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ABSORPTION AND MET ABOLISM OF CARBOHYDRATES BY THE SMALL INTESTINE by Benjamin M. Sahagian, M. S. A thesis submitted to the Faculty of Graduate studies and Research in partial fulfUment of the requirements for the degree of Doctor of Philosophy. Department of B iochemistry, . McGill University, Montreal. April 1963.
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ABSORPTION AND MET ABOLISM OF CARBOHYDRATES
BY THE SMALL INTESTINE
by
Benjamin M. Sahagian, M. S.
A thesis submitted to the Faculty of Graduate studies and Research in partial fulfUment of the requirements for the degree of Doctor of Philosophy.
Department of B iochemistry, . McGill University, Montreal. April 1963.
ACKNOWLEDGEMENTS
1 wish to express my sincere gratitude to Professor J.H. Quastel,
F. R. S. , of McGill University for his stimulating encouragement during the
course of this work under whose direction it was accomplished.
1 would like to thank Professor P. G. Scholefield for his valuable
assistance in the discussion of enzyme kinetics and competitive inhibition.
1 am indebted to Dr. W. Paranchych of The Wistar Institute, for
introducing me to the field of perfusion studies with the small intestine.
1 would like to extend my thanks to Dr. A.B. Haber for his par
ticipation in the discussions of intestinal absorption.
It is my pleasure to thank Mrs. H. Amsel for her excellent care
in typing this thesis.
iv
This work was supported by a U. S. Public Health Service fellowship
(GPM-13, 209-C2). For this assistance 1 am most grateful.
TABLE OF CONTENTS
Page
ABSTRACT .... "' ............................................. . ii
AC.K:N'OWLEOOEMENTS . • • • • . • • . . • • • . • • . • . . . . • • . . • • • • . • • • • • . • iv
TABLE OF CONTENTS . • . • • • • • • • . • • . • • . • . • • . • • • • . • • . • . • • • • • . v
LIST OF TABLES xii
LIST OF FIGURES xv
LIST OF ABBREVIATIONS . • • • • • • • • . . • . . • . • . . . . . • • . • . • • . • . • • . xvii
PARTI:
GENERAL INTRODUCTION
Historical
................................... 1
1
Histology and Morphology of the Small Intestine . . . . . . • . . . • • . • . 8
Absorption by the Small Intestine . . • . . . • . . • . . . • . • • . . • . . . . . . . . • 15
Absorption of fats .................................... Absorption of proteine and amino acids
Absorption of inorganic ions ..........................
15
18
2.6
v
Absorption of sugars .................................
Specificity of the sugar molecule ........................
Transport Mechanisms
Definition of terms
Simple diffusion
Mediated transport or facilitated diffusion .............
Pinocytosis
Carrier active-transport
Page
32
37
43
44
46
47
49
53
Effect of Conformation on Active Sugar Transport . . • . . . • . . • . . • 56
Definition of terms
Minimum requirements for active sugar transport
Isomerism of sugars
Instabllity factors
57
57
58
60
Inhibition Produced by Phlorhizin and Phloretin . . . . . . . . . . . . . . . 67
Nomenclature and chemistry 67
The inhibitory action of phlorhizin . . . . . . . . . . . . . . . . . . . . . 70
Inhibitory action of phloretin . . • . . • . . . . . . . . . . . . . . . . . . . . 7 5
Competitive inhibition by phlorhizin and phloretin . . . . . • . 78
Metabolism in the Intestinal Wall
The metabolism of amino acids ... • ................... . 81
87
vi
vii
Page
PART ll:
ACTIVE GLUCOSE UPT AKE BY THE INTACT-STRIP METHOD AND
COMPETITIVE INHIBITION BY PHLORHIZIN AND PHLORETIN • . . • 89
Introduction 89
Methods and Materials 92
Animals ............................................ 92
Perfusion procedure 92
Perfusion apparatus 93
Intact-strip method 93
Analytical methods 96
Re agents ............................................ 97
Treatm.ent of resulta 97
Experimental 99
1. Uptake of glucose by strips of golden ham.ster intestine.. 99
2. Uptake of glucose by strips of guinea pig intestinJ:l . . • • . • 101
3. Variation of glucose uptake activity along the length of the guinea pig intesti:qe . . . • • • • • • • • . • • • . . • • . • . • 103
4. Uptake of glucose by guinea pig intestinal strips with increase in the time of incubation • . . • • • • . • • • • . • • . . 103
5. Uptake of glucose by guinea pig intestine versus con-centration of glucose in the medium . • • . . • • . • • . • . . • 105
6. Inhibition by phlorhizin of the active uptake of glucose by strips of guinea pig intestine . . • • . . • • . • • . • . • . • . 105
7. Inhibition by phloretin of the active uptake of glucose by strips of guinea pig intestine . • • • • • . • .. • . . . • . • . . 108
8.
9.
10.
11.
Discussion
Summary
PART ID:
Determination of the range of phloretin concentrations which would serve as upper and lower limita of effective action on inhibition of glucose uptake by guinea pig intestinal strips ...•••....••......•.
Calculation of Km and Kt values from data obtained from the preceding experimenta ••..•..•••.••..•
The uptake of glucose by guinea pig intestinal strips using uniformly labelled glucose ..•.•.•.•.•.•••.
Perfusion apparatus for the active transport of labelled compounds •••••.....••.•..•.•...•••.•
................................................
............................................ " ..
THE INTRACELLULAR METABOLISM OF CARBOHYDRATES BY THE
Page
111
114
114
118
121
124
GUINEA PIG INTESTINE AND THE PRODUCTION OF AMINO ACIDS 126
Introduction 126
Methods and Materials ...................................... 130
Animals and procedure 130
Radioactive measurements 132
Ex:p:erimental . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . • . . . . . • 134
1. The amino acid content of guinea pig intestinal tissue preparations . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . 134
2. Study of o2 uptake and co2 output with guinea pig intestinal tissue preparations . . . • • • • • • • • • . . . • • . . 134
viii
Page
3. Synthesis of am:lno acids from glucose-u-c14 by intact-strips of gu:lnea pig intestine . . . • • • • • • . . . . 136
4. Time course study with glucose-u-c14 for the production of amino acids by the guinea pig intestinal strips . . • . . • • • . . • . • • • • • • • . • . . . . . . . . 138
5. Effect of increasing medium concentration ofglucose on production of am:lno acids by guinea pig :Intestinal strips . • . . . • • . • . • • . . . . . . . • • . . • . . . . . 138
6. A comparative study of am:lno ac ids produced by intact-strips. mucosal-sheets and serosal -sheets . . . . . . . . . . . • . . • . . . . . . . . . . . . . . . . . . . . . . 145
7. Comparison of formation of labelled amino acids by mucosal-sheets of gu:lnea pig intestine from glucose, fructose and sucrose . . . • • . . . . • . . • . • . . 149
8. Effect of starvation on the formation of am:lno acids from labelled glucose by guinea pig intestinal tissue . . . . . . . . . .. . . . . . . . .. . .. . .. . . . . . . . . . .. . . . . . .. 152
9. Interconversion of amino acids by intact-strips of guinea pig intestine . . • . • . . • . . • • . . . . • . . . • • . . . . 152
10. Formation of labelled amino acids from labelled acetate and formate
11. Effect of metabolic inhibitors or stimulators on the formation of am:lno acids by the guinea pig
154
intestine from glucose . . • • • . • • . • • • . • • . • . . • . . . . 157
12. The effect of high concentrations of potassium on the rate of formation of amino acids from glucose . . • . 160
13. The effect of addition of glutamine, asparagine or ammonium chloride on the rate of formation of amino acids from glucose by intact-strips of guinea pig intestine . . . • . . . • • • . . • . . • • . . • • . . . . 162
ix
14. The effect of formate on the rate of formation of amino acids from glucose by mucosal-sheets
Page
of guinea pig intestine . . • • . . • • • • . • .. • . . • . . • . . . • . 164
15. The formation of glutathione from glucose by guinea pig intestinal tissue ...••..•••.•••.••. ~ . . . . . . 166
16. Formation of proline from glucose by guinea pig intestinal tissue preparations . . • • . . . • . . • • . . . . . . 166
Discussion 170
Summary . .._ ..................................................... . 175
PART IV:
INFLUENCE OF COLCIDCINE ON THE INTESTINAL MUCOSA OF
THE GUINEA PIG 178
Introduction 178
Methods and Materials ...................................... 180
Preparation of tissues and procedure . . . • . . . . • • . . • • . . . 180
C alculation of results
Mate rials .......................................... Experimental
1. The incorporation of glucose carbon into amino acids, proteine, lipids and nucleic acids by mucosal
182
183
185
-sheets of GPI . . • • • • . . . . • • . . . . . • . . . . . . . • . • . . . 185
2. Formation of labelled amino acids from glucose by mucosal-sheets of GPI after the subcutaneous injection of colchicine . . . • • . • .. • • • . . • • . . • • . • • . . . 185
x
3.
4.
5.
Discussion
Sum.mary
Incorporation of carbon derived from labelled glucose into proteins, lipids and nucleic acids, by mucosal-sheets of GPI after subcutaneous injection of colchicine ........................ .
Formation of labelled amino acids from glucose by mucosal-she.ets of GPI after subcutaneous injection of increased dosage of colchicine ....... .
Incorporation of carbon derived from labelled glucose into proteins. lipids and nucleic acids by mucosal-sheets of GPI after subcutaneous injection of increased dosage of colchicine ..• ~ ...
.................... " ...... " " .................... .
CLAIMS TO ORIGINAL RESEARCH ....... , ................ .
BffiLIOGRAPHY .........................................
xi
Page
188
191
191
194
197
198
203
I
n
m
IV
v
VI
VII
VIII
IX
x
XI
xn
LIST OF TABLES
Uptake of glucose by strips of golden hamster intestme .................................. .
Uptake of glucose by strips of guinea pig intestine
~ value for glucose by ISM: and Ki_ values for phlorhizin and phloretin ....•.•.•..........
Uptake of glucose by strips of GPI using uniformly labelled glucose .............••.•....••..••
The amino acid content of GPI tissue preparations after incubation without added snbstrate .•..••...
Q0 and QCO values obtained with various tissue ~reparati~ of GPI .......•.••••••..•••.•...
Synthesis of amino acids from labelled glucose by intact-strips of GPI ....................•.....
Formation of labelled amino acids from uniformly labelled glucose by mucosal-sheets of GPI .•.••...
Formation of labelled amino acids from uniformly labelled glucose by intact-strips of GPI.Tissue homogenized with medium ...•..••..•....•.•....
Formation of labelled amino acids from uniformly labelled glucose by serosal-sheets of GPI
Formation of labelled amino acids from uniformly labelled glucose by intact-strips of GPI. Tissue separated from medium before homogenization
Comparison of amino acids produced by mucosal -sheets of GPI from labelled glucose, fructose an.d sucrose ................................. .
Xlli Effect of starvation on the formation of labelled amino acids from glucose by GPI
xii
Page
100
102
117
119
135
137
139
146
147
148
150
151
153
XIV Interconversion of amino acids by intact-strips of GPI ..................•..............
XV Formation of amino acids from labelled
XVI
xvn
xvm
XIX
xx
XXI
xxn
XXIll
xxv
acetate and formate by intact-strips of GPI
Effect of inhibitors on formation of amino acids by intact-strips of GPI from glucose ...•....
Effect of inhibitors or stimulators on formation. of amino acids by mucosal-sheets of GPI from glucose .......•.............•...
The effect of high concentrations of potassium on the rate of synthesis of amino acids by GPI tissue ........................ .
The effect of glutamine, asparagine and ammonium chloride on the formation of amino acids by intact-strips of GPI ....•.......•.....•......
Effect of formate on the formation of amino acids by mucosal-sheets of GPI ....•.•.....•.•....
Formation of glutathione from labelled glutamate and labelled glycine by intact-strips of GPI
Formation of labelled amino acids from glucose by mucosal-sheets of GPI ..•..........••...
Incorporation of carbon derived from glucose into amino acids, proteins, lipids and nucleic acids by mucosal-sheets of GPI .....••..••..
Formation of labelled amino acids from glucose by mucosal-sh.eets of GPI, after subcutaneous injection of colchicine .....•...••..•.•...•.
Incorporation of carbon derived from glucose into amino acids, proteins, lipids and nucleic acids by mucosal-sheets of GPI after subcutaneous injection of colchicine ..•...•.•...
xiii
Page
155
156
158
159
161
163
165
167
186
187
189
190
XXVI
:xxvn
Formation of labelled amino acids from glucose by mucosal.-sheets of GPI, after subcutaneous injection of increased amounts of colchicine ...•.
Incorporation of ·carbon derived from glucose into amino acids, proteins, lipids and nucleic acids by mucosal.-sheets of GPI after subcutaneous injection of increased amounts of colchicine ...•.
xiv
Page
19.2
193
LIST OF FIGURES
Page
1. nlustration of perfusion apparatus ...•...••.•••••... 94
2. Variation of glucose uptake activity along the length of tlle GPI .......................... . 104
3. Uptake of glucose by GPI strips with increase in the time of incubation ..••••..••..•..••.......• 106
4. Uptake of glucose by GPI versus concentration of glucose in the medium • . .. • . . • • . . • . . . • . . . • . . . . • • 107
5.
6.
7.
8.
9.
10.
11.
Inhibition by phlorhizin of active glucose uptake by strips of GPI ............................ .
Inhibition by phlorhizin of active glucose uptake by strips of GPI with varying medium concen-tration of glucose ...•••.•.....•..•....•.•.
Inhibition by phloretin of active glucose uptake by strips of GPI .•••...••••..•••.••....•.
Inhibition by phloretin of active glucose uptake by strips of GPI with varying concentrations of phloretin ....•............•.......
Variation in active uptake of glucose by GPI strips with increasing medium concentration of phloretin .••••..........•••....•..
Reciprocals of active glucose uptake by GPI strips plotted against medium concentration of phloretin . , ............... " ....... .
Variation in total production of amino acids with increasing time of incubation ..••.•....••...•..
109
110
112.
113
115
116
140
xv
12.
13.
14.
Variation in total production of amino acids with increasing medium concentration of glucose ..••
Variation in production of alanine with increasing medium concentration of glucose .......•.•. ~ ..
Variation in production of glutamate with increasing medium concentration of glucose •.....•.••....
xvi
Page
142
143
144
RNA
PNA
sRNA -
E/M
ATP
GTP
DPN
DPNH -
TPN
Co A
EMP
HMS
DNP
AMP
RBC
GOT
GPT
TCA
LIST OF ABBREVIATIONS
ribonucleic acid
pentose nucleic àcid
soluble ribonucleic acid
electron microscope or microscopy
adenosine triphosphate
guanosine triphosphate
diphosphopyridine nucleotide
reduced diphosphopyridine nucleotide
triphosphopyridine nucleotide
coenzyme A
Embden-Meyerhof-Parnas pathway of anaerobie glycolysis
hexose monophosphate shunt
2, 4 - dinitrophenol
adenosine monophosphate
red blood cell
glutamic oxaloacetic transaminase
glutamic pyruvic transaminase
trichloroacetic acid
xvii
xviii
GSH glutathione
F-acetate - fluoroacetate
KHBS Krebs-Henseleit-bicarbonate solution
GPI guinea pig intestine
GHI golden hamster intestine
ISM intact-strip method
PARTI
GENERAL INTRODUCTION
"La fixité du milieu intérieur est la condition de la vie libre." (45, 55, 56)
Claude Bernard, 1849.
1
It was not until the year 1839 that Theodor Schwannt a professor
of anatomy and physiology at Louvain, Germany, advanced his cell theory in
which he postulated that all living matter is made up of cells. Modem bio
chemistry has no quarre! with this concept and accepta the cell as the funda
mental organized unit of all living matter.
The living cell is composed of a number of intracellularly located
elements which are surrounded by an extremely thin and delicate membrane.
This membrane is a complex and fragile structure which is and must be con
sidered an integral part of the cell as a living unit. Cellular architecture or
structural compartmentation within the cell is also achieved almost entirely by
means of individual membranes surrounding each of the intracellularly located
parts. For example, there are nuclear, nucleolar. mitocondrial and micro
soma! membranes which surround these units and separate them as distinct
entities residing within a given cell.
For the living cell, substrates, enzymes and cofactors do not mix
readily as they may do so in a test tube. Enzymes and cofactors within a
cell are localized and tightly bound to interna! structures and hence are
spatially separated from substrates, so that, a certain degree of isolation
and specialization is exercised by each unit which resulta in an orderly
sequence of reactions carefully controlled at each step by the presence of
specifie enzymes and cofactors. Hence, the rate and direction of metabolic
reactions taking place in a particular part of a cell depend to a large extent
on the relative concentrations of substrates, enzymes and cofactors present
2
in that part, in an active form. Substrates which are the lowest in concen
tration therefore, and are in the greatest demand, as for example, ATP, DPN.
TPN, CoA and others will control the rate and direction of cell metabolism.
Reactions involving the oxidative metabolism of carbohydrates, fatty acids
and amino acids take place in the mitochondria because the organized enzyme
systems of the Krebs citric acid cycle, of electron-transferring in the respi
ratory chains, and of energy-coupling of oxidative phosphorylation are localized
in the mitochondria. In fact, this localization is so specialized that even in
minute structures auch as the mitochondria the matrix contains the enzymes
involved in the substrate-level transformations of the Krebs cycle and fatty
acid cycle, auch as glutamic, mal.ic dehydrogenases and crotonase and others
and the cristae contain the respiratory chain enzymes of the :flavoproteine and
the cytochromes. Simllarly, prote in synthe sis takes place in the microsomes
because RNA and amino acid activating enzymes are localized in these cellular
elements.
3
Experimental observations of many investigators have shown that
the cell membrane exists not only to act as boundary for the cell and its
contents but also to regulate the passage of materials in and out of the cell.
This latter function of the membrane far surpasses the former. It is vital
for the cell to have control over influx and effiux of substances. The cell
must be able to select substances which are needed to carry on its metabolic
activities and likewise, reject sorne of the products of enzyme catalyzed
reactions, which can act as inhibitors, in order to restore enzyme activity
to subsequent reactions. Homeostasie and hence viability of the cell depend
on the integrity of its membrane.
Cell membranes are selective and differentially permeable to various
substances or molecules which they encounter. Once a substance enters a
cell it may become attached or enmeshed in the colloïdal cytoplasm of the cell
or become converted into substances to which the membrane is not permeable
and thus is prevented from leaving the cell. The degree of permeability of
the cell membrane is subject to change. It depends on many factors which
we may he re place un der two general headings, environmental and hereditary.
Genetic material carried in the chromosomes of the cell nucleus. through
regulation of enzyme synthesis, gives primary direction to metabolism and
is fundamental to control of cellular activity. Concentration of substances
inside and outside of the cell, electrolyte balance, acidity, temperature, heat
and light may be regarded as environmental factors affecting metabolism.
The cumulative evidence based on the work of many investigators
in the field indicates that, whatever the nature of the cell membrane, the
passage or translocation of substances across it is not a matter of simple
diffusion or osmosis. The laws of diffusion and osmosis do not entirely
su:ffice to account for all the experimentally observable activities involved
in the passage of materials in and out of cella. The cell membrane is a
complex living unit which is highly organized to regulate metabolic activity
4
by reason of factors inherent in its physico-chemical, genetic and structural
make-up. Indeed, the phenomenon of the regulation of the passage of substances
across cell membranes is a complex process and in order to achieve some
measure of understanding as to what is happening within the membrane at
least one of the factors to consider must be the structure of the membrane
itself. For this, biochemists have turned to the electron microscope.
The electron microscope is an instrument of quite recent develop
ment. The first crude model came into existence in Germany in 1932 and was
constructed by Knoll and Ruska ( 23 ) . This was followed by rapid improve
ments and the first successful model built on this continent was in the Depart
ment of Physics of the University of Toronto by Prebus and Hillier in 1938
( 23 ).
The instrument now bas a resolving power of 20A or lesa or a mag
nification of some 100,000 fold. However no living material can be examined
with the E/.M. The material used must be subjected to rather drastic treatment
so that photographe obtained are to be cautiously interpreted making allowance
for many uncertainties.
5
Between the years 1949 and 1952 four major advances were made
in the techniques of electron microscopy which paved the way for the use of
E/M in the study of microstructures of cells. These advances were - the
development of "ultra" microtomes capable of cutting tissue sections into
slices in the range of one tenth to one fortieth of a micron thick; the dis
covery of an embedding medium such as n-butyl methacrylate, first introduced
in 1949 by Newman, Borysko and Swerdlow ( 5, 6); the improvements in the
fixative solution such as the use of buffered solutions of osmium tetroxide
introduced in 1952 by Palade (7); and the use of fracture edge of a piece of
plate glass as a cutting edge by Latta and Hartmann (8).
These advances and others have brought about a wider application
of the electron microscope to the study of the problems of histology and bio
chemistry as well as microbiology. For example, by means of the E/M Hall
and Slayter (9), and Huxby and Zubay (10) have been able to study the size and
shape of the 50-S ribosomes of Escherichia coli in frozen-dried and air-dried
preparations. These ribosomes are submicroscopic ribonucleoprotein particles
which apparently are present in ali living cells and serve as sites of protein
synthesis. Sjb'strand (11, 12) and Palade (13) have been able to determine the
salient features of mitrochondrial structure.
By means of ultra-centrifugation it has been possible to separate
the membranes of such microorganisms as Bacillus megatarium, Micrococcus
lysodeikticus, and staphylococcus aureus (20, 21, 22). The analyses of these
membranes have indicated that these membranes are essentially lipoprotein
in nature. They contain true protein in that hydrolysis yields ail the usual
L-amino acids found in proteins. They also contain about 20-30 percent
lipids.
6
Mitochon.drial membranes contain about 60 percent protein, a large
part of which is enzymatically active, and about 40 percent lipid in which the
phospholipid fraction is dominant. Recent work by Sj()strand (14) on mito
chondrial membranes indicated that the outer membrane consista of an ex:temal
monolayer of protein molecules about 25A in thickness, under which are two
opposed layers of oriented phospholipid molecules, the entire outer membrane
being about 90A thick. The inner membrane is considered to be identical
with the outer and a mirror image of the latter (14). It appears then that,
at least, in the mitochondrial membrane, the protein and phospholipid mole
cular entities are stratified in some manner. It may also be that each mem
brane unit is separated by a layer of protein or some other substance such
as a polysaccharide (14, 15). The work of Lehninger (16), and MacMurray
and Lardy (17), and others (24, 25, 26) have indicated that the electron-trans
ferring enzymes comprising the respiratory chains together with the energy
-coupling enzymes of oxidative phosphorylation exist presumably in or on the
membranes.
It is apparent from the experimental information now available to
us that, "mitochondrial membranes are not metabolically inert tsktnst but
rather complex fabrics of regularly spaced multienzyme systems, tt as stated
by Lehninger (18). We may also take notice of the statement made by Malek (19)
7
that the, "discoveries of the last ten to twenty years have clearly shown
that the cell can not be considered a simple osmometric sac but that trans
port phenomena are closely and directly coupled with metabolic processes.
It is a positive feature of this symposium that it strives for an integration
of our knowledge obtained through investigation of transport processes them
selves with concrete metabolic processes."
Histology and Morphology of the Smal.l Intestine
Certain cella in the body are particularly adapted to carry on the
process of absorption. Such specialized cells make up the tissues of the
mucosa of the smal.l intestine and of the proximal convoluted tubule of the
kidney.
8
The recognized functions of the small intestine are: the completion
of the digestive processes which have been started in the stomach. The
absorption of the products of digestion into the blood and lymph and the manu
facture of certain horiDones.
The siDal.l intestine displays great capacity toward selectivity in
absorption and differentiates closely among the various types of molecules
it encounters. Our primary concem here is with the capacities of selective
absorption and digestion at the striated border (46), therefore let us briefly
consider the histological and morphological features of this fragile tissue so
as to be able to anchor more readily the details of our subsequent discussion
with respect to the precise locus of absorption.
In the human being the small intestine is about 20-25 feet long, in
the guinea pig about 35-50 inches, in the golden hamster about 16-20 inches.
The first 10-12 inches of the human intestine constitute the duodenum which
pursues a horseshoe shaped course around the head of the pancreas. The
upper two-fi.fths of the smal.l intestine is called the jejunuiD and the lower
three-fifths the ileum. It tends to become narrower as it extends from the
pyloric end of the stomach to the ileo-caecal valve which marks the end of
the ileum. The rate of absorption of substances varies along its length,
being greater at the jejunal than at the ileal portion.
The wall of the small intestine is made up of four main layera of
tissue - the mucosa. submucosa. muscularis externa and the serosa. The
mucosa or mucous membrane in turn consista of three sub-layers - an
epithelial lining or epithelium, which faces the lumen of the intestine; a
supporting layer of !amin propria; and a thin layer of smooth muscle, the
muscularis mucosa. The epithelium is of the simple, columnar type. Its
cells do not al1 secrete mucus and are not al1 alike. Most of the cells are
9
of the tall columnar type each having a striated free border and are primarily
absorptive in function. These cells are subject to continuous and rapid replace
ment. Leblond and Stevens (1, 27 J 28) by means of subcutaneous injection of an
aqueous solution of colchicine, which arrests the mitosis in metaphase, have
demonstrated, in the rat ileum, that the turnover time of the crypt epithelium
is about 24. 2 hours and the complete renewal of the epithelium about 2 days.
By using the same technique McMinn (2) has shown the turnover time in the
ileal crypta of the cat to be about 2. 76 days.
The lamina propria which supports the epithelium structurally is
made up of areolar tissue sprinkled with lumphatic tissue which is non-encap
sulated presumably for filtering purposes. This layer carries both blood and
lymphatic capillaries close to the epithelial surface. Products resulting from
10
the digestion of food, once they have entered the epithelium, do not have to
diffuse any great distance through the tissue fluid of the lamina propria in
order to gain entrance to either type of capillary. The blood capillaries
lead into veina which drain into the portal system and into the liver sinuses.
The lacteals and lymphatic capillaries transfer lipid materials which are
absorbed into larger lymphatics and thence to the blood when the thoracic
lymph duct empties into the left subclavian vein.
The muscularies mucosa, which is the third outermost layer of the
mucosa, consista of two thin layera of smooth muscle fibers with varying
amounts of elastic tissue. The inn er layer of muscle fibers is circularly
disposed and the outer layer longitudinally disposed. The function of the
muacularis mucosa is probably to permit localized movements of the mucosa.
It throws the mucosa into circular folds and acts to relieve pressure on veina
in the submucosa caused by the action of the muscularis ex:terna.
The submucosa, which is a loose, pliable type of connective tissue,
comects the mucous membrane to the muscularis externa. It bouses a net
work of larger blood vessels and a network of nerve fi bers.
The muscularis externa consista of two substantial layera of smooth
muscle. The inner layer is circularly disposed and is somewhat thicker t han
the layer which is longitudinally dispos.ed. Both layera tend to pursue a some
w hat spiral course. The function of the muscularis externa is to propel the
contents of the tube downward. The state of tonus of the muscularis. ex:terna
is a very important factor in regulating the aize of the lumen of the gut.
11
The serosa is the fourth and outermost coat of the intestine. It
consiSta of areolar tissue in certain portions of the tube which are suspended
by mesenteries and in others merges into connective tissue associated with
the surrounding structures. It contains numerous adipose cells and neuro
vascular bundles.
The structure of the small intestine is specialized with regard to
both its digestive and its absorptive functions. Absorption occurs through
the epithelium of the mucosa and to accomplish this function efficiently the
epithelial surface is specialized or increase in a number of ways.
The mucosa is shaped into circularly or spirally disposed folds, the
plicae circulares which are crescentic for the most part and project into the
lumen about a third of an inch. These folds extend from the pylorus throughout
the entire length of the jejunum and end in the middle of the lower end of the
ileum. They ali have cores of submucosa and are not ironed out if the intes
tine ts full and, at first, are large and very close together, then in the upper
part of the jejunum they are smaller and farther apart.
The surface of the mucous membrane over the plicae circulares and
in the spaces between them is studded with tiny, leaf, tongue or finger like
projections that range from 0. 5 to 1. 0 mm or more in height. These are the
intestinal villi. In the upper part of the jejunum the villi are tongue shaped and
they become longer and finger shaped down along toward the ileum. In the latter
they are fewer in numbers and still narrower. In the duodenum the villi are
broader than those elsewhere and they are more leaf-like in shape.
The villi are highly specialized for absorptive function increasing
the surface of the mucosa many folds. Best and Taylor (3) cite that there
are some 5, 000,000 villi in the human intestine, providing a total of some
10 square meters of absorbing surface. But this is made even greater by
the countless number of tiny structures called microvilli that are present
on the free surfaces of the absorptive cells. The microvilli are so small
and so closely packed together that they can not be resolved as individual
12
units with the light microscope, thus appearing as striations or brush borders.
Each microvillus is covered with the cell membrane and has a core of cyto
plasm. There are goblet cells sprinkled among the absorptive cells which
secrete mucus and provide a protective film over the microvilli.
