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

Studies on fractionated purified materials
using ion exchange resins.
Michiro NAKASHIMAThe Fifth Division of Tuberculosis Research Institute. Kyoto University(Director : Prof. Shusuke TSUJI, M. D.)

INTRODUCTIONIn Part I, the presence of tuberculostatic substances in the crude materialsof rabbit's organ extracts was reported. The active substances are able to passthrough a cellophane membrane, heat stable and soluble in methyl alcohol but
insoluble in ether.

As it seems possible that the crude materials of rabbit'sorgan extracts may contain many other substances besides the active factors, it
may be desirable to remove the mixed inactive factors and to purify the active
factors. For this purpose, the procedures which have been reported by Oshima
or Fujita llZ )3) , that is, the fractionation procedures using ion exchange resins~
were used.
The investigations dealing with the fractionation procedures, the fractionatedmaterials, their antimycobacterial activities and chemical characteristics will bereported in this paper.
MATERIALS AND METHODSAs starting materials, dialyzed outer fluid of organ extracts from muscle,
liver, kidney and lung of normal or immunized rabbits were prepared as described in Part I.
were used.

In order to obtain enough material to fractionate, four rabbits

Technique of chemical fractionation of low molecular substances in rabbit'sorgan extract:
The adsorption technique was used, using ion exchange resins. As thetechnique was reported in a previous paper!> in detail, only a diagram is presented in Table 1 in this paper.
As shown in the table, the low molecular
substances of rabbit's organ extract were separated into four fractions as follows:

48

Michiro NAKASHIMA

Table 1. Procedures used in chemical fractionation of low moleculal substances inorgan extracts of rabbits.
Starting Material(Outer fluid of Dialyzed organ")

I

Amberlite lR-120
I

I

I

Absorbate 1

Fltrate 1

I Elution with NH 0H\

4

Amberlite lRA-400 (COa-Form)I

Amberlite lRA-400 (OH- Form)

I

I

I

Adsorbate 2

Fltrate 2

I Elution with Hel

Fraction II

Fraction I
acids)( AminoPolypeptides

Organic bases)( Anorganic kations

I

I

Adsorbate 3

Fltrate 3

I

Elution with N a2COg Fraction IV
Amberlite lR-120
I(Sugars)

I

Adsorbate 4I

Filtrate 4
Fraction III(Organic acids)\ Anorganic anions

Fraction I: adsorbed both on cation and antion exchange resins (aminoacids, peptides)
Fraction II: adsorbed on cation but not adsorbed on anion exchange resins(organic bases)
Fraction III: adsorbed on anion but not adsorbed on cation exchange resins(organic acids)
Fraction IV: adsorbed on neither cation nor anion exchange resins (sugars
etc.)
Each fraction was concentrated to dryness under reduced pressure and low
temperature Cat 10 mg Hg 50°C), was weighed, and dissolved in water of 1/8
the weight of the original organ. Thus, an eight-fold concentrated solution ofeach fraction was obtained.

Test for tuberculostatic activity:
By the same procedure as described in Part I, the tuberculostatic activity ofeach fraction was tested•.
As Fractions I and III were strongly acidic (pH undeJ; 1.2), they were tested
for their tuberculostatic activity after being neutralized with NaOH to pH 7.0.
As test bacteria, H37Rv strain, Bovine Rm strain and Avian Cho-kyo strain
were used.

Paper Chromatgraphy :Chemical analysis of each fraction was carried out by using the paper chromatography as follows:
Methods of developing
Ascending method (I~l the present investigations,Studies on Anti-tuberculous Low Molecular Factors in Various Organs of Rabbits

49

mainly one-dimension method was used) and sometimes circular chromatography
was used.
Solvents for developing; n-buthanol-acetic acid-water (4: 1 : 2) was mostly
used to delope each fraction. Besides, for Fraction III, ether-acetic acid-water(13: 3: 1), n-buthanol-methanol (1: 1) or n-buthanol-formic acid-water (10: 2: 15)
were used.
Detection;

Ninhydrin reaction (for Fraction I) Jaffe's reaction (for FractionII) ammoniac silver nitrate (for Fraction III and IV), fiuolescence by ultraviolet
ray or pH indicator (for Fraction III) were used.

