and once with
25
ethanol and dried in a desiccator in vacuuo. The dried BaC03 was then
scraped from the flasks and transferred to a tared sample bottle.
After seventeen hours in the chamber, the rat was anesthetized
with an intraperitoneal injection of 0.5 cc. of a five per cent nembutal
solution and sacrificed. The body was divided into three fractions, after
collecting the blood by draining as much as was possible from the dorsal
aorta using potassium oxalate to prevent clotting. These three fractions,
the liver, the viscera (all internal organs except the liver) and the
carcass, were immediately weighed and placed in hot thirty per cent KOH
solution and heated further oh a hot plate until completely dissolved.
The flasks were then placed on a steam bath until ready for further
treatment. The urine and feces, if any, were collected in a test tube
beneath the funnel which was removed and filtered into a tared round
bottom flask to remove fecal matter and the funnel and cage rinsed with
at least 100 ml. of distilled water which was similarly filtered into
the round bottom flask. The urine solution was then lyophylized without
further treatment and the total solids determined by weighing the residue.
The glycogen determinations were made on an aliquot of the
solution of the tissue by the method of Good et al (1933). The glycogen
was isolated from the remaining solution by precipitation with 1.1
volumes of 95 per cent ethanol in a 250 ml. centrifuge flask. The
solution was heated to boiling in a hot water bath and the precipitated
glycogen centrifuged*down. This precipitate was washed with sixty per
cent alcohol and was then purified by extraction of the glycogen with
three portions of ten per cent trichloroacetic acid solution and
26
precipitation of the glycogen from the extract with l.J volumes of absolute
alcohol. The precipitate having been washed three times with sixty per
cent alcohol was dried in vacuuo. Results of purity determinations
(Table III) on the isolated glycogen by the method of Good et al (1933)
using standard glucose solutions proved the glycogen to be, with but
two exceptions, no less than ninety-eight per cent pure, these exceptions
being a body glycogen sample of fifty-five per cent and a liver glycogen
of eighty-eight per cent which when purified by reextraction both gave
values above ninety-eight per cent.
The isolation of the lipids was carried out on the acidified
residue from the glycogen isolation plus washes which had been concentrated
on a steam bath. The lipid fraction consisted of all petroleum ether
soluble lipids but consisted mainly of fatty acids from hydrolyzed fat.
*
The procedure included the transfer of the residue from evaporation to a
250 ml. centrifuge flask, neutralization of the residue with $0% sulfuric
acid solution after overlaying with petroleum ether, extraction of the
neutralized residue with petroleum ether three times and finally drying
the extract over anhydrous sodium sulfate with filtration and removal
of the petroleum ether under N2 on a hot water bath.
Oxidation of the glycogen and urine samples in preparation
for their counting as barium carbonate was achieved through dry
combustion of the respective samples in the modified combustion train as
described above (Figure IV, page 1?) by the method of Pregl and Grant
(19U6). Lipids were not oxidized due to their low activity which would
be insufficient for counting if converted to barium carbonate.
TABLE III
27
PURITY OF THE GLYCOGEN ISOLATED FROM THE
FEEDING EXPERIMENTS
Glycogen
Sample
Experiment^
Notes
I II III IV V
Liver 100 100
— —
88 100 100
Values in Table are in
per cent purity based on
a standard glucose solution.
Carcass 53
100 98
97
98
The carcass glycogen in
experiment I. was repurified
and checked before oxidation.
*See footnote Table V.
28
Results of the isolation procedures for the four experiments
is summarized on Tables V and VI. Table IV gives the liver glycogen
control values for Experiment II. It may be noted that the liver glycogen
levels in all four cases were very close to the original control level
established. In some cases the quantity of glycogen isolated from viscera
was too small for oxidation and had to be counted by mounting the glycogen
on the planchett per se which is described below#
Radioactivity measurements were made on the barium carbonate
and lipid samples mounted on the metal planchetts previously described
(Chapter III) and prepared by the following procedure# The barium
carbonate was mounted by grinding the powder in an agate mortar under
9$% ethanol for a few minutes and allowing the course particles to
settle, the fine suspension so obtained was then taken up in a capillary
transfer pipet and dropped onto the previously tared planchett and dried
under an infra red heat lamp. By repeating the process a layer of
desired density could be obtained, In all cases where at all possible
the carbonate planchetts were prepared at infinite thickness in order
that no self absorption correction factor be needed in calculating the
specific activity. However, in some cases there was insufficient material
for an infinitely thick mount and the density had to be calculated and
the correction term applied. To obtain this factor a self absorption
curve was used which was prepared by counting a series of planchetts
mounted at different densities by the same method and the activity
plotted, against the corresponding density (Figure V). Lipid samples
were mounted by placing a sufficient quantity of the solid lipid for
TABLE IV
29
LIVER GLYCOGEN LEVELS FOR CONTROL ANIMALS
OF EXPERIMENT II
Group
II
Animal
222
203
21h
20k
213
228
216
211
2U U
225
251
223
198
Liver Weight
in grams
3*6
3.8
3.9
3.2
2.8
3.8
3.8
U.2
U.7
5.2
U.o
U.2
U.3
Glycogen
in mgs.