The crypts of Lieberkuh.n are the birthplace of the absorptive cella of
the epithelium. After a cell originates in the crypt it begins to migrate upward
unto the villus until it reaches the apical area of the villus, the extrusion zone,
where it is crowded out or ex.pelled by the adjacent cella. In this way the
entire epithelium is continuously replaced in an escalator fashion (1, 27, 28, 31,
32,33).
During this process of migration from the crypt to the apex of the
villus the young cella undergo progressive differentiation and growth resW.ting
in a number of cytochemical changes which considerably modify their functional
and absorptive characteristics. The undifferentiated cella in the crypt have
a large number of evenly distributed ribosomes which means a high concen
tration of ribonucleoprotein. As the young cells migrate they undergo a steady
13
losa of RNA (47,48). The young cells in the junction of the crypt al.so
undergo an abrupt differentiation which results in an increased capacity of
activities of phosphatases, esterases, and succinic dehydrogenase in the
cella.
These cytochemical changes undergone by the young cella are re-
:flected in their digestive and absorptive capacities as they move up the villus
reaching their maxima at the apex of the villus forming an epithelium with. a
brush border (row of tiny long slender microvilli) which is highly specialized
for digestion and absorption. The adult differentiated cella at the apex can
concentrate lipid,. sugars and amino acids to a much greater extent than the
young crypt cel.ls ( 46, 49, 29, 41, 50) .
To summarize brie fly, the outermost or the layer next to the lumen
of the intestine is the mucosa. The outermost sub-layer of the mucosa,
facing the lumen is the epithelium. The mucosa runs into circularly or spi-
rally disposed folds, the plicae circulares, which project into the lumen about
one third of an inch. The surface of the mucosa over these folds and between
them is studded with projections; these are the intestinal villi. The luminal.
surfaces of the villi are lined with the epithelium. This is made up of a
single layer of tal.l columnar cells; these are the absorptive cella. The re
are some goblet cella sprinkled among them. The free surfaces of the absorp-
tive cella over the villi have innumerable minute projections which appear as
striations when viewed with the ordinary light microscopej these are the micro-
vill. 1. With the E/M these striations or brush borders as they are sometimes
called, appear to be a row of closely packed microvilli. The microvüli
have a common membrane which is continuous with the membrane of the
absorptive cell bearing them. The microvilli of the absorptive cells are,
then, the first structural or cellular elements which are directly involved
14
in the process of absorption. Substances which are to be absorbed by the
intestine must first pass through the membrane lining the microvilli. Exactly
how various types of molecules pass through the membrane of the microvilli
will be the subject of discussion of later sections.
15
Absorption by the Small Intestine
Very little absorption tak:es place in the gastric mucosB.t even
substances which need not undergo hydrolysis such as glucose and water
are not absorbed to any appreciable extent, only alcohol and certain drugs
are readily absorbed by the stomach. The important site of the body for
absorption as well as digestion is the small intestine and, more precisely,
the mucosa of the intestine with its epithelium of absorptive columnar cells
with their brush borders or microvilli spread over the surface of the villi.
The large intestine has a singular absorptive capacity in that it absorba
water and th.us conserves the water supply of the body.
Absorption of Fats
In 1936 Verzar and McDougall (30) and others (40) earlier believed
that fats had to be completely hydrolyzed to fatty acide and glycerol before
they could be ahsorbed by the intestinal mucosa. They also upheld the view
that after absorption the fatty acide and glycerol formed reunited to form the
neutral fats via the phospholipide (43, 44) as intermediary products.
In 1948 ZUversmit (37) cast doubt on the hydrolysis-recombination
and the phospholipide as intermediate products concept of lipid absorption by
showing that the turnover rate and the total amount of labelled phospholipid
was not affected by the absorption of neutra! fats or of fatty acide.
16
In 1946 Frazer (36) showed that highly emulsified fat (particles of
0. 5 micron in diameter or lesa) could be absorbed by the intestine without
prior hydrolysis into fatty acids and glycerol. In this way a coœiderably
quantity of the ingested fat is absorbed as an emulsion. Frazer performed
his experimenta with the use of a detergent, sodium cetyl sulfonate, which
forma a fine emulsion with fats in water. This detergent also inhibits the
action of intestinal lipase so that no hydrolysis could take place during
absorption of the emulsified fat. Under normal conditions the bUe salta.
fatty acids and monoglycerides (38, 39) present in the lumen of the gut can
bring about the formation of a highly emulsified mixture of neutra! fats which
can then be readUy absorbed by the mucosa and then directed into the lym
phatics of the villi.
Pal.ay and Karlin (29) in their studies with the E/M have demonstrated
that small droplets of fat between 300 and 500 A in diameter appear between
the microvilli and in the cytoplasm close to the bases of the microvilli within
20 minutes after a fatty meal had been given to rats. After one half to one
hour, they found the droplets of fat deeper in the cytoplasm and each droplet
surrounded by a membrane. This may be suggestive that the mechanism of
absorption operating here is one of phagocytosis as by macrophages or pino
cytosis as by endothelial cella.
More recently Ladman, Padykula and Strauss (41) have demonstrated
in the human that during fat absorption the cella near the apex of the villus
(the oldest cella) accumulate lipid first and in greatest abundance as com
pared with the younger cella found nearest the crypta which were found to
17
be generally free of lipids at an interval. of about 20 minutes after ingestion
of 10 ml of corn oU by the subject. Apparently then, the most fully differen
tiated adult cella have the greatest capacity for lipid uptake.
Strauss (42) has further shown in his studies on fat absorption that
the isolated everted jejunal sacs of the golden hamster absorb emulsified fat,
in vitro, by the same pathway described by Palay and Karlin (29). Palay is
of the view (35) that the various components of the membrane systems present
are involved including those of the nuclear membrane.
Our present day knowledge of fat absorption, digestion and mobilization
by the absorptive cella of the mucosal epithelium is stlll in its infancy. The
mechanism of absorption of fats is far from complete and much remains to
be elucidated (34). For example, what are the mechanisms involved in the
absorption of fats? Do they enter the absorptive cell by simple diffusion or
by an "activett pro cess which is dependent on some source of metabolic energy?
What is the metabolic fate of the absorbed fat within the epithelial cell prior
to entry into the lymph or the porlal blood? How does the alteration of fats
within the absorptive cell affect the cytochemical. and morphological. pattern
of the absorptive cell and epithelium and bence the absorptive capacity of the
gut? These and other questions yet remain to be answered.
18
Absorption of Proteine and Amino Acide
It has been generally accepted that proteine are absorbed, after
hydrolysis into amino ac ids, by the same pathway as carbohydrates - namely,
through the absorptive cella of the mucosal epithelium and then passed on
into the portal blood. Two observations support this view. One is that the
amino acid concentration of the portal blood rises rapidly shortly after a
protein meal and the second is that nitrogen balance can be maintained upon
the intravenous administration of a suitable mixture of amino acids or a
protein hydrolysate which contains the necessary essential amino acide and
an adequate supply of calories.
These observations argue against the point that intact protein, per se,
may be necessary for adequate nutrition or maintenance of positive nitrogen
balance. Nevertheless, there is evidence to support the view that certain
proteine auch as those of egg white, shell fish, strawberries, milk and others
and certain small peptides can and do enter the blood stream - either through
absorption by the intestinal mucosa or through the respiratory passages without
undergoing prior hydrolysis. Su ch allergie reactions as those in hives, asthma
and hay fever have been attributed to them. It has also been shown that in
certain animais and man the young gets its immunity through absorption of
intact proteins or polypeptides (e. g. antibodies in colustrum) by the intestinal
mucosa (51). However these are absorbed in such minute quantities that they
can hardly be regarded as nutritionally significant as far as total protein
19
intake of the body is concerned. Yet, it is well established that these im.mu
nologically active proteine owe their activity to their complete structural
integrity (e. g. insulin, thyroglobulin and the placenta! proteine) so that there
is little doubt that they are absorbed by the gu.t intact at leaat in small quan
tities to achieve their specifie role. London and Kotchneva (57) have demon
strated the presence of polypeptides in various parts of the alimentary tract
and the blood coming from them during the digestion of protein.
The question to be raised then is - wha:t degree of hydrolysis must
the protein undergo, short of amino acids, before it can be absorbed by the
gut? In 1959 Newey and Smyth (52), and Wiggans and Johnston (53) demon
strated both by in vitro and in vivo techniques that very small amounts of
the dipeptide glycyl-glycine could pass through the intestinal wall. On the
basis of these findings and others these investigators concluded that dipeptides
can enter the epithelial cella intact as peptides - then are hydrolyzed intra
cellularly and subsequently emerge as amino acide.
Another question to be raised is:- how does the peptide enter the
cell, that is, wha:t is the mechanism of its entry - diffusion, or some "active"
process? TheJ:e is some evidence to indicate that a process other than simple
diffusion may be involved. One reason for this view is that anaerobie conditions
or the presence of 2, 4 dinitrophenol in the mucosal fl.uid impairs the entry of
the peptide into the cell, thus indicati:p.g the dependence of the process of
absorption on some source of metabolic energy. The other reason is tha:t
there is competition between peptide and amino acid molecules, which may
20
weil mean that there is a common carrier system operative for both types
of molecules.
As indicated, polypeptides and products of protein digestion other
than amino acids may be absorbed by the intestine in very small quantities.
What becomes of these molecular aggregates of the amino acids after entry
into the absorptive cells or the portal blood needs further investigation. The
possibllity that proteins may be reconstituted during their passage through the
absorptive cells and the mucosa must not be overlooked and has not been, as
yet, ruled out.
Delaunay (58), and V an Slyke and Meyer (59) were among the early
workers in 1913 to show that blood nitrogen increased during the digestion
of a protein meal and that the portal blood was particularly high in CIC'-amino
nitrogen. This meant that a large proportion of the ingested protein appeared
in the blood as amino ac ids. It was further demonstrated by these workers (59)
that amino acids could be concentrated agai.nst apparent concentration gradients
in tissues. Since then, amino acid uptake has been studied extensively in
microbial. cells (71, 72) and in various types of cells and tissues in higher
animais (73, 7 4, 7 5, 76, 77, 78, 79).
Let us now consider the information avai.lable for the concentrative
uptake of amino acids by the epithelial cells of the intestine. u
Rober and
tt Rober (60) in 1937 first advanced the idea that amino acids in the gut may
be absorbed by a process other than one of simple diffusion. In 1951 Gibson
and Wiseman (61) demonstrated that the D- and L-amino acids are absorbed
21
at different rates, the L-isomer being absorbed at much higher rates than
its enantiomorph. Wiseman (62) in 1953 using in vitro techniques showed
that the more rapid absorption of the L-forms were not due to the possi-
bility that the L-amino acids may be more rapidly metabolized upon entry
into the cell than the D-amino acids. When he placed racemic mixtures
into the mucosal solution he observed that the L-amino acids moved against
a concentration difference but the D-amino acids apparently did not. His
resulta were confirmed by Fridhandler and Quastel (63) and Agar. Hind and
Sidhu (64) by in vitro experimenta and by Matthews and Smyth (65) in vivo
experimenta. These findings established that the L-amino acids, alanine,
phenylalanine, methionine, histidine and isoleucine moved against a concen
tration gradient but the dicarbioxyllic acids L-glutamic and L-aspartic did
not. The conclusion drawn from this was that a process other than simple
diffusion must be operative with respect to the transfer of amino acids through
the gut. The next aspect of this problem was to ascertain whether the process
was linked with the existence of a carrier system which had stereospecific
properties. It had also been noted that lysine, ornithine and tryptophan did
not move against a concentration gradient. Not ali the amino acids which
were absorbed against a concentration gradient moved at equal rates - some
were slower than others - and the slower moving ones inhibited the movement
of the other more rapidly moving ones. Renee there appeared to be some
sort of competition for a carrier site among the amino acids.
More recent work by Jervis and Sm.yth (66), and Finch and Hird (67)
22
has indicated that D-methionine is capable of moving against a concentration
gradient when used alone. In the presence of L-methionine its passage is
impaired and does not accumulate against a concentration difference. These
findings as predicted by Wilbrandt (68) focus attention on the idea that the
uptake of both the D- and L- forma of the amino acids is probably by a
specifie carrier process in which the intracellular dissociation of the carrier
-amino acid complex is essentially irreversible (67). The L-amino acid shows
a greater affinity for the carrier system and hence can effectively compete with
the D-amino acid for a locus on the carrier. Kinetic studies by Fisher and
Parsons (69) and by Jervis and Smyth (70) also support this view and have
given added credence to the carrier-amino acid complex concept by showing
that the latter follows approximately the Michaelis-Menton type of kinetic
analysis, the Km value for L-methionine being about 9 x 10-3 M as compared
with 20 to 40 x 10-3 M for D-methionine.
Amino acid uptake in microorganisms has been studied by Gale (71)
and emphasizes the dependence of the uptake on energy metabolism. In 1958
the work of Britt and Gerhard (72) showed that bacterial protoplasts can
accumulate amino acids. One feature of amino acid transport appears to be
the unusually low specificity of this class of compounds as compared with the
sugars which display rather rigid stereochemical requirements as will be in
dicated in subsequent sections.
For the structural requirements of the amino acids for transport the
following characteristics have been noted. The amino acids can fall with
respect to their ability to be transferred, into three groups, the neutra!,
cationic and anionic amino ac ids. In the case of the neutral amino acids
the carboxyl and amino groups may be located on the same or adjacent
carbon atoms. The Cl( -carbon atom may have two hydrogen atoms on it
23
or two alkyl groups or one of each. However, the re seems to be a par
ticular advantage in having one of the hydrogena as a methyl group such as
in L-alanine (80, 81). Some evidence exists (80) for a correlation between
the capacity to concentrate and the pK of the amino group. It bas also been
suggested that the amino group must release a proton (80). Riggs (82, 83)
bas shown that pyridoxal and pyridoxal phosphate added to the media of
Ehrlich ascites cells stimulate their amino acid uptake, possibly these com
pounds acting as mediators of transport by forming complexes with the amino
ac ids. The presence of cellular potassium is needed to promote or maint ain
amino acid concentration. In Ehrlich ascites cells if potassium is deleted
from the cella the amino acid gradient is in the opposite direction (84).
Johnstone (85) and others (86, 87) have shown that requirements for energy
is not exclusively dependent upon oxidative processes, glycolysis provides
sufficient energy for concentrative uptake of amino acids in ascites cells.
ln the case of the other groups of the amino acids some unusual
observations have been noted (88). When the two amino groups of a diamino
acid are located close to each other for example as in « , Y diamino butyric
acid a stimulated uptake takes place. This bas been explained as a mani
festation of the ability of these charged molecules (Zwitter ions) to provoke
24
the migration of inorganic ions such as potassium ion. Amino acid move-
ments display great sensitivity toward K+ concentration and stimulate the
+ + movements of intracellular K and extracellular Na . The action of poi-
soning agents has been explained as being due to their ability in diminishing
the concentration of intracellular K +. Additional information has been obtained
by the study of the concentrative uptake of certain amines such as serotonin,
adrenaline and tryptamine whose uptake characteristics are considered similar
to those of the amino acids (89. 90, 91). In 1958 Born, Ingram and Stacey (91)
were able to show a correlation between the ATP content of the cell and its
uptake capacity for the amine. From these etudies and others (92, 93) has
emerged the suggestion that the nucleotides have the capacity to bind these
amines. Likewise in 1962 Abadom and Scholefield (96) were able to show
that severa! amino acid transport systems may exist in rat brain cortex
slices each with its own specificity with regard to substrate and with regard
to amino acids which produce inhibitory effects. They are also of the opinion
that, at least in the case of certain amino acids (e. g. glycine) the extent of
the concentrative uptake by rat brain slices on incubation in the presence of
glucose is directly proportional to the ATP content of the slice.
Kinetic studies have revealed that the higher the level of amino acid
in the cell the more rapid is the in:fl.ux, by ex change, of the labelled amino
ac id or an analog of the amino acid (94). If we assume, then, that the amino
acid is bound to the cell by a simple process or if a transport carrier exista
for the special purpose of carrying or binding the amino acid and then is
destroyed intracellularly with release of energy it would hardly account for
the exchange process observed. It seems more plausible~ therefore, that
if a complex is formed between the amino acid and a carrier molecule, it
is not of a simple kind which merely engages the amino acid, transfera it
to the other side and releases it intracellularly and returns to the outside
25
for a repeat performance. Kinetic studies support the view that an inter
mediate complex having special characteristics is formed. That is, one
which exchanges the complexed amino acid with a second analogous molecule
already in the cell more rearlily than it dissociates to release the amino acid
carried into the cell. If this is deemed credible then the cartier binding site
must possess special characteristics. For example, it may lack affinity for
the molecule just released or possess a 11peculiar insulationn (95) from the
forma of the delivered amino ac id molecules. Su ch insulation characteristics
may be visualized as being due to migration of the site, sequential donation
through a series of sites, or a rearrangement of the site.
To summarize - protein matter is absorbed by the intestinal epithelium,
to any appreciable extent, only as amino acids after the hydrolysis of the pro
tein in the lumen of the gut by proteolytic enzymes. Both the L- and D-amino
acids diaplay concentrative uptake characteristics, competitive phenomena (97,
98, 99) energy dependence, although not necessarily of the oxidative variety (85}.
Uptake is stimulated by intracellular K+ concentration and by coincidental iso
osmotic transfer of water. Correlations between uptake of amino ac ids and of
pyridoxal or pyridoxal phosphate have been noted as well as stimulation of
concentrative uptake by certain hormones as for example,. glycocorticoids,.
pituitary growth hormonet insulin and estradiol although some notable ex
ceptions have been found (e.g. deoxycorticosterone).
26
No particularly exacting stereochemical specificity requirements for
the concentrative uptake of the amino acids have as yet been designated with
the exception of the requirement of the presence of the amino and carboxyl
groups on the same or adjacent carbon atoms. In fact, a low stereochemical
specificity has been an outstanding feature of the transport of this class of
compounds. No one has be en able to show if the re is a direct change in
the amino acid molecule during transport. No one has unequivocally demon
strated whether there is one or many amino acid transport carrier systems
present each specifie for a particular amino acid. Experimental evidence
is now largely in favour of the hypothesis that a carrier-amino acid complex
with special characteristics exists which can undergo an exchange reaction
intracellularly and can modify its site subsequent to discharge of the amino
acid molecule carried into the cell.
Absorption of Inorganic Ions
The indispensability of the inorganic ions in the metabolism of the
yeast cell was observed by Pasteur in 1860. Osborne and Mendel (100) in
1919, working with rats, showed the importance of inorganic ions in the
27
metabolism of higher animais. and determined the requirement of the rat
for a particular salt mixture. Claude Bernard was one of the first to
recognize the importance of the maintenance of the constancy of the inter-
nal environment of the body or homeostasis in higher animais. And in
1924, the British physicist, Dounan (101) advanced his tb.eory on membrane
equilibrium depicting the influence of ions in colloidal systems. Thus, the
important role of the various inorganic ions in the maintenance of fl.uid
equilibrium in cella and tissues has been well recognized and early docu-
mented but the manner in which ionie equllibrium is established or the
manner in which inorganic ions pass across a living cell membrane is
still a matter of conjecture. To be sure a great deal of knowledge has
been accumulated since the time of Pasteur but still our present day know-
ledge about the movements of the inorganic ions in living cella is incomplete.
The principal inorganic cations and anions of the body fl.uids and
cella are: + + ++ ++ - - = -Na , K , Mg , Ca and Cl , HC03 , PO 4 and SO 4 . The
osmotic equilibrium of body fluids is largely determined by the variation
in the total concentration of these ions and their relative concentration
determines the integrity of the cell membrane and the bioelectric potentials
of the tissues. Moreover, intracellular hydrogen ion concentration and
those of the tissues is regulaied within narrow limita (e. g. blood pH, 7. 35 -7. 45)
by removal of the excess acid or alkali products resulting from cell metabolism
in which the total concentration and distribution of inorganic ions play a sig-
nificant role.
It has been established that K + is the principal inorganic cation
+ of the intracellular fl.uid and Na that of the extracellular fl.uid in a1l the
++ ++ higher animais. These two ions together with Ca and Mg play an
important role in the osmotic regulation of the body fiuids. possibly by
their capacity to control certain enzyme systems.
When a cell is at equilibrium there is a balance of ions inside
and outside of its membrane and also a balance in the electrical potential
on both sides of the membrane, the latter is referred to as the resting
potential or bioelectric potential. When an electrical impulse passes
through the membrane the electrical balance is disturbed and a new equi-
librium must be established. In this state the cell or membrane is said
to be in a state of excitement. Furthermore. it has been observed that
auch a state of excitement restùts in changes in the permeability of the
cell membrane (102).
Under normal or resting-state conditions the cell membrane does
not have the same degree of permeability toward ail ions. For example
bicarbonate ions are freely diffu.ible through most cell membranes whereas
the permeability of K+ and Na+ ions is restricted. The membrane is al
most impermeable to Na+ and permeable to K+. There is evidence that
28
the passage of K+ is linked with or dependent upon the energy derived from
the metabolic activities of the cell.
In the resting-state K+ and cr can pass through the membrane but
Na+ can not. Therefore it is said that an 11 active" process must be
operating to keep the Na+ from entering the cell since its concentration
29
is high outside and low inside the membrane. But, when an electrical
impulse passes, the bioelectrical potential is disturbed and the permea
bility of the membrane to the passage of Na+ increases considerably~ over
a short time interval, and bence Na+ rapidly entera the cell; and, in order
to maintain ionie equil:ibrium the K+ ions move out of the cell.
Now, in order to return to the resting-state, Na+ now located intra
cellularly must move out and K+ move in. In nerve tissue the extrusion of
Na+ and the uptake of K+ is said to be linked with the intracellular hydrolysis
of acetylcholine (103, 104), and in muscle, it has been demonstrated, that
if the action of acetylcholine estrase is blocked, Na+ exit is inh:ibited (105).
These and other experimental observations appear to make the
problem of transport of inorganic ions relatively simple. This is indeed a
paradox. We know very little about the conditions inside of a cell and much
lesa about the detailed architecture of the cell membrane through which these
ions must pass. Recent workers in this field are not at ali in agreement
as to exactly where and how metabolic energy is being obtained to drive
the active transport mechanisms. For example Conway states that there
is general agreement as to the storage of chemical energy (106, 107) in the
· form of high energy phosphate bonds as in ATP but he points out that a
supply of energy in the form of ATP is not the only requirement for the
30
normal functioning of the sodium pump in squid axons, since it appears to
be necessary for arginine phosphate to be present as weil. Nevertheless,
the idea that ATP is involved in active transport mechanisms either as a
supply of energy readily available or as a carrier providing a site has
gained impetus. The work of Skou (108) has shown that magnesium-activated
++ microsomal ATP-ase from crab nerves is activated, in the presence of Mg ,
by Na+ and inhibited by an ex cess of K+. He suggests th at the substrate
most readily attacked by the ATP-ase is Na-Mg-ATP, and that this enzyme
is somehow involved in the active transport of sodium.
Ernst (109) and others (110) have pointed out the fallacy in regarding
the excitation by an electrical impulse as being equivalent to a physiological
excitation - resulting in increa.sed permeability of the cell membrane. He
points out that the harmful by-effect of the electric current in itself could
cause increased ion shifts. He also argues that the con.tinuous maintenance
of a high concentration gradient of K+, in muscle, (50:1) can not be explained
solely on the basis of increased metabolism of the cell (111) and he leans
toward the postulate of Simon (112) that K + is held in the cell on adsorption
sites which are in a dynamic equilibrium with the external K+. Thus they
explain the unequal distribution of K+ and Na+ between the cell and its
surrounding fl.uid on the basis of the binding chara.cteristics of some non
-diffusible macromolecular anionic sites which bind or adsorb the slightly
more polarizable K+ ion (113, 114, 115, 116).
31
To summarize - certain inorganic cations and anions are essential
for the maintenance of the life of the cell and of the fluid equfiibria of cell
and tissues. These ions through their interaction with other molecules or
ionst chiefly proteine. and activation or control of certain enzyme systems
(e. g. ATP-ases) are capable of participating in the passage of other mole
cules auch as the sugars and amino acids across cell membranes. The
action of certain inorganic ions in this respect has been found to be quite
dramatic. For example, Ricklis and Quastel in 1958 using the surviving
guinea-pig intestine demonstrated (128) that the active transport of sugars
depend entirely on the presence of sodium ions. When Na+ is removed
from the medium bathing the mucosal and serosal aspects of the intestine
active transport ceases dramatically. The same investigators also found
(128) that potassium ions have a large stimulatory effect on the active trans
port of the sugars and amino acids. However in this instance K+ could be
replaced with rubidium ions but not with cesium ions, indicating that the
process is not absolutely dependent on K + and that the effect of K+ is to
bring about an acceleration in the rate of active transport of the sugar.
The presence of magnesium ions were found to be necessary to maintain
active transport and to secure the stimulation observed with K+ but those
of calcium ions and ammonium ions were found to be depressing. Experi
mental evidence to date, leans toward the concept that K+ and Na+ are
transported across cell membranes, against concentration gradients, re
quiring the expenditure of sorne form of metaholic energy - ATP and
32
other nucleotides being much in favor - although the nature and mode of
application of this energy and the mechanism of the transport systems are
still much in debate. Two hypotheses designed to help elucidate this problem
are, the concept of the changes in the permeahUity properties of the cell
membrane and the concept of the factors affecting the adsorption charac
teristics of intracellularly located non-diffusahle macromolecules which can
bind K+ preferentially.
In the case of sodium ion it is interesting to point out that the
full import of the indispensahility of this cation for active sugar transport,
as pointed out in 1958 by Riklis and Quastel (128), was not realized until
recently when supporting evidence gathered from other sources such as the
use of stophanthidin (129) and ouahain (130) showing inhibitory actions on
ion active transport in the intestine of the hamster and the frog, was re
viewed by Wilson (131).
Absorption of Sugars
The metaholism of carbohydrates plays a very important role in
body economy. The EMP and HMS pathways of glycolysis and the citric
acid cycle are almost entirely dependent for their operation on a constant
supply of carbohydrate. Glucose is by far the most important monosaccharide
33
in the body and the chief source of raw material for the above mentioned
pathways. In man, glucose and all other monosacharides are almost com
pletely absorbed from the intestine and pass into the portal blood. Poly
saccharides and oligosaccharides are broken down to monosaccharides by
the action of glycosidases in the lumen of the intestine prior to absorption.
Recently (1961) the work of Miller and Crane (160) has indicated the pre
sence of hydrolases, auch as invertase and maltase, within the epithelial
brush-border membrane which are capable of break.ing down disaccharides
prior to their entry into the transport activity region.
Cori (117) in 1925 studied the rate of absorption of sugars in rats
by feeding them with a stomach tube and reported his findings expressing
them as relative rates of absorption, assuming the rate of glucose as 100.
These rates of absorption are given as follows:
Sugar Relative rate of absorption
D-Galactose 110
D-Glucose 100
D-Fructose 43
D-Mannose 19
L-Xylose 15
L-Arabinose 9
34
It is apparent from the table that the relative rates of absorption
of galactose and glucose are considerably higher than the rest so that one
would suspect that the mechanism of entry of these sugars into the epithelial
cella of the intestine must be in some way different from mannose and the
pentoses. Fructose seems to take an intermediate position. This has
been clarified and accounted for by the work of Darlington and Quastel (139)
and later by Wilson and Vincent (155), in that.,part of the fructose is converted
to glucose and passes across the intestine partly as glucose and partly as
fructose. Cori also found that the rate of absorption of glucose was the
same whether the luminal concentration was 25, 59 or 80 percent. From
this it followed that the concentration of a particular sugar in the lumen
had little to do in determining the rate at which it will enter the epithelial
cell. Inatead, the rate of absorption was found to be characteristic of the
particular sugar and varied with its location of absorption in the gut, showing
a progressively diminishing rate from the pyloric end of the jejunum toward
the distal end of the ileum.
Since the work of Cori the passage of sugars across cell membranes
has been studied in many biological systems by numerous investigators. Active
transport has been investigated in auch cella and tissues as: erythrocytes
(142, 143, 144) ascites tumor cells (145) yeast cella (146) placenta (147) proxi
mal tubule of the kidney (148) and the mucosa of the small intestine in the
frog (149) the rabbit (150) the rat (151) the cat (152) the dog (153) the
35
hamater (154, 155, 156) the guinea pig (63, 139, 128) and man (157).
On the basis of the nature of the process of absorption the sugars
may be separated into two classes, those which pass across the cell mem-
brane by the process of t active-transportt and those which pass by the pro-
cess of tpassive-transportt, which is, primarily propelled by diffusion not '
requiring the expenditure of metabolic energy and not accumulating against
a concentration difference.
At the present time about fourteen sugars and related compounds
which conform to the criterion of active-transport have been found by use
of in vitro methods and two of these have also been tested by in vivo tech-
niques, namely, glucose (159) and 3-0-methyl-D-glucose (180). Thirty-five
other sugars and related compounds have been tested and found not to be in
conformity with the criterion of active-transport. These compounds will
now be listed.
