Method and apparatus for determination of the activity of cholesterol oxidase and method and apparatus for evaluation of the toxicity of chemical substances

A method for determination of activity of a cholesterol oxidase comprises the following steps. A monomolecule film comprising a sterol and a phospholipid is formed on a surface of a cholesterol oxidase solution. Subsequently, a surface pressure of the monomolecule film is measured in order to find a rate of increase in the surface pressure of the monomolecule film where a magnitude of the activity of the cholesterol oxidase is defined by the rate of increase in the surface pressure.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method and an apparatus for 
determination of the activity of cholesterol oxidase, which utilizes a 
gas-liquid surface mono-molecule film, and further relates to a method and 
an apparatus for evaluation of the toxicity of chemical substances, which 
also utilizes a gas-liquid surface monomolecular film. 
2. Description of the Related Art 
The conventional determination method for the activity of the cholesterol 
oxidase has been known as a biochemical analysis method for cholesterol 
oxidase to be used for causing quantitative reaction of cholesterol which 
is one of the clinical laboratory items. The following method is disclosed 
in Methods of Enzymatic Aualysis, vol. 1, Fundamentals, 3rd. ed., 1983. As 
shown in the following formula (1), cholesterol is oxidized in the 
presence of cholesterol oxidase to generate cholestenon and hydrogen 
peroxide. 
##EQU1## 
A first conventional method for determination of the activity of 
cholesterol oxidase is to subject hydrogen peroxide generated by the above 
oxidizing reaction to an UV measurement. 
A second conventional method for determination of the activity of 
cholesterol oxidase is as follows. Methanol is oxidized by both catalase 
and hydrogen peroxide generated by the above oxidizing reaction to 
generate formaldehyde which is then reacted with both acetylacetone and 
ammonia to thereby generate 3,5-diacetyl-1,4-dihydrolutidine which is 
subsequently subjected to a measurement of its absorbency in a wavelength 
in the range of 405-415 nm. 
A third conventional method for determination of the activity of 
cholesterol oxidase is as follows. Hydrogen peroxide generated by the 
above oxidizing reaction is reacted in the presence of peroxidase with 
phenol and 4-aminoantipyrine to generate a coloring matter which is then 
subjected to a measurement of its absorbency in a wavelength in the range 
of 600 nm. 
A fourth conventional method for determination of the activity of 
cholesterol oxidase is as follows. Hydrogen peroxide generated by the 
above oxidizing reaction is reacted in the presence of peroxidase with 
3-methyl-2-benzothiazolinonhydrazone and dimethylaniline to generate a 
coloring matter which is then subjected to a measurement of its absorbency 
in a wavelength in the range of 600 nm. 
A fifth conventional method for determination of the activity of 
cholesterol oxidase is as follows. Hydrogen peroxide generated by the 
above oxidizing reaction is reacted in the presence of peroxidase with 
2,2'-azino-di-3-ethylbenzthiazoline-sulfonic acid! ABTS to generate a 
cation radical which is then subjected to a measurement of its absorbency 
in a wavelengah in the range of 420-436 nm. 
A sixth conventional method for determination of the activity of 
cholesterol oxidase is as follows. Luminol is oxidized by hydrogen 
peroxide generated by the above oxidizing reaction in the presence of 
peroxidase to generate a chemical luminance energy to be measured. 
The above conventional determination methods have the following 
disadvantages. Since cholesterol is insoluble to water, surface active 
agent is used for dispersing cholesterol as substrate in water for 
determination of the activity of the cholesterol oxidase. The activity to 
be determined is an activity of the cholesterol oxidase to cholesterol in 
the presence of the surface active agent. Needless to say, the activity of 
the cholesterol oxidase to cholesterol in the presence of the surface 
active agent depends upon the surface active agent. The conventional 
method using the surface active agent, enzymes and coloring matters 
require complicated operations. 
A conventional method for evaluation of the chemical substance toxicity is 
carried out by using mammals except for human, animal cells and 
microorganisms in order to predict toxic influences-to-human of chemical 
substances since it is impossible to subject human to a direct test of 
toxicity influence of chemical substances to human. Actually, evaluations 
in the toxicity-to-human are carried out for medicine, food additive, 
industrial waste, biological products and the like. The above toxicity 
includes the general toxicity such as acute toxicity, short-period 
toxicity and long-period toxicity as well as specific toxicity such as 
variability, local stimulation, allergy, tumor igenicity, teratogenicity 
and reproducitvity, and additionally ensequestration such as breath, 
metabolism, accumulation, discharge, and yet additionally biological, 
pharmacological and cytological toxicity. 
In the Japanese patent publication No. 5-74358, the following cytological 
toxicity test is disclosed. Fluorescent substance and medication are 
introduced into cells which are on culturing so that a variation in 
fluorescence in cells is analyzed to determine indication for sensibility 
of cells to medication. 
If the conventional evaluation method for the toxicity of the chemical 
substances uses the animals, then it is required to breed the animals to 
be tested, for which reason this method is expensive, time-consuming and 
trouble-some. This method is not preferable from the zoophilic viewpoint. 
If the cells are used, it is required to culture the cells. This method is 
also time-consuming and trouble-some. For those reasons, the conventional 
toxicity evaluation method would be a complicated and time-consuming 
procedure. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the present invention to provide a novel 
method for determination of the activity of cholesterol oxidase to 
cholesterol without using any surface active agent. 
It is a further object of the present invention to provide a simple method 
for determination of the activity of cholesterol oxidase to cholesterol. 
It is a further-more object of the present invention to provide a method 
for determination of the activity of cholesterol oxidase to cholesterol, 
which is free from any time-consuming procedure. 
It is another object of the present invention to provide a novel method for 
evaluation of the toxicity of chemical substances without using any 
experimental animals and cells. 
It is yet another object of the present invention to provide a simple 
method for evaluation of the toxicity of chemical substances without using 
any experimental animals and cells. 
