JP2003171317A - Expiration test reagent, expiration test reagent kit, expiration test bag, expiration test device, and expiration test method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an expiration test reagent which is safe for human bodies, shortens a time from the administration of the expiration test reagent to the collection of exhaled air, and can clearly discriminate a patient having a liver disease and a healthy person, to provide an expiration test reagent kit, and to provide an expiration test method. <P>SOLUTION: The expiration test reagent is characterized by containing tyrosine whose 1-carbon atom is labeled with<SP>13</SP>C. The expiration test reagent kit is characterized by at least containing the expiration test reagent containing tyrosine whose 1-carbon atom is labeled with<SP>13</SP>C and an expiration test reagent at least containing phenylalanime whose 1-carbon atom is labeled with<SP>13</SP>C. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a breath test reagent,
The present invention relates to a breath test reagent kit, a breath test bag, a breath test apparatus, and a breath test method.

[0002]

2. Description of the Related Art At present, as a test of human liver function, the concentrations of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) in plasma are measured. Since these measurements are blood tests, they are a painful test for the patient as they require blood collection from the patient. In addition, when a patient has an infectious disease such as C infection, there is a risk of infection in the medical staff to be examined in blood collection, examination, and processing. In contrast to the liver function test by such a blood test, in recent years, as a test method that is non-invasive and painless to the patient and does not pose a risk of infection to medical personnel, a breath test method that tests human breath Has been studied.

The breath test method is a method in which a breath test reagent which is metabolized in the body by the liver or the like to produce a volatile substance is previously administered to a patient and the volatile substance diffused in the breath of the patient is detected. , A method of examining metabolic abnormalities in the body. Volatile substances generated in the process of metabolism of the administered breath test reagent are dissolved in blood and circulate in the body. When the vapor partial pressure of the volatile component in the blood increases, the volatile substance that reaches the lung diffuses from the capillaries of the lung into the alveoli through the alveolar membrane and is exhaled out of the body as exhaled air. By appropriately labeling the breath test reagent administered here with an isotope or the like, the volatile substance generated by metabolism of the administered reagent can be labeled and can be distinguished from the endogenous volatile substance. . That is, the volatile substances generated by metabolism of the breath test reagent are labeled, and the endogenous volatile substances are not labeled, so the metabolic function can be tested by measuring the content of the labeled volatile substances. can do.

As an example of a breath test method for liver disease
As a breath test reagent^ThirteenC-phenacetin was used
There is a way. This way the patient^ThirteenSolution labeled with C
Thermal analgesic^ThirteenC-phenacetin was administered, and after a certain period of time
The patient's exhaled breath was collected after
Check for minutes carbon dioxide. For example, 100
mg^ThirteenAdministration of C-phenacetin, if administered
It is possible to collect exhaled air after 1 hour 30 minutes to 2 hours.
desirable. Breath tests are specifically performed in the liver.
Of the volatile substances that are generated by being apologized^ThirteenCO_TwoAnd intrinsic volatilization
Is a substance^{1 Two}CO_TwoAnd the number of moles of (^ThirteenCO_Two/
¹²CO_TwoRatio). If the patient has liver disease such as cirrhosis
If you have a illness, administer^ThirteenOf C-phenacetin
Metabolism (detoxification) is inferior to healthy people,^ThirteenCO_TwoGeneration of
Less in exhalation^ThirteenCO _Two/¹²CO_TwoThe ratio is
It becomes lower than a healthy person. In this way^ThirteenC-phena
Administering cetin to the patient, collecting the patient's exhaled breath,^ThirteenC
O_Two/¹²CO_TwoNon-invasive and painless by measuring the ratio
For liver disease without risk of infection
You can

[0005]

[Problems to be Solved by the Invention] However, ¹³ C
-Since phenacetin is an antipyretic analgesic, it is not preferable in terms of safety to administer it to a patient who does not need antipyretic or analgesic. Further, in the examination of exhaled breath by administration of ¹³ C-phenacetin, there is a problem that it takes a long time from administration of the exhaled breath test reagent to collection of exhaled breath, which imposes a heavy time burden on the patient.

The present invention has been made in view of the above problems, and it is safe for the human body, the time from administration of a breath test reagent to collection of breath is short, and a healthy person and a patient with liver disease are clearly defined. An object is to provide a breath test reagent, a breath test reagent kit, and a breath test method that can be determined.

Another object of the present invention is to provide a breath test bag capable of easily collecting and discharging the breath before and after administration of a patient who has administered the breath test reagent, and storing the breath for a long period of time.

A further object of the present invention is to provide an exhalation test apparatus capable of accurately measuring the ¹³ CO ₂ abundance rate of exhaled air collected by the above exhalation test bag.

[0009]

The invention according to claim 1 is
A breath test reagent for testing liver disease by measuring the presence of ¹³ CO ₂ contained in breath, comprising:
It is characterized by containing at least tyrosine labeled with ¹³ C at the 1st carbon.

