STUDIES ON THE THIAMINE DEFICIENCY

DUE TO BACTERIAL THIAMINASE I.

INVESTIGATIONS ON INTESTINAL CONTENTS.

DANJI MATSUKAWA, SHOHAI CHANG, HIROTO MISAWA

MATSUTARO FUJIMIYA, NAOYA KOBAYASHI,
YOSHIO HORIKAWA AND KEIKO TAKATO.
DepartmentofMedicine,
NiigataUniversity
Shoolof Medicine,
Asahi-machi,
Niigata.
(Received
April9,1954)

In 1947 when Shibata and Chang (1, 2) were studying the thiamine absorp
tion, a strange phenomenon was observed that 10mg of thiamine, clysterized
into the sigmoidal colon of a patient with habitual constipation combined with
beriberi,
soon became impossibleto detectagain. This led to the discovery
of a thiaminase in the feces of that patient, and this enzyme was proved to

belong to thiaminase I according to the classification of A. Fujita (3).

This thiaminase can easily be demonstrated in some subjects, and in those

subjects decomposition of thiamine takes place in the intestinlal canal, possibly

resulting in thiamine deficiency. This condition may tentatively be called

"Thiamihase Disease"
.

About three per cent of ordinary subjects were proved to have this disease,
with no sex difference (4). It occurred in infants, except those below one

year of age, as well as in old people. It was seen more frequently in patients
suffering from beriberi or gastrointestinal disturbances. Fifteen per cent of

127 patients with beriberi showed this disease.

Under the presumption that there will be some bacteria responsible for

producing thiaminase in the feces, efforts were made to isolate them. At last
it was succeeded to discover a new bacterium in 1949 (5), and it was designated
by the Research Committee on Vitamin B as "Bacillus thiaminolyticus

Matsukawa et Misawa (BMM)" (6). This bacterium can regularly be found

in the feces of the patients with thiaminase disease.

In the present paper, a series of investigations concerning this problem,

which were made with intestinal contents, will briefly be reported.

METHODS

(1) For testing thiaminase in the feces or in intestinal contents, 1g each

of the materials was ground; water was added to make the total volume of
10ml and the whole was mixed well, extracted and centrifuged. To 1.0ml

of the supernatant was added 1 γ of thiamine and the whole was kept at 37°
and pH 5.6 for 1 to 2 hours.

43
 44 MATSUKAWA, CHANG ET AL. 1954

The degree of decomposition of the added thiamine was calculated by

determining the amount of thiamine still present in the fluid by means of the
thiochrome method.
(2) Ammonia-like nitrogen in the feces was determined by van Slyke's
method for measuring ammonia by distillation in vacuum at low temperature.

The procedures were as follows: 10ml of the feces extract (1:10) was taken

in a Claisen flask. Ten per cent suspension of Ca(OH)2 was added and the

produced ammonia as well as volatile amines, such as methylamine, ethylamine,

dimethylannine, etc. were aspirated at 40° and fixed in 1ml of 0.1 N H2SO4,

and the excess acid was titrated with 0.1 N NaOH.

(3) Amino nitrogen in the feces was mneasured by the formnol titration
method of Sorensen combined with the titration method of Willstatter and
Waldschmidt-Leitz, modified by Matsukawa (7).

RESULTS

The Distribution of Thiaminase in the Intestines of the Patients with Thiami

nase Disease.

With the purpose to observe the distribution of thiaminase along the whole
length of the intestinum of the patient with thiaminase disease, the thiamine

decomposing activity of intestinal contents obtained from different parts of

the intestinal tract was estimated. This was made in two autoptic cases of the

patients who deceased by other illnesses, but were occasionally found to have
been suffering from thiaminase disease. The results are summarized in Table Ⅰ.

Table Ⅰ

The figures indicate percentages of decomposition of the added 1 γ of thiamine

by 1ml of the intestinal content extract (1:10) at pH 5.6 and 37° in 2 hours.

I. K. K., a patient of dementia

paralytica combined with asca

riasis, 33 years old, male.

Ⅱ. K. S., a patient of meningitis

tuberculosa combined with asca

riasis, 10 years old, female.

As can be understood from this table, thiaminase seems to be present in

the whole distal portion of the alimentary canal, starting from the middle or
lower part of the small intestine. The portions of this distal canal arrange
themselves in descending order of the richness of thiaminase as follows: In
case 1, rectum, lower part of ileum, cecum, transverse colon and middle ileum;
in case 2, rectum, cecum, transverse colon and lower ileum.
It will be noted that the intestinal contents of these two cases had a
peculiar, irritating and offensive odor.
 Vol.1 THIAMINE DEFICIENCY 45

The Distribution of Thiamine in the Intestines.

The test of thiamine with intestinal contents was made in case 1 of the

patients mentioned in Table I and also in another autoptic case of the subject,
certainly normal from the viewpoint of thiaminase. The results of the
former are shown in Table Ⅱ and those of the latter in Table Ⅲ.

Table Ⅱ.

K. K., 33 years old, male.

Table Ⅲ.

E. T., 47 years old, male.

In the upper part of the small intestine where no thiaminase could be found
in the patient, there was no marked difference between the two cases with
regard to the content of thiamine. In the distal regions of the intestines,

especially in the colon, the thiamine content was higher in the ordinary subject

(Table Ⅲ). In the colon, the difference between the two was striking espe
cially in the amount of free thiamine.
Observations on the Discharge of Thiamine Ingested.
Ten mg each of thiamine a day was given orally in succcssion for seven
days, and the discharge of thiamine in urine and feces was determined during
this period of time. This was made on five patients suffering from thiami
nase disease and on five norml subjects. The results obtained from these two

groups of subjects are separately shown in Tables Ⅳ and Ⅴ.