Sugars and sugar derivatives actively-transported
D-gl ucose 6-deoxy-6-fl.uoro-D-glucose
3-û-methyl-D-glucose ct. -methyl-D-glucoside
3-deoxy-D-glucose 1, 5-anhydro-D-glucitol
6-deoxy-D-glucose D-galuctose
2-C-hydroxymethyl-D-glucose 6-deoxy-D-galactose
D-glucoheptutose 4-0-methyl-D-galactose
7 -deoxy-D-glucoheptose D-allose
Sugars and sugar derivatives passively-transporled
2-deoxy-D-glucose
2-0-methyl-D-glucose
3-0-ethyl-D-glucose
3-0-propyl-D-glucose
3-0-butyl-D-glucose
3-0 -hydroxyethyl-D-glucose
6-0-methyl-D-glucose
L-glucose
D-glucosamine
N -acetyl-D-glucosamine
1, 4-anhydro-D-glucitol
Gold-thioglucose
L-galactose
2-deoxy-D-galactose
.2, 4-di-0-methyl-D-galactose
6-deoxy-6-iodo-D-galactose
6-deoxy-L-galactose
D-gulose
Glycerol
D-fructose
3-0 -methyl-D-fructose
D-m anno se
6-deoxy-L-mannose
Mannitol
1, 5-anhydro-D-mannitol
D-mannoheptulose
L-arabinose
D-arabinose
D-lyxose
D-tal.ose
D-ribose
D-xylose
L-xylose
L-sorbose
Sorbitol
36
Specificity of the sugar molecule
In order to understand the differences in the capacity of sugar
molecules in relation to active-transport or passive-transport we need to
exainine their structures closely
The sugar molecule is a neutra! molecule and hence is different
37
in this respect from ions which carry positive or negative electric charges
and therefore can be influenced by electric fields or membrane potentials
in the ir transport activities.
The sugars are quite stable in their crystalline condition, however
when they are dissolved in water, particularly in the presence of acids or
alkalies, they can undergo many transformations The polar groups of the
sugars are highly solvated. In water solution the hydrogen atom of each
hydroxyl group is rapidly exchanged with hydrogen atoms of the sol vent.
Fredenhagen and Bonhoeffer (186) have shown that at room temperature the
exchange of the hydrogen atoms of the hydroxyl groups with deuterium atoms
of heavy water are quite rapid. However, the carbon bound hydrogena are
quite stable and do not exchange with tritiated water (154).
The sugar molecule is a polyhyd.rox:y aldehyde or polyhydroxy ketone.
The large number of hydroxyl groups on the molecule render it more hydro
philic and therefore lesa soluble in lipid media or lipid portion of membranes.
If solubility is deemed an important factor in passage through lipoprotein
38
membranes then reactions or changes which will make the sugar molecule
more lipid soluble must be recognized. Moreover, reactions involving any
or all of the hydroxyl groups as weil as the terminal aldehydic and ketol
groups must be taken into account as possible factors linked with transport.
Another important factor to be considered in active sugar transport
is that the sugar molecule can be metabolized during the process of trans
port into lactic acid, co2 and a very small amount of keto acid (181, 159,
158). This has been demonstrated by the in vivo experimenta of Atkinson,
Parsons and Smyth (159) and the in vitro experimenta of Newey, Smyth
and Whaler (158). It follows then, that if we are concerned with the intra
cellular concentration of the sugar at any given time we must be aware of
the rate of metabolism of that sugar both in the process of transport and
intracellularly after transport. If the metabolic rate is high as compared
with the rate of entry diffusion equilibrium can not be established and we
have instead a steady-state condition in which the rate of entry of the sugar
into the cell equals the rate at which it is being metabolized and hence the
intracellular concentration remains constant.
The exact detailed mechanism of sugar active transport by the
absorptive cells of the intestine is still unclear but the possible reactions
the sugar molecule may undergo during the process of transport have been
extensively studied by many workers in this field and as a result of this
effort it has become possible to correlate certain features of the sugar
molecule as common factors of structure which may be responsible for
active-transport. These common structural factors are:
1. A pyranose ring in the Cl conformation.
2. An hydroxyl group in an equitorial position on
c2. as in glucose.
3. At least one of the si de groups on carbone 3
and 4 in an equitorial position.
4. A methyl or substituted methyl group attached
to carbon 5.
One of the most probable and perhaps simplest reactions that a
sugar molecule can undergo is phosphorylation. It may have been this
39
very obvious simplicity which has brought about the so called "phosphorylation
-dephosphorylation hypothesis".
It was first proposed in 1933 by Lundsgaard (174) and resumed again
in 1936 by Verzar and McDougall (30). Since then it has been supported by
the work of Beek (175), Kjerulf-Jensen (176), and Gomori (163) and refined
and modernized by Drabkin (161), Hele (177, 178) and Csaky (179). In its
simplest version the hypothesis states that phosphorylation is the process
whereby the sugars are actively transported and accumulate against a con
centration gradient. According to this concept the sugar to be actively
transported is first phosphorylated at the luminal end of the epithelial cell.
The sugar phosphate thus formed passes across the cell membrane by a
40
process of diffusion and upon reaching the basal end of the cell is dephos
phorylated and the free sugar accumulates.
In order for phosphorylation to occur at the luminal end of the
absorptive cell it would be expected that this area would be relatively rich
in phosphorylating enzymes like hexokinase and similarly the basal end of
the cell rich in dephosphorylating enzymes or phosphatases (161). The
presence of any hexokinase at the luminal or brush-border end of the epi
thelial cell has not yet been demonstrated. On the contrary the work of
Clark (141) with the E/M has provided unmistakable evidence for the pre
sence of alkaline phosphatase in the plasma membrane lining the microvilli
(163). Furthermore Crane and Krane (162) in 1956 have demonstrated that
1-deoxy glucose and 6-deoxy glucose are actively transported. The one
and the six positions of the hexose molecule are considered the most pro
bable positions for phosphorylation and dephosphorylation to take place, hence
in view of the fact that these sugars are not actively transported the Verzar
hypothesis is unable to account for them. However, it must not be com
pletely overlooked that the transport of 1, 6-deoxy glucose has not been shown
and that the possibility of phosphorylation in one or the other position still
exista in the absence of one of them.
From the point of view of the structural specificity of glucose and
other actively transported compounds the hydroxyl group at carbon 2 is by
far the most essential and any chemical reaction involving the transport
41
mechanism should take place at this position. It has been shown by Crane
and Krane (154) and by Lee and Lifson (164) that in experimente with 1, 5
-anhydro-D-glucitol in H2018 and with glucose-2-018 in H20, no exchange
of o18 occurs during transport. They have also shown that the stable hy
drogen atom at carbon 2 is not exchanged with tritiated water. Wilson and
Landau (156) have demonstrated that 2-0-methyl-D-gluoose is not actively
transported. Sols (165) and Wilson and Crane (166) have shown that 2-deoxy
-D-glucose is not actively transported. But the fact remains that phospho
rylation at the carbon 2 position is still a possibility, and has neither been
proven or disproven, particularly when the occurrence of this reaction is
limited to the brush-border region and not taken for the absorptive cell as
a whole.
For the present the consensus of opinion on the adequacy of the
phosphorylation hypothesis is very much in the negative. Crane (140)
regards it, in the light of accumulated evidence against it, no longer
tenable. Smyth (159) maintain.s that since part of the sugar transported
is metabolized either to lactic acid or co2, the presence of phosphate
compounds or esters in the cell is to be expected and probably bears no
relationship to the active sugar transport process. Sols (184) has shown
that the specificity of intestinal hexokinase bears no relationship to the
specificity of active sugar transport. Specificity and mutual inhibition of
sugars observed by Crane and Krane (162) and the demonstration by Landau
42
and Wilson (185) that the absorbed glucose does not pass through the tissue
pool of glucose-6-phosphate has once again pushed this hypothesis into the
region of pure speculation and oblivion. Nevertheless the phosphorylation
hypothesis has been a useful one and has stimulated much thinking and
experimentation pertaining to the problems of absorption.
43
Transport Mechanisms
Having established, in previous sections, the important regulatory
role of the cell membrane as a living functional unit, the integrity of which,
to a large extent, determines the viability of the cell, let us now turn to
a consideration of the nature of this membrane in so far as its functional
characteristics are concerned in the mechanisms involved in its action
governing the influx and efflux of materials.
It has been intimated that multienzym.e systems with an elaborate
and intricate sequential array of cofactors regulate and coordinate cell
functions intracellularly and it does not appear too unrealistic to assume
that the same or similar enzyme systems probably dictate the functions of
the cell membrane itself in carrying out its regulatory function through its
complex architecture or structural mak.e up.
In the course of the past 60 years, since the early work of Van
Slyke and Meyers (59) with proteins and amino acids and that of Cori (117)
and V erzar and McDougal {30) with the sugars many attractive hypotheses
have found their way into the voluminous literature all attempting to explain
what may, at first glanee, appear to be a simple process, namely, the passage
or mechanism of entry and exit of substances into and out of cells. Such ex
planations as, simple diffusion, solvent drag, diffusion restricted by a lipid
barrier, mediated transport, pinocytosis, active carrier transport and others
have been listed by Park (118) and briefly discussed. It would be some
what repetitious and superfl.uous to discuss here, in detail, each and
everyone of these possible me chanis ms. Instead, it may suffi ce to con
sider a few selected ones so as to reveal on the one hand the complexity
44
of the problems of absorption, and on the other, what seems to be possibly
the best available explanation which can account for ail the presently known
experimental observations of numerous investigators in this field.
Definition of terms
A consideration of hypotheses of mechanisms of absorption may
best begin with the definition of terms which repeatedly occur in the litera
ture of this subject and are frequently misused or used with varying conno
tations.
In this respect the term "active-transport" occurs more frequently
and is very popular and has been given different meanings by different authors.
Since we do not yet know the exact nature of the mechanism of the passage
of substances across cell membranes, special terms like ittransport" are
difficult to fit into exacting conditions and are somewhat misleading.
There are fundamentally two types of passage of material.s across
cell membranes, one, termed "passive" is governed by electrochemical
forces or kinetic energies of the particles and therefore is subject to the
laws of diffusion and osmosis and the other, termed "active11 is directly
45
dependent upon the continuous expenditure of energy produced by the meta-
bolic activities of the cell. The "active" process also conforms to the
added stipulation that it is capable of transferring or accumulating sub-
stances against a concentration difference particularly when the direction
of the movement is from a very dilute solution to a very concentrated one.
For example, with K+, a concentration difference of fifty between muscle
and interstitial space, for the striated muscle of the gastrocnemius of
Rana esculenta has been recorded by Ernst (111) and with galactose-c14,
a concentration difference of ten-thousand fold higher than that of the exter-
nal medium has been observed by Horecker, Thomas and Monad (119, 120,
121) in the case of a certain strain of galactokinase-less mutant of E. coli
(119, 120, 121) and in the yeast cell, during active fermentation of glucose,
+ K transfer from the medium into the cell against a concentration difference,
as large as 5000 to 1, has been shown by Rothstein and Demis (122).
Wilbrandt (132) suggests that the term "active transport" be aban-
doned in favor of more descriptive terms such as, up-hill transport, down
-hill transport, metabolism linked transport and carrier transport and so on
in order to a void unnecessary confusion and misunderstanding. The re are
those who prefer to call all types of transport 11active" in which the activity
of the cell is in some way involved as for example in the formation of a
carrier-substrate complex; and, there are those who simply use the term
"transport" to mean what others mean by the term "active transport".
46
Rosenberg (133) defined the term "active-transport" in 1954 as
a selective, endergonic process. Quastel (134, 139) refera to it as a pro
cess by which sugars can be absorbed against a concentration gradient and
one in which the mechanism is controlled by cell oxidations. He points out
the unidirectional nature of the process in the guinea-pig-intestine (i.e. from
the mucosal surface to the serosal) and emphasizes the fact that the process
is energy-dependent possibly metabolic-energy in the form of ATP being re
quired.
At present there is no complete general agreement (132, 135) as
to what exactly is meant by "active-transport", but the definitions put forth
by Rosenberg and Quastel will be adhered to throughout the discussions in
this text. It may also be added that active transport is sensitive to tem
perature and pH changes, completely dependent on the presence of oxygen,
inhibited by the action of numerous metabolic inhibitors and displays com
petitive phenomena (167, 168,169, 155,63, 139, 128,172, 173).
Simple diffusion
The simplest process by which substances can pass through ali
membranes is by simple diffusion. The rate at which this diffusion would
proceed would depend to a large extent on the initial concentration of the
substance on the outside of the cell and also on the permeability of the
47
membrane to that substance. In the case of the surviving guinea-pig-intes
tine the passive diffusion rate, is directly proportional to the mucosal con
centration of the substance. There is no metabolic energy required to
maintain this process and it will continue until the concentration of the
substance is approximately equal on both aides of the membrane. However,
actually the process will continue because of the constant removal of the
substance by the metabolic activities of the cell.
The mechanics of simple diffusion do not account for a number
of experimentally observed factors. For example, in the case of the
sugars there is a high degree of stereochemical specificity shown by the
membrane. Glucose and galactose are very rapidly diffused but mannose,
sorbose and the pentoses are relatively quite slow in their movements. Why
should there be such discrimination or structural specificity if a11 are ab
sorbed by simple diffusion. The kinetics of sugar uptake are totally incon
sistent with the process of diffusion. That is they show Michaelis-Menton
type of relationship, in that their uptake reaches a maximum with increasing
extracellular concentration of the substance, suggesting an enzymatic or
adsorption type of process taking place rather than one of diffusion which
should follow Fickt s equation.
Mediated transport or facilitated diffusion
In this process the molecule to be transported combines with a
constituent, called a carrier, which is located on or near the surface of
the cell membrane to form a reversible complex that oscillates between
48
the inner and outer surfaces of the thin cell membrane. This combination
need not necessarily be enzymatically controlled. The carrier constituent
could be endowed with structural specüicity characteristics for the incoming
molecules and could conform to Michaelis-Menton kinetics (136). Moreover,
thermal agitation or molecular deformation of the carrier could account for
the binding and release of the transported molecule. This system also
does not require energy derived directly from the metabolism of the cell
and hence does not transfer against a concentration difference in the sense
defined earlier in this section but, if the transported molecule can be bound
to some intracellular site or if the counter flow of a transport competitor
is present it could lead to up-hill transport.
As in the case of the simple diffusion process the carrier concept,
as depicted here, can not account for a number of experimental observations.
For example, Rothstein, Meier and Hurwitz (123) have shown that in the
uptake of sugars by the yeast cell the uranyl ion (U02 +1 in low concentrations
completely blocks their uptake under anaerobie conditions. This appears to
indicate that the carrier surface is completely saturated or blocked but when
oxygen is allowed to enter the system sugar uptak.e is resumed at a rate
which is 40 percent of the normal rate, although it has been shown that
oxygen has no influence on the binding of uranyl ions by the cell. The
49
uptake of sugar is completely inhibited under aerobic conditions requiring a
higher concentration of uranyl ions. This is suggestive that the sugar finds
entry into the cell by two distinctly separate pathways one under anaerobie
conditions and one under aerobic conditions. This simple carrier system
visualized above can not adequately account for these observations and others,
chiefly stimulation on the surface reaction with inorganic ions.
Pinocytosis
This mechanism of absorption is a very interesting one and will
be described in order to help bring out a number of factors which are common
to most membrane phenomena. The study of pinocytosis promises to give
information on membrane activities and reactions which perhaps can not be
obtained in any other way. It is an important form of "active transport" and
the only one which has be en se en with E/M micrographs, with the exception
of studies carried out on the microvilli or the "brush-border" by Miller and
Crane (46, 137) and by Holt and Miller (138) and by Padycula et al (47).
The process was first observed in 1925 by J. G. Edwards of Johns
Hopkins University working with amoebae. In 1931 W. H. Lewis of the same
institution discovered the same phenomenon in cella grown in tissue culture.
He realized the importance of the process in that the cella were able to take
in substances that can not diffuse into them or be taken in by ordinary phago
cytosis. He promptly named the process pinocytosis meaning "drinking by
50
cells". Pinocytosis is initiated by the formation of depressions or indentations
of the outer membrane of the cell. These indentations are narrow channels
one or two microns wide and extend through raised cones or protrusions.
The channel is filled with the fluid of the surrounding medium and part of it
is pinched off near the bottom end forming a tiny pocket, about one micron
in diameter, or a "pinosome". After detachment the pinosome migrates into
the interior of the cytoplasm of the cell where its contents are emptied after
the membrane surrounding it is ruptured or digested. Although a great deal
of information about pinocytosis has been obtained through the study of amoebae
the process has been observed to take place in many different types of cells
including mammalian cells in tissue culture and certain plant cells. The
occurrence of pinocytosis has also been observed by Clark (182) and Halliday
(183) in the jejunum and ileum of suckling rats and mice and accounts for
the ability of the newborn of these species to absorb antibody and other pro
teins and colloidàl materials by this process.
Holter (125) studied pinocytosis in 1954 on a more quantitative
basis. He showed that amoebae could be induced to take in large amounts
of glucose, which they normally dontt, by introducing protein into the bathing
media. Other substances like salts and amino acids and even viruses and
ultra-violet light have been found to induce pinocytosis in amoebae.
We may at this point raise the question: are the channels in
pinocytosis produced by a series of complex biochemical reactions or are
they the simple physical response of the lipoprotein membrane around the
cell? It has been shown that cyanide and carbon monozide inhibit pinocy
tosis - apparently - the process requires ATP and therefore involves an
energy consumlng chemical reaction. It is also temperature sensitive,
channels appearing much slower and fewer in numbers at lower tempera
tures. Furthermore the process can take place after the removal of the
inducing agent, showing that it consista of at least two steps.
51
Brandt (126) using protein labelled with fluorescein and Schumaker
(127) using radioactive protein showed that in pinocytosis the cell surface
could bind a large amount of protein (50 times the amount of protein con
tained in its own volume of solution) indicating that a great deal more ma
terial is taken in into the cell through pinocytosis than would have been
predicted from the volume of fluid ingested by way of the pinosome. It is
significant to realize that the winding channels which have a very large sur
face area serve primarily as a means of entry for bound material rather than
for liquid. This surface binding suggests that in the case of more specialized
cella specifie binding sites or functlonal groups may be present on their mem
brane surfaces and thus selectively bind certain molecules.
Other studies by Rustad (124) using basic dyes such as toluidine
blue have shown that the dyes are attached to the surface of a living amoeba
the molecules of the dyes assuming a fixed, characteristlc orientation with
respect to the surface, probably by combining with acidic groups or sites on
52
the membrane. Furthermore the dyes induced pinocytosis and there was a
competition for the sites when the dyes were placed in the medium along
with proteine. From this it was concluded that the binding sites must be
on or very near the surface to have been accessible to proteins. Further
investigations with use of chemical techniques have shown the surface layer
to be mucopolysaccharide.
Another question to be answered was: what is the exact location
of the binding sites? Many cells have a very fine fringe area outside the ir
cell membrane surface. Am.oeba electron photomicrographs show this fringe
area to be made up of discrete fibrils that look like hairs with no apparent
internai organization. When ferritin, a very dense protein, is placed in the
medium bathing the amoebae, the molecules of this substance are rapidly
picked up by the fibrils or hairs showing that they are the binding sites. In
the case of cells with no fibrils or hairs, sites may be in extraneous coats
that do not show up in electron micrographs.
Pinocytosis is, then, a very complex process whereby a cell can
take in substances to which its membrane is ordinarily impermeable. The
surface area of the cell, the fringe area, is highly specialized having numerous
hair-like structures called fibrils which have important absorptive characteristics.
They are inducible, energy-dependent, selective binding sites which offer a
tremendous surface area for the attachment of on-coming molecules.
How channel formation is induced perhaps by the binding process
is still a moot question and is under current investigation.
Carrier active-transport
A very recent version of this type of mechanism of absorption
has been presented by Crane (140) as a possible working hypothesis and
applied to the process of sugar active transport by the small intestine of
the golden hamster.
In order to achieve spacial orientation pertaining to locus of
action of absorption, Crane has attempted to describe the brush border
53
pole of the epithelial cell as made up of two activity regions, one designated
as the hydrolase activity region, and the other as the transport activity region.
These regions or zones of activity are presumably located close to each other
at the apical or brush border area of the absorptive cell, the hydrolase acti
vity region being the outermost or located in or on the plasma membrane
(141) which lines the microvilli and the transport activity region being located
immediately adjacent to it or lying next to it closer to the cytoplamm of the
cell.
An incoming molecule, for example sucrose, fi.rst reaches the
hydrolase activity region where it comes in contact with invertase and is
split into glucose and fructose. The fructose moiety now approaches the
transport activity region and there combines with a mobile carrier molecule
to form a fructose-carrier complex. No sodium ions or metabolic energy
is necessary to render this complex mobile. Fructose traverses the trans
port activity region in the form of this complex and upon emerging from it
54
the complex dissociates the fructose molecule is released and the mobile
carrier returns to the hydrolase activity region leaving the fructose mole
cule behind. As long as the concentration of fructose is greater in the hy
drolase activity region the process continues with a net gain of fructose in
the cell.
In the case of the glucose moiety the carrier system requires the
presence of sodium ions to render the carrier mobile. Glucose in the pre
sence of Na+ forms a complex with a carrier which is rendered mobile. The
complex traverses the transport activity region and then dissociates into glu
cose and carrier molecules. The carrier returns to the hydrolase activity
region as in the case of the fructose carrier. The sodium ions transported
are now returned to the hydrolase activity region by an active process which
requires the expenditure of metabolic energy. Glucose accumulates be cause
of the absence of Na+ ions, required for· its return trip to the hydrolase
activity region or the medium.
This hypothesis appears to be consistent with a number of experi
mental facts available to us at the present time, however, as in the case of
the other hypothetical mechanisms proposed it fails to account for ali aspects
of intestinal absorption.
The structural or spacial relationships in or about the apical area
or so called brush border area are still far from being clear. Unfortunately
E/M studies have not, as yet, been able to show us the precise relationships
of the various parts or forms of the apical area. Crane speaks of a diffuse
55
terminal web being present at or in the borders of the microvilli, we know
nothing of the structure or nature of this web. The microvilli are preau
mably covered with the plasma membrane, the hydrolases are believed to
have been found in this membrane (163, 182). If this is true, then, how
does a molecule of sugar pass through the plasma membrane to come in
contact with the hydrolases? We are compelled to accept thai there are at
least two types of carriers present in the so called transport activity region,
one rendered mobile by Na+ ions, and the other unaffected by the ir presence.
The question of the stereospecificity of the incoming molecule for the carrier
site remains still unanswered.
Nevertheless the hypothesis is a useful one which can facilitate
the elucidation of the future problems of sugar active transport. It does
adequately account for the obligatory requirement of Na+ ions (128) and the
continuous supply of metabolic energy for active sugar transport. However,
in the final analysis it must be concluded that the mechanism of active
transport of sugars and other substances is still unknown and open to specu
lation and to experiment. The Crane mobile carrier hypothesis (140) is quite
new and unproved and must need to stand the test of time and the scrutiny of
other investigators in this field before it can be accepted or rejected.
56
Effect of conformation on active sugar transport
A general assumption on the part of many workers in the field of
intestinal absorption is that active sugar transport is carried on by a single
process. Since the phosphorylation hypothesis is no longer considered to be
consistent with experiment, the "carrier" mediated type of transport has
gained much favor. A recent version, by Crane (140), of this type of me
chanism operative in active sugar transport has already been described.
Any carrier-hypothesis of active sugar transport, however elaborately con
ceived must take into account, not only the structural differences among the
sugars in relation to their properties to be transported, but also steric fac
tors as weil. · It must in some plausible way explain why a given carrier
molecule or surface should prefer to be attached to one sugar molecule
rather than another. It has be en pointed out in a previous section that all
actively transported sugars have certain common structural features which
can not be altered without the loss of their capacities to be actively trans
ported. The indispensabllity of the hydroxyl group on carbon 2 as in the
glucose configuration has been pointed out. The partial formula summari
zing the minimum requirements for active sugar transport is given below.
All actively transported sugars tested are known to conform to these require
ments. In addition, another very important feature and one pertinent to the
1 -c-
OH
57
relationship of functional groups in space has been recognized. This is
known as conformation (constellation in German or Swiss usage).
In addition to structural isomerizations and geometrical isomerations
of the cis-trans variety and also those of the alpha. and beta. pyranose and
alpha. and beta. furanose types, the suga.rs with six-membered pyranose rings
are capable of another type of isomerization known as conformation. A par-
ticular shape or arrangement in spa.ce of a. molecule in which more than one
arrangement is possible by simple rotations about single bonds is referred
to as conformation. Conformation is, then, a type of isomerization which
involves changes in the ring shape. This change in the ring shape vastly
altera the relative position of various groups within the same molecule and
frequently influences reaction rate. To facilitate the orientation of the reader
some of the formulae showing the structures of glucose will be given, as
examples, on the next page.
In 1885 Adolf von Ba.eyer, in order to explain the differences in
rea.ctivity of compounds of various ring size, noted that if valences of the
carbon atom were directed toward the corners of a regular tetrahedron the
normal angle between any two pairs of linkages would be 109° 28' and he
58
postulated therefore that any deviation from this angle would result in a
condition of interna! strain. The linkage of two carbon atoms by a double
bond would then result in a very considerable distortion of the normal
valence angles with consequent strain and high energy content. This theory
adequately accounted for the reactivity of the Ql.efins but since it was based
on the concept of a planar model could not account for the behavior of six
mem.bered rings like cyclohexane.
In 1890 Sachse suggested that in cyclohexane the carbon atoms do
not lie in a plane, as postulated by Baeyer, but asswne a strain-free,
puckered configuration. When the valence angles are kept normal (109° 28t)
two space modela of the cyclohexane ring can be formed. These are cal.led
the ''boat" and "chair" forma.
In the realm of the sugars the existence of the alpha and beta iso-
mers or anomers has been one of the most important reasons for the formu-
lation of ring structures. For example, the isomerie alpha and beta glucoses
11
have quite different solubilities, melting points and rotations. Boeseken (187)
and his co-workers have shown that when adjacent ela hydroxyl groups are
present in a strainless six membered ring these groups will tend to repel
each other mutual.ly, that is the adjacent groups will tend to be oriented as
far apart as possible. Hukel (188) has shawn that the geometry of six-mem-
bered carbon-rings of the strainless type is auch that cis groups may be
oriented a maximwn of 72° apart, whereas trans groups may approach one
59
another as close as 48°. Additional complications arise for most sugars
other than glucose, in that, pairs of contiguous hydroxyls are present
in addition to those found on carbons 1 and 2. Il Boeseken (189) has shown
that also the furan ose forms may react preferentially.
Obviously the Fischer-Tollens formula representing an extended
chain of carbon atoms connected by an oxygen bridge between positions 1
and 5 is an impossible situation and can not at ali account for the different
reactions of the sugars. So, in order to provide a more understandable
picture of the structure and configuration of the sugar molecule, Haworth
proposed a three-dimensional perspective model, in which the pyranose ring
represented a single plane with all the atoms lying in it. This was a consi-
derable improvement over the older cyclic forms of the Fischer-Tollens for-
mulae but it too suffered from shortcomings and cau only be regarded as an
oversimplified model of a true molecular structure. It conceives of a single
coplanar ring which means that the valence angles would necessarily be appre-
ciably greater than those in a nstrainlessn structure having valence angles of
109° 28'.
Reeves (190, 191) working with cuprammonium glycoside complexes
has come to regard the pyranose ring as a regular skew hexagon theoreti-
cally capable of being oriented in any one of the eight Sachse strainless
ring conformations. He visualizes that small deviations from this regular
structure could exist and could produce minor but important changes in the
60
relative position of neighboring ring substituents. In a skew hexagon ring
of slight distortion, adjacent cis hydroxyl groups ~ould be closer together
than adjacent trans groups. The chemical reactions of the sugars are con
sistent with this stipulation.
Scattergood and Pacsu (192), and Hassel and Ottar (193) and others
(194) have suggested that boat-form pyranose rings are energetically unstable,
because of large repulsions both in the ring and among the subsidiary groups
and Hassel and Ottar have stated that tt all experimental evidence indicates
that the six-membered pyranose ring found in many sugars will generally
have the staggered or chair form11 • These pyranose ring forma are shown
on the next page.
Probably the repulsion due to the true cis relationship between
adjacent valences is responsible for the instability of boat form rings.
Whenever possible the adjacent carbon atoms take the position of "staggered
-valences" (195). Hence the conformation of the greatest stability is the one
in which the carbon atoms or groups attachetl to them are as remote as
possible, and the one of !east stability is that of greatest proximity.
