RIBGRLKVIN AND THIAMINE CONTENT OF THE INTESTINAL
TRACT OF CECECTOMIZED.AND NONFCECECTOMIZED WHITE RATS.

by
Violet Onerva Egtainen

AnTHESIS

Submitted to the School of Graduate Studies of Michigan
State College of A riculture and Applied Science
in partial rulr llment of the requirements
for the degree or

MASTER.OF SCIENCE

Department of Foods and Nutrition
School or Home Economics

19119

ACKNOWLEDGMEIT

The writer wishes to express her sincere
appreciation to Dr. Dena Oederquist and Dr. Margaret
Ohlson for the suggestion of a problem and for guidance
and constructive criticism throughout the study: to
Kiss Shirley McClure for doing the bacteriological work;
to Kiss Villa Brewer for her cooperation and helpful
suggestions; and to Mrs. Helen Tobey for her assistance

in the laboratory.

:2 83% L

TABLE OF CONTENTS

Introduction. . . . . . . . . . . . . . .
Review of Literature. . . . . . . . . . .
Introduction . . . . . . . . . . . .
The Significance of Coprcphagy . . .
The Phenomenon of Refecticn. . . . .
The Role of Diet . . . . .. . . . .
Availability of Synthesized Vitamins
Vitamin Excretion and Storage. . . .

Use of Sulfa Drugs in the Study. . .
of Vitamin Synthesis

Vitamin Synthesis in aninants . . .

Vitamin Synthesis in Human Subjects. . . . .

Problems Involved in the Study of Synthesis.

Experimental Procedure. . . . . . . . . . . . . .

Reauta and. Discussiono e e e e e e e o. e o e

Intestinal Concentrations of Riboflavin.

and Thiamine
Bacterial Counts . . . . . . . . . .

Comparison of Vitamin Concentrations
and Bacterial Counts

Balance Study. . . . . . . . . . . .
Growth cf.Animals. . . . . . . . . .
Summary. . . . . . . . . . . . . . . . .
Literature Cited . . . . . . . . . . . .

Appendix . . . . . . . . . . . . . . . .

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TABLES

Titlg

Experimental Design Showing
subgroups of Animals Within Each
Group of Milk~fed.Animals

Experimental Design Showing
subgroups of Animals lithin Each
Group of Stock-fed Animals

Average Concentration of Riboflavin
and Thiamine in the Intestinal
Tract of Rats

Vitamin Content of Hulk and Stock
Diets

Dietary Intake of Thiamine and
Riboflavin

The Bacterial Counts Found in the
Intestines cf Cecectcmized and

Control Rats Fed a Milk and Stock
Diet Arranged to Show the Effect
of a Particular Diet-on the
Bacterial Population of Each of
the Intestinal Sections

The Sum of the Bacterial Total and
Group Counts Found in the Intestines
of the Cecectcmized and Control Rats
Fed a Hilk and Stock Diet

The Dietary Intake and Excretion
of Riboflavin of Animals Fed “ilk
and Stock Diets

The Dietary Intake and Excretion of

Thiamine of Animals Fed Milk and
Stock Diets

Average Weight Gains of Rats Fed
Hilk and Stock Diets

18

19

23
25

25

27

29

33

3h

number

I

FIGURES

Title Essa

Intake and Output of Riboflavin
and Thiamine of Animals Fed Milk
and Stock Diets 35

Composite Growth Curves of
‘Animals Fed Milk and Stock Diets 39

INTRODUCTION

I INTRODUCTION

Studies concerned with the synthesis of vitamins
in the digestive tract of animals are becoming increas-
ingly important. The problem is of considerable .
nutritional importance not only as a contribution to the
nutrition of the animal but also as '... a complicating
factor in the interpretation of the data obtained in
feeding experiments“ (Peterson and Peterson, l9h5, p. 80).

Iumerous investigators (Herter and Kendall, 1909:
Porter and Rettger, 19M); Mitchell and Isbell, 19%) have
demonstrated that the intestinal flora is influenced by
diet. In a bacteriological study involving white rats,
LtcClure (1918) found that the total numbers of intestinal
bacteria were higher in cecectomized milk fed animals
than in the corresponding controls whereas the reverse was
observed in stock fed animals. From this interesting
observation a question arose concerning the effect of diet
and cecectomy on the intestinal synthesis of vitamins.

The purpose of this study was to investigate riboflavin
and thiamine concentrations in certain portions of the
intestinal tract of cecectcmized and non-cecectomized‘white
rats and to compare the vitamin concentrations with

bacterial counts of the corresponding segments of the tract.

 

 

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.IIJ‘III 1|

Since it has been shown that microorganisms are capable
of synthesizing B vitamins (Thompson, 19n2; Burkhclder
and neveigh, 19%). it was believed that a study of this
kind might present additional information to our know-e
ledge of intestinal synthesis.

REVIEW OF LITERATURE

REVIEW OF LITERATURE

Introduction

I'Pasteur, in 1885, suggested that animal life would
be impossible without the coécperaticn of the micro;
organisms found in the digestive tract“ (ElvehJem, l9h6).
The influence of the intestinal flora upon the nutrition
of the host has been studied by numerous investigators.
Conversely, the growth stimulating activity of some of
the vitamins for bacteria has also become important.

Peterson and Peterson (l9h5, p. #9) in discussing
the relation of bacteria to vitamins, illustrate the
merging of the fields of animal and microbiological
nutrition. They advocate a mutual exchange of ideas to
promote progress in both fields and '... predict that
several of the compounds now known to be required by
bacteria will some day become members of the vitamin family.I
Egg’Significance 2;,Coprcphagz

That the intestinal microorganisms are capable of
synthesizing “anti-neuritic substances' was suggested by
Cooper in 1914 when he found that alcoholic extracts of
the feces of a rabbit fed white bread and cabbage cured
pclyneuritis in pigeons.