It is still another object of the present invention to provide a method for 
evaluation of the toxicity of chemical substances without using any 
experimental animals and cells, which is free from any time-consuming 
procedure. 
It is an additional object of the present invention to provide a novel 
apparatas for determination of the activity of cholesterol oxidase to 
cholesterol without using any surface active agent. 
It is a further additional object of the present invention to provide a 
simple apparatus for determination of the activity of cholesterol oxidase 
to cholesterol. 
It is a further more additional object of the present invention to provide 
a apparatus for detemination of the activity of cholesterol oxidase to 
cholesterol, which is free from any time-consuming procedure. 
It is another additional object of the present invention to provide a novel 
apparatus for evaluation of the toxicity of chemical substances without 
using any experimental animals and cells. 
It is yet another additional object of the present invention to provide a 
simple apparatus for evaluation of the toxicity of chemical substances 
without using any experimental animals and cells. 
It is still another additional object office present invention to provide 
an apparatus for evaluation of the toxicity of chemical substances without 
using any experimental animals and cells, which is free from any 
time-consuming procedure. 
The above and other objects, features and advantages of the present 
invention will be apparent from the following descriptions. 
The present invention provides a method for determination of activity of a 
cholesterol oxidasc comprising the following steps. A monomolecule film 
comprising a sterol and a phospholipid is formed on a surface of a 
cholesterol oxidase solution. Subsequently, a surface pressure of the 
monomolecule film is measured in order to find a rate of increase in the 
surface pressure of the monomolecule film where a magnitude of the 
activity of the cholesterol oxidase is defined by the rate of increase in 
the surface pressure. 
The present invention also provides a method for evaluation of toxicity of 
a chemical substance which has an ability to reduce activity of a 
cholesterol oxidase, comprising the following steps. A monomolecule film 
comprising a sterol and a phospholipid is formed on a surface of a 
cholesterol oxidase solution added with a chemical substance. 
Subsequently, a surface pressure of the monomoleculc film is measured in 
order to find a rate of increase in the surface pressure of the 
monomolecule film where a magnitude of the activity office cholesterol 
oxidase is defined by the rate of increase in the surface pressure. The 
rate of increase in the surface pressure measured is compared to a 
reference rate wherein the reference rate has previously been found by 
measuring a surface pressure of the monomolecule film formed on a surface 
of the cholesterol oxidase solution free of the chemical substance. 
The present invention further provides an apparatus for determination of 
activity of a cholesterol oxidase comprising the following elements. A 
trough is provided, which has at least a cell which contains a cholesterol 
oxidase solution. A dropping unit is provided over the trough for dropping 
a monomolecule film developing solution onto a surface of the cholesterol 
oxidase solution in order to form a monomolecule film comprising a sterol 
and a phospholipid on the surface of the cholesterol oxidase solution. A 
measuring unit is provided over the trough and spaced apart from the 
dropping unit for measuring a surface pressure of the monomolecule film. A 
computing unit being electrically coupled to at least the measuring unit 
for receiving information of the surface pressure measured from the 
measuring unit in order to compute a rate of increase in the surface 
pressure of the monomolecule film and then compute a magnitude of the 
activity of the cholesterol oxidase on the basis of the rate computed. 
The present invention also provides an apparatus for evaluation of toxicity 
of a chemical substance which has an ability to reduce activity of a 
cholesterol oxidase, comprising the following elements. A trough is 
provided, which has at least a cell which contains a cholesterol oxidase 
solution. A dropping unit is provided over the trough for dropping a 
monomolecule film developing solution onto a surface of the cholesterol 
oxidase solution in order to form a monomolecule film comprising a sterol 
and a phospholipid on the surface of the cholesterol oxidase solution. A 
measuring unit is provided over the trough and spaced apart from the 
dropping unit for measuring a surface pressure of the monomolecule film. A 
computing unit is electrically coupled to at least the measuring unit for 
receiving infomation of the surface pressure measured from the measuring 
unit in order to compute a rate of increase in the surface pressure of the 
monomolecule film and then compares the rate measured to a reference rate 
already stored in the computing unit, wherein the reference rate has 
previously been found by measuring a surface pressure of the monomolecule 
film formed on a surface of the cholesterol oxidase solution free of the 
chemical substance.

DESCRIPTION OF THE PREFERRED EMOBIDIMENT 
The present invention provides a method for determination of activity of a 
cholesterol oxidase comprising the following steps. A monomolecule film 
comprising a sterol and a phospholipid is formed on a surface of a 
cholesterol oxidase solution. Subsequently, a surface pressure of the 
monomolecule film is measured in order to find a rate of increase in the 
surface pressure of the monomolecule film where a magnitude of the 
activity of the cholesterol oxidase is defined by the rate of increase in 
the surface pressure. 
The surface pressure of the monomolecular film may be measured by measuring 
a surface tension of the monomolecule film. 
Also, the monomolecule film maybe formed by dropping a monomolecule film 
developing solution onto the surface of the cholesterol oxidase solution. 
Additionally cholesterol oxidase solution may be a buffer solution 
including N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as the cholesterol 
oxidase is dissolved in the buffer solution, and that the monomolecule 
film development solution includes 70 mol % of 
L-.alpha.-dimyristylphosphatizircoline and 30 mol % of cholesterol. 
The cholesterol oxidas solution may be a buffer solution including 
N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the ion 
strenght is 0.1 and pH value is 7.45 as well as the cholesterol oxidase is 
dissolved in the buffer solution, and wherein the monomolecule film 
development solution includes 70 mol % of 
L-.alpha.-dipalmytylphosphatizircoline and 30 mol % of dihydrocholesterol. 
The cholesterol oxidase solution, may be a buffer solution including 
N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the ion 
strength is 0.1 and pH value is 7.45 as well as the cholesterol oxidase is 
dissolved in the buffer solution, and wherein the monomolecule film 
development solution includes 70 mol % of 
L-.alpha.-di-palmytylphosphatizir-ethanol amine and 30 mol % of 
pregnenolone. 
