1
STUDIES OF THE PURINE BIOSYNTHETIC
PATHWAYS IN THE GASTROINTESTINAL TRACT
by
Neal Simon LeLeiko
B.S.,
Brooklyn College of the
City University of New York
(1967)
M.D.,
New York Medical College
(1971)
SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE
DEGREE OF
DOCTOR OF PHILOSOPHY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
(June, 1979)
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Signature of Author
Department of Nutrition and Food Science,
- - - -- - - - -
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Certified by
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/11
Accepted by ..
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ARCHIVES
MASSACHUSE
S
OF TECHNOLOGY
OCT 1. 8 1979
LIBRARIES
V0...
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hesis Supervisor
a
2
STUDIES OF THE PURINE BIOSYNTHETIC
PATHWAYS IN THE GASTROINTESTINAL TRACT
by
Neal Simon LeLeiko
Submitted to the Department of Nutrition and Food Science
on June 29, 1979 in partial fulfillment of the requirements for
the Degree of Doctor of Philosophy.
ABSTRACT
A series of experiments was performed on rats in order to
determine the origin of dietary purines (nucleic acids, nucleosides
or bases) for the metabolism of the mucosal cells of the gastrointestinal tract.
The relative importance of the synthesis of
purine nucleotides by the de novo and by the salvage pathways was
assessed both by incorporation of isotopic precursors in each
pathway (glycine for de novo synthesis, adenine for salvage), and
also by measuring the activities of the rate-limiting enzymes in
each pathway (phosphoribosyl amidotransferase for the de novo
pathway, hypoxanthine-guanine phosphoribosyl transferase for the
salvage route).
De novo purine synthesis by small intestinal mucosal cells
incubated in vitro with 14 C-glycine was shown to be very slight
in contrast to the very active de novo pathway demonstrable in
colonic mucosal cells.
In contrast a salvage pathway in the mucosa
of the small intestine was demonstrated in vivo by oral administration of
3 H-adenine
and especially
3 H-adenosine,
which
3
resulted in restoration of some in vitro labeling of mucosal
RNA adenine from
4C-glycine by the de novo route.
The small intestine was found to contain significantly
less amidotransferase activity (first enzyme of the de novo
pathway) than either the colon or the liver.
This was observed
whether the animals were on a purine-free diet or on a purinecontaining diet.
Measurements of the hypoxanthine-guanine
phosphoribosyl transferase of the salvage pathway demonstrated
considerable activity in the mucosal cells of the small intestine.
Significantly more HGPRT activity was found in the
small intestine than in either the liver or colon when the
animals were receiving a diet containing purines.
The data thus demonstrates that the diet is a major
source of purines for the small intestine, whereas the de novo
pathway is not a major source of purine bases on diets containing purine bases as such or as nucleic acids.
However,
a modest increase in the de novo pathway (without enzyme
induction) was achieved by eliminating dietary sources of
purines.
This allowed the existing low activity of amido-
transferase to act more efficiently, probably through having
more substrate (PRPP) because less is diverted to salvage.
An additional mechanism could be that, on a purine-containing
diet, more free nucleotides may accumulate and result in
allosteric inhibition of the enzyme.
The significance of
these findings is discussed in relation to the participation
of dietary purines in the maintenance and repair of the intestinal mucosa.
Hamish N. Munro, M.B., D.Sc.
Professor of Physiological Chemistry
(Thesis Supervisor)
4
Acknowledgments
I wish to thank Dr. Hamish Munro for granting me the
privilege of working in his laboratory.
His patience and
interest made this work possible.
I wish to thank Dr. Richard Grand, Dr. Paul Newberne,
Dr. George Wolf and Dr. Vernon Young for their advice and
encouragement.
I wish to especially express my appreciation to Dr.
Richard Hamilton of Toronto for his crucial early assistance,
and to Dr. Richard Grand of Boston, not only for his contin uous advice and encouragement, but also for the tremendous
fairness and decency that he has shown in our relationship.
I wish to thank my many new friends who helped lighten
my burdens.
They provided many hours of stimulating debate,
heartening camaraderie and personal friendship.
I could ask
nothing more of Andy Bronstein, David P. Katz, Samuel AdenyiiJones and Kathleen Motil.
The lessons taught to me by Surendra Baliga will remain
with me throughout my life.
When I think of those whose
help and friendship were always available I will always think
most warmly of Suren.
I would be remiss if I did not acknowledge the financial
support supplied initially by the National Institutes of Health
for a joint nutrition training program at the Children's
Hospital Medical Center and M.I.T., and for the last two years
by the Nutrition Support Service of the Children's Hospital
Medical Center.
5
Finally, but certainly most importantly, I want to thank
my entire family, especially my wife Marilyn.
I wish that I
could adequately express how I feel about the help and understanding that she has provided.
I cannot do that, however,
and if I could she would probably edit it out of the text.
6
TABLE OF CONTENTS
TPage
Title Page ...............................................
1
Abstract................................................. 2
4
Acknowledgments .,........................................
Table of Contents .. ...................................... 6
List of Figures ......
................................... 8
List of Tables ...... ................................... 13
I.
II.
Introduction:
The Nature of the Problem ........ 17
Survey of Literature . ........................... 24
A. The effect of nutrition on metabolism in
24
the intestinal mucosa ...................
1 - Metabolism in the intestinal mucosa
and enteral nutrition -
............. .24
2 - The effect of parenteral nutrition
upon the intestinal mucosa .... -
3 - A trophic effect of pancreatic
... 26
secretions upon the intestinal
mucosa-----.-..
-..--...--.
B.
C.
D.
E.
F.
G.
III.
IV.
Purine metabolism and diet .......... ....
Intestinal RNA metabolism .............. Adenine metabolism ............
.........Glutamine Amidophosphoribosyl transferase
and de novo synthesis -- -o Hypoxanthine-guanine, phosphoribosyl
transferase; purine salvage; and LeschNyhan syndrome ..........................
Intraperitoneal parenteral alimentation...
30
32
38
40
42
45
47
Plan of Research ... .............................. 52
Materials and Methods ... .....
A.
B.
C.
D.
E.
.................
In vitro studies of the de novo and
salvage pathways ........................
The fate of absorbed versus injected
adenine and adenosine
..................
The influence of protein and purine content of the diet
............. ..........
The measurement of amidotransferase
activity in the small intestine, colon
and liver ........... ....................
Additional studies related to or influencing intestinal purine metabolism
55
55
58
64
6
69
7
Page
V.
Results .. ........................................... 75
A.
B.
C.
D.
E.
VI.
In vitro studies of the de novo and salvage
pathways .................................. 75
The fate of oral versus parenteral adenine
and adenosine .. ............................. 88
Influence of-the protein afid purin-e
content of the diet........................
The activity of the glutamine amidophosphoribosyl transferase in the small intestine,
colon and liver and the effect of dietary
130
purines ...... .............................
The activity of the hypoxanthine-guanine
phosphoribosyl transferase in the small
intestine, colon and liver and the effect
of dietary purines ........................
Results of Preliminary Studies ................... 169
A.
B.
C.
The effect of a self-emptying blind loop
of intestine on the activity of the de novo
pathway
................................... 169
Studies of the different intestinal cell
populations . ............................... 169
The effect of catheter placement and normal
saline infusion ............................ 171
VII.
Discussion and Conclusions ....................... 192
VIII.
Suggestions for Future Research ................... 202
References ...........--................................... 203
Biographical Note
.........................
214
8
LIST OF FIGURES
Figure No.
1
2
3
4
5
6
7
8
9
10
11
12
Title
Page
A general representation of the major
synthetic and catabolic pathways of
purine metabolism in mammalian cells...
18
The de novo biosynthetic pathway for
purine nucleotides......................
19
The sources of the atoms in the purine
ring......................................
20
The role of PRPP in purine nucleotide
biosynthesis,...........................
21
The incorporation of 4C-Glycine into
the RNA of small intestinal mucosa
(everted loops) and into liver (finely diced ).............................
77
The incorporation of 1C-Glycine into
small and large intestinal mucosal
protein (everted loops) after pancreatic duct ligation or sham surgery.....
80
The incorporation of 3 H-adenine into
mucosal RNA of everted loops of small
and large intestine with pancreatic
duct ligation and sham surgery........
83
The incorporation of 1C-Glycine into
the RNA in mucosal scrapings of small
and large intestines....................
85
The incorporation of 1 4 C-Glycine into
mucosal protein of small and large
intestinal mucosal scrapings..........
87
The design of the experiment to determine the fate of absorbed versus injected adenine and adenosine................
90
The incorporation of injected 3 H-adenine into the gastrointestinal tract
of animals on a regular chow diet.....
91
The incorporation of injected 3 H-adenosine into the gastrointestinal tract of
animals on a regular chow diet........
94
9
Figure No.
13
14
15
16
17
18
19
20
21
22
23
24
25
Title
Page
The incorporation of injected 3 H-adenine
into the gastrointestinal tract o'fanimals on a regular chow diet.........
96
The incorporation of injected 3 H-adenosine into the gastrointestinal tract
of animals on a regular chow diet......
97
The incorporation of gavaged 3 H-adenine
into the gastrointestinal tract of animals on a 0% protein diet................
99
The incorporation of gavaged 3 H-adenosine into the gastrointestinal tract of
animals on a 0% protein diet...........
100
The incorporation of injected 3 H-adenosine into the gastrointestinal tract of
animals on a 0% protein diet...........
102
The incorporation of injected 3H adenine
into the gastrointestinal tract of animals on a 0% protein diet..............
104
into
The incorporation of 3 H-adenine
the mucosal RNA of the stomach.........
105
The incorporation of 3 H-adenosine into
the mucosal RNA of the stomach.........
106
The incorporation of 3 H-adenine into
the mucosal RNA of the proximal small
intestine..............................
108
The incorporation of 3 H-adenosine into
the mucosal RNA of the proximal small
intestine................................
109
The incorporation of 3H-adenine into
the mucosal RNA of the middle small
...
intestine...............................
110
The incorporation of 3H-adenosine into
the mucosal RNA of the middle small
intestine,..............................
111
The incorporation of 3 H-adenine into
the mucosal RNA of the distal small
intestine.,.............................
112
10
Figure No.
26
Title
The incorporation of 3H-adenosine inr
to the ucosal RNA of the distal small
intestine.,,,
27
28
29
30
31
32
33
34
35
36
Page
9,,,.
113
9.,...,........,.,
The incorporation of 3H-adenine into
the mucosal RNA of the caecum...........
114
The incorporation of 3H-adenosine into
the mucosal RNA of the caecumn...........
The incorporation of 3H-adenine into
115
the mucosal RNA of the proximal colon..
116
The incorporation of 3H-adenosine into
the mucosal RNA of the proximal colon..
117
The incorporation of 3 H-adenine.into the
mucosal RNA of the distal colon........
118
The incorporation of 3H-adenosine into
the mucosal RNA of the distal colon....
119
The incorporation of 3H-adenine into
the mucosal RNA of the liver.............
120
The incorporation of 3H-adenosine into the mucosal RNA of the liver........
121
The correlation of the amount of glutamate formed per mole glutamine used as
substrate at different PRPP concentrations in the liver.......................
133
The correlation of the amount of glutamate formed as a function of PRPP concentrations in the reaction at different glutamine concentrations in the
liver...,.,..,...........--....-.-.-135
37
38
The correlation of the amount of glutamate formed per mole glutamine used as
substrate at a PRPP concentration of
.3mM in the liver......................
136
The correlation of the amount of glutamate formed as a function of PRPP concentration in the reaction at a glutamine concentration of .044mM in the
live ...,,,
....
,,..
...
....
...
.. ,
137
11
Figure No,
39
Title
Page
The amount of glutamate formed as a
fuction of the PRPP concentration and
glutamine concentration in the reaction mixture with liver amidotrans-
ferase,.,.,,.....................139
40
41
42
43
44
45
46
47
48
The correlation of the amount of
glutamate formed per mole glutamine
used as substrate at different PRPP
concentrations in the small intestine..
141
The correclation of the amount of
glutamate formed as a function of
the PRPP concentration in the reaction
at different glutamine concentrations
in the small intestine.................
143
The correlation of the amount of glutamate formed per mole glutamine used as
substrate at a PRPP concentration of
0.3mM'in'the small'intestine...........
144
The amount of glutamate formed as a
function of the PRPP concentration and
glutamine concentration in the reaction mixture with small intestinal
amidotransferase....................... ..
146
The amidotransferase activity (total
activity) in crude tissue extracts
from liver and small intestine.........
148
The amidotransferase activity (PRPP
dependent) in different tissues........
150
The amidotransferase activity in different tissues as a function of the
purine content of the diet.............
154
The liver HGPRT activity (using a crude
tissue extract) determined by spotting
10 lambda, 20 lambda or 40 lambda of
reaction-enzyme mixture on DEAE Cellulose Discs..,..........................
156
The liver HGPRT activity...............
158
12
Figure No.
49
50
51
52
53
54
55
56
57
58
59
Title
Page
The HQPRT reaction in different tissues using five lambda of enzyme source..
161
The HGPRT reaction in different tissues
using ten lambda of enzyme source.........
162
The HGPRT reaction in different tissue
using twenty lambda of enzyme source.....
163
The effect of dietary purines on HGPRT
activity in different tissues............
167
The incorporation of 1 4 C-glycine into
the protein and the nucleic acids in
different intestinal cell populations
(expressed as counts/min/mg protein.)....
172
The incorporation of 1C-glycine into
the protein and the nucleic acids in
different intestinal cell populations
(expressed as counts/mm/mg DNA)..........
174
3
The incorporation of H-adenine and
C-glycine into the RNA of different
intestinal cell populations.............
176
The average weight loss per day in animals receiving either intraperitoneal
amino acids or normal saline............
183
The average weight loss per day (expressed as percentage of initial weight) in
animals receiving either intraperitoneal
amino acids or normal saline............
184
The serum albumin of animals receiving
either intraperitoneal amino acids or
normal saline..............................
185
The liver (wet) weight of animals receiving either intraperitoneal amino
acids or normal saline....................
186
13
LIST QT TABLES
Table No.
1
Title
Page
The 0% protein and 0% purine diet for
the study of the fate of absorbed
versus in)ected adenine and adenosine..
60
2
The composition of Roger and Harpers
salt mixture.,.........................
61
3
The composition of vitamin mix employed in experimental diets...............
62
4
The composition of diets for diet
studies,,., ..
5
6
7
8
9
The incorporation of 1 4 C-Glycine into
the RNA of liver (finely diced) and
small intestine (everted loops)......
76
The incorporation of 1 4 C-Glycine into
small and large intestinal mucosal
protein (everted loops) after pancreatic duct ligation or sham surgery......
79
Incorporation of 1 4 C-glycine into small
and large intestinal mucosal RNA (everted loops) after pancreatic duct ligation
or sham surgery..........................
The incorporation of 3 H-adenine into
small and large intestinal mucosal RNA
(everted loops) after pancreatic duct
ligation or sham surgery................
81
82
The incorporation of 1C-glycine into
small and large intestinal mucosal RNA
(mucosal scrapings)..........
10
65
,.......................
..
......
The incorporation of 1 4 C-glycine into
mucosal proteins of the small and large
intestines(mucosal scrapings)..........
84
86
11
The fate of absorbed ve sus injected
adenine and adenosine ( H-adenine incorporation cpm/mg RNA)...................92
12
The fate of absorbed velsus injected
adenine and adenosine ( H-adenosine
incorporation cpm/mg RNA)...............
95
14
Table No.
13
Title
Page
The influence of protjin and purine
C-glycine incontent of diet upon
corporation into small intestinal
mucosal protein and mucosal RNA.....
125
14
The influence of protTin and purine
C-glycine incontent of diet upon
corporation into small intestinal
mucosal protein and mucosal RNA during refeeding.....................-.........127
15
The influence of protgin and purine
content of diet upon H-adenine incorporation into colonic mucosal RNA.
16
17
18
19
20
21
22
The influence of protgin and purine
content of diet upon H-adenine incorporation into colonic mucosal RNA
during refeeding..................- - The correlation
mate formed per
as substrate at
trations in the
of amount of glutamole glutamine used
different PRPP concenliver.................
128
.
129
132
The correlation of amount of glutamate
formed as a function of PRPP concentration in reaction at different glutamine
concentrations in the liver............
134
The glutamate formed as a function of
PRPP concentration and glutamine concentration in reaction mixture with
liver amidotransferase...................
138
The correlation of amount of glutamate
formed per mole glutamine used as substrate at different PRPP concentrations
in the small intestine...................
140
The correlation of amount of glutamate
formed as a function of PRPP concentration in reaction at different
glutamine concentrations in the small
intestine................................
142
The glutamate formed as a function of
PRPP concentration and glutamine concentration in reaction mixture with
small intestinal amidotransferase......
145
15
Table No.
Title
Page
PRPP amidotransferase activity (total
activity) in crude tissue extracts.....*
149
PRPP amidotransferase activity (PRPP
dependent) in different tissues......
151
Amidotransferase activity in different tissues as a function of the
purine content of the diet...........
153
The liver HGPRT activity (using crude
enzyme preparation) and spottinglO pil,
20pIl, or 40 jil of reaction-enzyme 'mixture on DEAE cellulose discs..........
157
27
The Liver HGPRT activity...............
159
28
The effect of concentration of HGPRT
enzyme source on enzyme reaction in
different tissues.........................
164
HGPRT activity as a function of the
purine content of the diet in different tissues...............................
166
The ratio of HGPRT activity/amidotransferase activity in different tissue
as a function of the purine content
of the diet...............................
168
The incorporation of 1 4 C-glycine into mucosal RNA-adenine and into mucosal protein in a self-emptying blind
loop of small intestine.............
170
The incorporation of 1 4 C-glycine into
the protein and nucleic acids in different intestinal cell population-5expressed as count/min/mg protein).............
173
23
24
25
26
29
30
31
32
33
34
The incorporation of 1C-glycine into
the protein and nucleic acids in different intestinal cell populations
(expressed as counts/min/mg DNA).......
14.
C-glycine into
The incorporation of
the RNA of different intestinal mucosal cell populations.....................
175
177
16
Table No,
35
'Title
The incorporation of 3Headenine into the RNA of different intestinal
mucosal cell populations.............
36
37
38
39
40
Page
178
The effect of intraperitoneal catheter placement and normal saline infusion (5.5 ml BI.D.)...................
18,0
The effect of intraperitoneal catheter placement and normal saline
infusion (19 ml B,ID.)................
181
The effect of intraperitoneal amino
acid infusions versus intraperitoneal
glucose infusions......................
188
The effect of control diets upon intraperitoneal study control animals.......
189
The incorporation of 1 4 C-glycine into
small intestine mucosal RNA and protein in parenterally alimented, and
control fed animals.....................
190
17
I. INTRODUCTION: THE NATURE OF THE PROBLEM
Adenine and guanine, the purine bases, are utilized in
the synthesis of nucleotides, nucleic acids, nucleoproteins
and co-enzymes.
The purine nucleotides can be synthesized
in most tissues by two routes:
cursors;
(1) de novo synthesis from pre-
(2) re-utilization of the free purine bases liber-
ated through catabolism of nucleic acids via the so-called
"salvage" pathway.
The identification of the individual enzymes
and metabolites of these pathways has provided a picture of the
major synthetic and catabolic pathways of the purine nucleotides
in mammalian cells (figure 1).
The recycling or "salvage" of
the purine bases is catalyzed by the enzymes hypoxanthine-guanine
phosphoribosyltransferase (HGPRT)(E.C.2.4.2.8 ) and by adenine
phosphoribosyltransferase (APRT)(E.C.2.4.2.7 ).
The de novo
formation of the purine ring is the result of a pathway (figure 2)
involving glutamine, aspartic acid, glycine,formate
and CO2,
while figure 3 concisely illustrates the final location of these
precursors in the purine ring.
The first committed step requires
the rate-controlling enzyme glutamine amidophosphoribosyltransferase
(amidotransferase)(E.C.2.4.2.14).
The competition between the salvage and de novo pathways
for phosphoribosyl pyrophosphate (PRPP) (figure 4) represents a
regulatory element in purine metabolism within the cell. APRT
has the highest affinity for PRPP, HGPRT has the next highest,
and the amidotransferase has the lowest (Wood and Seegmiller,
1973).
Amidotransfexase as. well as PRPP appear to regulate
ATP
PRPP
ADP
i
t
5 nucleotidose
ADENINE
ADENOSINE
AMP .0
adenosine
uodeny I
succ inate
deammnase
GTP
glutamine
PR PP
PRPP
synthetase
Ribose 5-P s
s
I
omido
PRPP
tronsferase
s
glutommne
ATP
uspartote
o
PRA
+
HYPOXANTHINE
INOSINE
IMP
gI ycne
nHGPRT
x
ilthine
I
XMP
oxicdose
PR PP
XANTHINE -4URIC
No XANTHOSINE-
ACID
GMP
GUANOSINE
-
GUANINE
HGPRT
GDP
PRPP
GTP
A
GENERAL REPRESENTATION OF THE MAJOR SYNTHETIC AND CATABOLIC
PATHWAYS OF PURINE METABOLISM IN MAMMALIAN CELLS
Figure 1
lw
(P)-0-CH,
5
HC
s5
(P)-o-CHI
H
NH 3
5
OH OH
H HCs
N
(P)-O-C
"
Ribose
NHH
5
o~gc
AMP
ATP
0
PiO-CHg
SYMITU"TA,7
5-P
Am I
s
iH
(3)
H OH
OH ON
PPriboseIP
5-1Phosphuoribosylaiminc
ATP
HM\
ADF
HC
4
Mg
HA
P,
H/
["
a u L
0
OH OH
puo. 5-P
Formylglyrinamide
ribnsyl-S-P
Glycinanide
ribosyl-5.-P
A
-00c
I
t
0 *.
A]
l
ATP
Not+
+
Aspartale HC-NH*
CH
-OC
-oOC0
N
0
SN
-O
-
H
-00C
-OOC
COHO's
v
0
C
N
C
0
H
CH
CN CN
H
I--,
0Avo ra
fmt
-CH'
CH
n
H1 0
N
RISOSE
H3N
5-P
rihosyl-5-P
N
RISOSE
P
carbhxylale ribosyI-5-
I
mm4 CLO* M
S-P
Aminsimidazole
ribosyl-S-P
Aminoiimsdaole
RM301K U-P
Formylglycinamidine
ribosyl-S-P
0
700
0o1r
4
11
H
\0)
T01-s1Oi1LAst
ATP. Mg +
H3 N
Uccinyl
I
O=
C
,
N
N
N
HNH
RIBOSE
5-P
-N
Ht,
C
--------
aH
AlSOSE
Aminmtiiid1d,
minoimidazic
carbnxamde
ocaas1
09
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REOSE 5-P
A
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11
(7)
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tle carbsxamide
()
'IIC
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C
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RMIE
riblliyl-5-P
I 5-P
IMP
THE DE
Novo
BIOSYNTHETIC PATHWAY
FOR PURINE NUCLEOTIDES
Figure 2
20
cO
:
C
ASPARTIC
GLYCINE
/
N
FORMATE
ACDN
FORMATE
' -N
/
NN:N
/
/GLUTAMINE
/I
I
GLUTAMINE
THE SOURCES OF THE ATOMS IN
THE PURINE RING
Figure 3
\,
21
RNA BREAKDOWN
G LY C INE
DE NOVO
<
feedback
PR pP
AMI DO .---------TRANS FERASE
AMP
IMP
GMP
PRPP
po ols
APRT
GLUTA MINE
ADENINE
HGPRT
GUANINE
HYPOXANTHINE
THE ROLE OF
PRPP
IN PURINE
[NUCLEOTIDE BIOSYNTHESIS
Figure 4
s a Iv ag e
22
the de novo pathway within the cell.