Osborne and Mendel (1913) noted improvement in the

performance of rats fed certain rations when they were
given access to their feces as a supplement. As research
in the vitamins and particularly in the synthesis of
vitamins has progressed the significance of this early
observation becomes interpretable.

The importance of preventing coprophagy in certain
types of experimental work has since been emphasized by
several investigators. Dutcher and Francis (1923) noted
that when screens were introduced in the cages of rats
receiving a ration deficient in vitamin B, a marked dc;
crease in food consumption and in body weight resulted.
That vitamin B was the factor supplemented by feces was
the belief of Steenbcck and his associates (1923). They
found that either ccprophagy or the addition of wheat
germ to the diet resulted in.an improved growth of rats
maintained on a purified diet low in vitamin B.

Heller, McElroy, and Garlock (1925, p. 263) from
observations made when feeding vitaminefree rations to
rats, suggested that '... the spore bearing organisms
present in the intestinal tract during the early part of
the experiment synthesize and store vitamin 3", Prolonged
life, and in some cases growth, were benefits attributed
by Roscoe (1931) to the feces consumed by rats receiving
diets deficient in the vitamin B complex.

\.

 

The Phenomenon gg_Refecticn

Refection refers to a condition in experimental
animals whereby the animals are able to grow, thrive, and
multiply on diets believed to be free of the undifferent-
iated vitamin B. The kind and amount of carbohydrate
present in the diet plays an important role in the
apparent synthesis of vitamins by the intestinal bacteria.
The investigative work which the accidental discovery of
refecticn stimulated in their laboratory has been described
and discussed by Fridericia et al (1927). The effects of
refecticn, which first arose spontaneously, could be trans-
mitted to. other young rats by. adding to their vitamin 13';
free diet the feces of other refected rats. These feces
were bulky and white and contained unusually large amounts
of 'vitamin B' and undigested starch.

Mendel and Vickery (1929) were unable to obtain
refecticn in rats but numerous other investigators have
confirmed Fridericia's original report (Roscoe, 1927: 1931;
Ken and Iatchorn, 1927: Parsons et al, 1933; Bliss and
Green, 1936).

Fridericia and his co-workers noted that the cecal
contents of refected rats resembled normal rats and differed
from rats suffering from vitamin B deficiency. The proposed
that '... the change in bacterial flora and in chemical

processes in the coecum cf refected rats may be correlated

with.the production of vitamin B in the intestine of
these rats'I (1927, p. 91 - 92).

Other investigators have postulated the cecum to be
the main site of vitamin synthesis by intestinal micro;
organisms (Porter and Rettger, l9h0; Schweigert et al,
l9#5: Day et al, l9h3: Taylor et al, l9h2). In contrast,
Griffith (1935) concluded that the cecum is not the
principal source of bacterial synthesis.
as: has 2:. 2.1.9.! +

These and other investigators studying synthesis have
pointed out the important role which diet plays in this
connection.

That the diet exerts a marked influence over the
bacterial flora of the intestinal tract of animals has
become a welleestablished fact. Herter and Kendall (1909)
were among the early investigators to demonstrate the
substitution of an acidophilic type of flora for a pro;
teclytic type when the diet was changed from one dominantly
protein to one high in carbohydrate. Other workers have
since confirmed this finding (Hull and Rettger, 1917:
Porter and Rettger, l9h0; Mdtchell and Isbell, 1952).

However, in studying the intestinal flora of dogs,
Torrey (1919) found that the type of carbohydrate or protein
food is also an important factor in determining the character
of the intestinal flora.

-7;

The superiority of certain carbohydrates in promoting
intestinal synthesis has been demonstrated repeatedly.
Morgan et a1 (1938) found lactose superior to cornstarch
and sucrose in the elaboration of B vitamins in the
intestines of the white rat. Mannering et a1 (l9hh),
Tepley et a1 (l9h7), and Guerrant et al (1935) found that
dextrinised cornstarch caused the greatest synthesis of
these vitamins while lactose appeared to occupy an inter;
mediate position.

Refecticn, which.has already been discussed, is
obtained with.diets having raw potato starch as the carbc¥
hydrate but, the beneficial effect is lost when cooked
potato starch is substituted for the raw starch.

Gall et al (l9h8a: 19%) studied both the intestinal
flora of mice fed different diets and the ability of the
predominating flora to synthesize riboflavin, niacin,
biotin, folic acid, and pantothenic acid in vitro. They
have shown that there is a flora, characteristic of mice
fed a dextrose diet,'which is different from that of
animals fed a dextrin diet. The coccus isolated from the
animal maintained on the dextrinecontaining diet was
capable of existing in a synthetic broth lacking folic acid
and at the same time was able to liberate this vitamin into
the environment. In contrast, the ooccus found in animals
fed the dextrose-containing diet exhibited little or no
growth in the deficient broth and liberated little if any
folic acid.

The addition of agar or other fibre to the diet
appears to favour growth of animals maintained on vitamin:
free rations (Heller et al, 1925) and to possess a sparing
effect on vitamin utilization (Guerrant and Butcher, 193a).
The latter workers believe the beneficial effect of fibre
to be due to the production of more favorable conditions
for the growth of microorganisms in the digestive tract.
Nielson, Shull, and Peterson (l9h2), who found a natural
stock dietato be far superior to synthetic diets in the
intestinal synthesis of biotin in the rat, stated that the
indigestible material contained in the stock diet pro;
bably favored bacterial growth.