1.8
2.8
1.2
3.U
2.2
3.U
l.U
U 9
80
56
65
U7
62
Per Cent Liver
Glycogen
0.05
0.07
0.03
0.02
0.08
0.09
0.0U
Average * * 0.05
1.17
1.70
1.08
1.60
1.10
1.U0
Average = 1.3U
^Group I received nothing except water ad libitum at the start of the
experiment. Group II were administered 1 cc. of 3M glycine (inert)
solution per sq. dm. of body surface. All rats were prefasted for
2U hours.
TABLE V
30
FRACTION RECOVERIES FROM RADIOACTIVE GLYCINE
FEEDING EXPERIMENTS
Experiment*
Glycogen Lipids (gms.) Urine
Solids
(gms.) Liver Body Viscera Liver Body Viscera
I
mg.
%
mg.
%
mg.
%
0.132 3.U80 0.762 0. 1*38 122 2.9 167
0,22 8.8 0.07
II Ui 1.114. Ihh
0.20 11 0.09 0.13U
5.U20 0.860
0.1*97
III 33
0.92 130 0.17 30 0.29 0.120 U.388 0.731 0.1*59
IV 70 1.32 216 0.19 23
0.12 0.168 6.212
2. 1U U 0.560
V 58 1.57 165
0.22
— -------
— -----
U.6S6
--------
0.373
^Carboxyl labeled glycine administered in experiments I and II, methyl
labeled glycine in III and IV and inert glycine plus C^ bicarbonate
in experiment V.
TABLE VI
31
EXPIRED CARBON DIOXIDE RECOVERIES
Experiment*
Expired Carbon Dioxide Per
Period*** as BaCOj (grams)
1 2
3 I * 5
6
7
8
I 1.371 1.572 1.585 2.651* 2.601* 2.118 2.617 2.310
II 1.312 1.885
2.301 2.388 2.681* 2.71*6 3.062
3.633
III 1.1*11* 1.666 1.552 2.182 2.207 1.667 2.331* 2.613
IV 1.669
2.51*7 2.31*1 3.71*3 2.923 2.610
2.252
1*.260
V
1.551*
1.810 1.51*7 1.61*2 1.51*9
2.115 1. 8U1
3.386
.
Carboxyl labeled glycine administered in experiments I and II, methyl
labeled glycine in III and IV and inert glycine plus C-^ bicarbonate in
experiment V.
Each period represents a two hour interval except period 8 which was
three hours*
FRACTION OF MAXIMUM ACTIVITY
* * d
H *
C'Q
VR
W
0 3
o
H
, °
Vo
C O
CD
H
V *
&
C O
o
2
H *
O
3
o
o
» - s
CD
o
c +
H -
O
O
£
<
CD
FRACTION OF MAXIMUM SPECIFIC ACTIVITY
Vo
ro
33
infinite thickness in the planchett well and then placing the planchett
under an infra red heat lamp which melted the solid material forming a
smooth layer. The planchett was then removed from the heat lamp and was
ready for counting when the lipid material had solidified. An absorption
curve was similarly obtained for lipids but in no case was an absorption
correction factor needed since all lipid mounts were at infinite thickness.
