Studies on Anti-tuberculous Low Molecular
Factors in Various Organs of Rabbits
Part I.

Studies on crude materials
Michiro NAKASHIMAThe Fifth Division 0/ the Tuberculosis Research Institute, KyDto University(Director: Prof. Shu8uke TSUJI, M. D.)

INTRODUCTION

The problems of resistance of various animals to tuberculous infection havebeen investigated from the viewpoint of the function of cells 1 ) 2)3) or of body
fluid 4 )fi). In general, although the function of cells has been thought to be moresignificant in the defense mechanism of animals against tuberculosis than thatof fluids, it has been recently demonstrated by our associates that body fluids ....
human urine and serum of various animal species
contains powerful tuberculostatic agents and that these agents may play a very important role in themechanism of noticeable resistance of animals 6 ).
In our laboratory, using several new experimental methods, (the slide culture
method 7), the chamber method 8), and the ring method 9 ) , ) the resistant factors ofanimal body fluids to tuberculous infection have been investigated and analysed.
It has been demonstrated that the low molecular substances (able to pass througha cellophane membrane) in animal body fluid can act to inhibit and the highmolecular substances to promote the growth of virulent tubercle bacilli. It has
been proved that there are some substances in human urine or in the serum ofvarious animal species which can inhibit the growth of tubercle bacilli in vitro.
And it has also been demonstrated that active substances may be some sorts-of amino acids, peptides or organic acids 1 0) •Subsequent investigations dealing with the tuberculostatic factors of various-organ extracts of normal or immune rabbits, will be reported in this paper. In
a series of investigations, this first part is concerned with crude materials of
various organ extracts of rabbits.
MATERIALS AND METHODS

Preparation of materials

Preparing of rabbit's organ extracts

A rabbit weighing 3 kg was bled toStudies on Anti-tuberculous Low Molecular Factors in Various Organs 0/ Rabtits

death by cutting the neck artery.

37

To promote blood flow, 1 ml of heparin solu-

tion (1,000 un.its) had been previously injected into an auricular vein.
Each organ (muscle, liver, kidney, brain and lung) was washed with physiologic saline solution through its artery and vein by pumping to remove
as much blood as possible until the washing water became perfectly blood free.
The spleen, however, could not be washed thus, because vessels were too thin
to insert an injection needle. The muscle was collected from the femoral muscles. All washed organs were weighed and recorded. After being cut grossly
with scissors, the organ tissue was homogenized for one or two minutes by adding an adequate amount of physiological saline solution (the amount is different
for each organ: the liver, kidney and brain could be homogenized without thesolution, but the muscle and lung needed the solution, as much as half thevolume of the original material. ')The homogenized organ was enclosed in acellophane membrane (for dialysis No. 300) and dialysed in fifteen- or twentyfold volumes of sterile distilled water at 4°C for 72 hours.
The outer fluid produced by dialyzing was concentrated under reduced pressure (less than lO mg Hg of pressure) and at 50°C in- temperature to dryness.
The dried materials looked like yellowish-brown caramel containing some crystals~
and were deliquescent. The color of the dried materials changed easily fromyellowish-brown to dark-brown at about 60°C in temperature. These dried materials are designated as "the crude materials" of organ extracts. The yield of
the crude material was not uniform according to the organ or the animal used.
Immunizaticn: Using the same procedure, we also prepared crude materials
from immunized rabbits. Normal rabbits weighing 3 kg were injected with 100
mg heat-killed H37Rv strain or 50 mg living BeG suspended in paraffin oil into
the gluteal muscle twice with an interval of two weeks. Two weeks after thelast injection, the rabbit showed a positive skin reaction to the ten-fold diluted
old tuberculin.

Culture method
Preparation of medium
The dried "crude materials" of organ extracts ofrabbits were dissolved in 1/8 the volume of the starting organ materials with

distilled water.
This solution was called "eight-fold concentrated solution of materials".
Test for tuberculostatic Getivity:

The slide culture method was used.

Twomilliliters of Kirchner's liquid, containing the serially diluted "crude materials",
was prepared. The composition of one of the serial media is shown in Table
1. Slides which were smeared with the test bacteria CH37Rv strain, unlessotherwise noted) and treated by the benzine method were immersed in the
media.

Cultures were continued for ten days at 3TC.

38

Michiro NAKASHIMA
Table 1.