The effect upon synthesis of varying amounts of fat
and of different fats in the diet has been studied. Whipple
and Church (1935) concluded that fat was essential for the
production of the antieberiberi factor in the gastro;
intestinal tract of the vitamin B-deficicnt rat. Guerrant
and Dutcher (193a) found that when increasing amounts of
fat (Crisco) were added to the basal diet, the rats became
depleted of their vitamin B reserve less rapidly but there
was no effect on the vitamin G requirement. However, in
further studies Guerrant et a1 (1937) did not find a more
favorable production of the B vitamins in the digestive
tract of the rat when 10 or 20 percent of Crisco was added
to a vitamin B complex deficient diet containing sucrose as

a source of carbohydrate. Ruth and his coawcrkers (l9h8)

found that the addition of a high level of corn oil to

a sucrose diet decreased the aerobic and anaerobic plate
counts as well as the numbers of coliforms in the coca of
rats. Supplementation of this diet with reticulcgen
(Lilly liver extract) tended to counteract the inhibitory
action of corn oil upon the growth of certain cecal micro;
organisms.

Czacakes and Guggenheim (l9h6) studied the influence
of high and low fat and high.and low protein diets on the
riboflavin metabolism of the rat. Rats kept on high
protein and high fat diets required more riboflavin than
rats on a normal diet but a low fat diet lessened the need
for riboflavin. The differences in riboflavin requirement
were shown to be due to differences in the amounts of
riboflavin which were synthesized in the intestines. That
high intakes of fat (25 to to percent) increased the ribo¥
flavin requirement of the rat has also been demonstrated
by Mannering et a1 (1999).

Elvehjem.and Irehl (19h?) have discussed the diver;
gence of opinion concerning the superiority of different
fats in the diet. Boutwell et a1 (l9h3) showed butterfat
to be superior to corn oil in effecting rat growth.where
lactose was used.as the sole carbohydrate.of the diet. With
a mixture of carbohydrates, the growth response was equal
for both fats. Tepley et al (191;?) found increased niacin
and folic acid synthesis in rats fed Isynthetic milk' diets
containing butterfat rather than corn oil.

~10;

Availability 2; Synthesized'Vitamins
The investigations discussed in the preceding para;

graphs have demonstrated the synthesis of vitamins by
microorganisms in the intestinal tract of animals. How;
cver,the availability of the synthesized vitamins to the
host has been questioned. Griffith (1935) showed that in
order to utilise the B vitamins present in the intestinal
contents rats must resort to coprophagy.

In refected rats, on the other hand, ccprophagy,
although it is practiced to some extent, is not necessary
for the continuance of the beneficial effects of the
condition.

The location of the vitamin production in the
digestive tract may account for some of the differences
observed. After studying the absorption of large concen;
trations of riboflavin in nephrectomized rats, Selye
(19h3) concluded that absorption takes place in the small
intestine while little if any absorption occurs in the
cecum and colon.

That synthesis occurs in voided feces kept at room
temperature has been demonstrated. Nielsen, Shull, and
Peterson (l9h2) found a 55 percent increase within a period
of two days in the biotin contents of the feces of rats
fed a stock diet. Studies by Lamoremx and Schumacher (l9h0)
revealed an increase in the riboflavin of the excreted

feces of fowl.

 

 

 

-11;

Diffusion of the synthesized vitamin from the beater;
ial cell is another factor to consider in availability.

Abde1&3alaam and Leong (1938) grew in vitro a mixed
flora taken from the rat cecum and were able to demonstrate
synthesis of thiamine but the vitamin did not diffuse into
the medium. Hitchell and Isbell (l9h2) also found a role;
tively small degree of diffusion of vitamins from the
bacterial cells into the surrounding medium. Czaczkes and
Guggenheim (l9b6) however, found that 90 percent of the
riboflavin produced by the intestinal bacteria of rats was.
extracellular and therefore accessible to the rat organism.
Vitamin Egcretion and Storage

In an attempt to explain the metabolism of vitamins,
studies of their excretion in the urine and feces of animals
have been conducted. Light and his cononkers (1938) found
that rats fed a daily intake of 15 to 515 micrograms of
thiamine excreted 25 to 30 percent of the ingested.vitamin
via the urine and 20 to 30 percent via the feces but the

remainder was unaccounted for. They stated that:

.,y,the explanation of this uniform distribution
may lie in a fixed relation between the
velocities of absorption elimination (by
urine), and destruction (utilization or
detoxication). There is no evidence to
indicate what may happen to the missin 'vitamin
other than storage of a portion. (193 , p. 3&0).

Subsequent studies by these same'wonkers (Schultz et
a1 1939) revealed that the tissue concentration was direct;

-12-

ly related to the intake when 10 to 65 micrograms were fed
daily but that the tissue concentrations were not further
increased with higher dosages. Leong (1937) had previously
reported similar results in the storage of vitamin Bl.

LL33 _o_f_ §_ul_f_5 m 19. 35.1.1.6. m 3; Vitamin Synthesis I

In more recent experiments designed to demonstrate
intestinal synthesis of vitamins, sulfa drugs have been
employed to inhibit the bacterial action. Black, Horibbin,
and ElvehJem (19u1) found that 0.5 percent sulfaguanidine
greatly reduced the growth rate of young rats on a purified
basal ration but that liver extract counteracted the effect
of the drug. Light et al (l9h2) demonstrated a similar
reduction in growth which was prevented by feeding yeast or
feces of rats kept on the same diet without the drug.

