CHEMOTHERAPY

17 6

ASSAY
BODY
PHITICMETHODFLUID

FORBIOLOGICALLYBY AGAR

MYCOBACTERIAMAYDIFFUSION

ACTIVEMETHODAS TESTISONIAZID
USING

1963

IN

SAPRO-ORGANISMYUTAKAKANAZAWA,M. D. * and TOSHIOKURAMATA,Ph. D. **Niigata Railway Hospital, Niigata, Japan(Received January 21, 1963)
Biologically active isoniazid (INH) levels in plasma
have been assayed by the tube dilution method' orby the vertical diffusion method2,8 using humantubercle bacillus as the test organism. The cylinder
plate method has been widely used as the standardprocedure for the assay in the antibiotic field, because of its simplicity and accuracy.
UMEZAWA et
a14 reported using this method with mycobacteria
607 as the test organism.
This only assays INH in
the ranges over 8 pgiml and was thus useless forclinical purposes.
In the present paper, the authors intend to reporttheir studies6'7'8'9 of the assay method for biologically
active INH in body fluids by the thin agar cylinder
plate method' using rapidly growing,
INH highly
sensitive acid-fast bacterium as, test organism.
Experimental
Material :Test organism : A saprophytic,
non-chromogenic
mycobacteria H-7 isolated from soil by the authors
was used.
Culture media : KIRCHNER's agar medium containing 10% serum or albumin and 1. 5% agar wasused for the assay plate.
The usual nutrient brothwith 0.05% Tween 80 or DUBOS's oleic-acid albumin
medium was used for the inoculum of the test organism.
Experimental
Method .
Preparation of samples : Normal plasma containing
INH in the following concentrations
8., 4., 2., 1., 0. 5,0. 25, O.125, 0. 0625, 0 pgiml were prepared as standards for testing of plasma taken after administration of INH.
Preparation of the inoculum of the test organism :Broth with Tween 80 or DUBOS's medium was inoculated with M. H-7 and incubated for approximately
24 hours at 37°C.
Preparation of the plates : KIRCHNER's agar medium melted and kept at 50°C was inoculated with 0. 2
% broth culture of M. H-7. Petri-dishes (inside diameter 90 mm) with plain bottoms were placed on alevel surface and 5 ml of the inoculated medium wasdistributed evenly and allowed to harden. If dishesof other sizes were used, the amount of the medium*Department

of Internal**Departmentof Pharmacy.

Medicineand Laboratory

used is altered to provide the thin layer of agar.
Assy procedure : Cylinders were placed on the inoculated agar plates and filled with standard samples
and test ones.
The plates were i ncubated at 37°C for approximately 48 hours.
Estimation of INH concentration : On semi-log pa
per, the diameter of the inhibition zone was plottedagainst the log of concentrationof INH and thestandard curve representing
the relation between
the diameter of inhibition zone and concentration ofINH was drawn.
Then the concentrationof testsamples was estimated by comparison with the standard curve.
Experimental
Results :The relation between the diameter of inhibitionzone and the logarithmic
concentration
was linear
in the range from O.125 to 8. 0 Agiml, as shown in
Table 1 and Fig. 1.
Consequently, the concentration
under test wascalculated from the formula :LU—Sh
og 0 — Sh —SL Log A
where 0 was the ratio of high and test dose, A wasthe ratio of high and low dose and Sh, Si and U
were the diameters of standard high, standard low
and test samples, respectively.
Table 1.

Relation betweenof INH and diameterzones in plasma

concentrationof inhibition

Concentration of INH Diameter of linhibition zone
in plasma (pg/ml)(mm) Mean ± Fiducial limit
858. 94 ± 1. 68

452. 66 ± 0. 33

244.31 ± 0.57

136. 97 ± 1. 20

0.5

30. 19±1.

0.25

23. 34 ± 1. 46

O. 125

14. 5±0.

0. 1

11.7

1073

0.0625
a=0.

01,

n=8

VOL.11NO.

CHEMOTHERAPY

3177

Table

3.

Concentration
eachFig1

calculated

from

plate

Plate

*gem'

**ggimi1

2.354

0. 252721.822

0. 231832.9080. 249942.0700. 2526

Te252.464

0.2338—c.