In some systems high levels
of adenosine monophosphate (AMP) and guanosine monophosphate
(GMP) tend to shut down the de novo pathway via allosteric
inhibition (Holmes et al.,1973). The apparent result of these
various interactions is the preferential use of PRPP for the
recycling or "salvage" of purine bases over the de novo synthetic
pathway.
The de novo pathway uses six high energy phosphate
molecules to produce either AMP or GMP whereas the salvage
pathway requires only one.
Thus intracellular conservation
of energy favors salvage over de novo synthesis.
The ability of various tissues to utilize each of the
synthetic pathways has not been systematically studied
A few studies or, isolated tissues suggested limited capacity
for the de novo synthesis of purines in cells of the bone
marrow (Lajtha and Vane, 1958), leukocytes (Williams,1962),
and erythrocytes
(Scott,1962; Smellie et al.,1958). Reem (1977)
reported, however, that circulating leukocytes are capable of
de novo synthesis.
McKeran and Watt (1976) recently suggested
that peripheral human granulocytes are not capable of purine
synthesis de novo. Brosh et al,(1976) found that mixed mature
peripheral human leukocytes synthesize nucleotides de novo,
It is interesting to note that they also reported a ten-fold
increase in the rate of synthesis in patients with Lesch.-Nyhan
Syndrome (HGPRT deficiency),
MacKinnon and Deller C1973) have
presented evidence to suggest that mucosa of the small intestine lacks the capacity to synthesize AMP and GMP via the
de novo pathway.
If the small
intestine is incapable of pro-
23
viding purine bases via the de novo synthetic pathway, then
the source of the preformed purines becomes a significant question.
The salvage pathway could use either exogenous purines provided
*as a result of digestion or endogenous purines provided by the
bloodstream.
If the dietary source is a significant or nec-
essary source of purines for the mucosal cell, then there would
be a role for dietary purines in the nutrition of this cell
population.
This concept can be extended to the question of
whether a change in the amounts of purine available from the
diet affects mucosal function in health and disease,
Intriguing
questions such as the relationship of dietary purines to mucosal
cell turnover and the role of purine lack in the mucosal atrophy
of parenteral nutrition are practical outcomes of the exploration of this problem.
Accordingly, before defining the experi-
mental protocols used, it is necessary to review the current
understanding of the response of the small intestinal mucosa
to dietary change, and also the details of purine metabolism.
24
II. SURVEY OF LITERATURE
A. The Effect of Nutrition on Metabolism in the Intestinal Mucosa
1. Metabolism in the Intestinal Mucosa and Enteral Nutrition
The concept that diet affects intestinal mucosal metabolism
is not new. The full extent of the dietary effect is, however,
unknown.
The effects of starvation and protein deprivation upon
the small intestine have been studied by many investigators.
Both have been found to decrease cell turnover, but their effect
upon intestinal cell size is not the same.
Protein deprivation
does not affect intestinal cell size, while starvation results
in decreased cell size.
purine-deficient as well.
Protein-deficient diets are usually
The role of purines in diet studies
of intestinal metabolism has been ignored, and therefore no
specific metabolic effect has been attributed to them.
Ju and Nasset (1959) demonstrated that diet could be a
major factor in the regulation of intestinal mucosal structure and cell metabolism.
They subjected rats to eight days of
fasting and found that the small intestine, pancreas, and
liver lost almost 50% of their protein content.
The stomach
lost only 10% of its protein content, Upon re-feeding, the
intestinal mucosa was rapidly restored.
Hooper and Blair
(1958) demonstrated that starvation decreases intestinal cell
division.
Munro and Goldberg (1964) found that a protein-
deficient diet also resulted in decreased cellular division.
Similar findings were reported by Durand et al. (1965) when
25
they fed rats a protein of low biological quality.
Hopper
et al.(1968) found that protein deficiency if allowed to
continue resulted in increased transit time up the intestinal
villi and Platt et al. (1964) observed that eventually
protein restriction led to a flattening of the mucosal surface.
Although Munro and Goldberg (1964) and Hill et al.(1968)
found that mucosal cell size is maintained in animals fed a
protein-deficient diet, Thaysen and Thaysen (1944) as well
as Steiner et al.(1968) have shown that starvation tends to
reduce the size of the average mucosal cell.
The rapidity with
which the small intestine responds to starvation has been
emphasized in the studies of McMannus and Isselbacher (1970).
They found that that the small intestines of a group of rats
that were fasted overnight contained 13% less mucosal
DNA than did fed controls.
They described a decreased number
of mucosal cells, decreased size of individual cells, and a
decrease in proliferative activity.
They suggested that their
observations were the combined result of an inhibitory effect
of the fast and a stimulatory effect of feeding.
At the cellular level Hagemann and Stragand (1977) found
during a two-to-three day fast the small intestinal crypt
proliferative compartment size and total cellularity declined
significantly.
This was accomplished by an increase in total
cycle time as a result of the lengthening of the G1 and S phases.
Following re-feeding there was a reduction in the cycle time and
a gradual return to the control values for the proliferative
compartment size and cellularity.
During a three-day fast
26
the small intestine lost about 40% of its cells.
In the colon, Hagemann and Stragand (1977) found that
fasting had no effect on the proliferative compartment size
or total crypt cellularity.
There was an approximate doubling
of the cycle time, however, due to an increase in G .
When the fast ended, and a normal diet was begun, there was a
hyperplastic burst of DNA synthesis in colonic crypts due to
the entry of the large number of cells blocked in Gi into the
S phase.
Peak levels of S cellularity exceeded four times
the fasting and two times the normal-fed control value.
It
is of interest to note that when the authors re-fed a "lowresidue" diet consisting of soluble casein, glucose, and corn
oil, there was a return to control values but no hyperplasia.
In the small intestine re-feeding was followed by return to
near control values with only a slight overshoot.
Thus it is clear that both starvation and protein deprivation
adversely affect cell turnover and that protein deprivation
affects cell size.
There is, however, no convincing data
on any effect of dietary purines on the intestine.
In the
studies performed, endogenous purines available from shed
mucosal cells would be expected to negate any effect of
dietary purine deprivation. (Furthermore the actual purine
content of the diets utilized in all studies above is unknown.)
2. The Effect of Parenteral Nutrition Upon the Intestinal Mucosa
Johnson et al.(1975) reported that parenterally-fed rats
underwent significant losses in small intestinal and pancreatic
27
mass.
In the small intestine there was a significant diminut-
ion of both RNA and DNA although the RNA:DNA ratio was nearly
double in the parenterally-fed group.
The authors attribute
much of the effect to "hormonal factors".
Eastwood (1977)
studied small bowel morphology and epithelial cell proliferation
in intravenously-fed rabbits,
He found that proximal small
intestinal mucosa was thinner in the parenterally-fed animals.
In addition the parenterally-fed group showed significantly
decreased incorporation of
3 H-thymidine
into crypt cells.
Eastwood (1977) also demonstrated increased goblet cell
number in the parenteral group.
All of the effects were most
definitive proximally and not present in the distal small
intestine.
Electron microscopy of the mucosa of parenterally
fed rabbits (Eastwood, 1977) demonstrated an increased number
of multivesicular bodies in the tip cells along with longer
microvilli.
These changes, which were most manifest in the
jejunum, may be interpreted as indicating an older cell
population and therefore decreased shedding and decreased
migration in addition to decreased proliferation.
The speed of change in intestinal and pancreatic structue
and function with the elimination of enteral nutrition was
assessed in parenterally-alimented rats by Hughes et al.
(1977).
They found that jejunal villus height fell quickly from 432 Mm
to 342 pm at three days, to 313 pm at six days, and 177 pm
at fifteen days.
Similarly, ileal villus height fell from
237 pm to 204 pm at three days, 197 pm at six days, and 177
pm at fifteen days.
There were corresponding changes in crypt
28
depth and mucosal protein, DNA content, md wet weight. In vivo
absorption studies in both jejunum and ileum disclosed significantly diminished galactose absorption after ten days. Pancreatic
mass fell from 500 to 307 mgm/100 gms body weight after fifteen
days.
Dowling and Gleeson (1973) and McDermott et al.
(1976)
have demonstrated that in rats resection of the proximal small
intestine produces mucosal cell hyperplasia and an increase
in the proliferative zone of crypt cells in the residual distal
intestine.
There is also villous enlargement and an increase
of the muscular wall.
Weser and Hernandez (1974) and Urban
and Pena (1974) demonstrated, after intestinal resection, that
stimulated cell proliferation occurs within five to six weeks,
Obertop et al. (1977) have shown that within two days of removal
of the proximal one-third of the small bowel in the rat, there
was a marked increase in the nucleic acid content of the remaining small intestine.
This increase became progressively
less marked in more distal sections.
Feldman et al.
(1976) showed that in dogs which were
alimented parenterally after jejunectomy, the adaptive mucosal
hyperplasia in the remaining small bowel was blunted,
The
villus height in residual ileum of parenterally-alimented
animals was lower than orally-fed control animals after six
weeks.
In addition, the villus height reduction was even
more prominent in intravenously-fed dogs whose bowels were
left intact (Feldman, et al.,
1973).
Other studies after
experimental intestinal resection (Altman, 1974; Dowling, 1974;
29
Johnson, et al., 1975; Johnson, 1976) all similarly demonstrated
that return of morphology and function is clearly related in
intraluminal nutrition.
Levine et al. (1974) fed an identical liquid diet to two
-groups of rats for one week.
One group received the diet
parenterally, the other via the enteral route.
Body weight
gains for the two groups were comparable, yet the group on
total parenteral nutrition had a 22% decrease in small intestinal
weight, 28% decrease in mucosal weight, 35% decrease in
mucosal protein, and a 25 % decrease in total DNA.
Of note is
that these effects were minimal in the distal small intestine
and maximal at the proximal end.
The net effect was a loss of
the proximal-to-distal morphological gradient.
This would
support the premise that this gradient was the result of the
preferential utilization of the luminal contents.
In addition to the studies of Levine et al.
(1974) cited
above, Dworkin (1976) compared intestinal mass between rats
receiving a formula orally and a group receiving the same
formula parenterally.
He found that the gut and mucosal
weights, DNA and protein contents and sucrose activity were
all significantly greater in the animals receiving enteral
nutrition.
Since there were similar though smaller differences
in sections of intestine that were defunctionalized, it is
possible that there is both a topical effect from food and
an effect that is mediated through the vascular system.
It is clear that the deprivation of enteral feeding has an
adverse effect upon the intestinal mucosa.
The exact mechanism
of this effect is unknown. An effect of dietary purine content
is an intriguing possibility.
30
3. A Trophic Effect of Pancreatic Secretions Upon the
Intestinal Mucosa
The primary cause of the loss of small intestinal mass which
is consistently reported during starvation and during total parenteral nutrition continues to be debated.
Ecknauer et al. (1977)
created self-emptying blind loops of intestine which although defunctionalized contained the pancreatico-biliary ducts.
They re-
ported that after thirty-two days the defunctionalized loops did
not lose any mucosal mass when compared to control sections of intestine.
When compared to the controls the defunctionalized loops
had similar protein and DNA content per dry weight, and similar
protein/DNA ratios.
The loops in vivo
had normal galactose ab-
sorption but showed low sucrase and normal maltase activity compared
to the control.
This would suggest a trophic effect of either the
pancreatico-biliary secretions or a trophic effect of a hormonal
factor released secondarily to eating.
Altmann (1971) transplanted a small segment of duodenum along
with the duodenal papilla to various parts of the small intestine.
He found that the presence of pancreatico-biliary secretion resulted in significant villous enlargement.
In addition, he found
that the major portion of the villous-enlarging influence was from
the pancreatic secretions rather than from the bile.
Hughes et al.
(1978) report that they were able to prevent the
small intestinal atrophy caused by total parenteral alimentation by
infusing daily CCK and secretin.
They were, however, unable to dis-
tinguish an intrinsic hormonal effect upon the intestine from a
secondary effect of either or both hormones acting primarily to
stimulate pancreatic secretions.
Thus, one might expect that any adverse effect from the exclusion
31
of intraluminal nutrition might be at least partially offset as a
result of pancreatic enzymic digestion of sloughed intestinal epithelial cells.
It is, however, well established that intravenous
hyperalimentation has an adverse effect upon pancreatic mass and results in decreased pancreatic secretions
Hughes et al.,
ton et al.,
(Kotler and Levine, 1979;
1978; Hughes et al., 1977; Towne et al., 1973; Hamil-
1971; Dudrick et al.,
1970; Johnson et al.,
1977).
Accordingly, it is plausible that the loss of intestinal mass as
a result of dietary deprivation may in large part be due to a deficiency of dietary purines or more specifically to a deficiency of intraluminally-available purines.
In protein-free diets where cell
size remains normal (Munro and Goldberg, 1964; Hill et al., 1968) the
dietary intake may provide enough pancreatic stimulus to allow utilization of endogenous purines (and protein) available from the
sloughed mucosal cells.
On hyperalimentation the decreased pancre-
atic protein secretion is inadequate to allow for the necessary reutilization of the endogenous purines (and proteins).
Whether the deprivation of the intraluminal supply of nutrients
via total parenteral nutrition has its adverse effect upon intestinal
mucosal integrity as a primary effect or as a secondary effect
through failure to stimulate hormonal secretion or as a combination
of these is unclear.
It seems, however, well established that intra-
luminal nutritional factors have a distinct role to play in the adequate maintenance of the gastrointestinal tract.
It is possible that under certain circumstances intestinal mucosal
cells are able to utilize endogenous proteins and purines,
The degree
to which the intraluminal protein and purines are available might ultimately affect cell turnover.
Hershfield and Kern (1969) have shown
that in the protein-depleted rat the mucosa made better use of orally-
32
administered amino acids than did the mucosa of normal controls.
Thus, one must conclude that amino acids from protein digested
in the gut are preferentially used for mucosal cell renewal and
-that a similar situation may exist for the endogenous purines.
In summary,
it
is established that pancreatic secretions
have a trophic effect upon the intestine.
effect is unknown.
The mechanism of that
That the endogenous purines liberated as a
result of the action of pancreatic ribonucleases are responsible
for the trophic effect of the pancreatic secretions is another
intriguing possibility.
B.
Purine Metabolism and Diet
If the small intestine is incapable of providing purines via
the de novo
synthetic pathway (MacKinnon and Deller, 1973), then
the actual source of the preformed purines becomes a very significant question.
There is evidence that purines are transported
from one tissue to another.
Studies by Pritchard et al.
(1970)
and by Lerner and Lowy (1974) suggest that adenosine is exported
from the liver.
Adenosine levels in hepatic venous blood were
reported (Pritchard et al.,
1970) to be ten times greater than
those in the portal and arterial blood.
The work of Pritchard
appears to establish adenosine as the major utilizable purine compound exported from the liver.
None of these studies contain data
concerning the quantitative aspects of purine supply.
Burnstock
(1977) states that rat lung takes up large amounts of adenosine.
This finding raises the possibility that the lung is an active
site of purine metabolism.
Should this be so, the importance of
hepatic venous adenosine will have to be reassessed.
33
The small intestine is ideally suited to make use of
ingested purines aRd to reuse digested desquamated mucosal cell
purines.
It may be that under certain conditions endogenous
purines as well as protein might be made available to the
-mucosa in significant amounts.
Ju and Nasset (1959) estimate
total endogenous protein in man to be of the order of 64 - 263
grams per day.
Specifically, they estimate 71 - 91 grams per day
of protein from desquamated cells.
Even at this lower level
there would be approximately 2 grams of available purine bases
potentially liberated from the shed mucosal cells.
The avail-
able pancreatic ribonucleases adsorbed to the small intestinal
membranes could, therefore, (even on a purine-free diet) make
a significant amount of purines available to the intestine.
It is well established (Clifford et al.,1976; Zollner
and Griebsch,1974; Zollner and Griebsch,1973; Rauch-Janssen
et al.,1977; Zollner, 1973; Griebsch and Zollner,1974) that
increased dietary intake of purine bases, purine nucleotides
or nucleic acids results in increased uric acid output.
Grobner
and Zollner (1977) found that after ten days of an iso-energetid,
purine-free diet, serum uric acid and urinary uric acid excretion
reached a minimum and then remained constant.
Addition of
purines yielded a linear relationship between dietary purines and
serum uric acid level and urinary uric acid.
They concluded
that their results were "consistent with a theory that purines
or purine containing compounds absorbed from the gut exert
little or no influence on purine biosynthesis...".
34
This "nitrogen balance" type of approach to purine metabolism seems inadequate with regard to the actual metabolic events
that occur as a result of dietary intake of purines.
et al.
Clifford
(1976) clearly demonstrated that, despite the great
biochemical similarity of the various purines, they produce
different alterations in uric acid metabolism when administered
Oral hypoxanthine, AMP, GMP, IMP, and
to human subjects.
adenine elevated serum uric acid levels while guanine and
xanthine did not.
Hypoxanthine, AMP, GMP, and IMP produced a
greater hyperurecemic effect in subjects with gout compared with
hyperureceicanfidnormoureceiic
controls. Urinary uric acid
levels were increased equally by all purines except for guanine
which did not alter urine uric acid levels.
Bowering et al.
(1969) studied the influence of different
dietary levels of protein and yeast RNA on uric acid metabolism
in humans.
They found that urinary urea, uric acid, alpha-amino
nitrogen, and ammonia varied directly with protein intake, but
the percentage contribution of individual compounds to total
nitrogen excretion differed at each dietary nitrogen level.
The
high protein and RNA diets produced identical urinary uric acid
excretion but only RNA increased serum urate levels.
used
1 5N
The authors
labeled uric acid with their different dietary regimens.
The increased uric adid excretion with the high protein diet
appeared to be the result of a two-fold increase in the uric
adid turnover rate without an alteration in urate pool size.
The similarly-increased uric acid excretion with the RNA feeding
was the result of a very slight increase in the turnover rate
35
and a 100% expansion of the miscible pool size.
Their data
do not appear to support a concept of inhibition of purine
synthesis by exogenous nucleic acid.
It does not, however,
demonstrate that the regulation of purine metabolism is
strongly interrelated with the nutrition of the organism.
This is consistent with the findings of Clifford et al.
(1972).
They found that de novo purine synthesis in the
livers of male rats was considerably stimulated by proteincontaining meals and conversely underwent a decrease following
a twelve-hour period of fasting.
In addition protein-con-
taining meals caused a small decrease in the concentration of
free adenine and guanine mucleotides in the liver and fasting
resulted in a transient increase in these nucleotides.
These
nucleotides, however, were eliminated as factors regulating
activity in the de novo pathway because they occurred some
hours after alteration in purine biosynthesis was evident.
A close correlation between alterations in purine biosynthesis
and changes in the state of polysome aggregation was observed.
They suggest that amino-acid-dependdnt
alterations in poly-
some aggregation determine the rate of RNA breakdown and that
an unidentified product of RNA degradation regulates purine
biosynthesis.
Sonoda and Tatibana (1978) studied the meta-
bolic fate of pyrimidines and purines in dietary nucleic
acids ingested by mice.
labeled diet.
Animals were fed a 14 C-adenine RNA-
The adenine was rapidly absorbed with 85%
excretion in eight hours.
Most of the exogenous nucleic
acid bases appeared to have been removed by both degradation
36
and utilization by the intestine and liver.
With increase
in dose of dietary nucleic acids, the amount of labeled purine
was increased in all tissues examined, but it was always greatest
in the intestines.
Not only do the authors document the active
utilization of the ingested bases by the intestine, but in
addition, they strongly suggest a limited capacity of the liver
and gastrointestinal tract to remove exogenous nucleic acid
precursors.
This would mean that with a large dose of purine
or pyrimidines the contribution to tissue synthesis of dietary
nucleotides and nucleic acids might be significant.
The question of the actual extent of the utilization of
purines derived from the diet continues to be debated.
et al.
Roll
(1949) reported that approximately 1% of the nucleic
acids of the combined viscera were derived from
fed to rats.
Burridge et al.
1 5 N-labeled
RNA
(1976) fed labeled yeast RNA to
mice and found 0.26% of the administered radioactivity in the
body tissues at six hours.
They noted that dietary nucleic
acid adenine appeared to be utilized somewhat more efficiently
than was dietary nucleic acid guanine.
Berlin et al.
(1968) found a large amount of xanthine
oxidase activity in the intestinal epithelial cell layer.
Furthermore, they found that hypoxanthine, xanthine and uric
acid were actively secreted in vitro into the lumen.
offered an extra-renal excretory mechanism for urates.
This
They
utilized isolated sacs of hamster small intestine in their
experiments and warn that in vivo, in
the presence of an active
blood circulation, absorption may be significantly increased.
The net balance of these factors in vivo needs experimental
37
evaluation.
Pritchard et al.
(1970) documented that the liver supplies
purines to other tissues.
In the introduction to their paper
they state, "the source of preformed purines has not been
conclusively established.
They are not derived from the diet,
since mammals may be maintained indefinitely on purine-free diets.
Moreover, dietary studies and in vitro examination of the
intestinal transport mechanism indicate that purines may, in
fact, be secreted rather than absorbed into the gut."
They
cited Berlin (1968) as their reference regarding purine secretion
in the gut.
Five years later Pritchard et al.
(1975) again
referred to the poor absorption of purines from the intestine
and also referred to the possibility of purines being secreted as
purine wastes by the intestine.
Wilson and Wilson (1962) studied absorption of purine
ribonucleotides by everted rat and hamster intestinal sacs.
They concluded that ribonucleotides do not appear to be absorbed
as such.
Rather the mucosa was capable of metabolizing them
along the following pathways:
>guanine 2'(3') GMP & 5'GMP -->guanosine --.
2'(3') AMP & 5'AMP
-o
adenosine --
inosine
-*
xanthine ->uric
hypoxanthine
acid,
-4
xanthine --- uric acid.
They further reported
that the absorption of the nucleosides and
bases appeared to occur via passive diffusion.
Khan et al.
(1975) could demonstrate no active transport
mechanism for uric acid, hypoxanthine or xanthine in rat or
hamster jejunum.
Berlin and Oliver (1975) in an excellent
38
and comprehensive review of membrane transport of purine
pyrimidine
and
bases and nucleosides in animal cells conclude that
purine bases and nucleotides seem to have their transport
mechanisms well established, and appear to enter animal cells
by facilitated diffusion.
Transport carriers are clearly
separate from the enzymes responsible for subsequent intracellular metabolism.
Harms and Stirling (1977) studied the
transport of purine nucleotides and nucleosides by in vitro
rabbit ileum.
Their data indicate that at least 85% of the
They
terminal phosphate of ATP is hydrolyzed during transport.
postulate hydrolysis of ATP followed by a preferred uptake of
the released adenosine at the membrane surface as explanation
of their data.
They provide support for a specific transport
path for the hydrolyzed nucleotides and for the nucleosides.
