BODY 900:. SIZE AND SECRETION
or ACETATE m ms. RAT

Thesis for the Degree‘ :of M. S.
MICHIGAN STATE UNIVERSITY
SHIRLEY CHlH-HSUAN CHEN

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THESIS Michigan State
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ABSTRACT

BODY POOL SIZE AND SECRETION
OF ACETATE IN THE RAT .

by Shirley Chih~13uan Chen

Twenty—four male Sprague~Dawley rats were used for the determina—
tion of Secretion rate and body pool size of acetate. Another nine rats
were used for the determination of tissue in vitro uptake or utilization
of VFA. The single~injection technique was used in the determination
of secretion rate and the body acetate pool size, in which potassium—l~1“C
acetate was the tracer. Different levels of acetate, prOpionate and
butyrate were incubated in cecal and large intestinal plus rectal tissue‘
'to determine utilization of VEA by these tissues.

Cecun, large intestine and rectum were the major sites of the me—
tabolism of acetate which came from the small intestine or directly from
the blood. Recovery of radioactivity in GI contents and VFA concentra—
tion in blood_indicated that the microflora in the cecum, large intes—
tine and rectum Were responsible for the presence of acids while the
secretion of acid from the blood is too small to be significant.

Body acetate pool size was 4.196 nmoles with a turnover rate of

0.1007 mmoles/nin. and half life of 3.69 minutes.

03

In viiru study rev;aled that there was no Uptake or utilization of

VFA by cecal or large intestinal plus rectal tissuet.

BODY POOL SIZE AND SECRETION

0F ACETATE IN THE RAT

137

Shirley Chih—Hsuan Chen

A THESIS

_ Submitted to
Michigan State University
in partial fulfillment of the requirements
‘ for the degree of

MASIER OF SCIENCE

Department of Foods and Nutrition

1969

A (3232

.-‘| ‘7

l—‘7'70

ACKNOWLEDGEMENTS

The writer expresses her hearty appreciation and gratitude to
Dr. Modesto G. Yang for his encouragement, guidance and invaluable
advice during the course of this work as well as the school years.

The writer also wishes to thank Drs. Robert M. Cook, Olaf
Mickelsen and Steven D. Aust for their kind suggestions, guidance in
the experimental procedures and generosity in providing the laboratory
instruments and chemicals.

Finally, the writer dedicates this work to her dearest parents.

ii

INTRODUCTION.

TABLE OF CONTENTS

REVIEVI OF LIIE&\TLTRE O O I O O O O O O O O O O O O O O O O I O O

I.

II.

III.

IV.

EXP ERIE—IE N TAL .

I.

II.

General aspects of volatile fatty acid produc—
tion and absorption in ruminants . .‘. . . . . . .

The metabolism of acetate, prepionate and butyrate
in the ruminant. . . . . . . . . . . . . . . . . .

The metabolism of acetate, prOpionate and butyrate
in t}le rat 0 O O I O O O O O O O I I. O 0‘ O O O C 0

Methods for analysis of volatile acids . . . . . .
Experimental animals . . . . . . . . . . . . . . 3
Experimental procedures. . . . . . . . . . . . . .
A. Chemical purification of sodium—l—IQC acetate.
B. Sample collection. . . . . . . . . . . . .

C. In vitro study of tissue uptake of VFA . . . .

D. Determination of VFA concentration of GI
contellts O O O O O O O O O O O O O O C O O I O

E. Determination of VFA concentration in blood.
F. Efficiency of liquid scintillation counting.

G. Radioactivity counting . . . . . . . . . . . .

RESULTSANDDISCUSSION.

I.

II.

Efficiency of liquid scintillation counting. . . .
Elution profile of standard acids: acetic, pr0*

pionic, butyric, isobutyric, valerlc and iso—
Valerie aCj.d O O O C I O O O O O O I O O O O O O 0

iii

Page

11

12

12

12

12

15

15

17

18

19

19

20

20

20

III

IV.

VI.

VII.

SUMMARY AND CONCLUSIONS

Distribution of gastrointestinal

VFA concentration of GI contents

Radioactivity of GI contents .

VFA concentration in blood, body
determination.

In Uitro stud‘7 of tissue untake of
J 1

acids.

LITERATURE CITED. .

APPENDIX.

iv

contents.

acetate pool size

volatile fatty

Face

22

22

25

31

36

4O

41

45

Table

10

11

ll

12

12

13

13

LIST OF TABLES

COHIpOSition Of basal diet. 0 O O O O O O O O O O O O O .

Supplemental mineral and vitamin per kilogram basal
diet 0 O O O C O O I O I O C O O O O O O I O O O O O O 0

Blood sampling schedule and parts of GI tract used for
VFA determination. . . . . . . . . . . . . . . . . . . .

Distribution of gastrointestinal and fecal contents. . .

Total and distribution of organic acids in the GI tract
and feces (micromole acid/gram digesta [wet]). . . . . .

Total counts in contents of each section of the GI tract

0 O O l O
and feces after injecting 1+C acetate into the heart
chamber (average of 4 saMplcs) . . . . . . . . . . . . .

Radioactivity in each section expressed as a percentage
of the total GI radioactivity found in each time inter—
val (average of 4 samples) . . . . . . . . . . . . . . .
Specific activity of GI contents (CPM/umole) . . . . . .
Blood Specific activity of acetate at various times. . .
Recovery of acids from the tissue incubation study . . .
Distribution of gastrointestinal contents (I). . . . . .
Distribution of gastrointestinal contents (II) . . . . .
Radioactivity in GI sections (I) . . . . . . . . . . . .
Radioactivity in GI sections (II). . . . . . . . . . . .

Specific activity of GI sections (I) (CPH/nmole) . . . .

Specific activity of GI sections (11) (CPM/nmole). . . .

Page

13

13

16

23

24

26

27
29
35
38
45
47
49
50
51

52

Figure

LIST OF FIGURES

Chemical purity examination on Na—l»

C14

acetate~~

elution and radioactivity profile. . . . . . . . . . . . .

Elution profile of standard acids:
n— and isobutyric, n~ and isovaleric acid.

Regression line of blood acetate specific activity and

time after injection .

vi

acetic, prepionic,

Page
. l4
. 21
. 34

INTRODUCTION

Volatile fatty acids (VFA) are the main products of cellulose and
other carbohydrate digestions in the rumen. These acids are valuable
energy sources for ruminants. VFA are also found in the gastrointes-
tinal (GI) tract of monogastric animals. In rats the concentration of
the.acids in the cecum is as high as that found in the rumen (18).
However, unlike ruminant species, the quantity of VFA derived from the
digestion of carbohydrates including cellulose is not known for mono-

‘ gastric animals. By extrapolating studies of ruminant animals, VFA in
rats are assumed to come from the digestion and/or fermentation of car-
bohydrates. It is also assumed that fermentation is the greatest source
of the acids in the GI tract of these animals.

The acids in the GI tract of monogastric animals could also have
come from secretion or conversion from other metabolites in situ or
both. There are probably some VFA utilized by the GI tissues as a
source of energy. The uptake and/or utilisation of VFA locally in the
tracts would lower the quantity of VFA reaching the circulatory system.
The quantity of VFA in the blood and the rate of disappearance of this
pool are unknown for rats. Furthermore, the literature provides very
little information concerning the quantity of VFA present in the rat,
the formation of these VFA and the uptake or the utilization of these
VFA.by the rat. Thus, this work was undertaken primarily for the fol—

lowing objectives:

2

1. To determine the body pool size of acetate in rats.

2. To determine the amount of this acid secreted into various
sections of the GI tract of the rat. The sections studied
were stomach, Upper and lower small intestine, cecum, large
intestine and rectum.

3. To determine whether VFA are utilized by cecal and large
intestinal tissues in vitro.

The single injection technique was used in this study and
sodium—l—IHC acetate was the tracer. The results showed that the body
pool size of acetate in rats is 4.196 mmoles. Half life was 3.69 minutes.
Secretion of acetate into the GI tract was present but quantitatively
insignificant. There was no indication that cecal or large intestinal

tissue utilizes VFA.

REVIEW OF LITERATURE

I. General aspects of volatile fatty acidAproduction and absorption

 

in ruminants.

 

As noted by Annison and Lewis (7), Tappeiner (38) as long ago as
1882 demonstrated that the fermentation of cellulose in the rumen of
the ox resulted in the formation of large amounts of volatile fatty
acids (VFA), which he concluded contained at least 50% acetic acid.
This observation was largely overshadowed, however, by the subsequent
work of Kellner (28) in which cellulose was found to be of the same
energy value as starch when fed to steers. For many decades VFA found
in the rumen were considered to be of little nutritional significance
and the digestion of cellulose was assumed to proceed no further than
a depolymerization to glucose, which was then absorbed and metabolized
as in monogastric animals. It is now established, however, that VFA
found in the rumen arise largely from the fermentation of dietary car-
bohydrates. These acids constitute the major source of energy to the
ruminants, since only a small prOportion of the ingested carbohydrate
escapes degradation in the rumen (7).

Acetate predominates in the mixtures of VFA.which are found in the
rumen under all dietary conditions and numerous in vitro studies have
shown that acetate is the major endproduct of the fermentation of carbo-
hydrates by rumen microorganisms. The following route for the produc-
tion of acetic acid from carbohydrates has been prOposed by Annison and

Lewis (7):

4

 

(C6)* -»> n.C6-—em-~«p C6 phosphate -»-~—$>pyruvate
acetate + CO2 lactate
*any kind of polysaccharide

Elsden (17) first conclusively demonstrated the presence of pro-
pionic acid in rumen contents and showed that it was produced during in
vitro fermentation of cellulose, glucose and lactate. Organisms respon—
sible for prepionic acid production in the rumen and from enrichment
cultures of rumen contents were identified as small Gram—positive cocci
of the genus Propionibaoteriun. These organisms fermented lactic acid
and glucose with the production of prOpionic acid and acetic acid and
carbon dioxide.