RESULTS
1. The inhibitory effect of the fractionated materials of several organ extracts of rabbits on the growth of H37Rv strain.
i) Fractionated materials from normal rabbits
Table 2.

The results are shown in

Table 2. The effect of the fractionated materials of several organ extracts fromnormal rabbits on the growth of tubercle bacilli.
Concen tra tion
Organs, Fractions
Muscle

8x

4X

2x

Ix

1/2 x

1/4X

n

+++

+++

+

IV

+++

+++

+++
+
+++

+++
+++
++
+++

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

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

++
++

+++
+++
+

+++
+++
+++

+
+++
+++
+++

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

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

+++
++
+++

+++
+++
+++

+++
+++
+++

++
+++
+++
+++

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

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

I

HI

Liver

I

n

m
IV
KidneyI
II
HIIV---"--------,--

Lung

I
11
HI

----------

+++

+++
Control: + + +
IV

+
+++
+++

++
+++
+++
+++

- - - - - ._,-------------

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

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

+++
+++
+++
+++
-----

-

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

Muscle;Fractions I and III could inhibit the growth of H37Rv in a concen-

tration of "4x" (four times the concentration of the starting organs), but Fractions II and IV could not inhibit growth even at "8 x" concentration.
Liver; Fraction I could inhibit the growth of H37Rv at "1 x" concentration(the same concentration as the starting organs), but Fractions II and III permitted the growth of H37Rv.

It may be worthy of note that Fraction IV could in-50

Michiro NAKASHIMA

hibit the growth of H37Rv at
as high concentrations as "8X".

~

Kidney; The results were
the same as obtained in theliver but Fraction IV could
not inhibit the growth of
H37Rv.
Lung; Although no inhibitive activity with thecrude materials at "2 X" con-

00

0
,...,

C'1
,...,

"'1'

......

centration was noted in Part
I, Fraction I could inhibit thegrowth of H37Rv at "8x"concentration, and Fraction
III at" 4 X " • The Fractions
II and IV could not inhibit~S......

---S
b.O

00

en

......

c;·C.....Q)

0

CIl

C'1

S

the growth of H37Rv.

4-4

0

.....

The titer of the tuberculostatic activity of the muscle was lower in the fractionated materials than in
the crude materials,
thecrude materials could inhibit

C'1
C'1

I::l

;:l
0

S

<r:

"'1'
C'1

~

C'1

the growth of H37Rv a t as
low concentration as "1 X ",but the fractionated materi-

++++
++++
+ +
+ +
++ +
+ +
+ +
++ +
+ +
+ +
++1+
+ +
+ +
+1+
+ +
+ +
+1+
+ +
+
1+
+
+
1+
+
+
I
1+
+
+
I
1+
+
+
I
1+
+

00

C'1

1

I

~

I

I

C'1

I

I

"'1'

I

I

als could not inhibit growtheven a t a concentration of"2 X"..... On the other hand,in the liver and kidney,
Fraction I could inhibit thegrowth of H37Rv strain at
the same concentration as
the crude material, that is"Ix ".
The real activity in mg
per ml of the active substances was calculated from
Table 2 and the weight of

C"J

C"J
Q)

,j:l

.....

++
++++
+++
+
+ ++
+ +
+
+1
+
+
+1
+
+
+1
+
+
I ++
+
+
1 +1
+
+
1 +J
+
+
I
+1
+
+
I
+1
+
+
I +1
+
+
1 +1
+
+
I
+1
+
+
I
+1
+
+
I
+1
+

++++
++++
++++
++++
++++
++++
++++
++++
+++
++-1-+
++++
++
++++
++++
++
+ ++
+ ++
+
+ ++
+ ++
+

I

++
+

++++
+-1-++
'++
++++
++++
++
++
+ ++
+ ++
++
++
++
+
+
+
+
+
+
+
,
+
+
I

++

I
I

4-4

++

I

I

++

I

1

++

I

1

++

I

I

I

++

I

I

I

++

I

I

I

o

-----

<+-<

o

I::l

.8
.....
u

-1=I1!5i!I1=:;-1=I1Eil1=:;-1=I1!5i!I1=:;-1=I1!5i!I1=:;

~

I

~

~

I::l

CIl
b.O
~

0

>.