In studies with ceoectomised rats, sulfasuxidine pre;
vented vitamin K synthesis but pagaEaminobenzoio acid
partially counteracted the effect of the drug (Day at al,
1918). Miller (1916) added succinylsulfathiazole and
phthalylsulfathiazole to the highly purified diet of rats.
Dietary deficiencies, lower fecal excretions or biotin,
folic acid, and pantothenic acid, and a drop in the number
of coliforms resulted. ,

m Bmthgsig _i_n Ruminantg

Although.most of the work with intestinal synthesis
has been conducted using rats as the experimental animals,
studies of the synthesis of Bevitamins in the rumen of sheep

and cows have received attention.

it Pennsylvania State College, investigations were
conducted on the rumen contents of a cow grown to maturity
on a vitamin B complex-deficient diet. From this experi;
ment Bechdel and his associates (1928) concluded that the
vitamin B complex was produced in the rumen of the cow
by bacterial fermentation. MoElroy and Goes have done
extensive work in this field and have demonstrated the pro;
ducticn and utilization of thiaminemiboflavin, pyridoxine,
pantothenic acid, and vitamin K in the rumen of sheep and
cows (McElroy and Goes, 1939: 19M)” l9h0b; l9hla: 191+lb).
Yegner et al (1941) studied the rumen synthesis of six
members of the vitamin B complex.

Hunt et al (191m: 191m) studied the thiamine and
riboflavin content of the rumen of cattle and found that an
increase in the amount of carbohydrate (corn) in the ration
' produced an increase in the riboflavin content of the dried
ingesta of the rumen. They did not obtain evidence of
thiamine synthesis as Judged by a comparison of the thiamine
content of the feeds and the dried rumen contents but they
did report evidence which suggested either that the thiamine
was rapidly absorbed by the animal or that it was destroyed.
Vitamin Synthesis in M Subjegfis

Experimental evidence of the microbiological synthesis
of vitamins in human subjects has also been reported.
Bauer and Holt (1918) measured the output of free and

 

 

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-1h§

combined thiamine in the feces of nine patients on a
completely thiamine free synthetic diet. Those subjects
which.developed deficiency symptoms had almost no free
thiamine in their stools, but those which remained free of
symptoms had large quantities of free thiamine in the feces.
That the fecal thiamine had its origin in the intestinal
bacteria was demonstrated by the reduction in free thiamine
in the feces of a subject given succinylsulfathiazole.
Iilliamson and Parsons (l9h5) found the fecal excretions of
thiamine in human subjects to be increased with a high fibre
diet. Since urinary excretions were approximately equal to
those obtained with a low fibre diet, limited availability
of the synthesized thiamine was suggested.

Hathaway and Strom (l9h6), who compared the thiamine
excretion of human subjects on synthetic and natural diets
reported evidence suggesting that the natural diet was more
favorable to bacterial synthesis of thiamine than the
synthetic diet. Similar conclusions were obtained in ribo;
flavin studies (Hathaway and Lobb, 19h6). Excretion studies
of normal adults by Denko et a1 (l9u6a; 19%b) implied
intestinal synthesis of certain B complex‘vitamins with
apparently slight absorption through the large intestine.
Problems Involved in the £132.51 2; Synthesis _

Hany problems have been encountered by the workers who
have studied bacterial synthesis. Even where cultural

studies of the predominant organisms are conducted, there is

-15;

the possibility '... that there might be other, unidentie
fied bacteria which, though present in smaller numbers,
might exert a strong influence on the nutrition of the
animal '(lath et a1, l9h8, p. 789). According to
Schwcigert et al (l9b5), the amount of any vitamin excreted
does not measure the synthesis since the amounts absorbed
or destroyed cannot be differentiated. These and other
problems including the possible symbiotic relationships
existing in a mixed flora have also been discussed by
Mannering, Orsini, and Elvehjem.(l9hb).

However, in spite of the many complicating factors
involved, our knowledge of the role of bacteria in the
nutrition of animals can be extended only through more

extensive and more intensive research in this field.

EXPERIMENTAL PROCEDURE

EXPERIMENTAL PROCEDURE

Eighty weanling male albino rats of the Sprague-
Dawley strain were used for this study. The initial
weight of the control animals was hh to 58 grams.
Gecectomized animals weighed 58 to 78 grams at the time
of the operation.

To reduce coprophagy to a minimum, the rats were
housed individually in cages containing false bottoms
made of galvanized wire screens (2 to 3 meshes to the inch).

Evaporated milk’, diluted with an equal volume of
distilled water and supplemented by a solution containing
iron pyrophosphate, manganese sulfate, and copper sulfate,
was fed to #0 of the animals. The other #0 animals were

fed a laboratory stock diet consisting of:

ellow corn meal ' 5,000 parts

inseed oil meal 1,600 parts
alfalfa meal 200 parts
casein 500 parts
wheat germ 1,000 parts
~yeast 500 parts
powdered milk 500 parts
sodium chloride 50 parts
calcium carbonate 50 parts

As dispensed, 25 grams of corn oil was
added to each 500 grams of stock diet.

Food and distilled water were given ad 11bitum and
records were kept of food consumption. The animals were
weighed at weekly intervals on two consecutive days. The

average of the two weighings represented the weekly weight.

* Pet Milk, Pet Milk Company, St. Louis, Missouri

—_—

Within each group one-half of the animals were
cecectomized and one-half were maintained as controls. The
cecectomy technique used in this study was devised by Dr.
Wade Brinker of the School of Veterinary Medicine, Michigan
State College. The method has been described in detail by
noelure (191m) .

The control animals were placed on their respective
diets immediately and were maintained on that ration for
R5 to #7 days before they were sacrificed. The animals to
be cecectomized were fed the appropriate diets for h to 8
days before cecectomy and for an to #8 days following the .
operation.

Since preliminary trials had shown that there was
insufficient material in the intestinal sections of one
animal for thiamine and riboflavin assays by the methods to
be used, pooled samples from four animals were used for
each determination. Tables 1 and 2 illustrate this grouping
of animals.