The present invention also provides a method of evaluation of toxicity of a 
chemical substance which has an ability to reduce acitvity of a 
cholesterol oxidase, comprising the follwoing steps. A monomolcule film 
comprising a sterol and a phospholipid is formed on a surface of a 
cholesterol oxidase solution added with a chemical substance. 
Subsequently, a surface pressure of a monomolecule film is measured in 
order to find a rate of increase in the surface pressure of the 
monomolecule film where a magnitude of the activity of the cholesterol 
oxidase is defined by the rate of increase in the surface pressure. The 
rate of increase in the surface pressure measured is compared to a 
reference rate wherein the reference rate has previously been found by 
measuring a surface pressure of the monomolecule film formed on a surface 
of the cholesterol oxidase solution free of the chemical substance. 
The surface pressure of the monomolecular film may be measured by measuring 
a surface tension of the monomolecule film. 
The monomolecule film may be formed by dropping a monomolecule film 
developing solution onto the surface of the cholesterol oxidase solution. 
The cholesterol oxides solution may be a buffer solution including 
N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the ion 
strength is 0.1 and pH value is 7.45 as well as the cholesterol oxidase is 
dissolved in the buffer solution, and wherein the monomolecule film 
development solution includes 70 mol % of 
L-.alpha.-dimyristylphosphatizircolinc and 30 mo1% of cholesterol. 
The cholesterol oxides solution may be a buffer solution including 
N-2-hydroxyethylpiperazine-N'-2-ethanc sulfuric acid, where the ion 
strength is 0.1 and pH value is 7.45 as well as the cholesterol oxidase is 
dissolved in the buffer solution, and wherein the monomolecule film 
development solution includes 70 mol % of 
L-.alpha.-dipalmytylphosphatizircoline and 30 mol % of dihydrocholesterol. 
The cholesterol oxides solution may be a buffer solution including 
N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the ion 
strength is 0.1 and pH value is 7.45 as well as the cholesterol oxidase is 
dissolved in the buffer solution, and wherein the monomolecule film 
development solution includes 70 mol % of 
L-.alpha.-di-palmytylphosphatizir-ethanol amine and 30 mol % of 
pregnenolone. 
The present invention further provides an apparatus for determination of 
activity of a cholesterol oxidase comprising the following elements. A 
trough is provided, which has at least a ccll which contains a cholesterol 
oxidase solution. A dropping unit is provided over the trough for dropping 
a monomolecule film developing solution onto a surface of the cholesterol 
oxidase solution in order to form a monomolecule film comprising a sterol 
and a phospholipid on the surface of the cholesterol oxidase solution. A 
measuring unit is provided over the trough and spaced apart from the 
dropping unit for measuring a surface pressure of the monomolecule film. A 
computing unit being electrically coupled to at least the measuring unit 
for receiving information of the surface pressure measured from the 
measuring unit in order to compute a rate of increase in the surface 
pressure of the monomolecule film and then compute a magnitude of the 
activity of the cholesterol oxidase on the basis of the rate computed. 
It is available to further provide at least a movable partition plate over 
the trough for partition of the cholesterol oxidase solution into a 
primary part and a subordinate part, wherein the surface pressure of the 
monomolecule film formed on the primary part is measured, and also provide 
a partition plate controller coupled to the movable partition plate for 
control movements of rise movable partition plate so as to set a surface 
area of the primary part at a predetermined value. In this case, the 
computing unit may compute a position of the movable partition plate on 
the basis of the predetermined value of the surface area of the primary 
part, and the partition plate controller is electrically coupled to the 
computing unit for receiving information of the position computed from the 
computing unit. 
The measuring unit may comprise a surface tensiometer for measuring a 
surface tension of the monomolecule film, and wherein the computing unit 
computes the surface pressure on the basis of the surface tension measured 
by the surface tensiometer. 
There may be further provided a movable stage provided under the trough for 
mechanically supporting and moving the trough in a horizontal plane, and 
also provide a stage controller coupled to both the stage for control 
movement of the stage. The stage controller is also electrically coupled 
to the computing unit for receiving information of the movement of the 
stage. In this case, it is available that the trough has a rectangular 
shape and the movable stage moves the trough on the basis of X-Y 
coordinates. Alternatively, the trough may have a circular shape and has a 
plurality of cells which are circumferentially aligned and different 
monomolecule films are formed, and the movable stage rotates the trough. 
The present invention also provides an apparatus for evaluation on toxicity 
of a chemical substance which has an ability to reduce activity of a 
cholesterol oxidase, comprising the following elements. A trough is 
provided, which has at least a cell which contains a cholesterol oxidase 
solution. A dropping unit is provided over the trough for dropping a 
monomolecule film developing solution onto a surface of the cholesterol 
oxidase solution in order to form a monomolecule film comprising a sterol 
and a phospholipid on the surface of the cholesterol oxidase solution. A 
measuring unit is provided over the trough and spaced apart from the 
dropping unit for measuring a surface pressure of the monomolecule film. A 
computing unit is electrically coupled to at least the measuring unit for 
receiving information of the surface pressure measured from the measuring 
unit in order to compute a rate of increase in the surface pressure of the 
monomolecule film and then compares the rate measured to a reference rate 
already stored in the computing unit, wherein the reference rate has 
previously been found by measuring a surface pressure of the monomolecule 
film formed on a surface of the cholesterol oxidase solution free of the 
chemical substance. 
There may be further provided at least a movable partition plate over the 
trough for partition the cholesterol oxidase solution into a primary part 
and a subordinate part, wherein the surface pressure of the monomolecule 
film formed on the primary part is measured, and also provide a partition 
plate controller coupled to the movable partition plate for control 
movements of the movable partition plate so as to set a surface area of 
the primary part at a predetermined value. In this case, the computing 
unit may compute a position of the movable partition plate on the basis of 
the predetermined value of the surface area of the primary part, and the 
partition plate controller is electrically coupled to the computing unit 
for receiving information of the position computed from the computing 
unit. 
