JPS6324253B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method and device for measuring amino compounds, and more particularly, to a method and device for measuring amino compounds that indirectly measures the concentration of amino compounds contained in blood etc. from the amount of change in conductivity of an absorption liquid. It is something. In recent years, advances in biomedical measurements have enabled the continuous telemetry of physiological variables such as body temperature, gastrointestinal pressure, blood pressure, rate of respiration, and biological potentials, as well as in vivo measurements of PO ₂ and PCO. _2. Measuring methods and devices for continuously measuring blood PH and electrolytes as well as gastric PH have been developed. As one of the above biomedical measurement methods, a specific enzyme is applied to an amino compound in the liquid to be measured, and the following formula (1) is used.
The enzymatic reaction of NH ₃ or
There is a method of indirectly measuring amino compounds in a liquid to be measured by detecting NH ⁺ ₄ with an NH ₃ gas electrode or an NH ⁺ ₄ cation electrode. However, in the above conventional example, the relationship between the output signal from the electrode and the NH ₃ concentration is an exponential relationship according to the Nernst equation as shown in equation (2) below, and the liquid junction potential difference of the reference electrode is The drawback was that minute changes could cause large errors in measuring NH ₃ concentration. E=Eo +2.303xRT/F(log[NH ₃ ]+loga) ...(2) [log[NH ₃ ]+loga = log([NH ₃ ]xa)=log[OH ^- ] ∵[OH ^- ]/[ NH ₃ ]=a E: equilibrium electrode potential, Eo: standard electrode potential,
R: gas constant, T: absolute temperature, F: Faraday constant, [ _NH3 ]: concentration of _NH3 , [OH ^- ]: concentration of OH ^- , a: constant] The present invention was made in view of these drawbacks. The purpose of this is to develop an amino compound that can accurately measure amino compounds in a sample solution even when there is little change in the concentration of amino compounds between normal and abnormal conditions, such as when measuring amino compounds during clinical tests. The purpose of the present invention is to provide a measuring method and a measuring device. A feature of the present invention is that in the method and apparatus for measuring amino compounds, the amino compound in the sample solution is decomposed by an enzyme reaction in which a specific enzyme acts to generate NH ⁺ ₄ or NH ₃ . A reagent is added to the measurement solution to make it alkaline and the NH ⁺ ₄ or NH ₃ is changed into NH ₃ gas, which is then introduced into a chamber on one side of the flow cell, and the acidic solution previously introduced into the chamber on the other side of the flow cell. Alternatively, a reaction is caused by contacting an absorption liquid made of a weakly acidic solution through an NH ₃ gas permeable membrane provided in a flow cell, and indirectly from the amount of change in conductivity of the absorption liquid due to the reaction. The objective is to quantify amino compounds in a liquid to be measured. Hereinafter, the present invention will be explained in detail using figures. FIG. 1 is a configuration explanatory diagram showing an embodiment of the present invention. In the figure, 1 is a flow cell.
A first chamber 3 and a second chamber 4 are adjacent to each other with an NH ₃ gas permeable membrane 2 in between. In addition, the first chamber 3 has an inlet 5.
The second chamber 4 is provided with an inlet 6 and an outlet 5'.
and an outlet 6' are provided. Furthermore, 21 is a sample liquid inlet, 23 is a sample liquid drain port,
In the middle of the flow path from the liquid to be measured inlet 21 to the inlet 6 of the second chamber 4, there is an immobilized enzyme 22 on which a specific enzyme is immobilized and a reagent addition port for adding a reagent to the liquid to be measured. 27 are provided. Furthermore, 24 is an absorption liquid introduction port, which is installed so as to be able to absorb the absorption liquid 26 (for example, 1/100N HCl) in the container 25. Reference numeral 10 denotes an absorption liquid outlet, and in the middle of the flow path from the outlet 5' of the first chamber 3 to the absorption liquid outlet 10, there is an electrode 7 connected to a power source 8 and a galvanometer 9. 7' is attached to a pipe with an inner diameter of about 1 mm and is located near the outlet 5'. The immobilized enzyme 22 may be replaced by a chromatography column filled with the enzyme or an enzyme-immobilized membrane bonded to the NH ₃ gas-permeable membrane 2. 2 is an exploded perspective view of the flow cell 1, and FIG. 3 is an assembled perspective view of the flow cell 1. In Figures 2 and 3, 11,1
7 is a block with inlets 11a and 17a, respectively.
and outflow ports 11b and 17b are provided.