Q)

U

en

;:l

::E

~

Q)

:>-

'"d

Q)

:J

I

I::l

b.O

~

;:l

I::l

~

Studies on Anti-tuberculous Low Molecular Factors in Various Organs 0/ Rabbits

the fractionated materials, andthe results are shown in Table 3
and Fig. 1. Fraction I could inhibit the growth of H37Rv at 22

Cl

010.
'" .++
~

;.

'" ++
~

'lQ

mg/ml in the muscle, 16 mg/
ml in the liver, 32 mg/ml in thekidney, and 18 mg/ml in the lung.

;.
~

'"

eo.

30

20

10

"

Fraction III could inhibit at 1240 mg/ml

mg/ml in the muscle, 23 mg/ml

Fraction I

in the lung, and Fraction IV
could inhibit at 14 mg/ml in theliver.
ii)
Fractionated materials~ ,0.

'" +++
~

;.

'"

~
0
.;

;.

\

~

'<:='

\

••

iF +

51

\

Muscle"-Kidney\ .,Lungfrom immunized rabbits: The
results are shown in Table 4.

\

"-

\

"-

Liver (lV)',,"-

'"

"-

\

eo.

"

No distinct difference between
the results obtained from normal
rabbits and those from immuniz-

40 mg/ml

30

20

10

The amount of the fractiOl~ated materials in Kirchner's medium

Fraction III

ed ones are noted. Although thetiter of activity in Fraction I of

Fig. 1. The effect of the fractionated materialsof the organ extracts from normal rabbits on
the growth of H37Rv strain.

the liver and the lung are higher
by one column of concentration in the immune animal than that in the normal
Table 4. The effect of the fractionated materials of several organ extracts from theimmunized raddits on the growth of tubercle bacilli-------·-··---·---·~~-----1

Concentration
Organ Fraction

8x

4x

2x

Muscle

IX

1/2x

1/4x

++
+++
+++
+++

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

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

+++
+++
+++

++
+++
+++
+++

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

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

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

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

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

Liver

- - - - - - _ . _ - - - - _ . ~ - - . - -- - - - _.. - - - -----_.

1II

+++
+
+

+++
++
++

+++
+++
+++

I
II
I
IV

++

+++

++
+++
+++
+++

++
+++
+++
+++

I

II

IV
Lung

- -

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

Kidney

___

I

I

+++

0 _ _ _ _ _ _- - - - - -+++._----------_ _ _ _ _ _ •----_.--~

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

52

Michiro NAKASHIMA

rabbit, the difference is so small that it cannot be thought to be the result of
immunity.
2.

Inhibitory effect of the fractionated materials of the muscle and theliver of rabbits on the growth of Bovine type and Avian type tubercle bacilli.
Fraction, I, both of the muscle and the liver, could inhibit the growth bothof Bovine type (Bovine Rm) and Avian type (Avian Cho-kyo) of tubercle bacilli
as Human type (H37Rv) (Table 5).