In summary, the literature on the role of dietary purines
No
in purine metabolism has stated that they are unimportant.
attention however has been given to the specific effect of
purines on the intestine.
In addition no consideration has
been made of situations where increased utilization or preceding
depletion of purines have been a factor.
This latter point is
extremely relevant to the study of disease states.
C.
Intestinal RNA Metabolism
Amano et al.
(1965) concluded that significant RNA synthesis
occurs only in the crypts.
They base this determination upon
auto-radiographic analysis after injection of
adult male rats.
3 H-cytosine
This is consistent with the findings of
in
39
Fontin-Magana et al.
(1970) and Herbst et al.
(1970) who
report that the enzymes of the pyrimidine biosynthetic pathways
are located primarily in the crypts.
Zaharko et al.
(1977)
noted that in mouse small intestine there is an anti-purine
effect upon DNA synthesis at high methotrexate plasma concentrations.
Altmann (1974) injected 3H-thymidine prior to
injection of high dose methotrexate in normal adult male rats.
He then maintained the high levels of methotrexate while
following the labeled DNA in the small intestine over several
days.
He observed that while the crypts became partially
emptied the labeled cells continued to move up the villi at a
At 48 hours the entire villus contained only
near normal rate.
the older labeled cells.
He concluded that since the metho-
trexate inhibited RNA synthesis the surviving cells must have
stored the RNA necessary for migration, differentiation, and
protein synthesis.
He felt that the length of cell survival
was linked to the amount of stored RNA.
Of related significance is the clinical observation of
Craft et al.
(1977) who reported on eighteen children with
acute lymphoblastic leukemia who were on various treatment
regimens, all of which included methotrexate.
They noted that
a progressive and significant increase in malabsorption (as
determined by xylose absorption test) directly correlated with
the
cumulative methotrexate dose.
Nakayama and Weser (1972) measured pyrimidine biosynthetic
enzyme activity
after proximal intestinal resection.
They
found an increase in total enzyme activity which appeared to
correlate with increased crypt length and cellularity and with
40
a general increase in the total proliferative population of
cells (as measured by methyl
3 H-thymidine
incorporation).
The
increase in the enzyme activity was taken to reflect an increase
in total nucleic acid synthesis.
In support of this conclusion
Nakayama and Weser (1972) reported findings in the small
intestine showing an increase in the percent of glucose metabolized
via the pentose phosphate pathway at three and at seven months
after a 50% resection.
Studies (Beaconsfield and Reading, 1964;
Beaconsfield and Rainsbury, 1964; and Beaconsfield et al. 1965)
have shown a definite relationship between the rate of the
pentose phosphate shunt pathway activity and RNA synthesis.
The literature suggests that RNA synthesis occurs entirely
(or at least mostly) in the crypt cells.
No studies to determine
if isolated intestinal cells or cell nuclei are capable of RNA
synthesis have been performed.
Thus, the ability of the enterocytes
to continue to synthesize RNA (and new proteins) as they age and
migrate to the villus tip has not been adequately assessed.
The
role of purines (and dietary purines in particular) in RNA synthesis
in the non-crypt enterocyte thus cannot be adequately evaluated.
D.
Adenine Metabolism
Ely and Ross (1950) studied the effect of adenine on rats.
They found that a dietary level of 0.17% adenine caused some
enlargement of the kidneys and a reduction in the size of the
liver and thymus.
The maximum level that had no influ6nce on
growth was 0.33% adenine.
Higher levels reduced growth in addition
to causing enlargement of the kidneys and reduction of the size
of the liver and thymus.
They noted crystalline deposits in the
41
rats' renal tubules at the doses they used.
Clifford and
Story (1976) studied the metabolic effects of several purines on
rats.
Male rats were fed purified amino acid diets supplemented
with adenine, guanine, hypoxanthine, and xanthine at a level
of 0.75gm/100 gm diet.
Guanine, hypoxanthine, and xanthine
did not affect the growth rate or the patterns of purine
excreted in the urine.
Adenin& suplem6ntation at that level
resulted in reduced growth rate, altered hepatic purine enzyme
activities and changes in purine excretion patterns in the urine.
Excretion of 2,8, dioxyadenine was first evident at 0.1% adenine
Increased urine volume, plasma urea nitrogen and
in the diet.
kidney 2,8, dioxyadenine were evident at 0.3% dietary adenine.
Under the conditions of their work Clifford and Story (1976)
concluded that the maximum safe level of adenine in rat diets
should not exceed 0.1%.
Bendich et al.
(1950) showed that the crystalline deposits
in the kidney tubules of the rats which were fed high doses of
adenine were 2,8, dioxyadenine.
They presented evidence to
suggest that 2,8,~dioxyadenine was the direct oxidation product
of adenine.
Philips et al.
(1952) studied adenine intoxication
in relation to in vivo formation and deposition of 2,8, dioxyadenine
in renal tubules.
They found that adenine was nephrotoxic as
the result of its in vivo oxidation to 2,8, dioxyadenine and the
subsequent deposition of the latter as crystalline occlusions in
renal tubules.
They showed that a threshold amount of adenine had
to be reached before deposition began.
All other effects previously
ascribed to adenine intoxication were probably the result of
42
complications secondary to uremia.
Bennett (1953) examined incorporation of adenine-4,6 1 4 C
into nucleotides and nucleic acids in C-57 mice.
He found that
intraperitoneally-administered isotope is rapidly and extensively
incorporated
into the nucleotides and nucleic acids of the
mouse liver, stomach and intestines.
Saviano and Clifford
(1978) found when they administered various free purine bases
that they were extensively absorbed.
Approximately 5% was
incorporated into tissues and the rest was excreted in the urine.
Adenine metabolism in man has been studied by deVerdier
et al.
(1972).
They found that the basal plasma level of
adenine varied about a mean of 70 nmole/l.
rate was around 30 nmole/minute.
The renal excretion
During intravenous infusion
of adenine about 2% of the dose appeared in urine as adenine
and less than 2% as 2,8, dioxyadenine.
The authors conclude
that adenine was quickly metabolized, primarily to the nucleotides.
The results of their oral loading test show that adenine
is readily absorbed and appears in the plasma.
Following the
oral loading test 3.3% of the adenine appeared in the urine
as adenine and 4.5% as 2,8, dioxyadenine.
Clifford and Story
(1976), extrapolating from their rat data, estimated that if
0.1% adenine is the safe intake for a rat then the maximum
safe intake for a 70 kg. adult human would correspond to about
3 grams of RNA per day.
E. Glutamine Amidophosphoribosyltransferase and De Novio Synthesis
Amidophosphoribosyltransferase (E.C.2.4.2.14) catalyzes
43
the reaction whereby phosphoribosylamine (PRA) is formed.
There
is some evidence that a sub-unit of this enzyme may utilize
NH3as a substrate in mammalian cells (Reem, 1974; Sperling
et al.,
1973).
Glutamine, however, is generally considered
as the usual nitrogen source
(Wyngaarden, 1972) and the major
substrate along with phosphoribosyl-pyrophosphate (PRPP) in the
reaction:
Glutamine + PRPP + H 2 0
Mg++
)
PRA + Glutamate + PP.
This is the first reaction that is unique to purine
nucleotide biosynthesis and it has been the main focus of
attention as far as end-point inhibition of purine biosynthesis
de novo
is concerned.
Various purine nucleotides have been
found to act as allosteric inhibitors of amidophosphoribosyltransferase from avian (Caskey et al.,
et al.,
1964), mammalian (Holmes
1973) and microbial (Nierlich and Magasink, 1965)
sources.
Only limited success with purification has been obtained
(Lewis and Hartman, 1978).
In spite of a great deal of work,
the kinetic and catalytic mechanisms of reaction are still
unclear.
This only contributes to experimental difficulties
that have been encountered in the study of the enzyme.
The inhibition of amidotransferase by ribonucleotides
appears competitive with respect to PRPP.
Caskey et al.
Hartman (1963) and
(1964) have demonstrated that the enzyme may be
totally desensitized to the action of nucleotide inhibitors
while retaining catalytic activity.
Thus it is clear that the
inhibitors bind at regulatory sites which are distinct from
44
sites for substrates.
The work of Holmes et al.
(1973) estab-
lished that in the human there are two forms of enzymes, a
270,000 molecular weight form and a 133,000 molecular weight
Glutamine does not appear to influence the change from
form.
one form to another but PRPP converts the large form to the
smaller active form.
LaLanne and Henderson (1975) found that the PRPP concentration in mouse liver varies in response to diurnal influences
as well as to hormones and drugs that influence carbohydrate
metabolism.
Clifford et al.
cant variation using rats.
(1972) did not find such signifiBagnara et al.
(1974) studied
Ehrlich ascites tumor cells incubated with purine bases.
They
observed that the inhibition of purine bi6synthesis de novo
in that system may be due largely to the decreased availability
of PRPP that resulted from its utilization by the purine phosThis would be consistent with the
phoribosyltransferases.
observed Michaelis constants in that system which are 5pM for
adenine phosphoribosyltransferase (E.C.2.4.2.7) (Ellis and
Scholefield, 1962),
20-4OpM for hypoxanthine-guanine phosphoribo-
syltransferase (E.C.2.4.2.8) (Hori and Henderson, 1966), and
1mM for amidophosphoribosyltransferase (E.C.2.4.2.17) (Bagnara
et al.,
1.974).
LaLanne and Henderson caution that the consequences of the
observed elevations of PRPP in mouse liver are uncertain and
that they may not lead to marked increases in the rates of any
PRPP-dependent reactions.
This is because studies in other
systems show that the rate of PRPP synthesis readily increases
when the concentration of purine bases is increased.
Thus the
45
amount of PRPP that is actually available for nucleotide synthesis can be considerably greater than that measured as "free"
PRPP (Henderson and Khoo, 1965; Bagnara et al.,
1974).
While the regulation of nucleotide biosynthesis de novo
can be assessed in terms of influences upon the amidophosphoribosyltransferase reaction, it must be remembered that the
actual activity of this enzyme in vivo
may be regulated by:
(1) metabolic effects, which may be remote or proximate, which
affect the concentration of either or both substrates
(PRPP or
glutamine); (2) changes in the concentration of any of the
nucleotides which may act as allosteric inhibitors; and (3) factors
that actually affect the amount of the enzyme (synthesis and breakdown).
F.
Hypoxanthine-Guanine Phosphoribosyltransferase; Purine Salvage; Lesch-Nyhan Syndrome
Hypoxanthine-guanine phosphoribosyltransferase (IMP: pyro-
phosphate phosphoribosyltransferase; E.C.2.4.2.8) (HGPRT) catalyzes the reaction:
Hypoxanthine or Guanine + PRPP
Mg
y
IMP or GMP + PP
Adenine phosphoribosyltransferase (APRT) catalyzes the reaction:
Adenine + PRPP
MgJ++
AMP + PP
Together they are responsible for the salvage of purine bases.
Although the phosphoribosyltransferases are generally referred to as "salvage enzymes" it is increasingly clear that
this term is too restrictive.
regulatory function as well.
They appear to serve an important
HGPRT deficiency results in massive
46
overproduction of uric acid.
APRT deficiency allows adenine
to be increasingly oxidized to its less soluble product 2,8,dioxyadenine.
Three possible functions of HGPRT have been suggested
(Rosenbloom et al.,
One is to utilize exogenously
1968).
A second role is to act in
supplied hypoxanthine and guanine.
the re-utilization of purine bases formed during turnover of
endogenous ribonucleic acid.
A third specific role is to
function in a cycle in which adenine ribonucleotides are regenerated via the series of reactions:
AMP
-
IMP
-
INOSINE
-
HYPOXANTHINE
HGPRT
P>
AMP
Inherited variation in HGPRT first became evident through
clinical observations of the Lesch-Nyhan syndrome.
In the most
severe form of this disease HGPRT activity is almost zero.
The
clinical syndrome then comprises mental retardation, choreoathetosis, compulsive self-mutilation, aggressiveness, hyperurecemia, hyperuric-aciduria, gout, and uric acid urolithiasis.
There are, in addition to partial and complete deficiencies, a
range of genetically-heterogenous states with varying traces
of residual enzyme activity (McKeran et al., 1975).
In the cours-e of studies on patients with Lesch-Nyhan
syndrome, Arnold and Kelly (1973) noted that with time each
patient seemed to display a wide range of HGPRT activity determinations.
They considered that environmental factors might
be important and studied the effect of diet upon HGPRT activity
in three patients.
During dietary purine restriction erythrocyte
HGPRT activity increased in all three patients.
With resump-
47
tion of either normal purine intake or the addition of only normal
The changes
adenine intake there was a fall of HGPRT activity.
were of the order of sixty to three hundred percent.
The
changes appeared to be the result of an activation or inactivation of the mutant enzyme, despite a constant amount of
immunoreactive HGPRT protein concentration.
McKeran et al.
(1975), in their studies on the diagnosis
of the carrier state for the Lesch-Nyhan syndrome, examined
HGPRT activities for several tissues including the jejunum in
seven patients with variations in their genotype.
Unfortunately,
while they comment upon the mucosal morphology of their controls,
they make no mention of the mucosal morphology of their subjects.
It is, however, clear that the intestinal tissue lacked HGPRT in
the severe forms of the disease.
G.
Intraperitoneal Parenteral Alimentation
Since Dudrick's study on total parenteral nutrition (Dudrick
et al.,
1969), parenteral nutrition has assumed an ever-increasing
role in the management of the extremely sick and debilitated
patient.
Problems remain, however, which inhibit the full
development of this therapy.
Among these problems are (1) the
absence of well-controlled laboratory experiments which could
provide a firm metabolic basis for parenteral nutrition and
(.2)
the absence of methods which would allow widespread pediatric
out-patient use of the therapy.
The absence of well-controlled laboratory experiments is
caused, in part, by the difficulty and high cost of maintaining
48
significant numbers of animals on intravenous parenteral
nutrition.
Intraperitoneal alimentation is a simpler and less
costly technique which would provide the well-controlled experiments required.
The use of this pathway for the administration
of nutrients has never been studied in a systematic way.
The use of intravenous parenteral nutrition at home has
proved to be a costly and difficult technique.
failed to gain widespread acceptance.
As such it has
By contrast, with the
development of a bacteriologically safe peritoneal access device
(Tenchoff and Schecter, 1968) maintenance home peritoneal
dialysis is already feasible (Counts, 1974).
This indicates
that the development of a similar home peritoneal alimentation
program would also be feasible.
Using the bacteriologically safe access device developed
by Tenchoff and Schecter (1968), Counts (1974) carried out
chronic home peritoneal dialysis in 12 children ages 2 10/12
years to 15 10/12 years.
During 179 dialysis months, peritonitis
occurred fourteen times in eight patients for an incidence of
less than one episode per twelve dialysis months:
four times
after a documented break in sterility; three times associated
with an exit-site infection; once in a piece of intracath free
in the peritoneal cavity; twice during a hospital Herella
epidemic; and four times for unknown causes.
One patient
accounted for more than one-third of all infections.
These
infections were milder and of a shorter duration than classical
peritonitis and treatment was sometimes carried out at home.
Petrie et al.
(1976) reported on the use of this technique
49
in 37 adult patients with end-stage renal disease.
The mean
duration of treatment was 14.4 weeks; the longest was 78 weeks.
During this time, 27 patients had one catheter, nine had two,
and one had three catheters.
Peritonitis occurred fourteen
times in ten of the 37 patients (27%).
Generally, the course
of those infections seems to have been benign, as dialysis was
continued in all infected patients.
is not necessarily contraindicated
That peritoneal dialysis
in peritonitis is confirmed
by Merrill (1963) who further mentions that it has been used
within one week of the performance of an appendectomy.
These studies suggest that while infection would be a
factor in peritoneal alimentation, there is reason to believe
that it may occur at an acceptable rate when compared to other
techniques of alimentation.
Overall, apart from metabolic complications, one must
consider the expense of maintaining a patient on a central
venous line or even with a peripheral line.
Skilled nursing,
extensive laboratory facilities, complicated infusing equipment
and full-time hospitalization (for infants and children) all
contribute to the cost, and this does not include the expense
of placing a central line.
Although successful home hyper-
alimentation is readily carried out in adults (Broviac and
Scribner, 1974;
et al.,
Shils, 1975; Jeejeebhoy et al.,
1973; Langer
1973) with good results, it is unlikely that with present
techniques this can ever become feasible on a large scale for
infants and children.
It is expected that absorption of nutrients from the
50
peritoneal cavity might be ultimately controlled not by an external infusion pump, but rather by the inherent absorptive
characteristics of the tissues.
This in turn might make home
hyperalimentation for infants and children a realistic alternative to prolonged hospitalization.
A reasonable model might
follow the one employed in chronic home peritoneal dialysis.
Furthermore, a peritoneal catheter would be less "fragile"
and subject to displacement than a catheter or needle placed in
a vein.
Because of the requirements for central venous alimentation, it is clear that only the tertiary care center is able to
undertake the challenge and expense of this treatment in
children.
The flexibility inherent in a system of peritoneal ali-
mentation might lessen geographic problems by allowing for
adequate follow-up in less specialized secondary care centers.
In addition, the economics of this method would likely bring
hyperalimentation technology to nations unable to afford the
sophisticated technology of central venous alimentation.
Furthermore, metabolic considerations may suggest another
advantage of the peritoneal route.
In central or peripheral
intravenous alimentation, large amounts of amino acids and
carbohydrates
(and possibly fat emulsion) are directly intro-
duced into the systemic circulation, the liver being initially
by-passed.
Whether this has an adverse effect is unknown, but
metabolic derangements such as abnormal liver function are very
common in the hyperalimentation experience.
It is likely, how-
ever, that the bulk of the fluid infused intraperitoneally
51
will be absorbed via the portal system, thus placing
the liver
back in its physiologic position.
Most studies of the peritoneal transport have concentrated
upon alimentation of substances from the body.
been documented about peritoneal uptake.
Very little has
In 1877, Wegner first
discussed the peritoneal surface as a membrane across which
The first report of peritoneal dialysis
solute could be removed.
was by Ganter in 1923.
Boen (1961) discussed the kinetics of
peritoneal dialysis and briefly mentioned absorption of bicarbonate and glucose.
He commented on the use of glucose as a
source of energy, but since he carried out only five investigations in four subjects, his data are very limited.
Cunningham (1920) studied the effects of intraperitoneal
dextrose upon the peritoneal lining of rats.
His studies were
also very limited, but it appeared to him that none of his rats
suffered any ill effects.
There was a suggestion that the
absorptive capacity of the peritoneum and its contents might
have increased with time.
Unfortunately, there is no further
elaboration of his work.
While there are a great many unanswered questions about the
use of the trans-peritoneal route, there is no reason to doubt
that the peritoneal cavity and its contents are capable of
absorbing enough nutrients to sustain life.
In the laboratory
this may well provide a relatively simple and efficient method
of studying parenteral nutrition.
value for the human patient.
Conceivably it may also be of
52
III.
PLAN OF RESEARCH
A series of experiments have been performed on rats to
determine the importance of dietary purine sources
(nucleic
-acids) for the metabolism of the mucosal cells of the gastrointestinal tract.
Purine nucleotides can be provided within cells
by de novo synthesis or by salvaging free purine bases, the key
enzymes being phosphoribosylamidotransferase and adenine or
hypoxanthine-guanine phosphoribosyltransferase (figure 1).
As
the diagram shows, both pathways use phosphoribosylpyrophosphate
(PRPP) as a major substrate.
The relative importance of the synthesis of purine nucleotides by the de novo and the salvage pathways was assessed both
by measuring the incorporation of isotopic precursors in each
pathway (glycine for de novo synthesis, adenine for salvage),
and also by measuring the activities of the rate-limiting
enzymes in each pathway (phosphoribosyl amidotransferase for the
de novo pathway, hypoxanthine-guanine phosphoribosyl transferase
for the salvage route).
Section I:
(A)
In vitro studies of the de novo and salvage pathways.
The activity of the de novo pathway was assessed
by examining the incorporation of 1C-glycine into
the mucosal RNA of the small and of the large
intestines.
(B)
The viability of the excised mucosa was checked in
part by examining the incorporation of
into the mucosal proteins.
1 4 C-glycine
This also served to show
53
that 14 C-glycine pool differences were not the
cause of differences in labeling of mucosal RNA.
The assumption is made in these experiments that the
glycine pool available for protein synthesis is either essentially the same pool as that available for purine nucleotide
synthesis or at least varies quickly and proportionately with
it.
The salvage pathway was assessed by examining
(C)
the incorporation of
3 H-adenine
into RNA
utilizing the same techniques as for the de
novo pathway.
Section II:
The fate of oral versus parenteral adenine and
adenosine.
(A)
The intestinal absorption of labeled adenine and
adenosine were studied in vivo in rats.
(B)
The results obtained by gavage feeding (oral) of
isotope were compared to those obtained by intraperitoneal injection (parenteral)
Animals received either a Purina Rat Chow diet, or a
synthetic, agar-based diet which contained no protein and
no purine source.
Section III:
The influence of protein and purine content of
the diet.
(A)
The effect of dietary protein and purines on the
de novo pathway in the small intestine was assessed.
(B)
The effect of dietary protein and purines on the
salvage pathway in the colon was assessed.
The actual incorporation experiments employed are
54
essentially the same techniques as those performed in
Section I.
Section IV:
The measurement of amidotransferase and salvage
activities in the small intestine, colon
and liver.
(A)
Verification of the above incorporation studies
was sought by assaying the amount of amidotransferase activity in the small intestine, colon and
liver.
(B) Modification of an assay for HGPRT activity and
use of this assay to assess the effect of dietary
purines on HGPRT activity in the small intestine,
colon and liver.
Section V:
Other conditions influencing purine utilization.
This group of experiments comprises a heterogenous series
of studies which are preliminary in nature but helpful in
understanding the data at hand and in suggesting avenues for
future research.
(A)
Studies of a self-emptying blind loop of intestine
free of pancreatic secretions.
(B)
Development of a technique to separate viable
enterocytes from villus tips as well as villus
crypts, along with results of incorporation studies
utilizing this technique.
(C) A preliminary assessment of the technique of alimenting
laboratory animals via an indwelling intraperitoneal
catheter.
55
-
A.
IV.
MATERIALS AND METHODS
In Vitro Studies of the De Novo and Salvage Pathways
Animals utilized in these studies were male 200-gram
white CD rats obtained from Charles River Breeding Laboratories
(Wilmington, Massachusetts).
On arrival the animals were
individually housed in standard screen-bottomed galvanized
cages measuring 25 cm X 18 cm X 18 cm.
The rats were main-
tained on standard Purina Rat Chow diets.
with fresh tap water daily.
was maintained at 22*C.
They were provided
The environmental temperature
Room lighting was automatically
controlled to provide fourteen hours of light and ten hours
of darkness.
Animals which were operated upon had their food removed
approximately six hours before surgery.
Animals were fasted
for the 24 hours following surgery but were allowed water ad
libitum.
Ether anesthesia was employed.
All animals were sacrificed in the cold room at 4*C by
decapitation.
Sections of small and large intestine were
rapidly dissected and flushed until clear with ice cold KrebsRinger bicarbonate buffer.
glass sheet.
The tissue was placed on an iced
Sections of intestine were everted over a glass
rod and then cut into 5 cm lengths.