According to the invention of claim 1, since at least the tyrosine having the carbon at the 1-position labeled with ¹³ C is contained, it is safe for the human body, and the time from the administration of the breath test reagent to the collection of breath is short, It is possible to provide a breath test reagent capable of clearly determining a healthy person and a patient having liver disease.

The invention according to claim 2 is the reagent for breath test according to claim 1, characterized in that the tyrosine having the carbon at the 1-position labeled with ¹³ C is provided in the form of being dissolved in an alkali.

According to the second aspect of the present invention, since the tyrosine having the carbon at the 1-position labeled with ¹³ C is given in a form that dissolves in an alkali, a necessary and sufficient dose of the breath test reagent can be obtained without waste. A breath test can be performed.

The invention according to claim 3 is included in exhalation.
¹³ A breath test reagent kit for testing liver disease by measuring the CO ₂ present rate, test reagent comprising at least one of carbon were labeled with ^{1 3 C} tyrosine, and 1-position carbon And a breath test reagent containing at least phenylalanine labeled with ¹³ C.

According to the third aspect of the invention, a test reagent containing at least tyrosine labeled with ¹³ C at the 1-position carbon and a breath test reagent containing at least phenylalanine labeled with ¹³ C at the 1-position carbon. , Which is safe for the human body, provides a short time from administration of a breath test reagent to collection of breath, and can clearly and easily determine a healthy person and a patient with liver disease. it can.

The invention according to claim 4 is the reagent kit for breath test according to claim 3, wherein the carbon at the 1-position is ¹³ C.
The tyrosine labeled with and the phenylalanine labeled with ¹³ C at the carbon at the 1-position are provided in the form of being dissolved in alkali.

According to the invention of claim 4, tyrosine in which the carbon at the 1-position is labeled with ¹³ C and the carbon at the 1-position are
^{Since 13} C-labeled phenylalanine is provided in a form that dissolves in alkali, it is possible to perform a breath test with a necessary and sufficient dose of the breath test reagent without waste.

In the invention according to claim 5, the carbon at the 1-position is ¹³
Respiratory test reagent containing at least tyrosine labeled with C or breath test reagent containing at least phenylalanine labeled with ¹³ C at the 1st carbon, for collecting, storing, and expelling exhaled breath A bag having a bag portion for collecting the exhaled air and a tubular mouth portion having both ends inside and outside the bag portion, the tubular mouth portion leaking the collected air from the bag portion. It is characterized in that it has a means for preventing it.

According to the invention of claim 5, it has a bag portion for collecting the exhaled air, and a tubular mouth portion having both ends inside and outside the bag portion, and the tubular mouth portion is collected. To provide a breath test bag capable of easily collecting and discharging breath before and after administration of a patient who has administered a breath test reagent and storing the breath for a long period of time, since the breath test bag has means for preventing the air from leaking from the bag portion. You can

[0019] According to a sixth aspect of the invention, the breath test apparatus for the exhalation collected by breath test bag according to claim 5, infrared analyzer, or by the mass spectrometer equipped with a gas chromatograph, ¹³ It is characterized by measuring the CO ₂ abundance.

According to the invention described in claim 6, ¹³ CO _{2 is} present in the exhaled air collected by the exhalation test bag according to claim 5 by an infrared analyzer or a mass spectrometer equipped with a gas chromatograph. Since we measure the rate,
About the exhaled breath collected by the above-mentioned breath test bag ¹³
It is possible to provide a breath test device capable of accurately measuring the CO ₂ presence rate.

[0021] The invention according to claim 7 is included in the breath after the administration of the breath test reagent containing at least tyrosine in which the carbon at the 1-position is labeled with ¹³ C in the breath test method.
It is characterized in that the ¹³ CO ₂ existing rate is measured.

According to the invention of claim 7, since the ¹³ CO ₂ abundance ratio contained in the exhaled breath after the administration of the exhalation test reagent containing at least tyrosine labeled with ¹³ C at the 1-position carbon is measured, It is possible to provide a breath test method that is safe for the human body, has a short time from administration of a breath test reagent to collection of breath, and can clearly determine a healthy person and a patient having liver disease.

The invention according to claim 8 relates to a breath inspection method.
And the first carbon^ThirteenLess tyrosine labeled with C
Included in exhaled breath after administration of breath test reagent
^ThirteenCO_TwoThe step of measuring the abundance and the carbon of the first place
^ThirteenA call containing at least phenylalanine labeled with C
Included in exhalation after administration of breath test reagent^ThirteenCO _Two
Measuring the abundance rate.
It

According to the invention of claim 8, the step of measuring the ¹³ CO ₂ abundance contained in the exhaled breath after the administration of the exhalation test reagent containing at least tyrosine labeled with ¹³ C at the 1-position carbon, Measuring the abundance ratio of ¹³ CO ₂ contained in the exhaled breath after administration of a breath test reagent containing at least phenylalanine whose 1-carbon is labeled with ¹³ C is safe for human body and exhaled breath. It is possible to provide a breath test method in which the time from the administration of a test reagent to the collection of breath is short and a healthy person and a patient having liver disease can be clearly and easily determined.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be described with reference to the drawings.