Food was withheld for 17 to 18 hours before the animals
were chloroformed. The visceral cavity was Opened up immed-
iately and the intestinal tract removed. Cecal contents
from the feur rats in one group were transferred to a
previously weighed beaker and mixed thoroughly with a glass
spatula. Samples from the small intestine, and from the

large intestine, were also combined for each group.

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For 8 of the 20 groups (Tables 1 and 2), samples of
the contents from the cecum, large intestine, and small.
intestine were taken aseptically for bacteriological
study. For this phase of the work, the method used by
McClure (19h9) was followed.

The material in each beaker was then weighed, trans-
ferred quantitatively with 0.2 N HZSOu to a 100 m1.
volumetric flask, and prepared for riboflavin and thiamine
assays by enzymatic hydrolysis using polidase S enzyme.

All determinations were made in duplicate.

The assay methods used were a modification of the
thiochrome method for determination of thiamine in urine as
developed by Mickelsen, Condiff and Keys (l9h5), and for'
riboflavin, the fluorometric procedure of Conner and
Straub (1901) as modified by Keys (by communication).
Readings were made with a fluorescence meter.‘ The linear
relationship existing between the vitamin concentration of
a solution and the instrument reading is shown for riboflavin
in Figure 3 and for thiamine in Figure it (Appendix).

In addition to the thiamine and riboflavin concentration
in the intestinal sections, the amount of these vitamins
ingested and excreted by certain of the animals during a
three-day period was measured. Tables 1 and 2 show the four
groups of rats, representing a control and cecectomised group
for each of the two diets, that were used for the balance
study.

 

'The Coleman Photofluorometer or the Lumetron
Photofluorescence Meter was used.

-21-

For three days preceding autopsy, these animals were
transferred to individual cages placed over large glass
funnels in order that their excreta could be collected for
assay. The urine was collected in a graduated cylinder
containing 5 ll. of 1 R HzSOh. .A plastic mesh.screen
placed under the cage prevented the feces from entering the
urine-collecting cylinder. Brown paper was draped around
the cages and funnels in order to prevent the destruction
of riboflavin by light. The urine was transferred daily
to a 100 ml. volumetric flask and stored in a refrigerator
until the three—day sample was complete. At the time of
urine collection, the funnel was washed once with 0.2 I
3230b and the washing added to the urine flask. Before the
vitamin determinations were made, the urine samples were
filtered.

The feces were collected once, at the end of the balance
period and weighed. Feces and weighed samples of the diets
‘were transferred quantitatively with.0.2 l 3280”,to volue
metric flasks and hydrolyzed with polidase S enzyme prior
to thiamine and riboflavin determinations.

 

RESULTS AND DISCUSSION

 

RESULTS AND DISCUSSION

Intestinal Concentration; 2;,Riboflavin 222 Thiamine

Table 3 shows the average concentrations of riboflavin
and thiamine in the intestinal tract of the four groups of
rats studied. The concentrations are expressed in micro;
grams per gram, moist weight, of the intestinal contents
and represent an average of five determinations. Since the
intestinal contents of four animals were pooled before
assays were made, the final value is an average for 20
animals.

in examination of the riboflavin and thiamine values
(Table 3) reveals that within each group the concentration
is lowest in the small intestine, highest in the large
intestine, and intermediate in the cecum. For example,
concentrations of riboflavin in the small intestine, large
intestine, and cecum of the milk control group‘were 8.75,
29.0h, and 15.68 micrograms per gram respectively. The
same general trend was found in the riboflavin and thiamine
values of each group studied with one exception, namely the
low riboflavin concentration in the cecum.of the stock
control group.

.A difference is noted in the vitamin concentrations

observed in animals on the two diets. The thiamine values

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n .23

 

in the small intestine are higher for the stock fed animals
than for the milk fed animals. Differences in the large
intestine and cecum are not marked.

The average daily intake of riboflavin was lower in
themilk fed than in the stock fed animals (Tables h and 5).
However, since the animals were fasted for 17 hours before
autopsy, the influence which the dietary intake of riboi
flavin may have had on the concentration of the vitamin in
the various intestinal sections should have been eliminated
to a large extent. Riboflavin.values in the small intestine
lie within a close range in both groups of animals. Higher
riboflavin values are found in the large intestine and
cecum of the animals fed a milk diet than in the stock diet
group. The high riboflavin content in the large intestine
and cecum of the milk fed animals suggests a higher degree
of intestinal synthesis for these rats than for the stock
fed animals, if absorption or destruction of the vitamin is
disregarded.

In both the milk fed and stock fed animals, the con;
centratiom of thiamine and riboflavin were higher in the
control group than in the corresponding cecectomized animals.
As indicated previously, several investigators have expressed
the belief that the cecum is the main site of intestinal
synthesis. (Porter and Rettger, 19h0: Schweigert et al,
l9h5; Taylor et al, l9h2). The data presented in this study
support the hypothesis that synthesis of riboflavin and

 

Table

1}

Vitamin Content of Milk and Stock Diets

 

 

 

 

 

 

 

 

 

 

Diet Thiamine Riboflavin
'V/gm.m ‘V/gm.
Milk a. lit 1.06
Stock 8.98 h.h0
Table 5
Dietary Intake of Thiamine and Riboflavin
. -.-=-1
Diet Condition Average‘ Daily Dietary Dietary
Food Consumption Thiamine Riboflavin
:f'f't. Ella. Y/day Y/day
Milk. Control 39.b 5.5 #2.
Hulk Cecectcmized no.5 5.7 #3.
Stock Control 11.? 105. 51.
Stock Cecectcmized 13.3 119. 59.

 

 

 

 

 

 

’ Average of the food intake of the total number of'

animals for a seven week period.

 

.25-

thiamine occurs in the cecum. However, it is difficult
to compare this work with previous studies since the
methods of investigation differ.