The everted intestinal
segments were placed in iced beakers with 10 mls of KrebsRinger bicarbonate buffer in 95% 02/5% C02 atmosphere, and
50 microcuries of either
3 H-adenine
or 14 C-glycine.
For experi-
ments involving mucosal scrapings the intestine was cut longi-
56
tudinally and scraped with a glass slide.
The scrapings
were placed into a tared flask and weighed.
An equal weight of
buffer was added and the scrapings were thoroughly mixed by
swirling the flask.
Isotope was added,
microcuries per gram of tissue, or
per gram of tissue.
either
3 H-adenine,
1 4 C-glycine,
10
25 microcuries
Each flask contained either everted
sections of tissue from four to six different animals or
mucosal scrapings from four to six different animals.
In
experiments utilizing everted loops, separate flasks were used
for each time point.
In experiments utilizing mucosal scrapings
samples were withdrawn from the same flask at each time point.
All samples were incubated at 37*C with gentle agitation
for ten, thirty or sixty minutes.
At the end of the incubation
period samples were precipitated with ice cold 0.6N~~PCA>
homogenized and centrifuged.
The precipitate was first washed
with 95% EtOH, then washed twice with a mixture of EtOH-chloroform
(3:1), then with EtOH-ether (2:1) and finally with ether.
The
residue was dissolved in 0.3N KOH brought to 37*C for one hour
to render the RNA acid non-precipitable (Fleck and Munro,
1962).
A sample was then placed aside for later protein
determination by the method of Lowry (1951).
was performed by the method of Burton (1956).
DNA determination
Incorporation
of isotope into protein and purines was determined by neutralizing with glacial acetic acid and counting an aliquot in
10 mls of ACS (Amersham, Arlington HeightsIllinQis},
Then
0.2 ml of 1ON PCA was added to the solution to precipitate
the protein and DNA (leaving the hydrolyzed RNA).
The pH
of the remaining solution was adjusted to pH 7 with PCA and KOH.
57
The RNA content was determined by the method of Fleck and
Munro (1962).
The solution was then made to 1N HC1 and
heated for one hour at 90'C to hydrolyze the RNA to purine
bases and pyrimidine nucleotides (Markham and Smith, 1951).
An aliquot was evaporated from an evaporating dish on a
sand bath.
The crystalline purine bases were redissolved
in water and spotted on Whatman 3mm chromatography paper.
The chromatography was run using a methanol:
water (50:25:6:19) solvent.
ethanol:
HC1:
The position of the purine
bases was determined using an ultra violet light source.
The respective spots were cut into uniformly small pieces and
placed in a scintillation vial.
To the vial was added 0.5ml
of Protosol (New England Nuclear, 549 Albany Street, Boston,
Massachusetts 02118) and it was then incubated at 50*C for
two hours.
The vials were then allowed to cool thoroughly in
the cold room, one to two drops of water were added and then
10 ml of toluene PPO-POPOP (12.2 gms PPO, 0.7 gms POPOP, 1 gal.
toluene) was added.
The vials were placed in tie cold room
Tabulations were
overnight and then counted the next morning.
made of 14 C-glycine incorporation into protein,
incorporation into RNA purines, and
3 H-adenine
1 4 C-glycine
incorporation
into RNA purines.
Isotope was obtained from New England Nuclear.
was:
The adenine
NET-350 Adenine (2-3 H), Lot Number, 1014-180, specific
activity 23 Curies/mmole.
The glycine was:
NEC-276 Glycine,
Lot Number, 1046-084, specific activity 98.7 millicuries/mmole.
Animals who underwent pancreatic duct ligation had this
1 4 C(U)]
58
procedure performed under ether anesthesia.
sterile technique was employed.
Clean but not
After shaving of the animals'
abdomens tincture of merthiolate was used to clean the surgical
A three to four cm midline abdominal incision was made
area.
with a sharp-pointed scissors,
was readily identified.
The pancreatico-biliary duct
Two 4/0 silk sutures were placed
tightly around the duct just proximal to its entrance into
the duodenum.
The duct was cleanly transected between the
ligatures, just proximal to the distal ligature, with a sharppointed scissors.
The intestine was carefully placed back in
proper position and the abdomen was closed with a single layer
of interrupted 3/0 silk sutures.
The entire procedure rarely
took more than 20 minutes.
Control animals underwent "sham" surgery involving an
identical procedure including identification of the pancreatic
ducts and manipulation of the area.
B.
The Fate of Absorbed Versus Injected Adenine and Adenosine
Animals utilized in this experiment were 200-gram white
male CD rats obtained from Charles River Breeding Laboratories,
On arrival the animals were individually housed in standard
screen-bottomed galvanized cages measuring 25 cm X 18 cm X
18 cm.
The rats were initially placed on Purina Rat Chow
diets and tap water ad libitum.
Room lighting was automatically
controlled to provide fourteen hours of light and ten hours of
darkness.
The environmental temperature was maintained at 224C.
Forty animals were divided into two groups of twenty,
one group for each isotope.
The animals were further divided
59
into one of two diet groups.
The "regular" diet was Purina
Rat Chow, the "0% protein" diet was an agar-based synthetic diet
mixed as follows:
The agar and water were brought to a full
boil in a large stainless steel pot.
The mixture was allowed
to cool in a large stainless steel mixing bowl.
When sufficiently
cool to allow comfortable holding the salt mixture and carbohydrates were added.
This was followed by the addition of
corn oil and then the protein source (if any).
vitamin mix and choline were added.
while electric mixing was proceeding.
Finally the
All additions were made
After all additions
were made the entire diet was mixed for an additional thirty
minutes.
The contents of the diet utilized in this study are
tabulated in table 1.
Isotope for this experiment was obtained from ICN
(Irvine, California).
One ml of
3 H-adenine
(ICN cat #27001,
lot # 853976) containing 1 mC/ml with a specific activity of
55 Ci/mM was diluted to a total volume of 22 cc's with sterile
normal saline.
One ml of the final solution containing
45 microcuries was either injected intraperitoneally or
infused intragastrically via a 5 french
polyethylene gavage
tube.
One ml of
3 H-adenosine
(ICN cat #24008, lot # 889572) con-
taining 1 mC/ml and with a specific activity of 50.2 Ci/mM
was diluted to 22 cc's and one ml of this solution containing
45 microcuries was either injected intraperitoneally or infused
intragastrically.
60
Table 1
0% Protein and 0% Purine Diet for
Study of the Fate of Absorbed
versus Injected Adenine
and adenosine
Components
Casein
267.4
Sucrose
225.4
Dextrin
275.0
Corn oil
150.0
Salt Mix*
40.0
Vitamin Mix**
10.0
Agar
Water
*
0.0
Dextrose
Choline
**
Grams/1000 grams dry wt.
See table 2.
See table 3.
4.0
35.0
1000.0
61
Table 2
Composition of Roger-Harper's Salt Mixture
Constituent
Gm. %
CaCO 3
29.29
CaHPO4 . 2H20
0.43
KH 2PO
34.31
NaCl
25.06
MgSO 4 . 7H20
Fe(C 6 H 5 ) 7 ).6H
9.98
20
0.623
CuSO 4
0.153
MnSO4 H 2 0
0.121
ZnCl 2
0.020
KI
0.0005
(NH4 )6 Mo 7 0 2 4 .4H 2 0
0.0025
Na 2 SeO 3. 5H20
0.0015
62
Table 3
Contents of Vitamin Mixture
Constituent
Amount (g/Kg
and IU/Kg)
Thiamine hydrochloride
1.0
Riboflavin
2.0
Niacin
5.0
Vitamin C
20
Pyridoxine
1.0
PABA
10
Biotin
0.05
Ca Pantothenate
5.0
Folic Acid
0.2
Inositol
20
Vitamin B1 2
Vitamin A
5.0
(As acetate)
500,000 IU.
Vitamin D 2
50,000 IU.
Vitamin E
10,000 IU.
Vitamin K
Sucrose
.005
910.25
63
After receiving the isotope the animals were kept
overnight in a special isolation room to prevent radioactive
contamination of the facility.
The animals were fasted
overnight but allowed water ad libitum.
Fourteen to twenty
hours after they received the isotope they were anesthesized
with ether and bled by cardiac puncture.
Sera and red blood
cells were separated by centrifugation.
The liver was rapidly
removed and placed on an iced glass sheet.
The entire gastro-
intentinal tract was removed rapidly and flushed with iced
saline until clear and then also placed on an iced glass sheet.
The gastrointestinal tract was divided into stomach, proximal
small intestine (the 10 cm of intestine just distal to the
ligament of treitz), middle 10 cm of small intestine, distal
10 cm of intestine (the 10 cm just proximal to the caecum),
caecum, proximal half of the colon and the distal half of the
colon.
All sections were slit longitudinally and scraped with a
glass slide to remove the mucosa.
A section of the median lobe
of the liver was diced with a sharp-pointed scissors.
Mucosal
scrapings and liver were placed in 10 X 75 mm glass test tubes
and then 5 ml of ice cold 0.6N PCA was added.
Each test tube
was thoroughly mixed on a vortex mixer and placed on ice.
Subsequently the test tubes were spun down and washed in 0.2N
PCA.
The precipitates were then hydrolyzed in 5 ml of 0.3N
KOH at 37*C for 60 minutes.
Then 0.2 ml of 10 N PCA was added.
The RNA in the supernatant was determined by the method of Fleck
and Munro (1962).
An aliquot of the supernatant was counted in
64
10 mls of ACS scintillation fluid.
CPM/mgm RNA for each sample
was determined.
C.
The Influence of Protein and Purine Content of the Diet
Male 200-gram white CD rats were obtained from Charles
River Breeding Laboratories.
On arrival they were individually
housed in standard, screen-bottomed galvanized cages measuring
25 cm X 18 cm X 18 cm.
For one week they were fed a standard
Purina Rat Chow diet. After this stabilization period they were
The animals had access to
weighed and assigned to diet groups.
tap water which was changed daily.
Their food cups were
weighed and changed every other day.
ature was maintained at 22*C.
The environmental temper-
Room lighting was controlled
automatically to provide 14 hours of light and ten hours of
darkness.
The diets were mixed three days before the start of the
experiment and stored at 4*C in tightly-sealed plastic bags.
The diets were mixed as per the procedure detailed in Section B
of Materials and Methods (page # 59).
listed in table 4.
The diet contents are
After seven days the animals were weighed
and killed by decapitation.
Incorporation studies were per-
formed as per the procedure in Section A of Materials and
Methods, with the following modifications:
1.
For each dietary group, proximal and distal sections
of both the small intestine and the colon were studied.
Mucosal
scrapings from four animals were pooled for each such organ
section.
Thus for each dietary group two incorporation studies
65
Table 4
Composition of Diets
Components
Diet
1
Diet
2
Diet
3
Diet
4
Diet
5
Diet
6
g/1000 g dry wt.
Dextrin
442
442
442
562
562
562
Sucrose
221
221
221
281
281
281
Lactalbumin
180
180
180
0
0
0
50
50
50
50
50
50
Vitamin Mix**
5
5
5
5
5
5
Choline
2
2
2
2
2
2
100
100
100
100
100
100
Adenine
0
1
0
0
1
0
Guanine
0
1
0
0
1
0
Cytosine
0
1
0
0
1
0
Uracil
0
1
0
0
1
0
Yeast RNA
0
0
1
0
0
1
30
30
30
30
30
30
1000
1004
1005
1000
1000
1000
Mineral Mix*
Corn oil
(wesson)
Agar
Water
*
**
See table 2.
See table 3.
66
were performed on each organ.
Each incorporation study con-
sisted of four or five individual samples at different time
points.
For each of these samples RNA, DNA and protein were
determined in addition to isotope incorporation.
2.
Animals were killed in the laboratory instead of the
cold room.
3.
Ten microcuries of 14 C-glycine/gram of mucosa were
added to the flasks containing small intestinal mucosa.
4.
Twenty-five microcuries of
3 H-adenine/gm
mucosa were
added to the flasks containing colonic mucosa.
5.
After identification of the purine spots on chroma-
tography paper the spots were cut out and eluted with 0.1N HCl.
An aliquot was taken and O.D. 2 6 3 was determined for adenine,
and O.D. 2 7 5 for guanine.
Molar extinction coefficients for
those bases in O.1N HCl were calculated and micromoles of base
per ml were determined.
6.
Aliquots were removed directly from'the spectrophoto-
metric curvettes and placed in scintillation vials with 10 cc's
of ACS and counted.
7.
Incorporation was expressed as cpm/micromole base.
8.
In the re-feeding experiments incorporation was ex-
pressed at cpm/mg RNA.
9.
In the re-feeding experiments the mucosa from eight
animals was pooled for each incorporation experiment.
67
D.
The Measurement of Amidotransferase Activity in the Small
Intestine, Colon and Liver
Animals utilized in these studies were male 200 to 300
gram white CD rats obtained from Charles River Breeding Laboratories.
On arrival the animals were individually housed in
standard screen-bottomed galvanized cages measuring 25 cm X
18 cm X 18 cm.
They were maintained on standard Purina Rat Chow
or on the special diets described below.
with fresh tap water daily.
maintained at 220 C.
They were provided
The environmental temperature was
Room lighting was automatically controlled
to provide fourteen hours of light and ten hours of darkness.
Animals were decapitated in the cold room.
Their liver
and intestines were immediately removed to an iced-glass sheet.
The intestinal tract was flushed clear with ice cold saline.
The proximal 15 cm of the jejunum and the entire colon were
separated.
They were slit longitudinally and then mucosal
scrapings were obtained with a glass slide.
Approximately 1 ml
of scrapings was placed in a 10 X 75 mm test tube.
A portion of
the median lobe of the liver was minced with a sharp-pointed
scissors and approximately 1 ml of this material was similarly
placed into a test tube.
To the tissue in the test tubes was
added 2 ml of homogenization buffer (10 mM Tris-HCl pH 7.8,
0.05 mM DTT).
The cells and buffer were homogenized with a
Potter-Elvehem homogenizer (while still in the iced homogenizer
flask) with four strokes for the intestine and approximately
twenty strokes for the liver.
27,000 X g for thirty minutes.
The mixture was then spun at
The supernatant was poured off
68
and kept on ice.
The pellet was resuspended in 1 ml of homo-
genization buffer and again spun at 27,000 X g for thirty minutes.
The supernatant was added to the first fraction.
One ml of the
combined supernatant was passed through a column containing
Sephadex G-25 to remove nucleotides and other small molecular
weight compounds.
Upon collection from the column the enzyme
source was ready for assay.
Final conc. in reactionenzyme mixture
Reaction mixture:
Tris-HC1 pH 8.0
(100mM)
12.5
Il
DTT
(100mM)
2.5
pl
(10 0mM)
MgCl
2
PRPP
L-Glutamine*
Sp.Ac. 45 mCi/mM
25
mM
0.50
mM
2.75 p1
5.5
mM
(10mM)
1.5
p1
0.30
mM
(0.1 Pc)
0.5
P1l
0.044 mM
0.25 V1
DD H 2 0
20.00 pl
30.00 p1
Enzyme source
50.00 p1
*Glutamine as L-Glutamine (U-1 4 C) Lot # 784273, cat. # 10068,
ICN Pharmaceuticals, 2727 Campus Drive, Irvine, California 91664
The amidophosphoribosyltransferase activity was determined.
(The general method of Tay et al.
(1969) was employed.)
The reaction was initiated by the addition of 30 p1 of
the enzyme source.
The 50 pl were vortexed and placed in a
water bath at 30C for 20 minutes.
The reaction was terminated
by adding to the test tube 0.2 ml of ice cold 2 mM glutamate in
69
80%
(v/v) propan-2-ol at O'C.
placed on ice.
The mixture was vortexed and
100 pl was spotted on a 1
inch wide strip of
Whatman 3 mm chromatography paper along with 10 pl of 10 mM
glutamine (to serve as a marker).
The chromatography paper was
run in an ascending system using isopropranol:
water (40:2:10) as a solvent.
with 0.3% ninhydrin in acetone.
99% formic acid:
The strips were dried and sprayed
The glutamate spots were cut
1 ml
out so as to have an equal amount of paper in each sample.
of 0.1N HC1 was added to the cut strips in scintillation vials
to elute the glutamate.
Fifteen ml of scintillation fluid (ACS)
was added and the samples were counted.
The counting efficiency
was 60% as determined by the channels-ratio method.
Control assays were carried out without PRPP and the control
rate was subtracted to give the net "PRPP dependent" rate.
All
assays were carried out in triplicate.
E.
Additional Studies Related to, or Influencing Intestinal
Purine Metabolism
1.
Hypoxanthine-guanine phosphoribosyltransferase was
assayed by the method of Olsen and Milman (1978) modified as
detailed below.
70
Conc.
in
reaction-
'enzyme., mix
Reaction mixture
Assay buffer:
Tris-HC1 pH 7.8
MgC1 2
DTT
*PRPP
Hypoxanthine
**Hypoxanthine-1 4 C
500
mM
5
mM
20
mM
10
mM
10 Al
.6mM
10 pl
10 Pl
.luCi
DD H 2 0
50
mM
0.5
mM
2.0
mM
1.0
mM
.060mM
.024mM
10 ;J1
40
il
60 pl
Enzyme source:
100 pl
*PRPP (Sigma Chemical Company, St. Louis, Mo., cat. # P-9758)
**Hypoxanthine (8-1 4 C), Lot # 1025-198, New England Nuclear
The reaction is initiated when the reaction mix and the
enzyme source are mixed together, vortexed and placed in a
37 C water bath.
The reaction is terminated by placing the
test tubes in boiling water for one minute.
Thirty lambda is
then spotted on DEAE discs (Whatman DE-81) and allowed to dry.
The discs are then washed three times in 1 mM ammonium formate
for fifteen minutes each wash, then washed two times in distilled
and deionized water for fifteen minutes each wash, and finally
in 95% ethanol for fifteen minutes and allowed to dry.
When dry
the discs are placed in scintillation vials to which 10 ml of
scintillation fluid is then added (Toluene PPO-POPOP; 12.2 gm PPO,
71
.9 gm POPOP, 1 gal. toluene) and counted.
Counting efficienty
was 82% as determined by the channel-ratio method.
All deter-
minations were made in triplicate.
The effect of a self-emptying blind loop of intestine
2.
upon the activity of the de novo pathway was assessed.
Animals were prepared as in Section B of Materials and
Methods.
On the day of surgery animals received 40 mgm/kg
of Nembuton i.p. for anesthesia.
The animals' abdomens were
shaved and washed with tincture of merthiolate and EtOH.
Surgery was performed using clean but not sterile technique.
A midline longitudinal incision was made three to four cm long
with a pointed scissors. The abdominal organs were displayed.
The ligament of Treitz was located as a landmark.
Approximately
10 cm distal to the ligament of Treitz the intestine was transected with a sharp scissors.
The distal cut intestine was
sewn with a purse-string invaginating suture.
Distal to the
transection, 15 cm on the anti-mesenteric side of the intestine,
a three to five mm incision was made along the longitudinal
axis.
The proximal stump was affixed so that its mesenteric
and anti-mesenteric borders were transfixed precisely at the
angles of the longitudinal incision with guiding sutures.
The
two suture lines created were then closed with invaginating seromuscular stitches using 6/0 silk suture.
The position of the
viscera and the respective blood supply was examined and the
abdomen was closed with a single layer of interrupted 3/0 silk
sutures.
72
Following recovery from surgery the animals were placed
back in their individual cages with rat chow and tap water.
Two weeks later incorporation studies were performed as outlined in Section A of Methods and Materials.
3.
The placement of an indwelling intraperitoneal catheter
Most experiments have employed CD male rats weighing 200
to 300 grams.
The rats were housed in galvanized suspension
cages, one per cage.
The animals were anesthetized with ether.
A two cm longitudinal incision through the skin was made in the
dorsal midline of the neck.
A second two cm incision was made
through the skin in the midline of the ventral abdominal wall.
A 20 cm length of silastic tubing (Dow-Corning o.d. 0.085 inch
and i.d. 0.040 inch) which had been fit with a 1 X 1.5 cm rectangular silastic "butterfly" at the proximal end and glued with
silastic cement one cm from the end of the tubing was used as
the catheter.
The distal end was fitted with a silastic grommet
to which a silk suture had been sewn.
The tubing was grasped
firmly in the jaws of a goose-neck forceps which had been
tunneled subcutaneously through the abdominal incision until
it protruded through the neck incision.
The forceps were then
withdrawn pulling the catheter through the subcutaneous tunnel.
The peritoneum was entered and the catheter tip (with grommet)
was placed into the peritoneum.
The peritoneum was sewn over
the grommet using the attached silk suture as an anchor.
abdominal wound was closed.
The
The incision in the neck was then
closed over the butterfly apparatus on the proximal end.
One
centimeter of tubing was left protruding from the dorsal aspect
73
Two milliliters of normal saline were infused
of the neck.
through the line into the peritoneal cavity to make certain
that the line was patent and a small plug was then inserted in
the protruding open end of the catheter.
4.
Studies upon different intestinal cell populations
Because the crypt cell compartment of the small intestine
represents the proliferative zone it was clear that the optimal
tissue to use for incorporation studies would be a crypt cell
population.
Several techniques to obtain differential separa-
tion of crypt and villus tip cells have been reported (Weiser,
1973; Culling, 1973; Kimmich, 1970; Harrison and Webster, 1969;
Iemhoff, et al., 1970; Imondi, et al.,
Cornell and Meisters,
1969; Gall, et al.,
1971;
1976).
The major criteria for the work at hand was to develop a
method of reliably obtaining viable cells.
Gall, et al.
(1977) was utilized.
The method of
This method was a modifica-
tion of the work of Harrison and Webster (1969) and Iemhoff,
et al. (1970).
The technique was further modified as follows:
(1) The isolation solution employed was the Krebs-Ringer
bicarbonate solution used in all incorporation studies.
(2) During the cell separation the buffer was continuously
gased with 95% 02/5% C02.
(3) After centrifugation the cells were re-suspended
in the same buffer with help of a vortex mixer.
(4) An aliquot was treated with Trypan Blue to check for
viability.
(5) The remainder was used for incorporation experiments.
74
(6) Isotope was added for incorporation studies,
glycine 10 pCi/ml of cell suspension and for
1 4 C-
3 H-adenine
25 pCi/ml of cell suspension.
(7) The rest of the incorporation protocol was the same
as that outlined in Section A of Methods and Materials with the
only other exception being that because of the small amount of
tissue the DNA assay had to be done by the method of Cerrioti
(1955).
(8) Cell viability by Trypan Blue exclusion on repeated
assays showed that after re-suspension of the pellet approximately 50% of the villus tip cells were viable.
approximately 92% of the crypt cells were viable.
However,
75
V.
RESULTS
A series of experiments has been performed on rats to
determine the influence of dietary purines (nucleic acids,
nucleosides or bases) on the metabolism of the mucosal cells
of the gastrointestinal tract.
The relative importance of the
synthesis of purine nucleotides by the de novo and salvage
pathways was examined.
3 H-adenine
In order to evaluate the two pathways,
(or adenosine) uptake into mucosal RNA was used to
measure salvage and
1 4 C-glycine
incorporation into RNA was used to
measure de novo synthesis.
A.
In Vitro Studies of the De Novo and Salvage Pathways
In the first studies, the procedure of MacKinnon and
Deller (1973) was followed.