The actual counting of the dried planchetts was done after the
counter had been checked with a standard beta ray source and a background
of at least one hour counting taken. Each planchett was counted for
a total of at least ten thousand counts except in the case of extremely
low activity* e,g, lipid samples where the sample was counted for at
least one hour. Background counts taken before and after completing
the counting checked within one count per minute in all instances. The
counting data (Tables Vila* b* c, and VIII) includes the total calculated
counts based upon the total weight of glycogen* lipid or barium carbonate
from expired carbon dioxide* the percentage of total counts administered*
the specific activity which is the counts per minute per milligram of
carbon on the planchett and the relative specific activity* i,e, relative
to the fed material placed at an arbitrary value of 100 for easier
interpretation. The specific activity of the fed material is based
upon the activity of the barium carbonate obtained upon oxidation of
a diluted sample by dry combustion as described above. The total counts
fed* included at the bottom of Table Vila* b and c* are similarly
calculated on the basis of the total weight of glycine administered.
This calculation which applies to any of the radioactive samples is
TABLE Vila
RADIOACTIVITY DATA AND BALANCE SHEET FOR THE CARBOXYL
LABELED GLYCINE ADMINISTRATION1 EXPERIMENT
Fraction
Glycogen Lipids Expired
Carbon Dioxide
Urine
Liver Carcass Viscera Liver Carcass Viscera
Experiment I II I II I II I II I II I II I II I II
Counts per Minute
per Planchett^ $68 829 189 107 16k 67
----
2
5 k 9 8
*2 *2
25UO
2993
Total Calculated
Counts 6780 3810 3160 1630 315
12$
----
h
230
287 91 91 235,000 273,000 73,000 83,000
Per Cent of Total
Counts Adm*4
1.5 0.85 0.70 0*36 0.07
0.03
----
<
0.01 .0$ .06 .02 .02 52.3 60.6 16.2
18.5
Specific Activity 123 180 ia 23
36
15
----
0,01 .21
.17 .37 .33
*2
.*2 5 1i 8 630
Relative Specific
Activity^ 5.8 8.5 1.9 1.1
1.7 0.7
<
0.01 *10 .08
.17
.16
*2 #2
26
30.7
1. Specific activity of fed glycine = 2100; x 10^ total counts were administered*
2* Refer to Table VIII. ( = ~
3* All activity corrected to infinite thickness count*
? 4* For calculation see Chapter III.
$. Specific activity of the material relative to the administered glycine taken as 100*
TABLE Vllb
RADIOACTIVITY DATA AND BALANCE SHEET FOR THE METHYL
LABELED GLYCINE ADMINISTRATION1 EXPERIMENT
Fraction
Glycogen Lipids Expired
Carbon Dioxide
Urine
Liver Carcass Viscera Li've r Carcass Viscera
Experiment III IV III IV III IV III IV III XV
III IV III IV III IV
Counts Per Min.
Per Planchett? 2900 2050
373
211 - 2 1 * 1 * 10 20 58
! 1 1
1A
I
1
1 *2 28 *2
*2
3396
1037
■Total Calcu
lated Counts 11,000 13,1*00 1*600 2 1 *1 *0 - ?2? 16 32 3 1 + 1 + 0 1+3*6 1*06 270 127,000 178,000 76,000 28,800
Per Cent of Tota
Counts Adm.^ 2.2 3.0
0.9 0.?
- 0.1 0.01
<
0.01 0.61+
0.85 0.08 0.06
2? 1 *0
lit.9 6.5
Specific
Activity 630
1 * 1 * ? 31 1 *6 -
?3
0.18 o.i* 1.0 1.3 0.7? o.so
■ *2 *2
7*0
225
Relative Spec.
Activity?
27 19 3.?
2.0 -
2.3
0.01 0.02 0.01+ 0.06
0.03 0.02
#2
#2
32 11
1. Specific activity of the fed glycine = 2300$ Total counts administered » 5*1 x 10? in Expt. III \ x >
and l * . l * x 10? in Expt. IV. ^
2. Refer to Table VIII.
3. See footnote 3., Table Vila.
1 * . See footnote 1 * * , Table Vila.