Veillonella gazofenes which was identified by Johns (27) was exten~
sively studied and the production of propionate from lactate was shown

to occur by a carbon dioxide fixation mechanism:

CO .
2 .
Lactate-"m“; Pyruvate~§emp Oxaloacetate-w~>rMalate

---«> Fumarate ...--..> Succinate ”v“; PrOpionate + CO2

Longer chain fatty acids in the rumen fluid are produced by secon»
dary reactions from both acetic and prepionic acid (24). There is much
less direct fermentative synthesis of butyrate other than from the con—
densation of acetate (30). The incorporation of the major VFA into the
branched—chain and longer chain acids is low (24).

The recognition of the importance of VFA as major sources of energy
to the ruminant has focused attention on the problem of measuring the

amounts of VFA produced in the rumen. Attempts have been made to asses

U)

the rates of production of the individual VFA after feeding by following

changes in the relative prOportions of acids in the rumen (23). The

5

system is complex, however, since the concentration of a particular acid
(or any rumen metabolite) at any one time is dependent on the rates of
its a) production in the rumen, b) absorption from the rumen, c) passa e
from the rumen to the omasum, d) dilution with saliva and food, 3) utili-
zation by rumen microorganisms and f) conversion to other rumen metabolites.

In 1966 Gray at al. (25) determined the rates of production of VFA
in the rumen. One part of the work measured the in viva production of
VFA by two isotope dilution procedures. The production of VFA by two
sheep was measured by two different methods involving continuous infusion
of C1”~labeled fatty acids into the rumen through a rumen fistula and
determining the concentration of 1“C in the rumen acids throughout the
feeding cycle. The infusion solution contained sodium acetate, prOpio~
mate and butyrate in proportions (75w80: 14~15: 6~lO%) which are nor—
mally found in rumen fluid. The acids were labeled with 1”C in the
C-1, C—2 and C—1 positions, respectively, and the total specific activity
was 0.03~0.04 uCi/ml. In one procedure, the varying rate of production
of volatile acids was matched by a varying rate of infusion so that a
constant concentration of label was maintained in the rumen acid. In
the other procedure, a constant rate of infusion was maintained and a
mean value was determined for the concentration of label during the
feeding cycle. Both procedures gave similar values, approximately 5
moles of VFA were produced per Kg of dry fodder. The next part of their
work (41) measured the production of the individual and total volatile
fatty acids. In sheep fed at 12 hour intervals a diet of lucerne and
Wheaten hay, production of volatile fatty acids was measured by infusion
of a mixture of 1L’C—labeled acids. They assumed that transfer of 1”C
from one acid to another was equal, thus total 1”C in each acid is con—

stant. The productions of individual acids were determined by infusion

6
of a single 1L‘C—labeled acid and measurement of the concentration of
14C in a composite sample of the acid in the rumen fluid collected
throughout the feeding cycle. With automatic sampling of rumen fluid,
they showed that this procedure was suitable for routine use. They also
showed that the composition of the acid mixture initially formed in the
rumen was acetic 77—83, propionic 15-18 and butyric acid 1*71. About
50-80% of the butyric acid was formed from acetic acid. Degradation of
luc-labeled butyrate formed from acetate-l—IQC in the rumen showed 93%
of the 1L'LC to be nearly equally divided between atoms C—1 and C-3.
Leng (31) used a constant intraruminal infusion of acetate—u—lkc,
propionate—u~1“C and butyrate 2,3—3H technique to measure simultaneously
the production rates ofacetic, prOpionic and butyric acid in the rumen
of sheep. There was a close correlation between production rates and
concentration of the individual acids in the rumen.

Barcroft, McAnally and Phillipson (10) first demonstrated the ab—
sorption of VFA from the rumen in anesthetized sheep, when they showed
that the concentration of these acids in blood draining the rumen was
higher than that of peripheral blood. Reid (36) using a chromato—
graphic method found that about QOZ of the VFA of sheep blood was acetic
acid and similar findings with respect to cattle blood were reported
by McClymont (34). Subsequent work using methods similar to Barcroft
et a1. suggested that the rate of disappearance of undissociated VFA
from the rumen increased with chain length (16) and that pH markedly
affects absorption rates (22). This dependence on hydrogen ion concen—
tration is related to the prOportion of acid present in the undissoci»
ated form. An increase of pH from the range normally associated with
rumen contents (5.5~6.5) to pH 7.0—7.5 leads to decreased VFA absorption

,

rates. Studies on the absorption of VFA from the cashed out and tied

off rumen of anesthetized sheep revealed a linear relationship between
acetic acid loss and movement of bicarbonate into the rumen, the absorp—
tion of two molecules of acetic acids being accompanied by the entry of
one molecule of bicarbonate (32). Ash and Dobson (9) confirmed this
result, and showed that the absorption of fatty acid from the rumen is
accompanied by a consumption of CO2 and by the production of bicarbonate
within the rumen solution, because of the incoming of unionized acid.
Near neutrality, about half of the fatty acid was left in unionized form.
The amount of VFA neutralized directly in this manner was about equal

to the amount neutralized by saliva. There is no evidence of active
transport of VFA across rumen epithelium and the concentration gradient
is the most important factor in determining rates of transfer. When
solutions free of fatty acids were placed in the isolated rumen, blood
VFA entered the rumen and the final concentration was close to that of

plasma.

II. The metabolism of acetate, prOpionate and butyrate in the ruminant.

 

 

Mature ruminants absorb relatively smaller quantities of glucose
from their digestive tract and depend to a large extent on gluconeo—
genesis for their glucose supply. Gluconeogenesis from prepionate is
of major importance (8). The data of Bensadoun at al. (11), however,
suggested that gluconeogenesis from precursors other than propionic
acid, e.g., lactate, could also be important. The mean value derived
from the data of Warner (40) based on estimates in the literature of
daily amounts of prOpionate absorbed by cattle, sheep and goats is
13 Kcal/Kg0°75/day. This SUpports the View that propionate is a major
source of glucose; however, that gluconaoeenesis from other sources
is likely to be important at least in the pregnant and lactating

ruminant (8).

8

Annison at al. (6) studied intensively the metabolism of acetic
acid, propionic acid and butyric acid in sheep. Acetateul—IQC,
prOpionate—l-th, prOpionate-2~1”C, butyrate—l-lqc, butyrate—Z—IHC and
butyrate-3~1”C were insufed into the portal vein of anesthetized sheep.
The result showed these labeled fatty acids were incorporated into blood
glucose, lactate and ketone bodies. The measured specific radioactivity
(S.A.) are summarized as follows:

1. ,The S.A. of plasma glucose was 2*5Z of the infused acetate or
butyrate, but 30% of the infused prOpionij2~1”C acid.

2. The S.A. of blood lactate was less than that of blood glucose
except after the infusion of butyrate—Z-IHC, when it was slightly higher.

3. The S.A. of blood B—hydroxybutyrate was about 50% of that of

14C, about 7—15% of infused acetate and about 2% of

infused butyrate—
the infused prOpionate~1”C.

4. The S.A. of blood formate was about 5% of that of infused
acetate—IHC, pr0pionate-luc or butyrate~1”C.

5. The S.A. of blood acetate was less than 1% of that of infused
propionate~1uc but about 6wlOZ of that of infused butyrate~1”C.

The infusiOn of acetate—l-IHC, prOpionate—l—IHC, butyrate—l—IHC
or butyrate—B—lqc gave rise to labelling mainly C—3 and C~4 of glucose
and C—1 of lactate. The infusion of acetate—2~1”C, propionate—2—1”C
or butyrate—Z—IQC labeled mainly C~l, C—2, C—5 and C—6 of glucose, and
C-2 and C43 of lactate. Thus, the metabolic pathways were concluded
to be Similar to those known to occur in other mammals.

The changes in blood volatile fatty acids after their infusion
into the rumen ofa cow were studied by Asfjes (l). A distinct and
rapid rise of a volatile fatty acid in the blood followed the introduc»

tion of the acid. However, butyrate gave not only a rise of the

9
butyrate content of the blood but also a marked rise in the blood pro~
pionate and acetate levels.

Acetate metabolism in ruminant tissues was studied by Hayfield at
al. (33). Acetate—l—IQC was incubated with various tissue homogenates
prepared from tissues of fed and 7-day fasted sheep to study the site
and routes of acetate metabolism in tissues of ruminants. Transfer of
11+C from acetate to C02, neutral lipids, free fatty acids, nondistil~
lable organic acids and proteins was followed. They found that acetate
utilization by ruminant tissues was most for adipose tissue, and de—
creased in order for kidney, muscle, heart, lung, liver and brain when
eXpressed on the basis of per unit of protein. The 7—day fasted sheep
tissues were slower in the overall acetate metabolism but CO2 production

from acetate was lowered only in liver and brain tissues.

III. The metabolism of acetate, propionate and butyrate in the rat.

 

Most herbivorous animals have an eXpanded part in their digestive
system where fibrous materials can be delayed in their passage through
the digestive tract; this allows fermentation to take place. In rumi—
nants the expanded part is the rumen, whereas in the rat the cecum is
the segment that is expended. Much work has been done to determine the
utilization of acetate, prOpionate and butyrate as energy sources for
the ruminant. However, similar investigations carried out on rats are
relatively few in number.

Elsden at al. (18) published a paper on the concentration and quan—
tity of volatile fatty acids in the digests of ruminants and other
animals in 1946. This is the only aper that could be found which
dealt with FA in the rat. The quantity of volatile acid in proportion

to the body weight is greatest in the ox and least in the rat. The

10
amount of volatile acids pre se.r nt in the digests depends on the rate at
which it is produced and the rate at which it disappears from any organ.
The regions of the aliment.ary tr .act, in x-.hich fermentation occurs, were
sharply defined and consist of runen and reticulum in the ruminant and
the large intestine in all other species. The most: impo ‘rtant pa) t of
this report was that on the basis of concentration the volatile acids
in cecal dige sta of or on, sheep and ate were alzvost the same (18). In
sheep the concentration was 4.3, in oxen 3.2 and in the rat 4.6% of the
dry ingesta.