First, the breath test reagent of the present invention will be described.

[0027] In the present invention, using 1-position carbon ¹² C stable isotope in a ¹³ C-labeled tyrosine, i.e. L ^[1-13 C] tyrosine as a reagent for breath tests. Further, in the present invention, L [1 as a breath test reagent is used.
- ¹³ C] L of the same reagent breath test and tyrosine
^{[1- 13} C] phenylalanine is used as a reagent kit for breath tests. This breath test reagent kit is administered to the same patient at regular time intervals.

[0028] With such a breath test reagent for the passage of a predetermined time by administering to a patient a breath test reagent administered L [1- 13 ^C] tyrosine and L [1- 13 ^C] phenylalanine in the liver Metabolized during the patient's exhalation
¹³ CO ₂ is detected. Molar ratio of an amount and endogenous volatiles ¹³ CO ₂ derived from the metabolism of the reagent for breath test in the exhaled ¹² CO ₂ ^{(hereinafter,} 13 _CO ^2/12
It will be referred to as a CO ₂ ratio. ) To determine the presence or absence of liver disease.

The inventors of the present invention have found that healthy people with normal liver function and liver disease
In patients with illness, L [1- ^ThirteenC] with tyrosine
[1-^ThirteenC] Breathing as a result of administration of phenylalanine
In ^ThirteenCO_Two/¹²CO_TwoHealthy people and liver disease in the ratio
The difference with the patient with the illness is L [1-^ThirteenC] tyrosine
When administered, [1-^ThirteenC] Phenylalanine
We got the fact that it was greater than when administered. Sanawa
L [1-^ThirteenC] tyrosine is [1-^ThirteenC]
Healthy people and patients with liver disease better than phenylalanine
It is suitable for determining the
Experimentally found to be a more effective breath test reagent
It was

In a healthy person, [1-^ThirteenC]
Administration of phenylalanine and L [1- ^ThirteenC] of tyrosine
Between administration,^ThirteenCO_Two/¹²CO_TwoAlthough the ratio is different,
In patients with liver disease, between these reagents
^ThirteenCO_Two/¹²CO_TwoThere is no big difference in the ratio, two amino
Examine the presence or absence of liver disease with a breath test reagent kit that administers acid.
The present invention was found by experimentally finding that it can be easily determined.
It was

Next, the result of the above experiment is shown in FIG.
- ¹³ C] it will be described in conjunction with phenylalanine and L ^{[1- 13} C] metabolic processes in the liver tyrosine.

[0032] L ^{[1- 13} C] phenylalanine, is mainly oxidized by cytochrome P-450 enzyme phenylalanine hydroxylase in the liver L ^[1-13
C] Becomes tyrosine. In mammals, this oxidation process is a rate-determining step and is an irreversible reaction. Then L
[1- ¹³ C] tyrosine undergoes a transamination reaction by the enzyme tyrosine aminotransferase to form ¹³ C hydroxyphenylpyruvate. This transamination reaction is the rate-limiting step in the presence of liver disease.
Further, ¹³ C hydroxyphenylpyruvate is decarboxylated by droxyphenylpyruvate oxidase to produce homogentisic acid. At this ^{time, 1 3} decarboxylated from 1-position of the carboxyl group of the C-hydroxyphenyl pyruvic ^acid, resulting in 13 CO _2. Homogentisic acid is further decomposed into carbon dioxide and water through the TCA cycle,
The carbon dioxide produced here is ¹² CO ₂ . Also, L
There is also a process in which [1- ¹³ C] phenylalanine is decarboxylated to form β-phenylalanine, and here also, ¹³ CO ₂ is produced by decarboxylation of the carboxyl group at the 1-position. This process is not the main metabolic pathway in healthy persons with normal liver function, but it is a relatively important reaction pathway in patients with liver diseases such as cirrhosis.

[0033] More L ^{[1- 13} C] tyrosine or more metabolic pathways, or L ^{[1- 13} C] phenylalanine for when administered to a patient will be described.

[0034] L in the case where the ^{[1- 13} C] tyrosine was administered to a patient, in normal healthy individuals of liver function, L ^{[1- 13}
There is no rate-determining step in the metabolic process of [C] tyrosine, and the metabolism proceeds rapidly, and ¹³ CO ₂ is diffused in the exhaled air.
On the other hand, when administered with L ^{[1- 13} C] tyrosine for patients with liver disease such as cirrhosis, ^{L [1-} 13 C]
The transamination reaction of tyrosine becomes the rate-determining step, the metabolic reaction is delayed, and the diffusion of ¹³ CO _{2 into} the breath is suppressed. That is, when administered to L [1- 13 ^C] tyrosine, towards healthy subjects can explain the fact that the ratio of ¹³ CO ₂ in the breath is higher than those with liver disease.

