JP2003065949A - Determination method for 12co2-13co2 isotope ratio in exhalation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a determination method for a<12> CO2 -<13> CO2 isotope ratio in an exhalation by the absorption of infrared rays in which an error caused by N2 gas is suppressed to a minimum. SOLUTION: In the determination method for the<12> CO2 -<13> CO2 isotope ratio in the exhalation by the absorption of the infrared rays, when a plurality of sample gasses are created in a known-sample creation process, a standard gas composed of an N2 gas component whose concentration is equal to that of the N2 gas in air,<12> CO2 gas component and a<13> CO2 gas component whose isotope ratios are known and a gas component which does not influence the absorbance of<12> CO2 and the absorbance of<13> CO2 is prepared, the standard gas is diluted with the air, a plurality of sample gases whose N2 gas concentrations are identical, whose isotope ratios are identical and whose CO2 gas concentrations are different are created, a plurality of sample gases are created from a plurality of standard gases whose known isotope ratios are different from each other, and a sample gas used to create a working curve is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the ¹² CO ₂ in breath by infrared absorption - ¹³ CO ₂ isotope ratio determination method, a rapid and accurate improvement of the particular process.

[0002]

2. Description of the Related Art Two types of isotopes ^{12 of} carbon dioxide CO ₂
By measuring the abundance ratio of CO ₂ and ¹³ CO ₂ with high accuracy, there is a possibility that various diseases can be diagnosed. One specific example is the urea breath test, which diagnoses a bacterium called Helicobacter pylori, which is considered as one of the factors of gastric ulcer. If Helicobacter pylori is present in the patient's stomach, it is necessary to treat the bacteria with antibiotics or the like. It is necessary to test whether Helicobacter pylori is present in the patient's stomach as a prerequisite for such treatment. Helicobacter pylori has a strong activity of decomposing urea into CO ₂ and ammonia by the enzyme urease. Then, urea labeled with ¹³ C isotope is administered into the patient, and if the isotope ratio of ¹³ CO ₂ and ¹² CO ₂ in the patient's breath, which is the final metabolite, can be accurately measured, the patient's stomach can be determined from the isotope ratio. Can be tested for the presence of Helicobacter pylori.

[0003] accurately isotope ratios in such diagnostic methods, e.g. 1δ per mille should be measured by the following accuracy (here, naturally ¹³ CO ₂ abundance ⁽¹³ CO in PDB
₂ abundance ratio) as a standard, and ¹³
As the deviation of the CO ₂ abundance ratio,

(Equation 2) Defined). Conventional¹²CO₂−^ThirteenCO₂Isotope ratio
There is a known method for determining
(Japanese Patent No. 2947742, JP-A-11-83)
739). With these methods¹²CO₂Ingredients and^ThirteenC
O₂Multiple samples with known isotope ratios and concentrations of components
First create the gas. And for each sample gas¹²C
O ₂as well as^ThirteenCO₂In the absorption band belonging to each
Measure the absorbance and calculate the absorbance and the known value of the isotope ratio.
Use to create a calibration curve. afterwards¹²CO₂Ingredients and ^Thirteen
CO₂The unknown sample gas whose component isotope ratio is unknown
hand,¹²CO₂as well as ^ThirteenCO₂Measure the absorbance and obtain
The unknown sample gas from the measured absorbance and the calibration curve created in advance.
Isotope ratio.

In these methods, a sample for preparing a calibration curve is used.
When getting gas,¹²CO₂Ingredients and ^ThirteenCO₂Same of ingredients
Isotope ratio, or¹²CO₂Ingredient or^ThirteenCO₂Ingredient
First, a standard gas having a known concentration is prepared. So
And then CO₂Dilute the standard gas with other gas components
To obtain the same isotope ratio and CO 2₂Different concentration
Multiple sample gases, or¹²CO₂Ingredient or^Thirteen
CO₂A sample gas with a known component concentration and a different concentration
It is usually created.

[0005]

[0006] However, ¹² CO ₂ in the unknown sample gas by infrared absorption - when determining the ¹³ CO ₂ isotopic ratio, the actual due to differences in N ₂ gas concentration in the unknown sample gas Was found to give different results. This, N ₂ gas mixed with CO ₂ is by intensifying the intensity of the infrared absorption spectrum of CO ₂ depending on the N ₂ concentration. However, the intensity change of the infrared absorption spectrum of CO ₂ due to the mixed gas is as follows.
It has also been considered to be due to mixed O ₂ gas.