Fraction III was more active in inhibiting

Table 5. The effect of the fractionated materials of muscle and liver of rabbits on
the growth of the bovine and avian type of tubercle bacilli.
~

1Concentration

Organ Fraction
Type of Bacilli

8x

4X

Ix

2x

1/2x

1/4x
._--

MuscleBovine TypeMuscleAvian TypeLiver
Bovine Type

I

HI
IV II

I

n

HI
IV

I

n

JR

IV

-

-

+

+++

+++

+++
+++

+

+

+++

-

I

n

-

+++
-

+++
-

-

-

+++
+++
+++

-

+++

+++
+++
++
+++

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

-

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

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

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

-

+
+++
+++
+++

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

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

+++
+++
+++

++
+

+++
+++

+++
+
+++

-

-

-

-

------

Liver
Avian Type

I
1I
JR

IV

+++
-

+++

+++
++
+++

+++
+++
+++

----_.------ - - - . _ - - - - - - - -

++
+++
+++
+++

++
+++
+++
+++

++
+++
+++
+++

Control : Bovine type + + +, Avian type + + +Slide culture in Kirchner's medium for ten days.

the growth of Bovine Rm than that of H37Rv, that is Fraction III of the muscle
could inhibit the growth of Bovine Rm even at a concentration of "1 x", while
it could hardly inhibit the growth of H37Rv at a concentration of "4x", andFraction III of the liver which could not inhibit the growth of H37Rv, could inhibit Bovine Rm at a concentration of "4x".

On the other hand, Fraction III

was less inhibitory to the growth of the Avian type than to that of the Bovine
type. It can only slightly inhibit the growth of Avian Cho-kyo at the high concentration of "8x".
Fraction IV of the liver could not inhibit the growth ofeither type of tubercle bacilli.
3. Effect of hydrolysis on the tuberculostatic activity of the active fractions.
As shown in Table 6, there were no distinct changes in tuberculostatic active fractions (Fraction I of the muscle, the liver and the kidney, Fraction III
of the muscle), after being hydrolysed by heating in boiling water with 6N Hel

Studies on Anti-tuberculous Low Molecular Factors in Various Organs oj Rabbits

53

Table 6. The effect of hydrolysis on the tuberculostatic activity of the active fractionsof muscle, liver and kidney of rabbits.
Hydrolysis was carries out by heating in boiling water with 6N HCl for 15 hours.

for 15 hours.

It may be assumed that the active fractions are very stable tohydrolysis.
4. Nature of the activity of the active fractions of tubercle bacilli.
To examine whether the tuberclo-inhibitory activity of the active fractions
was tuberculocidal or tuberc1ostatic, a series of test tubes, in which 2 ml ofsolution of the active fraction serially diluted with distilled water were contained,
were prepared.

Two slides on which the tubercle bacilli (H37Rv) were smearedby the benzine method were immersed in a test tube, and incubated at 3TC.
One of them was taken out after 24 hours and other after 48 hours. The slides
taken out from the incubator were transferred into 2 ml of ordinary Kirchner's
serum medium for cultivating for 10 days.

The results are shown in Table 7.

It can be noted that tubercle bacilli which were immersed in the solution of acTable 7. Nature of the activity of the active fractions (muscle and liver) on thegrowth of tubercle bacilli.
Organ Fraction
Immersed Period
MuscleI{2448 hours
hours

Concentration
8x

4x

2x

1x

1/2 x

1/4x

Control

++

+++
+++

+++
+++

+++
+++

+++
+++

++
++

+++
+++

+++
+++

+++
+++

+++
+++

----------------_._------- ------------

Liver

I {24
48

+
+

+++
+++

+++
+++

+++
+++

The bacilli (H37Rv strain) were immersed in the solution of active fractions for
24 or 48 hours, then the bacilli were tranferred into ordinary Kirchner's serum medium and caltivated for ten days. As the control, the physiologic saline solution
was used.

54

Michi1'O NAKASHIMA

tive fractions at high concentration for 24 or 48 hours were already incapable ofgrowth when transfrred to the test tube containing Kirchner's medium.
that the active fractions can act bactericidaly on the tubercle bacilli.

5.

It seems

Chemical investigations of each fraction.

The chemical characteristics of each fraction were examined. In order toconfirm the assumption (in Table 1.), some qualitative tests were carried out.
Results are shown in Table 8. From these results, it can be assumed that theTable 8.