[0035] The L If the ^{[1- 13} C] phenylalanine was administered to a patient, a healthy person ^{L [1-} 13 C]
The process of oxidation to tyrosine becomes the rate-determining step, and L [1
- ¹³ C] it is metabolized through the generation of ^tyrosine, 13 CO
₂ is diffused during exhalation. On the other hand, in patients with liver disease, L ^{[1- 13} C] simultaneously metabolism to tyrosine is suppressed, with L ^{[1- 13} C] transamination reactions rate-determining step of ^tyrosine, of ¹ 3 CO ₂ While diffusion into breath is being suppressed, L [1- 13 ^C] process of direct decarboxylation of phenylalanine has become important, generated when the
¹³ CO ₂ diffuses into the exhaled breath.

Therefore, L [1-^ThirteenC] Phenylalani
L [1-^{1 Three}C] throw tyrosine
Healthy subjects and patients with liver disease compared to when given
During exhalation between^ThirteenCO_TwoDifference in the amount of
L [1- ^ThirteenC] Phenylalanine
Than L [1-^ThirteenC] Tyrosine is more called a liver disease
It is explained that it is suitable as a reagent for a breath test.

For a healthy person, L [1-
^ThirteenC] There is no rate-limiting step in the metabolic pathway in tyrosine
But L [1-^ThirteenC] In phenylalanine, the rate-determining step is
Is present, so when both amino acids are administered^ThirteenC
O_TwoThere is a difference in the amount diffused during exhalation. Also the liver
In patients with the disease, L [1-^ThirteenC] throw tyrosine
When given, the transamination reaction becomes the rate-determining step.^Thirteen
CO_TwoAmount of L [1-
^ThirteenWhen [C] phenylalanine is administered, amino
In addition to the rate-controlling step of the group transfer reaction, L [1-
^ThirteenC] The metabolic pathway via tyrosine L is also suppressed
But instead of [1-^ThirteenC] Decarboxylation of phenylalanine
by ^ThirteenCO_TwoDiffusion occurs in both reagents
Exhaling^ThirteenCO_TwoIt seems that there is not much difference in the amount of. This
As a result, using the breath test reagent kit of the present invention, two
The individual breath test reagents can be administered separately to the patient
In^ThirteenCO_TwoBy measuring the difference in the content of
Explain that it is possible to determine whether it is a normal person or a patient with liver disease
To be done.

The breath test reagent L- of the present invention is used.
[1-^ThirteenC] tyrosine, L [1- ^ThirteenC] Phenyla
For Lanine, 99% of the 1st carbon^ThirteenIs C
L- [1- labeled with high purity^ThirteenC] tyrosine, L
[1-^ThirteenC] Phenylalanine is commercially available
It is possible.

Next, administration of the breath test reagent of the present invention will be described.

The breath test reagent of the present invention, L [1-
^{13 C]} tyrosine, L [1- 13 ^C] phenylalanine, since also present in normal food are both essential amino acids, the human body is harmless unless administered an excessive amount, an antipyretic analgesic conventional Unlike ¹³ C-phenacetin,
The safety of the test drug is high.

[0041] When administering only L [1- 13 ^C] tyrosine breath test is only once, because it is one of the reagents, the inspection is simple and inexpensive. When carrying out a breath test with a breath test reagent kit using two amino acids, the determination is clear and easy because the difference in the measured values relating to the ratio of ¹³ CO _{2 in} the breath should be observed.

The dose of the breath test reagent of the present invention is L
^{[1- 13} C] tyrosine, for both L ^{[1- 1} 3 C] phenylalanine, 50 mg or 200mg or less. If the dose is less than 50 mg, the ratio of ¹³ CO _{2 in} the exhaled breath produced by metabolism of the breath test reagent is small and it is difficult to detect. Also, if it is more than 200 mg, there is a safety problem for patients suspected of liver disease,
In addition, the cost of the drug is high.

The time for administering the breath test reagent of the present invention is preferably 2 to 12 hours after eating. If the time of administration is earlier than 2 hours after meal, ¹³ CO _{2 in} exhaled breath
Is small and difficult to detect. It is considered that this is because the breath test reagent that is an amino acid has a high blood concentration, and the administered breath test reagent is used for protein synthesis, and the metabolic process accompanying decarboxylation shown in FIG. 1 does not occur. . Also, if the time of administration is later than 12 hours after eating,
The patient is hungry for a long time and the examination is painful for the patient.