The Series of Cultur Medium.
Concentration

Component
8x

4X

2x

Ix

1!2x 1!4x

C

Crude material (8x)

1.6

0.8

0.4

0.2

0.1

0.05

0

Distilled water

0

0.8

1.2

1.4

1.5

1.55 1.6

Kirchner's Liquid (lOx)

0.2

0.2

0.2

0.2

0.2

0.2

0.2

Bovine Albumin

0.2

0.2

0.2

0.2

0.2

0.2

0.2

------------"1 x" means the same concentration as the orginal organ, and "2 x" means thetwo-fold concentration.

The degree of growth of bacilli is designated as follows:+ + + Colonies abhere to each other, and there are no distinct colonies.

+ + Very good proliferation, but there are distinct colonies+ Good proliferation (by oil immersion)- No bacillary growth.
Using the method described above, several strains of mycobacteria were cultured and the growth-inhibiting action of the adde1 "crude materials" fromvarious organs of normal or immunized rabbits was examined. Furthermore,stability against heat or hydrolysis, and solubility in several organic solvents ofthe effective factors in the crude materials were also examined.
RESULTS1.

The inhibitory effect of the crude materials on the growth of the H37Rv
strain.

a) The crude materials from the normal rabbits:

The results are shown in Table 2. It can be seen that while the muscle andthe liver showed an apparent inhibitory effect on the growth of the H37Rv strain.at a "Ix" concentration (i.e., the same concentration as in the living state),the lung and the spleen could not inhibit at a two-fold concentration ("2X") of
the starting organs. The brain and the kidney sometimes could and sometimes-could not inhibit at a two-fold concentration.
The relation, in one case, between the inhibitory effect and the calculated.amount (mg) of the crude material in Kirchner's medium (1 mI) is shown in
Table 3 and Fig. 1. The smallest amount capable of inhibiting the growth ofbacilli was 16 mg in the muscle, 24 mg in the liver, 40 mg in the brain and kidney,
but in the lung and the spleen, the smallest amount could not be determined inthis case.
Of course, there are individual differences in the inhibiting ability of ani-Studies on Anti-tuberculous Low Molecular Factors in Various Organs of Rabbits

39

Table 2. The effect of the crude materials of several organ extract of normal rabbiton the growth of tubercle bacHli.
- ~ _ . _ - - ~ - - - - - - - --._.~

Concentration_ _---.-

Organ

...

8x

4x

2x

IX

1/2x

1/4x

Muscle

+++

++
+++

Liver

+
++

++
++

++
++

+++
+++

+
++

Kidney- - - - ---~-----------~--------------- - - - - - - - - - - - - - - - _

_-

....

Brain

++

++

+
+++

++,
+'i l,

Lung

+
++

++
+++

+++
+++

+'i l
+++
.. ..

- - - - - - - - - - - ------~------~-++

SpleenI- ~ - - _

++
++

~

_-

+++
+'

++
+++

I
II

Control + ++Slide culture using H37Rv strain in Kirchner's medium for ten days
Table 3. The relation between the effect and the amount of the crude material
in Kirchner's medium on the growth of H37Rv strain.
- - - ------ - - -Dose of materials Cmg/ml)
Organs

40 35

30

25

20

15

10

Muscle

5

o

+++

+++

+++ ++++++ ++++++ +++

Kidney

++

++

++
+++

Brain

++

++

+++

++

++

+++

+++ +++

+++

++

++

+++

+++

+++

Liver

Lung
Spleen+++++mals, and the absolute value of the inhibiting dose of the crude material is not
uniform in all experiments, but tbere is always a distinct relationship between
the inhibiting ability and the organs. The grade of inhibiting ability is in thefollowing descending ordermusc1e, liver, brain, kidney_;~lung and spleen.
b) Crude materials from immunized rabbits:
The crude materials of the various organ extracts of rabbits which were im-Michiro NAKASHMA

40

munized by 100 mg heat-killed
H37Rv strain or 50 mg living
BeG were examined by the procedure described above. The results in one case are shown in
Table 4 and no significant difference between the data obtained
from the animals immunized bykilled bacilli or by living bacilli
was noted. Although the inhibiting ability of muscle and liver isalmost the same as that obtained

'lJ'l

:3
So
.g,

++

"

a:

+

~
<

en

OJ

w

S'

~

~

~

mg/ml

Contai;led dose of the crude materials in the Kirchner's medium

Fig. 1. The relation between the amount of
the crude materials (mg) in Kirchner's memium and their effect on the growth of the
H37Rv strain.