5. See footnote 5., Table Vila.
TABLE VIIc
36
RADIOACTIVITY DATA AND BALANCE SHEET FOR THE C1^ BICARBONATE
AND INERT GLYCINE ADMINISTRATION! EXPERIMENT
Fraction
Glycogen Lipids Expired
Carbon Dioxide
Urine
Liver Carcass Carcass
Counts Per Minute
Per Planchett^
3U5 1U7 3
1U23
Total Calculated
Counts
1950 2L6 185
386,000 29,000
Per Cent Total
Counts Adm,k 0.U3 0.5U 0.1*1
86 6.U
Specific
Activity 75
32 0.05
*2
310
1. See Chapter III on Methods for amounts administered,
2. Refer to Table VIII,
3. See footnote 3*} Table Vila,
U, See footnote lw, Table Vila*
TABLE VIII
RADIOACTIVITY DATA AND BALANCE SHEET FOR THE EXPIRED
CARBON DIOXIDE FRACTION
Experiment
Period^ C-^ Carboxyl Labeled Glycine
Administration
C"^ Methyl Labeled Glycine
Administration
C Bicarbonate Plus Inert
♦Glycine Administration
Expt, I Expt. II Expt, III Expt. 17 Expt. V
CP2 Sa3 rsa1 * CP2 Sa3 rsa* * CP2 SA3 rsa1 * CP2 sa3 rsa^ CP2 Sa3 rsa^
1 1383 301 l l * 11*83 325 16 209 1 * 5
2
253 55 3 1738 378 -
2 2100 1*56 22 2398 520 2 1* 581 126 6 668 11*5
7 1653 360 -
3 1903 1*15
20
2 1 * 1 1 * 525
2 1 * .
935 203 9 928 200 9 3133 680 -
h 1508 328
15
161*8 356 17 1093 238 10 101*3 228 10 5027 1100 -
5
802 175 8 679 ll*8
7
916 199 9 803 175 8 2913 630 -
6 628 137 7 37U 81 1 * 51*1 118
5 1 * 1 * 3 96 5 1323 288 -
7
1*81 105 5 259 56 3 1*15 90 1 * 307 67 3 791 172 -
8 375 81 1 * 185 33
1
307 67 3 227 1 * 5
2 1 * 1 * 8 98 -
1. Each period represents a two hour time interval of collection except No* 8 of three hours*
2. Counts per planchett (at infinite thickness),
3. Specific activity « counts per mg, of carbon on the planchett at infinite thickness.
U. Relative specific activity a specific activity relative to the fed glycine taken as 100*
38
as follows:
Total Counts - (f P°'kal Mgs, of Substance ) X (Counts/Min. at Reference Density)
(Mgs* of Material on Planchett at Reference Density)
the reference density being infinite thickness in this study but can be
*
any density desired if used consistently throughout with reference to
an absorption curve for corrections# The relative specific activity
is self explanatory and needs no further explanation than that cited above*
The experiment on carbon dioxide fixation was carried out on
a normal male albino rat weighing one hundred grams and fasted for
twenty-four hours# The animal was given at the end of this fast 1 cc,
of 3M glycine solution (inert) per square decimeter of body surface
and at the same time administered also by stomach tube a quantity of
radioactive sodium bicarbonate solution# This bicarbonate solution
was given in addition to the start at the end of each two hour interval
from the start by opening the chamber and removing the rat# The solution
was of such concentration and contained sufficient activity that no more
than three milligrams of sodium bicarbonate were in any one dose and were
contained in not more than 2.0 cc. of solution in order that the physio
logical effect be minimized. The quantity of bicarbonate to be adminis
tered during each period was determined by the number of counts excreted
in the corresponding period when the radioactive carboxyl labeled glycine
was administered, the number of counts being corrected by dividing by
the factor 0#60 since 60$ of the counts were recovered in the expired
39
carbon dioxide in that experiment, AH fractions taken at the end of
seventeen hours including the urine and expired carbon dioxide were identical
to the glycine feeding experiments, as was the analysis of the respective
fractions. Results of fraction isolations and activity analysis of the
carbon dioxide fixation experiment are included in the data for the
glycine feeding experiments (Tables I?, V, VI, VIIc and VIII)*
In order to more fully understand the metabolic pathway of the
administered glycine, it was decided that the glucose unit of the glycogen
molecule would have to be degraded. For this procedure the Yeast
Fermentation Method was chosen as it was the least time consuming and
just as efficient. The neutralized glucose solution resulting from the
hydrolysis of the glycogen with 0.6N HC1 was incubated in a 2f>cc.
Erlermeyer flask containing a vial held in place by a glass rod through
the flask stopper. The incubation media consisted of (1) the glucose
solution (10 mg, of glucose per cc.), (2) one cc, of 0.5>N KI^PO^ per cc*
of glucose solution and (3) sufficient washed yeast cells (bakers yeast)
to make a final concentration of 2% cells. The vial contained 1 cc. of
2N NaOH for absorption of the carbon dioxide released. The incubation
flask was placed in a Warburg bath at 37°C and the fermentation started
after first gassing the flask for E > minutes with nitrogen. The fermentation
was complete at the cessation of CC> 2 evolution as determined manometrically
by a control vessel consisting of a regular Warburg flask containing an
inert glucose solution, yeast cells and buffer of the same concentration.