2 and 15 are gaskets, respectively, which are connected to the first chamber 3;
and cavity portions 13 and 16 forming the second chamber 4.
is provided. Further, 14 is an NH ₃ gas permeable membrane. The operation of the embodiment of the present invention having the above configuration will be described below. In FIG. 1, the liquid to be measured introduced from the liquid to be measured inlet 21 passes through the immobilized enzyme 22, and the amino compound in the liquid to be measured is converted into the formula (1) by the action of a specific enzyme. undergoes an enzymatic reaction. After that, the reagent addition port 2 is added to the liquid to be measured.
A reagent such as NaOH is added from step 7 to make the liquid to be measured alkaline, and NH ₃ or NH ⁺ ₄ generated in the previous equation (1) changes to NH ₃ gas. The liquid to be measured reaches the second chamber 4 from the inlet 6, flows out of the flow cell 1 from the outlet 6', and is discharged to the outside from the liquid to be measured outlet 23. On the other hand, the absorbent introduced from the absorbent inlet 24 reaches the first chamber 3 through the inlet 5, flows out of the flow cell 1 through the outlet 5', and is discharged to the outside through the absorbent outlet 10. . Furthermore, the conductivity of the absorbent liquid is measured by electrodes 7, 7' in the middle of the flow path from the outlet 5' to the absorbent liquid outlet 10. Further, the NH ₃ gas in the liquid to be measured in the second chamber 4 passes through the NH ₃ gas permeable membrane 2 to reach the first chamber 3 and reacts with the absorption liquid. When the absorption liquid is HCl, the first chamber 3
An example of the reaction between the NH ₃ gas and the absorption liquid in is shown in equation (3) below. NH ₃ +H ₂ O+Cl ^- →NH ₄ Cl+OH ^- (3) Therefore, due to the reaction between the absorption liquid and the NH ₃ gas in the first chamber 3, the conductivity of the absorption liquid changes, and this change is The absorption liquid flows from the outlet 5' to the absorption liquid outlet 10.
It is detected by electrodes 7, 7' during the period of time. In this way, the amino compound concentration in the liquid to be measured is indirectly quantified from the detected amount of change in conductivity of the absorption liquid through predetermined signal processing or the like (not shown). In addition, in the enzymatic reaction of the above formula (1), it is necessary to maintain the PH value of the reaction system at a predetermined value (so-called optimum PH value) in order to allow the enzymatic reaction to proceed most efficiently with respect to each amino compound. The table below shows the enzyme name, optimal pH value, and enzyme reaction for each amino compound.

【table】

[Table] According to the embodiments of the present invention as described in detail above, the amino compound concentration in the liquid to be measured can be measured more accurately in a shorter time than in the conventional example. There is. FIG. 4 is a configuration explanatory diagram showing another embodiment of the present invention, and in the figure, 28 is an absorption liquid inlet.
In Figure 4, symbols with the same numbers as in Figure 1 (for example, 5'a, 5'b are symbols with the same numbers as 5') are used with the same meaning and will not be explained here. Omitted. In FIG. 4, the liquid to be measured is introduced from the liquid to be measured inlet 21, including the inlet 6a, the second chamber 4a of the flow cell 1a, the outlet 6'a, the immobilized enzyme 22, the inlet 6b, and the second chamber 4a of the flow cell 1b. The liquid to be measured is discharged to the outside through the second chamber 4b and the outlet 6'b from the outlet 23. Furthermore, when the liquid to be measured passes through the immobilized enzyme 22, a specific enzyme acts on the amino compound in the liquid to be measured, and the
The enzymatic reaction of formula (1) (specifically shown in the table above) occurs. Further, NH ₃ or NH ⁺ ₄ generated by the enzyme reaction is changed into NH ₃ gas by adding a reagent such as NaOH from the reagent addition port 27 to make the liquid to be measured alkaline. And the applicable
The NH ₃ gas passes through the NH ₃ gas permeable membrane 2b in the second chamber 4b and reaches the first chamber 3b. It should be noted that amino compounds coexisted in the liquid to be measured from the beginning.
NH ₃ gas is detected when the liquid to be measured is the second one of flow cell 1a.
When flowing through the chamber 4a, it passes through the NH ₃ gas permeable membrane 2a and reaches the first chamber 3a. On the other hand, a part of the absorption liquid 26 in the container 25 is introduced from the absorption liquid introduction port 24, flows through the inflow port 5b, the first chamber 3b of the flow cell 1b, and the outflow port 5'b, and flows through the measurement liquid outlet. 10b. In addition, the other part of the absorption liquid is
8, flows through the inlet 5a, the first chamber 3a of the flow cell 1a, and the outlet 5'a, and is discharged from the liquid to be measured outlet 10a. Furthermore, the first
In the chambers 3a and 3b, NH ₃ gas permeable membranes 2a,
It passed through 2b and reached from the second chambers 4a and 4b.