62.017

0. 2083

71.942

0. 22168

2.183

0. 2615

Mean

2.220

0.2390

fi
O

4.

atl

Experimental Error of the Assay Method :From the results obtainedby using 8 plates shownin Table 2, the couple of 4. and 1. pg/m1 or thecouple of 0.5 and 0. 125pg/m1 were consideredas
standard and 2. or 0. 25 pg/m1 were calculated as
test samples by means of the above mentioned formula, as shown in Table 3.
The mean (m) and reliable range wereM
N1=1
auF(0.01)
N2= 7/8
where, u was the sample standard deviationand n,
the number of plaiie‘ and F, the variance ratio.
And the probabli r1.aximumrange Colof standard
deviation was calculate . as flows
aU22 = F (0. =01)00
N2=7
Then the maximum range (Mr) of the standard
deviation of the mean obtained from the experiment
Table 2. Relation between
concentrationof INH in plasma and diameter ofinhibition zone
Concentration
Plate1842
1 1 0.5 0.25
pg/m1 itg/ml pg/m1 pg/m1 aug/m1
52.75

4537

31

24.5

1459

44.25

38

31

21.5

14.25

3

61

52.75

43.75

34.75

28.5

25.5

15.5

4

60

52.75

44.5

36

29. 5

22.75

15.25

52.5

44

37

30. 5

25

14.75

58.5

53

44

38.75

31

23

14.75

7

58

53

43.75

37.25

31.25

2213.5

8

59.5

52.5

45.25137 28. 75 22.5

Mean

58.94

52. 66 44.31

36.97

* *

**G.%

*

1

120-83

2

114-88133,-783112-90127-824110-92

123-84

As describedin Table146--6938idopicollyac/iJe. i5uniazidleI 1,1&rum.
1)1311-0!.10,7
aif
4'111<gafter oral CLIP

1'17
15 adults
50. 125526*ofusedlizg/m160

55.5

Numberplatesof INH

25*:Concetration calculated from diameter, 2 pg/
ml as the Test and 4, 1 pg/ml as the standard.
**Concentration calculated from diameter, 0. 25/./g/ml as the Test and 0. 5, 0. 125 pgiml as
the standard.
Table 4. Maximum range of the standard
deviation of the mean obtained fromthe experiments using 1-4 plates.

30. 1823.34

Ei0.5

1414. 5O.
Hours after Or

Aar7i,-;;,!trailon

hrsusing n plates were calculated from theformula .
a2
1±/TV-TU-17FTN-Mr
as shown in Table 4.
Biologically active INH in plasma .
After oral administration of INH, biologically active INH levels in plasmas weredetermined in 15 adults. As shown in Fig.
2, metabolic patterns of active INH wereascertained to differ individuallyas reported
previously by many workers.
Antimicrobial Activity of Metabolic
Substances of INH to the Test Organism :Antimicrobialactivity of metabolic substances of INH e. g. acetyl-INH, hydrazone
types of INHand isonicotinic acid, to the M.H-7,mm
were tested using the50
above mentioned agar

It seemedreasonable

INH as rapidly

bacillus

M.

100

INH
Pyruvic Acid Isonicotinyl Hydrazone
D-Glucose IsonicotinylHydrazone
cr-Ketoglutaric
Acid
Isonicotinyl Hydrazone
Isonicotinic Acid
1-Isonicotinyl-2-Acetylhydrazine

becauseactivity

bioassayforactivity

ofhighlyextentrapidlyin thegrow-sensitive

toincommonwith

appeared

tobemostslightly
Thee.g.

urineM.

paraamino-salicylic
the determination
combinedMoreover,therapy
a straintheconcentrationor sputum,even

H-7,resistantbeingwithpara-aminosalicylicof streptomycin
resistantin the laboratoryof active

INH after

is available

streptomycin

0.25

0.125

0.0625

36.25

30.0"

22.25'

14.5

35.25

28.25

21.0 14.00

20.5

14.0

000

35.5

27.5

20.75

12.0

000

SUMMARY

for theadministration.