At the end of the fermentation the flask is removed and the CO2 precipitated
from the solution in the vial as barium carbonate, this fraction representing
carbon atoms 3 and U of the glucose molecule* The alcohol contained in
the fermentation mixture was then recovered by distillation of the mixture
after removal of the yeast cells by centrifugation* The alcohol was
converted to acetic acid by oxidation with a chromic-sulfuric acid
mixture (0.68M K^CrO^ in 1:1 I^SO^) at a temperature of 80°C for h$
minutes. The acetic acid, containing carbon atoms 1, 2, £ and 6, was
recovered from the oxidation mixture by steam distillation and the
distillate neutralized with Ba(0H)2 solution and evaporated to dryness
on a steam bath. The barium acetate was then degraded by pyrolysis
by the method of Calvin et al (19U9) to give the carbon atoms 2 and 5 of
the glucose as barium carbonate and atoms 1 and 6 as acetone, which was
precipitated as iodoform and counted. The results (Table IX) are to
be compared with the specific activities of the starting glycogen
samples (Table Vila, b and c). All specific activities listed are
based upon infinite thickness counts using correction factors when needed.
TABLE IX
ill
DEGRADATION EXPERIMENTAL DATA
Specific Activities^-
Sample Glycogen Atoms
3 and U
Atoms Atoms2
1,6
Atoms^
1,6 and 2,£
Carboxyl Expt* Glycogen (CGA) 123 U30 - - 0
Methyl Expt. Glycogen (MGA)
Uh$ 160 590 U5o 580
C*^ Bicarbonate Expt. Glycogen (BA)
7 5
210 -
. 0
1. All values listed in the table are the specific activity in counts per minute*
, per mg* of carbon*
2. Counted as CHI^.
3* Counted as Barium acetate*
CHAPTER V
DISCUSSION
Tables Vila, b, c, VIII and IX, the tabulated activity data
for the experiments described above (Chapter III), constitute the basis
of the ensuing discussion. Since the relative specific activity (RSA)
of the respective fraction, i.e. glycogen, lipid, urine or expired
CO2* is an expression of the counts (disintegrations) obtained per
unit weight of carbon relative to the specific activity of the fed
material at the reference counting thickness, it is with these values
we are particularly concerned in evaluating the data. Hence the
degree of incorporation or per cent uptake of the carbon atoms of the
material administered can be measured through a comparison of the RSA
of the starting material, e.g. glycine or bicarbonate with the RSA
of the metabolic end product (glycogen, lipids etc.). However, in
interpretation of the degradation data (Table IX) it whould be
remembered that the comparison of the starting and end product materials,
i.e. the individual carbon atoms, presents a slightly different problem
since a molecule is being broken down into its constituent parts.
From a study of the tables, it should be noted that there are
several highly significant differences. A comparison of the RSA of the
liver glycogens from the carboxyl labeled glycine administration (CGA)
with those obtained from the administration of methylene labeled glycine
(MGA) shows a nearly four-fold activity in the latter over the former
U3
with an almost two-fold activity of the carcass glycogens over those carcass
glycogens from CGA. Similarly the RSA of the liver glycogen obtained from
the administration of inert glycine plus radio bicarbonate (BA) is not to be
overlooked since it is of considerable importance in the subsequent discussion
of carbon dioxide fixation. In terms of incorporation of carbon atoms of
the glycine administered, the data indicates the utilization of one methyl
carbon atom with every four carbon atoms being incorporated into glycogen
in the case of the MGA with a similar utilization of one carboxyl carbon
atom in every fifteen with CGA. The latter value, though in the range, is
twice the utilization found by Olsen et al (19U3) who could account for only
one out of every twenty-nine carbon atoms being incorporated into glycogen
as arising from the fed glycine carboxyl carbon.