It is worthy of note that the discharge of thiamine in feces is strikingly

less in the patients, while its renal elimination does not differ considerably

betwecn the patientsand the ordinarysubjects. In the patients,

only 7.6per
centof the ingestedthiaminewas found in feces, while the ordinarysubjects
discharged71.5per cent of iton an average.
 46 MATSUKAWA, CHANG ET AL. 1954

Table Ⅳ
10mg of thiamine are given orally for 7 successive days.

Table Ⅴ
10mg of thiamine are given orally for 7 successive days.

Observations of Ammonia-like Nitrogen in Feces.

Determinations of the contents of ammonia-like nitrogen and amino-nitrogen

in the feces were performed on 13 patients suffering from thiaminase disease

and on 19 healthy subjects. A marked difference between these two groups

of subjects could be noticed as shown in Table Ⅵ, namely the contents of

these nitrogens are definitely higher in the patients.

Table Ⅵ.
 Vol. 1 THIAMINE DEFICIENCY 47

BMM Carriers.
There are subjects in whom thiaminase cannot be found in their feces by

the ordinary way above mentioned, yet its presence can be verified by the
following procedures: A small piece of feces is put in the ordinary broth

culture; after heating at 80° for 20 minutes, it is cultivated aerobically at 37°

for two to three days. Thiaminase is then found in its centrifuged super

natant.
Such subjects are apparently healthy and to be discriminated from the

thiaminasic patients. They may be regarded as carriers of BMM. Such

carriers seem to form about ten per cent of the ordinary people.
The contents of ammonia-like nitrogen in the feces of eleven carriers were

estimated and found to be definitely higher than those of healthy subjects,

namely 64.0±16.9mg per 100g in the former and 27.1±8.8mg per 100g

in the latter. Compared with the figures in Table Ⅵ, we can notice that the
content of ammonia-like nitrogen of the carriers is approximately equal to that

of the patients.

Ingestion Test of BMM.

Twenty-nine healthy men and women were employed as subjects for

experiments. A BMM mass of about 4mg was swallowed by them every

day for several days or longer in succession. Their feces were carefully tested

for thiaminase. In only two out of the 29 subjects, thiaminase could be detected
in their feces two to three days after the beginning of the administrations of

BMM, and it disappeared spontaneously in 8 to 12 days after the cessation of

administration. BMM seems therefore to be not capable of growing in the

intestines of the majority of subjects.

DISCUSSION

The above observations indicate that, in the patients suffering from thia

minase disease, thiamine is decomposed in the intestines and that there is a

good possibility of causing thiamine deficiency in the body. On the other

hand, it has been shown that the growth of BMM in the intestines much

depends upon the disposition of the subject. This disposition may be in close

connection with the characteristics of the alimentary juices and/or with the
kinds of prevailing intestinal flora.

In another series of experiments which are not reported in this paper (2),
it was found that thiaminase disease could temporarily be cured by oral admi

nistrations of homosulfanilamide (2) or of preparations of lactic acid bacteria,

consisting mainly of Streptococcus faecalis (8). But the therapeutic effect

i
s usually not permanent, so that the patients relapse into the former conditions
soon after the treatments have been discontinued.

Recently Hamada (9) has demonstrated that the disposition of the BMM
carrier is in connection with the rate of secretion of bile acid and that putre
factive bacteria are found far more abundant in the intestinal canal of the

BMM carriers than in healthy subjects.

 48 MATSUKAWA, CHANG ET AL. 1954

SUMMARY

In patients suffering from thiaminase disease, thiaminase was found in

the lower half of the alimentary canal, i.e. from the lower part of the small  

intestine to the rectum, and thiamine was present less in amount there. A

very small portionof the ingestedthiamine could be found in feces. The

contentof ammonia-likenitrogenin the feceswas much higherin the patients,
comparedwith thatin normalsubjects.Particularsubjects
onlycan be aff
licted
temporarily
with thiaminasediseaseby oraladministration
of Bacillus
thiaminolyticus.
ACKNOWLEDGEMENTS
The authorswish to thankProf.S.Katsura,chairmanof thisDepartment
of Medicinefor hisencouragement,Prof.A. Fujitaforhisadviceand criticism
in thiswork,and Prof.K. Akazakiand Prof.T. Itoforsupplyingexperimental
materials. The investigationswere aided by a grant from the Fund of the
Department of Education.

REFERENCES
1) Shibata,T., and Chang,S., Proc. VitaminB Res. Comm. 21,2 (1948).
2) Chang,S., Vitamins2,174 (1950).
3) Fujita,A., Vitamins7,1 (1954).
4) Fujimiya,M., Chang, S., and Matsukawa, D., Proc. VitaminB Res. Comm. 38, 22
(1950).
5) Matsukawa, D.,and Misawa, H., ibid.31,16 (1949).
6) Kuno, Y., Proc.Japan Acad. 27,362 (1951);ibid.28,235 (1952).
7) Matsukawa, D., SaikingakuZasshi520,342 (1939).
8) Matsukawa, D.,and Fujimiya,M., Proc. Vitamin B Res. Comm. 34, 18 (1949);
ibid.36,14 (1950).
9) Hamada, K., Vitamins 6,951 (1953);ibid. 7,65 (1954).