The measuring unit may comprise a surface tensiometer for measuring a 
surface tension of the monomolecule film, and wherein the computing unit 
computes the surface pressure on the basis of the surface tension measured 
by the surface tensiometer. 
There may be further provided a movable stage provided under the trough for 
mechanically supporting and moving the trough in a horizontal plane, and 
also a stage controller coupled to both the stage for control movement of 
the stage. The stage controller is also electrically coupled to the 
computing unit for receiving information of the movement of the stage. In 
this case, it is available that the trough has a rectangular shape and the 
movable stage moves the trough on the basis of X-Y coordinates. 
Alternatively, the trough may have a circular shape and a plurality of 
cells which are circumferentially aligned and different monomolecule films 
are formed, and the movable stage rotates the trough. 
EXAMPLES 
According to the present invention, a monomolecule film development 
solution 13 is dropped on a surface of a cholesterol oxidase solution 12 
to form a monomolecule film on the surface of the cholesterol oxidase 
solution 12. A surface tension of the monomolecule film is measured by a 
surface-tensiometer 14. The measured variations in surface tensions of the 
monomolecule films versus time are shown in FIG. 2. A curve 21 represents 
a variation in a surface tension of a monomolecule film formed on a 
surface of a cholesterol oxidase solution 12 of 5 mU/ml. A curve 22 
represents a variation in a surface tension of a monomolecule film 
prepared on a surface of a cholesterol oxidase solution 12 of 1 mU/ml. A 
curve 23 represents a variation in a surface tension of a monomolecule 
film prepared on a surface of a cholesterol oxidase solution of 1 mU/ml in 
which 10 micro-Mol of lead acetate is dissolved. The surface pressure of 
the monomolecule film is gradually increased after the monomolecule film 
is prepared, wherein the increasing rate depends upon the cholesterol 
oxidase activity and the presence of toxicity substance. 
(Example 1) 
A first present invention directed to a method for determination in the 
activity of the cholesterol oxidase will be described with reference to a 
first example as follows. 
As the cholesterol oxidase solution 12, there was used a buffer solution 
including N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as 1 mU/ml and 5 mU/ml of 
the cholesterol oxidase are dissolved in the buffer solution. As the 
monomolecule film development solution 13, there was used a monomolecule 
film development solution comprising 70 mol % of 
L-.alpha.-dimyristylphosphatizircoline and 30 mol % of cholesterol. 
10 micro-1 of the monomolecule film development solution 13 was dropped on 
to a surface of 45 cm.sup.2 of the cholesterol oxidase solution 12 of 5 
mU/ml to form a monomolecule film on the surface of the cholesterol 
oxidase solution 12. A surface pressure of the monomolecule film was 
measured by a surface tensiometer 14 to obtain the curve 21 shown in FIG. 
2. The surface pressure of the monomolecule film was gradually increased 
after the monomolecule film was prepared. As shown by the curves 21 and 
22, the increasing rate depends upon the cholesterol oxidase activity. 
TABLE 1 
______________________________________ 
Cholesterol Oxidase 
Lead Acetate 
Linear Differential 
Curves 
(mU/ml) (.mu. Mol) 
Coefficient (mN/mmin.) 
______________________________________ 
21 5 0 25.5 .times. 10.sup.-2 
22 1 0 5.5 .times. 10.sup.-2 
23 1 10 4.2 .times. 10.sup.-2 
______________________________________ 
where the linear differential coefficient represents the rate of increase 
in the surface pressure of the monomolecule film. As described above, the 
activity of the cholesterol oxidase depends upon the rate of increase in 
the surface pressure of the monomolecule film. This means that the rate of 
increase in the surface pressure of the monomolecule film represents the 
activity of the cholesterol oxidase. Accordingly, the ratio of the linear 
differential coefficient represents of the curves 21 and 22 represents the 
ratio the activities of the cholesterol oxidase. At five minutes after the 
monomolecule film was prepared, the curve 21 has a linear differential 
coefficient which is larger five times than a linear differential 
coefficient of the curve 22. This means that the cholesterol oxidase of 
the curve 21 has an activity which is five times larger than an activity 
of the curve 22 since the linear differential coefficient represents the 
rate in the increase of the surface pressure, and wherein the rate in the 
increase of the surface pressure represents the activity of the 
cholesterol oxidase as described above. 
For the above reason, according to the present invention, the monomolecule 
film development solution 13 is dropped onto a surface of an unknown 
cholesterol oxidase solution 12 in order to form a monomolecule film on 
the surface of the unknown cholesterol oxidase solution 12 so that the 
surface pressure of the monomolecule film is measured to determine the 
activity of an unknown cholesterol oxidase included in the unknown 
cholesterol oxidase solution 12. In order to improve accuracy in the 
determination of the activity of the cholesterol oxidase, it is effective 
to previously measure the surface pressure of the already-known 
cholesterol oxidase activity and then prepare analytical curves on the 
basis of the measured surface pressure. 
A second present invention directed to a method for evaluation on the 
toxicity of chemical substances will be described with reference to the 
following second to sixth examples. Any chemical substrance to be 
evaluated on the toxicity is previously dissolved or dispersed in the 
cholesterol oxidase solution 12. A monomolecule film is formed on a 
surface of the cholesterol oxidase solution 12 by use of a monomolecule 
film development solution including any phospholipid and any sterol. The 
surface pressure of the monomolecule film is measured to find a rate in 
the increase of the surface pressure of the monomolecule film. If the 
toxicitic chemical substance is dissolved in the cholesterol oxidase 
solution 12, then the toxicity thereof reduces the activity of the 
cholesterol oxidase dissolved in the cholesterol oxidase solution 12. The 
reduction in the activity of the cholesterol oxidase results in a 
reduction in the increasing rate of the surface pressure of the 
monomolecule film. If the measured increasing rate of the surface pressure 
of the monomolecule film is smaller than that in case of the toxicity free 
solution, this demonstrates that the activity of the cholesterol oxidase 
dissolved in the cholesterol oxidase solution is reduced by the toxicity 
of the toxicitic chemical substance dissolved in the cholesterol oxidase 
solution. This means that the toxicitic chemical substance would be 
present in the cholesterol oxidase solution. 