Table 4. The effect of the crude materials of several organ extract from the immunized
rabbits on the growth of tubercle bacilli.
The rabbits were immunized with 100 mg heat killed H37Rv strain and 50 mg living
BCG suspended in paraffin oil injected into the gluteal muscle twice with an interval of
two weeks.
--_..

_--_._~-

I

Concentration

I

I

Organs Immunized

8x

4x

2x

Ix

1/2x

1/4X

{H37RV
BeG

-

-

-

-

-

++
++

+++
+++

{H37RV
BCG

-

-

-

-

-

-

+
++

+++
+++

-

-

+
++

+++
+++

+++
+++

-

+
++

+++
+++

+++
+++

+++
+++

+++
+++

I
I

Muscle

I

-

-

-

Liver

I

Kidney{H37RVBCG

Brain{H37RV
BCG

-

-

--

---------~-~._~--~-----~-~--------~---Lung{H37RV
BCG

+
+

++
++

Control + + +Slide culture using H37Rv stain in Kinchner's medium for ten days.

in normal rabbits, it can be noted that the other organs have a slightly more
powerful effect on the growth of bacilli than those obtained from normal rabbits.
But, as there is a problem of individual variation, comparing these three (normal
and two immunized) experimental results, it is almost certain that the degree ofgrowth-inhibiting power of various immur.e organs has not altered from that ofnormal organs.

Studi2S on Anti-tuberculous Low Moleculsr Factors in Various Organs of Rabbits

41

Heat stability

2.

Sterilization is indispensable in the process by which the antibacterial effectof these crude materials is examined. ~o it is necessary to examine how heatstable these crude materials are. For this puq;ose, the temperature for sterilization was changed as follows.
i. Sterization by Seitz's filter (no heating)ii. Sterilization by heating at 100°C for 30 min.
iii. Sterilization by heating at 120°C for 30 min.

These experimental results are shown in Table 5.

These crude materials are

so stable to heat that we usually can use sterilization by heat at 100°C for 30 min.
Table 5.

Heat stability of the crude material of muscle extract from the normal rabbit.
Concentration
Sterilization
4x

2x

Ix

Filtration with SEITZ's Filter· Heating lOO°C for 30 min.
I Heating 120°C for 30 min.
I

J

1/2 x

1/4x

+++

+++

+++

+++

+++

+++

------'_

Controle + + +Slide culture using H37Rv strain in Kirchner's medium for ten days.

3.

The solubility in several organic solvents of the crude materials oforgan extracts and their tuberculostatic effects.

The solubility of the crude materials of organ extracts in several organic
solvents was examined. As solvents, methanol, ethanol, ethyl ether and acetone
were used.
After the crude materials had been warmed and evaporated to dryness at
50-60°C, 50-100 ml of solvent was added to the dry material.
After being stirred and shaken the soluble and insoluble portions were separated by filtration or decantation. Both portions were again warmed and evaporated to dryness in order to remove solvents and were redissolved in distilledwater.
tested.

After sterilization, at 100°C for 30 min., the tuberculostatic activity was

In this case, we used only muscle and liver as testing materials, because
the other organs were too small in amount to get a large enough volume for theexaminations.
The results are shown in Table 6. It can be seen that the active portions
are soluble in water and methanol, while they are only very slightly soluble orentirely insoluble in ethanol, ethyl ether and acetone.

42

Michiro NAKASHIMA

Table 6. The solubility in organic solvents of the crude material of muscle fromthe normal rabbit and their effect on the growth of H37Rv strain.
Parts Soluble
or lnso luble

Solvents

WatersolubleMethanolsoluble{ insoluble

Ethanol

Concentration
4x

2x

Ix

1/2X

1/4x

++

+++
+++
+++

+

++

++
+++ +++

soluble{ insoluble+++++++ +++ +++++ +++

Ether

soluble{ insoluble+++++ +++ +++ +++++ +++

Acetonesoluble{ insoluble+++++++ +++
++

+++
++
-----

Control ++ +Slide culture in Kirchner's medium for ten days.