As shown in FIG. 1, in the CO ₂ infrared absorption spectra in each case using a mixed gas of N _{2 base} and O _{2 base} , the absorption intensities are different from each other, and the N _{2 base} has a slight absorption intensity. It can be seen that the absorption is strong. This means that O ₂ is C
It indicates that either the absorption of O ₂ is weakened or that N ₂ enhances the absorption of CO ₂ . Therefore, He-based mixed gas was further added as a comparison object. Since He has a monoatomic structure and does not have vibration and rotation levels and is not considered to be a factor affecting the CO ₂ vibration-rotation spectrum in the infrared region, N _{2 base} and O _{2 base are used.} In addition to the above, in the case of using a total of three types of mixed gases including a He base and an He base, the infrared absorption spectrum of 0.1% CO ₂ in the absorption band shown in FIG.
It was measured in a 00 mm cell. Then, the intensity of absorption in an appropriate wavenumber region was calculated and comparatively evaluated in three cases. The results are shown in Tables 1 and 2.

[0007]

[Table 1]

[0008]

[Table 2]

As is clear from Tables 1 and ₂ , the results of comparing the absorption intensity with the O ₂ base and the He base based on the N ₂ base show that the O ₂ base and the He base. Is about 4.5% compared to the case of N _{2 base.}
It was found that the absorption intensity was reduced. Therefore,
O ₂ does not weaken the absorption of CO ₂ , but N ₂
It is understood that the absorption of O ₂ should be interpreted as being enhanced. Thus, CO ₂ absorbance, further ¹² CO ₂ is determined on the basis thereof - the ¹³ CO ₂ isotope ratio, the cause is being greatly affected by the N ₂ gas concentration in the sample gas which does not know. The present invention has been made in view of the problems of the prior art, the purpose was to minimize the error caused by the N ₂ gas, ¹² CO ₂ in breath by infrared absorption - ¹³ CO ₂ isotope It is to provide a ratio determining method.

[0010]

[MEANS FOR SOLVING THE PROBLEMS] To achieve the above object
In the expiration due to the infrared light absorption of the present invention¹²CO₂−^{1 3}
CO₂The isotope ratio determination method is¹²CO₂Ingredients and^ThirteenCO
₂Create multiple sample gases with known component isotope ratios
And the known sample preparation process
About each sample gas¹²CO₂as well as^ThirteenCO ₂It
A known test for measuring the absorbance in the absorption band belonging to each
Sample measurement step and the absorbance measured in the known sample measurement step.
And a calibration curve for creating a calibration curve from the known values of the isotope ratios
During the process and during expiration¹²CO₂Ingredients and^ThirteenCO₂Ingredient
Absorption band for sample exhaled gas whose isotope ratio is unknown
In¹²CO₂as well as^ThirteenCO₂Not measuring the absorbance
The absorption sample measured in the unknown sample measurement step and the unknown sample measurement step
From the luminous intensity and the calibration curve created in the calibration curve creation step,
In exhaled gas¹²CO₂Ingredients and^ThirteenCO₂Component isotope
An isotope ratio determining step of determining a ratio;
During exhalation¹²CO₂−^ThirteenCO₂How to determine isotope ratio
A plurality of sample gases in said known sample preparation step.
When creating a₂N equal to gas concentration₂Moth
Component, and the isotope ratio is known.¹²CO₂as well as^ThirteenCO
₂Gas component, in the absorption band¹²CO₂as well as^Thirteen
CO₂Standard consisting of gas components that do not affect absorbance
Prepare a gas, dilute the standard gas with air and₂
Gas concentration and the same isotope ratio and CO 2₂Moth
Multiple sample gases with different gas concentrations
A plurality of the standard gases having different isotope ratios from each other
Similarly, by preparing a plurality of sample gases,
It is characterized in that a sample gas for creating a dose curve is obtained.

[0011] Further, the step of preparing a calibration curve includes the step of measuring ¹² C in the absorption band measured for the plurality of sample gases.
O ₂ and ¹³ CO ₂ absorbance, ¹³ CO ₂ absorbance at ¹² C
Preferably, the step is a step of creating a two-dimensional calibration curve for the isotope ratio from the absorbance ratio obtained by dividing by the O ₂ absorbance, the known value of the isotope ratio, and a model formula. Also,
One example of the model equation for the two-dimensional calibration curve is represented by the following equation.

[Equation 3] _{^{However, δ: 12 CO 2 - 13}} CO 2 isotopic ^x: 12 CO ₂ absorbance y: the absorbance ratio _{a ij:} coefficient n = 1~4, m = 1~4 Further, in the method, the air is CO It is preferable that the air is air from which ₂ has been removed.