(Example 2) 
As a cholesterol oxidase solution 12, there was used a buffer solution 
including N-2-hydroxyethylpiperzane-N'-2-ethane sulfuric acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as 1 mU/ml of the 
cholesterol oxidase is dissolved in the buffer solution. In the buffer 
solution, 10 micro-M of lead acetate as a toxicitic chemical substance was 
dissolved. As a monomolecule film development solution 13, there was used 
a monomolecule film development solution comprising 70 mol % of 
L-.alpha.-dimyristylphosphatizircoline and 30 mol % of cholesterol. 
10 micro-1 of the monomolecule film development solution 13 was dropped 
onto a surface of 45 cm.sup.2 of the cholesterol oxidase solution 12 of 1 
mU/ml, into which 10 micro-M of lead acetate as a toxicitic chemical 
substance was dissolved, in order to form a monomolecule film on the 
surface of the cholesterol oxidase solution 12. A surface pressure of the 
monomolecule film was measured by a surface tensiometer 14 to obtain a 
curve 23 shown in FIG. 2 and on Table 1. The surface pressure of the 
monomolecule film was gradually increased after the monomolecule film was 
prepared. The increasing rate of the surface pressure of the monomolecule 
film depends upon file cholesterol oxidase activity. As described above, 
the activity of the cholesterol oxidase depends upon the rate of increase 
in the surface pressure of the monomolecule film. This means that the rate 
of increase in the surface pressure of the monomolecule film represents 
the activity of the cholesterol oxidase. At five minutes after the 
monomolecule film was prepared, the curve 23 has a linear differential 
coefficient which is smaller than the linear differential coefficient of 
the curve 22. This means that the cholesterol oxidase of the curve 23 has 
an activity which is smaller than the activity of the curve 22. This 
proves that the lead acetate acts as a toxicitic chemical substrance which 
reduces the activity of the cholesterol oxidase. 
For the above reason, the monomolecule film development solution 13 is 
dropped onto a surface of a cholesterol oxidase solution 12, into which 
any toxicity chemical substance is dissolved, in order to form a 
monomolecule film on the surface of the cholesterol oxidase solution 12. 
The surface pressure of the monomolecule film is measured to confirm the 
presence of any toxicity chemical substance dissolved in the cholesterol 
oxidase solution 12. If the toxicitic chemical substance is dissolved in 
the cholesterol oxidase solution 12, then the toxicity thereof reduces the 
activity of the cholesterol oxidase dissolved in the cholesterol oxidase 
solution 12. The reduction in the activity of the cholesterol oxidase 
results in a reduction in the increasing rate of the surface pressure of 
the monomolecule film. If the measured increasing rate of the surface 
pressure of the monomolecule film is smaller than that in case of the 
toxicity free solution, this demonstrates that the activity of the 
cholesterol oxidase dissolved in the cholesterol oxidase solution is 
reduced by the toxicity of the toxicitic chemical substance dissolved in 
the cholesterol oxidase solution. This means that the toxictic chemical 
substance would be present in the cholesterol oxidase solution. 
(Example 3) 
As a cholesterol oxidase solution 12, there was used a buffer solution 
including N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as 1 mU/ml of the 
cholesterol oxidase is dissolved in the buffer solution. In the buffer 
solution, 10 micro-M of zinc chloride as a toxicitic chemical substance 
was dissolved. As a monomolecule film development solution 13, there was 
used a monomolecule film development solution comprising 70 mol % of 
L-.alpha.-dimyristylphosphatizircoline and 30 mol % of cholesterol. 
10 micro-1 of the monomolecule film development solution 13 was dropped 
onto a surface of 45cm.sup.2 of the cholesterol oxidase solution 12 of 1 
mU/ml, into which 10 micro-M of zinc chloride as a toxicitic chemical 
substance was dissolved, in order to form a monomolecule film on the 
surface of the cholesterol oxidase solution 12. A surface pressure of the 
monomolecule film was measured by a surface tensiometer 14 to obtain a 
curve 23 shown in FIG. 2 and on Table 1. The surface pressure of the 
monomolecule film was gradually increased after the monomolecule film was 
prepared. The increasing rate of the surface pressure of the monomolecule 
film depends upon the cholesterol oxidase activity. As described above, 
the activity of the cholesterol oxidase depends upon the rate of increase 
in the surface pressure of the monomolecule film. This means that the rate 
of increase in the surface pressure of the monomolecule film represents 
the activity of the cholesterol oxidase. At five minutes after the 
monomolecule film was prepared, the curve 23 has a linear differential 
coefficient which is smaller than the linear differential coefficient of 
the curve 22. This means that the cholesterol oxidase of the curve 23 has 
an activity which is smaller than the activity of the curve 22. This 
proves that the zinc chloride acts as a toxicitic chemical substance which 
reduces the activity of the cholesterol oxidase. 
For the above reason, the monomolecule film development solution 13 is 
dropped onto a surface of a cholesterol oxidase solution 12, into which 
any toxicity chemical substance is dissolved, in order to form a 
monomolecule film on the surface of the cholesterol oxidase solution 12. 