(1) The incorporation of
1 4 C-glycine
into the mucosal RNA
of everted small intestinal loops was compared to
1 4 C-glycine
incorporation into the RNA of finely diced liver tissue.
During
the sixty minutes of incubation there was a linear seven-fold
increase in incorporation into liver RNA.
In the small intestine
the increase was less than one and one-half times and not statistically different from the baseline reading (table 5, figure 5).
(2) The incorporation of
1 4 C-glycine
into mucosal RNA was
compared between everted loops of jejunum and everted loops of
colon.
To enhance the specific activity of the isotopic marker
and to minimize the possible role that pancreatic proteases and
ribonucleases might play, pancreatic duct ligations were performed
76
Table 5
Incorporation of 1 C-Glycine into the
RNA of Liver (finely diced)
and Small Intestine
(everted loops)
Time
Tissue
Liver*
10
148+40
243+60
Small
Intestine*
30
(min)
45
60
438+220 757+264 1018+381
335+103 323+85
363+64
Significance
p4.01
not sig.
w
L3
0
0
I
'1400
z
I
RAT LIVER
CHOW DIET
g1200
E
EIOQO
CL
I
I
I II
I
1
w
I
I
SMALL INTESTINE
CHOW DIET
z 0800
C-
600
0
z
w
z 200
A00
0
0
10
I
I
30
40
20
MIN UTES
INCORPORATION OF
I
50
60 0
I
I
10
20
I
I
30 40
MINUTES
50
I
C GLYCINE INTO THE RNA OF
SMALL INTESTINAL IUCOSA (EVERTED LOOPS) AND
INTO LIVER (FINELY DICED)
Figure 5
ij
60
mw
78
24 hours prior to the experiment.
In addition some animals
underwent sham surgery and served as controls.
Although the
pancreatic duct ligation resulted in an apparent increase in
incorporation into mucosal protein (figure 6, table 6) in both
tissues (when compared to the controls), the measurable incorporation into the mucosal RNA was nil (table 7).
(3) Using the identical system of everted loops of
intestine, the salvage pathway was assessed in the small
bowel and in the colon.
Again the experiment was carried out
with ligation of the pancreatic
which underwent sham surgery.
duct and there were controls
The results (table 8 and figure 7)
show that an active salvage pathway is demonstrable in both
tissues with the techniques employed.
It appears that the
demonstrable incorporation can be enhanced by performing pancreatic duct ligation 24 hours prior to the experiment.
(4)Because of the relatively low specific activity of
the
1 4 C-glycine
(98.7mC/mM) and because the incorporation into
the protein fraction was so low, the everted loop preparation
was dispensed with and mucosal scrapings were utilized for the
next set of experiments.
When mucosal scrapings were used
incorporation of labeled glycine into large intestinal mucosal
RNA was readily demonstrable.
However, significant incorporation
into small intestinal RNA could not be found (table 9 and
figure 8).
(5) When incorporation into the protein fraction of the
mucosal scrapings preparation was assessed, it was evident that
incorporation into both the small and the large intestine was
significant and similar (table 10 and figure 9).
79
Table 6
Incorporation of 14C-Glycine into Small and Large
Intestinal Mucosal Protein (everted loops) after
Pancreatic Duct Ligation or Sham Surgery
Time of incubation
10
Tissue
30
(min)
60
counts/min/mg RNA
Small*
Large*
Sham
76
94
118
Lign.
66
88
186
Sham
72
132
262
114
204
300
Lign.
*n=1
80
300
LIGAT ED
z
Ld
0
0r
CL
CONTROL
NTESTIN
200
LIGATED
SMALL INTESTINE
CONTROL
p
J00
al
0)
10
20
30
40
50
60
MINUTES
INCORPORATION OF
C GLYCINE INTO SMALL AND LARGE
INTESTINAL MIUCOSAL PROTEIN (EVERTED LOOPS)
AFTER PANCREATIC DUCT LIGATION
OR SHAM SURGERY
Figure 6
81
Table 7
Incorporation of 1 4 C-Glycine into Small and Large
Intestinal Mucosal RNA (everted loops) after
Pancreatic Duct Ligation or Sham Surgery
Time of incubation (min)
30
10
Tissue
60
counts/min/mg RNA
Small
(n=3)
Large
(n=2)
Sham
430+245
855+491
828+309
Lign.
446+187
400+28
414+176
Sham
709+144
1154+171
1105+290
742+109
884+254
Lign.
1035+74
82
Table 8
Incorporation of 3 H-Adenine into Small and Large
Intestinal Mucosal RNA (.everted loops) after
Pancreatic Duct Ligation or Sham Surgery
Time of incubation
10
Tissue
30
(min)
60
counts/min/mg RNA
Small*
Large*
Sham
3836
10825
18525
Lign.
9762
50670
32756
Sham
15934
14649
6321
8665
38075
24954
Lign.
*n=1
0
9
qP
I
SMALL
INTESTINE
LARGE
INTESTINE
15,000 -
16,000
4
zE'
z
LIGATED
LIGATED
1000
10,000
0
N
a-
a5,000
5,000
60
30
CONTROL
4a
CONTROL
10
0~
30
10
MINUTES
MINUTES
THE INCORPORATION OF 31 ADENINE INTO rUCOSAL RNA
OF EVERTED
Loops
OF SMALL AND LARGE
INTESTINE WITH PANCREATIC DUCT
LIGATION AND SHAM SURGERY
Figure 7
60
84
Table 9
Incorporation of 1 4 C-Glycine into Small and
Large Intestinal Mucosal RNA
(mucosal scrapings)
Time of incubation
10
Tissue
30
60
Significance
(CPM/mg RNA)
Small
*
1564+684
1209+190
4026+20
6020+85
2750+71
Intestine*
Large
Intestine*
*n =
2
-
-
15805+2553
Not sig.
pL.01
85
20000
I
I
I
I
I
I
INCORPORATION OF 14C GLYCINE
INTO PURINE BASES OF MUCOSAL RNACHOW DIET
(mucosal scrapings)
z
E
E
Q.
U
z0
LARGE
0
a. 10000
INTESTINE
z
z
0j 400
-
ISMALL
INTESTINE
-J
-I
2000
10
SI
20
I
I
30 40
MINUTES
THE INCORPORATION OF
RNJA
I
50
I
60
C-GLYCINE INTO THE
IN MUCOSAL SCRAPINGS OF SMALL
AND LARGE INTESTINES
Figure 8
86
I
Table 10
Incorporation of 14C-Glycine into Mucosal Protein of
the Small and Large Intestine (mucosal scrapings)
Time of incubation (min)
Tissue
10
30
60
counts/min/mg protein
Small
Intestine*
426
88,720
78,534
Large
Intestine*
20,852
27,640
69,504
87
90000
SMALL INTESTINE
83000--
7000000
400003 0000-
2~0000-.
10000-
10
30
MINUTE S
THE INCORPORATION
60
OF lC-GLYCINE INTO I'UCOSAL
PROTEIN OF SMALL AND LARGE INTESTINAL
UCOSAL SCRAPINGS
Figure 9
88
In view of the success in achieving satisfactory levels of
1 4 C-glycine
incorporation into mucosal protein when using
mucosal scrapings, the failure to accomplish this with the
everted loop preparation was attributed to the failure to
expose adequate numbers of cells, especially crypt cells, to
the isotope in the incubation media.
CONCLUSIONS
The final conclusion from this series of studies is
provided in experiments (4) and (5),
the results of which are
shown in tables 9 and 10 and in figures 8 and 9.
(figure 8) that only a small uptake of
1 4 C-glycine
It is apparent
occurs into
the RNA of mucosal scraping from the small intestine, contrasted
with a vigorous and prolonged uptake into the RNA of the large
intestinal mucosa.
In contrast, figure 9 shows no difference in
uptake into the protein of these two mucosal samples.
This
latter finding implies that differences in labeling of the
free glycine pool are unlikely to account for the low uptake of
1 4 C-glycine
into RNA.
This finding thus confirms and extends
the observations of MacKinnon and Deller (1973).
B.
The Fate of Oral versus Parenteral Adenine and Adenosine
Without an active pathway of de novo biosynthesis in the
small intestine, the possible use of dietary purine by the salvage pathway was considered.
Absorption of adenine and adenosine was examined in two
groups of twenty animals each.
Two dietary regimens
were
89
a regular Purina Rat Chow diet and a synthetic agar-
employed:
based 0% protein and 0% purine diet.
The design of the experiments is illustrated in figure 10.
The twenty rats were divided into two groups of ten and then
assigned to one of the diets.
were given 45 microcuries of
After thirty-one days, the animals
3 H-adenine
(or
3 H-adenosine)
by nasogastric gavage or by intraperitoneal injection.
either
The
animals were fasted overnight and then sacrificed fourteen to
twenty hours after receiving isotope.
Incorporation of label into the RNA of the entire gastrointestinal tract and liver was measured.
activity was expressed as cpm/mg RNA.
The tissue radio-
Within each tissue diff-
erences in incorporation were evaluated by one-way analysis of
Between different tissues graphic analysis was em-
variance.
ployed to illustrate differences as well as two-way analysis of
variance and testing for multiple comparisons (Tukey test).
Oral isotope - regular diet
In animals that were maintained on the chow diet but
received
3 H-adenine
by gavage the incorporation into the liver
was 2600 cpm/mgm RNA.
The distal small intestine (2700 cpm/mgm
RNA), caecum (2600 cpm/mgm RNA), proximal colon (1500 cpm/mgm RNA),
and distal colon (2100 cpm/mgm RNA) all incorporated label to a
similar extent.
The proximal small intestine (14,000 cpm/mgm RNA),
and the mid small intestine (15,000 cpm/mgm RNA) both incorporated
the label to a significantly (P(
.05)
greater extent than did
the distal small intestine, caecum, colon or liver (figure 11 and
table 11).
90
Study Design for Investigation of the Fate
of Absorbed versus Injected
Adenine and Adenosine
40 - 200 Gram White Charles River CD rats
3
20
20e
H-Adenosine)
(H-Ad enine )
10
10
Regular Chow
x 31 days
0% protein synthetic
agar based diet
x 31 days
5
i.p. isotope
5
p.o. isotope
fasted overnight
14-20 hours sacrificed
sto ach
1 2 3 -
dist.
p ox. mid.
small intestine
caecum
dist.
pr Ix.
colon
liver
assay RNA
assay CPMs/mg. RNA
analyze within each tissue by one-way analysis of variance
THE DESIGN OF THE EXPERIMENT TO DETERMINE THE FATE OF
ABSORBED
VERSUS INJECTED ADENINE AND ADENOSINE
Figure 10
0
0
0
45000
I
I
I
40000-
I
I
I
I
P
D
REGULAR DIET
Po. 3 H
ADENINE
35000-
z<r
3000025000-
E
E 20000-
15000
1000050001I
S
I
I
P
.
M
D
C
L
THE INCORPORATION OF GAVAGED 31-ADENINE INTO THE GASTROINTESTINAL
TRACT OF ANIMALS ON A REGULAR CHOW DIET
Figure 11
92
Table 11
3 H-Adenine
Incorporation cp/mg 1NA
Stan.
Small Intestine
Prox.
Mid.
Dist.
1
17421
39941
2
3
4
5
37757
77836
9386
5829
34866
63784
x
30014
+11002
6
7
8
9
10
Caecum
Prox.
Colon
Dist.
Colon
Liver
6067
3097
1826
3002
2704
3478
2178
1623
1732
2482
30666
5312
7137
7157
2116
2467
2642
2828
1976
3592
1001
3233
60520
+19157
12798
+12049
5864
+1914
2353
+506
2913
+385
2342
+864
2112
+961
35887
43291
20319
14956
16184
17314
5921
16364
16601
28145
4051
24138
10328
9050
1634
1251
1652
7582
1509
2099
1580
5489
3004
784
1114
1343
493
4152
384
2113
1573
2315
2372
2250
2019
3841
2656
1480
2727
x
26128
+12719
14051
+5435
15142
+10408
2726
+2719
2591
+1810
1498
+1538
2125
+323
2544
+885
11
12
0%
13
Prot. 14
Diet 15
287321
41978
152971
25664
17236
11499
14557
9279
9121
11777
9786
9283
12723
10813
47505
12224
7066
9510
12498
5711
29564
56561
16056
8109
69313
32430
21779
53562
6243
14871
7448
8363
-
-
126834
+121102
13143
+3483
Oral
0%
Prot.
Diet
Oral
Beg.
Diet
I.P.
x
I.P.
Reg.
Diet
-
9992
+1224
-
20816
+17811
-
8696
+2982
-
27573
+21258
-
44271
+21288
-
-
9231
+3854
-
16
-
-
-
-
-
17
18
19
20
8451
25754
43028
87919
4039
4794
6649
3290
1291
1500
7098
6525
1181
1817
5567
9730
4221
1173
1868
1530
1488
2859
1635
1739
841
837
4877
11034
795
863
5244
7805
x
41288
+34142
4693
+1441
4163
+3137
4574
+3945
2198
+1378
1430
+628
4397
+4817
3677
+3451
-
93
A second group of animals (figure 12 and table 12) which
were also maintained on a chow diet but which received
3 H-adenosine
by gavage had incorporation into the liver of
3200 cpm/mgm RNA.
The proximal small intestine (3400 cpm/mgm
RNA) -and the mid small intestine (4400 cpm/mgm RNA) both had
greater incorporation than did the rest of the gastrointestinal
tract but the differences were not statistically significant.
(The stomach incorporation was 44,000 cpm/mgm RNA.
Parenteral isotope - regular diet
A third group of animals maintained on the chow diet
and injected intraperitoneally with
3 H-adenine
demonstrated
incorporation into the liver of 3700 cpm/mgm RNA.
exception of the stomach (41,000 cpm/mgm RNA),
With the
all other tissues
had similar levels of incorporation (table 11, figure 13).
The average incorporation into the liver in a fourth
identical group of animals injected with adenosine was 2000 cpm/
mgm RNA, and similarly the other tissues (again with the exception
of the stomach, which was 22,000 cpm/mgm RNA) all incorporated
isotope essentially to the same extent (table 12, figure 14).
Thus, animals fed a chow diet and injected with
3 H-adenine
(or adenosine) demonstrated uniform incorporation in all gastrointestinal tissues except the stomach (see below for discussion
of the stomach).
The route of incorporation was clearly via
the blood stream, after absorption of the intraperitoneal in-
jection.
Animals on similar chow diets but who received isotope via
gavage had similar findings.
There was, however, significantly
v
v
0
0
0
45000
±38000
40000
REGULAR DIET
Po. 3 H ADENOSINE
35000
4
z
30000
0'
: 25000
E
L
E 20000
15000
10000
5000
S
P
M
P
D
THE INCORPORATION OF GAVAGED
3 H-ADENOSINE
D
INTO THE GASTROINTESTINAL
TRACT OF ANIMALS ON A 0% PROTEIN DIET
Figure 12
L
95
Table 12
3 H-Adenosine
Incorporation cpmVmg RNA
Prox.
Dist.
Colon
Colon
Liver
3092
2086
3020
1452
847
2850
2392
1605
3533
1124
5366
2032
1778
1345
2652
4170
4395
5140
2213
2444
9565
+2091
2094
+977
2301
+961
2635
+1599
3673
+1280
6075
2581
3879
3798
7947
995
909
1275
2406
2540
1021
1651
779
792
5645
912
563
706
886
1790
696
1070
860
1071
4477
2232
1721
906
4089
6857
3352
+776
4856
+2138
1625
+787
1978
+2081
971
+979
1735
+1819
3161
+2374
31 50448
32 24795
33 19247
34 152648
35 216835
6266
10817
5021
6851
14313
7620
15324
11766
10558
8593
11584
8668
9818
5815
6273
3947
7333
8230
11505
5730
12299
22181
23706
14322
16227
46753
34081
45677
34408
3755
6852
5925
2569
4008
x
93794
+86797
8654
+3836
10772
+3018
8432
+2420
7754
+3100
16648
+7512
35429
+12297
4618
+1727
36
37
38
39
40
15435
13701
22885
23725
32091
2170
3747
2573
2952
1411
2740
2030
2263
1489
1796
5871
1966
2265
4072
1834
1460
2611
2579
1670
4176
1762
1651
3847
3884
817
3393
7257
2230
5173
1107
2056
2107
2758
2057
1389
x
21567
+7359
2071
+720
2164
+363
3202
+1743
2449
+1072
2393
+1395
3832
+2435
2073
+484
Small Intestine
Oral
0%
Diet
Oral
Reg.
Diet
Mid.
Dist.
Caecum
66287
126885
117848
84036
48000
32867
17587
73911
6485
5994
10654
7855
12609
7584
9122
10585
+2749
88611
+33497
27369
+2818
26 18462
27 108000
28 47188
29 13365
30 34750
2338
4372
2983
3795
3274
44353
+38019
Stcrn.
Prox.
21
22
23
24
25
8872
10202
4628
8826
15396
x
x
I.P.
0%
Diet
I.P.
Reg.
Diet
9
0
4500C
I
40000
I
I
I
0
9
I
i
I
+34000
REGULAR CHOW DIET
35000
z<r
I P INJECTED
3H
ADENINE
3000025000-
E
E 20000-
150001000050001-
-I
s
P
M
D
THE INCORPORATION OF INJECTED
C
D
P
3 H-ADENINE
INTO THE
GASTROINTESTINAL TRACT OF ANIMALS ON
A REGULAR CHOW DIET
Figure 13
L
0
9
45000
-r 11
--
40000
--
I
I
REGULAR DIET
I. P INJECTION OF
3 H ADENOSINE
35000
z
0
0
30000
<r-
E
25000
E
'.0
E 20000
CL
U
15000
10000
5000
S
P
M
C
D
THE INCORPORATION OF INJECTED
P
3 H-ADENOSINE
D
INTO THE
GASTROINTESTINAL TRACT OF ANIMALS ON A
REGULAR CHOW DIET
Figure 14
L
98
(P/ 0.05) greater incorporation of
3 H-adenosine)
3 H-adenine
(but not
into the proximal and middle small intestine
than into the rest of the gastrointestinal tract.
Thus, under
the conditions of this experiment, adenine was significantly
incorporated into proximal small intestinal RNA after absorption
from the intestinal lumen.
Oral isotope - protein-free diet
A fifth group of animals which were maintained on the
synthetic agar-based protein-free and purine-free diet but which
were treated with
3 H-adenine
per os demonstrated a pattern of
incorporation that differed markedly from the earlier patterns
described (table 11, figure 15).
RNA was 2100 cpm/mgm RNA.
mgm RNA),
Incorporation into the liver
Incorporation into the caecum (2400 cpm/
the proximal colon (2900 cpm/mgm RNA), and the distal
colon (2400 cpm/mgm RNA) did not significantly differ from the
liver.
Incorporation into the proximal small intestine (61,000 cpm/
mgm RNA), the middle small intestine (13,000 cpm/mgm RNA) and the
distal small intestine (5900 cpm/mgm RNA) were all strikingly
(P<0.05) increased above the rest of the gastrointestinal
tract.
Incorporation into the gastric mucosal RNA was 30,000 cpm/
mgm RNA.
A sixth group of animals was maintained on an agarbased, protein-free and purine-free diet but was treated
with
3 H-adenosine
(instead of
3H-adenine
as was the fifth
6
9
0
50000
I
i
64000
±17000
I
0
--- T
0%
45000-
PO.
0
I
'4
D
L
PROTEIN DIET
3
H ADENINE
40000-
35000-
z 30000 _
E 25000E
0.
0
2000015000IOOOO-
5000II--
1-
S
P
D
M
THE INCORPORATION OF GAVAGED
I
C
3 H-ADENINE
P
INTO THE GASTROINTESTINAL
TRACT OF ANIMALS ON A 0% PROTEIN DIET
Figure 15
0
U-1
S
SS
50000
I
45000-
I
i
89000
33000
I
I
I
0% PROTEIN DIET
Po.
3
H ADENOSINE
40000-
35000-
z
30000-
E 25000E
2000015000OOO5000ki
S
i
I
P
.
M
I
D
I
C
P
I
D
L
THE INCORPORATION OF GAVAGED 3H-ADENOSINE INTO THE GASTROINTESTINAL
TRACT OF ANIMALS ON A 0% PROTEIN DIET
Figure 16
101
group discussed immediately above).
the
3 H-adenosine
The group received
per os and demonstrated a pattern of
incorporation that was essentially identical to that of
the fifth group.
Specifically, the liver incorporation
was 3700 cpm/mgm RNA, the caecum was 2100 cpm/mgm RNA,
the proximal colon was 2300 cpm/mgm RNA and the distal
colon was 2600 cpm/mgm RNA.
corporated
extent.
3 H-adenosine
All of these tissues in-
into mucosal RNA to the same
The incorporation into the proximal small in-
testine was 89,000 cpm/mgm RNA, into the middle small
intestine 27,000 cpm/mgm RNA and into the distal small
intestine 9600 cpm/mgm RNA.
Parenteral isotope - protein-free diet
A seventh group of animals maintained on the synthetic protein-free and purine-free diet received
adenosine intraperitoneally.
3H-
The pattern of incorpor-
ation into the RNA was completely different from any of
the earlier experiments (table 12, figure 17).
The incorporation into proximal small intestine
0
6
0
50000
I
I
6
w
w
w
Tf
I
99000
45000-
87000
00% PROTEIN DIET
I P INJECTION OF
40000-
3
H ADENOSINE
35000-
z
30000-
E 25000E
CL
0
20000150001oooo
5000[
I
S
I
P
I
I
M
D
THE INCORPORATION OF INJECTED
C
3 H-ADENOSINE
I
I
P
D
INTO THE GASTROINTESTINAL
TRACT OF ANIMALS ON A 0% PROTEIN DIET
Figure 17
L
w
103
(8700 cpm/mgm RNA), middle small intestine (10,800 cpm/mgm RNA),
distal small intestine (8400 cpm/mgm RNA) and caecum (7800 cpm/
mgm RNA) were somewhat greater than the incorporation into the
liver (4600 cpm/mgm RNA).
The proximal colon (17,000 cpm/mgm RNA)
and the distal colon (35,000 cpm/mgm RNA) however both demonstrated markedly greater incorporation than did the rest of the
gastrointestinal tract.
This unexpectedly high level of in vivo incorporation of
nucleoside into the colonic mucosal RNA was essentially mimicked
in an eighth group of animals identical to the seventh, except
that they received
3H-adenine
instead of
3 H-adenosine
(table 11,
figure 18).
COMPARISON OF GROUPS
The results of these experiments have been expressed in
a series of tables (tables 11 to 12) and graphs (figures 19 to 34)
which compare the incorporation of isotope in various tissues
within a particular dietary and treatment group.
An examination
of this data utilizing the four groups of diets within a particular organ was performed, comparing the results by one-way analysis
of variance.
Stomach
In the stomach there is no statistically significant
difference in incorporation into mucosal RNA among all groups
(figures 19 and 20).
Proximal Small Intestine
In the proximal small intestine there was a highly significant variance component among the groups for both adenini
Rw
w
qw
50000
w
w
I
I
45000~
130000
± 12 1000
40000-
v
w
w
I
I
0 % PROTEIN DIET
I P INJECTION OF
3 H ADENINE
I
I
w
I
Ii
2
35000-
z
I
30000-
E 25000E
20000-
1500010000 5000[S
I
P
M
THE INCORPORATION OF INJECTED
D
I
C
3 1-ADENINE
|I
P
18
I
L
INTO THE GASTROINTESTINAL
TRACT OF ANIMALS ON A 0% PROTEIN DIET
Figure
|I
D
w
105
0
H3
ADENINE
150,000
z<
100,000
50,000
00
8
o
0
00
P.O.