In the consideration of the stability of the pyranose ring shape a
number of factors have been noted which when present make for the insta
bllity of the ring. These factors have been evaluated in terms of instability
units. These factors are:
1. Pyranose rings will assume a chair form in
preference to any boat form whenever both are
FORMULAE OF GLUCOSE
FISCHER - TOLLENS FORMULAE
1
HO, ,.H
H-?-OH 1
CHO 1
H-C -OH HO-C-H 0 HO-C-H
1
H-C-OH 1
H-C-OH H-~-OH 1
H-C 1 1
CH 20H CH20H
(3- D- GLUCOSE ALDEHYDO-D-GLUCOSE
HAWORTH FORMULAE
__.. H,OH-+---
H OH 'c"' H-Ç~
HO-C 0
H-~-OH 1
H-C 1
CH 20H
oC -D- GLUCOSE
J-----0
61
OH
f-D-GLUCOPYRANOSE
OH
D- GLUCOPYRANOSE
OH
~-0-GLUCOPYRANOSE
CH 20H 1
HO-C-H 0
CH 20H 1
HO-C-H 0
H,OH
OH OH OH
~-D-GLUCOFURANOSE D-GLUCOFURANOSE ~-D-GLUCOFURANOSE
--
CONFORMATION
Cl
/ ' "' '
CONFORMATION
IC
' '
structurally possible. Any erected substituent other
than hydrogen on the pyranose ring introduces an ele
ment of instability into the ring conformation. This is
assigned an arbitrary value of one instability unit.
2. Hassel and Ottar effect - in a conformation in which
the primary carbinol group and one hydroxyl group
are in an erected position on the same aide of the ring
an energetically unfavorable or unstable situation arises.
This condition is designated by H.
3. A very special case of instability arises when the hydroxyl
group on carbon 2 is erected. In this case the C-0 valence
of this group is in such a position that it bisects the tet
rahedral angle of the two C-0 valences of carbon 1, as
for example in j3 -D-mannose; this arrangement places
three oxygen atoms very cise to one another. This re
sulta in a very unstable condition and has been designated
by 6. 2 (the 2 referring to carbon 2). The influence of the
A 2 condition seems to be greater than that of the insta
bility introduced by two ordinary erected groups in dater-
ring conformation and is given the value of 2. 5 instability
units. These conformational instability factors have been
determined for a1.1 the D-aldohexoses and the D-aldopentoses.
Those for a few of the D-aldohexoses will be listed.
62
63
"Instabllity Factors" for the various al.dopyranosides in the C 1 and lC
conformations.
Aldopyranoside Instability Factors
Cl lC
0( -D-Glucose 1 H1 A 2, 3, 4, 5
~-D-Glucose none JI[, 1, 2, 3, 4, 5
e<-D-Gal.actose 1, 4 H, A 2,3, 5
,# -D-Gal.actose 4 JI[, 1, 2, 3, 5
o(-D-Mannose 1,2 H,3, 4, 5
,.B-D-Mannose .A2 111, 1, 3, 4, 5
c<-D-Idose 1, 2, 3, 4 5
)9-D-Idose A2, 3,4 H, 1, 5
"'-D-Gulose 1, 3, 4 A2,5
j8 -D-Gulose 3,4 H, 1, 2, 5
The numbers refer to erected groups (other than hydrogen) on the carbon designated. A 2 refera to the exal.ted influence of an erected group on carbon 2.
H refers to the Hassel and ottar effect when an erected group on carbon 5 occurs on the same side of the ring with another erected group. With 2 erected groups the JI[ appears in bold face type. The effect of one erected group is one instability unit. The e:ffect of A 2 condition is 2. 5 instability units.
64
There are then, two possible "chair" forms or conformations of the py
ranose ring. These are designated as Cl and lC "chair" forms. In
solution the conformation with the !east sum of the instability factors will
predominate. For example, in jJ -D-glucose aU of the substituent groups
except the hydrogena are equitorially situated in the Cl conformation and
therefore there is no instabtlity in this form or the sum of the instabllity
units is zero and hence this conformation is quite stable. But in the lC
conformation all of the substituent groups except the hydrogena are axially
situated and two of them are on the same aide of the ring as the primary
carbinal. group at carbon 5. The sum of the instability units in this case
is 5 plus a double Hassel and ottar effect. Therefore the lC conformation
of ,;g-D-glucose is quite unstable, hence a solution of fi -D-glucose will
be entirely in the Cl conformation.
In the case of IX. -D-glucose the Cl conformation has only one
erected substituent on carbon 1 having a sum of one instabllity unit. But
the lC conformation has four erected groups one of which is at carbon 2
giving the .D. 2 effect and one on the same side as the primary carbinol
group giving the Hassel and Ottar effect so that the sum of the instabllity
units is 5. 5 plus the Hassel and ottar effect. Therefore the Cl confor-
mation for C( -D-glucose is definitely more stable and predominates in
solution. Glucose is probably entirely in the Cl conformation in solution,
however the j9 -isomer predominates in the equilibrium mixture ( ~ -isomer,
65
64%; « -isomer, 36%).
From the available data on the sugars which have been tested, to
date, the requirement as far as conformation is concerned seems to be for
the Cl chair form. It does not suffice therefore to say that the sugar to
be actively transported must be a D-sugar, al.though aJ.1 the sugars tested
up to the present time have been D-sugars. For example, D-gulose is an
aldopyranose which has a primary carbinol group at carbon 5 and a hydroxyl
at carbon 2 in the D-glucose configuration. One would expect that this sugar
would undergo active transport on the basis of L- and D- sugar criterion but
D-gulose is not actively transported. D-gulose ia the 3, 4 di-epimer of
D-glucose and hence has erected groups at 3 and 4 positions. D-galactose
which is the 4-epimer of D-glucose is actively transported as rapidly as
glucose, therefore it can be concluded that an erected group at carbon 4 has
no particular inhibitory effect. D-allose which is the 3-epimer of D-glucose
is only transported one-sixth as rapidly as glucose, hence, we can say that
the existence of an erected group on carbon 3 does have an inhibitory effect
on active sugar transport. It follows then that the existence of one erected
group at carbon 3 on D-gulose is one contributing factor for its not being
actively transported. The possibility must not also be overlooked that we
may have a special effect if there are erected groups on carbons 3 and 4
adjacent to one another in the ring. This may possibly introduce steric
hindrance factors for the site on the membrane or the carrier molecule.
66
The particular effect of an erected group at carbon 5 is not lmown.
The sugar L-idose is structural.ly identical. with D-glucose except that the
carbinol group at carbon 5 is axial.ly situated in L-idose. The primary
carbinol group in glucose is equitorial.ly situated. L-idose should be tested
in order to determine the effect of an erected group on carbon 5.
In conclusion it can be said that the significance of conformational.
considerations pertaining to sugar active transport is twofold. Conformation
is a very important factor involved in, the determination of the functional.
availability of the group present on the sugar molecule as weil as the shape
of the molecule itself as a whole (i.e. steric effect). Any consideration,
therefore, of physico-chemical. reactions involved in active sugar transport
must take into account the changes brought about by conformational. factors.
For example if a sugar molecule is attracted to a carrier site and if a
particular functional. group is not necessary, per se, in this reaction, but
plays a role only by reason of its positional. orientation on the molecule
(steric effect) then conformational. anal.ysis can certainly shed light on the
situation.
Inhibition Produced by Phlorhizin and Phloretin
Phlorhizin and phloretin deserve particular consideration because
of their extensive use in biochemical experimenta in which they display
rather unique inhibitory action toward absorption and metabolism of the
sugars by the intestine and reabsorption by the proximal kidney tubule.
Nomenclature and chemistry
Phlorhizin which is a classical example of a drug capable of in
ducing glycosuria appears in the chemical and biochemical literature under
various spellings. For example, phlorhizin, phlorizin, phloridzin and
phlorrhizin are interchangeably used. It is a glycoside or glucoside com
posed of phloretic acid, phloroglucinol and ;8 -D-glucose units. The for
mulae of these compounds are given on the next page. The com.bination
of these units as indicated makes up the phlorhizin molecule. Phloretin
67
is the aglycone of phloretic acid and phloroglucinol. It is interesting to
point out that the carbohydrate moiety of phlorhizin is the more abundant
fi -isomer of glucose and probably exista in solution in the C 1 confor
mation. It is also interesting to indicate that the phloroglucinol molecule
unlike a simple phenol which exista very little in the keto form or a simple
ketone which exista almost entirely in the keto form, behaves as if both
68
FORMULAE OF PHLORHIZIN AND DERIVATIVES
0 Il
CH -CH -C 2 2
OH
OH
PHLORETIN- 21-(3- GLUCOSIDE ( PHLORHIZIN)
l Dl LUTE H + OR
SACCHARASE, pH 4.45
OH OH
(3- ( p- HYDROXYPHENYL) PROPRIOPHENONE (3-D- GLUCOSE
(PH LORE TIN)
HO OH
OH H2
'ENOL' 1 KET0
1
PHLOROGLUCINOL
69
forms are present or are in very mobile equllibrium with each other. Both
the parent compound and the aglycone phloretin possess aromatic rings which
are not conformationally stable as the pyranose rings in the sugars. These
rings are rich in phenolic hydroxyl groups - even the keto group on the pro
prionic acid fragment might conceivably enolize under certain conditions and
provide an additional phenolfc hydroxyl group - all of which simply means
that both compounds offer a number of phenolic hydroxyl groups arranged
in a particular spacing pattern. It has been proposed by LeFevre (226)
that if the surface or membrane of erythrocytes possesses certain functional
groups or loci which are distributed in a particular recurrent spacing pattern
and that these loci are also capable of combining reversibly with phenolic
hydroxyl groups it is possible that the spacing pattern of the phenolic hy
droxyls as they occur in phloretin or phlorhizin and similar compounds
might weil fit the pattern and bind these sites, thus making them unavallable
for the sugar molecule.
The similarity of these compounds to alloxan (2, 4, 5, 6 - tetraoxy
pyrimidine) as far as abundance of phenolic hydroxyls or keto groups are
concerned is noticeable. As is weil known a single dose of alloxan can pro
duce permanent pancreatic diabetes in experimental animais by destruction
of the insulin producing beta cells of the islands of Langerhans in the pan
creas (196, 197). Phlorhizin diabetes is, however, actually a renal diabetes
in which glycosuria is produced by blocking or interference in some way of
reabsorption of glucose by the cells of the epithelium of the renal tubule
and not by destruction or inhibition of insulin production by the pancreas.
70
The parent compound exists as a crystalline dihydrate with a mo
lecular weight of 472.4 grams per mole (C21H24o10 . 2H20} and the agly
cone (C15H14o~ has a molecular weight of 274.3 grams per mole. Both
compounds are only sparingly soluble in water so that small amounts of
ethanol are added to take them into solution.
The Inhibitog action of phlorhizin
The discovery of the glucoside phlorhizin as a drug capable of pro
ducing profuse of glycosuria when injected into animais was made as early
as 1886 by von Mering (198). The e:ffect of the drug was so close in simu
lating the symptoms generally associated with pancreatic diabetes that for a
time it was believed that its action was one of inhibiting the utilization of
glucose. This view was strengthened by the findings of Ringer (199) and
Cori (200) that insulin given to phlorhizinized animais would diminish their
glycosuria and increase their respiratory quotients. However, it was later
shown by others (201, 202, 203) that when phlorhizin was given blood glucose
levels would go down. This observation could hardly be reconciled with
an inhibitory action of phlorhizin on carbohydrate utilization. Moreover,
when glucose was given to maintain blood levels the glucose was metabolized
with resultant disappearance of phlorhizin diahetes symptoms such as
hyperketonemia, gross ketonuria and nitrogen wastage. And perhaps the
last blow to this concept was delivered when Deuel (201) and Nash (202)
both were able to show that when animais were nephrectomized pblorhizin
diabetes could not be produced in them.
71
At the present time, it is weil documented that the action of pblor
hizin is separate from any pancreatic involvement and also not entirely con
fined to the kidneys, where the impairment of carbohydrate utilization, it
appears to produce, depends solely upon inhibition of the reabsorption of
glucose in the renal tubules. It is also well established that pblorhizin
does specifically show pronounced inhibitory action against the active absorp
tion of sugars from the intestine. This has been shown by the in vivo experi
menta of Bogdanov (204); Lundsgaard (174); Nakasawa (206); Ponz and Lluch
(207); and Wilson (209) and the in vitro experimenta of Fisher and Parsons
(210); Fridhandler and Quastel (63); Newey, Parsons and Smyth (211); and
Riklis and Quastel (128).
In 1933 Chasis, Jolliffe and Smith (212) were able to show that the
clearance of glucose under the influence of phlorhizin risee untll it becomes
identical with the clearances of sucrose and xylose. The glucoside the re fore
inhibits the active reabsorption of glucose so that glucose behaves like sucrose
and xylose, sugars which are now lmown to be passively transported by diffu
sion. It has been suggested that the action of pblorhizin consista in its
capacity to interfere or inhibit phosphorylation of glucose. However, both
Lundsgaard (205) and Lambrechts (213) found that the concentrations of the
72
drug required to induce diabetes are far lower than those required to inac
tivate the phosphatases. Lambrechts (214) also reported that when glycero
phosphate or parathormone was given phlorhizin sharply reduced the excretion
of phosphate in the urine without affecting its concentration in the blood.
This indicated that pblorhizin did not appear to interfere with phosphory
lation. Fleischmann (215) observed that in concentrations which induced
glycosuria phlorhizin did not affect the respiration of kidney slices. He
concluded from this and other experimenta that phlorhizin is a highly adsor
bable material and that when it is adsorbed by cella it blocks the absorption
of glucose. McKee and Hawkins (216) have shown that glucosuria can be
produced with phlorhizin without inhibiting the reabsorption of other sub
stances.
In 1922 Nakasawa (206) using tied-off loops of rabbit intestine showed
that phlorhizin inhibited the absorption of glucose. In 1946 Sols and Ponz
(217) conducted perfusion experimenta with cannulated intestines of rats and
dogs and discovered that if the phosphatases which are secreted into the per
fusion fluid were inactivated by heat intestinal absorption would markedly de
crease and when fresh phosphatases were added to the perfusion fl.uid absorp
tive activity would be restored. Shapiro in 1947 (218) suggested that the
action of phlorhizin is auch that it interferes with the production of high
energy bonds by preventing the phosphorylation of creatine. Bogdanov and
Barker in 1950 showed that when rats are injected phlorhizin, the absorption
of glucose and galactose from the small intestine are inhibited but not that
73
of fructose (204). In 1952 Ponz and Larral.de (219) in their studies of
glucose absorption from the intestine observed that phlorhizin at a concen
tration of 2 x 10-5 M had an inhibiting e:ffect on glucose transport, and also
that much greater amounts were needed to bring about inhibition of phos
phorylation reactions than were needed to inhibit intestinal absorption of
glucose. Hence they reached the conclusion that phlorhizin could not be
directly involved in phosphorylation reactions with respect to the transport
mechanism.
Parsons, Smyth and Taylor (220) in 1958 conducted a series of ex
perimenta using the everted sac technique of Wilson and Wiseman (227) in
order to determine wheth.er phlorhizin produced inhibition of glucose absorp
tion by a:ffecting metabolism or by a:ffecting the transport mechanism itself.
When no glucose was introduced into the incubation medium phlorhizin at a
concentration of 10-3 M was required to inhibit the metabolism of the endo
genous glucose and when the concentration of the phlorhizin was doubled
(2 x 10-3 M) they observed a marked decrease in metabolism. When la
belled glucose was added to the medium they found that a concentration of
phlorhizin as low as 10-6 M could produce an inhibition of glucose trans
port without a:ffecting the oxygen consumption or the utilization of glucose.
This probably meant that even as low a concentration of phlorhizin as ~0 -6 M
was preventing the entry of the labelled glucose into the cell. By using la
belled glucose in the medium on either the mucosal or serosal. side they
74
also were able to determine that pblorhizin had little effect on metabolism
of glucose initially present in the serosal fl.uid but considerably more in
hibitory effect on the metabolism of glucose present in the mucosal fl.uid.
These experimenta indicated that pblorhizin had two different sites of action
in the intestine - one on metabolism at high concentrations (10 -3 M or
higher) and one on active glucose absorption at low concentrations (10-6 M).
In 1958 Riklis and Quastel (128) in their experimenta with the iso
lated surviving guinea pig intestine showed that the potassium stimulated
active transport of both glucose and galactose were considerably more sen
sitive to inhibition by phlorhizin than at normal levels of potassium. They
have therefore drawn the conclusion that the active transport of these sugars
is not only absolutely dependent on the preseneeof Na+ ions and subject to
stimulation by K+ ions but that the inhibitory action of phlorhizin is also
cation-dependent. Fridhandler and Quastel (63) have also shown that phlor
hizin has no inhibitory effect on the conversion of fructose to glucose. It
al.so does not affect active amino acid transport. In this respect the action
of pblorhizin is different from that of DNP which not only interferes with
the conversion of fructose to glucose but also inhibits active amino acid
transport. They have concluded from these studies that since both potassium
stimulated active glucose transport and active galactose transport are highly
inhibited by phlorhizin there must be a common pathway or at least a common
step in their pathways of active absorption which is affected by phlorhizin.
75
Phlorhizin al.so inhibits the active transport of water al.though this
action may be regarded as an indirect effect. It has been shown by Smyth
and Taylor (221) that the absorption of water is dependent upon aerobic
metabolism, and others (220. 221, 222) too have shown that it al.so is depen
dent entirely on glucose metabolism, other sugars like galactose and fructose
being rouch less effective in supporting water absorption. Thus pblorhizin
by preventing glucose absorption interferes with water absorption, however,
this can occur only in vitro because in the intact animal sufficient glucose
1s present or available by way of the blood circulation so that no inhibition
of water absorption is observed.
Inhibitory action of phloretin
Phloretin, the aglycone of phlorhizin is less active in producing
glycosuria but rouch more effective as an inhibitor than the parent compound
in the transport of glucose into erythrocytes (170, 142, 223). But, in the in
testine phlorhizin is at !east ten times more effective an inhibitor of glucose
transport than phloretin. Why this is the case has not been satisfactorily ex
plained although suggestions have been made that the glucose moiety might be
playing a signüicant role in the inhibitory action of phlorhizin in the kidney
tubule and in the intestine, tissues which normally do accumulate sugar.
Phloretin is also known to inhibit the active transport of glycine
into excised mustard roots but not that of other amino acids such as
76
L-arginine, L-methionine and L-leucine (224). The fact that glycine and
glucose are mutually inhibited has been brought out by the experimenta of
Fridhandler and Quastel (63) with the isolated surviving GPI and with those
of Cori (228) with rats. The possibility of the existence of a common
carrier system or a common step in the pathways of active amino acid
and active sugar transport has been suggested by Quastel (63). However
this possibility has not yet been sufficiently explored. It may also be that
glycine, since it is the first member of this class of compounds, possesses
certain special characteristics different from other amino acids, which are
similar to the activities of the sugar molecule with respect to active trans
port.
To what does phloretin owe its specifie inhibitory potency has not
as yet been ascerta.ined. The work of LeFevre (226) has shown that frag
ments of the phloretin molecule such as phlorogucinol and phloretic acid
do not possess inhibitory potency comparable to the phloretin molecule ft
self, moreover1 they do not show any synergism when used together. Com
pounds such as diethylstilbestrol and hexestrol with similar functional groups
have shown only partial inhibitory potency compared with phloretin with re
spect to active sugar entry into RBC. Hence it has been concluded that
phloretin must owe its inhibitory capacity to the orientation and spacing of
the terminal groups .in the intact molecule.
The site of action and the precise mechanism of action of phloretin
and the glucoside phlorhizin is still undetermined. Crane (229) lists four
possible types of action in which phl.orhizin or phloretin may be involved.
These are:
1. Inhibition of enzymes and enzyme systema which
are dependent on nucleotides. (e. g. enzyme
systems involved in phosphorylative activity in
the tissues; hexokinase activity (Ehrlich ascites
tumor cells); ATP requiring enzymes; pyruvic
phosphokinase; and AMP stimulated rabbit muscle
phoaphorylase).
2. Inhibition of intestinal and kidney phosphotases.
3. Inhibition of mutarotase. (this enzyme found in the
membrane, presumably, catalyzes the change of the
sugar ring from a pyranose to some other trans
portable form)
4. Inhibition of sugar entry into cells. (brought about
by relatively low concentration of phlorhizin)
The concentrations of phlorhizin required to bring about inhibition
77
in the case of the first three categories is much higher (lo-3 M) than that
for the fourth whether the latter condition is met in the kidney or in the
intestine. From this fact alone the conclusion has been drawn that whatever
the real nature of the action of these inhibitors, as far as prevention of
active sugar entry into cella is concerned either in the intestine or in the
kidney tubule phosphorylation or dephosphorylation is not directly in vol ved
in their action. It has become increasingly clear that one role of phlor
hizin is its inhibition of active sugax entry into cells. Crane (140) has
postulated a possible mechanism of entry and of active sugar transport
78
into the epithelial cel! of the intestine in which he has linked the active
transport of Na+ ions - which was shown earlier by Riklis and Quastel
(128) to be absolutely necessaxy for active sugar transport - with the site
of action of phlorhizin, namely, that the drug in some way affects the dis
tribution of sodium ions possibly by immobllizing the carrier system.
Competitive inhibition by phlorhizin and phloretin
In 1955 Ponz and Lluch (207) working with the cannulated loop tech
nique measured the inhibitory action of a large number of compounds, in
cluding phlorhizin, on glucose absorption and came to the conclusion that
the action of phlorhizin was more specifically the inhibition of sugar entry
into the intestinal cella. They also showed that this inhibition could easily
be reversed by simply washing out the intestine containing the sugar and
phlorhizin and by refilling it with fresh sugar solution.
In 1956 using a similar technique Jervis, Johnson, She:ff and Smyth
(170) showed that absorption of glucose from a solution circulating through
the entire intestine of the rat could be inhibited by low concentrations of
79
phlorhizin and that this inhibition was reversible.
The fact that much lower concentrations of phlorhizin are required
for inhibition of active sugar entry into cella than are required for meta
boUc effects has been repeatedly confirmed. But, an additional experiment
which further clarified this point was carried out by Lotspeich and Woronkow
(230) who obtained maximal glycosuria in the dog by constant infusion of low
concentrations of phlorhizin (5-10 )'g/Kg/min. ).
Krane and Crane (231) showed that phlorhizin inhibits active galactose
entry into rabbit kidney cortex slices. Riklis and Quastel (128) have shown
that the rate of active galactose transport in the surviving isolated GPI is
also highly sensitive to the action of phlorhizin. Furthermore Riklis, Haber,
and Quastel (173) have shown that glucose competes with galactose for entry
into the cell and is highly inhibitory to galactose transport.
From these experimental findings and others it has become apparent
that the mode of action of phlorhizin is linked with a specifie step - probably
at the point of active entry into the cell, perhaps with the formation of a
complex between the sugar and a carrier molecule, in which the ionie en
vironment plays a significant role - Na+ ions being obligatory and K+ ions
highly stimulating to the transport system. From this point on it has be
come attractive to make the assumption that the inhibitory role of the drug
might be one of competition with the sugar molecule for a site on the surface
of the carrier molecule.
80
Wilbrandt (232) bas suggested that in order to have competition of
this kind one need not necessarily be compelled to visualize that the phlor
hizin molecule would occupy exactly the same locus or site on the carrier
molecule intended for the sugar molecule. It may be quite sufficient to
bind itself on a surface nearby where it would be close enough to prevent
the approach or binding of the sugar molecule. The significant point in
this is that the competitor molecule need not necessarily be structurally
sim.Uar to or an anal.og of the sugar molecule in order to achieve com
petition.
LeFevre (226) has pointed out, in his studies of competitive inhi
bition of active glucose transport across the red cell membrane, that a
two point attachment by the phloretin molecule to the surface of the carrier
molecule is necessary to prod:uce maximal inhibition, and that the terminal
phenolic hydroxyl groups as situated in the particular stereochemical. struc
ture of this compound are determinative factors in its capacity to attach it
self tightly to the carrier molecule and that its action is perhaps in this
respect sim.ilar to ion-exchange resins.
81
Metabolism in the Intestinal Wall
During the past severa! decades more effort has been put forth by
numerous investigators in the direction of the study of the problems of ab
sorption by the intestine and the elucidation of its mechanism than in the
direction of the study of metabolism. Both during the absorptive process
itself and during the period of regeneration and maintenance of the integrity
of the intestinal tissues a large number of synthetic as well as catabolic
processes must be taking place. But the relative importance of syntheses
by the intestinal wall compared with those of other tissues such as liver
and brain has not been recognized or evaluated.
The rapidly regenerating capacity of the intestinal epithelium has
been well accepted (27, 28). Recent studies (140) have also pointed out that
the primary locus of absorption is the epithelial cell and more specifically
its brush border area. Differentiation of the cells and their rapid replace
ment in the jejunal epithelium has been the object of severa! recent studies
(2, 47). Obviously any changes in the morphological and biochemical con
dition of the highly active or regenerative apex of the villus can have pro
found effects on its metabolism and its capacity to perform the important
absorptive function attributed to it. For example, pathological conditions
such as tropical sprue, in which there is a disturbance in differentiation
of cella and in the rate of replacement of the jejunal epithelium leading to
82
atrophy of the villi, have emphasized the fact that under certain conditions
the normal epithelium of the intestine can undergo degenerative changes and
result in an abnorma.l epithelium with considerably impaired absorptive and
metabolic capacities.
It has become increasingly apparent that much information can be
gai.ned througb. the study of the metabolic changes that take place within the
mucosa of the intestine and adjacent layera. The ex.tent of the metabolic or
biochemical changes occurring within the mucosal epithelium during its rapid
replacement and differentiation has not as yet been studied.
That the small intestine is involved in metabolic activities and can
take part in syntheses as well as catabolic reactions which for the most part
take place in the lumen has been early recognized. For example Petera and
V an Slyke in connection with the work of Sperry (238) have stated that, "the
intestine 1s to be regarded as an organ in which lipids are synthesized". In
1959 Bueil and Reiser (284) showed that the intestinal mucosa participates in
synthesizing neutra! fat by the incorporation of C-14 labelled fructose diphos-
phate into glycerol. This incorporation was carried out by the mitochondria
-free supernatant. In 1955 Fridhandler and Quastel (63) using isolated loops
of GPI were able to show that for the most part sucrose is hydrolyzed within
the epithelial cella after entry. Newey and Smyth (285) have also observed
• 4 the same with dipeptides. In a more recent study Miller an.d Crane (160)
havetshown that the hydrolyses of sucrose, maltose and glucose-1-phosphate
take place within the epithelial cells. They were also able to show that
the enzymes necessary to carry on these intracellular reactions such as
invertase and maltase were chiefly localized in the epithelial brush border
membrane.
Studies involving the intracellular enzymic reactions of the intes
tinal wall have been slow in development. One reason for this has been
the fact that the intestine is so rich in digestive enzymes that new and
83
more specifie assay methods (237, 238, 239. 240)' had to be developed to
differentiate between the digestive enzymes in the lumen and those located
intracellularly. The other reason is that mucosa of the intestine has not
been as readily avaüable for study as muscle, liver or brain tissue. The
mucosa of the intestine is an extremely fragile tissue and special techniques
had to be developed (160) in order to study its activities. Comparatively
little work has been done also on the fractionation of cytoplasmic particles
of the intestinal mucosa. In 1954 Morton (236) using the intestinal mucosa
of a 3-day old calf and O. 25 M sucrose solution was able to separate by
differentia! centrifugation and to identify by both E/M examination and bio
chemical tests the cytoplasmic particles in the various fractions and the
enzymes associated with them. His studies revealed some peculiarities
of mucosal cytoplasmic fractions which were different from sim il ar pre
parations of other tissues. For example, it was shown that mucosal
mitochondrial preparations were composed of large aggregates and that
the microsomes, in contrast to those obtained from liver homogenates,
did not contain any hemoproteins.
84
Mucosal preparations contain large amounts of mucus which modify
the behavior of the various cytoplasmic particles as far as differentiai
centrifugation is concerned and hence methods used for homogenates of
other tissues auch as liver, muscle_, and brain do not give satisfactory
resulta with mucosal homogenates. Schmidt, Bessmann and Thannhauser
(241) using isotonie salt solutions (O. 85% NaCl with 1 N sodium acetate)
have fractionated mucosal homogenates and have found an active cephalinase
in the mitochondrial fraction and Epstein and Shapiro (242) using KCI solutions
(1. 15%) for their fractionation have identified the presence of lecithinase and
lysolecithinase. Al.lard, de Lamirande and Cantero by similar methods of
fractionation have found the following distribution of alkaline phosphatase
activity in the various fractions (243): microsomal fraction 75% of total
activity; mitochondrial fraction 19%; supernatant fraction 10%; and nuclear
fraction 2%. Triantaphyllopoulos and Tuba (244) have been able to confirm
these findings for the most part,. the only exception being the nuclear fraction,
for which they obtained higher alkaline phosphatase activity (10%) in the rat
small intestine. These studies have also brought out the fact that the total
amount of alkaline phosphatase activity diminishes rapidly with the distance
from the duodenum to the end of the ileum, a finding which is not inconsis
tant with the observed absorptive characteristics of the smal.l intestine. The
intestinal mucosa is the tissue with the highest content of alkaline phospho
monoesterase and therefore it has been extremely tempting to assume that it
may be involved in active sugar transport in some way but the experimental
proof of this is still very mu ch lacking.