The chemical characteristics of each fraction.
FractionI

ReactionI
Sulfosalicilic acid
Ninhydrin
Molisch's R.
Jaffe's R.
pH

]I

III

]V

I

i
I

*

I

-Hit

I

+
<1.2

>8.0

<1.2

7.0

I

_I

materials contain no protein and each fraction consists of substance which havebeen assumed to be from Table 1.
The cheical analysis of each fraction using paper chromatography was asfollows:
Fraction I:
The dried material was colored yellowish-brown like caramel and the aqueous
solution of this material was strongAscending method :ly acidic (under pH 1.2). Wheno 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Rf.
neutralized with NaOH, a flaky precipitation occurred. This precipi- Muscle
tation appeared only under neutral
conditions (pH between 5.0-8.0) andconcentrated conditions (above 4fold). The separation of the precipitate from the supernatant failed
for technical reasons. Identification
by paper chromatography was donewith mixed materials. The chromatographic patterns of Fraction I
are shown in Figs. 2 and 3.
Muscle: As shown in Fig. 2,
six or seven spots were detected in
Fraction I of muscle. It can be as-

Liver

n

Lung

o

Kidney"-_'._e_e___...
__-__ _-O

Developer: n-buthanol-acetic acid-water(4 : 1 : 2)

Indicator: ninhydrine
Fig. 2. The chromatogram of Fraction Iof muscle, liver, lung and kidney ofrabbits.

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

sumed that they are histidine,
arginine, glycine, threonine, alanine, methionine or valine andleucine in the order of increasing
Rf. It was worthy of note that
histidine appeared in the muscle,
while it could not be found in theother organs.
Liver: More spots (about 12)

Controlr- Lung- Liver- Kidneyoccurred than in muscle.
The
spots between Rf. 0.1 and Rf.
0.24, however, were not well separated. Therefore, it was impossible to determine exactly
what the spots were which were
not present in the muscle. But
it is probable that they are cystineand lysine (Rf. smaller than histidine) and phenylalanine (ortryptophan) located between me-- Muscle

Developer: n-buthanol-acetic acid-water (4: 1 : 2)
Control: amino acids(1) Cystine(10) Threonine(2) Lysine(11) Aldnine(3) Arginine(12) Proline(4) Histidine(13) Tyrosine(5) Asparatic acid(14) ~etbionine(6) Serine(15) Valine(7) Oxiproline(16) Tryptophane(8) Glutamic acid(17) Phenylalanine(9) Glycine(I8) Leucine
Fig. 3. Circular chromatogram of Fraction
I of muscle, liver, kidney and lung.

thionine and leucine.
Kidney: It was believed that
there were 11 or 12 spots whichwere similar to the spots in theliver.
Lung
Nine spots were detected.
Fraction II
This fraction consists of organic or inogranic bases. Fraction II of the muscle could beobtained in the form of white
crystaline powder. The Fraction
II of other organs could not be
obtained in powder but in theform of white or non-colored amorphous substances. The yield of
this fraction was the smallest
among the four fractions.
It can be assumed that, as

55

Ascending method:
Rf.

0

0.1

0.2

0.3

0.4

0.5 '0.6

0.7 0.8

0.9

1.0

!

G'»
tr!J
'-----------

Muscle~-------[

Liver

Kidney

L--

Lung

~

@_ _

-'--I
I

L-------.:-Creatinine

Creatine

---_®

~I

Indicator: Jaffe's reagent
Control: Creatinine (directly)
Creatine (after heating at 100°C
for 1 hours)
Developer: n-buthanol-acetic acid-water(4 : 1 : 2)
Fig. 4. The chromatogram of Fraction II of
muscle, liver, kiclney and lund of rabbits.