O%PROTEIN
P.O.
REGULAR
DIET
DIET
I.P
IP.
STOMACH ( MUCOSA)
THE INCORPORATION OF 3 H-ADENINE INTO THE
MIUCOSAL RNA OF THE STOMACH
Figure 19
106
200000
-
0
H3 ADENOSINE
-
150.1000
0
z
100,000
-
o
-
50,000
I.
P.O.
OT.PROTEIN DIET
STOMACH
I.P.
Po.
REGULAR DIET
CMUCOSA
)
THE INCORPORATION OF 3 1I-ADENOSINE
THE MUCOSAL
RNA
OF THE STOMACH
Figure 20
INTO
107
and adenosine.
Examination of figures 21 and 22 clearly shows
the striking elevation of incorporation when the isotopes are
ingested by animals on the experimental diet.
Again the results
are similar whether the base or the nucleoside is employed.
The animals who received adenine even though on the regular
diet also had significantly increased incorporation of isotope
when the oral route was compared to the parenteral route.
Middle Small Intestine
The results in the middle small intestine were essentially
the same as those for the proximal small intestine but to a
lesser degree, being statistically significant only for the
adenosine group (PLC0.05) (figures 23 and 24).
Distal Small Intestine, Caecum, Proximal Colon, Distal
Colon and Liver
The distal small intestine (figures 25 and 26),
caecum
(figures 27 and 28), proximal colon (figures 29 and 30),
and
distal colon (figures 31 and 32) all have a similar pattern of
incorporation to that found in the liver (figures 33 and 34).
That is, greater incorporation when label was injected parenterally as opposed to when it was administered per os.
CONCLUSIONS
The presence of a salvage pathway in mucosa of the small
intestine was demonstrated in vivo
istration of
3 H-adenine
and
following the oral admin-
3 H-adenosine,
which resulted in
uptake of the label by the RNA of the small intestinal mucosa.
The finding that this was much greater on a diet devoid of
purines suggests that exclusion of a normal dietary source of
108
15000
-
-1
0
-
H 3 ADENINE
100,000
z
cr
0
T
504000 H
C
1.P.
P.O.
0% PROTEIN
DIET
PROXIMAL
THE
RNA
I.P.
P.O.
REGULA R
DIET
SMALL INTESTINE
INCORPORATION OF
MUCOSAL
t
3 H-ADENINE
INTO THE
OF THE PROXIMAL
SMALL INTESTINE
Figure 21
109
150,000
H3 ADENOSINE
0
0
z
100,00
502001
0
I.P.
1.2
RP0.
0% PROTEIN
DIET
PROXIMAL
REGULAR
DIET
SMALL
THE INCORPORATION OF
P.O.
INTESTINE
3 H-ADENOSINE
INTO
THE MUCOSAL RNA OE THE PROXIMAL
SMALL INTESTINE
Figure 22
110
r
60400
ADENINE
H3
-
0-
-
z 40,00
0
-
20000
IP.
P.O.
REGULAR
DIET
I.P.
P.O.
C / PROTEIN
DIET
MID SMALL INTESTINE
THE INCORPORATION OF
rIUCOSAL
RIJA
3 11-ADENINE
OF THE MIDDLE
SMALL INTESTINE
Figure 23
INTO THE
111
H3
-
ADENOSINE
60.00
z
40,000
-0
-
0
-
20O0DO
I.P
P 0.
0/ PROTEIN
DIET
I.p
P0
REGULAR
DIET
MID SMALL INTESTINE
THE INCORPORATION OF 31-ADENOSINE INTO THE
MUCOSAL
RNA
OF THE
MIDDLE SMALL INTESTINE
Figure 24
112
H3 ADENINE
..
.
20D00
0
0
o
-
10,000
CL
0.
0
U
-
P 0.
I.P
0% PROTEIN
I.P.
P.O.
REGULAR
DIET
DIET
DISTAL SMALL
INTESTINE
THE INCORPORATION OF 3 H-ADENINE
THE r'UCOSAL
INTO
RINA OF THE DISTAL
SMALL INTESTINE
Figure 25
113
H3 ADENOSINE
<20,000
cr
00
o
10,000C-b
P0.
I.P
REGULAR
DIET
P.O.
I.P.
0% PROTEIN
DIET
DISTAL
SMALL
INTESTINE
THE INCORPORATION OF 3H-ADENOSINE INTO
THE MIUCOSAL
RNA
OF THE DISTAL
SMALL INTESTINE
Figure 26
114
H3 ADENINE
10,000
5,000 -
1
0)
p P.O.
0%PROTEIN
DIE T
.
PO.
REGULAR
DIET
CAECUM
THE INCORPORATION OF
THE MUCOSAL
RNA
3 H-ADENINE
INTO
OF THE CAECUM
Figure 27
115
0
H3 ADENOSINE
< 10,0
z
0
0DO
0
--
00
.P
0/
P 0.
0PROTEIN
DIET
I.P.
P 0.
REGULAR
DIET
CAECUM
THE INCORPORATION OF
MUCOSAL
RNA
3 H-ADENOSINE
OF THE CAECUM
Figure 28
INTO THE
116
30,000 -
0
H3 ADENINE
20Z0
CL 10,000
-C)
.P P.O.
0% PROTEIN
DIET
PROXIMAL
THE INCORPORATION OF
PC.
I.P
REGULAR
DIET
COLON
3 H-ADENINE
THE MUCOSAL RNA OF THE
PROXIMAL COLON
Figure 29
INTO
117
30000
H3 ADENOSINE
<20,00
-8
z
0,00
CL
I.P.
PO.
o % PROTEIN
DIET
PROXIMAL
THE INCORPORATION OF
I.P
P. O.
REGULAR
DIET
COLON
3 H-ADENOSINE
INTO THE
MUCOSAL RNA OF THE PROXIMAL COLON
Figure 30
118
H3 ADENINE
60,000
-
Z
40,000
0
_
20,000
4,000
I.P
P.O.
0%PROTEIN DIET
DISTAL
THE INCORPORATION OF
JIUCOSAL
RNA
I.P
P.O.
REGULAR DIET
COLON
3 H-ADENINE
INTO THE
OF THE DISTAL DIET
Figure 31
119
H3 ADENOSINE
60000
z
cr
40,000
20,000
0
4,000
.p
P.O.
O/ PROTEIN DIET
DISTAL
THE INCORPORATION OF
MUCOSAL
RNJA
0
0
I.P
P.O
REGULAR DIET
COLON
3 H-ADENOSINE
INTO THE
OF THE DISTAL COLON
Figure 32
120
1500
H3 ADENINE
-
z 10,000
0
0
5200
CL
0
0
I.p
0% PROTEIN
T7
T
P0.
DIET
1.RP
-
o.
REGULAR DIET
LIVER
THE INCORPORATION OF
3 H-ADENINE
INTO THE
MUCOSAL RNA OF THE LIVER
Figure 33
121
H3 ADENOSINE
0,000 Iz
c)
5,000
0
F 18
mTtt
LP.
I.P.
PO.
0% PROTEIN DIET
P. 0.
REGULAR DIET
LIVER
THE INCORPORATION OF
3 H-ADENOSINE
RNJA
OF THE LIVER
MUCOSAL
Figure 34
INTO THE
122
purines resulted in less dilution of the labeled base.
This is
accordingly evidence that dietary purines are normally an important source of purines for the mucosa.
When the protein and purine depleted animals were studied,
there was significant (P L 0.05) utilization of gavaged isotope
by the proximal portion of the small intestine.
The caecum,
colon, and liver demonstrated an amount of incorporation clearly
attributable to the vascular supply.
The high level of small
intestinal incorporation was not the result of an increased
extraction by that tissue of vascular purines.
This is demon-
strated in the studies of identical animals who received isotope
by intraperitoneal injection.
In these latter studies, the
small intestine demonstrated incorporation essentially at the
same level as the liver.
Hence, the small intestine has no
unusual ability to extract vascular purines.
Striking, however,
is the extraordinary incorporation of vascular purines via the
colon.
It is concluded that the small intestine has no unusual
ability to utilize vascular purines.
When compared to the colon,
the small intestine even appears defective in its ability to
utilize purines from the vascular supply.
The unexpectedly large amount of isotope incorporated
into the mucosal RNA of the stomach is evidence of an extremely
active salvage pathway.
The stomach's ability to utilize purines
for the vascular supply appears to be the greatest of any gastrointestinal tissue.
123
C.
Influence of the Protein and Purine Content of the Diet
As a result of the above data, the role of dietary-purine
sources in relation to mucosal supply was further explored in
mucosa of the small intestine.
(1) The effect of dietary protein and purine content upon
the activity of the de novo pathway of purine nucleotide biosynthesis in the small intestine was investigated.
Groups of
four rats were placed on one of six experimental diets (table 4).
Diet # 1 was an 18% lactalbumin diet, free of purines; diet # 2
was identical to # 1 except that it contained 0.1% adenine,
0.1% guanine, 0.1% uracil and 0.1% cytosine; diet # 3 differed
from diet # 1 in that it contained 0.5% yeast RNA; diets # 4,
# 5, and # 6 were analogous to # 1, # 2, and # 3 respectively
except that they contained no protein.
After seven days on their respective diets, the animals
were sacrificed and intestinal mucosal scrapings were utilized
1 4 C-glycine
to determine in vitro incorporation of
protein and mucosal RNA.
into mucosal
RNA/DNA and protein/DNA ratios were
also measured.
The assumption is made that the intracellular pool of
14 C-glycine available for RNA synthesis via the de novo pathway
is essentially the same pool from which glycine is incorporated
into protein.
Furthermore, to the extent that the pools might
be different it is assumed that they
tionately.
vary. quickly and propor-
Thus any factor that tiight affect
1 4 C-glycine
uptake
by the cell will similarly affect incorporation into both RNA
and protein.
Therefore any effect upon
1 4 C-glycine
uptake and
124
resultant labeling of RNA can be eliminated by expressing
1 4 C-glycine
incorporation into RNA as a ratio of incorporation
of label into RNA/protein.
Thus a ratio of
1 4 C-glycine
incor-
porated into RNA (or RNA purine) to isotope incorporated into
protein was used to assess activity of the nucleotide synthesis
pathway in the various experiments.
Animals on the complete diet lacking only purines demonstrated significantly higher (P L 0.002) incorporation of
1 4 C-glycine
into mucosal RNA than did any of the groups of
animals on the other diets.
The ratio of incorporation into
adenine (isolated from mucosal RNA) to protein was similarly
significantly higher (P 4 0.01) for the group of animals of
diet # 1 (table 13).
Omission of purines from the synthetic diets used in these
studies resulted in restoration of some in vitro labeling of
mucosal RNA adenine from
1 4 C-glycine
changes in protein labeling.
without corresponding
Additionally the RNA/DNA ratio
is significantly reduced by adding free purine bases to the diet
whereas protein/DNA is reduced on the protein-free diet irrespective
of purine sources.
smaller.
This latter data implies that the cells are
The reduction in RNA/DNA on adding purines to the diet
could conceivably be the result of stimulation of mucosal cell
multiplication by the dietary purines with resulting increased
DNA.
(2) In another experiment the effect of dietary purine
content upon recovery from a protein-free diet was assessed.
Three groups of eight rats each were placed on diet # 4,
125
Table 13
Influqoe of Protein and Purine Content of the Diet upon
-''C-Glycine Incorporation into Smadl Intestinal
Mucosal Protein and Mucosal RNA
1 4 C-Glycine
Diet
Incorporation
RNA-Adenine
(cpVumloe)
(n=2)
Protein
(Cxng/n)
(n=2)
Aden. Incorp.
Prot. Incorp.
114750+15900
3200+ 170
35.5+ 3.5
2.30+ .20
26.29+ 3.76
BNA/DNA
Prot/DNA
(n=8)
(n=8)
Protein-containing
1.
No purines
2.
+ purine bases
11500+16300
1900+1100
4.4+ 6.2
1.51+ .42
24.86+ 3.98
3.
+ RNA
21700+13500
3700+ 250
6.5+ 5.2
2.01+ .28
27.65+ 9.94
26300+15800
2500+1100
13.9+11.1
2.12+ .19
23.92+ 3.41
14600+ 4400
5000+1600
3.9+ 0.3
1.48+ .13
16.68+ 6.18
37300+26000
8000+1440
5.0+ 4.2
2.68+1.02
17.28+12.06
p,0.002
not sig.
p4.01
pO0.001
Protein-free
4.
No purinas
5.
+ purine ba
6.
+ RNA
Significance
s
p<O. 05
126
a purine-free and protein-free synthetic diet, for seven days.
They were then switched to either diet # 1, @ 2, or # 3 for
seven days.
Overall
The same analyses were carried out as in (1) above.
incorporation was much lower in this experiment.
When incorporation of isotope into total mucosal RNA was
assessed and expressed as the ratio, incorporation into RNA:
incorporation into protein, the results were similar to those
obtained in (1).
Specifically, the incorporation ratio was
greater (P = 0.09) in the group that was re-fed the purinefree diet (table 14).
(3) The effect of purines and protein in the diet upon the
salvage pathway of the colon was assessed.
Diets # 1 through
# 6 as above were employed and after seven days the animals were
sacrificed.
Incorporation studies employing
3H-adenine
and
measuring its appearance as either RNA adenine or RNA guanine
were performed.
No significant difference in incorporation
into either adenine or guanine or their sum could be demonstrated
(table 15).
(4) The effect of dietary purines and protein upon the refeeding of a protein-depleted animal was assessed.
seven days of a 0% protein diet (diet # 4),
Following
the animals were
placed on either diet # 1, # 2, or # 3 for seven days and then
had repeat incorporation studies.
The incorporation of
3 H-adenine
into RNA guanine was not significantly different in the three
groups studied (table 16).
127
Table 14
Influence
upon
Protein and Purine Content of the Diet
C-Glycine Incorporation into Small
Intestinal Mucosal Protein and
Mucosal RNA during Refeeding
1 4 C-Glycine
RNA Incorporation
Protein Incorporation
Protein
cnVmg RNA
Incorporation
RNA
cpm/g RNA
(n=2)
(n=2)
1. Protein diet
alone
362+ 16
6076+1774
16.7+0.4
2. Sane + purine
bases
588+220
3181+2950
4.8+3.2
3. Same + IqA
409+ 93
2397+1670
5.5+2.8
Not sig.
Not sig.
Protein-free diet
followed by *
Significance
*7-day refeeding period
Not sig.
(p=0. 0 9 )
128
Table 15
Influence of Protein and Purine Content of Diet
upon 3 H-Adenine Incorporation into
Colonic Mucosal RNA
3 H-Adenine
Diet
Incorporation
into RNA-Guanine
RNA/DNA
Prot/DNA
cpn/umole
(n = 8)
(n = 8)
(n = 2)
Protein-containing
1. No purines
13254+ 5741
1.0 +0.4
26+ 9
2. + purine bases
15796+ 3384
1.2 +0.5
27+16
3. + RNA
25721+18344
2.0 +0.9
25+30
8268+ 2740
1.2 +0.2
20+ 6
22749+17673
1.3 +0.5
35+20
9251+ 2210
0.83+0.4
20+10
not sig.
not sig.
not sig.
Protein-free
4. No purines
5. + purine bases
6. + RNA
Significance
129
Table 16
Influence of Protein and Purine Content of Diet
upon
3 H-Adenine
Incorporation into
Colonic Mucosal MA during Refeeding*
3 H-Adenine
Protein-free diet
followed by
Incorporation
into RNA-Guanine
cpm/unnle
RNA/DNA
(n = 10)
Prot/DNA
(n = 10)
(n = 2)
1. Protein diet
alone
3321+3420
0.50+0.16
10.5+2.2
2. Same + purine
bases
8951+9044
0.61+0.25
10.4+2.1
12391+4580
0.73+0.32
9.3+3.7
not sig.
not. sig.
not sig.
3. Same + RNA
Significance
*7-day refeeding period
130
CONCLUSIONS
Animals receiving diets containing purine bases or RNA had
low de novo synthesis as judged by 14 C-glycine incorporation
into RNA.
However, with diets deficient in purines, labeling
by the de novo
pathway was much increased, especially when the
diet contained protein.
This is apparently independent of
labeling changes in the precursor pool of free glycine, since
protein labeling did not show these changes.
This suggests that
some stimulation of the de novo pathway in the small intestinal
mucosa occurs when purine sources are withdrawn from the diet.
Free purines may have an additional effect on cell division in
the crypt, since the RNA/DNA ratio and the protein/DNA ratio
both fall for ahiials fed purines as con'pared to those fed no
purines.
D.
The Activity of the Glutamine Amidophosphoribosyl Transferase
in the Small Intestine, Colon and Liver and the Effect
of Dietary Purines
(1) Validation of the glutamine-amidophosphoribosyl-transferase
assay
The method of Tay et al.
and Methods (page 68)
Experiment # 1:
(1969) as detailed in Materials
was utilized.
To test the effects of substrate concentra-
tions upon demonstrable liver enzyme activity the concentrations
of PRPP and
1 4 C-glutamine
were altered.
A grid pattern was con-
structed varying the amount of the PRPP in the reaction mixture
as follows:
0 mM, 0.3 mM, 0.6 mM, 1.2 mM, and 2.4 mM.
The gluta-
mine was varied utilizing 0.022 mM (0.05 pcurie), 0.044 mM (0.1
131
pcurie),
0.088 mM (0.2 pcurie)
and 0.17 mM (0.4 pcurie).
The amount of glutamate formed was directly proportional to the amount of glutamine used as substrate at
PRPP concentrations (table 17, figure 35) over the entire
range of glutamine concentrations employed.
The amount of
glutamate formed was also directly proportional to the amount
of PRPP used up to the concentration of 0.6 mM PRPP.
With
greater amounts of PRPP the amount of glutamate formed reached
a plateau (table 18,
figure 36).
For the experiments performed 0.3 mM PRPP (figure 37)
and 0.044 mM
employed.
1 4 C-glutamine
(0.1 pcurie) (figure 38) were
(Table 19 contains all the data in this experiment
and figure 39 represents that data on a single plot.)
Experiment # 2:
To test the effects of substrate con-
centrations upon demonstrable small intestinal enzyme activity,
the identical procedure was followed as in Experiment # 1.
The
amount of glutamate formed was directly proportional to the
amount of
1 4 C-glutamate
used as substrate at all PRPP concen-
trations (table 20, figure 40).
The amount of
1 4 C-glutamate
formed was proportional to the concentration of PRPP when
greater than 0.044 mM (0.1 pcurie)
1 3 C-glutamine
was used, and
then only when the concentration of PRPP did not exceed 0.6 mM
(table 21, figure 41).
For the experiments performed 0.3 mM PRPP (figure 42) and
0.044 mM 14C-glutamine (0.1 pcurie) (figure 41) were employed.
(Table 22 contains all the data in this experiment and figure 43
represents that data in a single plot.)
132
Table 17
Correlation of Amount of Glutamate
Formed per mole Glutamine Used as
Substrate at Different PRPP
Concentrations in the Liver
PRPP conc in
reaction mix.
r
0.0 mM
0.96
0.3 mM
1.00
0.6 mM
0.98
1.2 mM
0.97
2.4 mM
0.99
L
w
v
w
w
w
uJ
0
40,0001
[PRPP]
2.4
M
LIJ
30,0001
M
0.6
0
U-
-
20,000
1. 2 m
(A
(A
Iii
UOmnM
I00001
0.022
mM
0.044
0.088
ELUTAMINE]
IN
0.176
REACTION MIXTURE
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED PER MOLE GLUTAMINE
USED AS SUBSTRATE AT DIFFERENT
PRPP
Figure 35
CONCENTRATIONS IN THE LIVER
134
Table 18
Correlation of Amount of Glutamate Formed as a
Function of PRPP Concentration in Reaction
at Different Glutamine Concentrations
in the Liver
Glutamine
Conc.
Correlation coef. (r)
PRPP concentration
0-2.4 mM
0-1.2 mM
0-0.6 mM
0.022 mM
0.98
0.30
0.56
0.044 mM
0.99
0.95
0.89
0.088 mM
0.78
0.62
0.89
0.176 mM
0.97
0.34
0.46
135
S40,000 -7
GLUTAM IN E
z
7
30,000
Li
Ce
S20,00--
Io,000:-
4 4 pMy-
I-
22 PM
2,100
0.3
1.2
0.6
2.4
[PRPP]
rnM
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED
AS A FUNCTION OF
PRPP
CONCENTRATIONS IN THE
REACTION AT DIFFERENT GLUTAMINE
CONCENTRATIONS IN THE LIVER
Figure 36
136
I
I
30,000
LiJ
20,000
-=-
r 1.00
L)
22
44
88
176
y M GLUTAMINE
IN REACTION
MIXTURE
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED PER IOLE
GLUTAMINE USED AS SUBSTRATE AT A
OF ,3mMIN THE LIVER
Figure 37
PRPP
CONCENTRATION
9
9
8
a-12,500-
0
GL 10,000 U
7,500 -
(A-4
0
L
0.3
I
0.6
I
2.4
1.2
mM LPRPP]
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED AS A FUNCTION OF
PRPP
CONCENTRATION IN THE REACTION AT A GLUTAMINE
CONCENTRATION OF .014
Figure
MW IN THE LIVER
38
138
Table 19
Glutamate Formed as a Function of PRPP Concentration
and Glutamine Concentration in Reaction
Mixture with Liver Amidotransferase
Time
(min)
Glutamine
(mM)
PRPP Concentration (mM)
in Reaction Mixture
0.0
0.3
0.6
2.4
1.2
counts/min 14C-Glutamate formed
0
15
0.022
1511
2948
1903
2045
2158
0.044
3363
3842
3948
4034
4607
0.088
6511
6895
7500
7034
8767
0.176
12500
12925
14543
13681
12001
0.022
2509
4852
6080
3933
5976
0.044
3189
6983
9024
2509
12584
0.088
7789
15334
14163
14466
22536
0.176
10560
31316
40211
23765
35548
139
40,000-
30,000 -
U-j
U----
uj 2a,00
M176M
0,010,000 -
0
44 p.M
6000
4,000
2,000
2
M
15
MINUTES 0
[PRPP]
0
0
15
0
PRPP
[PRPP]
0.3
1
0.6
0
1
[PRPP
1.2
0
1
[PRPP
2.4
THE AMOUNT OF GLUTAMATE FORMED AS A FUNCTION OF THE
PRPP
CONCENTRATION AND GLUTAMINE CONCENTRATION
IN THE REACTION MIXTURE WITH
LIVER AMIDOTRANSFERASE
Figure 39
140
Table 20
Correlation of Amount of Glutamate
formed per mole Glutamine Used as
Substrate at Different PRPP
Concentrations in the
Small Intestine
PRPP conc. in
reaction mix.
r
0.0
.98
0.3 mM
.98
0.6 mM
1.00
1.2 mM
1.00
2.4 mM
.99
141
20,000
LPR PP .6mM
0.3m
15,000
1.2m
0
LU
A
10,000
0
0mM
0
aL0i
5,000
0.022 0.044
mM
0.088
0.176
GLUTAMINE
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED PER
MOLE GLUTAMINE USED AS SUBSTRATE AT
DIFFERENT
PRPP
CONCENTRATIONS IN
THE SMALL INTESTINE
Figure 40
142
Table 21
Correlation of Amount of Glutamate formed as a
Function of PRPP Concentration in Reaction
at Different Glutamine Concentrations
in the Small Intestine
Glutamine
Conc.