85
The small intestine is a unique tissue with a very high rate of meta
bolism and certain! y is not lacking in its variety and quantity of enzymE;.S. For
example, the presence of amine oxidases which are capable of deaminating a
number of important amines such as epinephrine, tyramine and tryptamine and
diamines such as cadaverine and putrescine have been reported (245). The
D and L-amino acid oxidases of the mammalian intestine do not seem to be
very active. Most of the deamination of amino acids in the intestine takes
place by the action of transaminases such as GOT and GPT. Oxidative dea
mination is considered to be relatively unimportant in the intestine except. for
the action of glycine oxidase and glutamic acid dehydrogenase, which is known
to cause coupling of transamination and deamination. Radhakrishnan and Meister
(246) have reported that L-amino acid oxidase and D-amino acid oxidase obtained
from snake venom and sheep kidney respectively can cause the reversai of oxi
dative deamination and thus bring about the syntb.esis of amino acids. Moreover,
the synthesis of D-proline from several o( -amino acid precursors by a D-amino
ac id oxidase from sheep li ver has been observed by the same investigators (246).
This finding is of particular interest to us because, as our experimental results
will show, the GPI also is capable of synthesizing proline from labelled carbo
hydrate.
A very interesting comparative study conducted by Nak.amura (247)
on the measurement of succinic oxi.dase activity of intestinal mucosa of the
rat guinea pig and man has revealed the finding that a great deal of species
difference exista in their Q0 (N). The GPI having a very high value and 2
the rat intestine a very low value. Investigation of the matter, however,
86
has indicated that the rat mucosal preparation contained free fatty acids par-
ticularly the unsaturated variety which acted as potent inhibitors to succinic
oxidase activity. The mucosal preparations from GPI and man did not have
free fatty acids. It is possible that the fact that the rat does not possess a
gall bladder is in some way linked with this finding.
The presence of amylases in small quantities in the mucosal homo-
genates of the rat have been reported (248, 249). Other enzymes found in in-
testinal mucosa are xanthine oxi.dase (250) a number of glycosidases, in addi-
tion to those already mentioned, auch as trehalase (251), -galactosidase (252)
and also phosphoglucose isomerase (287) and hexokinase (258).
In 1958 Paterson and Zbaraky (250) conducted experimenta using rat
mucosal suspensions and showed that this tissue possesses a very high de novo
purine synthesis rate. They measured the synthesis by estimating the incor
poration of formate-c 14, carbonate-c14 and glycine-c14 into the acid soluble
and nucleic acid purine fractions alter an incubation period of 3 hours. Their
resulta indicated that carbonate-c14 was readily oxi.dized but glycine-1-c14
only slightly. Syntheses of both acid soluble and nucleic acid purines occurred
and they were shown to contain c 14 from formate-c14 glycine-1-c14 and
bicarbonate-e 14
The metabolism of amino acids
In 1948 Friedberg, Tarver and Greenberg (254); and Tarver and
Morse (255) Bhowed the very rapid turnover of protein of the intestinal mu
cosa. This finding was not entirely unexpected because the intestinal tissue
87
is the locus for the synthesis of many enzymes and in addition the mucosa is
constantly being sloughed off so that a great deal of protein synthesis takes
place. Yet, the intestinal metabolism of amino acids has not received wide
attention such as it has for liver and brain tissue. However, some studies
have been made. Whaler (256) has shown that the intestinal mucosa of rats
and cats are capable of metabolizing N-acetyl amino acids. That is, they are
capable of deacetylating the N -acetylated compounds to produce free amino ac ids
with the exception of N-acetyl tryptophane. For some obscure reason this com
pound, although it has been shown to support growth in tryptophane-deficient
rats (257), is not attacked by the intestinal acylase. The deamination of
amino acids placed in mucosal and serosal. fl.uids was al.so observed by Whaler
(256). He was able to find o< -keto acids in the serosal solutions. From
all the amino acids tested he invariably obtained pyruvic acid and frequently
o< -keto glutaric acid and also the o(. -keto acid corresponding to the amino
acid used, indicating that transamination was not the only reaction taking
place. Using c14-carbox.yl labelled amino acids he also showed thal: decar
boxylation from the mucosal side was much greater than from the serosal
si de. In the synthesis of amino ac ids by the intestine Whaler reported that
a number of amino acids such as leucine, valine, methionine and glutamic
acid when placed in the mucosal :fl.uid resulted in what he believed to be
alanine. He also demonstrated that when « -keto-isocaproic and C'< -keto
-isovaleric acids were placed in the mucosal :fl.uid along with ammonia and
glucose he could obtain leucine and valine. In all his experimenta Whaler
used 0.4 per cent glucose (22.2 mM) in the medium. The possibility that
glucose itself could be metabolized to give amino acids does not seem to
have been suspected (256).
88
PARTIT
89
ACTIVE GLUCOSE UPTAKE BY THE INTACT-STRIP METHOD
AND COMPETITIVE INHIBITION BY PHLORIDZIN AND PHLORETIN
Introduction
By the use of the Intact-Strip-Method (ISM) the uptake of glucose was
studied both in the hamster and in the guinea pig. Crane (260) has shown by
the same method that active sugar uptake is restricted to the small intestine.
He has obtained ratios of final tissue to medium concentrations of 25. 4, 0. 62
and O. 61 for the small intestine, the stomach and the large intestine respec-
tively in the uptake of 6-deoxy-D-glucose by the hamster. Moreover the ISM
has shown 2-3 fold higher rates of active sugar transport than the reported
values for everted sacs. The rate of sugar concentration follows Michaelis
-Menton kinetics but the ~Sn values obtained by this method are significantly
different from those found by other methods (264). For example, in the ham
-3 ster for glucose uptake by the ISM the ~Sn value is 1. 5 x 10 M, in the
guinea pig by the perfusion method of Darlington and Quastel (128) the ~
value is 7 x 10-3 M and in the rat by the in situ perfusion method of Fisher
-3 and Parsons (265) the Km_ value is 8. 3 x 10 M.
Our primary objectives in this work were to study the uptake of glu-
cose by the guinea pig intestine (GPI) by adapting and using the ISM and to
elucidate the nature of the inhibitory action of phlorhizin and phloretin on
active sugar uptake by the GPI.
90
Quastel (134) has cited that since phlorhizin inhibits, the rate of
fermentation of glucose by acetone-dried yeast, phosphorylation by yeast and
muscle ex:tracts, and glycolysis in brain cortex slices it is probable that ac
tive sugar transport is affected either by an inhibition of the phosphorylation
step or a competition between phlorhizin and the sugar molecule for an acti
vating enzyme. :Moreover, as already indicated, Wilbrandt (232) is of the
opinion that the competing molecule need not be a sugar analog or structurally
similar to the sugar molecule or necessarily occupy exactly the same site on
the enzyme molecule or carrier in order to achieve competition.
For the work to be presented here we chose to concentrate our efforts
on the investigation of the possibility of competitive inhibition. That is we
attempted to show whether there was a competition between phlorhizin and the
sugar molecule for a site on the carrier molecule in the active transport of
glucose by the GPI.
Our views in this direction were strengthened by the fact that Ponz
and Lluch (207) in their experimenta bad shown that the action of phlorhizin
could be reversed by removing the phlorhizin and replacing it with glucose.
It was also supported by the fact that low concentrations of phlorhizin were
suffi cie nt to bring about inhibition of sugar entry into cells (220). In addition,
Fleischmann's (215) early concept that phlorhizin is a highly adsorbable
91
material. and that when it is adsorbed by cella it blocks the absorption of
glucose and that of McKee and Hawkins (216) that phlorhizin inhibition of
glucose did not interfere with reabsorption of other substances - indicating
the possible presence of a selective absorption mechanism - have al.so been
encouraging. Bence we have attempted to study the nature of phlorhizin and
phloretin inhibition of glucose uptak:e by GPI.
92
Methods and Materials
Animals
For some of the preliminary experimenta with the intact-strip-method
of Crane and Mandelstam (260) the small intestines from golden hamsters
(Gl:IT) of either sex weighing approximately 150-300 grams were used. For
the perfusion experimenta, the work with phlorhizin and phloretin and all ex
perimenta on metabolism the small intestines from guinea pigs were used.
These were male guinea pigs weighing approximately 300-550 grams. Ail
animais were kept on an ad libitum diet of Purina checkers, raw cabbage,
raw carrots and water. The animais were not starved except for certain
special experimenta as indicated in the text.
Perftunon procedure
The animais were killed with a blow on the head and decapitation at
approximately the same time of day, about 10 A. M. The abdomen of the ani
mal was opened by a mid-line incision and the entire small intestine removed
from the ileo-caecal end to the pyloric end. For perfusion experimenta the
first 10 inches from the pyloric end were discarded and the next two adjacent
sections each about 5 inches long were eut off and at once placed in a beaker
93
full of Krebs-Henseleit bicarbonate solution (258), containing no calcium, which
had been previously gassed for at !east 30 minutes at 37°C with a mixture of
6%C02- 95%02 for aerobic experimenta and 5%C02 - 95%N2 for anaerobie ex
perimenta. The segments were removed without delay and the mucosal sides
washed with a stream of KHBS and mounted on the apparatus in such a way
that the fluid circulated through the segment in the normal or sam.e direction
as in vivo. The substance to be tested was first dissolved or mixed in the
gassed perfusion fluid prior to introduction into the apparatus.
Perfusion apparatus
The perfusion apparatus of Darlington and Quastel (139) and its modi
fied form by Paranchych (259) have been adequately described elsewhere and
will not be repeated here. A more recent modification of this apparatus adap
ting it for the use of isotopically labelled compounds has been achieved. An
illustration of it is given in Fig. 1. The advantages of the new apparatus are
the small volumes of perfusion fl.uid required for its operation, mucosal volume
10 ml, serosal volume 40 ml and its ease of assembly. All joints are of
ground glass and the apparatus is sufficiently compact so that it can be readlly
immersed in dichromate-sulfuric acid solution for purposes of cleaning.
Intact-strip-method (ISM).
This method has been used for most of the experimenta and will now
PERFUSION APPARATUS
Fig. 1. Apparatus used for perfusion of radioactive substances. A segment of intestine is mounted between the prongs of part tA' and tied with surgical. sillc It is inserted into part 'B'; the perfusion fl.uid is poured in through the funnels; the long bulbous tubes are connected to a source of gas and the unit is ready for operation. The entire apparatus is situated in an incubator at 37°C.
94
95
be described in detail. The animals were sacrificed as indicated for the per
fusion procedure. Two or three adjacent sections were removed from the py
loric end of the jejunum after discarding the first 10 inches of intestine. The
segments were at once placed in a beaker full of ice-cold O. 9% saline which
had been previously oxygenated for about an hour. The segments were everted
with the aid of a long plastic probe (1. 5 mm in diameter) and the mucosa
washed clear of ali adhering material with a stream of oxygenated O. 9% saline
solution. The segments were placed in fresh oxygenated saline and kept oxy
genated during the entire cutting period. The segments were placed length
wise on a plastic plate embedded in cracked ice, held down with a pair of
forceps and eut into 2-4 mm strips with a sharp scalpel. They were returned
to the oxygenated saline solution until ali of the segments were similarly eut.
The strips thus collected were then gently mixed with the aid of a spatula or
glass rod, the saline solution decanted and the strips blotted with hardened
:fllter paper (Whatman No. 542). They were kept over cracked ice under a
gentle stream of oxygen and samples were weighed out on a torsion balance
as quickly as possible. Samples (about 10-12 strips per 400 mg) were trans
ferred immediately after weighing into respective 250 ml Erlenmeyer flasks
containing 10 ml of the appropriate incubation medium (258). The se fl.asks
were fitted with rubber stoppera and glass tubing lowered inside the fl.ask to
about 1-2 cm from the surface of the solution. A hypodermic needle of No.24
gauge was passed through the rubber stopper to allow for the exit of gases.
96
The flasks were placed in an incubator provided with a shaking deviee and
connected to a source of gas mixture (5%C02 - 95%02) by way of the inserted
glass tubing. The gas mixture was passed through a flask of distilled water
which was kept at 37°C prior to entry into the incubation flasks. The sam
pies were oxygenated at 37°C. with a gentle stream of gas mixture passing
through the flasks during the entire incubation period. The flasks were re
moved after incubation and immersed at once into a bath containing cracked
-ice. The tissue was separated from the medium by pouring the contents of
each flask over a coarse, perforated, :fi.lter paper and the tissue quickly re
covered with a pair of tweezers, gently blotted and transferred into a Potter
-Elvehjem homogenizer. Two ml of water and 1 ml of ZnSO 4 (0.19 M) was
added and the tissue homogenized. This was followed by addition of 1 ml
of Ba(OR)2 (0.3 N) and the ho~ogenate diluted to desired volume with distilled
water. The mixture was decanted through a fil ter paper and aliquots of the
clear, protein free filtrate were used for the determination of sugar.
Analytical methods
Glucose was determined by the method of Somogyi (261) and Nelson
{262, 263) using the Beckman Model B spectrophotometer at a wavelength of
565 millimicrons. A standard solution of glucose containing lOO micrograms
of glucose was routinely run in duplicata as well as a reagent blank carried
through the entire procedure along with the unknown samples.
Re agents
All reagents used were of high purity reagent grade chemicals.
Treatm.ent of results
For the calculation of tissue millimolarity the following formula
from Crane and Mandelstam (260) was used:
mM Tissue = mM (filtrate) x homogenate volume - 0.3 x mM (medium) wet weight of tissue x . 8
97
Total tissue water was taken as 80% of the wet weight. Tissue concentrations
were not corrected for extracellular space because this was considered of no
particular significance for our comparisons of tissue and medium concentrations.
The utilization of the sugar was also ignored because of its relatively small
effect on the error introduced upon the sugar uptake on a comparative basis.
The Lineweaver and Burk (266) plots were used to show the compe-
titive nature of the inhibition. The ~ and Ki values were calculated by the
aid of the following formulae:
! =!Sn 1 + 1 slope = Km and v Vmax s Vmax V max
v V max
(1+Q}J.!+! ( ~) s v max
slope = Km ( 1 + (1) ~ •
Vmax ( Ki
The statistical treatm.ent of results, whenever needed, consisted in
98
calculating, the mean, the variancet the standard deviation and the "normal"
range. These parameters were calculated with the aid of the following for-
mulae:
x = value of an individual measurement
N = total number of measurements
-mean = x = ~ S (x)
variance = s2 = 1 N-1
standard deviation = s. d. = ~ -+
"normal" range = X - 2 (s. d.)
99
Experimental
1. Uptake of glucose by strips of golden hamster intestine (GHI).
Incubation of Gill prepared according to the :lntact-strip method (ISM)
described under methods and materials was carried out using 5 mM glucose
in Krebs-Henseleit bicarbonate buffer without added calcium. Incubation time
was twenty minutes. Tissue strips were placed in 10 ml of medium :ln 250
ml flasks fitted for gassing as already described. They were gassed with a
continuous gentle flow of 5%C02 - 95%02 gas mixture at 37°C for the entire
duration of the incubation. Under these conditions the tissue strips swell up
with the uptake of glucose and fl.uid from the medium and take on the appearance
of "donutsu. The tissue strips were removed at the end of the incubation period
and processed as indicated under ISM. The resulta obtained by this procedure
are given in Table I. The values obta:lned are reasonably consistent and repro
ducible for a given set of conditions. The mean value obtained for glucose up
take by the Gill strips is 20.1 mM with a standard deviation of t 2.2 and a
statistical "normal" range extending from a low of 15.7 mM to 24.5 mM.
Two of the hamsters were starved for a period of 96 and 108 hours but the
uptake by these animais was not increased in fact the values are lower than
the mean value although they are well within the range of the statistical "normal".
This is indeed suggestive that the energy required for the active uptake of sugars
lOO
TABLE I
THE ACTIVE UPTAKE OF GLUCOSE BY STRIPS OF
HAMSTER INTESTINE
Experiment Net weight Tissue glucose Final medium number of tissue concentration concentration
(mg) (mM) (mM)
1 406 20.0 4.04
2 498 18.2 3.79
3 342 18.3 4.16
4 236 20.0 4.33
5 202 23.2 4.52
6 185 21.3 4.40
7 292 17.1 4.31
8 368 25.0 4.16
9 456 22.0 3.88
10 393* 18.6 4.10
11 408** 18.5 4.02
12 444 20.2 3.79
13 450 18.9 3.90
Note: The strips of Gm were incubated in 10 ml of 5 mM glucose in Krebs-Henseleit bicarbonate buffer for 20 min. at 37°C. The gas mixture used was 5%C02 - 95%02. The mean tissue glucose concentration is 20.1 mM, the standard deviation± 2. 2, the "Normal" range 15. 7 mM to 24. 5 mM.
* Animal starved 96 hours. ** Animal starved 108 hours.
101
is not necessarily dependent upon the utilization of the sugar from the medium.
Other sources such as the oxidation of fats or fatty acids may well supply the
needed energy. A compound like 3-0-methyl-D-glucose is not utilized by the
animal (267) although it is actively taken up by the tissue and similarly 1, 5
-anhydro-D-glucitol and 7-deoxy-D-glucoheptose are phosphorylated but not
metabolized any further (26 8) .
2. Uptake of glucose by strips of guinea pig intestine (GPI).
The GPI strips were incubated in Krebs-Henseleit bicarbonate buffer
with no added calcium. The medium was made 5 mM in glucose and 10 ml
of medium in 250 ml fl.asks were used as in the previous experimenta. Incu
bation time was 90 min. at 37°C. The tissue was gassed with 5%C02 - 95%02
mixture. The resulta obtained for the uptake of glucose are given in Table II.
The average or mean for the tissue molarity was 27. 3 mM, the standard de
viation "t 2.4 and the statistical "normal" range for this tissue 22.5 mM to
32. 1 mM. The se resulta indicated to us that the ISM was qui te sui table for
the study of active glucose uptake by GPI strips.
We also ran a tissue blank for endogenous glucose at this time. A
sample of GPI strips weighing 385 mg was incubated with 10 ml of medium
without any added glucose as substrate. The value obtained for the millimo
larity was 2,15 mM. The animal was not fasted.
Experiment number
1
2
3
4
5
6
7
8
9
10
11
TABLE il
THE ACTIVE UPT AKE OF GLUCOSE BY STRIPS OF
GUINEA PIG INTESTINE
Wet weight of tissue
(mg)
366
353
290
365
381
341
561
318
345
242
212
Tissue glucose concentration
(mM)
29.3
29.6
25.6
25.2
23.8
28.0
23.5
29.7
26.5
29.1
29.9
102
Note: The strips of GPI were incubated in 10 ml of 5 mM glucose in Krebs-Henseleit bicarbonate buffer for 90 min. at 37°C. The gas mixture used was 5%C02 - 95%02. The mean tissue glucose concentration is 27. 3 mM, the standard deviation ± 2. 4, the "Normal" range 22.5 mM to 32.1 mM.
3. Variation of glucose uptake activity along the length of the GPI.
In this experiment we were interested in ascertaining the relative
rate of active glucose uptake at different parts along the length of the small
intestine. Glucose at a concentration of 5 mM was used for all samples.
103
All incubations were carried out as in the previous experimenta. The entire
GPI was removed and eut into successive 5 inch segments and these in turn
separately eut into strips and incubated in duplicata samples. The resulta
obtained are summarized in Fig. 2. It can be seen from this graph that
there is a gradua! rise in the rate of active uptake of glucose by GPI star
ting with the segment 10 inches from the pylorus reaching a maximum value
at a point 25 inches from the pyloric end. For our subsequent experimenta
the 20 inch section between 10 and 30 inches from the pylorus was routinely
used as the are a having the maximum or optimum absorptive capacity.
4. Uptake of glucose by GPI strips with increase in the time of incubation.
The GPI strips were prepared and incubated as in the preceding ex
perimenta. The medium molarity used was 5 mM for all samples. The first
10 inch section was discarded and the next 20 inch section eut into strips.
After mixing the strips representative samples were removed and quickly
weighed and introduced into the incubation flasks containing 10 ml of medium.
The flasks were taken out at specified time intervals and tissue processed at
~ a -J;z;:l ~
t1l ~ t1l ..... < ....
>, ~ .tl
P-4 (1)
~ ~ (.) :::7 'Q ~ 0
20
15 /. /. 10 .'-= • -·
5
o~----L-----~----~----~----~ 10 15 20 25 30
DISTANCE
from pyloric end (in)
Fig. 2. Variation in the rate of uptake of glucose along the length of the guinea pig intestine. Incubation time 90 min. at 37oc.
104
105
once for glucose content. Fig. 3 illustrates the relationship of glucose accu
mulation in the strips with variation in time up to an interval of 90 minutes.
The resulta indicate a rapid initial uptake of glucose by the tissue up to about
60 minutes, then a graduai equilibration of influx and e:ffl.ux or a steady-state
condition within the next 30 minutes.
5. Uptake of glucose by GPI versus concentration of glucose in the medium.
To determine the effect of varying medium concentrations on the up
take of glucose by GPI strips concentrations of 2, 3, 5, 10 and 20 mM were
used. Incubations were carried out as before for a period of 90 minutes.
The ratios of tissue millimolarity to medium concentration were plotted against
the initial medium concentrations. The graph for this is given in Fig. 4. It
can be noted from the plot that as glucose is taken up actively by the tissue
the medium concentration decreases rapidly in the beginning and tissue con
centration of glucose rises very rapidly. But as tissue concentration rises
or as tissue becomes saturated with glucose, the less rapidly the tissue con
centration builds up or increases. Apparently with higher tissue concentrations
the e:ffl.ux or outward diffusion becomes large and begins to balance the active
uptake.
6. Inhibition by phlorhizin of the active uptake of glucose by strips of GPI.
Phlorhizin is not very soluble in water, so a solution of it in 95%
~ s -~ ~
IID ::i:l IID
< :;:; ~
E--c .0
p.. (1) 10
p 8 = 'bh 'ê)
106
30 .,..,-• ·-/. / . •
1 • 20 1 •
1 •
10
o~----~----~----~------~----~ 80 100 20 40 60
T lM E
of incubation (min)
Fig. 3. Incubations were carried out with GPI strips in 10 ml of 5 mM glucose in Krebs-Henseleit bicarbonate buffer, for each sample. Duplicate samples were run for each Ume interval. Incubation temperature 37°C.
z 0 -~ <t! ~ ~ ~ z a w -(,) Q)
r.o z 0 0 (.)
= (,) .... bO
::;s ..... 0
j;;:) -Q w ::;s
107
20 ' -
10 • \.
'-..... .. _ ---------·-·-OL---~2-----4L---~6-----8~--~10----~12
TISSUE UPTAKE/MEDIUM CONCENTRATION
Fig. 4. GPI strips were incubated in Krebs-Henseleit bicarbonate buffer with varying concentrations of glucose for 90 min. at 37oc.
108
ethanol having a concentration of 5 x 1o-3 M was prepared as stock solution.
Aliquote of this stock solution, usua.lly amounting to no more than 0.1 ml
were used to make up the desired concentrations of phlorhizin in the respec
tive media. Equivalent amounts of 95% ethanol were added to the control
samples to compensate for any ethanol effect.
In this experiment 2, 4 and 10 mM glucose concentrations, made up
in duplicate, were used for the media and the medium concentration of phlor
hizin was made 5 x 10-5 M for all the samples. Standards and control sam
pies contained the same amount of ethanol, all other conditions of incubation
were the same as in the preceding experimenta. The intact strips were se
parated from the media at the end of 90 minutes of incubation and processed
at once. The data obtained were plotted according to Lineweaver and Burk
(266) and the curves obtained were straight lines intercepting at a common
point on the 1/v-axis indicating the competitive nature of the inhibition. Fig. 5.
We next used 1. 3, 2, 4 and 10 mM glucose concentrations in the media
and the same concentration of phlorhizin for all the samples (5 x lo-5 M).
The results are given in Fig. 6 clearly indicating competitive inhibition.
7. Inhibition by phloretin of the active uptake of glucose by strips of GPI.
This study was made using a medium concentration of 2 x 10-4 M
phloretin for al.l of the samples. Ten ml of media of 1. 3, 2, 4, and 10 mM
concentrations in glucose were used. All incubations were carried out to 90
109
20
15 • - Controle
0 - Phlorhizin, 5 x 10-5 M
5
0 .__......~....-__ _._3 __ __._5 __ ---:7
1/s x 102
Fig. 5. Competitive inhibition of active glucose uptak.e by phlorhizin. GPI stripa were incubated in 10 ml of Krebs-Henseleit bicarbonate buffer 2, 4 and 10 mM in glucose concentration. Incubation time 90 min. at 37°C.
C'l 1 0 ..-4
><
-t ..-4
40
30
20
0
10 0/
0
110
• - Controls -5 0 - Phlorhizin, 5 x 10 M
0~--------~----~------~----~ 3 5 7 9
1/s x 102
Fig. 6. Competitive inhibition of active glucose uptake by phlorhizin. GPI strips were incubated in 10 ml of Krebs -Henseleit bicarbonate buffer 1. 3, 2, 4 and 10 mM in glucose concentration. Incubation time 90 min. at 37°C.
111
minutes. At this concentration of phloretin in the respective media and under
these conditions of incubation, the inhibitions of glucose uptake by phloretin
was found to be equally as effective as that obtained with phlorhizin. The re
sulta are given in Fig. 7. It can be readily seen that the straight lines ob
tained meet at a common point on the 1/v-ax.is as in the case of inhibitions
by phlorhizin.
In order to study the inhibitory action of phloretin more thoroughly
three different concentrations of phloretin were employed. That is, medium
glucose concentrations were 1.3, 2, 4 and 10 mM as before but now the phlo
retin concentrations were made 4 x 10-5, 10 x 1o-5 and 20 x 10-5 M. For
each concentration of glucose a control was run simultaneously in order to
establish a base value for active glucose uptake for that particular concen
tration. The resulta obtained are presented in graphie form in Fig. 8.
8. Determination of the range of phloretin concentrations which would serve
as upper and lower limita of effective action on inhibition of glucose
uptake by G PI strips.
For ali samples 10 ml of medium 2 mM in glucose concentration was
used. Triplicata samples were run for each concentration of phloretin employed
and for the controls. The mean of three values thus obtained for the inhibition
by phloretin was plotted against the concentration of the phloretin used. The
concentrations of phloretin used were: 4 x 1o-5; 8 x 10-5; 16 x 1o-5 and
N 1 0 ~
~
> ......._ ... ~
112
50
40 e - Co11i r; L
0- f:rJor~iir. 2·.10- 4 !\1
30 0
20
0
10 01 o~~------~------~----~----~
3 5 7 9 1/s x 102
Fig. 7. Competitive inhibition of active glucose uptake by phloretin. GPI strips were incubated with Krebs-Henseleit bicarbonate buffer 1. 3, 2, 4, and 10 mM in glucose. Phloretin concentration was the same for all concentrations of glucose (2 x lo-4 M). Incubation time was 90 min. at 37oc.
C\1 1
0 ......
x > -...,
......
1 60 0
• - Controls
50 0 - Phlcretin,
• - Phlorctin,
6 - Phlo .. etin,
40 0
30
20
10
o~~----~3----~5----~7-----9~--~,,
F . k. Con:pdttive inhibitiül' of active t-:lucose uptake of GPI strips, by phl(n·di:: ;.tt 3 diffen~nt conccutt·atwns. Incubation media werc made with Kn~bs-Her.SL'h:it bîcarbonatP buffm· in 1. 3, 2, 4 a1:d 10 n.M blucosL' concentratio11s. Incubation time 90 min. at 37°C.
113
-4 2 x 10 M
1 x 10- 4 M -5
4 x 10 1\1
114
20 x 10-5 M. The results obtained are plotted in Fig. 9 and 10.
It can be readily seen that as the concentration of phloretin increases
the uptake of glucose by the tissue proportionately decreases. When the data
are plotted as in Fig. 10 using the phloretin concentrations as abscissae and
the reciprocals of the glucose uptake as ordinates a straight line is obtained
indicating a proportional relationship between concentration of sugar uptake
and concentration of inhibitor.
9. Calculation of Km and Ki values from the data obtained from the
preceding experimente.
The Kzn for glucose uptake by GPI strips and the ~ values for phlor
hizin and phloretin have been calculated and are presented in Table Ill.
10. The uptake of glucose by GPI strips was also studied using glucose-U -c14.
In order to obviate the difficulties of the analytical procedure used for
sugar determination, the ISM was adapted to the use of glucose-U -c14. Incu
bations were conducted as before using 5 mM glucose as substrate. Approxi
mately O. 2 ;;- c per 10 ml of medium were introduced into each :fl.ask by way
of labelled glucose. Incubation time was 90 minutes at 37°C. Tissue strips
were homogenized and protein matter precipitated with barium hydroxide and
zinc sulfate. Aliquots of the clear filtrate were dried on planchets and counted
~ a -Q)
l'LI ~ tQ
~ tQ -< ..... ;>,
E-< ,.0
~ Q)
~ ::.> (.)