In addition, L [1-^ThirteenC] Tyrosine and L [1
−^ThirteenC] Where both phenylalanine is administered to the patient
If both drugs are administered, the time interval between administrations is 24 hours or more.
Preferably. The time interval between administrations of both drugs is 2
If it is less than 4 hours, the metabolism time of both drugs will overlap.
L [1--^ThirteenC] By metabolism of tyrosine
Occurs^ThirteenCO_TwoAnd L [1-^ThirteenC] Phenyla
By Lanin^ThirteenCO _TwoCoexisting with and without liver disease
May interfere with the determination regarding.

For administration of the breath test reagent of the present invention, the form of the breath test reagent may be in the form of powder together with water for convenience. The water temperature is preferably about 37 ° C., which is close to the body temperature, because the breath test reagent can be absorbed into the body well.

Further, L which is the breath test reagent of the present invention
^{[1- 13} C] tyrosine, L ^{[1- 1} 3 C] phenylalanine is an amino acid, these amino acids are absorbed from the small intestine. Therefore, it is desirable to administer in the form of tablets or capsules, which are not soluble in an acidic environment such as the stomach but are soluble in an alkaline environment such as the small intestine. For example, as a tablet that does not dissolve in acid but dissolves in alkali, hydroxypropylmethylcellulose phthalate (14th revision Japanese Pharmacopoeia Part 2 pharmaceutical) may be used as a coating agent. In this case, since the tablet or capsule containing the amino acid of the breath test reagent of the present invention is transported to the small intestine and dissolved, the breath test reagent of the present invention is efficiently absorbed in the body. Therefore, in the breath test using the breath test reagent of the present invention, the breath test can be performed with a necessary and sufficient dose without waste.

Next, the breath test bag of the present invention will be described with reference to FIG. FIG. 2A is a plan view of the breath test bag, and FIG. 2B is a side view thereof, and the internal structure of the bag is shown by a dotted line. The breath test bag of the present invention contains breath before administration of a breath test reagent, or L [1-
^A breath test bag capable of collecting the breath of a patient after administration of ¹³ C] phenylalanine or L [1- ¹³ C] tyrosine.

In the breath test bag of the present invention, a rigid tubular mouth portion 3 having a hole 4 is attached to the mouth portion of a bag portion 1 made of a laminated sheet of a plastic film and aluminum foil, which is removable. The cap 5 is attached. The bag portion 1 of the breath test bag is flatly folded before use, and the bottom portion 2 of the bag portion 1 of the breath test bag folded by the patient gripping the tubular mouth portion 3 and exhaling. The volume expands so that it stretches.
When the bottom 2 is stretched, the bottom 2 of the breath test bag has an area, and the breath test bag can be placed upright with the mouth facing upward.

The end of the tubular mouth portion 3 on the side of the bag portion 1 is sealed, and the exhaled breath is provided in the breath inspection bag of the tubular mouth portion 3 as shown by the arrow on the upper side of FIG. It is collected and discharged only through the two facing holes 4 facing each other. When exhaled breath is collected in the bag portion 1, the side portion of the bag portion 1 of the bag is held by manually holding the portion corresponding to the hole 4 provided in the tubular mouth portion 3 from the outside of the breath inspection bag by hand. The hole 4 is closed with, so that the exhaled breath does not leak from the breath inspection bag temporarily, and the cap 5 is tightened. Here the number of holes 4 is 2
However, the shape and number of the holes 4 may be any shape and number as long as they can be temporarily sealed by holding them down by hand.

The exhaled breath of the patient collected in the breath test bag is first closed in the same manner as when the exhaled breath is collected, and the cap 5 is attached to the mass spectrometer equipped with an infrared analyzer or a gas chromatograph described later. Connecting. To connect to such a device, attach a connecting adapter to the tubular mouth 3,
You may connect directly.

Further, an elastic sealing body through which an injection needle can be inserted is attached to the cap 5, and the elastic sealing body of the cap 5 is pierced with a syringe to extract a required amount of exhaled air, and the syringe is connected to a measuring device. Good.

As shown by the arrow on the lower side of FIG.
Exhaled air can be easily discharged from the bag portion 1 of the exhalation test bag through the two holes 4 provided in the.

By collecting the exhaled air in such an exhalation inspection bag, the exhaled breath can be exhaled even if it is temporally or spatially separated from an exhalation inspection device such as an infrared analyzer or a mass spectrometer equipped with a gas chromatograph. Can be collected, and the patient can be measured without being constrained in terms of time and space. Further, the breath test bag has a high hermeticity, and thus the collected breath can be stored for a long period of time and easily retested.

Next, a dedicated infrared analyzer (hereinafter referred to as IR analysis) which is a device capable of accurately measuring the isotope abundance ratio of carbon dioxide contained in the exhaled breath of the patient to whom the breath test reagent of the present invention is administered. Mass spectrometer) and a mass spectrometer equipped with a gas chromatograph (hereinafter referred to as GC / MS spectrometer).