Early in 1943, Buchanan at al. (12) Synthes sized acetate, propionate
and butyrate containing radioactive carbonyl—C and fed them with glu—
cose to fasted white rats. Evidence was presented which indicated that
prepionic acid and butyric acid were converted to liver glycogen, but
that acetate was not. Approximately 50% of the radioactivity of the
ingested fatty acids was excreted in the respiratory gases as C02 in a
2—hour period.

Wood at al. (42) isolated the glycogen of rat liver following intra—
peritoneal administration of heavy carbon bicarbonate and feeding glu-
cose by stomach tube. The position of the labeled carbon in glucose
from the glycogen was determined by bacterial and chemical degradation.
They found that CO2 carbon was fixed in 3 and 4 position of glucose,
thus proving CO2 fixation is involved in the synthesi is of glucose.

The incorpor ration of acetate and butyrate carbon into rat liver
glycogen was studied by Wood at al. (43). Evidence showed that these
acids could be converted to glycogen via pathways other than carbon

dio xid efixation. The result shcwed that aft er feeding labeled 13C—

r

acetate, doubly labeled acetate or labeled 13C~butyratr not only carbon

atoms 3 and 4 but all de gxedation fractions of glycogen contained

11
significant amounts of 13C. This furnished direct evidence that at
least the carbon atom of the VFA studied was incorporated into rat

liver glycogen by some means in addition to C02 fixation.

IV. Methods for analysis of volatile acids.

 

 

A major difficulty in the early studies of ruminal VFA and their
absorption and subsequent metabolism was the lack of reliable methods
for the analysis of mixtures of VFA. This difficulty was overcome by
Elsden (l7, 19), who devised a liquidwgel partition chromatography
technique which allowed the separation and estimation of a mixture of
acetic, prOpionic and butyric acids. The first accurate analyses of
volatile fatty acids of rumen contents thus became available, and
acetic, prepionic and butyric acids were invariably found to be present.

A second major advance in the analysis of VFA was the gas—liquid
partition chromatographic methods of James and Martin (26). This tech~
nique has revealed that in addition to acetic, prepionic and butyric
acid, small quantities of the naturally occuring branched—chain isomers
of butyric and valeric acid (isobutyric, 2~methylbutyric and isovaleric
acids) and n-valeric acid are usually present in the rumen (2).

Recently, Ramsey (35) used benzene, chloroform and the following
mixtures of tert~butanol in chloroform: l, 2, 5, 8, 12, 16, 24 and
30% (v/v) as the eluting solvents for a mixture of 17 pure organic
acids as well as protein free filtrates of ruminal blood saMples through
the silicic acid column. The result showed that the quality of resolu-
tion is satisfactory for most of the acids from capric acid to isocitric
acid. Ramsey's method is the main procedure which was used in the

present experiment to analyze for the different VFA.

EXPERINECL‘ETAL

1- £312.91"; 21:95:25.1. 3:15:18;

Thirty—three male SpraguenDawley rats having average body weight
of 350 grams were purchased from a local supplier.* The rats were housed
individually in wire bottomed cares and were fed ad Zibitum M—l diet
(Tables 1 and 2) and water. The rats were naintained on the diet until
their body weights reached 450 to 500 grams, which took approximately 3
weeks. At this time, 24 of the rats were used for the determination of

secretion rate and body acetate pool. The other 9 rats were used for

the determination of in 05530 tissue uptake or utilization of VFA.

I I- I Easement-32551-259939::Isc=5
A. Chemical purification of'sodium~l~1”C acetate.**
Sodium—l-IHC acetate was chromatOgraphed and the profile on purity
was checked by titration and by counting the radioactivity of the frac—
tions. There were traces of lucvprOpionate and formate besides acetate
(Fig. 1). Each batch of 20 uCi of the purchased sodium—l~lkC acetate
was run through silicic acid column and only tube numbers 19 to 26 were
pooled and made alkaline with 20% KOH solution. This solution was then

evaporated to dryness and redissolved in JO ml saline which ave a S.A.

O'
C)

of 2.9 x 105 CPN/O.l ml. The purified K~l~1”C acetate was used for

 

*Spartan Research Animals, Inc., Williawston, Michigan.

**Sodium~l+1“C acetate was purchased from Nuclear Chicago; its
Specific activity was 10.00 millicurie per millimole.

l2

13

Table 1. Composition of basal diet

 

 

 

Ingredients 1 2
Ground corn 60.8
Soybean meal (50% protein) ' 28.0
Alfalfa meal (17% protein) 0 2.0
Fish meal (16% protein) ' 2.5
Dried whey (67% lactose) ‘ ‘ 1.6
Limestone (38% Ca) 1.6
Dicalcium phosphate (18.5% P, 23.5% Ca) 1.7
Iodized salt ‘ . 0.5

 

Table 2. Supplemental mineral and vitamin per kilogram basal diet

 

 

 

. . Grams
Vit. A (Pfizer lOP, 100,000 USP/gm. 0.8088
Vit. D (9F Fleishmann in yeast, 9000 USP/gm. 0.0836
Choline chloride 0.6996
Ca pantothenate 0.0055
Niacin 0.0330
Riboflavin 0.0033
Vit. B12 (0.1% mannitol trituration) 0.0066
a-tocoPherol acetate 0.0044
Menadione 0.0022
DL-methionine 0.4994
Trace metal premix 0.9900

In 2: Mn 12.2, Fe 9.6, Ca 7.5, Co 0.26,
Cu 0.73, Zn 5.0, I 0.38

 

no; H.522 En. $228

 

CHEMICAL PIU4RITY EXAMINATION (N

FIG.
P l

 

 

 

  

 

2 N
I I w 8 6 4 2
m
E I..
E E .
H L m
T I """l' -
no I'I'lllllx’
\
w.”— m I'l“l"l|'|l‘|||||. 2
A |‘||I|'I'II _
T VI IIIIIII||I C I
G ..|.. .IllllH
A W II'III"IIIIII'IIII'II'OI'I'I'I-"""ll"""
a I
A
m m
. D 0.9
m m c
D D
w N
A u
u m
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P .H
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6. 5. 4. 3 2 1

0mm: 10x U_._OIOUJ< z _.o.._2

TUBE NUMBER (10 ML FRACTION)

15
injection, incubation and for standard curves use“ for identification

of unknown.

B. Sample collection.

The rats were fasted for 2 hours from 11 a.m. to l p.m. and then
anesthetized with ether. Heart puncture technique (13) was then used
to inject 0.125 pCi of potassiumwl—14C acetate in 0.5 m1 saline. After
the time intervals indicated on Table 3, ether anesthesia was given to
the rat again. A midventralincision was made to facilitate removal
of the whole GI tract. After removing the GI tract, it was immersed
in crushed ice immediately. Just prior to removing the GI tract, a
heparinized syringe was used to draw blood from the heart and portal
vein._ The blood was then deproteinized by a modified method of Folin
and Wu (21), centrifuged and the supernatant frozen until analyzed.

The GI tract was sectioned (Table 3) and digesta in the GI tract
were removed into separate chilled and tared containers and stored

frozen (~20°) until VFA‘were separated by column chromatography (35).

C. In vitro study of tissue uptake of VFA.

Nine rats which were fasted for 24 hours with water available at
all times were sacrificed by over etherization and bleeding. GI tissues:
a) cecum, b) large intestine and rectum were then cut into pieces of
15 to 20 mm2 squares and were then weighed on a Smith—Roller tissue
balance. All procedures mentioned above were performed in the cold.

From 85 to 125 mgs of the tissues were incubated in Krebs~Ringer bicar—
bonate solution (39) together with 100 units Penicillin G and 0.1 mg
streptomycin per ml and a known concentration of various volatile fatty
acids. They were a) 25 umoles acetic acid per ml, b) 5 Umoles prOpionic

acid per ml, c) 15 pmoles butyric acid per ml and incubated for 10, 20,

16

Table 3. Blood sampling schedule and parts of GI tract used for VFA
determination

 

 

Time after sodium-l-IQC acetate injection (min.)

 

 

 

 

10 15 20 3O 45 60

G1 section number of samples
Stomach 4 4 4 4 4 4
Upper small intestinel' 4 4 4 4 4 4
Lower small intestine1 4. 4 4 4 4 4
Cecum 4 4 4 4 4 4
Large intestine2 4 4 4 4 4 4
Rectum2 4 4 4 4 4 4
Feces3 4 4 4 4 4 4
4 Blood 4 4 4 4 4 4

 

1The small intestine was divided into two equal portions and the
anterior segment is referred to as U.S.I. and the posterior segment is
referred to as L.S.I.

2From the cecum to the first hard pellet in the large intestine.
The remaining large intestine, containing hard and well formed fecal
pellets, was considered to be the rectum.

3Feces were collected when drOpped between the time of injection
and during anesthesia.

17
40, 60 or 90 minutes. The tissue media and VFA were placed in 10 ml.
capped vials to prevent excessive evaporation. The vials were placed
in a shaking water bath at 37° and incubated for the designated time.
Two controls were included during each incubation: a) Krebs—Ringer
bicarbonate solution plus tissue but without acid, which served as
blank, b) Krebs-Ringer bicarbonate solution plus acid but no tissue,
which checked the possibility of loss of acid during incubation.

At the end of incubation, the tissues *ere taken out, and the media
were diluted ten to one with 0.7 N H2804. Ten ul of this diluted sample
'were injected with a Hamilton syringe into a F—M gas chromatograph. The
acid concentration was detected by flame ionization with helium as the

c with controls.

0

carrier gas. The concentration was determined by comparin

D. Determination of VFA concentration of GI contents.

Two grams of silicic acid which were acidified with four drops of
50% sulfuric acid were mixed thoroughly with 1.5 to 2 gms GI content.
The whole mixture was chromatOgraphed on a silicic acid column with
tertiary butanol in chloroform described by Ramsey (35). The flow rate
was adjusted to approximately 2 ml/min. with COZ—free—N2 pressure.
Twenty-six to twenty—eight tubes of 10 ml. fractions were collected
using a Gilson fraction collector. The fractions were titrated with
ethanolic KOH while being aerated and mixed with COZ—free—N2 gas. Two
drops of 0.004% thymolphthalein in ethanol was indicator; the endpoint
of this mixture was from colorless to violet at pH 9HlO. Blank titra-
tion was determined for and subtracted from each solvent. The identi~
fication of VFA : valeric acid + isovaleric acid, butyric acid +
isobutyric acid, prOpionic acid and acetic acid were achieved by com~

parinO with elution patterns from standard acids.