~ ...... bD
'a
6
2
QL---~4-----8~--~12-----1~6----2~0~--724
CONCENTRATION
of phloretin x 10-5
Fig. 9. GPI strips were incubated in 2 mM glucose in Krebs -Henseleit bicarbonate buffer with 4, s. 12, 16 and 20 x lo-5 M phloretin concentrations.
115
~ ~ < E-1 ..t"" P-t 1
::::> ~
~ g
0 Cl.) ltl 0
00 ~ ...:l -< b.O Cl.)
u :::.1 0 co
co ~
...... .... P-t 11;-4 - 0
u Cil ~
50
30 .~
10~• • ~
.~ .~
116
.-----
4 8 12 16 20 24
CONCENTRATION
of phloretin x 10-5
Fig.10. GPI strips were incubated in 2 mM glucose in KrebsHenseleit bicarbonate buffer. Phloretin in 4, 8, 12, 16 and 20 x lo-5 M concentrations were used. The straight line obtained clearly indicates competitive inhibition.
TABLE m
THE ~ VALUES. AND THE Ki VALUES FOR Plll.ORIDZIN AND
PHLORETIN WITH THE ACTIVE UPT AKE OF GLUCOSE BY GPI
USING THE INTACT STRIP METHOD
Phloretin Phlorhizin
Km Ki Ki (M) (M) (M)
4. 53 x to-3 5.31 x 10-5 1.01 x 10 -5
3.62 x 10-3 4. 56 x 10-5 1.00 x 10 -5
3.13 x 10 -3 3.41 x 10
-5 Mean: 1. 01 x 10-5
4. 39 x 10 -3 6. 20 x 10 -5
3.53 x 10-3 5.27 x 10 -5
Mean: 3.84 x 10-3 Mean: 4. 95 x 10-5
117
under a windowless contin:u.ous gas flow counter. Control solutions were
carried through the protein precipitation and subsequent steps and counted in
a similar mann er. The resulta obtained for GPI by this method are given
in Table IV. It is to be noted that the re is a reasonably good agreement
between the uptake of glucose by use of labelled glucose and that previously
gotten by the non-radioactive procedure.
11. Perfusion apparatus for the active transport of labelled compounds.
118
The apparatus of Darlington and Quastel (139) was altered in a number
of ways to render it more suitable for use in the study of transport and meta
bolism of labelled compounds. An illustration of the apparatus is given in
Fig. 1.
AH rubber connections have been eliminated by use of ground glass
joints. Ground glass fittings were also provided for the funnels and U tubes
which aid in the control of foaming of the solutions. Small bulbs were inserted
into the gas intet tubings to prevent sudden rise of the liquid when the gas
pressure is turned off and gas connections removed. The apparatus is very
compact, easily mounted and easily dismantled. In addition it has the advan
tage of small volumes - 10 to 13 ml for the mucosal side and 40 to 45 ml
for the serosal side. The volume capacity of the two circuits can be readily
altered or reversed - thus making the mucosal volume 40 ml and the serosal
volume 10 ml - by everting the gut segment before mounting it. These changes
119
TABLE IV
THE ACTIVE UPTAKE OF GLUCOSE-U-c14 BY STRIPS
OF GUINEA PIG INTESTINE
Experiment Wet Weight Tissue glucose number of tissue concentration
(mg) (mM)
1 160 29.5
2 156 29.6
3 193 24.6
4 282 24.6
5 240 27.6
6 227 32.9
7 241 31.4
8 270 31.3
9 265 26.7
10 246 28.5
11 222 26.9
Note: The strips of GPI were incubated in 10 ml of 5 mM glucose (labelled) in Krebs -Henseleit bicarbonate buffer for 90 minutes at 37°C. The gas mixture used was 5%C02 - 95%02. The mean tissue glucose concentration is 28. 5 mM, the standard deviation± 2. 9, the "Normal" range 22.7 mM to 34.3 mM.
make the apparatus very suitable for use with radioactive materials.
In order to test this apparatus and to see if we could obtain active
transport against a concentration gradient the following experiment was con
ducted.
120
A four inch segment of GPI was everted, washed and mounted - the
smaller volume (10 ml) being on the serosal side and the larger volume (40 ml)
on the mucosal side. A 5 mM solution of glucose containing glucose-U -c14
was made up in Krebs-Henseleit bicarbonate solution) o'o/genated and an ali
quot of 500~ plated and counted. The solution was introduced into both
circuits of the apparatus and properly gassed with 5%C02 and 95%02 mixture.
The initial concentration of glucose and specifie activity of the solutions
in both circuits were the same, namely, 5 mM and 3680 cpm per micromole
of glucose. After 60 minutes of perfusion 11.1 micromoles of glucose were
transferred from the mucosal to the serosal aide against a concentration
gradient.
It may be added that the apparatus lends itself to rapid perfusion
etudies. Samples can be easily removed from the funnel openings at desired
intervals of time. The in vitro perfusion achieved by this apparatus is a
alternative to the Wilson and Wiseman sac method (227).
121
Discussion
The intact strip method (ISM) of Crane and Mandestam (260) was
adapted for the study of the absorptive characteristics of the guinea pig
intestine. Since the absorptive capacity of the small intestine of the guinea
pig varies along its length, as has been shown by one of the experimenta
performed,. the ISM has been a distinct advantage in providing tissue in
which this factor is minimized. Another advantage of the ISM was that, at
least in our hands, the method was capable of yielding reasonably precise
and reproducible results. Our resulta both with the hamster and the guinea
pig intestine bear this out. The method is particularly useful for the study
of reaction kinetics and hence we have used it to study inhibition by phlor
hizin and phloretin of glucose uptake in the GPI.
No completely satisfactory explanation has as yet been advanced to
elucidate the mode of action of phlorhizin and phloretin nor have they been
linked with any particular reaction as far as their inhibitory role is concerned
in the active uptake of sugars. These inhibitors appear to play a double role
- at low concentrations they interfere in some way with sugar entry into cella
and at high concentrations they inhibit metabolic processes. It has been sugge
sted that the glucose moiety of phlorhizin plays a significant role in its capacity
to inhibit sugar entry into cells. Our results from the experimenta performed
122
with this compound and tts aglycone do not entirely support this suggestion. In
the use of IBM with GPI we have found phloretin at 2 x 10·4 M equally as effe-
tive in inhibiting glucose uptake as phlorhizin at 5 x 10-5 M. Moreover we find
both substances conforming to the criteria of competitive inhibition
For competitive inhibition it is assumed that the formation of the en-
zyme inhibitor complex (El) is reversible and that there is a competition be-
tween the substrate and the inhibitor for a locus on the enzyme surface. The
competition is then strictly dependent on the relative concentrations of substrate
and inhibitor. Our experimenta have shown that active glucose uptake by the
GPI is dependent on such relative concentrations of substrate and inhibitor and
hence is of the competitive type (Fig. 10).
The effective range of concentrations used in our studies have been,
4 x 1o-5 M to 20 x 1o-5 M. -4 It is to be noted that at the 2 x 10 M concen-
tration phloretin produces approximately 73% inhibition of glucose uptake.
The inhibition produced by phlorhizin and phloretin with the ISM appears
to be different from other ~vitro methods. There is a significant difference
in the Km values obtained by this method compared with those of the perfusion
method although the Km value for glucose with the GPI is in good agreement
with the KJn value reported for hamster intestine. The average Km value
obtained for active glucose uptake by ISM using the guinea pig intestine was
found to be 3. 84 x 10-3 M. The Ki value for phlorhizin (1 x lo-5 M) appears
to be lower, approximately by a factor of 5, from the Kt value obtained for
phloretin (4. 95 x lo-5 M).
123
When an inhibitor is competitive in its action its effect is to increase
the slope of the line obtained when 1/v vs. ,! are plotted, according to the
Lineweaver and Burk analysis,
ding change in the inte rcept ,! . v
s by a factor of ~1 + ~3 without a correspon-
In other words if the substrate concentration
is large enough the effect of the inhibitor can be diluted or overcome. The
curves we have obtained by plotting 1/v vs. ,! as weil as those of tissue glu-s
cose uptake or its reciprocal vs. inhibitor concentration clearly indicate that
the inhibition observed by these compounds is of a competitive type. Moreover
the competitive action displayed by these compounds resides not in the glucose
moiety but in the aglycone part.
124
Summary
1. The intact strip method (!SM) has been adapted for use with the guinea
pig intestine (GPI).
2. Active glucose uptake has been studied both in the hamster and the guinea
pig by the !SM. The average values of tissue molarity for Gill and GPI are
20. 1 mM and 27. 3 mM respectively.
3. Variation of tissue glucose concentration with time of incubation has
been plotted. A steady-state condition is reached within 90 minutes of incu
bation.
4. Accumulation of tissue glucose with varying medium concentration has
been studied. The curve indicates that a saturation level is attained in the
tissue in which effl.ux begins to equal the influx due to active transport.
5. The area of maximum rate of absorption in the guinea pig jejunum has
been shown to be, approximately, the segment between 15 and 30 inches from
the pyloric end.
6. Curves showing the competitive nature of phlorhizin and phloretin inhi-
bition have been obtained. When the phloretin concentrations are plotted
against the reciprocals of the glucose uptake a straight line resulte, clearly
showing the existing proportionality between inhibitor concentration and rate
of active glucose uptake or tissue glucose concentration.
125
7. The Km value for glucose uptak.e in the guinea pig intestine by the ISM
and the Ki values for phlorhizin and phloretin have been determined (Table III).
8. A new perfusion apparatus suitable for in vitro experimenta with radioac----tive substances has been designed. With the aid of this instrument, using the
isolated surviving guinea pig intestine. we have shown the active transport of
isotopical.ly labelled glucose against a concentration gradient.
PART ill
THE INTRACELLULAR MET ABOLISM OF CARBOHYDRATES
BY THE GUINEA PIG INTESTINE AND THE PRODUCTION OF
AMINO ACIDS
Introduction
126
It has been emphasized in the preceding sections, both with respect to
locus and functional capacity that the mucosa of the small intestine is the major
seat of active sugar transport. When the intact-strip method was used for study
of active sugar uptake it was shown that uptake is three times faster on a molar
basis than oxygen consumption, lactate formation or glucose utilization (264).
There is not rouch doubt that part of the actively absorbed sugar is uttlized
intracellularly although certain substances, notably, 3-0-methyl-D-glucose (270,
269, 267) are known to be actively transported but not utilized.
The metabolism of the sugars. lipids and amino acids have been exten
sively studied in the liver and brain but they have been relatively neglected in
the intestine itself as a site of metabolism. The absorptive importance of the
intestine has been so overwhelming that studies hawe been chiefly directed to
ward the elucidation of the absorptive processes and mechanisms rather than
metabolism, per se. What little has been learned about intracellular metabo
lism has come mostly through the study of the absorptive process itself. It
has been tacitly assumed that synthetic processes taking place within the
127
intestinal wall are of no particular significance and hence the relative impor-
tance of intestinal syntheses compared with tissues like the liver and brain,
under in vivo and in vitro conditions have not yet been established. Whether -~ ---
intestinal syntheses are merely for the sake of maintenance of this tissue
al.one - which is in itself of paramount importance - or that they go beyond
this point and contribute to the metabolism of the body as a whole is yet to
be revealed.
Recent etudies have been successful in showing that certain hydrolases
are intracellularly located (137), thus establishing the concept of intracellular
disaccharide and sugar phosphate hydrolyses (63, 235). The fact that lactic
acid and carbon dioxide are produced by the absorptive cells has been well
recognized and therefore the existence of large concentrations of intracellular
phosphate esters is not regarded as a surprising finding during the course of
the absorptive process. In fact no solid relationship can be established between
the existence of phosphate esters in the absorptive cells and Verzarts phospho-
rylation and dephosphorylation hypothesis (30) for active sugar transport, so
that. intracellular metabolism spart from active transport processes does exist
and may be of some consequence.
In the work to be presented here we have been more concerned with
the fate of the transported substance, in particular its conversion into amino
acids and proteins, rather than its mode of active transport. In vitro studies
by Newey, Smyth and Whaler (158); and in vivo studies by Atkinson, Parsons
128
and Smyth (159); and those by Kiyasu, Katz and Chaikoff (181) have established
that about 13% of the transported glucose is absorbed as lactic acid. They
have found that the amount of lactic acid produced by placing the glucose-U
-c14 containing medium on the mucosal aide is much greater than placing it
on the serosal aide and that more lactic acid is produced apparently in vitro
than in vivo. However the latter finding only appears so because in the in vitro
experimenta al.most ail of the lactic acid produced cornes from the glucose placed
in the medium whereas ~ vivo a large proportion of the lactic acid produced
cornes from sources other than the transported sugar. In any case they have
been able to account for ali of the transported glucose in terms of lactic acid,
carbon dioxide and very small amounts of keto-acid. This finding, very small
amounts of keto-acid, has been of particular interest to us because it establishes
the necessary link between carbohydrate utilization and the production of amino
acids by intestinal tissue.
In 1959 the work of Newey, Parsons and Smyth (211) showed that when
glucose-U -c14 was used as substrate and the total carbon dioxide produced
(C02 + c14o2) collected 50% of the total was c14o2 coming directly from the
glucose added to the medium and the other 50% was from endogenous sources.
They further established that of the 50% of the total CO coming from the added 2
glucose 80% was from the glucose initially added to the mucosal. side and 20%
from the serosal. aide.
These studies have clearly indicated that part of the transported sugar
is intracellularly metabolised and converted at least in part to a number of
substances. It is our aim in this section to show that part of the carbohy
drate thus utilized 1s converted into amino acids and proteins.
129
130
Methods and Materials
Animals and procedure.
In ali the experimenta in this section only male guinea pigs in the
weight range of 250-500 g. were used. The animals were kept and sacrificed
as described in the preceding section.
In ali cases the intestinal segment chosen was the twenty-inch section
between the 10 inch and 30 inch length of the upper jejunum. From this seg
ment three types of tissue preparations were obtained: intact-strips as des
cribed earlier; and mucosal sheets and serosal sheets prepared according to
the procedure of Dickens and Weil-Malherbe (271).
Tissue was washed with ice-cold oxygenated physiological saline and
kept under a steady stream of pure o2 during the entire procedure. Incubations
were carried out in Ringer's phosphate medium of the following composition
(millimoles/liter): NaCl 128, KCl 5, CaC12 3. 6, MgS04 1. 3, Na2HP04 10
(brought to pH 7. 4 with N HCl).
Tissue used was in the range of 50-150 mg per sample, incubated in
3 ml of medium containing the desired amount of radioactive substrate. The
flasks used for the purpose were 50 ml Erlenmeyer :tlasks fitted with rubber
stoppera, a glass tubing, and a hypodermic needle as described in the preceding
section under Methods and Materials.
Incubations were carried out for 90 min. at 37°C under a constant,
gentle stream of 0 2 with continuous shak.ing unless otherwise specified. At
131
the end of the period, the fl.asks were removed and at once chilled in a
crushed-ice bath. Sometimes the tissue was removed as indicated in the
preceding section and other times when total amounts were desired the tissue
was homogenized with the incubation medium. In both cases 2 ml of 95%
ethanol was added and the tissue was homogenized in a Potter-Elvehjem
homogenizer. The homogenizer was washed quantitative! y with small portions
of 80% ethanol and the washings were added to the original homogenate. The
homogenate was allowed to stand at 4° for 2 hours (usually overnight), then
centrifuged. The al.coholic extract was collected and the residue washed
twice with 2 ml al.iquots of 80% ethanol and finally with 1 ml of distilled
water. The total volume of the pooled extract usually did not exceed 12 ml.
The ethanolic extract was next passed through a "medium porosity" ion exchange
resin column (Dowex 50W-X8, 200-400 mesh, H+ form) to pick up the amino
acids present. The columns were about 1. 5 x 6 cm and were pretreated with
80% ethanol. The columns were washed with about 30-50 ml of distilled
water and the amino acids eluted with severa! portions of 2N NH40H made up
in 80% ethanol. The total eluate, about 12 ml, was evaporated down to dry
ness under a constant stream of clean, dry air at room temperature. The
residue was redissolved in 0, 4 ml of 80% ethanol. Fifty lambda of the re
sulting solution was plancheted and dried under an infra-red heat lamp and
counted for radioactivity. From the remainder of the solution 300 lambda
was spotted by means of a micropipette on Whatman No. 1 filter paper and
chromatographed two-dimensionally. The sol vents used for chromatography
were as follows:
phase I (methyl ethyl ketone; t-butanol; ammonia
sp. gr. 0.88; water (50/50/15/25 v/v/v/v))
phase n (sec-butanol; 90% formic acid; water (70/11/17
v/v/v) )
132
Paper chromatography was carried out from 16-18 hours in each di
rection. The chromatograms were dried at room temperature and placed in
contact with Kodak ''No Screen" X-ray film for 3-7 days. The films were
developed and the radioactive spots were located on the chromatograms. The
spots were measured for radioactivity, using a Tracerlah counter with a mica
window 28 mm in. diameter and a thickness of 1. 5-l. 8 mg/cm2• AU readings
were corrected for background. Chromatograms were then sprayed with nin
hydrin solution (0.1% in acetone) and allowed to dry. The amino acid spots
present were confirmed by comparison with standards of known amino acids
similarly chromatographed.
Radioactive measurements.
Ali readings were corrected for background. Calculations for the
amino acids were made as follows: for example, the incorporation of glucose
into amino acids from the metabolism of glucose-u-c14 was cal.culated as
mp-g atoms of c 14 from glucose incorporated per 100 mg wet weight of
tissue by use of the formula,
mp.-g atoms of glucose carbon incorporated into amino acids 100 mg wet weight of tissue
cpm of individual. amino acid spot on chromatogram total cpm of the sum of all spots on the paper
:: total cpm of ethanolic eluate from resin treatment
x
x Ill}.l-g atoms glucose total cpm of entire medium
x lOO mg Wet weight of tissue
Q0 and QC measurements were calculated by the use of the following 2 02
formulae:
133
Q0 =factor x difference in manometer height (mm) =pl of 0 2 consumed/mg dry wt./hr 2
factor = K0 of :tlask 2
dry weight of tissue (20% of wet weight)
QC = total cpm of Bae14o3 x 22. 4 pl/Jl mole 02 factor x 1000 x wet weight of t.issue x . 2
factor = total cpm of medium total mp.-g atoms of carbon
Bae14o8 is corrected for self absorption. For our work self-absorption loss
was about 28-30% for sample weights in the range of 14-15 mg i.e. 28-80%
of the count was added to the cpm of the sample (Bae14o3).
134
Experimental
1. The amino acid content of guinea pig intestinal tissue preparations.
Intact-strips and mucosal-sheets were incubated in 50 ml flasks con~
taining 8 ml of Ringer's-phosphate buffer with no added substrate for 90 mi
nutes at 87°C under a constant stream of 0 2 with shaking. At the end of
the period the intact-strips were separated from the medium, the mucosal
-sheets were left in the medium. Both sets of tissue were homogenized,
chromatographed and sprayed with ninhydrin solution as described under
methods and materials. The presence of the amino acids listed in Table V
were identified and confirmed.
For mucosal-sheets homogenized with the medium we obtained the
same pattern of amino acids on the chromatograms as we did for intact-strips
with the possible exception of tryptophan. In the intact-strips tryptophan
seemed to be missing or it was so low in concentration that we were not able
to detect it with the ninhydrin test. With mucosal-sheets the spots developed
were much darker indicating higher amino acid content. However it must not
be overlooked that in the latter case we were measuring the total amino acid
content, that is, tissue plus the medium concentration.
2. Study of 02 uptake and C02 output with GPI tissue preparations.
Three types of tissue preparations were used for the study of o2
TABLE V
AMINO ACID CONTENT OF GPI TISSUE PREPARATIONS
AFTER INCUBATION WITHOUT ADDED SUBSTRATE
Intact-Strips Mucosal-sheets
Alanine Alanine
Arginine Arginine
Aspartate AsPartate
Glycine Glycine
Glutamate Glutamate
Glutamine Glutamine
Glutathione G lutathione
Histidine Histidine
Leucine Leucine
Lysine Lysine
Methionine Methionine
Phenylalanine Phenylalanine
Proline Proline
Serine Serine
Taurine Taurine
Threonine Threonine
Tryptophan.
Tyrosine Tyrosine
Valine Valine
Note: Incubations were done in Krebs-phosphate buffer without added substrate for 90 min. at 37°C. Intact-strips were separated from the medium before homogenization, mucosal-sheets were homogenized with the medium.
135
136
uptake and co2 output, namely, intact-strips mucosal -sheets and se ros al
-sheets. Incubations were done in conventional Warburg vessels using 3 ml
of Ringerts-phosphate buffer containing 5 mM uniformly labelled glucose.
The center wells contained 0. 2 ml of 20% KOH with fluted filter paper to
pick up the evolved co2. The si de arms contained 0. 2 ml of 30% · TCA
which was tilted into the incubation mixture at the end of the period and
allowed to remain until ali of the co2 was evolved and trapped in the KOH
solution. The KOH solution was quantitatively removed and the co2 preci-
pitated as BaC03 . From the weight and count of the precipitate the amount
of c 14o 2 produced was estimated. The Q02 and Qc02
values were calculated
by the formulae given under methods and materials. These resulta are listed
in Table VI.
It can be readily seen that the rate of respiration (Q02
) is quite high
for GPI mucosal-sheets. However, the rate of oxygen consumption for the
oxidation of glucose is about the same in all the tissue preparations.
3. Synthesis of amino acids from glucose-u-c14 by intact-strips of GPI.
Intact-strips of GPI were incubated in Ringer's-phosphate buffer con
taining 5 mM glucose labelled with glucose-u-c14. Ali samples were placed
in 50 ml flasks containing 3 ml of medium and incubated for 30 minutes. They
were processed as indicated under materials and methods and the labelled
amino ac ids were determined by chromatography and radioautography. These
TABLE VI
Q0 AND QC VALUES OBTAINED WITH INCUBATION OF VARIOUS 2 02
Sample number
1
2
3
4
mean
TISSUE PREPARATIONS OF GPI IN 5 mM GLUCOSE
Intact-strips
Qo2 Qco2
7.45 1. 90
7. 85 2.19
8.15 2. 67
7. 78 1. 85
7. 82 2.15
27.5%
Mucosal-sheets
Qo2 Qco2
12.05 2.87
12.63 2.43
12.30 3.27
12.33 2.86
23.2%
Serosal-sheets
Qo2 Qco2
2. 83 0.63
2.89 0.70
2.60 0.55
2. 77 0.63
22.8%
137
Note: Q02
expressed as )1102/mg (dry wt.)/hr. i Qc02
expressed as )11C02/mg (dry
wt. /hr. Incubations were carried on in Warburg vessels with Ringerts-phosphate
buffer containing 5 mM labelled glucose for 1 hour at 37oc. c 14o2 was determined
as Bae14o3 .
138
results are given in Table vn. This pattern of the amino acids synthesized
by the gut wall has been repeatedly produced in all of our subsequent experi
menta so that there is not much doubt that the intestine is capable of produ
cing these amino acids from carbohydrate.
4. Time course study with glucose-u-c14 for the production of amino acids
by the GPI strips.
This experiment was done to study the variation in the production of
the total amounts of amino acids with increasing time of incubation. Incuba
tions were carried out as in the preceding experimenta using 5 mM labelled
glucose and varying the time from 5 min. to 75 min. of incubation. The re
sulta are summarized by the curve in Fig. 11. It can be seen that there is a
rapid initial rise in the rate of amino acid production with a gradua! decrease
beyond 60 minutes of incubation.
5. Effect of increasing medium concentration of glucose on production of
amino acids by GPI strips.
Incubations were carried out for 90 minutes at 37°C. The glucose
concentration of the medium was varied from 1.25 mM to 30 mM keeping the
specifie activity the same for ali concentrations. The total sum of all the
amino acids produced for each concentration of glucose in the medium was
139
TABLE Vll
SYNTHESIS OF AMINO ACIDS FROM LABELLED GLUCOSE
BY INTACT-STRIPS OF GPI
Labelled c derived from amino acid synthesized glucose
by strips (cpm)
Alanine 324
Glutamate 180
Aspartate 133
Glutamine 22
Serine 16
Glycine 11
Proline 30
Glutathione 24
Note: Incubations were done in Ringer's-phosphate buffer containing labelled glucose (5 mM). Incubation time was for 30 minutes at 37°C. Intact-strips were removed from the medium, blotted with filter paper and homogenized. Amino acid contents were determined as described under methods and materials.
Ail counts are based on 100 mg (wet weight) tissue.
r:t:J Q ..... u < ~
!Il
0 !Il ...... .... z bO ..... s ~ 0 < 0 .-!
H ........ s < Q..
f-4 C)
0 f-4
50
40
30
20
10
•
1 •
•
~·•
TI ME (min)
Fig. 11. Variation in the production of amino acids with time of incubation. Tissue was incubated with 5 mM labelled glucose in Ringer's·phosphate buffer at 37°C.
140
plotted against that concentration. The total sums were obtained by adding
the counts (cpm) for each amino acid produced after chromatography. The
results are given in Fig. 12. The curve indicates an initial rapid rise in
the production of amino acids with low concentrations of glucose in the me
dium followed by a more graduai rise with higher concentrations but even
tually a saturation point appears to be reached beyond which further intra
cellular accumulation of amino acids is proportionately very slow with in
creasing medium concentration of glucose and approaches a stead.y-state
condition.
141
Similar curves were obtained when values ( cpm) of the individual
amino acid were plotted against the medium concentration of glucose in the
case of alanine, aspartate, glutamine, glutathione and serine. Glutamate
and glycine behave differently. Glutamate concentration reaches a maximum
level at approximately the 2. 5 mM concentration and remains relatively con
stant or even decreases with subsequent higher concentrations. This may
be interpreted as an indication that the glutamate formed is rapidly utilized
or interconvertedJ so that, it does not accumulate intracellularly beyond a
certain lev el. It could also mean that the cell is not capable of retaining
the glutamate and that the latter leaks out into the medium. Glycine pro
duction is more difficult to analyze from our data. Glycine production is
quite low at ali concentrations of medium glucose.
The curves obtained with alanine and glutamate are given in Figs. 13
and 14 as typical examples.
-s fr r:J)
0 ..... u ~
0 z ..... ~ ~
~ ~ E-t 0 E-t
142
30
• • . ~·-20 / •
1 •
10
OL-~--~-------L------~------~------~----~ 2.5 5 10 15 20 25 30
MEDIUM CONCENTRATION
of glucose (mM)
Fig. 12. Effect of variation in medium glucose concentration on total amino acids produced by 100 mg wet weight of tissue.
160
140
- 120 ~ 0 -z 0
100 -~ ~ p::: ~ z
80 t:r:l u z 0 u
t:r:l 60 z -z ~ ~
40 ~
20
·-/ . ~· •
• 1
1 •
MEDIUM CONCENTRATION
of glucose (mM)
Fig. 13. Effect of variation of medium concentration of
glucose on alanine production by intact-strips of GPI.
143
240
-~ 200
-z 0 ....... E-t 160 <(
~ E-t z I:LI 120 u z 0 u I:LI 80 E-t <(
~ <(
E-t j:) 40 ...l ~
0
144
r·\ • ., .
~. ....... -· ·-
2.5 5 10 15 20 25 30 MEDIUM CONCENTRATION
of glucose (mM)
Fig. 14. Effect of variation in medium concentration of glucose on glutamate production by intact-strips of GPI.
6. A comparative study of amino acids produced by intact strips, mucosal
sheets and serosal sheets.
145
Intact strips were prepared as already described. Mucosal sheets
and serosal sheets were prepared by cutting longitudinally 1. 5 - 2. 0 inch seg
ments of everted jejunum and the mucosa was scraped off from the serosa
with the edge of a microscope slide. Ali tissues were incubated for 90 mi
nutes at 37°C in 3 ml of Ringer's-phosphate buffer containing 5 mM labelled
glucose. Ali tissues were homogenized with their respective media so that
the values given in Tables VITI, IX and X are for the total amino acids pro
duced, that is, tissue content plus the amount that bas passed out into the
incubation medium.
It can be seen from these tables that mucosal sheets are capable of
producing much higher totals of amino acids than either intact-strips or sero
sal sheets, however, most of this capacity seems to lie in the direction of
producing more alanine. It is interesting to note that serosal sheets produce
higher levels of glutamate thau either the mucosal sheets or intact strips.
Another significant feature of the mucosal -sheets is that its aspartate pro
duction is about the same as the other two tissue preparations.
Serine, glutamine, glycine and glutathione production in mucosal sheets
is higher on the average than the other tissues. Alanine production in serosal
sheets is by comparison significantly low. As might be expected it appears as
if for some of the amino acids the values attained by intact strips are inter-
TABLE VIll
FORMATION OF LABELLED AMINO ACIDS FROM UNIFORMLY LABELLED GLUCOSE
AS SUBSTRATE BY 100 mg OF MUCOSAL-SHEETS OF GPI
mu-g atoms of C from glucose incorporated into
Alanine Glutamate Aspartate Serine Glutamine Glycine Glutathione Totals
815 72 49 46 132 73 80 1267 696 134 119 29 44 18 36 1076 627 90 105 34 26 37 41 960 684 98 68 39 27 30 51 997 650 93 80 27 26 24 33 933 620 56 87 47 57 47 29 943 698 77 91 34 40 20 29 989 504 128 64 76 16 19 65 872 532 92 99 52 19 88 59 891
526 107 86 81 27 6 50 883
650 87 70 52 14 22 81 926
526 88 75 117 17 55 63 941 759 86 46 21 21 21 62 1016 530 94 91 56 82 18 48 869
Mean 630 93 81 51 36 31 50 969
Note: 100 mg of mucosal-sheets refera to wet weight of tissue. Incubations were for 90 min. at 37°C in Ringer's -phosphate buffer 5 mM in labelled glucose. Mucosal-sheets were homogenized with the incubation medium.