Bactgrial 92333;

Table 6 shows the counts of the bacterianound in the
various sections of the intestines. The counts are expressed
as the geometric means of bacterial counts obtained from the
four groups of animals. However it was possible to have
bacterial counts done on the intestinal contents of only 8 of

the 20 groups. Thus the values obtained represent 8 cececr

1 TI... Jflflm '

tamized and 8 control animals fed the stock diet, and the

same number of cecectomized and control rats fed a milk diet.
The bacterial counts obtained in this study were com;

pared'with those found in #0 rats maintained under similar

experimental conditions (McClure, 1959). McClure (l9h9 ,

p. 34) found that:

Irrespective of the diet, the coliforms were
the least numerous and the lactdbacilli the
most numerous organisms found in the intestinal
tract. The enterococci were lower in numbers
than the lactobacilli, but numerically exceeded
the coliforms.
In this study the lactobacilli were again the most numerous
organisms found in the intestinal tract of all groups. In
the control animals, the coliforms were the least numerous

organisms but in the cecectcmized groups, the streptococci
were the least numerous.

In.Mo01ure's study, the cecectomized milk fed animals
showed higher total counts than the control animals on the

 

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noneeq nonaonnn nonaonnn dean
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e .33

milk diet, whereas the reverse was observed in the stock
fed animals. Table 7 summarizes the total bacterial
counts obtained in this study. The results are in agree;
ment with the data of McClure although the differences
are not so marked. In both studies, the total counts and
lactobacillus counts show the same general trend.

Other similarities in the two studies are the
domination of the coliform group by E. coli, the higher
coliform counts in cecectomized animals. than in the
controls regardless of diet, and the relatively high
counts found in the small intestine.

then the variations in the counts of individual
animals under the same experimental conditions are consid;
ered along with the relatively small number of animals
used, theagreement found in the two sets of data is
surprisingly good.
chparison 2; Vitamin Concentrations and Bacterial _C_o_1_i_n‘_t_§_

Then the average concentrations of riboflavin and
thiamine.in the small intestine, large intestine, and
cecum (Table 3) are compared'with the total bacterial
counts in the corresponding intestinal sections (Table 6),
a similar trend in values is found. The one exception to
this similarity was mentioned previously, namely the ribo;
flavin concentration in the cecum of the stock control
group. The only bacterial group counts which did not follow

the trend of the vitamin concentration were the coliforms

and streptococci present in the milk control animals.

 

 

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on» ma canon cannon nacho nee Hones Hednoposm can no man one

a canon.

However, in all of the milk animals and in some of the
stock animals, the bacterial counts were lower in the
control groups than in the corresponding cecectomized
animals even though the vitamin concentration in each case
was higher in the control than in the cecectomized group.

Demonstration, by other workers, of vitamin synthesis
by intestinal microorganisms has been discussed in a
previous section. Since the total bacterial counts were
highest in those sections of the intestinal tract where
the concentrations of riboflavin and thiamine‘were also the
highest, there is a possibility that bacterial synthesis of
these vitamins occurred.

Miller (lets) found that the addition of sueoinyl;
sulfathiazole and phthalylsulfathiazole to the highly
purified diet of rats resulted in decreased coliform
organisms in the feces. Symptoms of dietary deficiency
accompanied the drop in the number of organisms. Czaczkes
and Guggenheim (19%), who studied the riboflavin metabolism
of the rat, were able to show that the riboflavin content
of the feces was a function of the numbers of intestinal
bacteria and of the amount of riboflavin which they
synthesized. The validity of this conclusion was strength;
sued by measurements of the amount of riboflavin that could

be produced by the intestinal flora of rats.

 

 

-31;

The interesting and complex relationship existing
between bacteria and vitamins has been reviewed by

Peterson and Peterson (l9h5). During the seven year

period preceding the publication of their'paper, about

13 new compounds had.been added to the list of accessory

growth substances for bacteria. At that time, five of

these additions, biotin, pantothenic acid, pagaf

aminobenzcic acid, pyridoxine, and choline, had found a

place in animal nutrition. .
Campbell and Hucker (l9hh) studied the riboflavin

 

requirements of.a large number of strains belonging to
the genus Lactobacillus and a few strains belonging to
the genera Leuconostoc and Streptococcus. The effect of
riboflavin upon the growth of Streptococci was found to
be strain variable. Niven and Sherman (19%) tested 19
strains of streptococci and found only two that were
able to grow well without added riboflavin. All grow
well in the absence of added thiamine.

Hoepke, Libby, and Small (19kb) found E. coli ~
sensitive to slight changes in the growth medium.
Although E. coli usually grows well in a synthetic
medium containing only inorganic salts and glucose,
mutant strains appeared to have lost the ability to syn;
thesize thiamine.

Studies on the synthesis of thiamine by four strains
of an g. goli freshly isolated from human and animal
sources, and a stock strain of the same organism were
undertaken by Genung and Lee (l9hfl). Among the results
‘which they have reported is the observation that I'... there
is an optimum level of thiamin in the presence of which the
organism.will synthesize a maximum quantity of this vitamin“
(19M, p. #35).

Burkholder and McVeigh (l9h2), studied the production
of riboflavin, thiamine, nicotinio acid, and biotin by pure
cultures of six species of common intestinal organisms in:
cluding E. coli. Under the conditions of the experiment, the
species of bacteria studied synthesized some of the B vita;
mine in greater amounts than were needed in their metabolism
and the residues accumulated in the cultures.

These investigations demonstrate the many factors in;
volved in the interpretation of data such as is being pre;
sented in this study.

Balancelggggz,

In order to study the possibility of intestinal
synthesis of thiamine and riboflavin and to investigate their
metabolism within the body of the rat, a threegday balance
study was conducted with four animals from each group. The
results obtained are presented in Tables 8 and 9. The same

data are shown graphically in Figure l.