[0012]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, ¹² C
O ₂ - ¹³ CO ₂ isotope ratio can be minimized by simple means errors caused by N ₂ gas in determining the infrared absorption. Hereinafter, a preferred embodiment of the present invention will be described for each process.

1. And _{N 2} gas concentration (79%) equal to _{N 2} gas component known sample preparation step in air, absorbance range to be CO ₂ gas (measurement consisting isotope known ^{1 2} CO ₂ and ¹³ CO ₂ gas component And the amount of ¹² CO ₂ and ¹³ CO ₂
A standard gas consisting of a gas component that does not affect the absorbance in the absorption band belonging to each is prepared. Next, the standard gas is diluted with air to prepare a plurality of sample gases. Here, the N ₂ gas concentration of the standard sample in air N ₂
Since the same 79% as the gas concentration, even if the standard gas is diluted with air, N ₂ gas concentration is always constant.

For example, CO ₂ = 10%, O ₂ = 11%, N
Considering the case where the standard gas of ₂ = 79% is diluted 10 times with air, since the composition of air is O ₂ = 21% and N ₂ = 79%, CO ₂ = 1% and O ₂ = 20% , N ₂ = 79%
, And the N ₂ gas concentration is constant before and after dilution. Similarly, N ₂ = 79 even when diluted at an arbitrary ratio.
% Sample gas is obtained. In addition, it is preferable to use air from which CO ₂ has been removed by a method such as passing through a CO ₂ adsorbent.

If the N ₂ concentration of each sample gas is unified to the concentration in the air as described above, the N ₂ gas concentration in the exhaled gas from which the isotope ratio is determined is the same as the value in the air ( 79
%), It is standardized to a constant N ₂ gas concentration over the entire measurement sample. Therefore, the error in determining the isotope ratio due to the change in the CO ₂ absorption intensity due to the N ₂ gas can be minimized. Further, since the dilution gas may be air, the preparation of the sample gas can be performed extremely easily.

In this way, the same N ₂ gas concentration and the same isotope ratio for one standard gas
_{2. After a} plurality of sample gases having different gas concentrations have been prepared, a plurality of sample gases are similarly prepared for known standard gases having different isotope ratios.

2. Known sample measurement process For each sample gas prepared in this way,
In the sample gas¹²CO ₂Ingredients and^ThirteenCO₂Each component
Absorbance of the absorption band belonging to¹²CO₂Absorption
Degree and^ThirteenCO₂Measure the absorbance.

3. Calibration curve creation process As the calibration curve, a normal XY axis type calibration curve can be used, but the two-dimensional calibration curve described below is preferably used because the processing is quick and easy and the accuracy is good. It is. First, ¹² CO ₂ absorbance obtained by measurement and ¹³ CO ₂
From the CO ₂ absorbance, data for creating a two-dimensional calibration curve (x _i ,
y _i) ^{= (12} CO ₂ ^absorbance, 13 CO ₂ absorbance ^{/ 1 2}
CO ₂ absorbance). In this way, the sample gas, respectively of the two-dimensional calibration curve data _{(x i, y i, δ} i)
= ⁽¹² CO ₂ ^absorbance, 13 CO ₂ absorbance ^{/ 1} 2 CO ₂
Absorbance, isotope abundance ratio).

Next, the coefficients of the model equation for the two-dimensional calibration curve are obtained from the data by the ordinary least square method. The order of the model equation for the two-dimensional calibration curve is appropriately determined so as to best match the measurement result of the sample gas, and is expressed by the following equation, for example.

(Equation 4) _{^{However, δ: 12 CO 2 - 13}} CO 2 isotopic ^x: 12 CO ₂ absorbance y: the absorbance ratio _{a ij:} coefficient n = 1~4, m = 1~4 coefficients _{a ij} of the model equation, the Is determined from the solution derived by the ordinary least squares method using the data for creating a two-dimensional calibration curve obtained from the sample gases at n points. That is,
and [delta] _i, as in the model equation (x _{i, y i)} the squared difference between the values obtained by substituting, the sum obtained by adding the squared value for each i is minimized, mathematical Each coefficient is determined from the solution derived in. Such a coefficient a _ij can be obtained by simply performing a simple matrix operation by substituting the value of each data point (x _i , y _i , δ _i ) into the determinant shown below. That is, for example, if the model equation is

Δ = a ₀ + a ₁ x + a ₂ x ² + a ₃ x ³ + a ₄
In the case of ^{_{y + a 5 y 2 + a}} 6 xy + a 7 x 2 y is

(Equation 6) A that minimizes e in the above determinant is

(Equation 7) By determining the coefficient a _{ij of the} model equation from the above equation,
A two-dimensional calibration curve as shown in FIG. 3 is created.