18

There was a yellow color in the fractions from the column, usually
tubes 2, 3 and 4 but sometimes all tubes, which interfered with the
titration (endpoint was less sharp). The color of the fraction probably
partially quenched the radioactivity counting. This problem was taken
care of by dividing those fractions with yellow into two portions and
to one, a known amount of Rel—1&0 acetate (0.005 uCi) was added. Both
were then treated and counted as described in Section G, Radioactivity
counting. ‘The difference in CPM from these 2 divided by CPM correspond—

ing to 0.005 “Ci gave the percentage of radioactivity recovered.

E. Determination of VFA concentration in blood.

The blood sample was deproteinized by following the method of Folin
and Wu (21) with little modification. One volume of whole blood was
laked into 2 volumes of distilled water, one volume of sodium tungstate
(10%) was added followed by adding drOpwise 1.2 volume of 0.78 N sulfuric
acid. The mixture was shaken vigorously and allowed to stand for 15
minutes at room temperature. The mixture was then centrifuged and the
SUpernatant filtered through a Whatman no. 41 filter paper. The clear
filtrate was made alkaline (pH 10) with a few drOps of 20% KOH and
stored in a freezer until analyzed. The frozen, deproteinized blood
samples were thawed and the samples from 4 rats of each time interval
were pooled; this amounted to about 45 ml which were then evaporated to
about 1—2 ml in a flash rotating vacuum evaporator at 55°. The concen—
trated sample was acidified by adding a few drOps of 50% H280” to about
pH 2. (It was triturated with 2 gms of silicic acid and chromatographed

using the same method as described for digesta.

19
F. Efficiency of liquid scintillation counting.

Efficiency of the liquid scintillation counting which took into
consideration the instrument capabilities and quenching by ethanol which
is part of the solvent system was determined by the following procedures:

a) 0.05 mCi methyl—1“C~toluene (Nuclear Chicago) were dissolved
in 100 ml redistilled reagent toluene (Mallinckrodt) to form a stock
solution.

b) The stock solution was diluted with redistilled reagent toluene
to form a working solution of 5 x 10"3 nCi/ml.

c) One ml of working solution, 1 ml ethyl alcohol and 14 ml liquor
(counting solution) were then used for determining the counting effi-

ciency of the instrument.

G. Radioactivity counting.

The fractions eluted from column chromatographs were transferred
quantitatively to glass spectrovials after being titrated with 0.01 N
ethanolic KOH to determine total VFA. The mixture was then evaporated
(n1 a 60° hotplate to dryness. The radioactivity was determined by

adding to the vial 1 ml absolute ethanol and 14 ml scintillation liquor

:‘c 3’:

of 0.5% PPO* and 0.05% POPOP (Nuclear Chicago) in toluene and count—
ing in a scintillation counter (Nuclear Chicago). Blank counting was
made each time with 1 m1 absolute ethanol and 14 m1 scintillation
liquor. Quenching by ethanol was determined with toluene«1”Ci (methyl)

as described in the preceding section. Channel settings were at upper

F 62 and lower F 48.

 

 

*POP: 2,5-diphenyloxazole

**POPOP: pnbis(2—(5-phenyloxazolyl))"bcnzene

RESULTS AND DISCUSSIONS

I. Efficiency of liquid scintillation counting.
Efficiency of liquid scintillation counting, which took into con-

.sideration the instrument capabilities and quenching by ethanol, was

calculated as follows:

CPM of (5 x 10‘3 pci1”c toluene -
‘ + 1 ml absolute ethanol
- + 14 m1 counting solution)
DPM of 5 x 10‘3fiuC1an toluene x 100%

= 8000 CPM = .
5 x 10‘3 x 2.22 x 106 DPM 72°7ZA

This value was used in correcting all the values from radioactivity
counting.

II. Elutiongprofile of standard acids: acetic, propionic, butyric,

180butyric, valeric and isovaleric acid.
Sixty m1 benzene, 100 m1 chloroform, 100 m1 1% t—butanol in chloro-

form.were used in this order to elute the standard acetic, prOpionic,

butyric, isobutyric, valeric and isovaleric acids. One tenth m1 of each

standard acid of 0.1 N was chromatographed to obtain the profile. Re-

:covery of the standard acids was 100 :_52. Valerie plus isovaleric,

‘rutyric, isobutyric, prOpionic and acetic acid are distinguishable from

one another (Fig. 2). This profile was used as the standard elution

(curve for identification of unknowns.

20

Azo:U<mE .42 O. u mwmznz

mu ON m.

o_U<
U_mu.d<>-z oz< Om_ .U.m>._bm.z oz<

-Om. .o_zo_n_oE.o_m_o< “we“:
omézfim to 3:93 zoEjm __ .o:

q

1

q

j

 

O.

 

3-8.

4 l‘

j

 

 

 

mm“:

 

 

 

 

moém.

 

 

0350 H0)! DI'IOHOD'IV N I'O'WW

22

III. Distribution of gastrointestinal contents.

 

 

The wet digesta from the same GI portion from different rats varied
considerably in weight. In order to minimize this variation, the wet
digesta per 100 gm body weight was calculated (Table ll). Per unit of
body weight, cecal content was the highest followed in order by stomach,
lower small intestine, rectum, large intestine and upper small intestine
(Table 4).

In the whole tract, cecal digesta accounted for 50.0% of the total
wet digesta weiaht (1.3/2.6 = 0.50) (Table 4B). The cecum is thus the
"eXpanded" part of the rat GI tract. As will be shown in the following
section, it also contained the highest quantity of acetic acid eXpressed
as micromole acid per gran wet digesta. No doubt this is the most

active section as far as fermentation is concerned in the whole tract.

IV. VFA concentration of GI contents

 

Table 5 gives total concentration and distribution of organic acids
(in the GI tract contents. Stomach, small intestine and feces have more
valerate than other acids, whereas cecum, large intestine and rectum
have acetate as the major acid.

Several attempts were made to remove the yellow color present in
the fractions from the silicic acid column. Activated—carbon adsorp—
tion, steam distillation of the eluent, and changing the polarity and
amount of the solvent system proved unsatisfactory, for they also removed
a large quantity of the radioactivity. The adding of external standard
was adopted finally. This took care of the color quenchinO. However,
as the endpoint of thymolphthalein was from colorless to violet, the
yellow color interfered with the location of the exact endpoint; this

introduced an error in the concentration determination, especially on

23
Table 4. Distribution of gastrointestinal and fecal contents

.——-.-—-- _—- “C. —.~_. -...--- aw»— ...W -.—-—_.--.. ._.- v- ._-.... ,,_. _..‘ _.. -_.__.-.-- . ..._.——_ - _. -0. .. -__ .._.__._.--..___....._-_...-..
o _- . - . 2 . -r , .- .

 

Sections Range Average Number of anivals

A~
Wet weight of digesta or feces (mm.)

 

 

l

.N

,_
I.
”\

UleUJNO\}-—‘.

3.57 24
0.71 24
3.26 24

Stomach
Upper small intestine
Lower small intestine

Qi—‘O‘
HWHOJ-L\N\l

’30 C?- (D ‘3.) O
\DN

OOOLOF-‘OO

Cecum 6 7 6.28 24
Large intestine .1 ~ .9 1.08 24
Rectum .40~ 6 1.59 24
Feces .27— .6 0.63 24

r 100 grams of body weight (gm.)

Wet weight of digest:

‘2
is?

II,"
.n
.9
‘1)

l

0.75 24
0.18 24

Stomach
Upper small intestine

J—\ U1
..“.\\D

0 12 1.

0.03w0
Lower small intestine 0.24-1.02 0.68 24
Cecum 0.85~l.76 1.30 24
Large intestine 0.04—0.45 0.22 24
Rectum 0.09-0.83 0.33 24
Feces 0.06~0.32 0.13 . 14

c
W? i 81‘ t (am -> 0 f £9,111. -£53512.sextant.

 

Range: 12.67—20.00 Average: 16.80

D
Weightmof total :33C§_E9§Ff;UeJ¥{E_1QQMSTQK§ body weight (Em.)

e: 2.39—4.16 Average: 2.60

Ran

(r
O

 

 

 

...— v.-

Table 5.

24

feces (micromole acid/gram digesta [wet])*

 

 

GI section or feces

Total and distribution of organic acids in the GI tract and

 

 

 

Carbon chain length

C

C

 

3 4 5
Stomach 16.01 3.42 5.80 45.08
Upper small intestine 7.54 3.04 1.48 10.39
Lower small intestine 5.58 2.87 2.31 8.08
Cecum 44.26 13.98 23.62 13.19
Large intestine 29.13 15.91 14.12 14.13
Rectum " 24.81 13.72 15.18 25.16
Feces 17.32 7.86 15.51 30.05
Sum 144.64 60.80 78.02 120.83

 

 

-—-.~.

*Each value is the average of four samples.

25 .
valerate, since the color usually appeared at tubes 2, 3 and 4. This
yellow color probably came from bile or feed color.

The molar distribution of the acids in the cecum was 46.6% acetate,
14.7% prOpionate, 24.9% butyrate and 13.8% valerate and acids of higher
chain lengths. Weller at aZ. (41) reported that the distribution was
77—83% of acetate, 15~18E prOpionate and 1—7% butyrate and traces of
higher acids were in the rumen of sheep. The difference in the acid
distribution pattern could be an indication of different microorganisms

present in the two species.