...... If;>. CD
TABLE 1X
FORMATION OF LABELLED AMINO ACIDS FROM UNIFORMLY LABELLED GLUCOSE
AS SUBSTRATE BY 100 mg OF INTACT-STRIPS OF GPI
mu-g atome of C from glucose incorporated into
Alanine Glutamate Aspartate Serine Glutamine Glycine Glutathione Totale
400 112 56 28 28 8 12 685
427 95 50 26 86 4 9 639
482 98 58 30 28 5 16 662
412 101 68 29 19 9 21 654
409 89 70 24 .22 10 28 647
419 lOO 73 20 27 1.2 18 666
Mean 417 99 62 25 25 8 17 651
Note: 100 mg of intact-strips refera to wet weight of tissue. Incubations were done in Ringer' a-phosphate buffer containing 5 mM labelled glucose. Incubation time 90 min. at 87°C. Intact-strips were homogenized with the incubation medium.
..... ~
TABLE X
FORMATION OF L.ABELLED AMINO ACIDS FROM UNIFORMLY L.ABELLED GLUCOSE
AS SUBSTRATE BY 100 mg OF SEROSAL-SHEETS OF GPI
mu-g atoms of C from glucose incorporated into
Alanine Glutamate Aspartate Serine Glutamine Glycine Glutathione Totals
374 109 85 67 14 12 13 674
294 128 79 29 3 29 42 604
222 179 67 34 14 11 26 553
340 150 81 29 8 18 32 658
351 140 89 27 12 15 37 671
289 142 72 31 15 9 27 585
Mean 311 142 79 36 11 16 30 624
Note: 100 mg of serosal.-sheets refers to wet weight of tissue. Incubations were done in Krebs-phosphate buffer 5 mM in labelled glucose. Incubation time 90 min. at 37°C. SerosaJ:...sheets were homogenized with the incubation medium.
.... lflo. 00
mediate to those of mucosal sheets and serosal sheets. For example this
is true in the case of alanine, and glutamine formation.
149
Table XI shows the values obtai.ned for intact-strips when these are
first removed from the medium immediately after incubation and processed.
The labelled amino acids found then, are the intracellular or those located
within the tissue which have not passed out into the incubation medium. The
interesting observation is made when one compares the results of Table XI
with those of Table IX that alanine, serine and glutamine are the amino acids
which leak out of the tissue into the medium, alanine to the extent of approxi
mately 50%.
7. Comparison of formation of labelled amino acids by mucosal sheets of
GPI when incubated with uniformly labelled glucose, fructose and sucrose.
Incubations were run for 90 minutes at 37°C with 5 mM concentration
of the respective sugar used as substrate. All three sugars were uniformly
labelled. The resulta are given in Table XII. The amino acid levels for fruc
tose are significantly higher than those of glucose. It appears as if fructose
is more readily converted into amino acids than glucose. The values obtained
with sucrose are almost twice those of glucose but this is only apparently so
and does not mean that sucrose is more efficiently utilized than glucose or
fructose. Sucrose is uniformly labelled therefore on an equimolar basis it
has twice the number of c14 atoms as compared to the hexoses, hence twice
TABLE XI
FORMATION OF LABELLED AMINO ACIDS FROM UNIFORMLY LABELLED GLUCOSE
.AS SUBSTRATE BY 100 mg OF INTACT-STRlPS OF GPI
mu-g atoms of C from glucose incorporated into
Alanine Glutamate Aspa.rtate Serine Glutamine Glycine Glutathione Totals
218 137 97 10 7 17 30 516
258 115 92 5 6 7 6 507
251 112 64 7 3 2 20 459
239 106 46 8 6 6 21 432
224 109 54 8 3 8 15 421
213 65 45 4 9 4 11 351
181 86 78 8 6 10 20 388
172 75 64 4 5 3 27 349
Mean 220 101 68 7 6 7 16 428
Note: 100 mg of intact-strips refera to wet weight of tissue. Incubations were done in Ringer's-phosphate buffer containing 5 mM labelled glucose. Incubation Ume 90 min. at 37°C. Intact-strips were separated from the medium before homogenization.
1-' tn 0
151
TABLE XII
COMP ARISON OF AMINO ACID PRODUCTION BY MUCOSAL-SHEETS
OF GPI BY INCUBATION WITH UNIFORMLY LABELLED GLUCOSE
FRUCTOSE AND SUCROSE AS SUBSTRATE
Labelled mp-g atoms of C derived from amino acids
formed Glucose Fructose Suc rose
Alan:lne 743 909 1180
Glutamate 115 162 238
Aspartate 104 123 183
Serine 36 51 95
Glutamine 37 71 83
Glycine 24 85 81
Glutathione 42 107 170
Totals 1100 1912 2030
Note: All incubations were done with 5 mM uniformly labelled glucose, fructose or sucrose in Ringer's-phosphate buffer. Incubation time 90 min. at 37°C. Values given for each amino acid are in mp-g atoms of C from the respective labelled substrate which has been incorporated into that amino acid. The resulta are the average of at least 5 determinations done on different days with intestine from different guinea pigs.
All values given are based on 100 mg (wet weight) tissue.
as much radioactivity wotùd be expected to be incorporated into the amino
ac ids deri ved from it.
152
8. Effect of starvation on the formation of amino acids from labelled glucose
by GPI tissue.
A 5 mM concentration of glucose was used for al.l incubations. Both
intact-strips and mucosal.-sheets were used at no starvation, 24 hour, and 48
hour starvation levels. All conditions for the incubations were otherwise the
same as in the preceding experimenta. The restùts obtained are given in
Table XIII. It is apparent from the table of values that 24 or 48 hour star
vation has no signifi.cant effect on the amino acid forming capacity of the gut
wall. There appear to be slight reductions in the formation of amino acids
partictùarly alanine when intact-strips are used but mucosal. sheets do not sub
stantiate this apparent reduction. Perhaps it can be concluded that the effect
of 24-48 hour starvation is, if significant, of reducing the capacity of the
gut to synthesize amino acids, possibly by limiting a. cofactor or enzyme
necessa.ry for the reactions.
9. Interconversion of the amino acids by intact-strips of GPI.
Intact-strips were incubated with Ringer's-phosphate buffer made 5 :mM
in the respective labelled amino acid. Glutamate-u-c14, aspartate-u-c14,
153
TABLE :xm
EFFECT OF STARVATION ON THE FORMATION OF LABELLED
AMINO ACIDS FROM LABELLED GLUCOSE BY GPI
Labelled No starvation 24-hr. starved 48-hr. starved amino aclds Intact Muoosal Intact Mucosal Intact Mu cos al
formed strips sheets strips sheets strips sheets
Alanine 798 827 514 946 531 912
Glutamate 115 96 109 61 75 66
Aspartate 82 72 75 51 63 63
Serine 25 62 22 45 19 39
Glutamine 27 55 28 28 35 34
Glycine 9 34 23 18 16 19
Glutathione 51 68 67 38 47 42
Totals 1107 1214 838 1187 786 1175
Note: All tissues were incubated in Ringerts-phosphate buffer oontaining 5 mM labelled glucose. Incubation time 90 min. at 37°C. All tissues were homogenized in their respective medium. Each value given is the mean of two separate determinations.
All values given are mp-g atoms/100 mg (wet weight) tissue.
154
glycine-2-c14 and glycine-1-c14 were used as tracera. Conditions for incu
bation were the same as the preceding experimenta. The tissues were re
moved at once after incubation and processed rapidly as described in methods
and materials. The resulta obtained are given in Table XIV. In the case
of glycine there was hardly any interconversion, only very small amounts of
aspartate and serine were formed. The values obtained for glutathione can
not be regarded as true interconversions except for aspartate but rather in
corporation into the structure of this tripeptide. Both glycine-1-c14 and
glycine-2-c14 were used giving identical resulta. The interconversion of
glutamate is quite high (about 45%). The only amino acid into which gluta
mate failed to be converted was, significantly enough, glycine. Aspartate
seems to be converted into substantial amounts of alanine and glutamate
and mu ch sm aller amounts of the other amino acids, however, the values
are rouch lowered compared with glutamate interconversions (about 16%).
10. Formation of labelled amino acids from labelled acetate and formate.
Intact-strips of GPI were incubated in 5 mM. solutions of labelled
acetate (acetate-1-C1~ and labelled formate (formate-e14) in Ringer's-phos
phate medium. The resulta are given in Table XV. The only labelled amino
acid derived from formate is serine. Acetate seems to give rise to substantial
quantities of all the amino acids listed and glutathione, the only exceptions are
serine and glycine.
155
TABLE XIV
INTERCONVERSION OF AMINO ACIDS BY INTACT-STRIPS OF GPI
Labelled mp-g atoms of C derived from labelled amino acids
formed Glutamate Aspartate Glycine
Alanine 281 117 0
Glutamate 740 43 0
Aspartate 203 1037 6
Serine 28 4 8
Glutamine 27 8 0
Glycine 0 7 725
Glutathione 59 11 18
Total Conversion (%) 44.7 15.5 4.2
Note: Incubations were carried out with Ringer' a-phosphate buffer containing 5 mM respective labelled amino acid as substrate. Incubation time 90 min. at 37°C. The labelled amino acids used were glutamate-u-c14, aspartate-u-c14 and glycine-2-c14. Values given are the mean of 4 separate determinations.
Ali values given are based on 100 mg (wet weight) tissue.
TABLE XV
FORMATION OF AMINO ACIDS FROM LABELLED ACETATE
Labelled amino acids
formed
Alanine
Glutamate
Aspartate
Serine
Glutamine
Glycine
Glutathione
Totals
AND FORMATE BY INTACT-STRIPS OF GPI
mp-g atoms of C derived from labelled
Acetate Formate
238 0
645 0
324 0
0 27
91 0
0 0
159 0
1456 27
156
Note: Acetate-1-c14 and Formate-c14 were used as tracera in 5 mM concentration of acetate and formate in Ringer' a-phosphate buffer solution. Incubations were with intact-strips of GPI for 90 minutes at 37°C.
Al1 values given are based on 100 mg (wet weight) tissue.
11. Effect of metabolic inhibitors or stimulators on the formation of amino
acids by the GPI from glucose.
157
Ali incubations were done with 5 mM labelled glucose. Conditions of
incubation, unless otherwise noted. were the same as those in the preceding
experimenta. The concentrations for the inhibitors or stimulators are given
under the respective compound and refer to their initial concentration in the
media. The resulta obtained are given in Tables XVI and XVII.
It may be noted from Table XVI that amytal increases alanine pro
duction but decreases glutamate, aspartate and glutathione production. This
is to be expected since amytal is an effective inhibitor of the oxidation of
DPNH and its associated phosphorylations. The formation of acetyl-CoA is
prevented and the citric acid cycle slows down and the production of oC -keto
glutarate and oxaloacetate is impaired and bence the ensuing reduction in as
partate and glutamate. Likewise there will be an accumulation of pyruvate
which will transaminate to alanine thus increasing its formation.
Salicylate and DNP are weil known uncoupling agents and by preventing
the formation of ATP they influence the production of amino acids. Salicylate
reducea the formation of alanine, glutamate, and glycine. DNP likewise lowers
the alanine, glutamate and gluta.mine values.
The action of phlorhizin is interesting, at the effective metabolic con
centration (lo-3 M) it produces more effective inhibition than any of the other
compound&. It is to be noted however that not al.l of the amino acids are
158
TABLE XVI
EFFECT OF 1NIDBITORS ON FORMATION OF AMINO ACIDS BY
1NTACT-STRIPS OF GPI FROM 5 mM LABELLED GLUCOSE
Labelled Dl}l-g atoms of C derived from labelled glucose
amino acids Control Amytal Salicylate Phlorhizin Malonate formed (5mM) (1 mM) (5mM) (1 mM) (10 mM)
Alanine 245 344 173 117 243
Glutamate 109 44 70 65 50
Aspartate 55 20 124 58 60
Serine 8 6 5 7 8
Glutamine 5 4 4 4 5
Glycine 4 3 0 5 6
Glutathione 21 5 14 13 16
Totals 447 426 390 269 378
Note: Ali incubations were done with intact-strips of GPI in Ringerrs-phosphate buffer containing 5 mM labelled glucose. Incubation time 90 min. at 37°C. Inhibitor concentrations of the media were as indicated. The values given are the mean of 4 separate determinations.
Ali values given are based on 100 mg (wet weight) tissue.
159
TABLE XVIT
EFFECT OF INHIBITORS OR STIMULATORS ON FORMATION OF AMINO
ACJDS BY MUCOSAL-SHEETS OF GPI FROM 5 mM LABELLED GLUCOSE
Labelled m)l-g atoms of C derived from labelled glucose amino acids
formed Control F-acetate* DNP Insulin Colchicine (1 mM) (1 mM) (11. 5units/ml) (1mM)
Alanine 770 732 490 790 763
Glutamate 121 64 80 103 119
Aspartate 79 29 84 67 81
Serine 43 22 29 53 44
Glutamine 47 14 0 71 82
Glycine 18 14 13 25 18
Glutathione 45 17 15 43 36
Totals 1123 892 711 1152 1093
Note: Ail incubations were done with mucosal-sheets of GPI. Incubation time 90 min. at 37°C. Concentrations of the inhibitors refer to initial medium concentrations.
Ail values given are based on 100 mg (wet weight) tissue. * Fluoroacetate
160
equally affected by phlorhizin. Alanine and glutamate are inhibited signifi.cantly
but not the others.
Insulin a.t the concentration present has no particularly stimula.tory
effect as was anticipated although serine alanine and glutamine show slight
gains.
Colchicine also a.t 1 mM concentration has no signüicant observable
effect in vitro. Fluoroacetate and malonate are effective inhibitors of the
citric acid cycle reactions. Fluoroaceta.te inhibits the action of aconitase and
malonate tha.t of succinic dehydrogenase. These effects are observable from
the tables. The formation of glutamate, aspartate and glutamine are signifi-
cantly low with fluoroacetate inhibition. The resulta with malonate are less
pronounced, the only signüicant inhibition seems to be with the formation of
glutamate, which probsbly can be explained by the fact that the citric acid
cycle is slowed down because of the impairment in the formation of oxaloa-
cetate due to malonate inhibition of the succinic acid to fumaric acid step.
12. The effect of high concentrations of potassium on the rate of formation
of amino acids from glucose.
In order to make a comparative study of the effect of K+ concentra-
tion both intact-strips and mucosal-sheets were incubated in Ringer's-phosphate
buffer 5 mM in glucose with varying concentrations of potassium as indicated
in Table XVIU. It can be readily observed that with 15 mM potassium the
161
TABLE XVIll
THE EFFECT OF HIGH CONCENTRATIONS OF K+ ON THE RATE OF SYNTHESIS
OF AMINO ACIDS FROM LABELLED GLUCOSE BY GPI TISSUE
m)l-g atoms of C derived from labelled glucose
KCl KCl KCl Labelled (5 mM) (15 mM) (105 mM)
amino acids Intact Mucosal Intact Mucosal. Intact formed strips sheets a trips sheets strips
Alanine 93 348 117 640 96
Glutamate 53 56 72 54 51
Aspartate 58 32 57 36 57
Serine 3 56 2 46 5
Glutamine 3 19 5 24 2
Glycine 3 27 2 0 2
Glutathione 25 55 30 34 20
Total a 238 593 285 834 233
Note: Intact-strips were separated from the medium before homogenization. the mucosal.-sheets were not. Ail tissues were incubated in 5 mM labelled glucose for 90 minutes at 37°C.
Ali values given are based on 100 mg (wet weight) tissue.
162
total amount of amino acids elaborated by mucosal-sheets is about 834 lDJ.l-g
atoms of glucose carbon as compared with 593 for the total by mucosal-sheets
incubated with 5 mM potassium. There is also a smal.ler but noticeable sti
mulation by 15 mM potassium when intact-strips are used. It is apparent
from both types of tissue preparation that the stimulation by 15 mM potassium
is mostly in the formation of alanine. No stimulation was observable with
intact-strips incubated in 105 mM potassium.
13. The effect of addition of glutamine, asparagine or ammonium chloride
on the rate of formation of amino acids from glucose by intact-strips
of GPI.
Ali tissues were intact-strips of GPI incubated in 5 mM labelled glu
cose. Asparagine, glutamine or NH4CI were also added to make the concen-
tration 5 mM. The resulta are given in Table XIX. Gl:utamine and aspa-
ragine show significant stimulatory e:ffects on amino acid formation from glu
cose but NH4Cl does not except in the formation of alanine where a slight
stimulation seems to be present. Asparagine stimulation is greater than that
of glutamine. As in the previous cases alanine production is affected the most
although here in the case of asparagine aspartate formation al.so appears stimu
lated. Also both glutamine and asparagine show slight stimulatory effects on
formation of glutamine from glucose.
163
TABLE XIX
THE EFFECT OF GLUT AMINEt ASPARAGINE AND AMMONIUM CHLORIDE ON
THE RATE OF FORMATION OF AMINO ACIDS FROM LABELLED GLUCOSE BY
JNTACT-STRIPS OF GPI
Labelled m}l-g atome of C derived from labelled glucose
amino acide Controle Glutamine Asparagine NH4Cl formed (5 mM) (5 mM) (5 mM)
Alanine 120 203 308 169
Glutamate 90 92 103 59
Aspartate 63 73 113 55
Serine 3 6 2 5
Glutamine 3 16 21 5
Glycine 4 9 3 6
Glutathione 22 21 14 11
Totale 305 420 564 310
Note: All tissue was intact-strips of GPI incubated in Ringer's-phosphate 5 mM in labelled glucose. In addition the test flasks were made 5 mM in· tm.labelled glutamine, asparagine or NH4Cl. Incubation time 90 min. at 37°C.
All values given are based on 100 mg (wet weight) tissue.
14. The effect of formate on the rate of formation of amino acids from
glucose by mucos.al-sheets of GPI.
164
Mucosal-sheets of GPI were incubated with Ringer's-phosphate buffer
containing glucose and formate as indicated in Table XX. After incubation
tissue was homogenized with the medium. The controls run with glucose-U
-c14, as usual, indicate higher values for serine, glutathione and glycine.
This factor seems to be borne out or valid whenever we compare the pro
duction of these amino acids by mucosal-sheets with those obtained by intact
-strips, that is, mucosal-sheets seem to give considerably higher values for
these amino acids as weil as for the others.
When formate-c14 is used with unlabelled formate as substrate al
most .ali of the labelling occurs in the serine. Labelling in the other amino
acids is very small and probably not significant.
When labelled glucose is used as tracer with unlabelled formate as
substrate a rather large amount of labelling occurs in .alanine. That is, the
presence of excess formate enhances the production of alanine. The labelling
in the other amino acids particularly glutamate and serine is also increased.
When unlabelled glucose is used as substrate with labelled formate
as tracer extremely large counts are obtained in serine and some small in
creases in glutamine and glutathione.
165
TABLE XX
EFFECT OF FORMATE ON THE RATE OF FORMATION OF AMINO
ACIDS FROM GLUCOSE BY MUCOSAL-SHEETS OF GPI
cpm/100 mg wet weight tissue
Labelled Glucose 5mM Glucose 5mM Formate 5m:M Formate 5m:M amino acids + + + +
formed Glucose-U -c14 Formate-c14 Formate-c14 Glucose-U -C
Alanine 728 0 15 15, 125
Glutamate 133 73 91 1t 750
Aspartate 138 137 0 965
Serine 176 73,400 1, 850 1, 540
Glutamine 44 3,640 90 362
Glycine 94 0 0 401
Glutathione 68 2,640 62 790
Note: All incubations were done in Ringerta-phosphate buffer containing the respective substrate and tracer for 90 minutes at 37°C. All tissue was homogenized in the ir respective media. Background has been subtracted from all counts given.
14
166
15. The formation of the tripeptide glutathione from glucose by GPI tissue.
Glutathione in its characteristic triplet spots has occurred in every
chromatogram that has been run with our solvent systems. The Rf values of
these spots correspond exactly to the Rf values of standard chromatograms
run with known samples of glutathione. Yet, we have felt that this might
still be an artifact and hence we have decided to confirm the presence of
this substance found in our preparations. Hydrolysis of the eluted spots
from the chromatograms has not given us conclusive evidence because of the
difficulties inherent in hydrolysis and subsequent recovery of the individual
amino acids making up the tripeptide ( Y -glutamyl-cysteinyl-glycine). However
the results obtained in Table XXI show clearly that glutamate and glycine are
definitely incorporated into the molecule of glutathione, the labelling obtained
with glutamate being much higher. We have also applied the alloxan test (272)
to the pooled eluate of the spots obtained from a number of chromatograms
and found this test to be positive. According to Kay and Murfitt (272) alloxan
and glutathione interact to form a compound characterized by an absorption
band at 305 mp. This test is considered specifie for glutathione under speci
fied conditions.
16. Formation of proline from glucose by GPI tissue preparations.
Proline has occurred repeatedly in all our chromatograms. Its Rf
value corresponds exactly to the Rf values obtained with known samples of
TABLE XXI
FORMATION OF GLUTATHIONE FROM LABELLED GLUTAMATE AND LABELLED GLYCINE
BY INT ACT-STRIPS OF GPI
Cysteine 2. 5 mM Cysteine 2. 5 mM Labelled Cysteine 2. 5 mM Cysteine 2. 5 mM Glycine 2. 5mM Glutamate 2. 5mM
amino acids Glucose 5 mM Glutamate 2. 5 mM Glycine 2. 5 mM Glucose 5mM Glucose 5mM form.ed Glucose-u-c14 Glycine-2-C 14 Glutamate-U -cl4 Glutamate-u-c14 Giycine-2-c14
Alanine 205 0 91 79 0
Glutamate 118 0 372 423 0
Aspartate 84 8 208 132 0
Serine 11 6 14 12 3
Giutamine 9 3 21 17 0
Glycine 5 536 28 0 505
Glutathione 12 21 110 49 21
-Note: Intact-strips were separated from the incubation medium before homogenization. Ali incubations were done in Ringer's -phosphate bu.ffer containing the respective substrates. Incubation time 90 min. at 37oc.
Ali values are given in mu-g atoms and are based on lOO mg wet weight of tissue. 1-' Q). -::J
HIS
proline and hydroxyproline. However we have not determined its value rou
tinely because its separation from alanine is not good when the solvent sys
tem for chromatography is t-butyl alcohol, methyl-ethyl-ketone, ammonia and
water. An excellent separation of proline is obtained when the phenol, am
monia., water system is employed. However this was not suitable for the
separation of the serine, glycine and glutamine combination, therefore we
were compelled to use the former sol vent system and abandon the routine
determination of proline.
To ascertain that proline is actually present in our chromatograms
we have relied upon a number of colorimetrie tes~ which are considered
quite specifie for proline.
When a solution of ninhydrin in acetone is applied to the spot sus
pected of being proline the spot turns to a canary yellow color upon drying.
Other amino acids usually give a blue or purple col or.
When the spot is sprayed with a solution of isatin in butanol-a.cetic
acid, dried and allowed to remain in the dark for 24 hours, the spot turns
blue-green.
When the spot is sprayed with a solution of vanillin in n-propanol,
dried, heated to 110°C, then treated with ethanolic KOH, heated at 110°C
and then allowed to remain at room temperature, the spots develop red
colora.
The spots on our chromatograms have re.sponded po.sitively to the
169
ninhydrin, isatin and vanlllin tests. In addition its R-f value in two different
solvant systems corresponds exactly to the Rf values obtained with known
samples of praline and hydroxyproline. Therefore we have felt that, in the
absence of any evidence to the contrary. the spots in question must be praline
or hydroxyproline.
In addition we have noticed that praline values consistently go up
whenever formate is present in the incubation medium. Praline values are
usually in the range of 10-30 IIlJl-g atoms/100 mg tissue but in the presence
of formate they may go up as high as 150 mp-g atoms/100 mg tissue. .Appa
rently the gut is capable of synthesizing praline possibly from glutamate by
way of glutamic semi-aldehyde and pyrolline carboxylate.
170
Discussion
In these studies we have been able to show that the intestinal wall
of the guinea pig is capable of synthesizing amino acids from carbohydrate
moieties. The syntheses of alanine, glutamate, aspartate, serine, glycine,
glutamine, proline and the tripeptide glutathione from glucose, fructose and
sucrose has been demonstrated. There is not much doubt that these amino
acids are produced from carbohydrate and that the gut does not need to have
an already formed carbon chain or skeleton such as the acetylated amino
acids or ol:.-keto acids to convert them into amino acids. Some of the amino
acids formed such as alanine, glutamate and aspartate are sufficiently high
in their rates of syntheses to be significant not onl.y for maintenance and
repair of the gut tissues themselves but also to contribute to the general me
tabolism of the body itself. It would be interesting to investigate the amino
acid synthesizing capacity of the small intestine under .2:!: vivo conditions.
With an adequate blood supply and rapid removal of amino acids formed the
total production capacity of the intestinal wall may be quite large and com
parable to that of the liver and brain tissues.
It is also significant that the intestinal tissues are capable of uttli
zing for amino acid synthesis substances other than the sugars, for example,
acetate and formate are readily metabolized. Acetate by entering the citric
acid cycle becomès readily available to yield almost all of the amino acids
synthesized by the gut. Formate does not seem to be as versatile but it
is defi.nitely involved in the formation of serine.
171
Our experimenta have shown that the intestinal mucosa is, as in the
case of active sugar transport. the most active layer of the intestinal wall
and is capable of synthesis of amino acids at much higher rates than either
serosal sheets or intact-strips. Formate incorporation and glutathione for
mation are also significantly at higher rates with mucosal-sheets.
In the interconversion of the amino acids it is to be noted that glu
tamate is most readily converted into all the other amino acids except gly
cine and that it has the highest percentage of conversion ( 45% as compared
to 16% for aspartate and 4% for glycine conversions).
We have been able to show that the rates of amino acid formation
are subject to the action of various known metabolic inhibitors and stimu
lators. This finding, first of all, establishes clearly that the formation of
amino acids by the gut wall is not a matter of simple exchange of groups
auch as deacetylations or transaminations as has been demonstrated by Whaler
(256). although these reactions do take place in the gut. Secondly, it has
been demonstrated that from simple carbohydrate molecules and acetate the
gut wall can manufacture its own amino acids which simply means that the
glycolytic and citric acid cycle pathways of metabolism are operative in the
gut providing this tissue with sufficient quantities of pyruvate, «.-ketoglutarate,
oxaloacetate and other molecules which can be readily converted into amino
acids with transamination and amination reactions. The fact that inhibitors
like amytal, DNP • phlorhizin and :fluoroacetate can inhibit amino acid for
mations selectively shows that these metabolic pathways are operative in the
gut and are the basic avenues of amino acid production.
172
It is interesting to note that serosal-sheets also are capable of pro
ducing substantial quantities of amino acids. Apparently this capacity is not
confined solely to the mucosal layer as is the case with active sugar trans
port. Diffusion provides limited but probably sufficient influx of carbohydrate
into the serosal-sheets to be converted into amino acids. The intermediate
role of intact-strips in the rates of formation of amino acids is also of in
terest. In every case the values obtained by intact-strips have been lower
than those obtained with mucosal sheets but higher than serosal-sheets. In
addition intact-strips provide a convenient means of separating the tissue from
the incubation medium and hence one can study the rate of influx and ef:flux
of the amino ac ids.
The stimulatory effect observed by 15 mM potassium is worthy of
further comment. It has been established de:finitely by Riklis and Quastel
(128) that 15 mM potassium concentration in the medium bathing the serosal
and mucosal aspects of the surviving guinea pig intestine does stimulate active
sugar uptake. It has not been shown however what potassium stimulation pre
cisely involves. It may be that it influences the action of pyruvic kinase and
speeds up the conversion of phosphoenol pyruvate to pyruvate and the latter
is readily converted to alanine by transamination. Our experimenta with
potassium certainly bear this out, that is, potassium stimulation is essen
tially confined to increased alanine formation. This, then, provides a link
between utilization and enhanced active sugar uptake and the essential role
played by the cation as cofactor of pyruvic kinase.
173
The fact that the intestinal wall is capable of synthesizing the tri
peptide glutathione is most interesting. The first step in the synthesis of
glutathione is lmmvn to be the combination of cysteine and glutamic acid
forming Y -glutamyl cysteine, which then condenses with glycine to give
glutathione. These reactions require the expenditure of ATP and are lmown
to take place chiefly in the li ver. The metabolic turnover of glutathione is
said to be extremely rapid (273, 27 4) and may play a significant role in syn
thesis of proteins. It is known to participate in transpeptidations. The en
zyme glutathione glutamotransferase can pass the glutamyl radical from glu
tathione to various natural amino acid acceptors thus forming alpha peptide
linkages during protein synthesis.