When an ordinary XY axis type calibration curve is used, the isotope ratio is determined, for example, as follows. That is, first ¹² CO ₂ - ¹³ CO ₂ isotope ratio ([delta]%)
Are known and the sample gas with the same concentration of CO ₂
^{The 12} CO ₂ and ¹³ CO ₂ absorbances were measured and ¹² CO ₂
A calibration curve is created by the least squares method as shown in FIG. 4 (A), with the absorbance on the horizontal axis and the absorbance ratio between ¹³ CO ₂ and ¹² CO _{2 on} the vertical axis. Further, as shown in FIG.
¹² CO ₂ - ¹³ CO ₂ isotope ratio creates Similarly a calibration curve of ¹² CO ₂ absorbance and the absorbance ratio for the case differ from each another.

In the subsequent unknown sample measurement step, ¹² CO
₂ - ¹³ for CO ₂ isotope unknown exhaled gas ¹² C
O ₂ and ¹³ CO ₂ absorbance are measured. Then, in the next isotope ratio determination step, ¹³ CO ₂ and ¹² CO _{2 of the} plurality of calibration curves in the value of the ¹² CO ₂ absorbance obtained by the measurement are used.
To prepare a calibration curve by the least square method as shown in FIG. 4 (B) using a ¹³ CO ₂ isotope ratio - the absorbance ratio and ¹² CO _2. Then, ¹³ obtained by measuring the isotope ratio unknown sample was obtained.
Determining the ¹³ CO ₂ isotope ratio - corresponding to the absorbance ratio of CO ₂ and ¹² CO _2, from the value of the calibration curve ¹² CO _2.

4. Unknown sample measurement process In the sample breath gas¹²CO₂Ingredients and^ThirteenCO₂Same of ingredients
Absorption of the sample exhaled gas whose isotopic ratio is unknown
Method in the sample gas¹²CO₂Ingredients and^ThirteenCO ₂Ingredient
Absorbance of the absorption band belonging to each, that is,¹²C
O₂Absorbance and ^ThirteenCO₂Measure the absorbance.

5. (X, y) obtained by isotope ratio determination step measurement = ( ¹² CO ₂ absorbance, ¹³
(CO ₂ absorbance / ¹² CO ₂ absorbance) is substituted into a two-dimensional calibration curve equation in which the coefficient is determined in advance in the above step, to determine the isotope ratio (δ per mil) of the unknown sample.

[0024]

EXAMPLE A two-dimensional calibration curve prepared with a plurality of sample gases having the same N ₂ gas concentration and a two-dimensional calibration curve prepared with a plurality of sample gases having different N ₂ gas concentrations were prepared by the following methods. Then, two breath samples having substantially the same isotope ratio (δ per mille) and a difference Δ per mille thereof near zero are prepared, and in the case of a plurality of CO ₂ concentrations obtained by diluting both samples with air, the two breath samples are obtained. The relationship between the difference in CO ₂ concentration and the difference in isotope ratio (Δpermil) obtained from the created two-dimensional calibration curve was plotted on a graph.

[0025]Creating an air-based two-dimensional calibration curve N₂Gas, CO₂Gas and O₂Consisting of gas, N₂gas
The concentration is 79%, the same as that of air,¹²CO₂−^Thirteen
CO₂A standard gas with a known isotope ratio is
The isotope ratio was prepared. And the standard gas is CO₂
N obtained by dilution with air passed through the adsorbent₂Same concentration
A plurality of sample gases were prepared. Based on these sample gases
And the two-dimensional calibration curve

The following equation, which is an aspect of the model equation of

Δ = a ₀ + a ₁ x + a ₂ x ² + a ₃ x ³ + a ₄
It was created from the ^{_{y + a 5 y 2 + a}} 6 xy + a 7 x 2 y.

[0026]Creation of the N _2-based two-dimensional calibration curve The standard gas is N₂Diluted with gas, N₂Different concentration
A plurality of sample gases were prepared. Based on these sample gases,
And the two-dimensional calibration curve

It was created from the model equation CO _{2 of} both samples
FIG. 5 is a graph plotting the difference in concentration and the difference (Δpermil) between the isotope ratios obtained from the prepared two-dimensional calibration curve.

As shown in FIG. 5A, when an air-based two-dimensional calibration curve in which the N ₂ gas concentration of the sample gas is constant is used, Δ per mil hardly changes with respect to the CO ₂ concentration difference. I understand.