Correlation coef (r)
PRPP concentration
0-0.6 mM
0-1.2 mM
0-2.4 mM
.022 mM
0.21
0.15
0.26
.044 mM
0.99
0.37
0.11
.088 mM
0.91
-0.31
-0.63
.176 mM
1.00
0.02
0.06
143
Li
00
0
LlU
GLN]
176 pL M
G 20OO
=2,000
-j
8 8 p. M-
310,00
4 4 y M
,0 C,
2 2M
-
0.3
1.2
0.6
mM
2.4
LPRPP]
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED AS
A FUNCTION OF THE PRPP CONCENTRATION IN THE
REACTION AT DIFFERENT GLUTAMINE
CONCENTRATIONS IN THE SMALL INTESTINE
Figure 41
144
15,000
U-
-LJ
r=
100
-S
00
-
-.
1000
0.022 0.044
0.088
0.176
GLUTAM INE CONCENTRATION ( m M )
THE CORRELATION OF THE AMOUNT OF GLUTAMATE FORMED PER
MOLE GLUTAMINE USED AS SUBSTRATE AT A
CONCENTRATION OF .3Mt IN
THE SMALL INTESTINE
Figure 42
PRPP
145
Table 22
Glutamate Formed as a Function of PRPP Concentration
and Glutamine Concentration in Reaction Mixture
with Small Intestinal Amidotransferase
Time
(min)
PRPP Concentration (mM)
in Reaction Mixture
Glutamine
(iM)
0.0
0.3
0.6
2.4
1.2
counts/min 14C-Glutamate formed
0
15
0.022
889
1094
1222
983
1171
0.044
1947
1716
2221
2807
2088
0.088
4491
3840
4644
4301
4730
0.176
9207
8607
9434
1027
8615
0.022
1426
2930
1755
2118
2330
0.044
2892
3819
5163
3739
3768
0.088
7599
7767
9313
6692
6339
0.176
11375
16639
20423
12287
15679
146
020,000
0
176 w.
LD
M
-88
6,000
4,000 -44
0
M
2,000
0
15
0
15
0
I5
0
is
0
IS
MINUTES
EPRPG o
[PRPP]O.3
[PRPPj.6
[PRPpl 1.2 [PRPP] 2.4
THE AMOUNT OF GLUTAMATE FORMED AS A FUNCTION OF THE
CONCENTRATION AND GLUTAMINE CONCENTRATION IN THE
REACTION MIXTURE WITH SMALL INTESTINAL
AMIDOTRANSFERASE
Figure 43
P RPP
147
Experiment # 3:
The PRPP amidotransferase activity was
measured utilizing a crude enzyme extract (that had not been
passed through the Sephadex G-25 column).
Only total enzyme
activity (PRPP dependent and independent) activity was measured
in both the small intestine and in the liver.
Over thirty
minutes the reaction was linear (figure 44, table 23).
No
significant amount of amidotransferase activity was demonstrable in the small intestine.
Experiment # 4:
The assay was next performed utilizing an
enzyme source prepared as in Materials and Methods (page 68).
Here the high speed supernatant was passed through a Sephadex
G-25 column.
PRPP dependent activity was determined and is
plotted in figure 45.
Data for total activity as well as PRPP
independent activity are tabulated in table 24.
14 C-glutamate
The amount of
formed was linear over the sixty minutes for
both liver and colon.
There was no significant demonstrable
activity in the small intestine.
(2) The effect of dietary purines upon amidotransferase activity
Experiment # 5:
Glutamine amidophosphoribosyltransferase
activity was measured in four animals who were on a synthetic
agar-based diet
free of purines and in four animals on an
identical diet with 0.1% of each purine and pyrimidine base
added (diets # 1 and # 2 from table 4, respectively).
Regard-
less of the diet the enzyme activity in the small intestine was
found to be significantly lower than in either the liver or the
colon (P < 0.025 in the purine-added group and P < 0.005 in the
purine-free group by one-way analysis of variance).
The colon
148
II
I
I
I
I
LIVER
i
wMIT000-
C
30
,00 -0
INTESTINE
0SMALL
10
20
6
4-0
30
40
50
60
MINUTES
THE AMIDOTRANSFERASE ACTIVITY (TOTAL ACTIVITY)
IN CRUDE TISSUE EXTRACTS FROM LIVER AND
SMALL INTESTINE
Figure 44
149
Table 23
PRPP Amidotransferase Activity (total activity)
in Crude Tissue Extracts
Time of incubation (min)
60
30
15
5
cpm 1C-glutamate formed
Small Intestine
348
351
510
463
Liver
428
856
1669
1572
150
4,000
X
LIVER
i3,000
-
COLON
<
2,000
CD
l-
0~0
1000-00
0
SMALL INTESTINE
10
20
30
40
MINUTES
50
60
THE AMIDOTRANSFERASE ACTIVITY (PRPP DEPENDENT)
IN DIFFERENT TISSUES
Figure 45
151
Table 24
PRPP Amidotransferase Activity (PRPP Dependent)
in Different Tissues
Tissue
Min.
Total
PRPP
PRPP
Correlation
Indep.
Depend.
Coefficient
counts/min of
Small
Intestine
0
5
15
30
1464
1612
1602
1713
1361
1429
1491
1509
102
182
111
203
1811
1597
213
0
5
15
30
60
1470
2248
2754
3445
5961
1441
1535
1484
1740
2302
29
713
1265
1705
3658
r =0.99
0
5
15
30
60
1586
2106
2392
3601
5653
1420
1588
1650
1851
2769
166
518
742
1750
2883
r =0.99
60
Liver
Colon
4C-Glutamate formed
~
r =0.71
152
and the liver had essentially similar amounts of enzyme
activity.
The purine-free diet resulted in a decrease in the
enzyme activity of the liver from 2.11±0.52 micromoles glutamate formed/gram protein/minute (diet with bases) to 1.27±0.50
micromoles glutamate formed/gram protein/minute (diet without
bases).
This difference had a p value of 0.06 by t-test.
The
small intestine had a small (but not statistically significant)
increase in activity when switched to the purine-free diet.
The
colon had a slight (but not statistically significant) decrease
in activity when on the purine-free diet.
This data is
summarized in table 25 and figure 46.
Thus, the small intestine contains significantly less
amidotransferase than either the colon or the liver.
This was
found to be true on a purine-free diet (P Z 0.05) and on a
purine-added diet (P 4 .025).
There was no significant diff-
erence in the amount of small intestinal enzyme with the two
diets.
Similarly, the colon showed no significant change in
activity with the diet.
On the purine-free diet, however,
there was a 66% increase in the liver amidotransferase activity
which was significant at the P = 0.06 level.
concluded that the increased
1 4 C-glycine
Therefore, it is
incorporation into
RNA observed in the experiment described above is not due to
more enzyme molecules but must be due to a change of some
factor influencing enzyme activity, i.e., the supply of substrate.
As figure 4 shows, this could well be PRPP, the level of which
is regulated by utilization in the competing salvage pathway.
Thus, the presence of purine in the diet could rapidly reduce
153
Table 25
Amidotransferase Activity
Diet
Small Intestine
Liver
Colon
Significance
umoles glutamate formed/g Protein/min
Purine
Added
0.064+.025
0.019+.020
0.105+.032
p<0.0 2 5
Purine
0.106+.026
0.020+.014
0.086+.032
p<0.005
not sig.
not sig.
Free
(t test)
(n=4)
p=.06
9
v
0
0
0
0
0
0.140
LUJ
0.120
0.100
0
N. S.
N =4
0.080
PURINE
FREE
ci: 0.060
0
DIET
U-
DIET
DIETDIET
WITH
BASES
LUJ
0.040
0.020
PURINE
FREE
DIET
DIET
WITH
BASES
WITHt
BASES
PURINE
FREE
DIET
N.S.
N =4
N.NS.
N 4
LIVER
SMALL
INTESTINE
COLON
THE AMIDOTRANSFERASE ACTIVITY IN DIFFERENT TISSUES AS A
FUNCTION OF THE PURINE CONTENT OF THE DIET
Figure 46
0
155
the availability of PRPP for the de novo pathway and regulate it as is shown in figure 4.
E.
The Activity of the Hypoxanthine-Guanine Phosphoribosyl
Transferase in the Small Intestine, Colon and Liver
and the Effect of Dietary Purines
(1)
Validation of the HGPRT assay
The HGPRT assay was performed as per Materials and Methods
(page 70).
Experiment # 1:
To test the capacity of the DEAE cellu-
lose (Whatman DE-81) discs to hold the reaction-enzyme
mixture,
HGPRT activity was assayed in a crude liver enzyme mix.
Ten pl;
20 pl and 40 pl of the original reaction-enzyme mixture were
spotted on the discs.
The results are illustrated in figure 47 and tabulated
along with the relevant correlation coefficients in table 26.
The correlation coefficients confirm the linear relationships
expected and demonstrate that the capacity of the discs was
not exceeded.
As a result of this experiment it was decided
to spot 30 pl of enzyme-reaction mixture for future experiments.
Experiment # 2:
Liver HGPRT-activity was assayed
utilizing the reaction mixture and enzyme source detailed
above.
The enzyme source was a high-speed supernatant, passed
through a Sephadex G-25 column.
The reaction was linear over
the sixty minutes (figure 48, table 27).
Experiment # 3:
The effect of concentration of enzyme
in enzyme preparation was assessed in liver, colon, and small
156
11,000
I0000
HGPRT LIVER
8,000 7,000
CPM
-
4O
r
1.00
6,030 -
4~O0O
4,00=\.0
r =1.00
10 A
3,000 -
_
2,000 1,000,
I
20
I
10
30
I
40
I
50
60
MINUTES
THE LIVER
HGPRT
ACTIVITY (USING A CRUDE TISSUE
EXTRACT) DETERMINED BY SPOTTING 10 LAMBDA,
20
LArBDA OR 110 LAMBDA OF REACTION-ENZYME
MIXTURE OR
DEAE
CELLULOSE DISCS
Figure 47
157
Table 26
Liver HGPRT Activity (Using Crude Enzyme
Preparation) and Spotting 10 Lambda,
20 Lambda, and 40 Lambda of
of Reaction-Enzyme Mix on
on DEAE Cellulose Discs
Minutes
10
Lambda Spotted
20
40
r
0
623
800
1035
0.99
5
834
1190
2130
1.00
15
1371
2026
3415
1.00
30
2193
3171
5565
1.00
60
3417
5763
11032
1.00
1.00
1.00
1.00
2794
4964
9998
(60-0)
1.00
158
5O0C0
400
3.000
HGPRT LIVER
= 1.00
-r
CPM
IWOO
400
10
20
30
50
40
MINUTES
THE LIVER
HGPRT
60
ACTIVITY
Figure 48
70
159
Table 27
Liver Hypoxanthine-Guanine
Phosphoribosyltransferase Activity
Time
(min)
Total
Minus
Blank
Minus
Zero
counts/min 14C-Hypoxanthine
converted to nucleotide
Blank
617
(0)
-
0
800
183
(0)
5
1190
573
390
15
2026
1409
1226
30
3171
2554
2371
60
5763
5146
4963
160
intestine.
The assay was performed using five ul, ten ul,
and twenty ul of enzyme preparation.
The time points chosen
were 0, 2, 5, 10, 15, 20, and 30 minutes.
The results are
illustrated in figures 49, 50, and 51, and tabulated in
table 28.
The reaction is linear over 30 minutes for the
liver, small intestine and colon when 5 ul of the enzyme
source is used (figure 49).
When 10 ul of the small intes-
tinal enzyme source is used the reaction reaches a plateau
after 15 minutes.
When 10 ul of the liver enzyme source is
used a plateau is reached by 5 minutes (figure 50).
When
20 ul of the small intestinal enzyme source is used the
reaction reaches a plateau after approximately 10 minutes.
When 20 ul of liver enzyme source is used the reaction
reaches a plateau in less than 5 minutes (figure 51).
(2) The effect of dietary purines upon HGPRT activity
Experiment # 4:
Based upon the foregoing data, the
assay was applied to the same animals and tissues that were
studied in experiment # 5 of the amidotransferase group.
Specifically, a group of animals that had been on a purinefree diet for seven days and a group that had been on an
identical agar-based synthetic diet with 0.1% of the purine
and pyrimidine bases added were studied.
The small intestine
was found to have significantly (P < 0.01) more HGPRT activity
than either the liver or colon when the animals were on a
purine-containing diet.
By comparison, when the animals
were on a purine-free diet their intestinal HGPRT activity
was less while their liver HGPRT activity was increased.
The
161
LU
LU
o
15,000
LIVER
0
0
0
r =0.96
LU
Zi 0,000
* SMALL
INTESTINE
r =1.00
o
5,000
COLON
.
2
5
10
x
r
Is
20
=0.95
MINUTES
THE HGPRT REACTION
IN DIFFERENT TISSUES
USING FIVE LAMBDA OF ENZYME SOURCE
Figure 49
30
162
M
t--
0
i 5,-00-
LIVER
z
u-j
z
SMALL
INTESTINE
0
5,000
COLON
0
2
5
10
15
20
30
MINUTES
THE
HGPRT
REACTION IN DIFFERENT TISSUES
USING TEN LAMBDA OF ENZYME SOURCE
Figure 50
163
9-
UJ20,000
L0IVER
SMALL INTESTINE
0
0
<I0,000--
I0...00
|
S5,000--
COLON
2
5
10
15
20
30
MINUTES
THE HGPRT REACTION IN DIFFERENT TISSUE USING
TWENTY LAMBDA OF ENZYME SOURCE
Figure 51
164
Table 28
The Effect of Concentration of HGPRT Enzyme on
Enzyme Reaction in Different Tissues
Amt. of
Enzyme
Time of
Incbn.
Small
Intestine
cpm
(min)
14
Liver
Colon
C Hypoxanthine converted
to nucleotides
5 pl
0
2
5
10
15
20
30
543+0
1210+71
2246+93
4283+28
6624+20
9060+282
12802+764
r
10 Pl
20 j1
0
2
5
10
15
20
30
*0
0
2
5
10
15
20
30
=1.00
546+39
804+65
1162+240
1633+147
1661+117
2084+234
2287F50
r
=
.95
1168+80
2869+346
4668+0
8187+326
9991+363
15620+1085
15573F56
r
= .96
1083+114
2508+20
4490+187
8307+704
11755+342
16899+725
15397F860
524+106
1178+277
1608F95
2323+45
2986+173
3326+58
36377271
3594+342
7016+76
12221+149
13207+345
14137+620
16412+163
16712F2976
r 3 0 = .94
r 2 0 =1.00
r 3 0 = .95
r20= .90
r 1 5 = .99
r 5 =1.00
r 10= .92
r 1 5 = .89
r 2 0 = .91
r30= .87
552+66
1737+221
5495+139
9571+586
13057+864
15023+554
14529+1894
15061+1328
555+174
848+18
2030+3
3266+649
3888+23
3839+200
4567+102
5145+13
570+3
10423+3748
14206+578
12342F869
13008F822
11400+2454
14770+202
11395~404
r
r 1 5=
r20=
r 3 0=
.98
.0=
.96
.91
.84
r5 =
r1 0 =
r 1 5=
r 2 0=
r 3 0=
.99
.94
.88
.90
.90
r5
* Enzyme boiled before addition to reaction mixture.
.41
165
data are tabulated in table 29 and illustrated in figure 52.
In each organ for each animal a ratio of HGPRT;amidotransferase activity was computed.
For the liver (n = 2) the
ratio was 46 ± 1 in the group fed the purine-containing diet.
In the group fed the purine-free diet the ratio was 130 t 69.
In the small intestine (n = 2) the purine-containing group
had a ratio of 2413 ± 1326 versus 1081 ± 1326 for the purinefree group.
These were not statistically significant at the
P = 0.05 level.
There was only a minimal change in the colon.
On the purine-free diet (n = 4) the ratio was 62 t 31 and on
the purine-containing diet it was 52 ± 26.
The data are
tabulated in table 30.
Thus in the liver the purine-free diet appears to be
associated with a shift towards increased salvage as compared
to de novo synthesis.
In the small intestine the purine-free
diet is associated with either a shift toward de novo synthesis or at least a shift away from salvage.
These results
are completely consistent with the results obtained in the
incorporation studies reported above.
166
Table 29
The HGPIRI Activity in Different Tissues
as a Function of the Purine Content
of the Diet
Diet
Liver
Small
Colon
Significance
Intestine
pmole glutamate formed/g protein/min
(anova)
Purine
added
4.06+ .47
9.45+0.85
5.59+0.54
p(.01
Purine
free
5.62+2.43
7.90+1.4
5.06+1.82
not sig.
(t-test) not sig.
not sig.
not sig.
qp
0
Vw
w
w
Vw
z
N.S.
N=2
Uj
N.S.
N=2
--
0
cr
0
w
N.S.
N4
-
0~
IT
0
BASES
9-j
_
t
z
PURINE
FREE
DIET
0
-
DIET
WITH
~ BASES
PURINE
FREE
DIET
LIVER
DIET
WITH
BASES
SMALL
INTESTINE
THE EFFECT OF DIETARY PURINES ON
IN DIFFERENT TISSUES
Figure
52
PURINE
FREE
DIET
COLON
HGPRT ACTIVITY
168
Table 30
Ratio of HGPRT Activity/Amidotransferase
Activity in Different Tissues
Diet
Purine Free
Diet
Purine Added
Diet
Liver
S.I.
Colon
(n=2)
(n=2)
(n=4)
130+69
46+1
Not sig.
1091+986
-
62+31
2413+1326
-
52+26
-
Not sig.
Not sig.
169
THE RESULTS OF PRELIMINARY STUDIES
VI.
A.
The Effect of a Self-Emptying Blind Loop of Intestine on
the Activity of the De Novo Pathway
A self-emptying blind loop of intestine that excluded
pancreatic secretions was studied two weeks after surgery.
An adult male 300 gm rat that had been on a regular Purina
Chow diet was operated upon and continued to gain weight normally after surgery.
In this single experiment there appears to have been
unequivocal incorporation of
3 H-adenine
1 4 C-glycine
into RNA purines.
incorporation was not unequivocally demonstrated,
the actual values in fact showing a high initial value with decreased values over the time course of the experiments (table 31).
B.
Studies of the Different Intestinal Cell Populations
Experiment # 1:
Animals in this study were 250 gm white
male CD rats obtained from Charles River Breeders and were
maintained precisely as in the other studies above.
mals were on Purina Rat Chow diets.
The ani-
They were not fasted be-
fore killing.
On the day of the experiment following decapitation the
small intestine was rapidly dissected and flushed until clear
with normal saline at 370 C.
The entire small intestine be-
ginning approximately 10 cm distal to the ligament of Treitz
was utilized.
It was cut into four sections approximately
15 cm long each.
The segments were everted and shaken as
170
Table 31
14
C-Glycine into Mucosal
Incorporation of
RNA-adenine and into Mucosal Protein
in a Self-emptying Blind
Loop of Intestine
Time
(min)
10
30
60
90
RNA (cpm/)imole
adenine)
5345
15206
68125
35882
Protein (cpm/mg
1094
1046
2042
2835
Incorporation
protein)
171
described by Gall et al.
(1977).
The results are illustrated in figure 53.
It is evident
that labeling in the acid precipitable pool (which is mainly
protein and nucleic acids) is much greater in the fraction of
cells obtained from close to the crypts.
Histologic examin-
ation revealed that after shaking there was still an appreciable
amount of cells left in the crypts although many crypts were
empty.
The data are expressed as cpm (in acid insoluble fraction)
per mg protein in figure 53 and table 32 and as cpm per mg DNA
in figure 54 and table 33.
Experiment # 2:
This study was carried out the same way
as Experiment # 1 except that only the specific incorporation
of both
1 4 C-glycine
mined.
The results demonstrate that there is a distinct
and
3 H-adenine
into RNA purines was deter-
gradient of purine salvage activity increasing in the cells
closer to the crypts (figure 55 and tables 34 and 35).
Examin-
ation of the data on de novo activity shows that there is an
approximate doubling of incorporation into the cells of the
the intermediate and crypt fractions as compared to the villous
fraction (table 34).
It is not certain that this represents
meaningful incorporation via the de novo Dathway.
C.
Effect of Catheter Placement and Normal Saline Infusion
Experiment # 1:
Two groups of animals were used.
The
test group was prepared as per Materials and Methods, section E.
The other group served as an unoperated control.
The test group
received two bolus injections of 5..5 cc's each of normal saline
172
200,000
CRYPT
CELLS
z
S100,000
INTERMEDIATE
60.00040,000-
TIP CELLS
20,000-
10
20
30
40
MINUTES
50
60
THE INCORPORATION OF LC-GLYCINE INTO THE PROTEIN AND
THE NUCLEIC ACIDS IN DIFFERENT INTESTINAL CELL
POPULATIONS (EXPRESSED AS COUNTS/
MIN/MG PROTEIN)
Figure 53
173
Table 32
14
C-glycine into
The incorporation of
the protein and nucleic acids in different
intestinal cell populations
Cell
Population
Time of incubation
10
30
(min)
60
counts/min/mg Protein
Villus tip cells
14530
13490
14386
Intermediate cells
82010
76780
75030
Crypt cells
81000
178210
190830
174
3co0o0
00-
~20,000
CRYPT CELLS x
10000
-
x
2,000
TIP .ELeS
10
THE INCORPORATION OF
30
MINUTES
1 4 C-GLYCINE
60
INTO THE PROTEIN
AND THE NUCLEIC ACIDS IN DIFFERENT
INTESTINAL CELL POPULATIONS
(EXPRESSED AS COUNTS/MIN/MG
Figure 54
DNA)
175
Table 33
The incorporation of
14
C-glycine into
the protein and nucleic acids in different
intestinal cell populations
Cell
Population
Time of incubation (min)
10
30
60
counts/min/mg DNA
Villus tip cells
Crypt cells
480
1000
1300
10000
12700
19000
176
3 H-ADEN INE
'
4
C-GLYCINE
o CRYPT CELLS
a INTERMEDIATE
CELLS
* TIP CELLS
1,500
<\,000
z
500
a_ 4 00
300
300
200
100
200
100
10 20 30 4050 60
MINUTES
THE INCORPORATION OF
THE
RNA
3 H-ADENINE
10203040 50 60
MINUTES
AND 1 4C-GLYCINE INTO
OF DIFFERENT INTESTINAL CELL POPULATIONS
Figure 55
177
Table 34
Incorporation of 14C-Glycine into the RNA
of Different Intestinal Mucosal
Cell Populations
Cell
Population
10
Time of incubation
30
(min)
60
counts/min/mg RNA
Villous tip
54
48
74
Villous intermediate
60
75
185
Villous crypt
55
-
164
178
Table 35
3
Incorporation of H-Adenine into the RNA
of Different Intestinal Mucosal
Cell Populations
Cell
Population
Time of incubation
10
30
(min)
60
counts/min/mg RNA
Villous tip cells
105
255
340
Intermediate cells
160
710
850
Crypt cells
440
1655
1750
179
per day.