The surface pressure of the mononmolecule film is measured to confirm the 
presence of any toxicity chemical substance dissolved in the cholesterol 
oxidase solution 12. If the toxicitic chemical substance is dissolved in 
the cholesterol oxidase solution 12, then the toxicity thereof reduces the 
activity of the cholesterol oxidase dissolved in the cholesterol oxidase 
solution 12. The reduction in the activity of the cholesterol oxidase 
results in a reduction in the increasing rate of the surface pressure of 
the monomolecule film. If the measured increasing rate of the surface 
pressure of the monomolecule film is smaller than that in case of the 
toxicity free solution, this demonstrates that the activity of the 
cholesterol oxidase dissolved in the cholesterol oxidase solution is 
reduced by the toxicity of the toxicitic chemical substance dissolved in 
the cholesterol oxidase solution. This means that the toxicitic chemical 
substance would be present in the cholesterol oxidase solution. 
(Example 4) 
As a cholesterol oxidase solution 12, there was used a buffer solution 
including N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as 1 mU/ml of the 
cholesterol oxidase is dissolved in the buffer solution. In the buffer 
solution, 10 micro-M of calcium chloride as a toxicitic chemical substance 
was dissolved. As a monomolecule film development solution 13, there was 
used a monomolecule film development solution comprising 70 mol % of 
L-.alpha.-dimyristylphosphatizircoline and 30 mol % of cholesterol. 
10 micro-1 of the monomolecule film development solution 13 was dropped 
onto a surface of 45 cm.sup.2 of the cholesterol oxidase solution 12 of 1 
mU/ml, into which 10 micro-M of calcium chloride as a toxicitic chemical 
substance was dissolved, in order to form a monomolecule film on the 
surface of the cholesterol oxidase solution 12. A surface pressure of the 
monomolecule film was measured by a surface tensiometer 14 to obtain the 
same curve as the curve 22 shown in FIG. 2 and on Table 1. This means that 
calcium chloride has no toxicity. 
(Example 5) 
As a cholesterol oxidase solution 12, there was used a buffer solution 
including N-2-hydroxyethylpiperazine-N'-2-ethane sulfufic acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as 1 mU/ml of the 
cholesterol oxidase is dissolved in the buffer solution. In the buffer 
solution, 10 micro-M of mercury chloride as a toxicitic chemical substance 
was dissolved. As a monomolecule film development solution 13, there was 
used a monomolecule film development solution comprising 70 mol % of 
L-.alpha.-dipalmytylphosphatizircoline and 30 mol % of dihydrocholesterol. 
10 micro-1 of the monomolecule film development solution 13 was dropped 
onto a surface of 45 cm.sup.2 of the cholesterol oxidase solution 12 of 1 
mU/ml, into which 10 micro-M of mercury chloride as a toxicitic chemical 
substance was dissolved, in order to form a monomolecule film on the 
surface of the cholesterol oxidase solution 12. A surface pressure of the 
monomoleoule film was measured by a surface tensiometer 14 to obtain the 
same curve as the curve 23 shown in FIG. 2 and on Table 1. The surface 
pressure of the monomolecule film was gradually increased after the 
monomolecule film was prepared. The increasing rate of the surface 
pressure of the monomolecule film depends upon the cholesterol oxidase 
activity. As described above, the activity of the cholesterol oxidase 
depends upon the rate of increase in the surface pressure of the 
monomolecule film. This means that the rate of increase in the surface 
pressure of the monomolecule film represents the activity of the 
cholesterol oxidase. At five minutes after the monomolecule film was 
prepared, the curve 23 has a linear differential coefficient which is 
smaller than the linear differential coefficient of the curve 22. This 
means that the cholesterol oxidase of the curve 23 has an activity which 
is smaller than the activity of the curve 22. This proves that the mercury 
chloride acts as a toxicitic chemical substance which reduces the activity 
of the cholesterol oxidase. 
For the above reason, the monomolecule film development solution 13 is 
dropped onto a surface of a cholesterol oxidase solution 12, into which 
any toxicity chemical substance is dissolved, in order to form a 
monomolecule film on the surface of the cholesterol oxidase solution 12. 
The surface pressure of the monomolecule film is measured to confirm the 
presence of any toxicity chemical substance dissolved in the cholesterol 
oxidase solution 12. If the toxicitic chemical substance is dissolved in 
the cholesterol oxidase solution 12, then the toxicity thereof reduces the 
activity of the cholesterol oxidase dissolved in the cholesterol oxidase 
solution 12. The reduction in the activity of the cholesterol oxidase 
results in a reduction in the increasing rate of the surface pressure of 
the monomolecule film. If the measured increasing rate of the stufface 
pressure of the monomolecule film is smaller than that in case of the 
toxicity free solution, this demonstrates that the activity of the 
cholesterol oxidase dissolved in the cholesterol oxidase solution is 
reduced by the toxicity of the toxicitic chemical substance dissolved in 
the cholesterol oxidase solution. This means that the toxicitic chemical 
substance would be present in the cholesterol oxidase solution. 
(Example 6) 
As a cholesterol oxidase solution 12, there was used a buffer solution 
including N-2-hydroxyethylpiperazine-N'-2-ethane sulfuric acid, where the 
ion strength is 0.1 and pH value is 7.45 as well as 1 mU/ml of the 
cholesterol oxidase is dissolved in the buffer solution. In the buffer 
solution, 1 micro-M of sodium dodecylsulfate as a toxicitic chemical 
substance was dissolved. As a monomolecule film development solution 13, 
there was used a monomolecule film development solution comprising 70 mol 
% of L-.alpha.-di-palmytylphosphatizir-ethanol and 30mol % of 
pregnenolone. 