 

V. Radioactivity of GI contents

Cecal digesta had the highest radioactivity among the GI sections,
followed in order by rectal and large intestinal digesta (Tables 6 and
7) throughout the whole experiment. The radioactivity in the upper small
intestine, stomach and feces was negligible. In general, the recovery
of the injected radioactivity in the GI content was rather low (Table
6). There are several possibilities for the low recovery:

a) Low secretion rate and most of the radioactive sodium acetate
still remained in the circulatory blood or was metabolized before secre-
tion could take place. This is likely to happen, since the metabolism
of acetate by rats is rather high.

b) The radioactive sodium acetate remained in the GI tissue. Spe~
cific activity of GI contents is eXpressed as CPM/ moles of acid (Table
I
8). Since only radioactive acetate was injected, radioactivity of pTO*
pionate, butyrate and valerate in the contents must have come from this
acid. The formation of propionate, butyrate and valerate probably
involves condensation reactions. These could be nonenzymatic, as well
as through intestinal microfloral fermentation. It is possible that

both took place at the same time.

26

Table 6. Total counts in contents of each section of the GI tract and
feces after injecting 1b'C acetate into the heart chamber

(average of 4 sap-plea)1c

 

 

———--.—

 

Time after injection (min.)

._.-- “gm”

 

 

10 15 20 3O 45 60
CI section Counts per minute
Stomach 58.1 41.3 24.1 46.4 24. 25.8
Upper small
intestine 47.9 6.3 13.7 9.6 20. 26.0
Lower small
intestine 248.8 313.5 412.3 132.7 280. 275.0
Cecum 2394.6 3930.6 3343.8 3061.8 3306. 2787.9
Large
intestine 296.6 154.0 413.1 290.3 625. 381.8
Rectum 305.9 849.8 238.5 459.3 369. 484.8
Feces 70.2 41.3 4.3 110.0 22. 19.8
Sum 3422.0 5336.9 4449.8 4111.0 4085. 3657.5

 

*Average of the sum =

4177.0 CPM

Injection dose = 1.4 x 106 CPR

% recovery of radioactivity in GI content

I!

(4177.0/1.4 x 106) x 100%

0.3%

27

Table 7. Radioactivity in each section expressed as a percentage of
the total GI radioactivity found in each time interval
(average of 4 samples)

 

 

Time after injection (min.)

 

GI section 10 15 20 30 45 60
Stomach 1.69 0.79 0.54 1.10 0.50 0.64
Upper small

intestine 1.40 0.01 0.31 0.20 0.40 0.66
Lower small - .

intestine _ 7.27 6.03 9.27 3.32 6.05 ,6.87
Cecum 69.95 75.61 75.14 74.40 71.20 69.68
Large

intestine 8.67 2.96 9.28 7.05 13.36 9.54_
Rectum 8.95 16.35 5.36 11.16 7.95 12.12
Fices 2.05 0.79 0.10 2.70 0.47 0.49

 

28

Data on the Specific activity (S.A.) of the contents in different
parts of the GI tract of each rat are tabulated in Table 12 in the
Appendix. Specific radioactivities in all sections of the GI tract
were low. However, the following observations are apparent:

a) Stomach: The S.A. of acetate increased with time and reached
a plateau at 15 to 30 minutes and then decreased. The decreasing S.A.
of acetate suggested that l) acetate passed to the lower section, 2)
there was recycling of acetate of lower S.A. to the stomach. The preSe
ence of radio—labelling in pr0pionate and valerate at 60 minutes indi—
cated the formation of prOpionate and valerate from acetate.

b) Upper small intestine: Highest S.A. of acetate was found at
10 minutes after injection. It then decreased with time, suggesting
that acetate was the major acid secreted into this section and that the
Upper small intestine is a major metabolizing site. As S.A. of acetate
decreased, S.A. of prOpionate, butyrate and valerate increased. A maxim
mum S.A. of butyrate was found at 20 minutes, which then decreased
immediately. At the same time, S.A. of prOpionate increased and peaked

in 30 minutes, suggesting the following reactions took place:

2 C2 -«~uwvm> C4 fast

CO 'I’ C "“‘w--'*) C3 slow

and prOpionate and butyrate were absorbed or passed down to the follow—
ing section.

‘c) Lower small intestine: Comparable to the upper small intes-
tinal S.A., the S.A. of acetate in this section peaked at 10 minutes.
The S.A. of acetate and prOpionate fell with time, suggesting that they
were passed down to the cecum. There is an extremely high S.A. of

butyrate at 45 and 60 minutes; this could be an exPerinental artifact,

.meeEmm Meow mo mumum>m we» we esaw> comm

 

29

 

0N.s am.a 0m.m 0N.H 00.0 00.0 "N0

N0.0 am.m Ne.m N0.N 00.0 mm.0 "m0

ma.0 e0.m 0a.0 NH.0N 0s.a 00.0 “:0

0a.0 00.N 0H.m 00.0 se.m 0H.0 "mo 00

00.0H ~0.a no.0 0H.N 00.0 em.0 “N0

0a.ma am.0 am.a N0.m 0a.0 00.0 "m0

00.0a 0N.HH ma.0 00.0w 00.0 00.0 ":0

am.0 as.a 00.m ma.m 0a.m 00.0 “0 me

m0.m ~0.n 05.a ma.n ma.0 a0.a "N0

mq.0 NH.0 am.0 sq.m mq.0 00.0 “m0

0a.0 ms.s Nq.a 00.0H 00.0 00.0 ":0

0a.a r0.0 Ne.a am.0 Ne.0 00.0 "00 00

a0.0 aa.0 0a.m sa.a am.m 0s.a "N0

0H.N 0m.e 00.0 a0.0a as.“ 00.0 "m0

N0.m 09.0 0m.0 mm.aa 0a.s 00.0 ":0

00.a Ha.0 aa.0 0a.m am.0 00.0 ”m0 0N

a0.0 mm.0 a0.a ma.0 sm.m wm.a “N0

aa.m m0.s a0.0 a0.a 00.0 00.0 ”00

50.5 00.0 5N.0 00.a 00.0 ea.0 "40

00.m 40.H 0e.n 00.e 05.N (0.0 "00 na

00.m 0H.s 00.m aa.aa am.~a 0N.0 "N0

HN.H Na.a a0.a am.0 00.0 00. u.

00.N Hm.N an.“ 50.5 00.0 00.0 ":0

e0.a 0N.m 00.e a0.. 00.0 ma.0 "m0 0a

.suuem mcfiumOusfi Esoou ecflumeucfl mafiueeuefi zowsoum cowuoehCfl
swung HHeEm HHeEm umume

uezoq amen: eEfiH

 

 

Ammaoan\2mov museucoo H0 mo mufi>fiuum ofiwfiommm .w wanes

30
for at 45 minutes 4 determinations for S.A. of butyrate were 0, 0, 0
and 229.5 CPM/umole acid. This latter value is most likely due to
contamination (Table 13). If this is true, then there should be a
sharp decrease instead of a sharp increase of butyrate S.A. The S.A.
of valerate stayed constant throughout the whole time.

d) Cecum: S.A. of acetate was constant almost throughout the
whole time. The S.A. of prOpionate increased steadily with time. Cor—
relation :as 0.72 for the regression line of the ln S.A. versus time.
There was no significant change in the S.A. of butyrate. The S.A. of
valerate decreased at 30 minutes with a concomitant increase of valerate
in the large intestine. This is a good indication of the passage of
valerate from the cecum to the large intestine. All the above data sug—
gest that the rate of microfloral fermentation of VFA is in equilibrium
with secretion, absorption, chemical degradation and chemical recombina-
tion. It also suggests that the cecum is a very active section of the
GI tract.

e) Large intestine: The S.A. of acetate and propionate increased
steadily and appeared similar to that of the cecum. A decreased buty—
rate and valerate S.A. in this section and a concurrent increase in the
S.A. of butyrate and valerate in the rectum indicated that the acids
passed from the cecum to the rectum.

f) Rectum: The S.A. of VFA in the rectum matched the trend of the
S.A. of VFA in the large intestine (Table 8).

The auove results showed that among these sections, the stomach
\contents were the most inactive in converting acetate to other VFA,
while the contents in the cecum, large intestine and rectum functioned

as if they were a homogeneous mass and were the most active in this

31

 

VI. VFA concentration in b102§3—§9§X_§E§£?£§MP391lEEZEnéE£93§£E§EEQE°
Since most of the free VFA of the body are in the entracellular
fluids, injecting rapidly a tracer dose (negligible weight, high Speci—
fic activity) of sodium acetate assumed rapid dilution of the radio~
active material, which can be measured using the following equation:

ln x = 1n x0 - at = b - at, where x = Specific activity of blood acid

at time "t", a= constant = the reciprocal of the turnover time, and
b = ln x0 = zero time Specific activity.

The zero time specific activity is obtained by extrapolation.
Plotting In x against time will give a straight line with lepe of «a
and ordinate intercept of b. The body pool size is then equal to the
amount of radioactivity injected divided by the zero time Specific
activity.

In applying single~injection technique, several assumptions were
made:

a) The animals were assumed to be in a steady state, that is, the
concentration of blood and tissue components is held at nearly constant
concentrations uhile net synthesis and breakdown of chemical constitu—
ents proceed (44). In a Situation in which the rate of entry of a
molecule "A" by synthesis or transport equals the rate of exit by break—
down or transport, the concentration of "A” remains constant and a steady
state is said to exist. An increase in the concentration of "A” may
result not only rom an increase in the rate of "A” formation but also
from a decrease in the rate of "A” loss or both.

\ A nonsteady state exists whenever the influx of material does not
balance the outflux. Thus, a growing person or person in negative

nitrogen balance is not in a steady state.

32

b) An instantaneous mixing of injected labeled substance with the
pool.

c) The extent of labeled substance recycled is negligible. That
is, the degradation product of this substance will not be utilized to
resynthesize this substance in the pool.

The rat was fasted for 2 hours before injection in order to obtain
a constant level of blood acetate. Since the interval between injection
and blood sampling was rather short, a relatively steady state should
still be in existence.