The relatively high RSA of the glycogens from the CGA and the
BA are indicative of several things. First, the relatively high activity
of the latter means that there is definitely an incorporation of carbon
atoms into glycogen by carbon dioxide fixation alone, these results
being compatible with the present concepts of CO2 fixation. In addition
the activity is of such order of magnitude that it cannot be concluded
that all of the carbon atoms of the glycogen arising from carboxyl glycine
are not incorporated by CO2 fixation. Even under the carefully controlled
conditions and the seemingly high C^Og available for CO2 fixation as
shown by the rate of C-^02 expiration (data Table VIII) many control
experiments of this type would have to be run in order to establish an
accurate control level which could probably never be ideal. Examination
of the data (Table IX) from the degradation experiments indicates further
Uli
that the probable pathway of incorporation of the carboxyl carbon atom
of glycine into glycogen is by carbon dioxide fixation a^-one since all
of the activity of the glycogen by CGA is found in carbon atoms 3 and U
of the glucose molecule (carbon atoms 3 and k represent one-third of the
total atoms present, therefore, they should have an activity three times
as great as the total molecule if they contain all of the activity)#
The relatively low uptake of carboxyl carbons of the fed glycine
compared to the uptake of labeled methyl carbon atoms suggests separate
pathways of incorporation of the respective atoms with limited incor
poration of the carboxyl and methyl carbons directly as a two carbon
fragment using the classical Krebs cycle as the pathway to glycogen
via pyruvate. That this is the case is even more strongly reflected in
the rate of carbon dioxide elimination and total recovery resulting
from the administration of carboxyl labeled glycine as compared to that
obtained from MGA.
Refering again to Table IX, it may be noted that the RSA of
the carbon atoms 3 and U obtained from the degradation of MGA liver
glycogen contained only one-sixth or less of the RSA of the source glycogen
which may be interpreted as additional evidence of a split in the glycine
molecule with each fragment (carboxyl or alpha carbon) entering the
carbohydrate cycle via a separate route. On the basis of the activities
of the remaining carbon atoms of the MGA glycogen it is possible to
postulate several possible pathways of incorporation of the methylene
carbon atom into the glycogen molecule. The high RSA of carbon atoms
1, 2, 5 and 6 immediately suggest that the methylene carbon was incorporated
U5
into the glucose unit by more than one pathway or mechanism. To account
for this, it is necessary to recall the work of Sakami (19it.8, 19k9) who
showed the in vivo conversion of glycine to serine (and glycogen) by
condensation with a formate residue which by the later work was shown to
be capable of having been derived from the methyl carbon of glycine
itself under certain conditions. Using this evidence along with the
data obtained in the present work which shows an incorporation of the
alpha carbon into the 1, 6 and 2, £ positions in nearly equal amounts
from MGA, it is quite conceivable that the alpha carbon could have been
first incorporated into serine by the proposed mechanism of Sakami (19U8)
and that the serine in turn gave rise to pyruvate and ultimately glycogen.
Supporting evidence for the latter mechanism has been presented by various
investigators including Butts, Blunden and Dunn (1938), Schofield and
Lewis (19U7) and Chargaff and Sprinson (19li3)*
That the alpha carbon gave rise to formate for the condensation
reaction to form serine is evident on the basis of the high or nearly
equal RSA of the 1, 6 positions as compared to the 2, 5» Additional
support for this action is similarly evidenced in the activity found in
the 3) U positions which is more logically explained on a split molecule
basis. Some of the activity in the 1, 6 positions can, however, be
accounted for by the reversible transformation of oxalacetic acid in the
carbohydrate cycle to the symmetrical succinate molecule in which form
the alpha and beta carbons would loose their identity.
This evidence, however, does not preclude the possibility that
other pathways may exist. Important evidence by Lifson et al (19l±8)
U6
presents another possible pathway namely via acetate or a similar two
carbon fragment entering the Krebs cycle through pyruvate to glycogen
or as proposed by Sprinson (l9k9) the serine formed from the glycine and
formate (which could be derived from glycine) upon conversion to pyruvate
(Chargaff and Sprinson 19U3) could give rise to acetate or oxalacetate
by decarboxylation or COg fixation respectively and hence enter the
carbohydrate cycle and glycogen, Sprinson fed alpha labeled glycine and
found upon isolation of acetic acid (and aspartic acid) that the labeled
atom was incorporated into the alpha and carboxyl positions in equal
amounts* Thus is afforded a possible explanation of the presence of
significant activity in the 3S l i positions of the MGA glycogen, the
acetate carboxyl providing the source. Another proposed mechanism by
Sprinson is that oxidative deamination of glycine to glyoxylic acid gives
rise, by condensation of the glyoxylic acid, to a four carbon compound
labeled in the alpha and beta positions which is in equilibrium with
aspartic acid. The aspartic acid could then conceivably give rise to
oxalacetic acid and hence glycogen*
Of considerable importance regarding the above proposed pathways
is the activities of the lipid fractions, especially in the case of the
MGA. All three fractions contained considerable activity in view of the
widely accepted belief in the antiketogenicity of glycine* It would seem
on comparison of the activities of the lipids resulting from the two types
of glycine administration that the five-fold or greater activity of the
MGA over the CGA lipids could be considered supportive evidence for the
acetate scheme of incorporation. Acetate itself being a fatty acid precursor
hi
tends to establish this route as a possible pathway of incorporation of
glycine into lipids. However, it should be noted that no attempt was
made to carry out further fractionation of the lipids which may or may
not be important in view of the fact that acetate has been shown to be
incorporated into cholesterol which would be included in the total lipid
fraction, and thus could carry the activity (the acetate having been
derived from glycine). Although the total counts and RSA of the lipid
fraction appear quite low, their significance is not to be completely
overlooked especially in view of the shortness of the experimental period
in terms of fat deposition.