10 micro-1 of the monomolecule film development solution 13 was dropped 
onto a surface of 45 cm.sup.2 of the cholesterol oxidase solution 12 of 1 
mU/ml, into which 1 micro-M of sodium dodecylsulfate as a toxicitic 
chemical substance was dispersed, in order to form a monomolecule film on 
the surface of the cholesterol oxidase solution 12. A surface pressure of 
the monomolecule film was measured by a surface tensiometer 14 to obtain 
the same curve as the curve 23 shown in FIG. 2 and on Table 1. The surface 
pressure of the monomolecule film was gradually increased after the 
monomolecule film was prepared. The increasing rate of the surface 
pressure of the monomolecule film depends upon the cholesterol oxidase 
activity. As described above, the activity of the cholesterol oxidase 
depends upon the rate of increase in the surface pressure of the 
monomolecule film. This means that the rate of increase in the surface 
pressure of the monomolecule film represents the activity of the 
cholesterol oxidase. At five minutes after the monomolecule film was 
prepared, the curve 23 has a linear differential coefficient which is 
smaller than the linear differential coefficient of the curve 22. This 
means that the cholesterol oxidase of the curve 23 has an activity which 
is smaller than the activity of the curve 22. This proves that the sodium 
dodecylsulfate acts as a toxictic chemical substance which reduces the 
activity of the cholesterol oxidase. 
For the above reason, the monomolecule film development solution 13 is 
dropped onto a surface of a cholesterol oxidase solution 12, into which 
any toxicity chemical substance is dissolved, in order to form a 
monomolecule film on the surface of the cholesterol oxidase solution 12. 
The surface pressure of the monomolecule film is measured to confirm the 
presence of any toxicity chemical substance dissolved in the cholesterol 
oxidase solution 12. If the toxicitic chemical substance is dissolved in 
the cholesterol oxidase solution 12, then the toxicity thereof reduces the 
activity of the cholesterol oxidase dissolved in the cholesterol oxidase 
solution 12. The reduction in the activity of the cholesterol oxidase 
results in a reduction in the increasing rate of the surface pressure of 
the monomolecule film. If the measured increasing rate of the surface 
pressure of the monomolecule film is smaller than that in case of the 
toxicity free solution, this demonstrates that the activity of the 
cholesterol oxidase dissolved in the cholesterol oxidase solution is 
reduced by the toxicity of the toxicitic chemical substance dissolved in 
the cholesterol oxidase solution. This mans that the toxicitic chemical 
substance would be present in the cholesterol oxidase solution. 
A third present invention will be described with reference to seventh to 
ninth examples, which is directed to an apparatus for both determination 
in the activity of the cholesterol oxidase and the and evaluation of the 
toxicity of chemical substances. 
(Example 7) 
An apparatus for both determination in the activity of the cholesterol 
oxidase and the evaluation on the toxicity of chemical substances 
comprises a trough 11 and a surface tension controller 30. FIG. 3 
schematically illustrates the apparatus in this embodiment. The trough 11 
receives the cholesterol oxidase solution 12 on which a monomolecule film 
is formed by dropping a monomolecule film development solution onto the 
surface thereof. The surface tension controller 30 is provided to set, at 
a constant value, an initial surface pressure of the monomolecule film at 
a time when the surface pressure measurement is commenced. The surface 
tension controller 30 has a partition plate 31 which is positioned over 
the cholesterol oxidase solution 12. The surface area of the monomolecule 
film formed on the surface of the cholesterol oxidase solution 12 is 
defined by the position of the partition plate 31. The position of the 
partition plate 31 is adjusted by a partition plate positioning controller 
32. The partition plate positioning controller 32 is mechanically 
connected to the partition plate 31 for adjusting the position of the 
partition plate 31. The partition plate positioning controller 32 is 
electrically connected to a control unit 33 for receiving electrical 
information about the surface pressure of the monomolecule film formed on 
the surface of the cholesterol oxidase solution 12 so as to adjust the 
position of the partition plate 31 on the basis of the information about 
the surface pressure from the control unit 33. The control unit 33 is 
electrically connected to a surface tensiometer 14 which is positioned 
over the cholesterol oxidase solution 12 and adjusted to measure a surface 
tension of the monomolecule film formed on the surface of the cholesterol 
oxidase solution 12. The control unit 33 receives, from the surface 
tensiometer 14, electrical information about the surface tension of the 
monomolecule film, which has been measured by the surface tensiometer 14. 
The monomolecule film development solution 13 is dropped onto a surface of 
the cholesterol oxidase solution 12 to form the monomolecule film. 
As described above, the surface tension controller 30 is provided to 
control at a constant value the initial surface tension of the 
monomolecule film at an initial time of the measurement thereof in order 
to improve accuracy in the measurement of the surface pressure of the 
monomolecule film. This allows a correct and accurate determination of the 
activity of the cholesterol oxidase or a correct and accurate evaluation 
on the toxicity of chemical substance dissolved or dispersed in the 
cholesterol oxidase solution. 