On the other hand, when an N ₂ -based two-dimensional calibration curve is used as shown in FIG. 5B, the N ₂ concentration in the sample gas used for preparing the calibration curve is different from each other, so that an error is included. It becomes a calibration curve, and it can be seen that Δ Permil clearly changes with respect to the CO ₂ concentration difference.

[0029]

As described above, according to the present invention, ¹² CO ₂ in breath by infrared light absorption of the present invention - the ¹³ CO ₂ N ₂ concentration in the gas sample for calibration curve creation according to the isotope ratio determination method Having unified concentration in air, CO due to N ₂ gas
₍₂₎ An error in determining the isotope ratio due to a change in absorbance can be minimized. Further, since the dilution gas may be air, the preparation of the sample gas can be performed extremely easily.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of CO ₂ gas when the mixed gas is N ₂ and O ₂ , respectively.

FIG. 2 is an infrared absorption spectrum of CO ₂ gas at 2300 to 2380 cm ⁻¹ .

FIG. 3 is a graph showing an example of a two-dimensional calibration curve created in the present invention.

FIG. 4 is a graph showing an example of an XY axis type calibration curve created in the present invention.

FIG. 5 is a graph plotting the difference in CO ₂ concentration between two breath samples and the difference in isotope ratio (Δpermil) between the samples obtained from a two-dimensional calibration curve created on an air or N ₂ basis. It is.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Masao Yamazaki             5 Japan, 2967 Ishikawacho, Hachioji-shi, Tokyo             Within Spectral Co., Ltd. F term (reference) 2G045 AA25 CB22 DB01 FB01 GC10                 2G059 AA01 AA05 BB01 BB12 CC04                       CC16 DD04 EE01 FF08 HH01                       MM01 MM12

Claims (4)

[Claims] (1)¹²CO₂Ingredients and^ThirteenCO₂Component Isotope
A known sample that produces multiple sample gases with known body ratios
Creation process, About each sample gas created in the known sample creation process
¹²CO₂as well as^ThirteenCO ₂To the absorption band belonging to each
A known sample measurement step of measuring the absorbance in Absorbance measured in the known sample measurement step and the isotope ratio
A calibration curve creating step of creating a calibration curve from known values of Exhaling¹²CO₂Ingredients and^ThirteenCO₂The isotope ratio of the component is
The unknown exhaled gas in the absorption band
¹²CO₂as well as^ThirteenCO₂Unknown sample measurement to measure absorbance
Fixed process, Absorbance measured in the unknown sample measurement step and the calibration curve
From the calibration curve created in the synthesis process,¹²C
O₂Ingredients and^ThirteenCO₂Isotope that determines the isotope ratio of a component
Ratio determining step, and comprising:¹²
CO₂−^ThirteenCO₂In the isotope ratio determination method, When creating a plurality of sample gases in the known sample creation step
And N in the air₂N equal to gas concentration₂Gas components and before
Known isotope ratio¹²CO₂as well as^ThirteenCO₂Gas component
And in the absorption band¹²CO₂as well as^ThirteenCO₂Absorption
Prepare a standard gas consisting of gas components that do not affect the temperature
And The standard gas is diluted with air to obtain the same N₂Gas concentration and
One said isotope ratio and CO 2₂Gas concentration is different
Create multiple sample gases, Further, the plurality of known isotope ratios are different from each other.
Creating multiple sample gases from a standard gas
And obtaining a sample gas for preparing the calibration curve.
Inhalation due to infrared light absorption¹²CO₂−^ThirteenCO₂same
Isotope ratio determination method. 2. A method according to claim 1, wherein the calibration curve generating step is, ¹² CO ₂ and ^{1 3} CO ₂ absorbance in the absorption band determined for the plurality of sample ^gas, 13 CO ₂ absorbance ¹² CO A step of creating a two-dimensional calibration curve for the isotope ratio from the absorbance ratio obtained by dividing by _two absorbances, the known value of the isotope ratio, and a model formula. ¹³ CO ₂ isotope ratio determination method - ¹² CO ₂ in. 3. The method according to claim 2, wherein the model equation is: _{^{However, δ: 12 CO 2 - 13}} CO 2 isotopic ^x: 12 CO ₂ absorbance y: the absorbance ratio _{a ij:} coefficient n = 1 to 4, red, characterized by being represented by m = 1 to 4 ¹² during exhalation due to outside light
CO ₂ - ¹³ CO ₂ isotope ratio determination method. 4. The method of claim A method according to any one of 1 to 3, the air ¹² CO in the exhaled by infrared light, characterized in that an air removal of the CO _{2 2} - ¹³ CO
_2. Isotope ratio determination method.