 

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1351

Figure l

Intake and Output of Riboflavin and Thiamine
of Animals Fed Milk and Stock Diets

j
‘I:

Hilk fed control animals:

Riboflavin intake #
Riboflavin output MILL—.4
Thiamine intake

Thiamine output a

Bill: fed cecectomized

 

 

 

 

animals:
Riboflavin intake
Riboflavin output
Thiamine intake I
Thiamine output

Stock fed control animals:
Riboflavin intake ”.1
Riboflavin output
Thiamine intake H

Thiamine output

Stock fed cecectomized
animals:

Riboflavin intake m

Riboflavin output

Thiamine intake

Thiamine output H
166 266 36s dfifib

Micrograms of vitamin

 

 

— Intake of vitamin
Fecal excretion of vitamin
L :1 Urinary excretion of vitamin

 

 

 

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Us! .

fair ' a
_

It appears that riboflavin was synthesized in the
intestinal tract of the control group of the milk fed
animals. The average intake of riboflavin for the three;
day period was 17h micrograms. For the same period, the
average total excretion was 292 micrograms.

The large quantity of riboflavin in the feces, 198 We
micrograms, as compared with the urine, 9h micrograms, L :1
suggests several possible explanations. Firstly, the '
synthesized riboflavin may have been held within the

 

luv - .t' -

bacterial cell, thereby rendering it unavailable for use
in the metabolism of the animals. Secondly, the riboflavin
may have been synthesized in the lower part of the intestinal
tract where absorption is prdbably limited, if at all possible.
Lastly, the riboflavin may have been synthesized in the feces
after excretion, since the feces were collected only once
during the three-day period. Lamoreux and Schumacher (l9h0)
have demonstrated an approximate 100 percent increase in ribo-
flavin in the feces of fowl held at room temperature for
2h hours and a 300 percent increase or more when they‘were
held one week.

The excretion of riboflavin, 121 micrograms, was
greater than the intake, 99 micrograms, in the cecectomized
milk fed animals also.

A comparison of the riboflavin balance of the control
and cecectomized.milk fed animals suggests that the cecum

‘was an important site of intestinal synthesis in the control

animals, but that the riboflavin was in an unavailable
form. However, the possibility of synthesis in the
excreted feces cannot be disregarded.

Intake and output of thiamine were equal in the milk
control animals but most of the vitamin was excreted in
the feces (Table 9). lo apparent synthesis occurred in - 9-?

the cecectomized group.

Apparently riboflavin‘was synthesized in both the
control and cecectomized animals fed the stock diet ‘
(Table 8). With dietary intakes of 178 and 192 micrograms Er

 

respectively for the control and cecectomized groups, the
excretions were 222 and 2&2 micrograms. In these animals
a greater percentage of the eliminated riboflavin was
voided in urine than was excreted in feces.

The thiamine intake for the stock diet animals was
much.greater than the output (Table 9). However, synthesis
of this vitamin is not necessarily precluded. One of the
results reported by Genung and Lee (l9hh, p. #35) in their
study of m. coli was that '... the stock strain in the
presence of a relatively large amount of thiamin tends to
use the vitamin rather than synthesize it'. Further investi;
gations‘with thiamine and with other vitamins may reveal
that under certain conditions other bacteria and strains of
E. coli isolated from the intestinal tract will also display
a similar tendency to utilize rather than to produce vitamins.

m 9; Animals

figure 2 and Table 10 show the growth curves and
weight gains of the four groups of rats. The animals
receiving the stock diet grew better than those on the
milk diet, but there were no striking differences in the ,
control and cecectomized animals on one ration. These F'
results are in agreement with those obtained by Dhanda
(19b?) and HcClure (19M).

 

250

N
O
0

Weight in grams
[.1

\n

O

100

50

figure 2

Composite Growth Curves of Animals
Fed Milk and Stock Diets

 

 

team»

1 2 3‘ f

Time in weeks

b
D

Stock cecectomized
Stock control
Milk control

Milk cecectomized

 

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0H 0.3.69

 

 

sums!

 

SUMMARI

Eighty young albino rats were divided into two
groups: one group was fed a laboratory stock diet and
the other group was fed a milk diet. Uithin each group,
onenhalf of the animals were cecectomized.

After a feeding period of about seven weeks, the
animals were sacrificed and thiamine and riboflavin
determinations made on the contents of the cecum, large
intestine, and small intestine. Intestinal contents
from four animals‘were pooled for each determination.

In certain cases, bacterial counts were also made
on the intestinal contents.

With the exception of one group of animals, the
concentration of thiamine and riboflavin within each group
was found to be lowest in the small intestine, highest in
the large intestine, and intermediate in the cecum. In
all cases, the vitamin concentrations were higher in the
control group than in the corresponding cecectomized
animals.

The lactobacilli were the most numerous organisms
found in the intestinal tract of all groups of animals.
The coliforms and streptococci, respectively, were the
least numerous organisms in the control and cecectomized

groups. Total counts were lower in the cecectomized

‘ ' n ‘

 

42.

stock fed animals than in the controls but little differ;
ence was noted in the milk fed animals.

The total bacterial counts were highest in those
sections of the intestinal tract where the concentration
of thiamine and riboflavin were the highest.

.1 three day balance study was conducted.with four
animals from each group. The total excretion of ribo;
f1avin.was higher than the intake for each group of
animals but the greatest increase appeared in the milk'
control animals. In this group the thiamine excretion
equalled the intake, but in all others, the amount
excreted was lower than the intake.

The possibility of intestinal synthesis of thiamine'
and riboflavin in these animals is discussed.