First, an IR analyzer for analyzing the breath of a patient will be described with reference to FIG. As shown in FIG. 3, the infrared light emitted from the IR light source 11 as divergent light passes through the chopper 12, becomes parallel light by the collimator lens 13, and is applied to the gas in the reference cell 14 and the sample cell 15. It The infrared light that has passed through the reference cell 14 is reflected by the first mirror 16, and the first filter 18
And then enters the first Gorace cell 20. Similarly, the infrared light that has passed through the sample reference cell 15 is reflected by the second mirror 17, passes through the second filter 19, and enters the second Goule cell 21.

Here, the first filter 18 is¹²CO
_Two238 is a filter for measuring absorption by
0 ± 10 cm^-1It transmits infrared light with a bandwidth of. Well
The second filter 19^ThirteenCO_TwoAbsorption by
It is a filter for setting, 2280 ± 10 cm^-1
It transmits infrared light with a bandwidth of. That is, the first
-At race cell 20,¹²CO_TwoThe absorption amount by the second
In Goracel 21 ^ThirteenCO_TwoAbsorption by
It

At the time of measurement, nitrogen gas was made to flow from the N ₂ cylinder 22 into the reference cell 14, and the third and fourth valves 28 and 29 were opened before measuring the exhaled breath, and the sample cell 15 was also subjected to the measurement. Flow nitrogen gas. This gives ¹² CO
The reference value of the infrared light intensity for measuring the absorption of infrared light by ₂ , ¹³ CO ₂ can be obtained by the first Gore cell 20 and the second Gore cell 21.

Next, the third valve 28 is closed, the first (or second) valve 26 (or 27) is opened, and the first (or second) valve is opened.
A pump 25 for exhalation of the exhalation inspection bag 23 (or 24)
Is used to pour into the sample cell 15. ¹² CO ₂ in the sample cell 15, ^{1 3} CO _2, respectively 23
80cm ^-1, to absorb the infrared light of wavelength in the vicinity of 2280cm ^-1. The first Gorace cell 20 detects infrared light absorbed by ¹² CO _{2 at} around 2380 cm ⁻¹ . The amount of infrared light absorbed by ¹² CO ₂ is calculated from the above infrared light reference value. Similarly, the second go race 21
Detects infrared light absorbed by ¹³ CO _{2 in the} vicinity of 2280 cm ⁻¹ . ¹³ from the standard value of infrared light above
The absorption amount of infrared light by CO ₂ is calculated. It is possible to calculate the ¹³ CO ^_2/12 CO ₂ ratio from absorption of infrared light as described above.

Since the infrared analyzer does not require cooling with liquid nitrogen or the like, and uses a Goule cell as an infrared detector, the device is smaller and lighter than the GC / MS device. .

Next, a GC / MS analyzer for analyzing the breath of a patient will be described with reference to FIG. As shown in FIG.
First, before exhalation analysis, the measurement value is calibrated from the referer lens cylinder 39 using carbon dioxide containing a natural isotope as a reference gas. Further, since the measurement sample is carbon dioxide, it is necessary for the mass spectrometer to remove the air in the apparatus by using a pump and keep it in vacuum. The exhaled air collected in the exhalation test bag is transferred to the test tube 31, and passed through the thin capillary while passing through the injection needle 32 and the valve 33 and passed through the gas chromatograph device 34. The thin capillary through which the exhaled air passes maintains the vacuum state of the mass spectrometer.

The gas chromatograph device 34 removes the water contained in the exhaled air so that the carbon dioxide of the exhaled air is not absorbed by the water. The exhaled air after removing the water is an open branch system 35.
Through the ionization-ion accelerator 36 of the mass spectrometer. In this device, fragments, which are carbon dioxide ions in the exhaled breath, are generated by the electron gun, and an electric field is applied to accelerate the carbon dioxide fragments toward the permanent magnet 37. The accelerated fragment of carbon dioxide is bent by the permanent magnet 37 and detected by the ion detector 38.

Here, due to the difference in mass of the fragments,
The fragments pass through different trajectories, and different ion detectors 38 detect the fragments having different masses. Carbon dioxide is mainly composed of ¹² C and ¹³ carbon atoms.
C, the oxygen atom is isotopes ¹⁶ O and ^{1 8} O,
It has become possible to measure the fragments generated by the combination of these isotopes. As described above to determine the amount of ¹³ CO ₂ and ¹² CO ₂ in the ^breath, it is possible to calculate the 13 _CO ^2/12 CO ₂ ratio.

It should be noted that the GC / MS device uses ¹³ C during exhalation.
The O _^2/12 CO ₂ ratio can be measured more accurately than infrared spectrometer.

[0064] Next, the ¹³ CO ^_2/12 CO ₂ ratio as measured by the inspection system of the present invention will be described in detail.

[0065] The ¹³ CO ^_2/12 CO ₂ ratio measurements for which have long been used in the environmental field, are determined international standards. By international standards, arrowhead stones (PD
B: Show in ¹³ CO ^_2/12 CO ₂ ratio δ units relative to the generated calcium carbonate) when treated with 100% phosphoric acid in a vacuum (per mil, ‰). The ¹³ C abundance ratio in exhaled air is first converted to δ units by the following formula.