4. Chemical properties examined in these crude materials.
The following chemical reactions were examined using these crude materials.
- --------------

Reaction
Sulfosalicilic acid R.
Ninhydrin R.
Millon's R.
Jaffe's R.
Molisch's R.
Nylander R.

I

- - -

Meanings

Results

(-)

C+)
C+)
(+)
(+)

(+)

No proteinsAmino acids present
Amino acids present
Creatinine presents
Active reducing substances present
Active reducing substances present

These results may' indicate that these crude materials are really low molecular and containing no protein or other high molecular substances.
DISCUSSION

We have proved that there are substances, which can inhibit the growth oftubercle bacilli in vitro in various organs (muscle, liver, kidney, lung and spleen)in rabbits. These substances are low molecular substances which are able topass through a cellophane membrane.
The grade of the inhibiting activity isdifferent according to the organ, the substances from muscle being strongest, followed by, in descending order, liver, kidney and brain, lung and spleen~
These substances are heat stable and their activity is not -decreased even byheating at 120°C for 30 min. or by hydrolysis with heating with 6N HCI inStudies on Anti-tuberculous Low Molecular Factors in Various Organs of Rabbits

43boiling water for 15 hours 33 ). They are easily soluble in water and methanol
but almost insoluble in ethanol, ethyl ether or acetone.
It maybe worthy ofnote that the activities of these substances did not increase after immunization,that is, that tuberculous immunity is not conferred by these low molecular antituberculous substances in organs. These characteristics of the substances invarious organ extracts of rabbits which can inhibit the growth of tubercle bacilli
in vitro are quite similar to the tuberculostatic substances which were previously
found by Fujita 10 )11l in the low molecular substances of rabbit's serum. Oshima10) 121
also proved the presence of tuberculostatic substances in human urine. But
the activity in urine is not so stable to heat or hydrohysis as that in organ extract or serum, so it seems to us that there are some differences between theeffective substances of organ extracts or serum and those in human urine.
Previously, BjornesjolS)-20) rer:orted the presence of tuberculostatic substancesin human urine, and also the presence of similar substances in the bovine spleen,kidney, lung, liver and muscle. He stated, however, that the muscle substance
was the weakest in activity as compared with that of other organs. That fact
is opposite to our finding that muscle is the most active.
Dieh1 21l showed that tubercle bacilli which were incubated with the organ·emulsions were inhibited either in their power to infect guinea pigs or in their
growth on Hohn's medium. He implied that this fact demonstrated the presenceof some tuberculocidal substances.
Ozasa 22 ) examined the tuberculostatic effect of alcoholic extracts of lung,
liver, spleen and kidney of guinea pigs and found they contained substances
which inhibited the growth of tubercle bacilli, while the other organs had growthpromoting substances.
The contradictory results reported by the last-mentioned two authors, may
be resolved if we consider that the substances should be divided into two categories, low and high molecular substances. As a matter of fact, it has been
demonstrated by our recent investigations that the low molecular substances inthe serum of various animals act to inhibit and the high molecular substances topromote the growth of tubercle bacilli.

Therefore, it may be inadequate to deal

with both high and low molecular substances together in examining the growth
inhibiting activity.
Dubos et a1. 2S )24) demonstrated fairly pure substances in several organs, thatis spermin in the kidney and thymus-peptide in the calf thymus as antituberculous
factors in the organs.
There are some other investigations dealing with antituberculous substances
in various chemical factors in animal organs, in serum, in urine, and even inmilk25) -32) .
In short, although it may be natural that there are many substances which

44

Michiro NAKASHIMA

are capable of acting on the growth of tubercle bacilli in so complicated a substance as one taken from an organ extract, it may still be noteworthy that no
distinct relationship exists between these substances and the organ disposition to
tuberculosis.
On the other hand, in this investigation we can demonstrate asomewhat reasonable parallelism between the antituberculous activity of an organ
substance and the resistance of these organs to tuberculosis.

SUMMARY
1. The tuberculostatic activity of low molecular substances (able to passthrough a cellophane membrane) in various organ extracts, (muscle, liver, kidney"
brain, lung and spleen) of rabbits was investigated. And it was confirmed that
substances which can inhibit in vitro growth of the H37Rv strain of tubercle bacilli were actually present.
The activity of these substances is, in descendingorder, muscle (strongest), liver, kidney and brain, lung and spleen.
2. These substances are soluble in water and methanol but insoluble inethanol, ethyl ether and acetone.
3. These substances are stable to heating at 120°C for 30 min.
4. The activity of these substances is not increased by immunization of the
rabbits.