An inherent error in the single—injection technique, as pointed out
by Cook (15), is the rapid metabolism of the tracer dose of acetate~14C
before it mixes with the body pool of acetate. This can result in low
values for the Specific activity of blood acetate and a smaller value
for b in the equation In x = —at + b and consequently, a higher value
for the body pool size than what it really should be. Another important
point has to be made in applying singlewinjection technique for pool
size determination, that is, the slope of the curve should be obtained
from the early part of the specific activity versus time curve (29),
since recycling of the label might occur. And if the latter portion of
the curve is used for SIOpe determination, the pool size of the acetate
will appear higher than it actually is. In this study, a regression
line based on all the six time—S.A. points (Fig. 3) was used for calcu—
lation of body pool size and half—life of acetate. It is obvious, then,
that the values obtained here are higher than when the lepe was ob—
tained from the first three points of the graph.

Blood specific activity in CPR/pmoles acetate for each time was
determined by pooling blood from 4 rats (Table 9). On an average, there

was 37.35 pmoles acetate/100 ml blood.

33
T = log Y = 1.57292 — 0.02405 X was obtained by regressing specific
activity Y (CPM/umoles) against time X (min.) after injection. The
correlation is —0.88, which is statistically significant (P < 0.01).
From the above equation, body acetate pool, turnover rate, turnover
time and half life can be calculated:

(1.4 x 106 CPM)/(anti log 1.5202 CPM/umole)

Body acetate pool

37.43 umoles

2.245 grams

Turnover rate (body pool size) (slope of the curve)

= 37.43 x 0.02405

= 0.8983 mmoles/min.

I!

Turnover time l/lepe of the curve
= l/0.02405
= 41.58 min.

Half life turnover time X log 2

14.44 min.

If only the first three values of Fig. 3 were used, then:
.,' I
Body acetate pool = 4.196 mmoles
Turnover rate = 0.1007 mmoles/min.

18.47 min.

Turnover time

Half life = 3.69 min.

These two sets of data vary greatly from each other. The regres-
sion line puts equal weight on the first three points, which fall on a
line (10, 15, and 20 minutes after injection) and the last three points
(30, 45, and 60 minutes after injection). Recycling of the labeled
acetate inevitably occurred at the latter stage. The values obtained
by applying the regression line are obviously too high. Extrapolating

the line linking the first three points to zero time wives a specific

SPECIFIC

ACTIVITY (CPM/MICROMOLE ACETATE)

 

FIG.lll LINEAR REGRESSION ON BLOOD ACETATE
SPECIFIC ACTIVITY AND TIME AFTER
INJECTION: BODY ACETATE POOL SIZE
200}, DETERMINATION
‘|
.‘
I.‘ _ LINEAR REGRESSION
10051, .----- EXTRAPOLATION OF THE FIRST
. I‘, 3 POINTS
I |
I ‘
\
I |
|
I |
I ‘I
I |
I ’5
|
\
I
I |
I |
20‘ ' ‘I
I
I
I
I
I I
I \
'3 . I. -
3T I \‘ Y= LOG v: I.S7292—O.024OS x
74- I ;
6‘ : "‘ (Rs-0.88 )
5 I ‘.
I \
4‘ : ‘x
3‘ I \
I \
I |
2‘ ' ‘I
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| \
I

 

 

 

 

: i J.
IO 1 20 30
MINUTES AFTER INJECTION

35

Table 9. Blood Specific activity of acetate at various times

 

 

 

--..-- ~ —~.-..—~o

 

 

_-._... —_.._._- — .— h...

 

 

Time (min.) Pooled blood CPM of Specific
after volume from Micromoles the pooled activity
injection 4 rats (ml) of acetate blood (CPH/umoles)
. 10 36.4 15.00 576.5 38.45
15 43.8 16.00 218.6 13.68
20 48.9 _11.17 75.1 6.72
30 45.5 15.72 141.1 8.98
45 44.0 21.26 51.2 2.41
60 39.8 17.30 29.1 1.68

 

 

.—- ~v _.._.-—.—--

Average of pooled blood volume = 43.0 ml

.‘
u)

1.

Averac micromole of acetate = 16.07

C
(

Acetate concentration in blood = (100 x l6.074)/43.02

= 37.35 micromeles/lOO ml blood

36

activity of 233.33 CPM/umole; this gives the acetate pool size of 4.196
mmoles (251.76 F3) and half life of 3.69 minutes. Assuming there is
an equal distribution of acetate between blood and extracellular fluid,
then the average concentration of acetate in the blood and extracellu—
lar fluid is 2.241 mg/100 gm body fluid (Table 9, blood acetate concen—
tration = 37.35 umoles/lOO ml blood), which, when divided by the size
of acetate pool, will give the weight of the blood and extracellular
fluid, in this case 111.8 gm. This is about 22% of the body weight for
a 500 gm rat and is a reasonable value.

Since blood contained little if any prOpionate, butyrate or valer—
ate, the presence of tlTe.e acids in the GI contents suggested that a
quick dissociation or cleavage and reassociation could have taken place
in the blood be tween acetate and its derivative which gave acid of
various kinds, i.e., acetate to valerate

There is a relatively high concentration of VFA in each section of
the GI tract at each time interval. Cecal content averaged 6.28 gm
wet material and the acetaLe co ncentratIon from the average of six ani—
mals was 44. 26 up oles/gm vet weight. This amounted to 277.95 “moles
acetate with a spe ecific activity maintained constant at 5 6 GNP/pmole.
Thus, microiloral fermentation was probably responsible for the acetic
acid presence in each section .shil e secretion of this acid from the

blood was too small to be quantitatively sig m1 icant.

VII. In Ditto SLuviy of tissue Uptake of volatile fatty acids.

~—-._.—.._.»_.-—_- fi

 

One control containing buffer and tissue served as the blank (0%
acid). A second control which had buffer and one of the acids served
as another control (100% acid). There was no uptake or uti]_ization
of VFA by cecal or large intestinal and rectal tis: us under thz presc:nt

experimental couditiors (Table 10).

37
It is possible that conditions were not conducive for tissue uptake
of VFA. This could include imprOper temperature, amount of acid in the
incubation media, the length of incubation, the kind of buffer, the
amount of antibiotics and the balance of C02-02~N2 surrounding the
media. If there was any acid utilization or u take by the GI tissues,

it was too little to be detected by the present methods.

38

 

 

 

Table 10. Recovery of acids from the tissue incubation study
.Incubation Acid incubated

Tissue time (min.) with Z of recovery Average

cecum 10 acetic acid 95.6 102.0 97.3 98.3
20 ‘97.8 99.5 96.8 ' 97.7
40 96.6 99.2 97.2 97.6
60 97.1, 98.7 100.3 98.7
90 99.3 101.4 97.4 99.4
10 * propionic acid 100.4 97.3 98.2 98.6
20 100.8 98.5' 96.7 98.7
40 102.2 97.4 98.4 99.3
60 97.5 96.7 95.2 96.5
90 98.7 98.1 97.6 98.1
10 butyric acid 99.3 96.7 95.6 97.2
20 102.4 97.8 9817 99.6
40 100.5 99.7 98.3 99.5
60 99.4 98.8 103.6 100.6
90 98.2 94.9 97.5 96.9

Large 10 acetic acid 94.0 99.6 95.7 96.4

intestine

and 20 97.0 99.4 97.4 97.9

rectum ’
40 94.9 100.1 95.3 96.8
60 100.2 99.2 100.9 100.1
90 100.4 97.2 98.4 98.7

Table 10 (cont'd.)

39

 

 

 

Incubation Acid incubated

Tissue time (min.) with Z of recovery Average

/ 10 prOpionic acid 100.3 98.9 - 96. 98.6
20 ’ 100.5 .97.2 99. 99.0
40 99.3 96.9 95. 97.2
60 96.8 99.8 98. 98.1
90 97.4 98.5 98. 98.0
10 'butyric acid 94.8 99.8 98. 97.6
20 100.2 99.2 ' 97. 98.9
40 99.6 96.5 102. 99.5
60 98.7 98.2 95. 97.4
90

99.1

98.5

98.

98.8

 

SUflflrRY AND CONCLUSIONS

Twenty-four male Sprague-Dawley rats were used for the determina~
tion of secretion rate and body pool size of acetate. Another nine
rats were used for the determination of tissue in vitro Uptake or
utilization of VFA. The single—injection technique was used in the
determination of secretion rate and the body acetate pool size, in
which potassium~l—1”C acetate was the tracer. Different levels of
acetate, prOpionate and butyrate were incubated in cecal and large
intestinal plus rectal tissues to determine utilization of VFA by
these tissues.

Cecum, large intestine and rectum were the major sites of the
metabolism of acetate which came from the small intestine or directly
from the blood. Recovery of radioactivity in GI contents and VFA
concentration in blood indicated that the microflora in the cecum, large
intestine and rectum were responsible for the presence of acids while
the secretion of acid from the blood is too small to be significant.

Body acetate pool size was 4.196 mmoles with a turnover rate of
0.1007 mmoles/min. and a half life of 3.69 minutes.

In vitro study revealed that there was no uptake or utilization

of VFA by cecal or large intestinal plus rectal tissues.

40

LITERATURE C IT ED

10.

11.

12.

LITERATURE CITED

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their infusion into the rumen ofa cow. Brit. Vet. J. 120(10):
487.

Annison, E. F. -1954. Some observations of VFA in the sheep's
rumen. Biochem. J. 57: 400.

Annison, E. F. and D. B. Lindsay. 1958. Acetate utilization in
Sheep. Biochem. J. 69: 33.

Annison, E. F. and D. B. Lindsay. 1961. Acetate utilization in
Sheep. Biochem. J. 787: 777.

Annison, E. F. and R. R. White. 1962. Further studies on the
entry rates of acetate and glucose in sheep with special reference
to endogenous production of acetate. Biochem. J. 84: 546.

Annison, E. F., R. A. Long, D. B. Lindsay and R. R. White. 1963.
The metabolism of acetic acid, prepionic acid and butyric acid
in sheep. Biochem. J. 88: 248.

Annison, E. F. and D. Lewis. 1963. Metabolism in the rumen.
Published by Methuen's monographs on biochemical subjects.
Armstrong, D. G. 1965. Carbohydrate metabolism in ruminants and
energy supply. p. 272 in The Phys€QZOgy and Digestive Reaction
of’the Emmsn, edited by R. W. Dougherty at aZ., published by
Butterworths.