The major role of glutathione, hmvever, is considered to be that of
being the prosthetic group of the enzyme phosphoglyceraldehyde dehydrogenase
and in this capacity it forms an addition product with the carbonyl group of
glyceraldehyde-3-phosphate, which is, in turn, oxidized to a thioester by
hydrogen transfer to DPN followed by phosphorolysis.
It has been shown by Sinex (275) that insulin promotes the incor
poration of labelled alanine into proteins of rat diaphragm and by Krahl
(276) that glutathione synthesis by rat liver slices from fasted diabetic rats
is below normal values and can be partially restored by the administration
of insulin and of carbohydrate. Other experimenta not reported here with
glucose and fructose have shown us that insulin does increase alanine and
glutathione formation by mucosal strips of GPL
174
175
Sum.mary
1. An in vitro procedure using Erlenmeyer flasks, pure oxygen in Krebs
-Ringer phosphate medium and three different types of tissue preparations
from GPI has been used to study the metabolism of carbohydrates. A pro-
cedure for the separation and identification of amino acids synthesized by
the gut wall has been initiated.
2. Glucose, fructose and sucrose are metabolized by GPI preparations and
yield the amino acids alanine, glutamate, aspartate, serine, glycine and pro-
line, and glutamine and glutathione.
3. The Q02
and QC values for mucosal-sheets, serosal-sheets and intact 02
-strips of GPI have been determined. Mucosal-sheets have high Q0 • 2
4. Normal GPI tissue has been analyzed for its amino acid content.
5. A curve showing the rate of amino acid formation from glucose with in-
creasing time has been constructed.
6. The effect of change of medium concentration of glucose on the rate of
amino acid formation has been determined.
7. A comparative study showing the variation in the rates of amino acid
formation by three types of GPI tissue, namely, mucosal-sheets, serosal
-sheets and intact-strips has been made. The rate of formation of amino
acids is much higher with mucosal-sheets.
176
8. It has been shown that in the GPI prolonged starvation does not lead to
increased rates of amino acid production but rather to decreased rates possi
bly beca:use of depletion of important enzymes or cofactors needed for amino
acid syntheses.
9. The interconversion of the amino acide glutamic, aspartic and glycine has
been studied. Glycine is not readily converted to other amino acide by the
gut wall, some serine glutamine and aspartate were formed. Also, glycine
is incorporated into glutathione. Glutamate is converted to all the other amino
acide encountered in gut syntheses of amino acids except glycine. Aspartate
is converted essentially into glutamate and alanine.
10. That acetate and formate take part in amino acid synthesis by the gut
wall has been shown. Acetate is synthesized into all of the amino acids ex
cept serine and glycine. Formate essentially goes to serine.
11. The action of a number of metabolic and respiratory inhibitors have been
studied. Amytal increases alanine production but decreases glutamate, aspar
tate and glutathione production. Phlorhizin at 10-3 M concentration is the most
effective inhibitor among those studied. Salicylate and DNP showed inhibition
of alanine, glutamate and glutamine formation.
12. The stimulating effect of 15 mM ~ on amino acid synthesis with mucosal
-sheets and intact-strips of GPI has been shown.
13. Glutamine and asparagine show significant stimulatory effects on amino
acid formation from glucose. Ammonium chloride only seems to stimulate
the formation of alanine.
14. Glutathione production is enhanced when glutamate glycine and cysteine
are made available to the gut wall. It has been shown that the PGI is ca
pable of synthesizing glutathione.
15. The GPI wall can also synthesize proline or hydroxyproline from glu-
cose.
16. The presence of glutathione and proline as a result of synthesis from
carbohydrate by GPI tissue preparations has been identified and confirmed
by a number of tests in addition to syntheses and paper chromatography.
177
PART IV
178
INFLUENCE OF COLCIDCINE ON THE INTESTINAL
MUCOSA OF THE GUINEA PIG
Introduction
It has been intimated in the general introduction and elsewhere that
the epithelium of the mucosal layer of the small intestine is an extremely
fragile tissue with a relatively short span of life and is constantly sloughed
off during digestive episodes and hence needs to be, and is, rapidly replaced
by new tissue. This reconstruction or regeneration process calls forth upon
" the ability of the crypta of Lieberkiilm to provide the necessary infant cella
which by gradual migration upon the aides of the villi reach the apical area
and eventually cause the extrusion of the more matured or older cells which
probably have lost a good ahare of their absorptive and digestive capacities
(47). This entire process of rapid epithelial replacement must require the
syntheses of amino acids which serve as building blocks for the necessary
protein parts of the abaorptive cells.
It has also been reported by Leblond and Stevens and others ( 1, 2)
that since the structure of the epithelial layer remains relatively constant
there must be a balance between production and losa of cella and renewal
must therefore be continuous. The maintenance of auch a balance demanda
the read.y manufacture and supply of amino acids to meet the demanda of
protein synthesis which is taking place in these cella at a rapid pace.
One of the techniques used to study the rate of renewal of the mu
cosal epithelium makes use of the finding by Brues (277) that colchicine in
certain dosage causes the arrest of mitosis in metaphase in liver paren
chymal cella. It has occurred to us that if mitosis is arrested this pro
cess must be accompanied by, or result in, certain metabolic and bioche
mical changes which will be re:flected in the blocking of pathways of nucleic
acid and protein syntheses. We, therefore, have been intrigued by the pro
spect of investigating or finding the possible biochemical changes that may
occur as a result of colchicine administration.
179
With the incubation techniques ad.apted for use with the GPI prepa
rations it has been possible for us to study the changes manüested, by col
chicine action in mucosal tissue, pertaining to amino acid patterns, proteins,
lipids and nucleic acids. The resulta obtained with colchicine ad.ministered
subcutaneously to guinea pigs will be the subject of discussion of this section.
180
Methods and Material.s
Preparation of tissues and procedure
In all the experimenta in this section mucosal. -sheets from GPI
prepared according to the procedure of Dickens and Weil-Malherbe (271)
will be used. The changes necessary in this technique to suit our needs
has al.ready been described in the preceding section and will not be repeated
here.
M&le guinea pigs weighing approximately 400-500 grams were injected
subcutaneously in their hind quartera with varying amounts of (0. 4 to 4 mg)
• colchicine dissolved in sterile, distilled water. The animais were sacrificed
·,
8 hours after injection and the entire small intestine removed at once and
mucosal.-sheets prepared as al.ready described.
Ail tissues were incubated in Krebs-Ringer-phosphate buffer as des-
cribed by Umbreit et al. (278, 54) made 5 mM with labelled glucose. Incu-
bations were done in an atmosphere of pure o2 for 90 minutes at 87°C. After
completion of incubations the tissue was homogenized with the incubation me-
dium so that resulta reported here are in every case total amounts (tissue
and medium contents).
The methods used for the chromatographie and autoradiographie de-
termination of the free amino acids derived from labelled glucose has been
presented under materials and methods of the preceding section (279).
The homogenates were brought to a volume of 8 ml by 80-percent
ethanol and were allowed to remain at 5°C for at least 2 hours. The etha
nolic extracts were used for the determination of the free amino acids by
paper chromatography after the removal. of salta and glucose by treatment
with the ion exchange resin Dowex 50W-X8 (200-400 mesh).
The amino acid free protein residue was next extracted with 5 ml
181
of ethanol-ether mixture (3:1) by heating the mixture in a 60°C water bath
for 10 minutes with occasional. stirring with a glass rod. The supernatant
after centrifugation was transferred (hot) to a clean test tube and the residue
washed thrice with 2 ml portions of ether and this added to the supernatant.
The residue after this treatment was considered free of lipids.
The procedure followed for the separation of the nucleic acids from
the lipid-free protein precipitate was that of Schneider (280) in which advan
tage is taken of the fact that nucleic acids can be separated from other tissue
compounds by their preferential solubfiity in hot trichloroacetic acid. Accor
dingly the lipid-free protein residue was suspended in 3 ml of 5-percent TCA,
heated to gooc in a water bath for 15 minutes with occasional stirring. The
DN A and PN A which splits off by this treatment was recovered by way of the
supernatant and the residue was treated twice more with 3 ml portions of
5-percent TCA and all washings were combined with the original supernatant
thus constituting the nucleic acid fraction.
182
The insoluble protein residue from the nucleic acid separation was
next washed twice with 2 ml portions of ethanol (and these washtngs returned
to the nucleic acid fraction) to remove the residual TCA in the precipitate.
Finally the residue was suspended in O. 5 ml of chloroform-ether mixture
(4:1), plated on tared aluminum planchets with a Pasteur pipet and allowed
to dry. Calculations were made after the determination of the radioactivity
and the weight of the precipitate.
The nucleic acid fraction (DNA and PNA) was evaporated dawn to 1
or 2 ml and 3 ml of water added to the mixture. The TCA was extracted
with three successive 3 ml portions of ether, then the water layer was eva
porated down to almost dryness and the residue made up to 1 ml volume with
water. It was thoroughly mixed with a glass rod to dissolve the residue and
O. 5 ml of this solution was plated, dried, and counted.
The combined lipid-extracts. and washings were evaporated down to
almost dryness and the lipid residue made up to 1 ml volume with chloroform
-ether mixture (1:1) and O. 5 ml of this lipid mixture was plated1 dried, and
counted.
Calculation of resulta .
. Ali planchet counts were made to at least 5000 cpm or 3 minutes,
depending upon the activity of the sample, under a thin window continuous
188
gas flow Tracerlab counter. For the counting of the radioactivity of the spots
on the paper chromatograms a Tracerlab counter with a mica window 28 mm
in diameter and a thickness of 1. 5-1.8 mg/cm2 was used. Ail counts were
made to at !east three minutes. Background counts were subtracted from ali
readings. The free amino acids were calculated as indicated in the preceding
section under methods and materials. The proteins were weighed and their
counts were corrected for self-absorption. Calculations were made for total
proteins, expressing resulta as mp.-g atoms of C from glucose incorporated
into the prote in structure by incubation of 100 mg wet weight of tissue.
The lipids and nucleic acids were calculated in a similar manner
all values being based on mp.-g atoms C derived from glucose and incorporated
into the respective fraction per 100 mg wet weight of tissue incubated under
the conditions already described.
Materials.
Colchichine used for subcutaneous injections was treagent grader
purity obtained from Abbott and Company. This compound has been first
extracted from Colchicum autumnale L., Liliace:ane but more recently was
synthesized by van Tamelen et al. (281). It has the empirical formula of
C22H25N06 with a molecular weight of 399.43 grams. It is pale yellow
scales or powder which darkens on exposure to light. One gram of the
substance dissolves in 22 ml of water.
184
Colchicine has been used in research in plant genetics for doubling
chromosomes (282). Medically it is used to relieve the discomforts of gout.
All radioactive substances used were of 'reagent grade'.
Experimental
1. The incorporation of glucose carbon into amino acids, proteins, lipids
and nucleic acids by mucosal-sheets of GPI.
185
Mucosal-sheets of guinea pig intestine were prepared and incubated
in 5 mM glucose solution for 90 minutes at 37°C. The contents of the flask.s
(tissue and medium) were homogenized and the proteins precipitated with 80
-percent ethanol at 5°C for two hours. The supernatant was removed and
analyzed for its amino acid content and the protein residue was processed
for lipids and nucleic acid separations as indicated under methods and ma
terials. The radioactivities of each fraction were determined and values of
carbon derived from labelled glucose and incorporated into the respective
fractions were calculated in mu-g atoms of C/100 mg wet weight of tissue.
These resulta are given in Table XXII and Table XXIll.
It can be noted from the tables that there is very good precision
and reproducibility when resulta are compared both with one another and
with free amino acids obtained in the preceding section.
2. Formation of labelled amino acids from glucose by mucosal-sheets of
GPI after the subcutaneous injection of colchicine.
One guinea pig was injected O. 4 mg of colchicine in water and allowed
TABLE XXII
FORMATION OF LABELLED AMINO ACIDS FROM GLUCOSE BY MUCOSAL-SHEETS OF GPI
mp-g atoms of C derived from labelled glucose incorporated into
Alanine Glutamate Aspartate Serine Glutamine Glutathione Glycine Totals
684 98 68 39 27 31 30 977
650 93 80 27 26 33 24 933
620 56 87 47 57 29 47 943
698 77 91 34 40 29 29 998
Mean 633 81 82 37 38 31 33 962
Note: Ali incubations were done in Krebs-Ringer-phosphate buffer 5 mM in uniformly labelled glucose for 90 min. at 37°C in an atmosphere of pure o2. Ali values are total amounts based on 100 mg wet weight of tissue.
1-' CIO 0')
TABLE XXID
INCORPORATION OF C DERIVED FROM LABELLED GLUCOSE INTO AMINO ACIDS,
PROTEINS, LIPIDS AND NUCLEIC ACIDS BY MUCOSAL-SHEETS OF GPI
ID)l-g atome of C derived from glucose incorporated into
Proteine Lipide DNA-PNA Amino acide
170 73 62 977
156 66 71 933
174 78 62 943
142 55 44 998
Mean 161 68 60 962
Note: Ali incubations were done in Krebs-Ringer-phosphate buffer 5 mM in uniformly labelled glucose for 90 minutes at 37°C in an atmosphere of pure 0 2• All values given are total amounts based on 100 mg wet weight of tissue.
....... 00 -:1
to stand for 2 hours before sacrifice. Another guinea pig was injected O. 5
mg of colchicine and allowed to stand for 3 hours before sacrifice. Tissue
of mucosal-sheets were prepared, incubated and amino acids determined as
already described. The resulta obtained are reported in Table XXIV. The
following are to be noted:
There is an increase in the amounts of total free amino acids pro
duced with increasing time of standing and quantity of colchicine injected.
Most of this increase is due to an enhanced production or accumulation of
alanine.
3. Incorporation of carbon derived from labelled glucose into proteins,
lipids and nucleic acids by mucosal-sheets of GPI after subcutaneous
injection of colchicine.
188
The proteins, lipids and nucleic acids were separated and their ra
dioactivities determined as indicated under methods and materials. The re
sulta are given in Table XXV. It can be readily noticed that as the amount
of colchicine injected is increased and the time of standing is increased there
is a defi.nite decrease in the amount of protein produced. There is also some
decrease in the amount of nucleic acid produced.
TABLE XXIV
FORMATION OF LABELLED AMINO ACIDS FROM GLUCOSE BY MUCOSAL-SHEETS OF GPI,
AFTER SUBCUT ANEOUS INJECTION OF COLCHICINE AS INDICATED
mp-g atom.s of C derived from labelled glucose incorporated into
Alanine Glutamate Aspartate Serine Glutamine G lutathione Glycine Total.s
692 85 50 21 27 56 9 940 Controle: 664 83 47 34 23 38 13 902
674 79 49 24 29 44 10 909
Colchicine 836 74 46 25 31 40 13 1065 injected 815 65 35 21 31 28 13 1008 0.4 mg 924 101 58 30 26 35 11 1185
Colchicine 1070 70 37 18 23 33 8 1259 injected 1000 78 52 20 21 27 10 1208 0.5 mg 990 79 41 24 32 41 6 1213
Note: Ail incubations were done in Krebs-Ringer-phosphate buffer 5 mM in uniformly labelled glucose for 90 minutes at 37°C in an atmosphere of pure 02. Ali values given are total amounts based on 100 mg wet weight of tissue.
...... 00 co
TABLE XXV
INCORPORATION OF C DERIVED FROM LABELLED GLUCOSE INTO AMINO ACIDS,
PROTEINS1 LIPIDS, AND NUCLEIC ACIDS BY MUCOSAL-SHEETS OF GPI AFTER
SUBCUT ANEOUS INJECTION OF COLCHICINE AS INDICATED
mp-g atoms of C derived from glucose incorporated into
Proteins Lipids DNA-PNA Amino acids
145 55 146 940 Controls 130 77 .223 902
156 57 157 909
Colchicine 108 73 121 1065 injected 108 46 122 1008 0.4mg 114 57 129 1185
Colchicine 58 90 106 1259 injected 72 113 75 1208 0.5 mg 96 77 101 1213
Note: All incubations were done in Krebs-Ringer-phosphate buffer 5 mM in uniformly labelled glucose for 90 minutes at 87°C in an atmosphere of pure 0 2 . All values given are total amounts based on 100 mg wet weight of tissue.
...... 4:0 0
4. Formation of labelled amino acids from glucose by mucosal-sheets of
GPI after subcutaneous injection of increased dosage of colchicine.
In order to obtain a more pronounced effect on the levels of the
various fractions separated after injection of colchicine we increased the
dosage. One guinea pig was injected 2 mg subcutaneously and the other
4 mg. Both were allowed to stand for three hours before sacrifice. The
free amino acids were determined as usual. These resulte are given in
Table XXVI. There is a definite increase both in the total amounts of free
amino acids and in alanine. There also seems to be an increase in the
amount of glutamate formed.
5. Incorporation of carbon derived from labelled glucose into proteine,
lipids and nucleic acids by mucosal-sheets of GPI after subcutaneous
injection of increased dosage of colchicine.
The effects of larger amounts of colchicine injected into guinea pigs
191
as reflected in the protein, lipid and nucleic acid fractions are given in Table
xxvn. Ali guinea pige were allowed to stand 3 hours after injection of col
chicine. It can be readily seen that there is a sharp drop in the values ob
tained for proteine and for nucleic acide. There also appears to be an increase
in the amounts of lipid produced.
TABLE XXVI
FORMATION OF LABELLED AMINO ACIDS FROM GLUCOSE BY MUCOSAL-SHEETS OF GPI,
AFTER SUBCUT ANEOUS INJECTION OF COLCHICINE AS INDICATED
m)l-g atoms of C derived from labelled glucose incorporated into
Alanine Glutamate Aspartate Serine Glutamine Glutathione Glycine Totals
677 107 75 45 25 48 22 999 Controls 740 159 110 48 32 76 24 1189
Colchicine 770 197 115 50 27 79 22 1160 injected 740 183 112 56 29 109 13 1242 2mg 1060 168 108 42 84 72 14 1493
Colchicine 1110 185 83 46 81 80 15 1550 injected 1010 144 77 28 18 67 8 1352 4mg 1040 202 112 51 31 46 12 1494
1020 120 121 29 24 58 28 1400
Note: Ali incubations were done in Krebs-Ringer-phosphate buffer 5 mM in unüormly labelled glucose for 90 minutes at 37°C in an atmosphere of pure 0 2 . Ail values given are total amounts based on 100 mg wet weight of tissue.
,.... <0 !):)
TABLE XXVIT
INCORPORATION OF C DERIVED FROM L.ABELLED GLUCOSE INTO AMlNO ACIDS,
PROTEINS, LIPIDS, AND NUCLEIC ACIDS BY MUCOSAL-SHEETS OF GPI AFTER
SUBCUTANEOUS INJECTION OF COLCIDCINE AS INDICATED
mp-g atoms of C derived from glucose incorporated into
Proteins Lipids DNA-PNA Amino acids
Controls 295 29 136 999 248 42 122 1189
Colchicine 111 92 49 1160 injected 157 44 73 1242 2mg 38 77 28 1493
Colchicine 43 29 30 1550 injected 38 46 -31 1352 4mg 48 77 -39 1494
42 79 28 1400
Note: Ail incubations were done in Krebs-Ringer-phosphate buffer 5 mM in uniformly lahelled glucose for 90 minutes at 37°C in an atmosphere of pure 0 2• All values given are total amounts based on 100 mg wet weight of tissue.
!-'«> Cl:!
194
Discussion
In the study of the effects of colchicine on the capacity of guinea
pig mucosal-sheets to incorporate carbon from glucose into amino acids,
protein, lipids and nucleic acids, we have demonstrated that colchicine inter
feres with the metabolic pathways in the synthesis of proteins and nucleic
acids. Apparently it does not interfere, to any great extent, with the pro
duction of amino acids from glucose. That ist wh en tissue is incub ated
with colchicine the free amino acids are either at the same levels or at
higher levels than the control samples. The fact that alanine accumulates
might be indicative that the citric acid cycle is slowed down and pyruvate
is not rapidly utilized and hence alanine concentration increases. If this
reasoning were correct we would expect an increase in acetyl-Co A as weil
and this should eventually lead to increased lipid production. There is some
evidence for this, that is, lipids do seem to show some increase but the evi
dence from our present data is rather undecisive.
An approach to explain the accumulation of alanine is the possibility
that the amino acid activating enzyme specifie for alanine, and ATP supplies,
which are necessary for the carboxyl activation of alanine and its conversion
to an amino-acyl-adenylate are interfered with by the action of colchicine
thus preventing the incorporation of alanine into proteins and hence the re
sulting increase.
195
Another approach and perhaps one better supported by our data
might be that colchicine is interfering in sorne special way with either the
formation of sRNA specifie for alanine incorporation into proteine or inac
tivating it by destroying or removing the 2' or 31-hydroxyl group from AMP,
the terminal adenosine residue of sRNA, involved in the binding of the acti
vated amino acid.
This possibility can be tested if radioactive amino-acyl-adenylates
are brought together with sRNA in the presence of a purified activating en
zyme and the effect of colchicine on such a system observed. It is quite
possible that the inhibitory action of colchicine consista of affecting this
very binding-step of activated alanine to the terminal AMP of 8RNA.
The fact that our data indicate a very sharp decrease in the nucleic
acid fraction with a concomitant decrease in the protein fraction certainly
supports the argument that the accumulation of alanine may well be due to
the decrease or lack of the specifie 8RNA which must be present in order
to bind the activated alanine. The attachment of activated amino acids to
8 RNA is an intermediary step in protein synthesis and it has been shown
that when radioactive amino acids are injected into an animal they label the
8RNA earlier than any other fraction of RNA or protein. The sRNA bound
to the amino acid then transfers the amino acid to microsomal particulate
RNA and not directly to protein. However it has also been shown that
sRNA contains a natural sequence of other amino acids ail attached to the
196
terminal adenine and the transfer to the microsomes occurs simultaneously
as a group. These transfera require the mediation of a number of soluble
enzymes, ATP, GTP and vitamin B12. It would suffice to add at this point
that colchicine inhibition could be involved in any one or all of these steps.
The medical use of colchicine in the treatment of gout has shown
that prolonged use of the drug produces aplastic anemia and agranulocytosis
resulting in ulcerati ve lesions of the gastrointestinal tract and other mucous
membranes and it is not unlikely, therefore, that these changes observed are
no more than manifestations of disturbance of protein and nucleic acid syn
theses or relationships for wbich colchicine is responsible by its ability to
interfere with a key step in the sequence of synthe ses of these compounds.
It would be interesting indeed to carry on further research work with colchi
cine in order to identify its focal point of action and associate it with a par
ticular or specifie step in protein or nucleic acid synthesis.
To summarize, then, we can state that first of all we have been
able to show that mucosal-sheets of guinea pig intestine are capable of me
tabolizing glucose and incorporating the carbon atoms of this sugar into the
amino acids, proteins, lipids and nucleic acids of this tissue. Secondly, we
have demonstrated that the drug colchicine appears to have a specifie inhi
bitory action and interferes with or prevents the formation of proteins and
nucleic acids.
197
Summary
1. The incorporation of carbon from uniformly labelled glucose into amino
acids, proteins, lipids and nucleic acids has been studied.
2. We have been able to show that there is an increase in the total amounts
of free amino acids produced and in alanine and glutamate when colchicine is
injected subcutaneously into guinea pigs.
3. There is a pronounced drop in the production of proteins when colchicine
is injected into guinea pigs in the range of 2-4 mg.
4. There is an equally sharp drop in the production of nucleic acids after
2-4 mg colchicine injections.
5. There is also some increase in the lipid fraction with higher amounts of
colchicine injected.
CLAIMS TO ORIGINAL RESEARCH
1. The intact-strip method (ISM) was modified for use with the guinea
pig intestine (GPI) and active uptake of glucose studied.
198
2. When 5 mM glucose is used in the medium the average value of intact
-strip tissue molarity for GPI is approximately 27.3 mM.
3. The variation in tissue glucose concentration with time of incubation
appears to follow Michaelis-Menton kinetics. A steady-state condition
is reached within 90 minutes of incubation.
4. Accumulation of tissue glucose with varying medium glucose concen
trations has been studied. The curve obtained indicates that a satu
ration level is attained in the tissue in which efflux begins to equal
the influx of glucose due to active transport.
5. The area of maximum rate of absorption in the guinea pig jejunum
has been shown to be the 15 inch segment between 15 and 30 inches
from the pyloric end.
6. Inhibition of active glucose uptake by 5 x 10-5 M phlorhizin at varying
medium glucose concentrations was studied. It has been clearly shown
that under these conditions phlorhizin is a competitive inhibitor.
7. Inhibition of glucose uptake by phloretin with concentrations ranging
from 4 x 10-5 M to 2 x 10-4 M at varying medium glucose concentrations
has been studied by the IBM. Inhibition by phloretin was found to be
definitely of the competitive type.
8. The inverse proportionality between inhibitor concentration (phloretin)
and rate of active glucose uptake or tissue glucose concentration has
been shown.
9. From the data obtained by the ISM,. the ~Sn value for glucose uptake
and the Ki values for phlorhizin and phloretin have been calculated.
199
The Km value for glucose is 3. 84 x lo-3 :M; the Ki value for phlor
hizin is 1. 00 x lo-5 :M; and the Ki value for phloretin is 4. 95 x lo-5 M.
It is clear from these data that phlorhizin is approximately 5 times
more effective in its inhibitory action than an equal concentration of
phloretin.
10. A new perfusion apparatus suitable for in vitro studies with radioactive
substances has been designed and constructed. With the aid of this in
strument using the isolated surviving GPI we have shown the active
transport of isotopically labelled glucose against a concentration gra
dient.
11. The IS:M has been used to study the metabolism of carbohydrates with
three different types of tissue preparations from GPI. A procedure for
the separation and identification of amino acids synthesized by the gut
wall has been initiated.
12. Glucose, fructose and sucrose are metabolized by GPI preparations
13.
and yield the amino acids alanine, glutamate. aspartate, serine, gly-
cine and proline, and glutamine and glutathione.
The Qo2
and Qc02
values for mucosal-sheets, serosal-sheets and
intact-strips of GPI have been determined. Mucosal-sheets have a
high Q 0 value (12. 33). However the rate of oxygen consumed is 2
about the same for the oxidation of glucose in all types of tissue
preparations.
14. Normal GPI tissue has been analyzed for its amino acid content.
15. A curve showing the rate of total amino acid formation from glucose
with increasing tlme has been constructed.
16. The effect of change of medium concentration of glucose on the rate
of amino acid formation has been determined.
17. A comparative study showing the variation in the rates of amino ac id
formation by three types of GPI tissue, namely, mucosal-sheets,
200
serosal-sheets and intact-strips has been made. The rate of formation
of amino acids is much higher with mucosal-sheets.
18. It has been shown that in the GPI prolonged starvation does not lead
to increased rates of amino acid formation but rather to decreased
rates, possibly because of depletion of important enzymes or cofactors
needed.
19. The interconversion of the amino acids glutamic, aspartic and glycine
has been studied. Glycine is not readily converted to other amino
acids by the gut wall, however, it does form some serineJ gluta
mine and aspartate. Glycine is al.so incorporated into glutathione.
Glutamate is converted into ali the other amino acids formed by the
gut except glycine. Aspartate is converted essential.ly into glutamate
and alanine.
20. That acetate and formate take part in amino acid synthe sis by the
gut wall has been shown. Acetate is converted into al.l of the amino
acids except serine and glycine. Formate essentially goes to serine.
201
21. The action of a number of metabolic and respiratory inhibitors on the
formation of amino acids by the gut has been studied. Amytal. increases
alanine production but decreases glutamate, aspartate and glutathione
production. Phlorhizin at 10-3 M concentration is the most effective
inhibitor among those studied. Salicylate and DNP show inhibition of
alanine, glutamate and glutamine formation.
22. The stimulatory effect of 15 mM pbtassium on amino acid formation
by mucosal-sheets and intact-strips of GPI has been shown.
23. Glutamine and asparagine show significant stimulatory effects on amino
acid formation from glucose. Ammonium chloride seems to stimulate
only the formation of alanine.
24. Glutathione production is enhanced when glutamate, glycine and cysteine
are made available to the gut wall. It has been shown that the GPI is
capable of synthesizing glutathione.
25. The presence of glutathione and proline as a result of synthesis from
carbohydrate by GPI tissue preparations has been confirmed by a
number of tests in addition to synthesist and paper chromatography
with two different sets of solvants.
26. The incorporation of glucose carbon into free amino acids. proteinst
lipids and nucleic acids by mu cos al -sheets of GPI has be en shown.
27. The inhibitory action of colchicine on the incorporation of carbon
derived from glucose into free amino acids, proteins and nucleic
acids by mucosal -sheets of GPI, after the subcutaneous injection of
varying amounts of colchicine, has been demonstrated.
202
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
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