56

Michiro NAKASHIMA

shown in Fig. 4, although there may be some other substances, it is almost certain that in the muscle and the kidney, Fraction II contains creatinine and creatine as its main components, when tested by chromatography.
to be neither creatinine nor creatine in the liver or the lung.
Fraction III
This is the fraction consistingof organic acids. The detection of
the components of Fraction III was
not so easy as of Fraction 1. In
general, as indicators of acids, inAscending method:.Rf.

o

0.1 0.2 0.3

0

C>

0.4

0.5

0.6

0.7 0.8

0.9

1.0

Muscle1

ether-acetic acid-water (13 : 3: 1)o01

chromatography, ammoniac silver
nitrate, pH indicator, fluorescenceby ultraviolet ray, etc. are used.
Fig. 5 shows that the muscle Fraction III always showed two spoteagainst the ammoniac silver nitratesolution regardless of the sort of

There appeared

methanol-n-butnanol (1 : 1)

I

o

0

n-buthanoHormic acid-water (10 : 2 : 15)
Liver

°

_

1_ _

n-buthanol-formic acid-water (10: 2 : 15)

c===>

1

phenol-formic acid-water (75 : 1 : 25)

developer used and the spots coinLung

cided with the fluerescent spots
detected by ultraviolet ray.
In the lung, also, two spots weredetected but they were not the sameas in the muscle.
Fraction IV :This is the fraction containing"sugars". The monosaccarides canbe detected by ammoniac silver
nitrate solution in paper chromatography. As the developer, n-butanolacetic acid-water was used, and theresults obtained are shown in Fig.
6. There are three spots in themuscle, two spots in the kidney but
only one in the liver.
The spots
appearing near the Rf 0.2 arethought to be glucose.

o

1

0

---'

ether-acetic acid-water (13 : 3: 1)

Ieon-buthanol-formic acid-water 00 : 2 : 15)

Developer:
Indicator: ammoniac silver nitrate solution
or fluorescence of ultra violet ray
Fig. 5. The chromatogram of Fraction III of
muscle, liver, lung of rabbits.

Ascending method:
Rf.

0

0.1

0.2 0.3

0.4

0.5 0.6

0.7

0.8

0.9 1.0

MuscleLung

KidneyGlucose\Developer: n-buthanol-acetic acid-water(4 : 1 : 2)

DISCUSSION

Several investigators have re-

Indicator: ammoniac silver nitrate solution
Fig. 6. The chromatogram of Fraction IV ofmuscle, liver and kidney of rabbits.

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

57

ported on antituberculous factors in animal body fluid.
Dubos4)5) reported that there was an antituberculous substance which was
extracted from the kidneys of guinea pigs with ethanol.

It was soluble in ethanol

and insoluble in acetone. This substance inhibited the growth of virulent tubercle bacilli but was inactive against avirulent acid fast bacilli. It was identified as spermin or spermidin, a sort of organic base or amine. He also reported
that a sort of peptide which was obtained from calf thymus possessed a definite
antimycobacterial activity in vitro 6 ). The thymus peptide could also be extracted from calf spleen, sheep thymus, beef lymphnode and calf pancreas but notfrom calf lung and calf liver.
End0 71 also obtained a similar peptide to Dubos's thymus peptide by using
the same procedure in beef.
Myrvik~) investigated a tuberculostatic substance posessing lysozyme-like
properties in the serum of immune rabbits. He also assumed that ascorbic acidmight be the antituberculous factor in human urine 9 ). But this was denied by
Bj6rnesj6 later 101 .
Patnodel l l was able to obtain some antituberculous substances from rabbit's
lungs. As they were soluble in alcohol and acetone they were assumed to beprobably fatty acid.
Franc 12 ) investigated antituberculous factors in milk. The active substancecould pass through a cellophane membrane.

It was fractionated by using active

charcoal, cation exchange resin, or organic solvents.

He assumed that the active

fraction was a sort of fatty acid.
Bjornesj(13)-17) obtained an antituberculous factor from human urine. The
active substance was heat stable, dialysable by cellophne membrane, adsorbableon charcoal but not adsorbable on cation exchange resin Dowex 50, and only
very slightly soluble in organic solvents. From these facts, he assumed that the
tuberculostatic substances in human urine might be som.e organic acids otherthan ascorbic acid.
As described above, these investigators obtained various antituberculous factors in different ways from different organs.
In short, it is assumed that antimycobacterial factors in animal body fluid may be peptides, amines, (organic
bases) or organic acids (fatty acid or not). There, however, has been no systematic investigation in which antituberculous agents have been systematicallyexamined in all organs.
The idea of using ion exchange resins to purify the active factor fromthe crude materials of organ extracts, serum or urine, have previously beenreported by a few investigators2) 16) . But the systematic fractionation procedureby useng cation and anion exchange resins for isolating antituberculous agentsfrom animal body fluid was first tried by our associates 1). Using this fractiona-