Ash, R. W. and A. Dobson. 1963. The effect of absorption on the
acidity of rumen contents. J. Physio. 169: 39.

Barcroft, J., R. A. McAnally and A. T. Phillipson. 1944. Absorp—
tion of VFA from the alimentary tract of the sheep and other
animals. J. Esp. Biol. 20: 120.

Bensadoun, A., O. L. Paladines and J. T. Reid. 1962. Effect of
level of intake and physical form of the diet on plasma glucose
concentration and volatile fatty acid absorption in ruminants.
J. Dairy Sci. 45: 1203.

Buchanan, A., M. John, B. Hastings and F. B. Nesbett. 1943. The

role of carbOXyl~laheled acetate, prOpionic and butyric acid in
liver glycogen formation. J. Biol. Chem. 150: 413.

41

14.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

42

Burhoe, 3.0. 1940. Methods of securing blood from rats: As
deve10ped in a study of blood groups and their inheritance. J.
Hered. 31: 445.

Carroll, E. J. and R. E. Hungate. 1954. The magnitude of the

microbial fermentation in the bovine rumen. Appl. Microbiol. 2:
205.

Cook, R.‘M.. 1966. Use of C1“ to study utilization of substrate
in ruminants. J. Dairy Sci. 49: 1018.

Danielle, J. F., M. W. M. S. Hitchcock, R. A. Marshall and A. T.
Phillipson. 1945. The mechanism of absorption from the rumen

' as exemplified by the behaviour of acetic, prOpionic and butyric

acid. J. Exp. Biol. 22: 75.

Elsden, S. R. 1945. The fermentation of carbohydrates in the
rumen of the sheep. J. Exp. Biol. 22: 51.

Elsden, S. R., M. W} M. S. Hitchcock, R. A. Marshall and A. T.
Phillipson. 1946. Volatile acid in the digesta of ruminants and
other animals. J. Exp. Biol. 22: 191.

Elsden, S. R. 1946. The application of the silica gel partition
chromatogram to the estimation of VFA. 'Biochem. J. 40: 252.

Feller, D. D., E. H. Strisower and I. L. Chaikoff. 1950. Turn-
over and oxidation of body glucose in normal and alloxan—diabetic
rats. J. Biol. Chem. 187: 571. -

Folin, 0. and H. wu. 1919. A system of blood analysis. J. Biol.
Chem. 38: 81. '

Gray, F. V. 1948. The absorption of volatile fatty acid from the
rumen. II. The influence of pH on absorption. J. Exp. Biol.

25: 135.

Gray, F- V. and A. F. Piligrim. 1951. Fermentation in the rumen
of the sheep. V. The production and absorption of volatile
fatty acids during fermentation of Wheaten hay and lucerne hay

in the rumen. J. Exp. Biol. 28: 83.

Gray, F. V., A. F. Piligrim, H. J. Rodda and R. A. Weller. 1952.
Fermentation in the rumen of the sheep. VI. The nature and origin
of the volatile fatty acids in the rumen of the sheep. J. Exp.
Biol. 29: 57.

Gray, F. V., R. A. Weller, A. F. Pilgrim and G. B. Jones. 1966.
The rates of production of VFA in the rumen. III. Measurement

of production in vivo by two isot0pe dilution procedures. Aust.
J. Agr. Res. 17(1):

James, A. T. and A. J. P. Martin. 1952. Gas~liquid partition
chromatograph: The separation and microestimation of VFA from
formic acid to dodecanoic acid. Biochem. J. 50: 679.

27.

28.

29.

30.

31.

32.

33.

‘34.

35.

36.

37.

38.

39.

40.

2.1.

42.

43

Johns, A. T. 1951. Isolation of a bacterium, producing propionic
acid from the rumen of sheep. Gen. Microbiol. 317: 326, 337.

Kellner, 0. 1900. Landw. Versuchs-Stationen 53: 394.

Lee, S. D. and W. F. Williams. 1962. Acetate turnover rate as
determined by two superimposed techniques. J. Dairy Sci. 45: 517.

Leng, R. A. and G. J. Leonard. 1965. Measurement of the rates of
production of acetic, propionic and butyric acids in the rumen of
sheep. Br. J. Nutr. 19: 469.

Leng, R. A. 1966. VFA production in the rumen of sheep. Proc.
Aust. Soc. Ani. Prod. 6: 389. '

Masson, Ms J. and A. T. Phillipson.' 1951. The absorption of
acetate, propionate and butyrate from the rumen of sheep. J.
Physio. 113: 189.

Hayfield, E. D., A. Bensadoun and B. C. Johnson. 1966. Acetate
metabolism in ruminant tissue. J. Nutr. 89: 189.

McClymont, G. L. 1951. Identification of the volatile fatty acids
in the peripheral blood and rumen of cattle and the blood of other
species. Aust. J. Agr. Res. 2: 92.

Ramsey, H. A. 1963. Separation of organic acids in blood by
partition chromatography. J. Dairy Sci. 46: 480.

Reid, R. L. 1950. Studies on the carbohydrate metabolism of
sheep. II. The uptake by the tissues of glucose and acetic acid
from the peripheral circulation.— Aust J. Agr. Res. 1: 338.

Steele, R., J. 8. Wall, R. DeBodo and N.JA1tszuler.1956.Measure—
ment of size and turnover rate of body glucose pool by the isotOpe
dilution method. Am. J. Physiol. 187: 15.

Tappeiner, H. 1882. Ber. Deutsch. Chem. Gas. 15: 999.

’Umbreit, W. W., R. H. Burris and J. F. Stauffer. 1964. 4th edi-

tion,.Man0metric Techniques, p. 132. Preparation of Krebs-Ringer
Phosphate and Bicarbonate Solutions.

Warner, A. C. I. 1964. Production of volatile fatty acid in the
rumen: Methods of measurement. Nutr., Abstr. and Rev. 34: 339.

Weller, R. A., F. V. Gray, A. T. Pilgrim and G. B. Jones. 1967.
The rates of production of VFA in the rumen. IV. Individual and
total volatile fatty acid. Aust. J. Agr. Res. 18(1): 107.

Wood, H. G., N. Lifson and V. Larber. 1945., The position of fixed

carbon in glucose from rat liver glycogen. J. Biol. Chem., 159:
475. 4

43.

44.

44

Wood, H. G., N. Lifson, V. Larber and W. Sakami. 1948. The in-
corporation of acetate and butyrate carbon into rat liver glycogen
by pathway other than carbon dioxide fixation. J. Biol. Chem.
176: 1263.

Zilversmit, D. B. 1960. The design and analysis of isotope ex-
periments. Am. J. Med. 29: 832.

APPENDIX

Table 11. Distribution of gastrointestinal contents (1)

45

 

 

 

Upper Lower
small , small
Rat ' Body wt. Stomach intestine intestine Cecum
No. (gm.) 1* 2* 1* 2* 1* 2* 1* 2*-
1 496 7.02 1.42 0.45 .090 3.50 ..706 5.80 1.17
2 492 3.47 .704 .961 .195 4.33 .880 7.40 1.50
3 470 3.43 ".730 .630 .134 4.14 .880 .4.81 1.02
4 h 486 3.44 .708 -.420 .086 4.43 .912 6.40 1.62
5 469 2.75 .586 .700 .149 4.78 1.02 4.33 .923
6 468 7.44 1.59 .510 .109 .3.35 .716 5.74 1.23
7 412 2.58 .623 .840 .204 1.22 .296 5.02 1.22
8 433 4.17 .963 .490 .113 3.63 .838 3.66 .845
9 422 3.17 .751 .240 .057 1.00 .237 3.67 .870
10 482 2.93 .608 .270 .056 3.39 .703 4.82 1.00
~11 493 ' 1.99, .404 .680 \.138 '3.02 .613 8.21 1.67
12 490 1.21 .247 .380 .078 1.82 .371 6.47 1.32
13 508 4.77 .939 2.21 .435, 2.05 .404 7.17 1.41
14 531 2.80 .527 .710. .134 3.23 .608 8,37 1.58
15 499 3.70 .741 1.47 .295 ' 2.63 .527 7.15 1.43
16 518 3.58 ..691 .780 .150 3.54 .683 8.72 1.68
17 516 2.38 .461 1.02 .198 4.96 .961 6.46 1.25
18 510 .600 .118 .610 .120 2.84 .557 5.81 1.14
19 445 4.89 1.09 .300 .067 4.21 .946 5.87 1.32
20 452 3.93 .869 .130 .029 3.33 .737 .5.94 1.31
21 544 4.37 .803 .730 .134 4.04. .743 6.28 1.15

46

Table 11 (cont'd.)

 

 

 

Upper Lower

small small
Rat Body wt. Stomach* intestine ingesting gecum *
No. (gm.) 1* 2 1* 2 1 2 1 2
23 _ 437 2.01 .460 .860 .197 2.67 .611 7.57 1.73
24 469 , 7.40 1.58 1.32 .281 2.10 .448 7.10 1.51
Ave. 3.57 .749 .707 .175 3.26 .678 6.28 1.31

 

*1 Weight (gm.)
2 Weight (gm.)/100 gm. body weight

Table 11. Distribution of gastrointestinal contents (II)

47

 

 

 

3.22

. Large Total

:zt lintesgine 1§ectu§* lgece82* 1E1 trag£
1 .625 .126 1.74 .350 .600 .121 19.74 3.98
2 .764 .155 1.61 .327 .460 .093‘ 18.99 3.86
3 1.60 .340 .720 .153 -** -** 15.33 3.26
4 1.17 .241 2.21‘ .455 .400 .082 18.47 3.80
5 1.56 .333 1.44 .307 .270 .058 15.83. 3.37
6 .940 .200 .400 .085 --** --** 18.38 3.93
7 1.08 .262 .930 .226 1.34 .325 13.01- 3.18
8 1.43 .330 .620 .143 .530 .122 14.53 3.36
9 .310 .073 1.19 .281 .510 .121 10.09 2.39
10 1.10 .228 .160 .034 --** -—** 12.67 2.63
11 1.32 .268 1.53 .310 '.640 .130 17.39 3.53
12 .530 .108 1.89 .386 .460 .094 12.76, 2.60
13 1.43 .281 1.76 ‘.246 .300 .059 19.69 3.88
.,14 1.37 .258 2.03 .382 --** --** 18.51 3.49
15 .530 .106 1.77 .355 .590 .118 ”17.84 3.58
16 .500 .096 4 _2.88 .556 --** --** 20.00 3.86
17 .950 .184 .930 .180 --** —-** 16.70 3.24
18 1.37 .269 1.92 _.376 1.17 .324 14.80 2.90
19 1.98 .445 .550 .123 —-** --** 17.80 4.00
20 ' .180 .040 .900 .200 .580 .128 14.89 3.32
21 1.02 .187 .592 —-** --** 19.66 3.61

48

ll (cont'd.)