(Example 8) 
An apparatus for both determination in the activity of the cholesterol 
oxidase and the evaluation on the toxicity of chemical substances 
comprises not only a trough 11 and a surface tension controller 30 but 
also a stage control system 40. FIG. 4 schematically illustrates the 
apparatus in this embodiment. The trough 11 receives the cholesterol 
oxidase solution 12 on which a monomolecule film is formed by dropping a 
monomolecule film development solution onto the surface thereof. The 
surface tension controller 30 is provided to set, at a constant value, an 
initial surface pressure of the monomolecule film at a time when the 
surface pressure measurement is commenced. The surface tension controller 
30 has a partition plate 31 which is positioned over the cholesterol 
oxidase solution 12. The surface area of the monomolecule film formed on 
the surface of the cholesterol oxidase solution 12 is defined by the 
position of the partition plate 31. The position of the partition plate 31 
is adjusted by a partition plate positioning controller 32. The partition 
plate positioning controller 32 is mechanically connected to the partition 
plate 31 for adjusting the position of the partition plate 31. The 
partition plate positioning controller 32 is electrically connected to a 
control unit 33 for receiving electrical information about the surface 
pressure of the monomolecule film formed on the surface of the cholesterol 
oxidase solution 12 so as to adjust the position of the partition plate 31 
on the basis of the information about the surface pressure from the 
control unit 33. The control unit 33 is electrically connected to a 
surface tensiometer 14 which is positioned over the cholesterol oxidase 
solution 12 and adjusted to measure a surface tension of the monomolecule 
film formed on the surface of the cholesterol oxidase solution 12. The 
control unit 33 receives, from the surface tensiometer 14, electrical 
information about the surface tension of the monomolecule film, which has 
been measured by the surface tensiometer 14. The monomolecule film 
development solution 13 is dropped onto a surface of the cholesterol 
oxidase solution 12 to form the monomolecule film. The stage control 
system comprises the above control unit 33, an X-Y stage 41 and an X-Y 
stage controller 42. The X-Y stage 41 is provided to support the trough 11 
so that the trough 11 is placed on the X-Y stage 41. The X-Y stage, on 
which the trough 11 receiving the cholesterol oxidase solution 12 is 
placed, is adjusted to move the trough 11 in a horizontal plane along 
X-axis and Y-axis which are vertical to each other. The X-Y stage may be 
mechanically connected to the X-Y stage controller 42 so that the X-Y 
stage can be mechanically moved by the X-Y stage controller 42. Otherwise, 
the X-Y stage may be electrically connected to the X-Y stage controller 42 
for receiving electrical signals as instructions of what amounts of 
distances the trough 11 should be moved in the X and Y directions so that 
the X-Y stage can move by itself on the basis of the electrical signals. 
As described above, the surface tension controller 30 and the stage control 
system 40 are provided to control at a constant value the initial surface 
tension of the monomolecule film at an initial time of the measurement 
thereof in order to improve accuracy in the measurement of the surface 
pressure of the monomolecule film. This allows a correct and accurate 
determination of the activity of the cholesterol oxidase or a correct and 
accurate evaluation on the toxicity of chemical substance dissolved or 
dispersed in the cholesterol oxidase solution. 
(Example 9) 
An apparatus for both determination in the activity of the cholesterol 
oxidase and the evaluation on the toxicity of chemical substances 
comprises not only a circulation trough 51 and a surface tension 
controller 30 but also a stage control system 50. FIG. 5 schematically 
illustrates the apparatus in this embodiment. FIG. 6 is a plane view 
illustrative of a circulation trough 51, a circulation stage 52 and a 
circulation stage controller 53. The circulation trough 51 has a plurality 
of cells, each of which receives the cholesterol oxidase solution 12 on 
which a monomolecule film is formed by dropping a monomolecule film 
development solution onto the surface thereof. The surface tension 
controller 30 is provided to set, at a constant value, an initial surface 
pressure of the monomolecule film at a time when the surface pressure 
measurement is commenced. The surface tension controller 30 has a 
partition plate 31 which is positioned over the cholesterol oxidase 
solution 12. The surface area of the monomolecule film formed on the 
surface of the cholesterol oxidase solution 12 is defined by the position 
of the partition plate 31. The position of the partition plate 31 is 
adjusted by a partition plate positioning controller 32. The partition 
plate positioning controller 32 is mechanically connected to the partition 
plate 31 for adjusting the position of the partition plate 31. The 
partition plate positioning controller 32 is electrically connected to a 
control unit 33 for receiving electrical information about the surface 
pressure of the monomolecule film formed on the surface of the cholesterol 
oxidase solution 12 so as to adjust the position of the partition plate 31 
on the basis of the information about the surface pressure from the 
control unit 33. The control unit 33 is electrically connected to a 
surface tensiometer 14 which is positioned over the cholesterol oxidase 
solution 12 mad adjusted to measure a surface tension of the monomolecule 
film formed on the surface of the cholesterol oxidase solution 12. The 
control unit 33 receives, from the surface tensiometer 14, electrical 
information about the surface tension of the monomolecule film, which has 
been measured by the surface tensiometer 14. The monomolecule film 
development solution 13 is dropped onto a surface of the cholesterol 
oxidase solution 12 to form the monomolecule film. The stage control 
system 50 comprises the above control unit 33, an circulation stage 52 and 
an circulation stage controller 53. The circulation stage 52 is provided 
to support the circulation trough 51 so that the circulation trough 51 is 
placed on the circulation stage 52. The circulation stage, on which the 
circulation trough 51 receiving the cholesterol oxidase solution 12 is 
placed, is adjusted to rotate the circulation trough 51 in a horizontal 
plane. The circulation stage 52 may be mechanically connected to the 
circulation stage controller 53 so that the circulation stage 52 can be 
mechanically rotated by the circulation stage controller 53. Otherwise, 
the circulation stage 52 may be electrically connected to the circulation 
stage controller 53 for receiving electrical signals as instructions of 
what amounts of distances the circulation trough 51 should be rotated in 
the horizontal plane so that the circulation stage 52 can rotate by itself 
on the basis of the electrical signals. 
As described above, the circulation trough 51 has a plurality of cells 
which are individually designed to receive different cholesterol oxidase 
solutions in order to allow short-time determinations of the activity of 
the different cholesterol oxidase or short-time evaluations on the 
toxicity of different chemical substances dissolved or dispersed in the 
cholesterol oxidase solutions. The surface tension controller 30 and the 
stage control system 50 are provided to control at a constant value the 
initial surface tension of the monomolecule film at an initial time of the 
measurement thereof in order to improve accuracy in the measurement of the 
surface pressure of the monomolecule film. This allows a correct and 
accurate determination of the activity of the cholesterol oxidase or a 
correct and accurate evaluation on the toxicity of chemical substance 
dissolved or dispersed in the cholesterol oxidase solution. 
Whereas modifications of the present invention will be apparent to a person 
having ordinary skill in the art, to which the invention pertains, it is 
to be understood that embodiments as shown and described by way of 
illustrations are by no means intended to be considered in a limiting 
sense. Accordingly, it is to be intended to cover all modifications which 
fall within the spirit and scope of the following claims directed to the 
subject mater of the present invention.