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Michiro NAKASH/MA

tion procedure, the crude materials were separated into four fractions. In eachfraction, antituberculous activity was examined. And it was ascertained thatthe active factors inhuman urine or rabbit's serum were contained in Fractions
I and III (Oshima, Fujita). These active fractions in the serum or urine werevery similar to the fractions found in this present investigation dealing withorgan extracts, from the following points of view, heat stability, attitude against
hydrolysis, solubility in organic solvents, adsorbability in ion exchange resins.
From this point of simHarity, Fujita 3 ) considered that these tuberculostatic substances might be made first in tissue cells, and then transported by circulating
blood or serum and then to the urine.
There are, however, some differences between the antit.vberculous substancesin organ extracts and these in the serum or human urine.

The former are more-complicated than the latter. That is, while Fractions I and III of the muscle
and the lung are active, Fraction III of the liver and the kidney are not active.
It may be worthy of note that· Fraction' IV of the liver (sugars) is inhibiting tobacilli.
Fraction III, organic acids as antituberculous factors, may not be ascorbic
acid or fatty. acids because of the physicochemical property described above.
Oshima 19 ) obtained active organic acid as crystals from human urine, but
has not yet identified them. The active organic acids of organ extracts may be:similar to those in urine, but the identification has not been accomplished at
this stage.
Fraction II of all organs has no antituberculous activity. As one of thereasons, it may be pointed out that the absolute amount of active substances
contained is small.
Anyway, Fraction II cannot inhibit growth of tubercle
bacilli even at an eight-fold concentration of the original organ, so it appears
that there is no significant relationship between the mechanism of the resistanceof the animal body to tuberculous infection and Fraction II.
Fraction I, the fraction of peptides or amino acids, commoply has antituberculous activity not only in each organ but also in the serum and urine. Therefore, it seems that this fraction may play the most important role in the:antituberculous mechanism of low molecular substances in animal body fluid .
.As one of the antituberculous factors of peptides in organ extracts, Dubos' found:an antituberculous substance in the form of peptide from calf thymus, and called it "thymus peptide". However, in this present investigation, from the factthat tuberculostatic factors in Fraction, I could not easily be decreased byhydrolysis, it may be assumed that the active factors in this fraction are notpeptides or are peptides which are very resistant to hydrolysis. On the other
hand, since the tuberculostatic activity of each amino acid has been denied by<>ther investigators 13118 ), it is hard to assume that the tuberculostatic activity ofStudies on Anti-tuberculous Low Molecular Factors in Various Organs of Rabbits

Fraction I is related to its content of free amino acids.

59

The chemical structure

must be thoroughly investigated later.
Sometimes Fraction I of muscle or Fraction IV of the liver showed no tuber,culostatic activity even at an eight-fold concentration of the original organs.
On the other hand, the crude materials of these two organs have the strongest
activity of any of the organs. Therefore, Fraction III may be the most important substance in muscle, and Fraction I in the liver. Fraction I in the muscle
and Fraction IV in the liver may play no significant role in tuberculostatic activity.
We observed in Part I, that the difference in antituberculous activity ineach organ was related to its content of active substances. And in the present
investigation, we observed a lack of uniformity in the activity of fractions
according to organs as follows ;(1) While Fraction I ordinarily showed an apparent activity in each· organ, inthe muscle a few cases were noted in which no activity was detected in Fraction I. (2) Fraction III in the muscle and the lung always showed activity but
not in the liver and the kidney. (3) Fraction IV had no activity in the muscle,
the kidney and the lung, but sometimes the activity was noted in the liver.
This means that there is a qualitative complicacy in each organ fraction rather
than simply a quantitative difference formed in the crude materials. In conclusion, antituberculous agents of each organ extract lack uniformity not only
-quantitatively but also qualitatively. Thus, although it may be difficult todetermine clearly, this lack of uniformity may be concerned to some extent
organ disposition to tuberculous infection. And it cannot be negated that thepresence of such antituberculous substances in animal organs may play somerole in the mechanism of resistance of each organ to tuberculous infection.
Therefore, to discover the really active substance, further investigations must
be continued.
SUMMARY