 

 

 

Table
Large Total

Rat intestine Rectum Feces GI tract
No. 1* 2* 1* 2* 1* 2* 1* 2*
22 1.62 .358 3.34 .737 -** -—** 18.81 4.15
23 .410 .094 3.63 .831 .460 .105 17.61 4.03
24 .880 .188 .69 . .147 --** -—** 19.49 4.16
Ave. 1.03 .215 1.59 .328 .627 .134 16.79 3.64

 

*1 Weight (gm.)
2 Weight (gm.)/100 gm. body weight

**No fecal sample was obtained during the experimental interval.

49

 

 

 

Table 12. Radioactivity in GI sections (I)
Time count Z of Z of ‘ Z of
(min.) CPM CPM total CPM total total CPM total
Upper 10 47.9 27. 56.6 1.3 18.5 10.6 22.2
small 15 6.3 0 0 0 0 6.3 100.0
intes— 20 13.7 2.8 20.5 0 6.5 10.0 73.0
tine 30 9.6 2.1 22.3 4.7 0 2.7 28.5
45 20.5 8.4 40.9 3.3 0 8.8 42.9
60 26.0 0 r0 0 6.8 24.2 93.2
Lower 10 248.8 198.3" 79.9 19.5 8 6.9 14.0 5.6
small 15 313.5 154.2 49.2 37.1 .9 16.6 ’70.1 22.4
intes- 20 412.3 70.9 17.2 127.0 .8 30.7 88.0 21.3
tine 30 136.7 85.5 62.6 9.2 .7 12.1 25.4 18.6
45 280.8 44.3 15.8 25.1 .9 46.0 82.1 29.3
60 275.0 24.0 8.7 52.6 .2 35.6 100.3 36.5
Stomach 10 58.1 42.6 70.4 0 0 15.4
15 41.3 36.2 86.8 0 13.2 0
20 '24.1 24.1 100.0 0 0 0
’ 30 46.4 46.4 100.0 0 0 0
45 24.4 24.4 100.0 0 0 0
60 25.8 14.6 56.8 2.7 .5 0 8.7
Cecum 10 2394.6 1648.8 68 9 254.1 .6 .5 15.0 131.1
15 3930.6 2093.7 53.3 240.9 .1 .0 25.7 587.2
20 3343.8 1706.0 51.0 294.9 .8 .9 36.8 110.0
30 3061.8 1792.3 58.5 518.0 .9 .0 21.9 79.5
45 3306.2 1487.0 44.9 448.0 .6 .0 36.8, 154.1
60 2787.9 1103.6 39 6 416.6 .9 .8 36.4 254.0
Large
intestine 10 296.8 180.9 61.0 26.0 .6 12.3 53.0
15 4154.0 89.6 58.2 28.4 .0 10.7 19.5
20 413.0 220.5 53.4 52.7 0 28.1 23.8
30 290.3 100.0 34.5 63.3 .9 21.0 65.9
45 620.5 213.4 34.1 114.7 0 43.5 25.9
60 381.8 163.2 49.3 61.2 2 29.7

 

50

Table 12. Radioactivity in GI sections (11)

 

 

Total C2 C C“ CS
Time count 2 of g of Z of Z of
(min .) CPM CPM total CPM total CPM total CPM to tal

 

Rectum 10 305.9 207.8 67.9 33.9 11.1 34.2 11.2 29.1 9.8
15 849.8 849.6 22.3 70.9 8.3 137.0 19.1 426.6 50.2
20 238.5 116.0 47.9 47.6 19.9 52.4 21.9f 24.1 10.1
30 459.3 216.0 47.0 106.0 23.1 110.0 24.1 26.5 5.8
45 369.2 163.3 44.2 76.2 20.7 91.7 24.9 37.0 10.2
60 484.8 168.0 34.7 77.7 16.0 220.2 45.4 18.7 3.9'
_ Feces 10 70.2 52.4 74.6 2.2 3.2 15.6 22.2 0 0
15 41.3 30.4 73.3 0 0 5.8 14.1 5.1 12.5
20 4.3 0 0 0 0 0 0 4.3 100.0
30 111.0 68.4 61.7 13.8 12.4 27.0 24.4 1.6 1.5
45 22.1 14.5 65.8 2.6 11.7 4.9 22.6 0 o
60 19.8 8.3 42.8 0 o 2.6 13.6 8 5 43.7

 

All values are the average of four samples after correcting for
background. '

CPM of one acid at one time at one section
CPM of acid total at one time at one
section

 

percentage of total % = x 100

\

51

Table 13. Specific activity of GI sections (I) (CPM/uM)

 

 

 

 

Upper Lower

small small
Time Animal Stomach intestine ‘ intestine
(min.) NO. C5 CL. C3 C2 C5 CL} C3 C2 _ C5 C1, C3 C2
10 14 0 0 0 18.75

18

_ 1.5 9.9 22.0 13.4
13 1.5 19.0 1.9 9.4
0.4 0 0 0.6 0 0 0 20.0
15 . 4.7 2.1 1.2 3.0
15 23 ' 14.5 20.6 5.3 10.3
0 0 0 .33 .8 0 1.9 4.5
24 . 0 0 0 8.97
11 .45 23.7 13.1 8.20
0 .3 0 2.4 4.7 0 0 0 p
1 _ -. 4.98 0 .82 5.10
20 17 _ 1.5, 0 0 6.98
0 _ 0 0 2.3 42.2 0 14.4 4.3
‘ 3 2.9 0 0 0
22 ' 7.9 58.1 42.4 1.87
0 0 0 .71 4.3 14.9 0 2.8
2 . .46 0 0 2.62
30 12 2.3 33.3 0 4.21
0 0 0 2.7 .47 0 9.5 .75
7 2.4 9.1 14.4 6.98
20 ” ‘ 0 0 0 13.50
0 0 0 .55 -— -- - --
9 8.3 0 7.4 3.92
45 ‘ 19 . 1-1 0 9.7 3.90
0 0 0 18 2.2 0 7 4 3 4
8 .- __ .. ..
6 9.8 0 0 1.14
- 0 0 0 .3 2.8 0 5 5 4 5‘
21 - 6.3 229 8.1 1.27
60 . ‘ 16 ' 2.9 0 0 0
0 0 .7 5 4.6 2.8 0. 0 '
10 2.9 12.5 .76 1.33
5 0 40.6 3.69 2.6
.3 0 0 .3 6.3 0 0 0
4 9.9 63.5 3.86 1.23

 

—-= sample was missing
*When data appear between 2 samples, they are pool values from
both.

Specific activity of GI sections (11) (CPM/0M)

Table 13.

 

 

 

 

Large intestine Rectum

Cecum

 

C5

Animal
Number

 

Time
(min.)

3.5

5.8
1.4

7.43
.33

6.0

2.10
1.24
2.2

2.29
.87

1.95 10.7
3.08

3.51
11.6

16.73 1.33 .70‘

14
18
13

10

3.3'

.61

.83
7.1

.28 1.05
1.13

.76
1.6

1.98
7.1

2.1
2.2

2.29
4.6

.85

4.4

3.62

1.35

.84 7.19 5.53

15

5.3
1.1

2.08 3.81 2.51 1.3 6.75 1.52 35.0 25.4 6.51
13.2 .99 2.6

4.7

23 13.11 3.5

24

15

13.8

15.7

1.54

QB
Nl-n

.53 1.72 2.2
3.7 2.1

2.47
_6.44

3.07
5.4

1.08
1.44

.95 1.89 2.26
6.85 3.42 8.84

1.16

52

6.6 5.1 3.7
1.7

1.94

2.95
6.5

3.4 13.8 1.3
8.0

5.3
6.2

2.2

6.3

1.77

17

20

22.5

1.99
2.0
.97

1.04
.23

11.0
5.2

19.7

1.36

3.0

1.6
1.0

4.1
3.6

3.3 1.1 2.7
4.4

2.6

4.9
8.5

22

2.2

6.3

.76

2.4 4.1 3.7 15.4 10.9 6.2 8.9 3.0 7.7 7.0 3.5
6.7 2.9 ‘4.0

12 4.21

30

5.2
6.0

16.2

9.3
4.5

3.9 4.5 7.7
3.4

4.2

4.8

5.3
9.5

3.0

2.2
2.6

9.6

2.8
7.7

20

16.5“

7.9

7.1

7.5’

5.0

1.48

6.2 7.7 2.8 2.8 6.9 8.2 8.2 6.9 12.3 ‘19.8 30.7
8.8
6.8

4.39

19

45

15.3

18.7 15.9 3.3 14.5 15.7
6.3 6.9 - -‘ '-

25.4
9.5

2.7
.68

5.8 5.8 .85 3.0 3.9 5.4 .82 3.3 4.0 3.6

6.8

21

5.7 .82 6.0 5.6 4.5
4.7

4.2

6.9
5.3

6.8 6.0 4.2
4.7
4.7

14.78 10.7 7.7
4.6 5.4

16

60

4.0

11.5

5.5

6.2
7.9

6.7

10

3.3
4.4

2.3
3.8

4.3
3.4

4.6

4.3 4.6
7.6 5.4 4.4 .72

7.4

2.79
3.22

3.4
11.8.

.93

 

- = sample lost

 

..
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14.-:51