[0066] ^{δ 13 C (‰) = {} (13 CO 2/12 C
O _{2) expiratory ^{- (13 CO 2/12 CO}} 2) PDB} / (
¹³ CO _^2/12 CO ₂₎ exhaled fasting human in a state in which not administered test reagent _PDB × 1000 present invention is about δ ¹³ C = -20.8 per mil. In addition, even if about 100 mg of the breath test reagent of the present invention concentrated to an abundance ratio of 99% is administered, ¹³ CO
The ^_2/12 CO ₂ ratio is very small value even in healthy individuals, in the breath of patients with at least liver function, the value of [delta] ^{13 C} is normally a negative value.

In the medical field, determining negative liver disease as positive may cause confusion during diagnosis. Therefore, the value of δ ¹³ C in the breath of the patient measured before administering the breath test reagent of the present invention δ ¹³ C ₀
Δ in exhaled air after a certain period of time after administration
The ¹³ C value [delta] ¹³ C _t, evaluation value so that a positive value, it is desirable represented by increment delta ¹³ C of ¹³ CO ^_2/12 CO ₂ ratio in the breath represented by the following formula.

Δ^ThirteenC (‰) =^ThirteenC_t−^ThirteenC₀ Therefore, the detection of liver disease using the breath test reagent of the present invention is performed.
As for the examination, the distinction between a healthy person and a patient with liver disease is actually
As shown in the examples below,^ThirteenC
This is done by measuring the value.

[0069]

[Example] 99 of the 1st carbon used in this example
% Is a ¹³ C L- phenylalanine, and 99% of 1-position carbon of L- tyrosine is ¹³ C, Cambridge
Obtained from Isotope Laboratories.

L which is the breath test reagent (kit) of the present invention
^{[1- 13} C] implementation results of the breath test using tyrosine and L ^{[1- 13} C] phenylalanine are shown in Figure 5. The horizontal axis of FIG. 5 shows L [1- ¹³ C] tyrosine and L [1-
The elapsed time (minutes) after administration of ¹³ C] phenylalanine to a patient under examination is shown. Further, the vertical axis is the value of Δ ¹³ C (permill) based on the value before administration described above.

The patient breakdown is L [1-^ThirteenC] Tyrosine
4 healthy subjects were treated with L [1- ^ThirteenC] Phenyla
Fifteen healthy people were treated with Lanin, L [1-^ThirteenC]
4 patients with cirrhosis who received rosin, L [1-^ThirteenC]
14 patients with cirrhosis who received phenylalanine
It

In all of the above cases, the dose of each breath test reagent was 100 mg, and with 100 ml of water,
It was administered 7 hours after eating. First, the exhaled breath of each patient before administration was collected in a breath test bag and used as a standard for Δ ¹³ C value. 1 after administering each breath test reagent to the patient
After 0, 15, 20, 30, 45, 60 minutes, the sample was taken into a breath test bag and the Δ ¹³ C value was obtained using the infrared analyzer. From the obtained delta ^{13 C} values for each classification of a combination of a patient and the breath test reagent administered, by calculating the average value of delta ^{13 C} values were plotted, it was added standard deviation as error bars.

In any case, the breath test reagent was administered.
In a short time of 10 to 20 minutes, Δ ^ThirteenC value is maximum
It was. Looking at each breath test reagent, breath test reagent
At 10 to 20 minutes after administration of^ThirteenHealth at C value
The difference between normal and cirrhotic patients is L [1-^ThirteenC] Tyrosine
Is L [1-^ThirteenC] Larger than phenylalanine
L [1-^ThirteenC] Tyrosine is better as a breath test reagent
And shows that it is excellent.

Further, Δ ¹³ C was determined for each healthy person and each patient with cirrhosis.
Looking at the results of the value, in healthy persons L ^{[1- 13} C] tyrosine and L ^{[1- 13} C] Although the difference of the reagent breath test with phenylalanine in delta ¹³ C value at 10 to 20 minutes after administration exits In cirrhosis patients, there was almost no difference between the two breath test reagents. From this administered separately L ^{[1- 13} C] a tyrosine and L ^{[1- 13} C] phenylalanine into the same patient, the breath was collected at the same time of 10 to 20 minutes after the administration, the delta ¹³ C value It can be seen from the difference that it can be determined whether the patient is a healthy person or a patient with cirrhosis.

[0075]

EFFECTS OF THE INVENTION According to the present invention, a breath test reagent that is safe for the human body, has a short time from administration of the breath test reagent to collection of exhaled breath, and can clearly determine a healthy person and a patient with liver disease. A breath test reagent kit and a breath test method can be provided.

Further, according to the present invention, it is possible to provide a breath test bag capable of easily collecting and discharging the breath before and after the administration of the patient who has administered the breath test reagent, and storing it for a long time.