1) Extracts from the muscle, the liver, the kidney and the lung of rabbits
were fractionated by using systematically the ion exchange resins (Am berlite
IR-120 and IRA-400) into four fractions, that is, Fraction I ; amino acid or
peptide, Fraction II ; organic base, Fraction III ; organic acid, Fraction IV ; sugar.
2) The fractions which have tuberculostatic activity, more or less, were as
follows; Fraction I in these four organs, Fraction III in muscle and lung, Fraction IV in liver.
3) The active fraction (muscle, liver) acted more inhibitingly on the growthof bovine type tubercle bacilli and less on the avian type, than on the human
type.

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Michiro NAKASHIMA

4) The tuberculo-inhibitory action of the active factors was thought to be·bactericidal rather than bacteriostatic.
5) The tuberculostatic activity of each active fraction was not increased byimmunization.

6) Chemical examinations of each fraction were carried out by using chromatography, but identification of each component could not be finished.

REFERENCES
1)

Oshima, S., Fujita, Y., Takeoka, A., Nakajima, M. and Tsuji, S. : Chemical analysisof the role of humoral factors in native and required resistance to tuberculosis, Am.
Rev. Tuberc., 78, 884, 1958.

2)

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

3)

Fujita, Y. : Low molecular antituberculous factor in the serum of various animal

Kyoto Uhiv., 7, 82, 1959 (In Japanese)
specieses, Rep. Tuberc. Res. Inst. Kyoto Univ., 7, 7, 1959 (In Japanese)
4)

Dubos, R. ]. : A tuberculostatic agent present in animal tissue, Am. Rev. Tuberc ...
63, 119, 1951.

5)

Hirsch, J. G., Dubos, R. J. : The effect of Spermin on tubercle bacilli, J. Exper. Merl ...
95, 191, 1951.

6)

Dubas, R. J., Hirsch, J. G. : The antimycobacterial activity of a peptide preparation
derived from calf thymus, J. Exper. Med., 99, 55, 1654.

7)

Endo, M., Takayama, K., Taguchi, S. : The antimycobacterial activity of peptide
preparation derived from bovis muscle, Studies Chemotherap. Inst. Med. Res., 10, 21,
1956 and 11, 32, 1957. (in Japanese)

8)

Myrvik, Q., Weiser, R. S. : A tuberculostatic serum substance possessing lysozyme-

like propaties, Am. Rev: Tuberck., 64, 669, 1951.
9)

Myrvik, Q. : Studies on the tuberculoinhibitory properties of ascorbic acid derivatives and their possible role in inhibition of tubercle bacilli by uirne, Am. Rev ..
Tuberc., 69, 406, 1954.

10)

Bj5rnesjo, K. B. : Tuberculostatic factors in normal human urine, Am. Rev. Tuberc ...
73, 697, 1956.

11)

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.,
69, 710, 1954.

12)

Franc, Z. : Antituberculous factors in milk. Nature, 128-4639, 884, 1958.

13)-15)

Bjornesjo,' K. B. : On the effect of human urine on tubercle bacilli, Acta Tuberc_

Scandinav., 25, 425, 447, 457, 1951.
16),17)

Bjornesjo, K. B. : Acta Tuberc. Scandinav., 27, 116, 123, 1952.

18)

Morita, M. : Tuberculostatic effect of human urine, Progr. Obst. GynaecoI., 6, 335..

19)

Oshima, S. : in printing

1954 (In Japanese)