.Animals receiving the stock diet grew better than
those on the milk diet, but cecectomy had no effect on
growth.

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LITERATURE CITED

LITERATURE CITED

Abdel-Salamm,.i., and Leong, P. G. 1938, Part I GXXIX

Synthesis of vitamin Bl by intestinal bacteria of
the rat. Biochem. J., 32:958-963.

Bechdel, S. 1., Honeywell, H. E., Butcher, R. A., and
Inutsen, H. H. 1928 Synthesis of vitamin B in the
rumen of the cow. J. Biol. Chem., 80:231-238.

Black, S., McKibbin, J. M., and Elvehjem, G. A. l9hl I
Use of sulfaguanidine in nutrition experiments. Free. +
Soc. Exp. Biol. and Med., #78308-310. .

Bliss, S., and Green F., 1936 Refection in the rat. h;
J. Nutrition, 11:1-19. r

Boutwell, R. 1., Geyer, R. P., ElvehJem, G. A., and Hart,
E. B. l9h3 Further studies on the comparative value
of butterfat, vegetable oils, and oleomargarines.

J. Nutrition, 26:601-609.

Burkholder, P. R., and HcVeigh, I. 19h2 Synthesis of
vitamins by intestinal bacteria. Proc. Ratl. Acad.
8010 Us Se, 28:285'2890

Campbell, T. E., and Hacker, G. J. 19h# Riboflavin
requirements of certain lactic acid bacteria. Food

Research, 9:197;205.

Conner, R. T., and Straub, G. J. l9h1 Combined deter;
minations of riboflavin and thiamine in food products.
Inde Enge Chane, m1“ Ede, 13:385-3880

Cooper, E..A. 1914 On the protective and curative
properties of certain foodstuffs against pclyneuritis
induced in birds by a diet of polished rice. Part II.
J. Hygiene, 1&812-22. s

-Jr-yfl‘

 

 

 

Ozaczkes, J. l., and Guggenheim, I. 19h6 The influence of
diet on the riboflavin metabolism of the rat. J. Biol.
Chem. , 163267-271». .

Day, R. G., flakim, K. G., Irider, H. 3., and O'Banion, E. E.
19h3 Effects of cecectomy, succinylsulfathiazole, and
aminobenzoic acid on vitamin K synthesis in the
ntestinal tract of rats. J. nutrition, 26:585-600.

Denko, O. V., Grundy, W. E., Porter, J. R., Berryman, G. R.,
Friedemann, T. E.,and Ioumans, J. B. 19h6 The
excretion of B-complex vitamins in the urine and feces
of seven normal adults. Arch. Biochem., 10:33-hO.

. , Hheeler, N. 0., Henderson, 0. R.,
Berryman, G. R., Friedemann, T. E., and Ioumans, J. B.
19h6 The excretion of B-oomplex vitamins by normal
adults on a restricted intake. Arch. Biochem.,
11:109-117.

Bhanda, M. R. 1947 Effect of cecectomy on the fecal flora '~,
of white rats. unpublished H. S. Thesis. East Lansing,
Michigan, Michigan State College Library.

Dutcher, n. A., and Francis, 1:. 1925-21; Vitamin studies. 1:.

Feeding technique in vitamin studies. Proc. Soc. Exp.
BiOle and Made, 213189.193.

ElvehJem, O. A. 19h6 The role of intestinal bacteria in ;:
nutrition. J. Amer. Dietet. A., 228959-963.

, Krehl, U. H. 19h? Imbalance and dietary
inter-relationships in nutrition. J. Am. Hed..Assoc.,

Fridericia, L. S., Freudenthal, P., GudJonnsson, S.,
Johansen, G., and Schoubye, N. 1927 Refection, s
transmissible change in the intestinal content, enabling
rats to grow and thrive without vitamin B7 in the food.
J. Hygiene, 27:70-102.

Gall, L. S., Fenton, P. R., and Gowgill, G. R. l9h8a. The
nutrition of the mouse II. Effect of diet on the
bacterial flora of the intestine and the cecum. J.
Nutrition, 35:13.25e

, Illingworth, B. A., Oowgill, G. R., and Fenton,
F. F. l9b8 b. The nutrition of the mouse III.
Relation of diet to the synthetic activity of the
predominating flora isolated from the small intestine
and cecum. J. Nutrition, 35:27-38.

Genung, E. P. and Lee H. E. 19kt Studies on the synthesis
of thiamin by certain strains of Escherichia coli.
Jo BECte ' h7gh3£kh35e

Griffith, W. H. 1935 Studies in Growth III. B and G
avitaminosis in cecectomized rats. J. Nutrition,
10 3 667-671}.

 

 

 

 

 

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Guerrant, R. B., and Dutcher, R. A. 193k Some effects of
the composition of the diet on the vitamin B and the
vitamin G requirement of the growing rat. J. Nutrition,

. . and Brown, R. A. 193? Further
studies concerning the formation of the B-vitamins in_
the digestive tract of the rat. J. Nutrition, 13:305-315.

, , and Tomey, L. P. 1935 The
eTfect of the type 6?_carbohydrate on the synthesis of
B vitamins in the digestive tract of the rat. J. Biol. .
Chem., 110:233-2u3. E

 

Hathaway, H. L, and Lobb, D. E. l9h6 A comparison of
riboflavin synthesis and excretion in human subjects on
synthetic and natural diets. J. Nutrition, 32:9-18.

. and Strom, J. E. l9h6 A comparison of

thiamine synthesis and excretion in human subjects _ r
on synthetic and natural diets. J. Nutrition, 32:1-8. f‘

 

Heller, V. 0., McElroy, O. R., and Garlock, B. 1925 The
effect of the bacterial flora on the biological test for

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4
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APPENDIX

 

 

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