_NH3 gas reacts with the absorption liquid. Furthermore, in the flow paths from the outlet ports 5'a, 5'b to the outlet ports 10a, 10b, electrodes 7a, 7'a, 7'a, to which power supplies 8a, 8b and galvanometers 9a, 9b are connected, respectively, are connected.
The conductivity of the absorption liquid is detected by 7b and 7'b. In addition, by guiding the electrical signals of the galvanometers 9a and 9b to an arithmetic circuit (not shown) and differentially amplifying them, it is possible to detect amino compounds when an amino compound and NH ₃ gas are present in the liquid to be measured. Measurements are taken. According to other embodiments of the present invention as described in detail above, even when an amino compound and NH ₃ gas coexist in a liquid to be measured such as blood or urine, the influence of the coexisting NH ₃ gas can be suppressed. This method has the advantage that the concentration of an amino compound in a liquid to be measured can be accurately measured without any exposure.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing an embodiment of the present invention;
Figure 2 is an exploded perspective view of the flow cell, Figure 3 is
FIG. 4, an assembled perspective view of the flow cell, is a configuration explanatory diagram showing another embodiment of the present invention. 1, 1a, 1b...flow cell, 2, 2a, 2
b...NH ₃ gas permeable membrane, 3, 3a, 3b...
1st room, 4, 4a, 4b... 2nd room, 5, 5a,
5b, 6, 6a, 6b...inflow port, 5', 5'a,
5'b, 6', 6'a, 6'b... Outlet, 7, 7
a, 7b, 7', 7'a, 7'b... Electrode, 8, 8
a, 8b...power supply, 9,9a,9b...galvanometer,
10, 10a, 10b, 23... discharge port, 21,
24, 28...Inlet, 25...Container, 26...
Absorption liquid, 27...Reagent addition port.

Claims (1)

[Scope of Claims] 1. Means for causing an enzymatic reaction by causing a specific enzyme to act on a test solution containing an amino compound, and a means for producing an enzyme reaction by adding a reagent to the test solution to make it alkaline. NH ₃ or NH ⁺ ₄
means for converting into NH ₃ gas, and the NH ₃ gas
A means for contacting and reacting with an absorbing liquid consisting of an acidic or weakly acidic solution by permeating an NH ₃ gas permeable membrane, and a power source and a galvanometer connected to measure the amount of change in conductivity of the absorbing liquid due to the reaction. 1. A method for measuring an amino compound, the method comprising: detecting the amino compound in the liquid to be measured indirectly from the amount of change in conductivity of the absorption liquid. 2. The measuring method according to claim 1, wherein the amino compound is glutamine and the specific enzyme is el-glutamine amide hydrolase. 3. The measuring method according to claim 1, wherein the amino compound is elasparagine, and the specific enzyme is elasparagine amide hydrolase. 4. The measuring method according to claim 1, wherein the amino compound is L-amino acid and the specific enzyme is L-amino acid oxidase. 5. The measuring method according to claim 1, wherein the amino compound is a deamino acid, and the specific enzyme is a deamino acid oxidase. 6. The measuring method according to claim 1, wherein the amino compound is plasma amine and the specific enzyme is spermine oxidase. 7 A first chamber in which an absorption liquid that absorbs and reacts with NH ₃ gas is introduced from the inlet and flows out from the outlet, forming a channel on one side of the flow cell, and a liquid to be measured is introduced from the inlet. At the same time, it flows out from the outflow port, forming a flow path on the other side of the flow cell, and is connected to the first chamber through an NH ₃ gas permeable membrane to the adjacent second chamber, which acts on the amino compound in the liquid to be measured. an immobilized enzyme on which a specific enzyme that causes an enzymatic reaction is immobilized; a reagent addition port provided in the middle of a flow path from the immobilized enzyme to the inlet of the second chamber; An electrode is provided in the flow path downstream of the outlet of the chamber and is connected to a power source and a galvanometer to detect the conductivity of the absorbing liquid. An amino compound measuring device characterized by indirectly quantifying an amino compound. 8 A first chamber in which an absorption liquid that absorbs and reacts with NH ₃ gas is introduced from the inlet and flows out from the outlet, forming a channel on one side of the flow cell, and a liquid to be measured is introduced from the inlet. At the same time, it flows out from the outlet and constitutes the flow path on the other side of the _flow cell. An electrode is provided downstream and connected to a power source and a galvanometer to detect the conductivity of the absorption liquid, and is additionally disposed upstream of the immobilized enzyme in the flow path through which the liquid to be measured flows to immobilize the enzyme. Claim 7, characterized in that the amino compound in the liquid to be measured is quantified from the difference in the amount of change in conductivity of the absorption liquid detected by two electrodes upstream and downstream of the enzyme.
Amino compound measuring device described in Section 1.