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Rich, A. R. : The Pathogenesis of Tuberculosis Ed. 2, Charles C. Thomas, Springfield"
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2)

Suter, E. : The multipulication of tubercle bacilli within normal phagocytes in tissue
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. 3)

Mackaness, G. E. : The growth of tubercle bacilli in monocytes from normal andvaccinated rabbits, Am. Rev. Tuberc., 69, 479, 495, 1954.

4)

Kling, C. A. : Untersuchungen tiber die bakterientatenden eigenschaften der wissen
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5)

Hanks, J. H. and Evans, B. : The action of serum, cells and blood on acid-fast bacteria in vitro, Am. Rev. Tuberc., 41, 605, 637, 1940.

6)

Tsuji, S., Ito, K., Oshima, S. : The role of humoral factors in native and aquired
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7)

Yamamoto, H. : Study on the slide culture method (S. C. M.), J. J. C. P., 1 58,.
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8)

Tsuji, S., Ito, K. : An in vitro method of culturing tubercle bacilli-The chambermethod, Am. Rev. Tuberc.,72, 393, 1955.

9)Tsuji, S., Oshima, S., Takeoka, A. : The ring method: A method for analyzing theeffect of serum on the growth of tubercle bacilli in vitro, Am. Rev. Tuberc., 77, 524,.
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Studies on Anti-tuberculous Low Molecular Factors in Various Organs of Rabbits
10)45

Oshima, S., Fujita, Y., Takeoka, A., Nakashima, M., Tsuji, S. : Chemical analysisof the role of humoral factors in native and aquired resistance to tuberculosis, Am.
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Fujita, Y. : Low molecular antituberculous factor in the serum of various animal
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12)

Oshima, S. : Antituberculous factor in human urine (Part 1-2). Rep. Tuberc. Res.
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13)

Bjornesjo, K. B. : On the effect of human urine on tubercle bacilli, Acta tuberc.
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14)

Bjornesjo, K. B. : II. The tuberculostatic effect of various urine constituents, Acta
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Bjornesjo, K. B.: III. The solubility of the tuberculostatic factor in organic solvents,
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Bjornesjo, K. B. : On the effect of human urine on tubercle bacilli. IV : Some attempts
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Bjornesjo, K. B. : V. Experiments with the tuberclostatic factor purified from urine,
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Bjornesjo, K. B. : On the occurrence of tuberculostatic factor in human urine, Acta
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Bjornesjo, K. B.: Tuberculostatic factors in normal human urine, Am. Rev. Tuberc.,
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20)

Bjornesjo, K. B. : On the occurrence of tuberculostatic substances in bovine organ
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'21)

Diehl, K. : Uber ein antituberkulose Prinzip in Organen, Beitr. zur Klin. der Tuberk.,
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22)

Ozasa, S. : Effect of the extracts from animal tissues on the growth of M. tuberc.,
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23)

Dubos, R. J. : A tuberculostatic agent present in animal tissue, Am. Rev. Tuberc.,
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Dubas, R. ]. : Descussion on treatment of tuberculous meningitis and survival of
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Hirsch, J. G., Dubas, R. ]. : The effect of spermin on tubercle bacilli, J. Exper. Med.,95, 191, 1952.
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Dubas, R. J. : Viability of tubercle bacilli in vivo with and without chemotherapy,
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Dubas, R. J., Hirsch., ]. G. : The antimycobacterial activity of a peptide preparation
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'28)

Pathonode, R. A. : Tissue fatty acids and their possible relationship to the naturalresistance of rabbits to infection with human type tubercle bacilli, Am. Rev. Tuberc.,
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:29)

Endo, M., Takayama, K., Taguchi, S. : The antimycobacterial activity of peptide
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46." Michiro NAKASHIMA
1956 and 11, 32, 1957. (in Japanese)

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Myrvik, Q. and Weiser, R.S.: A tuberculostatic serum substance possessing lysozymlike properties, Am. Rev. Tuberc., 64, 669, 1951-

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Myrvik, Q.: Studies on the tuberculoinhibitory properties of ascorbic acid derivatives
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32)

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33)

Part II.

Antituberculous factors in milk, Nature. 182-4639, 884. 1958.