Both groups were allowed a complete synthetic agarover 18 days.
based diet ad libitum
There was no significant
difference in the average daily food intake of the animals, or
Histopathology as well as gross path-
in their daily weights.
ology was normal.
There was no significant difference in
their serum albumin levels (table 36).
The
Two groups of animals were used.
Experiment # 2:
test group was prepared as above.
an unoperated control.
The other group served as
The test group received two bolus
injections of 19 cc's each of normal saline for fourteen days.
(Diets were as above in Experiment # 1.)
the animals were sacrificed.
After fourteen days
Weight gain expressed as
weight gained per day, liver weight and spleen weight at
autopsy and serum albumin on day fourteen all showed no significant differences between the groups.
Histopathology as
well as gross pathology was normal (table 37).
Experiment # 3:
Both
Two groups of animals were used.
were prepared as per Materials and Methods, section E.
Both
were placed on similar synthetic agar-based 0% protein diets.
The groups were pair-fed so that their oral intakes were comparable
2).
(34 kcal/day for group 1; and 32 kcal/day for group
In addition animals in group 1 received 3 grams of
crystalline amino acids injected as two bolus injections of
19 cc each per day.
Animals in group 2 received the same
volume of normal saline.
Animals in group 1 lost 10% of their initial weight
compared to 18% for group 2 (P < 0.05).
The actual daily
186
Table 36
The Effect of Intraperitoneal Catheter Placement
and Normal Saline Infusion (5.5cc twice a day)
Init.
Wt.
Final Wt.
Change
in wt/day
Average Daily
Food Intake
Serum Albumin
Day 18
(gm)
Control
369+48
469+112
5.56
47+10
3.5+.24
362+54
465+60
5.72
45+10
3.3+.17
not sig
not sig
not sig
not sig
(n = 4)
Injected
(n = 3)
Sig.A
not sig
11
Table 37
The Effect of Intraperitoneal Catheter Placement
and Normal Saline Infusion
(19cc twice a c-y)
Init.
Wt.
Final Wt.
Change in
Wt/day
Wt.
Liver Wt
Spleen Wt.
Serum
Day 14
Day 14
Albumin
(gm)
Day 14
(gn)
Control
(n
=
Injected
Sig.
394+42
465.5+54
62.5+12.9
4.46
15.19+2.26
.81+.12
3.36+.21
383+59
463
79.8+14
5.70
16.92+1.72
.91+.13
3.33+.19
not sig.
not sig.
8)
+49
not sig.
not sig. not sig.
not sig.
not sig.
182
weight losses averaged 2.59 gms for group 1 and 5.49 gms for
group 2 (P < 0.01) (figures 56 and 57).
When sacrificed the serum albumin of group 1 averaged
2.89 gms versus 2.63 gms for group 2 (P <
0.02) (figure 58).
The liver weights for the two groups were 13.1 gm (group 1)
and 10.2 gm (group 2) (P < 0.05) (figure 59).
Despite the
hypertonicity of the amino acid solution (approximately
780 mosm/1) the only consistent adverse tissue reaction observed was some focal fibrosis directed to the silk suture
that was placed to anchor the catheter grommet in the peritoneum.
Experiment # 4:
Two groups of animals were used.
Both
groups had intraperitoneal catheters placed at least one week
prior to the beginning of the experiment.
Both groups re-
ceived an agar-based synthetic diet that consisted only of
water, agar, anhydrous dextrose, and NaCl.
Group "AA" (amino acid infused) received approximately
1.7 grams of amino acids as crystalline amino acid solution,
divided into two bolus injections.
The other group "GLU"
(glucose infused) received an equivalent amount of glucose
in two daily intraperitoneal injections.
After fourteen
days the animals were matched for total caloric intake.
The animals thus had the same total caloric intake, but
group AA received 1.7 grams of intraperitoneal amino acids
per day and group GLU received approximately the same amount
of intraperitoneal glucose.
Both groups selected an amount
of food that was hypocaloric and therefore their weight loss
183
AVERAGE WEIGHT
LOSS PER DAY
5.4
5p <.01
4-
0
-J*
2.59
Il
c/) 2-
IPAA
IPN S
THE AVERAGE WEIGHT Loss PER DAY IN ANIMALS
RECEIVING EITHER INTRAPERITONEAL
AMINO
ACIDS OR
INORMAL
Figure 56
SALINE
184
WEIGHT LOSS
20
18%
C!,
10%
101-J
0L
THE AVERAGE WEIGHT
IPAA
P NORMAL
SALINE
Loss
PER DAY (EXPRESSED AS
PERCENTAGE OF INITIAL WEIGHT) IN ANIMALS
RECEIVING EITHER
AMINO
INTRAPERITONEAL
ACIDS OR NORMAL SALINE
Figure 57
185
SERUM ALBUMIN
N.S.
Oak
4
3
ap1
c
3.57
H1
p <.02
2.89
3.28
-
IPAA
PNS
2f
2.
C,
1
0
S
Ills',
2
0
CONTROL
4
6
8
DAYS
10
12
14
THE SERUM ALBUMIN OF ANIMALS RECEIVING EITHER
INTRAPERITONEAL
AMINO
ACIDS OR NlORMAL SALINE
186
LIVER WEIGHT
20 p <.05
15 13.1
0%
0
10.2
10 I
5
0LNO PROT
DIET
IPAA
NO PROT
DIET
IPNS
THE LIVER (WET) WEIGHT OF ANIMALS
RECEIVING EITHER INTRAPERITONEAL
AMINO
ACIDS OR NORMAL SALINE
Figure 59
187
was expected.
Group AA (amino acid infused), however, lost
only 56% as much weight as group GLU (glucose infused).
was significant at the P < 0.01 level.
This
All parameters are
listed in table 38.
Experiment # 5:
Concurrent with the running of groups
AA and GLU in Experiment # 4 two additional groups were
studied:
group "C" and group "S".
Group C received a diet
of NaCl, glucose, and amino acids and never had any surgery.
Group S received the same diet as group C except that NaCl
was omitted.
In addition group S received normal saline via
intraperitoneal injection twice a day.
The original goal was
for all four groups to have matched intakes of NaCl and glucose with the key variable being intraperitoneal amino acids.
The animals in groups C and S consistently ate more than those
in groups AA and GLU.
Therefore the pair feeding aspect of
The data for group C and
the entire study was invalid.
group S are listed in table 39.
After completion of the above study incorporation
experiments were performed on groups C, AA, and GLU to assess
1 4 C-glycine
uptake into RNA.
Table 40 summarizes the data.
Biologically significant incorporation via the de novo
pathway is present in the group receiving the agar-based,
amino acid, glucose diet.
The level of incorporation in the
other groups was essentially at baseline.
A one-way analysis of variance utilizing the three
groups of data has an F value of 7.96 which is significant
at approximately the P = 0.08 level.
188
Table 38
The Effect of Intraperitoneal Amino Acid Infusion
versus Intraperitoneal Glucose Infusion
Amino Acid
Infused
Effect
Initial wt.
Final wt.
(gms)
(gms)
Change in wt.
(gms)
Oral calories
Sig.
Glucose
Infused
371
+28
393
+21
Not Sig.
295
+18
264
+44
Not Sig.
75
+36
128.8 +37
37
+11
36
p<.01
+12
Not Sig.
Not Sig.
Intraperitoneal Protein
calories/day
6.8 + 0.6
0
Intraperitoneal Glucose
calories/day
0
8.7 + 0.8
Total calories
44
+10
44
+11
-
Not Sig.
Average daily wt. loss
5.36+ 2.56
9.20+ 2.67
p<.01
Serum albumin
(Day 14)
3.1 + 0.2
3.0 + 0.3
Not Sig.
BUN (Day 14)
7.5 + 2.7
6.0 + 2.2
Not Sig.
9.4 + 1.1
10.7 + 0.6
Not Sig.
Liver weight
(Day 14)
(gm)
Spleen weight (Day 14)
(gm)
7.0 +
.23
.84+
.05
Not Sig.
189
Table 39
The Effect of Control Diets upon Intraperitoneal
Study Control Animals
Effects
Unoperated
Controls
(C)
Operated & Saline
Infused Controls
(S)
63.0 + 5.0
54.0 +4.0
Average wt loss/day
2.1 + 0.8
2.6 +1.0
Serum albumin
3.72+ 0.38
2.73+0.29
Total Calories
BUN
11.9 + 1.2
11.5 +1.9
Liver weight
11.3 + 1.4
10.4 +1.4
Spleen weight
Interperitoneal saline
infused/day/gm
.64+ 0.07
-
.73+0.13
21.9 + 1.9
*
9
9
qP
10
4P
w
Table 40
Incorporation of 1C-Glycine into Small Intestinal Mucosal
RNA and Protein in Parenterally Alimented,
and Control Fed Animals
Total
Protein
Average
wt. loss
per day
(gms)
Serum
albumin
day 14
Incorp
into Protein
day 14 cpm/mg Prot
Oral
Diet
I.P.
Infusion
Total
Calories
Unoperated
Controls
Amino Acids
Glucose
NaCl
(purine free)
none
63
2.2
Amino Acid
Infused
Glucose
NaCl
(purine free)
amino
acids
44+10
~
1.7+0.1
~
5.4+2.6
~
3.1 +0.2
Glucose
Infused
Glucose
NaCl
(purine free)
glucose
44+11
0
9.2+2.6
3.0 + 0.3
Sig.
2
3.72
BUN
Incorp
into RNI
Purines
cpn'mg IA
11.9
6639+54
43520+14730
7.5
2162+653
3450+ 4879
1300+285
8468+10800
+2.7
6.0
+2.2
p<.-08
'~0
0
191
This data shows incorporation of
1 4 C-glycine
into RNA
when the animals were on an amino-acid-containing but proteinfree diet and is consistent with the previous data.
192
VII.
DISCUSSION AND CONCLUSIONS
A series of experiments has been performed on rats
to determine the influence of dietary purines (nucleic acids,
nucleosides or bases) on the metabolism of the mucosal
cells of the gastrointestinal tract.
Purine nucleotides
can be provided within cells by de novo synthesis or by
salvaging free purine bases, the key enzymes being phosphoribosyl amidotransferase and adenine or hypoxanthineguanine phosphoribosyl transferase.
Both pathways use
phosphoribosyl pyrophosphate (PRPP) as a major substrate.
In order to evaluate these two pathways for purine nucleotide formation,
3 H-adenine
(or adenosine) uptake into mu-
cosal RNA was used to measure salvage and
1 4 C-glycine
in-
corporation into RNA was used to measure de novo synthesis.
Experiments were conducted when the animals were on an
unrestricted chow diet.
Initially, everted loops of intes-
Pancreatic duct ligation was performed
tine were utilized.
with the expectation that this would decrease the available
pool of endogenous glycine and adenine.
poration of
4C-glycine and
3 H-adenine
The actual incorinto the mucosal
cells was enhanced, but no significant incorporation of
14C-glycine into small intestinal RNA was demonstrable.
Therefore, the pancreatic duct ligation was not performed
in other experiments.
The everted loops of intestine were replaced by
mucosal scrapings.
The use of scrapings was found to offer
193
the most reliable system for obtaining adequate incorporation
of isotope into cells.
This was probably because with the
use of scrapings an increased concentration of cells, especThe mucosal
ially crypt cells, was exposed to the isotopes.
scrapings were employed for the incorporation studies reported.
De novo purine synthesis by small intestinal mucosal
cells incubated in vitro with 1C-glycine was shown to be
very slight in contrast to the brisk de novo incorporation
by colonic mucosal cells.
In contrast, uptake of
1 4 C-glycine
by the mucosal protein was equally active for the small and
large intestine and continued throughout 30 to 60 minutes of
incubation, an indication of continuing viability.
The
experiments thus show the absence of an active de novo pathway in the small intestine of rats on a normal diet and its
presence in the colonic mucosa.
The final conclusion from this series of studies is
shown in tables 9 and 10 and in figures 8 and 9.
apparent (figure 8) that only a small uptake of
It is
14 C-glycine
occurs into the RNA of mucosal scrapings from the small
intestine, contrasted with a vigorous and prolonged uptake
into the RNA of the large intestinal mucosa.
In contrast,
figure 9 shows no difference in uptake into the protein of
these two mucosal samples.
This latter finding implies
that differences in labeling of the free glycine pool are
unlikely to account for the low uptake of 1C-glycine into
RNA.
This finding thus confirms and extends the observa-
tions of MacKinnon and Deller (1973).
194
Since there was no active pathway of de novo biosynthesis, the use of dietary purines by the salvage pathway
was considered.
Animals on chow diets who received isotope
via gavage provided evidence of salvage of dietary purines.
There was significantly (P < 0.05) greater incorporation of
3 H-adenine
(but not
3 H-adenosine)
into the proximal small
intestine than into the rest of the gastrointestinal tract.
Thus adenine was incorporated into proximal small intestinal
RNA after absorption from the intestinal lumen.
When animals
deprived of dietary protein and purine were studied, there
was significantly (P K 0.05) greater utilization of both
3 H-adenine
and
3 H-adenosine
by the proximal and middle
portions of the small intestine when compared to the rest
of the gastrointestinal tract.
The incorporation of label
into the caecum, colon and liver was attributable to salvage of purines derived from the vascular system.
The high level of small intestinal incorporation when
isotope was fed was not the result of an increased extraction by that tissue of vascular purines.
This is demon-
strated in the studies of identical animals who received
isotope by intraperitoneal injection.
In these studies, the
small intestine demonstrated incorporation essentially at
the same level as the liver.
Thus the finding that the
uptake of label by the RNA of the small intestinal mucosa
was more extensive on a diet devoid of purines suggests
that exclusion of a normal dietary source of purines resulted in less dilution of the labeled base.
This is accord-
195
ingly evidence that dietary purines are normally an important source of purines for the mucosa.
Animals fed a chow diet and injected with
3 H-adenine
(or adenosine) demonstrated uniform incorporation in all
gastrointestinal tissues except the stomach (see below for
The route of incorporation
discussion of the stomach).
following absorption of the intraperitoneal injection was
clearly via the blood stream.
Animals fed a purine-free and
protein-free diet and injected with isotope demonstrated
markedly elevated incorporation into the RNA of the colon.
Thus, intraluminal purines are utilized by the small
intestine for RNA synthesis.
On a protein and purine
deficient diet, the small intestine is capable of deriving
a highly significant amount of purines for RNA synthesis
from the intestinal lumen.
The small intestine extracts
less purines for RNA synthesis from the vascular compartment than does the colon, and indeed the colon has a special
ability to extract vascular purines for RNA synthesis.
As a result of the above data, the role of dietary
purine sources in relation to mucosal supply was further
explored in the mucosa of the small intestine.
The effects
of six diets, all identical except for protein and purine
contents, were studied with regard to the activity of the
de novo pathway, and the data was analyzed by two-way
analysis of variance and the Tukey test.
When animals
were fed a complete synthetic diet without purines
196
there was a restoration of some in vitro labeling of
14
C-glycine without corresmucosal RNA adenine from
ponding changes in protein labeling.
This change in
purine labeling through de novo synthesis could be due to
less dilution by a reduced pool of free adenine nucleotides
when purines are absent from the diet, but such a large
change seems unlikely.
A more probable cause is an increase
in de novo biosynthesis when purines are excluded from the
diet and a near obliteration in the activity of this pathway when they are available from the diet.
The supply of
purines thus regulates de novo synthesis. Specifically,
14
C-glycine (via the de novo pathway)
the incorporation of
in the group fed the adequate protein but no purine diet
was significantly (P (
0.05) greater than the incorporation
in any of the other groups.
Detailed analysis showed that
this was the result of three significant effects:
(1) the
effect of the protein content of the diet (P < 0.001);
(2) the effect of the purine content of the diet (P(
0.01);
and (3) the interaction effect between the two (PK(0.01).
The exa'mination of the RNA/DNA ratio by the same methods
as above reveals that there is a demonstrable purine effect
(P 4, 0.001) but no significant protein or interaction effect.
Multiple comparison testing, however, shows that the only
significant (P
(
0.05) differences were between the animals
fed the protein-free and purine-free diet on the one hand
and both groups (with and without protein) receiving the
purine bases on the other hand.
If this represents a true
197
biological phenomenon rather than chance causing an elevation
of the RNA/DNA ratio in one group, then an explanation will
demand further work.
Similar analysis of the protein/DNA
ratios revealed a significant (P < 0.05) variance component as
a result of the dietary-protein content.
Multiple comparison
testing utilizing the Tukey test and the Student-Newman-Kuel's
test, however, revealed no significant differences among the
group means.
The lower ratios in the protein-free diets
containing purine bases or RNA are suggestive of a possible
biological effect.
It is however only speculation that they
represent a true decrease in cell size.
This speculation is
not entirely unwarranted if based upon the premise that the
dietary purines have a role to play in the regulation of cell
turnover.
The increased cell turnover in the presence of
protein deprivation would result in a reduction in cell size.
This decrease in cell size however would be offset, at least
in part, by the increased number of young, large cells.
(Intestinal cells decrease in size as they age and migrate
up the villus.)
After animals had been fed a protein-free and purinefree diet for seven days, the effect of re-feeding with a
protein-containing diet, varying in its purine content, was
evaluated.
The de novo activity was demonstrated only in
the group re-fed on a purine-free diet (P = 0.09).
The
lower level of statistical significance is attributed to the
small number of experiments performed and the resultant
decreased degrees of freedom available in statistical analysis.
198
No significant effects of the various diets on the salvage pathway in the colon was demonstrated in either the
feeding or re-feeding studies.
The demonstration of the presence of a weak de novo
pathway in the small intestine when purines were excluded from
the diet 5uggested that the key enzyme in de novo purine biosynthesis, amidophosphoribosyl pyrophosphate transferase, might
be present in low concentrations but inhibited under normal
dietary conditions.
In an effort to verify this and to try
to determine possible mechanisms by which this would be
explained, a method for the assessment of the enzyme in
liver and small intestine was developed.
of Tay et al. (1969) was employed.
assay was confirmed.
The general method
The validity of the
The small intestine was found to con-
tain significantly less amidotransferase than either the
colon or the liver.
This was true on a purine-free diet
(P / 0.005) and on a purine-added diet (P 4 0.025).
There
was no significant difference in the amount of small intestinal enzyme between the two diets.
Similarly, the colon
showed no significant change in activity related to the
diet.
On the purine-free diet, however, there was a 66%
increase in the liver amidotransferase activity which was
significant at the P = 0.06 level.
cluded that the increased
Therefore, it is con-
1 4 C-glycine
incorporation into
RNA observed in the experiment described above is not due to
more enzyme molecules but must be due to a change of some
factor influencing enzyme activity, i.e.,
the supply of sub-
199
strate.
As figure 4 shows, this could well be PRPP, the level
of which is regulated by utilization in the competing salvage
pathway.
Thus, the presence of purines in the diet could
rapidly reduce the availability of PRPP for the de novo pathway and regulate it as is shown in figure 4.
Measurements of the hypoxanthine-guanine phosphoribosyl
transferase demonstrated a very active salvage pathway in the
small intestine.
Significantly more (P4( 0.01) HGPRT activity
was found in the small intestine than in either the liver or
the colon when the animals were being fed a diet containing
purines.
Animals on a purine-free diet showed opposite
enzyme -activity changes in the small intestine and liver.
The liver HGPRT activity increased on the purine-free diet,
while the small intestinal activity decreased.
These latter
studies just failed to reach statistical significance
probably because of the limited number of animals involved.
Preliminary studies have been done to explore certain
aspects of purine metabolism in the mucosa of the small
intestine.
The de novo pathway appears to be more active
when diet and digestive juices are excluded from a selfemptying blind loop of small intestine,
In contrast, in-
corporation by this pathway was inhibited in the mucosal
atrophy caused by parenteral feeding.
Of considerable im-
portance was the finding of inactivity of the de novo pathway in all cell types (crypt as well as villus), indicating
that possible selection of villous cells in the mucosal
preparation is not the reason for the failure to observe an
200
active de novo pathway.
It is thus shown:
(1) Under normal dietary con-
ditions, no de novo pathway for purine biosynthesis is
demonstrable; (2) The small intestine utilizes dietary
purines for RNA synthesis;
(3) The colon utilizes intravascular
purines to a much greater extent than does the small intestine;
(4) The purine content of the diet has an effect upon the
ability to demonstrate an active de novo pathway of purine
biosynthesis, the pathway being demonstrable only on a purinefree diet;
(5) There is low amidotransferase activity in the
small intestine compared to either the colon or liver and it
does not significantly change with alterations in the purine
content of the diet;
(6) There is a very active salvage path-
way in the small intestine.
In conclusion, the data reported here show that
the diet is a major source of purines for the mucosa of the
small intestine, and that the de novo pathway is not a major
source of purines on diets containing purine bases as such
or as nucleic acids.
However,
a modest increase in this
pathway (without enzyme induction) can be achieved by eliminating dietary sources of purines.
This allows the existing
low-level of PRPP amidotransferase to act more efficiently,
probably through having more available substrate (PRPP)
because less is used for salvage.
It is also possible that
on the purine-containing diet there may be more free nucleotides present resulting in a greater level of allosteric inhibition.
201
This work confirms that of MacKinnon and Deller (1973)
and goes significantly beyond their study.
A new area
for the study of intestinal metabolism has been opened.
The results presented suggest many new questions for
further work.
202
VIII.
1.
SUGGESTIONS FOR FUTURE RESEARCH
The essential findings of this work should be
confirmed in the human gastrointestinal tract.
2.
Studies of intestinal cell turnover should be
done on diets of varied purine content.
3.
The effect of dietary purine content upon the
mucosal hyperplasia that occurs after intestinal resection must be examined.
4.
The relationship of dietary purine content to
the development of carcinoma in the stomach, small
intestine and colon should be examined after feeding
carcinogens.
5.
The relationship between dietary purine content
and purine biosynthetic enzyme activities in the liver
and gastrointestinal tract should be further studied.
6.
During total parenteral nutrition the effect upon the
intestinal mucosa of both feeding and infusing purines
should be studied.
7.
The clinical effects of defined formula diets
devoid of purines should be studied.
8.
The use of purines in tissue culture media should be
studied in relation to efforts to culture mature enterocytes
in vitro.
203
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BIOGRAPHICAL NOTE
Neal Simon LeLeiko was born in Brooklyn, New York on
October 26, 1946.
He attended Brooklyn College of the City
University of New York and received his Bachelor of Science
Degree (Chemistry) in June, 1967.
He received his M.D.
degree from New York Medical College in June, 1971.
He did
his internship, residency and chief residency in Pediatrics
at The Mount Sinai Hospital in New York City from July, 1971
to June, 1974.
From July, 1974 through June, 1976 he served
in the United States Air Force as Chief of Pediatrics at the
Rickenbacker Air Force Base Hospital.
From July, 1976 to
June, 1979 he served as a Fellow in Medicine (Gastroenterology and Nutrition) at the Children's Hospital Medical
Center.
He married Marilyn Bush on August 20, 1967.
They
have two daughters, Sarah (four years old) and Rebecca (eight
months old).