The activity of the expired carbon dioxide fractions (Table VIII)
brings to light several important points. The high rate of elimination
of C-^02 from CGA with the much slower rate of excretion of 0^02 in the
case of the MGA is indicative of a preferential utilization of the carboxyl
carbon for combustion with a reservation of the methyl carbon for other
metabolic functions. It should also be noted that there is in both cases
a rapid drop in the elimination of 0^*02 after the peak level during
the seventh and eighth hours. This can possibly be explained upon the
basis of mobilization of glycine for other functions, e.g. incorporation
into proteins or formation of carbohydrate. These findings are in
agreement with the work of Olsen et al (19U3) in all instances except
that these workers found a more complete elimination in the first eight
hours. However, this may possibly be accounted for by their inability
to detect the low concentrations of in the later periods.
Finally, an analysis of the balance sheet is to be desired since
U8
without this information it would be impossible to evaluate the experiment
as a whole. First to be noted is the percentage total counts contained
in the material fed which were recovered in a given fraction. Addition
of these values for the individual fractions shows a recovery of forty-
five per cent from MGA, seventy-five per cent from CGA and approximately
ninety-four per cent in the case of BA* The differences in the two latter
recoveries with the former can for the most part be explained by the
great contrast in values for the expired CO2 amounting to approximately
thirty and fifty per cent respectively. When it is remembered that the
total recovery for the MGA experiment is only of the order of two-thirds
that in the CGA experiment and that this recovery represented only one-
half of the material fed, the significance of the high RSA of the glycogen
is even greater. The recovery of counts in the urine is not easily
interpreted since the urine was not fractionated but it is suspected that
the counts are for the most part in the form of urea with some of the
counts as glycine per se. Glycine given in such massive dose could
result in an exceeding of the renal threshold with glycine spilling over
into the urine* That this might be the case is even more strongly
suggested by noting the consistent values for urine recovery regardless
of what type of labeled glycine was administered.
In conclusion it may be said that there is a much greater
utilization of the methyl as compared to the carboxyl carbon atom of
glycine for the synthesis of glycogen in fasted rats and on the basis of
the data, there is apparently a limited amount of direct incorporation
of glycine as a two carbon unit into the glycogen molecule*
CHAPTER VI
SUMMARY
A short historical review of the literature with correlation
of the evidence pertinent to the study has been presented*
The preparation of carboxyl and methylene labeled glycine
has been described using C-^ carboxyl and methylene labeled acetic acid
as the starting material. Overall yields of 2£-30 per cent were obtained.
The carboxyl and methylene labeled glycines were adminis
tered separately to fasted rats and the metabolic end products isolated
and analyzed for radioactivity. The degradation of glycogen was described
and the radioactivity data presented for the constituent carbon atoms
of the glucose.
From the data it was concluded that there is a much higher
utilization of the methyl as compared to the carboxyl carbon of glycine
for the formation of glycogen and that the respective carbon atoms of
the glycine molecule are incorporated to a greater degree via separate
pathways which have been discussed*
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Asset Metadata

Creator Barnet, Harry Nathan (author)

Core Title The comparison of the metabolism of C¹⁴ carboxyl and methylene labeled glycine in the intact rat

School Graduate School

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1949-08

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Mehl, John W. (committee chair), Wick, Arne N. (committee member), Wingler, Richard J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c20-301205

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Legacy Identifier EP41302.pdf

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