agarcylinder‑Platemethod,inatwodayperiod,
usingthesaprQphyticmycobacteriaH‑7asthetest◎rgamsm。
(2)Thetest'organismwasmoresensitiveto
free‑INHthanthemetabolichydra20netypesofINH
a簸dresistanttoacety1‑INHandisonicQtinicacid.
(3)Theexperimentalerrorsinherentintheforin tissue

1)thoughtoacid, was eminently
usefulof active
INH in the period

obtained

0.5mm
42.5

meth◎dwerealsodiscussed.

for assaying
INH.
method is also applicablecontaminated.
test organism

10toproperties

or in body fluids

in Aqgenii(1)BiologicallyactiveINHwasassayedtothe

theH-7,of the drug

metainhibi-

0.1
Concentrat

somedetermination

ofofkinds

diameterConcentrationbacillus

thevarioustheCompounds

humansuitable
test organism
The cylinder-plate

betweenbolites of INH andtion zone

INH and having
tubercle

Relation

Nydrazone x

thefree-INH
mightdeteriorate
to somemedium with the lapse of time.
Theacid-fast

5.

1963

lowerlimitofO。125μ91mlinplasmabythethin

Discussion
to complete

as possible,

Table

INN
PjruvicAcid
Hydrozone
D-Gh.L.-ose
sr:nicofinyl
149dro,zonp
a-Keto)(ularic Acid

diffusion
method.
As40r,
shown in Table 5 and Fig.
3, pyruvic acid isonico:CZ 30
tiniyl hydrazone, ce-ketoglutaric acid isonicotinyl
hydrazone and D-glucose
s. 20—
isonicotinyl
hydrazone
were approximatelyand 1/5 in antimicrobial
ca10—
activityin comparison
with free-INH.
Isonicotinyl acid and acetyl-INH,
however, aia
nowever,
did notnot snow
show any remarkableremaricaoie
M. H-7M.
H-7 strain.
strain.

ing

MAY

CHEMOTHERAPY

178

forof2)acid.
M. H-7assay3)

BIBLIOGRAPHY
MANDEL, W., COHN, M. L., RESSELL, W. P.
and MIDDLEBROOK,G.: Effect of para-aminosalicylic acid on serum isoniazid levels in man.
Proc. Soc. Exper. Biol. & Med. 91 . 409, 1956.
OGAWA,M.: Vertical diffusion method for sensitivity test of tubercle bacillus and for assayof active INH level.
Japanese Clinical Tubercle, 16 : 417, 1957.
SCHMIEDEL, A.. Der Verticaldiffusiontest
alsMethode zur Resistenzbestimmengen
von Tuberkelbacterienand zur INH Spiegeltesungt.

VOL.

11NO.

3

CHEMO"rHERAPY

Zent. Tbk. 112 (1) : 8, 1958.
4) UMEZAWA,H., OIKAWA,K. and SUZUKI,M.:Cylinder plate assay and some studies of isoniazide with M. tuberculosis 607. Japanese Journal of Medical Science & Biology 6 (1) : 11,1953.
5) MIYAMURA,S. and KANAZAWA,Y.: Assay forpenicillin and streptomycin in body fluids
using thin agar cylinder plate method. J.
Antibiotics, Ser. B 13(5) : 295, 1950.
6) KANAZAWA,
Y. and KURAMATA,T.: Assay
method for biologically active INH in bodyfluids by agar diffusion method (1st report).
Chemotherapy 8(5) ; 451, 1960.
7) KANAZAWA,
Y. and KURAMATA,T.: Assay

179

method for biologically active INH in body
fluids by agar diffusion method using nonpathogenic acid-fast bacillus. Clinics of Respiratory Organs 16(11) : 681, 1961.
8) KANAZAWA,
Y. and KURAMATAT.: Assaymethod for biologically active INH in bodyfluids by agar diffusion method using nonpathogenic acid-fast bacillus.
Chemotherapy
10(3) : 198, 1962.
9) KANAZAWA,Y.: Assay method for biologically
active INH in body fluid by agar diffusion
method using non-pathogenic acid-fast bacillus.
(5 th report). Kekkaku (Tubercle) 37(9-10)531, 1962.