Further, according to the present invention, the breath test
About the exhaled air collected by the bag ^ThirteenCO_TwoExistence rate
To provide a breath test device that can measure accurately.
You can

[0078]

[Brief description of drawings]

[1] L ^{[1- 13} C] phenylalanine and L [1
It is a figure explaining the metabolic process in the liver of- ^13C ] tyrosine.

FIG. 2 is a view of a breath test bag for collecting breath of a patient who has been administered the breath test reagent of the present invention, (a) is a plan view, and (b) is a side view.

FIG. 3 is a diagram of an infrared analyzer for analyzing the breath of a patient to whom the breath test reagent of the present invention has been administered.

FIG. 4 is a diagram of a mass spectrometer equipped with a gas chromatograph that analyzes the breath of a patient to whom the breath test reagent of the present invention has been administered.

FIG. 5 is a graph showing the time course of the Δ ¹³ C value in the exhaled air of the patient to whom the breath test reagent of the present invention was administered.

[Explanation of symbols]

1 Bag Part 2 Bottom Part 3 Tubular Part 4 Hole 5 Cap 11 IR Light Source 12 Chopper 13 Collimator Lens 14 Reference Cell 15 Sample Cell 16 First Mirror 17 Second Mirror 18 First Filter 19 Second Filter 20 First Gole cell 21 Second gole cell 22 N ₂ cylinder 23 First breath test bag 24 Second breath test bag 25 Pump 26 First valve 27 Second valve 28 Third valve 29 Fourth valve 31 Test tube 32 Injection Needle 33 Valve 34 Gas chromatograph 35 Open branch system 36 Ionization-ion accelerator 37 Permanent magnet 38 Ion detector 39 Reference cylinder

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 21/35 G01N 21/35 Z 4C085 27/62 27/62 C V 30/72 30/72 A 33/497 33 / 497 A (72) Inventor Hiroto Yamashita 2-1-1 Omorikita, Ota-ku, Tokyo Omori NM Building 7F, F-Term (Reference) 2F045 AA25 BB02 CB22 DA77 DB01 FA36 FB06 FB08 HA06 2G052 AA34 AB06 AD02 DA14 DA27 GA11 GA24 GA27 2G057 AA01 AB02 AB06 AC03 BA01 BB06 BB07 2G059 AA05 AA06 BB12 CC16 EE01 FF08 FF09 GG10 HH01 HH06 JJ02 JJ11 JJ13 JJ24 MM05 4C038 SS00 ST00 ST04 SU19 SX02 SX20 4C085 HH17 HH20 JJ03 JJ11 KA30 KA36 KB45 LL05

Claims (8)

[Claims] 1. A breath test reagent for examining liver disease by measuring the rate of ¹³ CO ₂ contained in exhaled breath, which comprises at least tyrosine labeled with ¹³ C at the 1-position carbon. A breath test reagent characterized by: 2. The breath test reagent according to claim 1, wherein the tyrosine having the 1-position carbon labeled with ¹³ C is provided in a form that dissolves in an alkali. 3. A breath test reagent kit for testing liver disease by measuring the rate of ¹³ CO ₂ contained in breath, comprising at least tyrosine labeled with ¹³ C at the 1-position carbon. A breath test reagent kit comprising a test reagent and a breath test reagent containing at least phenylalanine labeled with ¹³ C at the 1-position carbon. 4. A labeled with the 1-position of the carbon ^{13 C-labeled} tyrosine and the 1-position carbon ^{13 C} of phenylalanine, according to claim 3, characterized in that it is provided in the form that dissolves in alkaline Respiratory test reagent kit. 5. A breath test reagent containing at least tyrosine in which the 1-position carbon is labeled with ¹³ C, or the 1-position carbon
A breath test bag for collecting, storing, and discharging breath before and after administration of a breath test reagent containing at least ¹³ C-labeled phenylalanine, comprising: a bag portion for collecting the breath, and an inside of the bag portion. A tubular mouth portion having both ends on the outside, and the tubular mouth portion has means for preventing the collected air from leaking out from the bag portion. 6. The ¹³ CO ₂ abundance ratio of the exhaled air collected by the exhalation test bag according to claim 5 is measured by an infrared spectrometer or a mass spectrometer equipped with a gas chromatograph. Breath test device. 7. A breath test method, which comprises measuring the ¹³ CO ₂ abundance contained in the breath after administration of a breath test reagent containing at least tyrosine labeled with ¹³ C at the 1-position carbon. 8. A step of measuring an abundance ratio of ¹³ CO ₂ contained in exhaled breath after administration of a breath test reagent containing at least tyrosine labeled with ¹³ C at the 1st carbon, and ^{13C at the} 1st carbon. Measuring the presence rate of ¹³ CO ₂ contained in the exhaled breath after administration of the exhalation examination reagent containing at least phenylalanine labeled with.